Effects of insulin on IGF-1 receptors in equine lamellar tissue in vitro.

Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubated in cell culture medium for between 2 min and 48 h. In this system, a low physiological concentration of IGF-1 (10 nM; 1.31 ng/mL) caused a marked increase in the appearance of phosphorylated IGF-1R after 5 min (P < 0.05), and this effect was blocked by a human anti-IGF-1R monoclonal antibody (mAb). However, a high concentration of insulin (10 nM; 1,430 µIU/mL) appeared to cause dephosphorylation of the IGF-1R after 5 min (P < 0.01), 15 min, and 30 min (P < 0.001). Using 3H-thymidine as a marker, it was also demonstrated that insulin and IGF-1-stimulated cell proliferation in lamellar explants over the same concentration range as each other (1-100 nM), implying that each peptide acts via its own receptor (P < 0.001). Conversely, the effect of both peptides could be blocked using a selective anti-IGF-1R mAb (P < 0.001), implying that insulin acts via IGF1-R (either directly or indirectly). Notwithstanding this conundrum, the results demonstrate that insulin acts directly on lamellar tissue and suggest that a therapeutic anti-IGF-1R mAb could be useful in treating or preventing endocrinopathic laminitis.

Ultrastructural examination of basement membrane pathology in horses with insulin-induced laminitis.

The third phalanx of the equine digit is suspended within the hoof capsule by a specialized interdigitating dermoepidermal layer called the lamellae, which fails during laminitis. Pathology of the basement membrane (BM), which interfaces epidermis and dermis, is evident during acute laminitis. However, BM damage appears to be less prevalent in ponies with the insulin-associated form of laminitis. The aim of the present study was to investigate changes to the ultrastructure and morphometry of the lamellar BM in the acute phase of insulin-induced laminitis in horses. Lamellar tissue from the left forefoot of 3 horses with acute hyperinsulinemic laminitis was examined with transmission electron microscopy and compared with tissue from normal horses. Lamellar BM width and hemidesmosome (HD) density were assessed every 5 µm along ∼200 µm of secondary epidermal lamellar BM. The BM zone of treated horses was extensively disorganized with loss of uniformity of the lamina lucida and lamina densa, fragmentation and disorientation of HDs, and cytoskeletal disengagement of the HDs. The mean (±SD) lamellar BM was twice as wide in treated (0.25 ± 0.05 µm), compared with control (0.14 ± 0.02 µm), horses. The HD density (HDs/µm) was reduced by half in the treatment group (1.88 ± 0.37), compared with controls (3.6 ± 0.13). The reduced number of HDs in horses with laminitis may contribute to the weakening of the dermoepidermal junction and lamellar failure. Disassembly of HDs during excessive cellular proliferation, secondary to hyperinsulinemia, may account for HD loss. Further investigation of the underlying etiopathogenesis of BM dysfunction during hyperinsulinemic laminitis in horses may facilitate an improved understanding of the disease.

The oral glucose test predicts laminitis risk in ponies fed a diet high in nonstructural carbohydrates.

The aim of this study was to investigate the relationship between laminitis development in ponies and insulin/glucose concentrations in response to the oral glucose test (OGT) and a dietary challenge high in nonstructural carbohydrates (NSCs). After undergoing an OGT (1 g dextrose/kg BW in feed), 37 ponies with 2-h serum insulin concentrations ranging from 22 to 1,133 µIU/mL were subjected to a diet challenge period (DCP), consuming 12 g NSC/kg BW/d for up to 18 d. Insulin and glucose responses were measured on day 2 of the DCP. Clinical laminitis was diagnosed by blinded experts and confirmed radiographically. Basal ACTH levels and clinical signs were assessed to investigate concurrent putative pituitary pars intermedia dysfunction (PPID). The diet induced Obel grade 1 or 2 laminitis in 14 ponies (38%). The ponies that developed laminitis had higher maximum concentrations of blood glucose (P = 0.04) and serum insulin (P = 0.02) in response to the diet. The geometric mean (95% CI) blood glucose concentration for laminitis cases was 14.9 (12.9-17.2) mM, compared to 10.7 (9.2-12.5) mM for ponies who did not develop laminitis. Similarly, the geometric mean (95% CI) for serum insulin was 396 (301-520) µIU/mL for laminitis cases, compared to 216 (148-316) µIU/mL for ponies who did not develop laminitis. Laminitis incidence was likewise associated with insulin concentrations measured during the OGT. Laminitis occurred at frequencies of 0% (0/7) if postdextrose insulin (µIU/mL) was <50; 35% (8/23) if insulin was 50 to 195; and 86% (6/7) if insulin was >195 µIU/mL. Basal ACTH concentrations were above seasonally accepted reference ranges in 16/37 ponies, and 8 of these animals (50%) developed laminitis. This included all 5 ponies in the study that had clinical signs of PPID (100%). In contrast, hyperinsulinemia and laminitis occurred in only 3/11 ponies (27%) with elevated ACTH concentrations and no clinical signs of PPID (P = 0.009). Thus, laminitis occurrence was associated with higher glucose and insulin responses to both the OGT and challenge diet, and the frequency of laminitis can be predicted based on insulin and glucose hyperresponsiveness to these oral carbohydrate challenges.

