Evaluation of the Integrity of Horse Hoof Dermal and Epidermal Tissues Collected by Dorsal Transmural Access

The purpose of this study was to evaluate the integrity of dermal and epidermal tissues collected by dorsal transmural access. Eight healthy adult horses were subjected to two biopsies of the right hoof, with a 24-hour interval. To ensure hoof integrity, a radiographic evaluation with dorsopalmar and lateromedial positions was performed. The animals were sedated, and the pedal tissue was collected from two sites that were 2 cm distal to the coronary band. For each biopsy, the hoof wall was perforated using an aluminum oxide drill bit, 4.8 mm in diameter that was connected to a drilling machine, yielding a hole of approximately 9.0 mm. The perforation procedure was performed slowly in the stratum medium until 1 mm of it remained, which was determined by evaluating the movement in response to pressure applied with a Halstead forceps. Laminar deep incisions were performed with a number 11 scalpel blade until the distal phalanx was reached. The laminar tissue was then removed using a Frahm scaler. A cuboid laminar sample measuring approximately 5 mm at the base and 7 mm in height was obtained. The samples were suitable for analysis by histology and transmission electron microscopy showing that the ends of the secondary epidermal laminae were round, the basement membrane was intact between the secondary epidermal laminae and secondary dermal laminae, and the ultrastructural analysis of the cells showed their structural integrity.     

Morphological spectrum of primary epidermal laminae in the forehoof of Thoroughbred horses

Reasons for performing the study: Hoof health is a major concern of horse owners as well as the equine industry. However, many questions remain concerning regional variations of laminar junction and its potential to remodel. Hypothesis: To examine regional variations in the morphology of the laminar junction and thickness of the hoof wall in Thoroughbred horses. Methods: The forefeet of 25 Thoroughbred cadavers were examined. Each hoof was divided into 20 blocks through 4 proximodistal slices (below the coronary band, each 1 cm apart) and 5 circumferential positions (toe, medial and lateral quarters and heels). In each block, 25 central primary epidermal laminae (PEL) were considered. Orientation of each lamina in relation to the hoof wall (LO), degree of bending (IA) and the spaces between the adjacent laminae (LS) were measured. Thickness of the hoof wall and number of branched PEL were also measured. Data were analysed using a split‐block design in ANOVA. Results: There were significant differences between the 2 proximal and 2 distal slices in LO and IA data, but not in LS data. Circumferentially, toe blocks were different from heel and quarters blocks. Lateral and medial heels as well as the quarters were mostly different. The hoof wall was slightly thicker laterally than medially. There were more branched PEL on the lateral side of the left hooves and on the medial side of the right hooves. Conclusions: These data add to the circumstantial evidence supporting the hypothesis of adaptive remodelling in the laminar junction. Results of this study signify the capability of PEL to remodel in response to applied stress to the regions of the hoof. Potential relevance: A deeper understanding of the gross and cellular processes of laminar remodelling may well prove to be complementary to an understanding of their failure in laminitis.     

Characterisation and distribution of epidermal growth factor receptors in equine hoof wall laminar tissue: comparison of normal horses and horses affected with chronic laminitis

Epidermal growth factor (EGF) receptors were detected in plasma membrane preparations of equine hoof wall laminar tissue at concentrations comparable to that of equine liver. Scatchard analysis of the equilibrium binding data suggested the presence of two classes of EGF binding sites in most of the controls (plasma membranes from clinically normal horses); a high-affinity class and a more numerous low-affinity class. The dissociation constant of the low-affinity class of EGF-specific receptors (KD = 1 x 10(-9)M) is in reasonable agreement with other values established for the EGF receptor. The variability between individual estimates for the KD of the high-affinity receptor class precluded an accurate estimate for those sites. A possible explanation is discussed. The high-affinity binding sites were uniformly absent in plasma membranes prepared from horses affected by chronic laminitis. Autoradiographic analysis localised the EGF receptors primarily to the secondary epidermal laminae, with an apparent greater density over the proliferative basal keratinocytes. Little label was associated with the dermal or the keratinised primary epidermal laminae. Tissue from horses with chronic laminitis had EGF receptors located uniformly over the hyperplastic epidermal keratinocytes. These data suggest that an EGF-mediated response may be involved in the hyperproliferative response that is characteristic of chronic laminitis.     

