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Thursday November 23, 2017
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Category: Hoof Anatomy & Physiology
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Summary

Thirty-two dorsal, seek 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. Earliest changes were rounding of the basal cell nuclei and elongation of secondary epidermal lamellae (SELs). Secondary epidermal lamellae tips were pointed instead of round and the basement membrane had separated from the lamellae. In early Grade 1 lesions, this was obvious at the tips of the SELs where the BM had lifted to form teat-shaped bubbles. The absence of BM at the tips of secondary dermal lamellae, along with varying amounts of connective tissue, was considered a progression in severity and classified as Grade 2. Eventually, even the primary epidermal and primary dermal lamellae separated from each other and the empty shells of isolated BM, in what was once the tip of the primary epidermal lamella, signified that a global separation of the epidermal and dermal lamellae had occurred (Grade 3 lesion). The histopathological grading system correlated well with the degree of lameness at the time of euthanasia, (r2=0.94) and apparently described the severity of laminitis accurately, Disintegration of the BM and failure of its attachment to the basal cells of the epidermis appears to be one of the earliest pathological events to occur in acute laminitis and could be the change that initiates the collapse of the lamellar architecture. Hislopathological diagnoses of laminilis arc strengthened when based on sections stained with at least PAS, in addition to routine H&F and should exhibit evidence of the BM pathology described here.

Introduction

The histopathology of equine laminitis at various stages in its development has becn well described (Obel 1948; Garner et al. 1974; Kameya et al. I980; Roberts et al. 1980; Linford 1987). Sudden changes in key cytoplasmic organelles led Obel (1948) and, more recently, Ekfalck et al. (1992) to conclude that the lamellar epidermis was the site of the primary lesion of laminitis. The severity of lamellar epidermal pathology was considered by Roberts et al. (1980) to be directly correlated to clinical signs. Biopsies taken from the dorsal hoof wall as horses developed laminitis induced by carbohydrate overload allowed Linford (1987) to describe the sequence of epidermal lamellar stretching and deformation as the severity of lameness increased. Using light microscopy of haematoxylin and eosin (H&E) stained sections of the lamellar tissues, these authors concentrated on describing the changes which occur within the epidermal cells of the primary and secondary epidermal lamellae. No reference was made to basement membrane pathology and no special connective tissue stains were employed.

In the investigation described here, 32 dorsal, mid-hoof wall, lamellar sections from 8 horses were examined by light microscopy to gain additional information about the nature of the lamellar changes at an early acute stage of laminitis. Because the basement membrane (BM) co-ordinates a number of critically important biological processes between the dermis and the epidermis, attention was focussed on changes to the anatomy of the lamellar BM, attributable to laminitis, as suggested by Pollitt (1994). This was achieved by staining sections with the connective tissue and basement membrane stains periodic acid- Schiff (PAS), Azan and periodic acid silver methanamine (PASM), as well as with H&E. A correlation was sought between the degree of lameness and the severity of the lamellar histopathology. The results of this detailed histopathological study form the basis for discussing the possible biological pathways by which changes in the large intestine and the gut wall alter the lamellar epidermis and lead to laminitis.

Materials & Methods

Sections were obtained from 8 horses used in an experiment to test whether a patented formulation of virginiamycin (Founderguard) would protect horses from developing laminitis after alimentary carbohydrate overload (Rowe et al. 1994). Four of the horses were predosed with Founderguard (2.47 g Virginiamycin/day) for 2 days prior to carbohydrate overload. Each of the 8 horses received 12 g of ground wheat/kg body weight administered into the stomach via a nasogastric tube. It was given as a slurry, in 2 equal amounts, 6 hours apart. The wheat slurry given to the 4 horses in the Founderguard group contained an additional 4.8 mg virginiamycin/kg dose of virginiamycin. The animals were examined and blood samples for L-lactate, D- lactate, blood pH and blood bicarbonate analysis were taken every 8 hours. Rectal temperature, heart rate and faecal pH were also recorded (Rowe et al. 1994). Forty-eight hours after the first administration of the wheat slurry the horses were humanely killed by captive bolt.

Results

Normal lamellar epidermis Characteristics of transverse sections of the lamellar epidermis from horses not in the experiment, and those in the experiment but with no apparent abnormalities.