Effect of Continuous Digital Hypothermia on Lamellar Inflammatory Signaling When Applied at a Clinically-Relevant Timepoint in the Oligofructose Laminitis Model.

BACKGROUND: Although continuous digital hypothermia (CDH) protects lamellae from injury in the oligofructose (OF) model of sepsis-related laminitis (SRL), conflicting results exist from these studies regarding effects of CDH on lamellar inflammatory events. HYPOTHESIS/OBJECTIVES: To determine the effect of CDH on lamellar inflammatory events in normal and OF-treated horses when instituted at a clinically relevant time point (onset of clinical signs of sepsis in this model). ANIMALS: Standardbred geldings (n = 15) aged 3-11 years were used. METHODS: In a randomized, controlled discovery study, animals were administered either OF (OF group, n = 8) or water (CON group, n = 8) by nasogastric tube and CDH was initiated in one forelimb (ICE) 12 hours later. Lamellar tissue samples were collected 24 hours after initiation of CDH (ICE and ambient [AMB] forelimbs). Lamellar mRNA concentrations of inflammatory mediators and lamellar leukocyte numbers were assessed using qPCR and immunohistochemistry, respectively; values from four sample groups (CON AMB, OF AMB, CON ICE, and OF ICE) were analyzed using mixed model linear regression. RESULTS: Although lamellar mRNA concentrations of multiple inflammatory mediators (IL-1ß, IL-6, CXCL1, MCP2, COX-2) were increased after OF administration (OF AMB group versus CON AMB; P < 0.05), only 2 inflammatory mediators (IL-6 and COX-2) and lamellar leukocyte numbers were decreased with CDH (OF ICE versus OF AMB; P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: Continuous digital hypothermia initiated at a time point similar to that commonly used clinically (clinical onset of sepsis) resulted in a more focused inhibition of inflammatory signaling.

Effect of Delayed Digital Hypothermia on Lamellar Inflammatory Signaling in the Oligofructose Laminitis Model.

BACKGROUND: In the oligofructose (OF) model of sepsis-related laminitis (SRL), digital hypothermia ("cryotherapy") initiated before the onset of clinical signs is reported not only to limit lamellar injury, but also to cause marked inhibition of lamellar inflammatory signaling. HYPOTHESIS/OBJECTIVES: Because hypothermia also has been reported to be protective when not initiated until the onset of lameness in the OF model of SRL, we hypothesized that the lamellar protection conferred by hypothermia is caused by local lamellar inhibition of inflammatory signaling as described when hypothermia was initiated earlier in the disease process. ANIMALS: Eight Standardbred geldings aged 3-11 years with no lameness and no abnormalities of the feet detectable by gross or radiographic examination. METHODS: Using the OF model of SRL, lamellar mRNA concentrations of proinflammatory cytokines, chemokines, and endothelial adhesion proteins were compared between samples from treated limbs (CRYO, submerged in ice water for 36 hour starting at the onset of lameness), untreated limbs (NON-CRYO, opposite limb from CRYO limbs maintained at ambient temperature), and untreated limbs from normal horses in which laminitis was not induced (CON). RESULTS: Although OF administration resulted in increases in lamellar mRNA concentrations of several inflammatory mediators in NON-CRYO limbs (vs CON), digital hypothermia had no significant effect on these increases. CONCLUSIONS AND CLINICAL IMPORTANCE: The lack of inflammatory inhibition in lamellar tissue samples in our study indicates that the protective effects of digital hypothermia instituted at the onset of clinical signs of laminitis do not arise from inhibition of inflammatory pathways.

Sustained, Low-Intensity Exercise Achieved by a Dynamic Feeding System Decreases Body Fat in Ponies.