Unilateral basement membrane zone alteration of the regenerated laminar region in equine chronic laminitis

Between the laminar epidermis and the laminar dermis of laminar region (LR) in equine foot, it can be observed the basement membrane zone (BMZ), which is composed of a basement membrane and its accompaniments like the hemidesmosome and anchoring fibril. Alteration in the BMZ in equine laminitis is possibly related with not only development but also recovery outcome and recurrence of this disease. However, there is little known about the structure of the BMZ during the recovery phase of this disease. To assess the condition of the BMZ of LR affected by chronic laminitis, the tissue was examined in three cases at two weeks, four weeks and three months after the onset of laminitis, using pathological, immunohistochemical and electron microscopic techniques. Histologically in all laminitis cases, there was a regenerated laminar epidermis with proliferating keratinocytes between the Stratum medium and the dermis, but it included the undeveloped secondary epidermal laminae (ud-SELs) structure in one side of the primary epidermal laminae, especially in the part of the deep area of LR. Immunohistochemical results were positive for the anti-type IV collagen, anti-type VII collagen and anti-laminin 5 antibodies in the most BMZs. However, partial BMZs adjacent to the ud-SELs were negative for the anti-type VII collagen and anti-laminin 5 antibodies. Ultrastructurally, in the BMZ of the ud-SEL, the lamina densa and the lamina lucida were present. In contrast, the anchoring fibrils and the hemidesmosomes were either absent, or present at lower than normal levels. In conclusion, the present study indicated that the part of regenerated LR in chronic laminitis was not able to fully restore to construct the BMZ for a long time, especially in the unilateral side of laminar epidermis. It might be related with recurrence of this disease.     

A scanning electron microscopical study of the dermal microcirculation of the equine foot

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 (40 mu diameter) were found clustered close to the origin of the axial arteries. The density of the laminar AVAs was estimated to be 500 AVAs/cm2. Blood vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog shared a basic structural organisation. 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. AVAs were always present at the base of the papillary units and anastomoses connected the central artery and vein. AVAs 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.     

Light and electron microscopy of keratinization in the laminar epidermis of the equine hoof with reference to laminitis

The laminar epidermis (epidermis parietis) of hooves from 14 clinically normal horses, 6 months to 15 years old, was examined by light and electron microscopy and immunofluorescence to measure the contributions of this region to the formation of the hoof wall. By their progressive keratinization to form primary epidermal laminae, the secondary epidermal laminae ultimately contributed about 20% of the thickness of the hoof wall (as revealed in the white line [zona alba]). The keratinized, primary epidermal laminae were developed to a height of 4 mm during their proximodistal-course, much of this obscured because of their basal portion being embedded in the cap horn epidermis. From evaluation of structural changes accompanying keratinogenesis in the cell and determination of the contribution of the laminar epidermis to the formation of laminar horn, cap horn, connecting horn, terminal horn, and the white line, we concluded that the sterile bed concept of a nongerminative role for the secondary epidermal laminae is no longer tenable.     

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.     

The effect of hoof angle variations on dorsal lamellar load in the equine hoof

Reasons for performing study: In the treatment of laminitis it is believed that reducing tension in the deep digital flexor tendon by raising the palmar angle of the hoof can reduce the load on the dorsal lamellae, allowing them to heal or prevent further damage. Objective: To determine the effect of alterations in hoof angle on the load in the dorsal laminar junction. Methods: Biomechanical finite element models of equine hooves were created with palmar angles of the distal phalanx varying from 0–15°. Tissue material relations accounting for anisotropy and the effect of moisture were used. Loading conditions simulating the stages in the stance where the vertical ground reaction force, midstance joint moment and breakover joint moment were maximal, were applied to the models. The loads were adjusted to account for the reduction in joint moment caused by increasing the palmar angle. Models were compared using the stored elastic energy, an indication of load, which was sampled in the dorsal laminar junction. Results: For all loading cases, increasing the palmar angle increased the stored elastic energy in the dorsal laminar junction. The stored elastic energy near the proximal laminar junction border for a palmar angle of 15° was between 1.3 and 3.8 times that for a palmar angle of 0°. Stored elastic energy at the distal laminar junction border was small in all cases. For the breakover case, stored elastic energy at the proximal border also increased with increasing palmar angle. Conclusions and potential relevance: The models in this study predict that raising the palmar angle increases the load on the dorsal laminar junction. Therefore, hoof care interventions that raise the palmar angle in order to reduce the dorsal lamellae load may not achieve this outcome. See also correspondence by Redden See also correspondence by Curtis     

A preliminary study into the correlation of stiffness of the laminar junction of the equine hoof with the length density of its secondary lamellae