Haematoxylin and eosin stained sections, which were graded normal, conformed to the descriptions of lamellar histology published by Stump (1967), Leach (1980), Linford (1987) and Pollitt (1994). The following key features of normal lamellar histology were identified.


basementmembranepathology_1

Fig 1: Diagram showing the protocol for obtaining tissue blocks of the lamellar epidermis and dermis. The dotted lines 1-7 represent cuts made with a bandsaw and lines 8 and 9 are cuts made with either a scalpel or reinforced razor blade. It is important to trim all but 1 mm of the hard inner hoof wall to facilitate successful sectioning with a microtome.


In sections stained with H&E, most of the nuclei of lamellar basal cells were oval and situated at the apical pole of the cell. The apical pole is furthest from the BM. The long axis of the basal cell nucleus was oriented at right angles to the keratinised axis of the SEL (inset Fig 2). The range of SEL widths was mean +s.e.13.5 +2.5 Nm.


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Fig 2: A section of normal sectioning epidermal lamellae. The PASM stain identifies collagen, the major protein and structural backbone of the basement membrane. The BM of each secondary epidermal lamellae (SEL) is outlined as a dark brown line (arrowheads). The tips of the SELs are round and club-shaped and the tip of each wafer thin secondary dermal lamellae (SDL) is sharp and tapered. The tip of each SDL is lose (mean s.e. 12 +3um) to the keratinized axis of the primary epidermal     lamellae (PEL). Bar=25 um. PASM. Inset nuclear detail and orientations. The long axis of each oval nucleus is at right angles to the long axis of the SEL.  H&E. Bar=10 um.


PAS stained sections located the glycoprotein constituents of the basement membrane, for example laminin. The basement membrane stained magenta. The BM of normal lamellae penetrated deeply into the crypt between pairs of SELs and clearly outlined the tapered tip of each SDL. The proximity of the SDL tip to the keratinised axis of the PEL was, therefore, readily appreciated; it was always a distance equivalent to the length of one or 2 epidermal basal cells (range of basal cell length = mean ± s.e. 12 ± 3 m). The club-shaped tips of the SELs, as outlined by the BM, were always rounded and never tapered or pointed.

The basement membrane of SDLs, stained with periodic acid silver methenamine (PASM), was a fine black line (Fig 2). The PASM stain identifies collagen, the structural backbone of the BM. In the connective tissue matrix of the SDLs, very fine, black staining, collagen fibres merged with the black-staining collagen bundles of the PDL (Fig 2).

The Azan stain is a relatively poor nuclear and cytoplasmic stain, but clearly showed the nature of the connective tissue of the PDLs and SDLs. The SDL connective tissue consisted of very fine, blue fibres which filled and extended to the very tip of every tapered SDL. The fibres merged into the dermal side of the clearly delineated, blue BM. Staining the connective tissue with the Azan stain emphasised how much narrower the SDLs  were compared to their adjacent SELs. Nevertheless, fine, thin walled capillaries, the diameter of 1 or 2 erythrocytes, were located within the filamentous connective tissue of most of the wafer thin SDLs. Azan stained sections were superior than PAS and PASM for showing the density of the SDL connective tissue.

Grade 1 Lamellar Lesions

H&E stain: At low magnification the keratinised axes of the PELs and SELs appeared normal. However the SELs had decreased in width (mean ± s.e.13 ± 3 gm), were elongated and their tips were less rounded (more pointed) than normal. The nuclei were round instead of oval and more centrally located in the basal cell cytoplasm, abnormally close to the BM (Fig 3). When oval nuclei were located they were abnormally orientated with their long axes parallel to the long axis of the SEL (Fig 3 inset). The outline between adjacent SELs was indistinct as if the structures had become confluent. This early change was difficult to appreciate with H&E stained sections because the BM and the SDL connective tissue did not stain clearly.


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Fig 3: A stained section of secondary epidermal lamellae (SELs) affected by Grade 1 laminitis. The SELs have an attenuated appearance, are smaller than normal in width (mean + s.e.13 +3 um) and have pointed tips. Many of the basal cell nuclei are round instead of oval. When an oval nucleus is located its long axis is abnormally oriented parallel to the long axis of the SEL (arrows). Bar=25 Nm. HEE. Inset shows detail of round nuclei and abnormal nuclear orientation. Bar=10um. H&E.