BACKGROUND: Obesity in horses is increasing in prevalence and can be associated with insulin insensitivity and laminitis. Current treatment strategies for obesity include dietary restriction and exercise. However, whether exercise alone is effective for decreasing body fat is uncertain. HYPOTHESIS: Our hypothesis was that twice daily use of a dynamic feeding system for 3 months would induce sustained, low-intensity exercise thereby decreasing adiposity and improving insulin sensitivity (SI). ANIMALS: Eight, university-owned, mixed-breed, adult ponies with body condition scores (BCS) ≥5/9 were used. METHODS: Two treatments ("feeder on" or "feeder off") were administered for a 3-month period by a randomized, crossover design (n = 4/treatment). An interim equilibration period of 6 weeks at pasture separated the 2 study phases. Measurements of body mass (body weight, BCS, cresty neck score [CrNS], and morphometry), body fat (determined before and after the "feeder on" treatment only), triglycerides, and insulin sensitivity (SI; combined glucose-insulin test) were undertaken before and after treatments. RESULTS: The dynamic feeding system induced a 3.7-fold increase in the daily distance travelled (n = 6), compared to with a stationary feeder, which significantly decreased mean BCS (6.53 ± 0.94 to 5.38 ± 1.71), CrNS (2.56 ± 1.12 to 1.63 ± 1.06) and body fat (by 4.95%). An improvement in SI did not occur in all ponies. CONCLUSIONS AND CLINICAL IMPORTANCE: A dynamic feeding system can be used to induce sustained (daily), low-intensity exercise that promotes weight loss in ponies. However, this exercise may not be sufficient to substantially improve SI.

Microdialysis measurements of lamellar perfusion and energy metabolism during the development of laminitis in the oligofructose model.

REASONS FOR PERFORMING STUDY: Failure of lamellar energy metabolism, with or without ischaemia, may be important in the pathophysiology of sepsis-associated laminitis. OBJECTIVES: To examine lamellar perfusion and energy balance during laminitis development in the oligofructose model using tissue microdialysis. STUDY DESIGN: In vivo experiment. METHODS: Six Standardbred horses underwent laminitis induction using the oligofructose model (OFT group) and 6 horses were untreated controls (CON group). Microdialysis probes were placed in the lamellar tissue of one forelimb (all horses) as well as the skin dermis of the tail in OFT horses. Dialysate and plasma samples were collected every 2 h for 24 h and concentrations of energy metabolites (glucose, lactate, pyruvate) and standard indices of energy metabolism (lactate to glucose ratio [L:G] and lactate to pyruvate ratio [L:P]) determined. Microdialysis urea clearance was used to estimate changes in tissue perfusion. Data were analysed nonparametrically. RESULTS: Median glucose concentration decreased to <30% of baseline by 8 h in OFT lamellar (P = <0.01) and skin (P<0.01) dialysate. Lactate increased mildly in skin dialysate (P = 0.04) and plasma (P = 0.05) but not lamellar dialysate in OFT horses. Median pyruvate concentration decreased to <50% of baseline in OFT lamellar dialysate (P = 0.03). A >5-fold increase in median L:G compared with baseline occurred in OFT lamellar and skin dialysate (P<0.03). From a baseline of <20, median L:P increased to a peak of 80 in OFT skin and 38.7 in OFT lamellar dialysates (P<0.02); however, OFT lamellar dialysate L:P was not significantly different from CON. Urea concentration decreased significantly in OFT lamellar dialysate (increased urea clearance) but not in OFT skin or CON lamellar dialysate. CONCLUSIONS: Increased lamellar perfusion occurred during the development of sepsis-associated laminitis in the oligofructose model. Glucose concentrations in the lamellar interstitium decreased, suggesting increased glucose consumption but there was no definitive evidence of lamellar energy failure.

The effect of weightbearing and limb load cycling on equine lamellar perfusion and energy metabolism measured using tissue microdialysis.

