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.     

Histopathology of insulin-induced laminitis in ponies

Reasons for performing study: Ponies with laminitis associated with insulin resistance and hyperinsulinaemia lack systemic and/or intestinal inflammatory signs, suggesting a different pathogenesis potentially reflected in differing histopathology. Objectives: To describe the histological appearance and quantify morphological changes in primary and secondary epidermal lamellae (PEL and SEL) of laminitis lesions from ponies with insulin‐induced laminitis. Methods: Equine hoof lamellar tissue was obtained from 4 control ponies and 5 ponies with laminitis induced following infusion of insulin (1036 ± 55 µU/ml) while maintaining euglycaemia for 55.4 ± 5.5 h. Sections from all 4 hooves were stained and examined by a veterinary pathologist. Measurements of lamellar length (PEL and SEL) were made in mid‐dorsal sections of the right forefeet by 2 blinded observers. Immunolabelling for calprotectin was performed using a monoclonal antibody. Results: No lesions were detected in normal ponies. Lesions detected in ponies with laminitis were variable in severity between ponies. Within ponies, SEL lesions were more severe along the axial region of PEL. Lesions included swelling, disorganisation and abnormal keratinisation of epidermal cells, increased mitotic activity and apoptosis. Separation of basement membranes was minimal. Immunostaining revealed inflammatory cells within the lamellar dermis. SEL were significantly elongated in laminitic hooves relative to controls, with the greatest elongation in those attached to abaxial and middle regions of PEL. Conclusions: Laminitis induced by prolonged infusion of insulin lacked widespread basement membrane disintegration, and increases in epidermal cellular proliferation at axial aspects were marked for this acute stage of disease. Potential relevance: Defining equine laminitis entirely in terms of separation of the basement membrane may not be appropriate for laminitis associated with hyperinsulinaemia.     

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation.These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis.     

Equine laminitis: Ultrastructural lesions detected in ponies following hyperinsulinaemia

Reasons for performing study: Anatomical changes in the hoof lamellar tissue induced by prolonged hyperinsulinaemia have not been described previously. Analysis of the induced lesions may promote understanding of hyperinsulinaemic laminitis pathogenesis and produce clinical benefit. Objectives: To use light and transmission electron microscopy (TEM) to document hoof lamellar lesions in ponies clinically lame after prolonged hyperinsulinaemia. Methods: Nine clinically normal, mature ponies were allocated randomly to either a treatment group (n = 5) or control group (n = 4). The treatment group received insulin via a modified, prolonged euglycaemic hyperinsulinaemic clamp technique (EHCT) and were subjected to euthanasia when clinical signs of Obel grade II laminitis occurred. The control group was sham treated with an equivalent volume of 0.9% saline and killed at 72 h. Lamellar tissues of the right front feet were harvested and processed for TEM. Results: Lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamellar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. In 2 ponies, the lamellar BM was thickened. The number of hemidesmosomes/μm of BM was decreased in all insulin treated ponies. Conclusions: Prolonged hyperinsulinaemia causes unique lamellar lesions normally characteristic of acute and chronic laminitis. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. Potential relevance: The study shows that insulin alone, in higher than normal circulating concentrations, induces profound, changes in lamellar anatomy. Medical control of insulin resistance and hyperinsulinaemia may ameliorate lesions and produce clinical benefit.     

Equine laminitis: ultrastructural lesions detected 24-30 hours after induction with oligofructose