Reason for performing study: The relationship between mechanical behaviour and microscopic structure of the laminar junction of equine hooves under testing conditions requires elucidation. Objectives: To determine mechanical parameters and 2D length density of profiles of secondary lamellae of the laminar junction in the dermal region and to assess possible correlations. Methods: Specimens (25 samples in total) of the laminar junction were taken from front, quarter and heel parts from 3 equine hooves and exposed to a uniaxial tensile test until rupture to obtain Young's moduli of elasticity, ultimate stress and strain. Neighbouring specimens to those used for the biomechanical experiment were processed histologically to assess the length density of laminar junction basement membrane using stereological grids. Results: The estimated median (interquartile range) length density of the laminar junction basement membrane was 0.024 (0.020–0.027)/µm. Young's modulus of elasticity was 0.15 (0.11–0.35) MPa in the small deformation region, and 7.58 (6.14–8.68) MPa in the linear region was. The ultimate stress was 1.67 (1.41–2.67) MPa, and the ultimate strain was 0.50 (0.38–0.70). The Young's modulus of elasticity in the region of small deformations has a moderate correlation with the length density of the laminar junction basement membrane. Conclusions: As with most soft biological tissues, the laminar junction has a nonlinear mechanical behaviour. Within the range of small deformations, which correspond to physiological loading of the laminar junction, a higher length density of the laminar junction basement membrane is correlated with a higher resistance of the laminar junction against high stresses transmitted from the distal phalanx to the hoof wall. Potential relevance: The condition of the laminar junction apparatus may be easily quantified as the length density of profiles of secondary dermal lamellae. This quantification provides a simple tool that could be used for comparing the proneness of the various parts of the laminar junction to initial stages of laminitis.     

Presence of mononuclear cells in normal and affected laminae from the black walnut extract model of laminitis

Reasons for performing study: There is increasing evidence of involvement of inflammatory cells in acute laminitis. Objective: To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)‐induced laminitis. Methods: Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recognise lymphocytes (T and B) and monocytes/macrophages, respectively. Results: Mononuclear cells were present in laminar tissue of normal horses. The majority of CD3‐ and CD20‐positive lymphocytes were localised around the deep dermal vessels but were also evident around vessels of the primary dermal laminae. CD163‐positive macrophages were primarily perivascular in deep dermis or in dermal laminae. No changes in the number of laminar B or T lymphocytes occurred at any time point post BWE administration. However, increases (P = 0.0016) in laminar CD163‐positive cells occurred in the secondary dermal laminae (SDL) in the 1.5H DTP and 3H DTP groups, returning to basal values in LAM group. Conclusions: Lymphocyte and macrophage populations are present in the laminar tissue of clinically normal horses and BWE administration induces an increase in CD163‐positive macrophages in SDL. Potential relevance: Both the host tissue population of mononuclear cells and the influx of monocytes may play an important role in the pathophysiological changes leading to laminar injury.     

Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study

BACKGROUND: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis. HYPOTHESIS: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis. ANIMALS: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5). METHODS: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2. RESULTS: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.     

Leukocyte emigration in the early stages of laminitis

The mechanisms that initiate the pathophysiologic changes in the digital laminae in equine laminitis are poorly understood. Due to the fact that (1) the horse at risk of laminitis has many similarities clinically to the human sepsis patient and (2) our recent finding of marked laminar proinflammatory cytokine expression at the developmental time point of the black walnut extract (BWE) model of laminitis, we tested the possibility that, similar to organ damage in human sepsis, leukocyte emigration is an early event in laminitis. Using immunoperoxidase methods with an anti-equine CD13 monoclonal antibody that recognizes neutrophils and monocytes, we discovered that, whereas the dermal microvasculature of the skin commonly has a marginal pool of leukocytes, the normal laminar dermal microvasculature has minimal to no perivascular leukocytes. However, increases in leukocyte numbers occurred around the dermal vasculature of both the laminae and the skin in the majority of BWE-treated horses in the developmental stage and at the onset of clinical signs of lameness in the BWE model. These findings indicate that, similar to organ failure in human sepsis, leukocyte emigration is likely to play a significant role in initiating numerous pathophysiologic mechanisms that lead to the development of laminitis.     

