Oral oligofructose challenge reduces expression of glucose transport-1 and 5'-adenosine monophosphate-activated protein kinase in lamellar wall of Holstein heifer claw.

The laminar tissue of bovine laminitis may undergo energy failure. The expression of glucose transport protein-1 (GLUT-1) and 5'-adenosine monophosphate-activated protein kinase (AMPK) affects the energy metabolism of digital laminar tissue. This study aimed to determine the expression of glucose uptake and AMPK in laminar wall corium of Holstein heifer claw by oral administration of oligofructose. A total of twelve clinically healthy Holstein heifers were selected and divided into two groups, including control (CON, n = 6) and experimental (OF, n = 6) groups. The heifers of OF group were given 17 g/kg BW oligofructose dissolved in water (20 mL/kg BW) and the heifers of CON group were given water only (20 mL/kg BW). The laminar tissues were collected after euthanasia. The amount of protein and transcript expression of AMPK and GLUT-1 were determined by western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Expressions of phosphoenolpyruvate carboxy-kinase (PEPCK), receptor-c coactivator1-α (PGC-1α) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were determined by qRT-PCR. The heifers of OF group showed no significant change in the expression and concentration of AMPK. The phosphor-(Thr172) AMPK and GLUT-1 were significantly decreased, while the gene contents of PPAR-γ and PGC-1α were significantly increased. The activation of AMPK and GLUT-1 in digital laminar tissues of heifers was inhibited, which may contribute to digital laminar tissue damage.

Building a dishonest signal: the functional basis of unreliable signals of strength in males of the two-toned fiddler crab, Uca vomeris.

Males of many species use signals during aggressive contests to communicate their fighting capacity. These signals are usually reliable indicators of an individual's underlying quality; however, in several crustacean species, displays of weapons do not always accurately reflect the attribute being advertised. Male fiddler crabs possess one enlarged claw that is used to attract females and to intimidate opponents during territorial contests. After the loss of their major claw, males can regenerate a replacement claw that is similar in size but considerably weaker. As this inferior weapon can still be used to successfully intimidate rivals, it represents one of the clearest cases of unreliable signalling of strength during territorial contests. We investigated the functional mechanisms that govern signal reliability in the two-toned fiddler crab, Uca vomeris. Male U. vomeris exhibit both reliable and unreliable signals of strength via the expression of original and regenerated claw morphs. We examined the morphological, biomechanical and biochemical characteristics of original and regenerated claws to establish the best predictors of variation in claw strength. For a given claw size, regenerated claws have less muscle mass than original claws, and for a given muscle mass, regenerated claws were significantly weaker than original claws. The mechanical advantage was also lower in regenerated claws compared with original claws. However, the activity of three catabolic enzymes did not differ between claw types. We conclude that the structural and physiological predictors of force production influence the frequencies of reliable and unreliable signals of strength in U. vomeris. This study furthers our understanding of the proliferation of unreliable signals in natural populations.

Effect of dietary nonstructural carbohydrate content on activation of 5'-adenosine monophosphate-activated protein kinase in liver, skeletal muscle, and digital laminae of lean and obese ponies.