REASONS FOR PERFORMING STUDY: The pathology of equine laminitis has been well-documented 48 h after dosing with oligofructose when clinical lameness and lamellar disintegration is well advanced. Further analysis of the earliest lesions, by collecting lamellar samples at the first sign of foot lameness after oligofructose dosing is required in order to increase understanding of the disease. OBJECTIVES: To investigate lamellar epidermal hemidesmosome damage and basement membrane dysadhesion by transmission electron microscopy (TEM). METHODS: Eight clinically normal, mature Standardbred horses were divided randomly into 2 groups of 4. The treatment group were dosed with oligofructose (10 g/kg bwt) and subjected to euthanasia when shifting weight from one foot to other commenced and at the first sign of lameness during walking and turning. This occurred at 24 h in 3 horses and 30 h in one. The sham treatment control group were dosed with water and subjected to euthanasia after 48 h. Lamellar tissues of the front feet were harvested and processed for ultrastructural study using TEM. RESULTS: Examination by TEM showed excessive waviness of the basement membrane zone and pointed tips of some secondary epidermal lamellae, an ultrastructural lesion typical of laminitis. The average number of hemidesmosomes/microm of basement membrane was decreased and their distance from the centre of the lamina densa of the basement membrane was increased. CONCLUSIONS: Laminitis lesions are detectable 24 h after oligofructose administration. POTENTIAL RELEVANCE: Hindgut events occurring in the first 24 h after dosing have begun the destruction of the hoof lamellar interface. Prevention and treatment strategies should precede lameness if they are to be efficacious.     

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

REASONS FOR PERFORMING STUDY: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. OBJECTIVES: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. METHODS: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. RESULTS: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. CONCLUSIONS: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. POTENTIAL RELEVANCE: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.     

Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion

REASONS FOR PERFORMING STUDY: The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. OBJECTIVES: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). METHODS: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were studied using LM and IFM. Plectin, integrin alpha6 and BP230 antibody was used to label hemidesmosome intracellular plaque proteins and anti-BP180 and anti-laminin 5 (L5) was used to label anchoring filament (AF) proteins. Cytoskeleton intermediate filaments were labelled using anti-cytokeratin 14. The primary antibodies of selected sections were double labelled to show protein co-localisation. RESULTS: Laminitis caused reduction of transmembrane integrin alpha6, the AF proteins BP180 and L5, and failure of co-localisation of BP180 and L5. Proteins of the inner hemidesmosomal plaque, plectin and BP230, were unaffected. CONCLUSIONS: Loss of co-localisation of L5 and BP180 suggests that, during the acute phase of laminitis, L5 is cleaved and therefore, the AFs connecting the epidermis to the dermis, fail. Without a full complement of AFs separation at the lamellar dermo-epidermal junction occurs. POTENTIAL RELEVANCE: Suppressing or inhibiting metalloproteinase activity may prevent L5 cleavage and therefore the lamellar dermo-epidermal separation of laminitis.     

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro

REASONS FOR PERFORMING STUDY: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. OBJECTIVES: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. METHODS: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). RESULTS: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. CONCLUSIONS: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). POTENTIAL RELEVANCE: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.     

Histological and morphometric lesions in the pre-clinical,developmental phase of insulin-induced laminitis in Standardbred horses

Lamellar pathology in experimentally-induced equine laminitis associated with euglycaemic hyperinsulinaemia is substantial by the acute, clinical phase (～48 h post-induction). However, lamellar pathology of the developmental, pre-clinical phase requires evaluation. The aim of this study was to analyse lamellar lesions both qualitatively and quantitatively, 6, 12 and 24 h after the commencement of hyperinsulinaemia. Histological and histomorphometrical analyses of lamellar pathology at each time-point included assessment of lamellar length and width, epidermal cell proliferation and death, basement membrane (BM) pathology and leucocyte infiltration. Archived lamellar tissue from control horses and those with acute, insulin-induced laminitis (48 h) was also assessed for cellular proliferative activity by counting the number of cells showing positive nuclear immuno labelling for TPX2.Decreased secondary epidermal lamellar (SEL) width and increased histomorphological evidence of SEL epidermal basal (and supra-basal) cell death occurred early in disease progression (6 h). Increased cellular proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and BM damage occurred later (24 and 48 h). Some lesions, such as narrowing of the SELs, were progressive over this time period (6–48 h). Cellular pathology preceded leucocyte infiltration and BM pathology, indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.     

