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.     

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.     

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.     

MAGNETIC RESONANCE IMAGING OF THE EQUINE DIGIT WITH CHRONIC LAMINITIS

Chronic laminitis is a severe disease affecting the equine digit. It was hypothesized that magnetic resonance (MR) imaging would improve visualization of structures within the foot and pathology associated with chronic laminitis. This study aimed to describe the MR imaging findings in chronic laminitis, compare different pulse sequences for visualization of pathology, and to compare MR imaging with standard radiography. Twenty (10 forelimb, 10 hindlimb) cadaver limbs from 10 horses clinically diagnosed with chronic laminitis (group L) and 10 limbs without laminitis (group N) were used. Lateromedial radiographs and sagittal and transverse MR images of the foot were obtained. Radiographs and MR images were evaluated for anatomic definition and evidence of pathology. Dorsal hoof wall thickness and angle of rotation and displacement distance of the distal phalanx were measured. Comparisons were made between group L and N, forelimb and hindlimb within each horse, and MR imaging and radiography. Features consistently noted with MR images in group L, but not detected using radiography, included laminar disruption, circumscribed areas of laminar gas, laminar fluid, and bone medullary fluid. Other findings seen only on MR images included increased size and number of vascular channels, alterations in the corium coronae, and distal interphalangeal joint distension. Magnetic resonance imaging allowed better definition of laminar gas lines and P3 surface irregularity observed on radiographs. Based on measurements, group L had a greater angle of rotation, distal displacement, and dorsal hoof wall thickness than group N; forelimb hoof wall thickness was greater than hindlimb; and distal displacement and hoof wall thickness measurements were smaller using MR imaging than radiography, but had a similar pattern. It is concluded that there are features of chronic laminitis consistently observed using MR imaging and that these may be additional to features observed radiographically.     

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.     

Equine laminitis induced with oligofructose

REASONS FOR PERFORMING STUDY: Experimental induction of equine laminitis with a reliable and clinically relevant model should facilitate understanding of the disease. Successful induction with oligofructose (OF) could link pasture consumption to laminitis. OBJECTIVES: To determine whether alimentary administration of OF induces laminitis. METHODS: Twelve horses were dosed with OF and 6 received a sham (placebo) treatment. Clinical observations were made and blood collected at 4 h intervals over a 48 h study period. Stained sections of the hoof wall lamellae, examined by light microscopy, were graded for laminitis severity. RESULTS: All horses administered OF, but no sham-treated controls, developed clinical and histological laminitis. CONCLUSIONS AND POTENTIAL RELEVANCE: Alimentary overload with OF is a valid induction model for studying the pathogenesis of laminitis. A link is therefore established between field cases of laminitis and pasture fructan content.     

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.     

Hoof wall wound repair

REASONS FOR PERFORMING STUDY: Surgical stripping of the hoof wall results in a wound that heals remarkabley well. In contrast, lamellae recovering from laminitis are often deformed. Investigating lamellar wound healing may aid understanding of laminitis. OBJECTIVES: To document temporal changes in the lamellar basement membrane (BM), dermis and epidermis after surgery. METHODS: Wall strips were made in the dorsal hoof wall midline of 6 mature horses. Immunohistochemistry was used to document changes in the basement membrane (BM) and detect proliferation of epidermal cells in lamellar tissues harvested at intervals. A conforming metal plate was screwed to the hoof wall to maintain alignment of the wound edges. RESULTS: Wall stripping caused lamellar tips to snap and remain behind in the dermis along with the majority of the lamellar BM and some lamellar basal cells. Three days later the BM was intact and new lamellae had been reconstructed by proliferation of surviving epidermal cells. By 5 days the surface of the stripped zone was covered with yellow epidermis that subsequently thickened and hardened. Eventually the hoof wall deficit was replaced by new wall growing down from the coronet. The conforming metal plate and post operative analgesic ensured minimal lameness. CONCLUSIONS AND POTENTIAL RELEVANCE: In wall stripped lamellae the BM survives virtually intact and is used as a template for proliferating cells, from snapped-off lamellar tips, to migrate and quickly achieve repair to near normality. In laminitis epidermal dysadhesion and lamellar BM destruction occurs and lack of a functional BM template may explain the prolonged and abnormal repair of affected lamellae.     