Equine laminitis model: Lamellar histopathology seven days after induction with oligofructose

Reasons for performing study: The histopathology of laminitis during its transition from the acute to the chronic phase has not been previously documented. Studying hoof lamellar tissues 7 days after induction of laminitis may provide insight into the intractable nature of the chronic phase of the disease. Objectives: To induce laminitis and investigate hoof wall lamellar tissues 7 days after dosing. Methods: Laminitis was induced using oligofructose in 6 normal Standardbred horses. The dorsal hoof lamellar tissues of these and 12 normal horses were processed and examined by light microscopy. Serial sections of a lamellar tip affected by laminitis were used to create a 3 dimensional reconstruction. Results: Transverse sections of dorsal hoof wall lamellae were significantly longer than normal. Many secondary epidermal lamellae were not connected to primary lamellae and existed as spherical or ovoid, discrete islands isolated in the lamellar dermis. The lamellar basement membrane was intact. Conclusions: Lamellar tissue has the ability to reorganise rapidly following an episode of acute laminitis. Although histopathological evidence of ongoing acute laminitis was absent by 7 days, there was marked disruption of lamellar architecture. Potential relevance: The architecture and subsequent strength of the resultant lamellar interface could be greatly influenced for the better by strategies that minimise mechanical displacement during the acute phase of laminitis.     

Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof

Reasons for performing study: Abnormal epidermal stem cell regulation may contribute to the pathogenesis of equine chronic laminitis. Objective: To analyse the involvement of p63, a regulator of epidermal stem cell proliferative potential, in chronic laminitis. Methods: Epidermal tissues from skin, coronet and lamellae of the dorsal foot were harvested from 5 horses with chronic laminitis and 5 control horses. Tissues were analysed using histopathology, immunofluorescence microscopy and quantitative immunoblotting Results: Hoof lamellae of laminitic horses had a lower frequency of p63 positive cells than control lamellae, particularly in the distal region. Quantitative immunoblotting confirmed reduced p63 expression in the laminitic distal lamellar region. The decreased p63 expression in laminitic epidermal lamellae was most apparent in the abaxial region adjacent to the hoof wall and highly associated with the formation of terminally differentiated, dysplastic and hyperkeratotic epidermis in this region, whereas lamellae from control horses maintained high p63 expression throughout the axial‐abaxial axis. Conclusions: Expression of p63 in equine skin resembles that reported in other species, including man and rodents, suggesting that p63 can serve as a marker for the proliferative potential of equine epidermal stem cells. p63 expression was significantly lower in the chronic laminitic hoof than in that of control horses, suggesting laminitic hoof epithelium has more limited proliferative potential with a shift towards differentiation. This may reflect reduced activity of epidermal stem cells in laminitic hoof. It is proposed that p63 contributes to the maintenance of hoof lamellae and that misregulation of p63 expression may lead to epidermal dysplasia during lamellar wedge formation. Potential relevance: This study suggests that loss of epidermal stem cells contributes to the pathogenesis of equine laminitis. Autologous transplantation of p63‐positive epidermal stem cells from unaffected regions may have regenerative therapeutic potential for laminitic horses.     

Equine laminitis caused by distal displacement of the distal phalanx: 12 cases (1976-1985)

Clinical data from 12 cases of equine laminitis characterized by distal displacement of the distal phalanx (P3) were reviewed. Clinical features of horses that survived the syndrome were compared with the nonsurvivors to obtain prognostic indicators. Animals affected included 8 Quarter Horses, 2 Welsh ponies, 1 Thoroughbred, and 1 Arabian. Eight of the animals were females (67%), 2 were stallions, and 2 were geldings. The mean age of affected animals was 8.6 years (2 to 14 years), and the mean body weight was 442 kg. The survivors weighed less than the nonsurvivors (384 kg vs 473 kg, respectively), suggesting that body weight may be of prognostic value for horses affected with distal displacement of P3. Ten of the 12 animals (83%) were admitted because of a disorder other than laminitis, but subsequently developed laminitis during the treatment period. All affected animals had clinical evidence of endotoxemia and/or sepsis before the onset of laminitis. Cavitation or depression of the dorsal coronary band was detected in all animals and was the most reliable clinical indicator of distal displacement of P3. Five horses had fluid (blood or serum) ooze from their coronary bands and 2 of these sloughed one or more of their hooves. Necropsy findings of the 8 horses that were euthanatized included severe hemorrhagic, congested laminae and complete detachment of P3 from the hoof wall. Histologic examination of affected laminae revealed vascular thrombosis and multifocal areas of hemorrhage and necrosis. Radiography failed to reveal distal displacement of P3 in 8 animals, but the remaining 4 animals had an accentuation of the dorsal proximal hoof wall and cavitation of the coronary band visible on lateral radiographs.     

Is there a characteristic distal tarsal subchondral bone plate thickness pattern in horses with no history of hindlimb lameness?