BACKGROUND: In EMS-associated laminitis, laminar failure may occur in response to energy failure related to insulin resistance (IR) or to the effect of hyperinsulinemia on laminar tissue. 5'-Adenosine-monophosphate-activated protein kinase (AMPK) is a marker of tissue energy deprivation, which may occur in IR. HYPOTHESIS/OBJECTIVES: To characterize tissue AMPK regulation in ponies subjected to a dietary carbohydrate (CHO) challenge. ANIMALS: Twenty-two mixed-breed ponies. METHODS: Immunohistochemistry and immunoblotting for total AMPK and phospho(P)-AMPK and RT-qPCR for AMPK-responsive genes were performed on laminar, liver, and skeletal muscle samples collected after a 7-day feeding protocol in which ponies stratified on body condition score (BCS; obese or lean) were fed either a low-CHO diet (ESC + starch, approximately 7% DM; n = 5 obese, 5 lean) or a high-CHO diet (ESC + starch, approximately 42% DM; n = 6 obese, 6 lean). RESULTS: 5'-Adenosine-monophosphate-activated protein kinase was immunolocalized to laminar keratinocytes, dermal constituents, and hepatocytes. A high-CHO diet resulted in significantly decreased laminar [P-AMPK] in lean ponies (P = .03), but no changes in skeletal muscle (lean, P = .33; obese, P = .43) or liver (lean, P = .84; obese, P = .13) [P-AMPK]. An inverse correlation existed between [blood glucose] and laminar [P-AMPK] in obese ponies on a high-CHO diet. CONCLUSIONS AND CLINICAL IMPORTANCE: Laminar tissue exhibited a normal response to a high-CHO diet (decreased [P-AMPK]), whereas this response was not observed in liver and skeletal muscle in both lean (skeletal muscle, P = .33; liver, P = .84) and obese (skeletal muscle, P = .43; liver, P = .13) ponies.

Expression and activity of collagenases in the digital laminae of horses with carbohydrate overload-induced acute laminitis.

BACKGROUND: Matrix metalloproteinases (MMP) are hypothesized to degrade structurally important components of the laminar extracellular matrix (ECM) in horses with laminitis. OBJECTIVE: To compare levels of expression of stromelysin-1 (MMP-3), collagenases (MMP-1, -13), and membrane type-MMPs (MMP-14, -15, -16), and the distribution of their ECM substrates, in laminae of healthy horses and horses with carbohydrate overload laminitis. ANIMALS: Twenty-five adult horses. METHODS: Gene and protein expression were determined in extracts of laminae using real-time quantitative polymerase chain reaction and Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis. Distribution of MMP-13 and ECM components was determined using indirect immunofluorescent microscopy of nonfixed frozen sections. ECM morphology was assessed by hematoxylin and eosin staining. RESULTS: Of the genes studied, only those encoding MMP-1 and -13 were upregulated in CHO-induced laminitis; MMP-1 at Obel grade (OG)1 lameness and MMP-13 at OG3 lameness. Laminar MMP-1 was present as 52 kDa proenzyme only. MMP-13 was present as pro- (61 kDa) and processed (48 kDa) enzyme. MMP-13 localized to the basal epithelium of the secondary epidermal laminae and its increased expression were accompanied by the appearance in secondary dermal laminae (SDL) of multiple foci that were devoid of collagen I, fibronectin, chondroitin and keratan sulfate glycosaminoglycans, and eosin-staining material. CONCLUSIONS AND CLINICAL RELEVANCE: MMP-13 is upregulated in laminae of horses with CHO-induced OG3 lameness and, by degrading components of the ECM, may contribute to the formation of ECM-free lesions (gaps or tears) that appear in the SDL with OG3 lameness.

Hypoxia and a hypoxia mimetic up-regulate matrix metalloproteinase 2 and 9 in equine laminar keratinocytes.

The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity.

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n=4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n=4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6h (n=4), 12h (n=4), 24h (n=4) and 48 h (n=4), and in normal control horses (n=4). The only change in gene expression observed was an upregulation of MMP-9 (p<0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p<0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract.

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5-15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5h (n=5), 3h (n=6) or 12h (n=10) after BWHE administration. Control horses (n=7) were euthanized 12h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3h BWHE horses, in 6 of the 12h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p<0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8h compared to the control group and at 8 and 10h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.

Leukocyte-derived and endogenous matrix metalloproteinases in the lamellae of horses with naturally acquired and experimentally induced laminitis.