Clinical Outcome of 14 Obese,Laminitic Horses Managed with the Same Rehabilitation Protocol

A specific method of rehabilitation was used to manage obese horses with laminitis, and clinical outcome was evaluated after 5 to 20 months. Clinical data from 14 similar laminitis cases were statistically analyzed to evaluate response to rehabilitation. Data were analyzed using repeated measures or logistic regression methodologies. Each horse presented as obese and laminitic with no history of a systemic inflammatory disease. The rehabilitation method emphasized a mineral-balanced, low nonstructural carbohydrate diet; daily exercise; hoof trimming that minimized hoof wall loading; and sole protection in the form of rubber hoof boots and/or hoof casts. Distal phalanx alignment within the hoof capsule was significantly improved, and hoof wall thickness was significantly decreased (P < .0001) following treatment. Solar depth was significantly increased (P < .0015). Reduction of palmar angle measurements was detected in acutely and chronically affected horses. This treatment effect was statistically greater for horses with chronic laminitis than for horses with acute laminitis (P interaction < .0001). Horses were 5.5 times more likely to be sound post-treatment than before treatment. Daily exercise, dietary modification, and removal of ground reaction force from the hoof wall were foci of the rehabilitation program. Hoof care and husbandry as applied to these horses may be an effective method of rehabilitation of horses from obesity-associated laminitis.     

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.     

Low‐field magnetic resonance imaging of keratomas of the hoof wall

Standing low‐field magnetic resonance imaging (MRI) was used to image the feet of 21 horses affected by keratomas. The animals had variable clinical histories including acute, chronic or recurrent lameness. Of the 21 horses, only 14 (66%) had radiological changes suggestive of keratoma. Standing low‐field MRI revealed a smoothly demarcated hoof wall lesion in all horses, with deformity of the adjacent surface of the distal phalanx in 15 cases. The signal characteristics of the keratomas varied, with most showing either hypointensity or heterogeneous mixed signal intensity in T1‐ and T2*‐weighted images and hypointensity in STIR sequences. An intermediate or high STIR signal intensity was present in the trabecular bone of the adjacent region of the distal phalanx in 5 cases. Surgical debridement was performed in 16 horses. Recurrence was common, especially in cases where the lesion was ill‐defined; the prior MRI appearance of these cases tended to be of heterogeneous signal intensity.     

The treatment of laminitis in horses

The structural and vascular anatomy of the healthy equine foot is compared with the pathologic changes in the foot of horses with acute and chronic laminitis. The structural and vascular abnormalities present in the foot of horses with laminitis are demonstrated in order to explain the abnormal manner in which their feet grow. The medical, surgical, dietary, and endocrine management of acute and chronic laminitis is discussed. Various forms of hoof trimming beneficial to the reestablishment of normal digital perfusion, normal hoof growth, and normal spatial orientation among the distal phalanx, hoof wall, and sole are described. Guidelines for the provision of frog support provided by adjustable heart-bar shoes are presented.     

Equine laminitis: Induced by 48 h hyperinsulinaemia in Standardbred horses

Reasons for performing study: Hyperinsulinaemia is known to induce laminitis experimentally in healthy ponies with no history of the condition. Horses are more insulin sensitive than ponies and whether prolonged hyperinsulinaemia and euglycaemia would have a similar laminitogenic effect requires study. Objectives: To determine if laminitis results when the prolonged euglycaemic hyperinsulinaemic clamp technique (p‐EHC) is applied to clinically normal Standardbred horses, and to monitor hoof wall temperature seeking an association between vascular activity and laminitis development. Methods: Eight young, clinically normal Standardbred horses were assigned into 4 pairs and within each pair, one was assigned randomly to either treatment (n = 4) or control (n = 4) groups. Treated horses received continuous infusions of insulin and glucose until clinical signs of laminitis developed, at which point the horses were subjected to euthanasia. Control horses received an equivalent volume of a balanced electrolyte infusion for the same period. Hoof wall surface temperature (HWST) was monitored continuously throughout the experimental period. Results: All horses in the treatment group were calculated to have normal insulin sensitivity. All treated horses, and none in the control group, developed laminitis (P = 0.01). Pronounced digital pulses were a feature of the treatment group, while insignificant digital pulses occurred in control horses. HWST was higher and less variable in treated horses once hyperinsulinaemia was established. Conclusions: Healthy Standardbred horses subjected to prolonged hyperinsulinaemia develop laminitis within 48 h, demonstrating that laminitis in horses can be triggered by insulin. Potential relevance: Insulin resistance and the associated hyperinsulinaemia place horses and ponies at risk of developing laminitis. This study demonstrates a need for prompt management of the persistent hyperinsulinaemia seen in some endocrinopathies.     