REASONS FOR PERFORMING STUDY: Osteoarthritis (OA) is a common cause of distal tarsal pain, but disease development is poorly understood. Awareness of normal tarsal structure and function is important in order to understand the pathogenesis of OA. Thickening of the subchondral bone (SCB) plate has been related to the development of OA, but SCB plate patterns in the equine tarsus have not been documented. HYPOTHESES: There is a repeatable pattern of SCB thickness across the distal tarsal joints, and specifically that thickness would be greatest dorsally and laterally. METHODS: Twenty cadaver tarsi were collected from mature horses that had undertaken low-level exercise only with no history of hindlimb lameness. Magnetic resonance images were acquired using a high-resolution sagittal 3-dimensional T1-weighted spoiled gradient echo sequence. Subchondral bone thickness was measured on sagittal images at dorsal and plantar locations on the proximal and distal aspects of the central (CT) and third (T3) tarsal bones and proximal aspect of the third metatarsal bone (Mt3). RESULTS: On the proximal aspect of CT, medial and lateral SCB thickness were significantly greater than midline. On the distal aspect of CT and T3 and proximal Mt3, lateral SCB thickness was significantly greater than medial and midline sites. Dorsal SCB thickness was greatest on the proximal and distal aspects of CT and proximal Mt3. Subchondral bone accounted for a greater proportion of CT and T3 on the dorsal aspect than the plantar. CONCLUSIONS: There is a repeatable pattern of SCB thickness in the distal tarsal bones of horses with no history of hindlimb lameness. This reflects the pattern of loading across the joints. POTENTIAL RELEVANCE: This study provides evidence of a consistent osteochondral pattern in the equine tarsus for reference in identification of osteoarticular pathologies.     

Effects of the addition of endotoxin during perfusion of isolated forelimbs of equine cadavers

Objective-To examine the effect of endotoxins on metabolism and histopathologic changes of isolated perfused equine forelimbs. Sample-Forelimbs (comprising the metacarpus and digit) were collected from cadavers of 12 healthy adult horses after slaughter at an abattoir (14 limbs; 1 forelimb of 10 horses and both forelimbs of 2 horses). Procedures-Forelimbs were perfused for 10 hours with autologous blood, with and without the addition of endotoxin (80 ng of lipopolysaccharide [LPS]/L). Two limbs of the endotoxin exposure group and 2 nonperfused limbs were loaded to failure of the suspensory apparatus of the pedal bone to evaluate the effect of body weight. Metabolic and histologic variables were evaluated. Results-Blood pressure increased during the first hour and did not differ between groups. Lactate dehydrogenase activity was similar in both groups and increased significantly during the 10-hour period; glucose consumption at 5 hours and lactate concentration at 8 hours were significantly higher in limbs exposed to endotoxin. The width of secondary epidermal lamellae was greater in LPS limbs. In the primary dermal lamellae of LPS limbs, there were significantly more vessels with an open lumen and aggregates of intravascular neutrophils. Conclusions and Clinical Relevance-In the blood-perfused isolated forelimbs of equine cadavers, exposure to LPS led to significant changes in the laminar tissue as well as to metabolic changes. Therefore, endotoxin should be considered as a causative factor for laminitis and not merely as a risk factor.     

Ultrastructure of the horse tongue: further observations on the lingual integumentary architecture

This investigation examined primarily epidermal specializations of the adult horse tongue by light, scanning and transmission electron microscopy. Samples were collected from seven regions of the normal tongue of various breeds of horse. The filiform papillae, present on the dorsal and lateral aspects but not the ventral aspect of the tongue, were short, slender and finger-like structures with variable-shaped terminae. The epidermal thickness and height of dermal ridges were reduced on fungiform and vallate papillae, but tissue architecture and keratinocyte ultrastructure of most of the lingual epidermis corresponded to the common mammalian epidermal paradigm. One unique finding was the highly localized clustering of epidermal cells with exceptionally high content of PAS-negative trichohyalin cytoplasmic granules at a location atop the dermal ridges and beneath the base of filiform papillae. These granular cells were immediately subjacent to clusters of clear, non-granulated epidermal cells. It is believed that this integumentary specialization may enhance the structural strength at this localized site of the tissue architecture, in relationship to the mechanical papillae.     

Calprotectin in Myeloid and Epithelial Cells of Laminae from Horses with Black Walnut Extract-Induced Laminitis

Background: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin.
Hypothesis: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis.
Animals: Twenty adult horses.
Methods: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed.
Results: Laminar epidermal CP signal was significantly increased ( P = .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups ( P = .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group.
Conclusions and Clinical Importance: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.