RATIONALE: Inflammation and dysregulation of endogenous matrix metalloproteinase (MMP) production are implicated in the development of equine laminitis. In this study, we examine quantitative relationships among levels of leukocyte-derived proMMP-9 and MMP-9, lamellar proMMP-2 and MMP-2, and expression of proMMP-2 processing enzymes, MT1-MMP/PACE4, as steps towards determining whether inflammation and dysregulation of endogenous MMP production are independent or co-dependent processes. ANIMALS: Archived samples of lamellae from horses with naturally acquired laminitis (n = 12), and from horses administered a pro-laminitic gastric bolus of starch gruel were used, the latter horses falling into two groups: (i) responders (CHO-R, n = 7), which developed Obel grade 3-lameness and (ii) non-responders (CHO-NR, n = 4), which did not become lame. METHODS: Lamellar tissue extracts were analyzed by gelatin zymography to determine gelatinase content and by a myeloperoxidase ELISA to quantify relative monocyte/neutrophil content in the tissue. Real-time PCR was employed to measure gene expression of MT1-MMP and PACE4. RESULTS AND CONCLUSIONS: Extracts of lamellae from control horses, CHO-NR and horses with chronic (non-aggravated) laminitis had similarly low levels of pro and processed MMP-9 and MMP-2. In contrast, proMMP-9 was significantly elevated in extracts of lamellae from CHO-R and horses with naturally acquired acute and aggravated chronic laminitis. Lamellar MMP-2 was also increased significantly in the CHO-R and aggravated chronic laminitis groups, although not in the horses with naturally acquired acute laminitis. Concentrations of proMMP-9 correlated directly with myeloperoxidase content in lamellar extracts, suggesting production/induction by inflammatory leukocytes. In contrast, concentrations of proMMP-2 and MMP-2 were unrelated to concentrations of myeloperoxidase or proMMP-9 suggesting that leukocyte infiltration and dysregulation of endogenous MMP-2 are independent processes most likely with distinct inducers. Neither MT1-MMP nor PACE4 gene expression was elevated relative to controls in any group; this is discussed with respect to proMMP-2 processing in disease. In addition, variability in relative concentrations of lamellar MMPs observed among horses with Obel grade 3-lameness is discussed in the context of laminitis risk assessment and disease outcome.

Equine laminitis: membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset.

REASONS FOR PERFORMING STUDY: Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. OBJECTIVES: To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. METHODS: Tissue samples were obtained from an oligofructose induced model of laminitis. Total RNA was isolated, amplified by RT-PCR, cloned into a vector and sequenced. Real-time PCR was used to quantify MMP-14 and TIMP-2 expression. Rabbit anti-equine MMP-14 antibody was developed to analyse MMP-14 proteins from hoof tissues. RESULTS: Immunohistochemistry detected MMP-14 in the cytoplasm of normal lamellar basal and parabasal cells in close proximity to the lamellar basement membrane. In laminitis affected tissue MMP-14 immunostaining was depleted in lamellar basal cells. Quantitative real-time PCR showed MMP-14 and TIMP-2 expression significantly (P<0.05) elevated and lowered respectively in laminitis affected tissues. CONCLUSION: MMP-14, located in the cytoplasm of normal lamellar basal cells, disappears during laminitis development. The pathology of laminitis is associated with increased and lowered transcription of MMP-14 and TIMP-2, respectively. POTENTIAL RELEVANCE: Enzymes have a role in laminitis pathology and inhibition of their activity may prevent laminitis.

Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance.

We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of the same tissues were screened for SDS-renaturable and native MMP-9 activities by denaturing and non-denaturing gelatin zymography. Samples were also screened for MMP-2 and MMP-9 gene expression by RT-qPCR. Control laminae were devoid of both MMP-9 and neutrophils, whereas neutrophils and SDS-renaturable MMP-9 activity were detected in laminae from BWE-treated horses and were strongly correlated at the acute stage of the disease at which time laminar MMP-9 gene expression was significantly (15-fold) elevated. In contrast, BWE-treatment did not significantly elevate MMP-2 gene or protein expression in the laminae. Interestingly, MMP-9 that was present in extracts of laminae from BWE-treated horses at both the prodromal and acute stages of the disease was mainly in the zymogen form, suggesting that the accumulation of the MMP did not contribute to pathology during these stages. However, elevated presence of the MMP-9 zymogen in the tissue would predispose it to catastrophic damage should conditions arise that cleave the regulatory propeptide domain.

Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses?

To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired laminitis. In addition, the effects of laminitis-inducing treatment have not been prevented or reversed by treatment with an MMP inhibitor or a vasoactive antagonist. It is possible that there is simultaneous activation of the vascular and enzymatic pathways and/or other inflammatory processes. Moreover, the third theory involving mechanical factors cannot be discounted simply because strong evidence for vascular and enzymatic changes exists. It is common for horses with severe musculoskeletal disease affecting weightbearing on a limb to develop laminitis in the contralateral limb. It remains to be determined what factors are responsible for initiation of laminitis in these individuals. Evidence has not been presented that precludes the possibility of coincident occurrence of vascular and enzymatic changes. In fact, many of the inflammatory mediators (e.g. interleukin-1beta) found in laminitic tissues can concurrently stimulate synthesis of vasoactive substances and activate MMPs. Because enzymatic action on proteins is largely dependent on the concentrations of proteins and enzyme, the enzymatic theory is not dependent upon increased delivery of enzymes via increased capillary flow. Likewise, because vascular changes can alter tissue function via increased capillary flow and oedema formation, the vascular theory is not dependent upon decreased capillary flow. It is true that naturally acquired laminitis is widely variable in severity and predisposing diseases. Therefore, most probably there are multiple mechanisms involved in the initiation and propagation of the pathophysiologic cascade(s) and, therefore, successful intervention will necessitate multiple treatment modalities.

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase.

REASONS FOR PERFORMING STUDY: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. OBJECTIVES: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. METHODS: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. RESULTS: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P < 0.01) elevated in laminitis affected tissues. CONCLUSION: The lamellar pathology of laminitis is associated with increased transcription of MMP-2. POTENTIAL RELEVANCE: Real-time PCR analysis of lamellar MMP-2 accurately monitors laminitis development at the molecular level and can be used diagnostically and for testing preventive strategies. Controlling increased MMP-2 transcription may ameliorate or prevent laminitis in high risk clinical situations. Our findings represent a warning to clinicians that the basement membrane lesion of laminitis is insidious and well under way before clinical signs are apparent.

Equine laminitis: cryotherapy reduces the severity of the acute lesion.

REASONS FOR PERFORMING STUDY: The hypometabolic and vasoconstrictive effects of cryotherapy could prevent the development of laminitis. OBJECTIVES: To use distal limb cryotherapy to prevent laminitis induced by alimentary carbohydrate overload. METHODS: Laminitis was induced in 6 Standardbred horses that had one front limb continuously cooled in an ice/water mixture. Lameness evaluation, blinded lamellar histological grading and analysis for lamellar matrix metalloproteinase-2 (MMP-2) mRNA expression were used to evaluate the severity of laminitis. RESULTS: Cryotherapy was well tolerated and effective in cooling the feet. In each horse no lameness was observed in the treated limbs. Laminitis histology scores in the treated limbs were significantly less than those of the corresponding untreated forelimbs (P < 0.05). Laminitis histology scores in the treated limbs were also significantly less than those of the untreated limbs (fore- and hind) as a group (P < 0.05). Expression of MMP-2 mRNA in the iced feet was significantly (P < 0.05) less than that detected in the untreated feet. CONCLUSIONS: Cryotherapy, when applied to one foot, markedly reduced the severity of acute laminitis in this study. We propose that vasoconstriction (preventing delivery of haematogenous trigger factors) and hypometabolism (reduction in lamellar MMP activity) were the primary therapeutic mechanisms. POTENTIAL RELEVANCE: Although further research is needed, we suggest cryotherapy as a potentially effective prophylactic strategy in horses at risk of developing acute laminitis.