PAS stain: The clear symmetry of the BM, outlining the alternating rows and SELs and SDLs, was lost (compare Fig 4 with Fig 2). The BM at the base of many of the SELs was absent and the SELs adhered to each other. At this stage of laminitis, most of the SDL tips were still close (12 to 3 pm) to the keratinised PEL axis. Where the fine connective tissue of the PDL was absent, PAS-positive BM was still present, attached to the remaining SEL basal cells. However, the PAS-positive line of the BM was often wavy in outline, blurred and sometimes broken. Isolated elements of the broken wavy BM were sometimes present in the otherwise empty SDL. The most consistent lesion revealed by the PAS stain was at the tips of the SELs (Fig 4). The BM did not adhere to the epidermal basal cells of the SEL tip, but was separated from them by a cytoplasm-free space resembling a teat-shaped bubble. In some cases, the BM covering the bubble was confluent at its tip, forming a thin taper. This teat-shaped bubble, formed by the BM and present at the tip of many SELs, was always angled in the direction of the PDL base. The fragmented, PAS-positive BM of capillaries, with endothelial cells still attached, was occasionally seen.


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Fig 4: A stained section of secondary epidermal lamellae (SELs) affected by Grade 1 laminitis. The basement membrane (BM) is blurred, thickened and in parts faint in outline (arrows). The SELs are elongated with pointed tips. The tips of most of the secondary dermal lamellae (SDLs) are still close (mean ± s.e. 12 ± d) to the keratinised axis of the primary epidermal lamella (PEL). At the tips of the SELs the BM is often not attached to the epidermal basal cells and appears to have separated from the epidermal basal cells to enclose a cytoplasm free space resembling a teat shaped cap (arrowhead.s). Bar=25 Nm. PAS. Inset shows detail of pointed SEL tip and blurred BM. Bar=10um. PAS.


PASM stain Staining the collagen component of the basement membrane with PASM showed that the apparent loss of BM collagen, based on its failure to stain, was one of the first changes to occur in acute laminitis. In general, the changes in the BM resembled the changes observed with the PAS stain. However, the magnitude of the difference between normal and Grade 1 laminitis appeared greater remaining was around the bases of the SDLs at the tips of the SELs. Wavy bundles of collagen stained densely in the PDL, confirming that the proper staining reaction had occurred.

Azan stain: In contrast to sections stained with H&E, examination of Azan-stained sections in the Grade 1 category gave an immediate impression of abnormality (Fig 5). Blue staining connective tissue had disappeared from many of the SDLs, leaving many SDLs separated from their neighbours by spaces. Where such spaces were absent the SELs adhered to each other, often in bunches.


basementmembranepathology_5

Fig 5: A stained section of secondary epidermal lamellae (SELs) affected by Grade 1 laminitis. There is no blue staining connective tissue in most of the secondary dermal lamellae (SDLs). The SELs are separated from each other by empty spaces instead of connective tissue. The tips of the SELs are pointed as if stretched in the direction of the primary dermal lamella (PDL). Much of the connective tissue in the PDL appears to be elements of basement membrane (arrows) no longer attached to the epidermal basal cells. Bar=25 pm. Azan. Inset is detail of SEL tip and free BM in PDL. Bar=10 um. Azan.


The remains of the fine SDL connective tissue, apparently no longer attached to the BM, accumulated in bundles around the bases of the SDLs, adjacent to the tips of the SELs. Interwoven amongst the strands of fine, nonattached, connective tissue were free, pink-staining nuclei apparently derived from epidermal basal cells. Many capillaries appeared to have ruptured and there were many erythrocytes scattered throughout the connective tissue elements. The lumina of some capillaries contained uniform orange/red staining proteinaceous material.

Grade 2 Lamellar Lesions

H&E stain: At low magnification the PELs appeared relatively normal. However, the PEL tips had a stretched and wavy appearance. The absence of connective tissue between SELs was widespread and the rounding of the SEL basal cell nuclei was more obvious (Fig 6). Neutrophils were present, particularly around vessels adjacent to the PEL tips.