REASONS FOR PERFORMING STUDY: Lamellar perfusion is thought to be affected by weightbearing and limb load cycling; this may be critical in the development of supporting limb laminitis. OBJECTIVES: To document the effects of unilateral weightbearing and altered limb load cycling on lamellar energy metabolism and perfusion. STUDY DESIGN: Randomised, controlled (within subject), experimental trial. METHODS: Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate. Samples were collected every 15 min for a 1 h control period, then during periods of unilateral weightbearing (opposite limb held off the ground for 1 h); enhanced static limb load cycling (instrumented limb lifted every 10 s for 30 min); reduced limb load cycling activity (i.v. detomidine sedation) and continuous walking (30 min). Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios were calculated. For each intervention, values were compared with baseline using nonparametric statistical testing. RESULTS: Lamellar dialysate glucose increased and L:G decreased significantly during enhanced static limb load cycling. Glucose and pyruvate increased, and L:G, L:P and urea decreased significantly during walking. Simultaneous skin dialysate values did not change significantly. There were no significant dialysate changes during unilateral weightbearing or after detomidine administration, but only the latter resulted in a significant decrease in limb load cycling frequency. CONCLUSIONS: Increases in limb load cycling frequency (particularly walking) caused dialysate changes consistent with increased lamellar perfusion. Unilateral weightbearing (1 h) and a sedation-induced reduction in limb load cycling frequency did not have a detectable effect on lamellar perfusion. More research is needed to confirm the role of hypoperfusion in supporting limb laminitis, but strategies to increase limb load cycling may be important for prevention.

Microdialysis measurements of equine lamellar perfusion and energy metabolism in response to physical and pharmacological manipulations of blood flow.

REASONS FOR PERFORMING STUDY: A suitable method for evaluating lamellar perfusion changes and their metabolic consequences is currently lacking. OBJECTIVES: To examine perfusion changes in lamellar tissue using serial microdialysis measurements of urea clearance and energy metabolites. STUDY DESIGN: Randomised, controlled (within subject) experimental trial. METHODS: Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate and its clearance was used to estimate local perfusion. Samples were collected every 15 min for a 1 h control period, then during application of a distal limb tourniquet, during periods when norepinephrine or potassium chloride (KCl) were included in both skin and lamellar perfusates, and after systemic (intravenous) acetylpromazine. Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios calculated. Values were compared with pre-intervention baseline and also between simultaneous skin and lamellar samples using nonparametric statistical methods. RESULTS: Lamellar glucose decreased and lactate, urea, L:G and L:P increased significantly with tourniquet application, without significant changes in skin dialysate values. Lamellar and skin glucose decreased and L:G increased significantly during norepinephrine infusion, but mild increases in urea were not significant at either site. KCl caused significant decreases in lamellar and skin L:G, and an increase in skin glucose, but did not affect urea clearance. Acetylpromazine caused profound decreases in lamellar glucose and L:P, with increased L:G and pyruvate, but did not affect urea clearance or any skin dialysate values. CONCLUSIONS: Significant changes in microdialysis urea clearance only occurred with severe lamellar hypoperfusion. However, changes in dialysate metabolite concentrations reflected less profound fluctuations in perfusion. This method may be useful for examining lamellar perfusion and energy balance during laminitis development and for the evaluation of vasoactive therapeutics.

The suspensory apparatus of the distal phalanx in normal horses.

REASONS FOR PERFORMING STUDY: The suspensory apparatus of the distal phalanx (SADP) is functionally and clinically important. OBJECTIVES: To investigate SADP form and function and the microanatomy of its insertion zone. STUDY DESIGN: Descriptive gross and microanatomy. METHODS: The feet of 6 normal Standardbred horses were sectioned into blocks along the traditional perpendicular transverse axis and along functional axes of the SADP, decalcified and processed for staining with haematoxylin and eosin, Jones' periodic acid silver methenamine or Masson's trichrome stains. RESULTS: In traditional midline toe transverse plane sections SADP collagen bundles were irregular with an unstructured appearance. In sections made transversely along planes (70° and 30°) aligned with the long axis of the SADP, collagen bundles were arranged in linear rows. The linear bundles were continuous from their origin on parietal ridges of the distal phalanx through to the secondary epidermal lamellar basement membrane. At the parietal ridge interface the collagen bundles coalesced into smaller, strongly silver staining, linear structures that penetrated the cortical bone and merged with adjacent osteons. In proximal sagittal sections collagen bundles were also linear, angled at 70° to the ground surface. In distal sagittal sections collagen bundles were also arranged linearly but in a multi-angled, 'spokes of a wheel' arrangement, centred on the distal phalanx apex. CONCLUSIONS: Sectioning along functional axes demonstrated the true suspensory nature of the SADP connecting the parietal surface to the lamellar hoof wall. SADP/distal phalanx insertions showed penetrating fibres extending through the chondral-apophyseal interface up to and between distal phalanx osteons. Lamellar measurements made from sections perpendicular to the dorsal aspect of the distal phalanx are underestimations but if made along the longer, functional midline 70° transverse plane would accurately reflect the suspensory function of the lamellae. Laminitis pathophysiology correctly viewed as SADP degradation should inform logical, future therapeutic strategies.