Evaluation of hoof wall surface temperature as an index of digital vascular perfusion during the prodromal and acute phases of carbohydrate-induced laminitis in horses.

OBJECTIVE: To evaluate the use of hoof wall surface temperature (HWST) as an indirect indicator of digital perfusion and to describe HWST patterns during the prodromal and acute phases of carbohydrate-induced laminitis in horses. ANIMALS: 30 adult horses without foot abnormalities. PROCEDURES: Three experiments were performed. In the first, HWST was measured in 2 groups of horses acclimatized to hot (n = 6), or cold (6) environments and exposed to cold (15 C) ambient temperature. In the second experiment, HWST were measured in both forefeet of 6 horses before and after application of a tourniquet to 1 forefoot to induce vascular occlusion. In the third experiment, HWST were recorded in 12 horses before and during the prodromal and acute phases of carbohydrate-induced laminitis. RESULTS: Mean HWST of hot-acclimatized cold-challenged horses was significantly less than that of cold-acclimatized cold-challenged horses at all times. Transient episodes of high HWST were observed during prolonged cold-induced vasoconstriction. Hoof wall surface temperature significantly decreased during arterial occlusion and increased during reperfusion. Digital hypothermia was observed during the prodromal phase of carbohydrate-induced laminitis. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of HWST is a valid technique to evaluate digital perfusion under appropriate controlled conditions in horses. Digital hypothermia detected during the prodromal phase of laminitis is consistent with decreased digital vascular perfusion or metabolic activity. If administered to horses during the prodromal phase, agents that enhance digital perfusion may prevent development of laminitis.     

Computed Tomography or Magnetic Resonance Imaging‐Assisted Partial Hoof Wall Resection for Keratoma Removal

Objectives: To (1) describe the computed tomography (CT) and magnetic resonance imaging (MRI) appearance of keratomas; (2) describe a CT‐ or MRI‐assisted partial hoof wall resection technique for removal of keratomas; and (3) evaluate the morbidity and postoperative outcome of these horses. Study Design: Case series. Animals: Horses (n=10) with keratoma. Methods: Data retrieved from medical records included signalment, lameness duration and grade, physical and diagnostic evaluation findings, CT and MRI technique and findings, surgical details, histopathologic diagnosis, postoperative treatment, and complications experienced. Long‐term outcome was obtained by telephone interviews of owners. Results: Complications including excessive granulation tissue formation and infection were seen in 2 horses (20%). No keratoma recurrence occurred. Follow‐up information was available for 8 horses; 7 were sound and had resumed work. Mean time until they became sound was 2.7 months, and mean time until work resumed was 3.6 months. Conclusions: CT and MRI can be used to accurately identify the location of keratomas. Postoperative complications may be decreased by creating smaller hoof wall defects, filling the defects with antimicrobial‐impregnated polymethylmethacrylate, and placing a shoe early in the postoperative period.     

