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

In 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.

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.

Introduction

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.

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.

Materials & Methods

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.

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.

Sections to be examined by scanning electron microscopy (SEM) were prepared in 3 ways:

  1. 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).
  2. 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.
  3. 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.

Results

Light Microscopy

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).

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).

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).

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).

basement_membrane_1

Fig 1: 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


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).


basement_membrane_2

Fig 2: 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.


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.

Transmission Electron Microscopy

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).


basement_membrane_3

Fig 3: 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.


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.


basement_membrane_4

Fig 4: 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.

Scanning Electron Microscopy

  1. 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.
  2. 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.

basement_membrane_5

Fig 5: 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.


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).


basement_membrane_6

Fig 6: 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


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).


basement_membrane_7

Fig 7: 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.


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).


basement_membrane_8

Fig 8: 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


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).


basement_membrane_9

Fig 9: 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.


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.


basement_membrane_10

Fig 10: 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.


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).


basement_membrane_11

Fig 11: 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.


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).


basement_membrane_12

Fig 12: 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.


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).


basement_membrane_13

Fig 13: 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.


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).


basement_membrane_14

Fig 14: 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


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).


basement_membrane_15

Fig 15: 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.


basement_membrane_16

Fig 16: 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.


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.


basement_membrane_17

Fig 17: 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.

Discussion

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).

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).

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Acknowledgments

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.

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© C. C. Pollitt

posted here with the permission of the author
first published in Equine vet. J., (1994) 26 (5) 399-407

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