At high magnification, many of the basal cell nuclei, particularly those adjacent to the SDLs in which connective tissue was absent, were small and darkly stained. Nearly all the basal cell nuclei were round instead of oval and were situated close to the BM rather than in the normal apical position. The basal cell cytoplasm was pale, cloudy and vacuolated. Many of the SELs had tapered tips that streamed off into the PDL in the direction of the distal phalanx (Fig 6). Cellular vacuolation, small round nuclei and lamellar attenuation were most pronounced near the tips of SELs. The regular symmetrical arrangement of the lamellar anatomy had disappeared. The basal cells at the tips of PELs were the least abnormal. Many had oval, normal staining nuclei, situated in the normal position close to the apex of the cell and there were mitotic figures suggesting a proliferative response.


basementmembranepathology_6

Fig 6: A stained section of secondary epidermal lamellae (SELs) affected by Grade 2 laminitis. The SELs have tapered tips and a stretched and wavy appearance. Some of the SEL tips contain only single basal cell nuclei arranged in rows (arrows). The BM is no longer close to the keratinised axis of the primary epidermal lamella (PEL) and most of the secondary dermal lamellae (SDLs) are depleted of connective tissue particularly at their tips (arrowheads). Many of the basal cell nuclei are round instead of oval. Bar=25 um. H&E. Inset shows detail of tapered SEL tip. Bar=10 um. H&E.


PAS stain: The PAS stain confirmed that the BM between the SEL bases (and hence the tips of the SDLs) was no longer close to the keratinised axis of the PEL. The distance between BM and the edge of the PEL axis had increased from 12 ± 3 to 52 ± 17 um. This change in the relationship of the BM to the keratinised axis of the PEL was the major difference between Grade 1 and Grade 2 laminitis. Apart from being wavy in outline and intermittently blurred, the BM was relatively intact in the SEL mid-region and near the tips. However, the characteristic BM enclosed, teat-shaped bubble, was present at the tips of most SELs.

PASM stain: Changes seen in PASM stained sections resembled those in PAS stained sections, but PASM staining was useful to confirm that the changes to the BM involved the collagen components of the BM and PAS-positive glycoproteins (Fig 7).


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Fig 7: A stained section of secondary epidermal lamellae (SELs) disected by Giade 2 laminitis. The BM (arrowed), which is normally close to the keratinised axis of the PEL, is now some distance away from it (mean ± s.e. 52 ± 17 instead of 12 ± 3 um). There is virtually no connective tissue in the SDL tips and the epidermal basal cells of adjacent SEIs, apparently denuded of BM in this region, have become confluent. At the tip of each SEL the BM has separated from the basal cells and formed a BM enclosed, teat shaped bubble (arrowheads). Bar=25 um: PASM. Inset shows detail of BM enclosed teat shaped bubble and blurring of BM staining. Bar=10 um. PASM.


Azan stain: The lack of stainable connective tissue between adjacent SELs was evident with the Azan stain. Most of the SDLs were empty of connective tissue except at the PEL tip where the SELs were confluent.

At high magnification, the lack of the SDL connective tissue was confirmed and the BM stained as a broken, sometimes blurred, blue line. Between the bases of the SELs there was no BM and adjoining SELs were confluent. Therefore, the tip of the SDL was no longer close to the keratinised PEL axis as it was in normal lamellae and in Grade 1 laminitis lesions.

Isolated BM, which appeared to have separated from the bases of the SEL basal cells, was present in wavy bundles near the tips of the SELs and along the edges of the PDLs. The tips of the SELs were tapered instead of rounded and many of the tapered SEL tips showed the BM enclosed, teat-shaped bubble.

Grade 3 Lamellar Lesions

H&E stain: The keratinised axial cores of the PELs were still intact, but instead of the PEL tips having an overall rounded shape, the tips were tapered and shrunken. The axial cores of the SELs were barely recognisable as distinct architectural entities. Most of the epidermal cells formed an amorphous mass between alternating primary epidermal and dermal lamellae. The few SELs that were recognisable were devoid of connective tissue attachments.

At high magnification, the tapered PEL tips consisted of wavy empty tubes containing pink material and the occasional epidermal basal cell nucleus. Some of the epidermal cell nuclei were pyknotic. Further from the original PEL tip, the anatomy was recognisable as the remnants of SELs. The cytoplasm of the SEL basal cells was light pink, cloudy and vacuolated. There was extreme elongation and stretching of the SELs. Many of the nuclei were spindle shaped and elongated in the direction of the lamellae. There was no recognisable connective tissue between SELs, most of the epidermis appeared as an amoiphous mass of elongated nuclei and basal cell cytoplasm interspersed between the remnants of the keratinised axes of SELs. Although there were many pyknotic nuclei in the epidermal structures, many seemed intact and relatively unaffected by surrounding disarray.