Magnetic resonance imaging metallic artifact of commonly encountered surgical implants and foreign material

Magnetic resonance imaging (MRI) artifacts secondary to metallic implants and foreign bodies are well described. Herein, we provide quantitative data from veterinary implants including total hip arthroplasty implants, cranial cruciate repair implants, surgical screws, a skin staple, ligation clips, an identification microchip, ameroid constrictor, and potential foreign bodies including air gun and BB projectiles and a sewing needle. The objects were scanned in a gelatin phantom with plastic grid using standardized T2-weighted turbo-spin echo (TSE), T1-weighted spin echo, and T2*-weighted gradient recalled echo (GRE) image acquisitions at 1.5 T. Maximum linear dimensions and areas of signal voiding and grid distortion were calculated using a DICOM workstation for each sequence and object. Artifact severity was similar between the T2-weighted TSE and T1-weighted images, while the T2*-weighted images were most susceptible to artifact. Metal type influenced artifact size with the largest artifacts arising from steel objects followed by surgical stainless steel, titanium, and lead. For animals with metallic surgical implants or foreign bodies, the quantification of the artifact size will help guide clinicians on the viability of MRI.     

Glucocorticoids and laminitis in the horse.

The administration of exogenously administered GCs and syndromes associated with GC excess are both attended by increased risk for the development of laminitis in adult horses. However, there exists substantial controversy as to whether excess GCs cause laminitis de novo. If true, the pathogenesis of laminitis arising from the effects of GC excess is probably different from that associated with diseases of the gastrointestinal tract and endotoxemia. Although a satisfactory explanation for the development of laminitis as a consequence of GC action is currently lacking, numerous possible and plausible theoretical mechanisms do exist. Veterinarians must exert caution with respect to the use of GCs in adult horses. The extent to which individual horses are predisposed to laminitis as a result of GC effect cannot be predicted based on current information. However, the administration of systemic GCs to horses that have been previously affected by laminitis should be used only with extreme caution, and should be accompanied by careful monitoring for further signs of laminitis. The risk of laminitis appears to be greater during treatment using some GCs (especially dexamethasone and triamcinalone) compared with others (prednisone and prednisolone). Whenever possible, to reduce the risk of laminitis, GCs should be administered locally. For example, the risk of GC-associated laminitis is evidently considerably reduced in horses affected with chronic obstructive pulmonary disease (COPD) if GC treatment is administered via inhalation. We have hypothesized that structural changes in the equine hoof that resemble laminitis may arise as a consequence of excess GC effect. Although these changes are not painful per se, and are not associated with inflammation, they could likely predispose affected horses to the development of bona fide laminitis for other reasons. Moreover, the gross morphological appearance of the chronically GC-affected hoof resembles that of a chronically foundered hoof in some respects. Further investigation into the effect of GC on the hoof lamellar interface is clearly needed.     

Expression of endothelin in equine laminitis.

Biosynthesis of endothelin-1 (ET-1), the most potent endogenous vasoconstrictor yet identified, is increased following myocardial infarction (MI) in man. Pathological events which occur in the connective tissues of the equine hoof during laminitis are similar in some respects, to changes occurring in the myocardial connective tissues following MI in man. The objective of this study was to determine whether ET-1 expression in connective tissues obtained from the hoof of laminitic horses is increased compared with tissues obtained from healthy horses. Expression of ET-1 in connective tissues of the equine hoof was measured following tissue extraction from 3 groups of horses: horses in which acute laminitis had been induced by the administration of starch; chronically foundered horses; nonlaminitic horses. The concentration of ET-1 in laminar connective tissues obtained from all laminitic horses (1573.0 +/- 392.8 pg/g of tissue; n = 10) was increased when compared with tissues obtained from nonlaminitic horses (392.5 +/- 117.4 pg/g of tissue; n = 5) (P<0.05). The concentration of ET-1 in laminar connective tissues obtained from the experimentally induced, acute laminitic horses (1043.6 +/- 254.4 pg/g of tissue; n = 7) and from the spontaneously affected, chronic laminitic horses (2808.3 +/- 878.6 pg/g of tissue; n = 3) was increased compared with the control group (P<0.05, P<0.01, respectively). The concentration of ET-1 in laminar connective tissues obtained from the chronic laminitic horses was greater than that of the experimentally induced, acute laminitic group (P<0.05). It is suggested that the data provide a strong argument that increased ET-1 expression in the connective tissues of the equine hoof represent a potentially important and hitherto unrecognised component of the pathophysiology of equine laminitis. Further studies are needed to determine whether inhibitors of ET-1 converting enzyme or antagonists of ET-1 receptors might be useful in the treatment and prevention of laminitis in horses.