Tissue-specific dysregulation of cortisol metabolism in equine laminitis

REASONS FOR PERFORMING STUDY: The role of glucocorticoids (GCs) in the pathogenesis of laminitis is incompletely understood. Local tissue activity of GC is regulated by the steroid converting enzyme, 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1). Changes in integumentary (skin and hoof lamellar) 11beta-HSD activity occurring during laminitis could affect the extent to which GCs are involved in its development. HYPOTHESIS: That changes in integumentary 11beta-HSD-1 activity associated with the laminitic condition would lead to elevated local tissue levels of GCs, which could subsequently contribute, through paracrine and autocrine mechanisms, to the further development of laminitis; and that similar changes in 11beta-HSD-1 activity would be evident in both skin and hoof lamellar tissue. METHODS: Activity of 11beta-HSD-1 was determined in skin and hoof lamellar tissue specimens obtained from normal and laminitic horses using a radiometric assay. Skin samples were obtained from 10 normal horses and from 10 horses before and after induction of acute laminitis following administration of starch via nasogastric tube. Hoof lamellar samples were obtained from 10 normal horses, 10 horses following induction of acute laminitis and 4 chronically-foundered horses. Bidirectional 11beta-HSD-1 activity was measured in both skin and lamellar tissues. RESULTS: 11-ketoreductase activity exceeded 11beta-dehydrogenase activity in both skin and lamellar tissues. Cutaneous activity was higher than lamellar 11beta-HSD-1 activity in all groups. Both ketoreductase and dehydrogenase activity increased in skin and lamellae following experimental induction of acute laminitis, but the increase in ketoreductase activity was substantially greater than that for dehydrogenase in the lamellae. Induction of acute laminitis was attended by increases of 227 and 220% in cutaneous dehydrogenase and ketoreductase activity, respectively, and 173 and 398% in lamellar dehydrogenase and ketoreductase activity, respectively (P<0.05). CONCLUSIONS: The 11-ketoreductase moiety of 11beta-HSD-1 plays a role in equine skin and hoof lamellae regarding the regulation of local glucocorticoid activity. Increased 11-ketoreductase activity will lead to increased local tissue GC activity by virtue of conversion of cortisone to cortisol. POTENTIAL RELEVANCE: The laminitic condition is attended by integumentary biochemical changes that enhance the local concentration of cortisol, especially in the hoof lamellar interface. Through multiple and diverse actions, increased local GC activity contributes to the pathogenesis and morbidity associated with laminitis. Pharmacological manipulation of 11beta-HSD-1 deserves further investigation regarding the prevention and treatment of laminitis.     

Magnetic resonance microscopy of the equine hoof wall: a study of resolution and potential

REASONS FOR PERFORMING STUDY: Obtaining magnetic resonance images of the inner hoof wall tissue at the microscopic level would enable early accurate diagnosis of laminitis and therefore more effective therapy. OBJECTIVES: To optimise magnetic resonance imaging (MRI) parameters in order to obtain the highest possible resolution of the structures beneath the equine hoof wall. METHODS: Magnetic resonance microscopy (MRM) was performed in front feet from 6 cadaver horses using T2-weighted fast spin echo (FSE-T2), and T1-weighted gradient echo (GRE-T1) sequences. RESULTS: In T2 weighted FSE images most of the stratum medium showed no signal, however the coronary, terminal and sole papillae were visible. The stratum lamellatum was clearly visible and primary epidermal lamellae could be differentiated from dermal lamellae. CONCLUSION: Most structures beneath the hoof wall were differentiated. Conventional scanners for diagnostic MRI in horses are low or high field. However this study used ultra-high field scanners currently not available for clinical use. Signal-to-noise ratio (S/N) increases as a function of field strength. An increase of spatial resolution of the image results in a decreased S/N. S/N can also be improved with better coils and the resolution of high field MRI scanners will increase as technology develops and surface array coils become more readily available. POTENTIAL RELEVANCE: Although MR images with microscopic resolution were obtained ex vivo, this study demonstrates the potential for detection of lamellar pathology as it occurs. Early recognition of the development of laminitis to instigate effective therapy at an earlier stage and may improve the outcome for laminitic horses. Clinical MR is now readily available at 3 T, while 4 T, 7 T and 9 T systems are being used for human whole body applications.     

Glucose transport in the equine hoof

Reasons for performing study: Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin‐responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. Objectives: To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. Methods: Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2‐deoxy‐D‐[2,6‐³H]glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin‐induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushing's syndrome. Results: Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin‐independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. Conclusions: The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1‐mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. Potential relevance: Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.     