Thermolysin activates equine lamellar hoof matrix metalloproteinases.

Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinase-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact when tested on a calibrated force transducer, but when treated with an MMP activator, developed "in-vitro laminitis", separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated MP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and batimastat. These findings suggest that thermolysin-mediated gelatinase activation is not dependent on membrane-bound matrix metalloproteinase (MT-MMP) activation, providing further evidence that bacteria can produce potent MMP activators that probably facilitate host invasion.

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.

Activation of extracellular matrix metalloproteinases in equine laminitis.

Samples of connective tissue obtained from the hoof of six laminitic and eight non-laminitic adult horses were analysed zymographically to investigate whether connective tissue matrix metalloproteinases are activated or induced during laminitis. The activity or matrix metalloproteinases was substantially greater in the tissues from the laminitic horses than in the tissues from the non-laminitic horses. A comparison of the collagenolytic activity in the laminitic and control tissues showed that collagenolytic activities corresponding to the 92 kDa (P < 0.001), 72 kDa (P < 0.01) and 66 kDa (P < 0.01) bands were induced in the laminitic tissues.

Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis.

A method for culturing explants of lamellar hoof was developed to investigate the process of lamellar separation that occurs in laminitis. Explants, consisting of hoof wall, dermal and epidermal lamellae and the adjacent sub-lamellar connective tissue remained intact when cultured in tissue culture medium for 2 days. However, when cultured in the presence of the matrix metalloproteinase (MMP) activator aminophenylmercuric acetate (APMA), the lamellae separated when tension was applied by pulling the hoof wall in an opposite direction to the connective tissue. The separation occurred between the epidermal basal cells and the basement membrane therefore mimicking the lesion of laminitis. Electrophoresis of culture medium from control hoof explants into gradient polyacrylamide gels co-polymerised with gelatin revealed that the explants had produced 2 gelatinases of molecular weight 92 and 72 kDa corresponding to EqMMP-9 and EqMMP-2 respectively. Minor bands of lower molecular weight were the active forms of these enzymes. The zymograms of culture medium from APMA treated explants revealed an increase in the amount of active MMPs. Equine polymorphs cultured for 2 days produced only EqMMP-9. Lamellar explant medium from horses with acute laminitis contained increased amounts of zymogen and active EqMMP-2 and EqMMP-9 particularly in explants from the fore hooves. Zymography of homogenates of normal lamellar hoof tissue revealed only EqMMP-2 and a minor active band. However, homogenates of lamellar tissue from horses with laminitis showed that EqMMP-9 was present as well as increased EqMMP-2 in both zymogen and active forms. Addition of the MMP inhibitor batimastat (BB-94) to the culture medium of APMA treated explants prevented lamellar separation. BB-94 incubated with polyacrylamide strips containing the MMPs from laminitis affected lamellar explants inhibited enzymatic activity at a concentration of 1 mmol/l. It is concluded that activation of MMPs may be responsible for the lamellar separation seen in laminitis and that MMP inhibitors may be useful clinically for preventing this process.

Structural changes in intercellular junctions during keratinization of the stratum medium of the equine hoof wall.

Cells of the intertubular horn of the stratum medium of the equine hoof wall are joined by three types of junctions. Desmosomes and gap junctions were present in all strata. Septate-like junctions and an intercellular line were seen in the distal stratum spinosum subsequent to extrusion of the contents of membrane-coating granules. At a later stage of keratinization, non-membrane-bound acid phosphatase reaction product appeared to leak into the intercellular space except into areas occupied by septate-like junctions and the intercellular line. It is proposed that the formation of septate-like junctions and the intercellular line prior to leakage of acid phosphatase into the intercellular space created stable areas of intercellular adhesion between fully keratinized cells.