This, the most severe of the laminitis lesions, was not characterised by oedema. Blood vessels adjacent to the tips of the PELs were surrounded by leucocytes but the inflammatory response was minimal. Blood vessels in the sublamellar dermis appeared normal. A few lamellar capillaries contained proteinaceous, amorphous, pink staining material but this was by no means generalised.

PAS stain: The PAS stain confirmed that the remains of the PEL tips were devoid of epidermal cells and consisted almost entirely of PAS-positive BM (Fig 8).


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Fig 8: A stained section of secondary epidermal lamellae (SELs) affected by Grade 3 laminitis. The PEL tip consists entirely of collapsed tubes of BM (arrows) devoid of epidermal cells. There are many neutrophils in the surrounding dermis and some appear to have crossed the BM and are within the epidermal compartment (arrowheads). Bar=25 um. PAS. Inset shows neutrophils in BM enclosed epidermal compartment Bar=10 um. PAS


Many of the PEL tips resembled the collapsed fingers of an empty glove. The accumulation of stain was particularly dense where the 2 BM layers of the collapsed tubes were side by side. There were PAS-positive neutrophils surrounding the PEL tips and some appeared to be between layers of BM and, therefore, within the epidermal compartment (Fig 8). The tapered tips of a few SELs in the mid-PEL region still had a BM covering but the bulk of the BM appeared to have been stripped from the epidermal basal cells and was lying free beside the connective tissue of the PDL (Fig 9).


basementmembranepathology_9

Fig 9: A stained section of the mid lamellar region of epidermal lamellae affected by Grade 3 laminitis. Lamellar BM is still present, but is now a long distance from the axis of the primary epidermal lamella (PEL). Most of the epidermal cells have coalesced into an amorphous mass and the BM of each SEL appears to have separated from the epidermal basal cells and lies free, in strands, among the connective tissue of the PDL (arrows). Bar=25 um. PAS. Inset shows strands of free BM in PDL. Bar=10um. H&E.


PASM stain: Examination of PASM-stained sections revealed the same changes as those in PAS-stained sections.

The tapered PEL tips consisted mainly of wavy, hollow, tubes of blue connective tissue within which were remnants of a few pink epidermal cells and their nuclei. High power examination of the tapered PEL tips showed that the bulk of connective tissue consisted of wavy tangled masses of isolated BM. Where the BM was still arranged in tubes the epidermal cells appeared as remnants of vacuolated epidermal cytoplasm with an occasional nucleus.

In the mid-PEL region a similar reaction was seen. Basement membrane was recognisable around the tapered stretched tips of a few SELs but the bulk of the BM occurred as free wavy strands along the edges of the PDLs. The remaining SELs, stripped of their BM, coalesced on either side of the axial core of each PEL. The stretched broken remnants of a few SEL keratinised cores were present amongst the epidermal basal cells. The BM of the SDL, a prominent feature in normal tissue stained with the Azan stain, was largely unrecognisable in these sections. Relationship between histological changes and the clinical assessment of lameness prior to euthanasia.


basementmembranepathology_10

Fig 10: The score for lamellar histology regressed against the Obel grade lameness of the 8 horses used in the study.

The graded lamellar lesions, for the 4 hoof samples taken from each horse, were added to give the lamellar histology score (Table 1). The score for the lamellar lesions (x axis), determined by histological examination for each horse, correlated with (Fig 10) the Obel lameness score (y axis), determined by clinical examination prior to euthanasia 48 hours after the administration of ground wheat (Table 1 ) and was described by the following equation: y = - 0.19 + 0.262 (rz = 0.94).

Discussion

The close correlation between histological changes and clinical signs of lameness indicate a good agreement between the 2 assessment techniques. The results suggest that the assessment of lamellar histological damage by the 4 stage grading system accurately describes the severity of equine laminitis. The novel histological findings, particularly the changes to the lamellar BM, suggest new contributions to the pathogenesis of laminitis.