Cytokeratins of the matrices of the chestnut (torus carpeus) and periople in horses with acute laminitis

OBJECTIVES: To determine whether there is a change in the expression of cytokeratins in the epidermal cells of the non-weight-bearing parts of the limb in horses with acute laminitis and thus determine whether the morphologic changes that develop in the periople and chestnut (torus carpeus) of horses early in acute laminitis are caused by inhibition of keratinocyte differentiation. ANIMALS: 8 horses with acute laminitis. PROCEDURE: Tissue specimens were obtained from the chestnuts of all 8 horses and from the stratum externum of the hoof wall of 3 horses. Tissue specimens were obtained within 48 hours of the first clinical signs of laminitis. The cytokeratins were characterized by 1- and 2-dimensional gel electrophoresis, and the tissue distribution of the cytokeratins was studied by immunohistochemical staining. RESULTS: The biochemical findings indicated that the epidermal cells of tissues from horses affected by laminitis contained the same set of cytokeratins as corresponding tissues from clinically normal horses. Immunohistochemistry on sections from specimens of horses with laminitis versus clinically normal horses indicated a difference in the expression of cytokeratin in the basal cells in the matrix of the stratum externum of the hoof wall and in the matrix of the chestnut of horses with laminitis in which the most severe morphologic changes were observed. CONCLUSIONS: Inhibition of keratinocyte differentiation, as observed by immunohistochemical changes, in cells in parts of the chestnut and periople may indirectly indicate that the observed epidermal changes in horses with laminitis are primary and are unaffected by weight-bearing.     

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.     

Cytokeratins of the stratum medium and stratum internum of the equine hoof wall in acute laminitis

The cytoskeleton of living keratinocytes consists mainly of cytokeratins that have polymerised into intermediate filaments. The aim of this study was to describe the expression of cytokeratins in the living epidermal cells of the weight-bearing parts of the equine hoof wall during acute spontaneous laminitis. A total of 9 hooves from 3 horses subjected to euthanasia within 48 h of the first clinical signs of laminitis were sectioned and examined. The cytokeratins in the stratum medium and stratum internum of the hoof wall were characterized by 1- and 2-dimensional gel electrophoresis, and the tissue distribution of the cytokeratins was studied by immunohistochemical staining. The biochemical results showed the same set of cytokeratins as was seen in 8 normal horses, reported on previously, used as controls. The immunohistochemical results indicated a difference between normal horses and horses with acute laminitis in the content of cytokeratins in the basal cells of the matrix of the stratum medium of the hoof wall and in the basal and suprabasal cells in the stratum internum at the mid level of the hoof wall. However, no conclusion could be drawn as to whether this change in the cytokeratin distribution in laminitis was primary or was caused by the initiation of the local tissue-repairing process.     

The timeline of metalloprotease events during oligofructose induced equine laminitis development

Reason for performing study: The role of matrix metalloproteases (MMPs) and the timeline of proteolysis during laminitis development are incompletely understood. Objectives: To determine the temporal progression of selected MMPs and protease regulators during laminitis development. Methods: Five clinically normal Standardbred horses received, via nasogastric intubation, an oligofructose (OF) bolus (10 g/kg bwt). Laminitis induction proceeded for 48 h followed by euthanasia. Lamellar biopsies were obtained prior to dosing and at intervals during the treatment period for analysis (12, 18, 24, 30 and 36 h and at 48 h following euthanasia). Tissue samples were analysed by real‐time PCR, zymography and western blotting. Results: Activation of proMMP‐2 occurs either simultaneously or at least 12 h following lamellar basement membrane (BM) damage, while no activation of proMMP‐9 is seen during OF laminitis induction. Aggrecanase gene expression increased initially at 12–18 h post OF dosing, similar to BM changes. Gene expression of TIMP‐2, a MMP regulator, decreases during laminitis development. Conclusions: The MMP‐2/MT1‐MMP complex may not play a major role in initiating lamellar BM damage. Aggrecanase and TIMP‐2 gene expression appear related to BM lamellar changes. Potential relevance: MMPs, historically thought to cause laminitis, do not appear to play an initiating role in the lamellar lesion. Other host derived proteases and degradation of alternative lamellar matrix components need to be considered.