An abattoir survey of equine dental abnormalities in Queensland, Australia.

OBJECTIVE: A cadaver study to estimate the prevalence of dental disorders in horses presented at an abattoir in Queensland, Australia. METHODS: Cadaver heads at a Queensland abattoir were examined for the presence of dental abnormalities and categorised into age groups. The prevalence of abnormalities was analysed by binomial observation of observed proportion, Pearson's Chi-square test or Fisher's exact correlation test. Strength of association was evaluated using Cramer's V test. RESULTS: Heads from horses (n=400) estimated to be between 1 and 30 years of age were placed into four age groups. The most common abnormalities were sharp enamel points (55.3%) and hooks (43%). The highest frequency of dental diseases and abnormalities were in horses 11-15 years old (97.5%). CONCLUSIONS: Common abnormalities were found in all groups and the prevalence increased with age. This study suggests that all horses should have regular complete dental examinations to detect and treat dental disorders in order to limit more severe dental pathologies later in life.

Regional intravenous limb perfusion compared to systemic intravenous administration for marimastat delivery to equine lamellar tissue.

Pharmaceutical agents with potential for laminitis prevention have been identified. Many of these, including the MMP inhibitor marimastat, are impractical for systemic administration. This study compared local delivery of marimastat by regional limb perfusion (RLP) to systemic intravenous bolus dosing (SIVB), and established whether RLP results in local lamellar drug delivery. Six adult horses received 0.23 mg/kg of marimastat by RLP followed by 0.23 mg/kg marimastat by SIVB, with a 24-h washout period. Lamellar ultrafiltration probes sampled lamellar interstitial fluid as lamellar ultrafiltrate (LUF). LUF and plasma marimastat concentrations (LUF[M] and P[M] respectively) were measured for 24 h after each treatment. Regional pharmacokinetic parameters were calculated using noncompartmental analyses. The LUF C(max) following RLP was 232 [34-457] times that following SIVB. LUF[M] after RLP were higher than those obtained after SIVB for 18 h (P < 0.03). Median LUF[M] were > IC(90) of equine lamellar MMP-2 and MMP-9 for 9 h after tourniquet removal. RLP appeared superior to SIVB for lamellar marimastat delivery (higher LUF C(max),, AUC and T > IC(90) of lamellar MMPs). However, frequent dosing is necessary to achieve therapeutic lamellar concentrations. RLP could be used to investigate whether marimastat prevents experimentally induced laminitis. Further refinement of the technique and dosing interval is necessary before clinical application.

The impact of prolonged hyperinsulinaemia on glucose transport in equine skeletal muscle and digital lamellae.

REASONS FOR PERFORMING STUDY: An increased incidence of metabolic disease in horses has led to heightened recognition of the pathological consequences of insulin resistance. Laminitis, failure of the weightbearing digital lamellae, is an important consequence. Altered trafficking of specialised glucose transporters (GLUTs), responsible for glucose uptake, is central to the dysregulation of glucose metabolism and may play a role in the pathophysiology of laminitis. OBJECTIVES: We hypothesised that prolonged hyperinsulinaemia alters the regulation of glucose transport in insulin-sensitive tissue and digital lamellae. Our objectives were to compare the relative protein expression of major GLUT isoforms in striated muscle and digital lamellae in healthy horses and during marked and moderate hyperinsulinaemia. STUDY DESIGN: Randomised, controlled study. METHODS: Prolonged hyperinsulinaemia and lamellar damage were induced by a prolonged euglycaemic-hyperinsulinaemic clamp or a prolonged glucose infusion, and results were compared with those of electrolyte-treated control animals. Protein expression of GLUTs was examined with immunoblotting. RESULTS: Lamellar tissue contained more GLUT1 protein than skeletal muscle (P = 0.002) and less GLUT4 than the heart (P = 0.037). During marked hyperinsulinaemia and acute laminitis (induced by the prolonged euglycaemic-hyperinsulinaemic clamp), GLUT1 protein expression was decreased in skeletal muscle (P = 0.029) but unchanged in the lamellae, while novel GLUTs (8 and 12) were increased in the lamellae (P = 0.03) but not in skeletal muscle. However, moderate hyperinsulinaemia and subclinical laminitis (induced by the prolonged glucose infusion) did not cause differential GLUT protein expression in the lamellae compared with control horses. CONCLUSIONS: The results suggest that lamellar tissue functions independently of insulin and that insulin resistance may not be an essential component of the aetiology of laminitis. Marked differences in GLUT expression exist between insulin-sensitive and insulin-independent tissues during metabolic dysfunction in horses. The different expression profiles of novel GLUTs during acute and subclinical laminitis may be important to disease pathophysiology and require further investigation.