The most significant finding in this study is damage to the lamellar basement membrane. The apparent loss of collagen from the BM of the SELs, as detected by the PASM stain, represents a serious early insult to the integrity of this key lamellar constituent. Type IV collagen forms the axial scaffold of the BM around which the glycoproteins laminin, heparin sulphate proteoglycan and other molecules are arranged (Leblond and Inoue 1989). The changes in the PASM-stained sections reported here provide evidence that disintegration of the BM and detachment from the epidermal basal cells is one of the earliest pathological events to occur in acute laminitis. It seems probable that the altered BM could be a change which initiates acute laminitis and leads to failure of the lamellae. The strands of separated BM, clearly identified in this study, were always oriented towards the base of the PDL, as if being stretched in that direction (perhaps by the pull of the descending distal phalanx). The results presented here are consistent with the observation of Roberts et al. (1980) that fibrillar material was present at the tips of the PELs in H&E stained sections. The fibular material, although unidentified at the time, was probably the residue of isolated BM as detected by the PAS and PASM stains shown here in Figure 8. While Roberts et al. (1980) assumed the spaces between adjoining SELs provided evidence of oedema, an altenative explanation is that the failure of epidermal cells to adhere to a compromised BM has simply left the SDLs empty of connective tissue. When fresh hoof wall tissue from horses affected by Grade 3 histological lesions is trimmed prior to fixation, the spaces between the lamellae can be seen with the naked eye. The spaces appear dry and contain no discernible fluid (C. C. Pollitt, unpublished data). These gas/air filled spaces may be responsible for the radiolucent line which appears palmar to the lamellar hoof in acute cases of laminitis examined clinically. The lack of histological evidence of oedema reported here supports the conclusion of Robinson et al. (1976) that the vascular responses of laminitic animals is normal and that oedema formation is not involved in the pathophysiology of acute laminitis.

Considering the rapidity with which the lamellar BM lesions develop (there can be global separation of the dermal and epidermal lamellae 48 hours after the administration of a laminitis- inducing carbohydrate), it seems unlikely that ischaemia could be a primary event. If enzymatic autodegradation of the lamellae is the initiating lesion then most of the vascular mechanisms, (Hood et al. 1993), must occur as secondary and/or parallel events.

While the cause of the observed BM changes remains speculative, there are a number of mechanisms by which damage could occur. Firstly, it is well established that laminitis is usually preceded by a metabolic crisis elsewhere in the body. Evidence for this hypothesis is provided by the recent study of Weiss et al. (1994) on the role of microvascular thrombosis in carbohydrate induced laminitis. These authors reported a systemic coagulopathy prior to the onset of lameness. This is consistent with elevated concentrations of plasmin initiating fibrinolysis. Apart from its action in fibrolysis, plasmin is a known activator of the metalloproteinase collagenase IV and could activate metalloproteinase destruction of the lamellar BM.

Secondly, BM changes could be initiated through pathologically elevated concentrations of tumour necrosis factor (TNF). Elevated concentrations of TNF are known to circulate in the blood of horses with severe gastrointestinal diseases (May et al. 1992). This can induce the production of collagenase in peripheral tissues (Tracey et al. 1989) which would have destructive effects on the integrity of the basement membrane and consequently the anatomy of the epidermal basal cells of the hoof wall lamellae. Keratinocytes are now known to be competent immunocytes and rapidly produce the potent cytokines TNF and interleukin-lß (IL-Iß) in response to diverse pathological stimuli (Nickoloff and Turka 1993). Alterations to the properties of epidermal and intercellular adhesion molecules (ICAMs) can be induced by cytokines and the dramatic changes in the shape of the SELs, the epidermal basal cells and the orientation of their nuclei, may well be attributable to the cytopathic effect of a systemic and/or locally produced TNF, IL-1b cascade.

A third possibility is that a critical proteolytic event occurring during the development of gut acidosis and laminitis may initiate the degradation of the lamellar BM. Specific zinc-containing type IV collagenases are in neutrophils and a wide variety of mammalian tissues including epithelia. They are metalloproteinases, named for their selective ability to degrade basement membrane collagen type IV (Stetler-Stevenson 1990). Currently, two types of type IV collagenase are known with molecular weights of 72 and 92 kDa. Each has a specific inhibitor known as TIMP-1 and TIMP-2 respectively, after tissue inhibitor of metalloproteinase (TIMP). In normal tissues, type IV collagenase is involved in BM remodelling and turnover. These activities are important in the equine hoof wall, given the BMs large surface area and complexity (Pollitt 1994). The presence of enzymes capable of digesting supporting connective tissue would compromise the integrity of the equine foot. As in all vertebrate systems (Woessner 1991), there is strict regulation of secretion: enzymes are produced in zymogen form, are activated by a multistep process only when required and there are multiple tissue and blood inhibitors to prevent damage. It can be inferred from the data presented here that the changes to the BM and its associated connective tissue, is evidence that proteinase action may be damaging lamellar extracellular matrix. In laminitis, the offending proteinases may be produced in excess within the foot or they may arrive via the circulation from remote sites such as a diseased gastrointestinal tract, uterus or lung. Concomitantly, there may be consumption of tissue inhibitor of metalloproteinase, (TIMP) in the foot or of the collagenase inhibitor, a2-macroglobulin in the blood. The initial activation of the proenzyme form of collagenase IV by enzymes such as plasmin, may set off an autocatalytic process and a `permanently' active form of the enzyme.

It is clear that even 48 hours after the initiation of laminitis the epidermal lamellae were strongly chemotactic to neutrophils. Degranulating neutrophils liberating collagenase IV and other proteases, cytokines and free radicals, could further damage the lamellae. In the Grade 3 laminitis lesions reported here, neutrophils had left the dermis and were within the BM-enclosed epidermal compartment of the PEL tips. To do this they probably lysed the BM barrier by the local production of collagenase IV, just as malignant tumour cells do when they invade and metastasise (Stetler-Stevenson 1990).

The presence and location of the metalloproteinases of the equine hoof has yet to be established. This normal cell product may be manufactured by dermal fibroblasts, the epidermal basal cells or the suprabasal cells adjoining them. Evidence that they exist may be inferred be drawn from the ease with which the equine foot can be exunguilated. It is a simple matter to remove the hoof from an amputated horse's foot if it is kept for a few hours at 60C. Heat is a known activator of tissue collagenase IV. Similarly, trypsin, which activates metalloproteinase by cleaving cysteine from the zinc- containing active site, triggers autolysis and causes the epidermis to separate along a single plane of cells (C. C. Pollitt, unpublished data). The results of an immunohistochemical study, currently underway in this laboratory, should more precisely locate the tissue metalloproteìnases of the equine hoof.

The horses in this study had a variety of lamellar lesions, ranging from mild to severe, despite all being killed 48 hours after the administration of the laminitis-inducing diet. This variability can be explained in part because peak blood D-lactate concentrations occurred at different times in different horses. There were also different concentrations of lactic acid and, therefore, different degrees of acidosis between horses. The grading system described in this study represents damage to the lamellar BM of increasing severity. It may be that it also represents the progression of changes that leads to laminitis and ultimately the breakdown of the connective tissue bond between the distal phalanx and the inner hoof wall. Horses with Grade 3 lesions presumably progressed rapidly through stages 1 and 2.

Therefore, it is suggested that the initial changes in the development of laminitis are rounding of the basal cell nuclei and elongation of SELs whose tips become pointed. The basement membrane detaches from the epidermal cells and begins to separate from the lamellae. Initially, this is apparent at the tips of the SELs where the BM lifts to form teat-shaped bubbles. This also occurs at the tips of the SDLs where the BM, and varying amounts of connective tissue, leave the epidermal lamellae. Without the controlling influence of an intact, functional BM, the SELs distort and elongate further. The BM either disintegrates or separates from the epidermal lamellae in ever increasing quantities until eventually even the primary epidermal and dermal lamellae begin to separate from each other. The empty shells of isolated BM, in what was once the tip of the PEL, signify that global separation of the epidermal and dermal lamellae has occurred. Clinically observable pain occurs when a critical mass of lamellae fail and the anatomy of the inner hoof wall disintegrates.

It would seem advisable that future histopathological research of laminitis should include the examination of sections stained with the PAS stain and should exhibit evidence of the BM pathology described here. The activity of the tissue metalloproteinases (particularly collagenase IV) has recently been shown to correlate strongly with the degree of malignancy and invasiveness of tumours such as malignant melanoma, breast and colon cancer (Goldfarb and Liotta 1986; Stetler-Stevenson 1990). This research in neoplasia has generated a wide range of agents designed to block or enhance tissue metalloproteinase activity both in vitro and in vivo. The identification of BM pathology in the current study suggests that such agents could ultimately be useful in the management of early laminitis.

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