Equine lamellar energy metabolism studied using tissue microdialysis.

Failure of lamellar energy metabolism may contribute to the pathophysiology of equine laminitis. Tissue microdialysis has the potential to dynamically monitor lamellar energy balance over time. The objectives of this study were to develop a minimally invasive lamellar microdialysis technique and use it to measure normal lamellar energy metabolite concentrations over 24 h. Microdialysis probes were placed (through the white line) into either the lamellar dermis (LAM) (n = 6) or the sublamellar dermis (SUBLAM) (n = 6) and perfused continuously over a 24 h study period. Probes were placed in the skin dermis (SKIN) for simultaneous comparison to LAM (n = 6). Samples were collected every 2 h and analysed for glucose, lactate, pyruvate, urea and glycerol concentrations. LAM was further compared with SUBLAM by simultaneous placement and sampling in four feet from two horses over 4 h. Horses were monitored for lameness, and either clinically evaluated for 1 month after probe removal (n = 4) or subjected to histological evaluation of the probe site (n = 10). There were no deleterious clinical effects of probe placement and the histological response was mild. Sample fluid recovery and metabolite concentrations were stable for 24 h. Glucose was lower (and lactate:glucose ratio higher) in LAM compared with SUBLAM and SKIN (P < 0.05). Pyruvate was lower in SUBLAM than SKIN and urea was lower in LAM than SKIN (P < 0.05). These differences suggest lower perfusion and increased glucose consumption in LAM compared with SUBLAM and SKIN. In conclusion, lamellar tissue microdialysis was well tolerated and may be useful for determining the contribution of energy failure in laminitis pathogenesis.

Continuous digital hypothermia initiated after the onset of lameness prevents lamellar failure in the oligofructose laminitis model.

REASONS FOR PERFORMING STUDY: Prophylactic digital hypothermia reduces the severity of acute laminitis experimentally but there is no evidence for its efficacy as a treatment once lameness has already developed. OBJECTIVES: To investigate the therapeutic effects of digital hypothermia, applied after the onset of lameness, in an experimental acute laminitis model. STUDY DESIGN: Randomised, controlled (within subject), blinded, experimental trial. METHODS: Eight Standardbred horses underwent laminitis induction using the oligofructose model. Once lameness was detected at the walk, one forelimb was continuously cooled (CRYO), with the other forelimb maintained at ambient temperature (NON-RX). Dorsal lamellar sections (proximal, middle and distal) harvested 36 h after the onset of lameness/initiation of cryotherapy were analysed by 2 blinded observers: laminitis pathology was scored (0 [normal] to 4 [severe]) and morphometric analyses performed. RESULTS: Median (interquartile range) histological scores were greater (P<0.05) in NON-RX (proximal 2.8 [2.5-4]; middle 3.5 [2-4]; distal 2.5 [2-3.8]) compared with CRYO limbs (proximal 0.5 [0.5-1.4]; middle 1 [0.6-1]; distal 0.75 [0.5-1]). There was complete physical separation of lamellar dermis from epidermis (score of 4) in 4 of the NON-RX feet at one or more section level(s), which was not observed in any CRYO sections. Histomorphometry was thus limited to sections that remained intact; there was a trend of increased total (TELL) and secondary (SELL) epidermal lamellar length and decreased secondary epidermal lamellar width (SELW) in NON-RX limbs compared with CRYO at all 3 levels; differences were significant (P<0.05) for SELL and SELW in the distal sections. CONCLUSIONS: Digital hypothermia reduced the severity of lamellar injury and prevented lamellar structural failure (complete dermoepidermal separation) when initiated at the detection of lameness in an acute laminitis model. This study provides the first evidence to support the use of therapeutic digital hypothermia as a treatment for acute laminitis.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses: implications for laminitis.

Equine laminitis, a disease of the lamellar structure of the horse's hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Toll-like receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-α and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-α, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

The feral horse foot. Part B: radiographic, gross visual and histopathological parameters of foot health in 100 Australian feral horses.

BACKGROUND: It has been proposed that the feral horse foot is a benchmark model for foot health in horses. However, the foot health of feral horses has not been formally investigated. OBJECTIVES: To investigate the foot health of Australian feral horses and determine if foot health is affected by environmental factors, such as substrate properties and distance travelled. METHODS: Twenty adult feral horses from five populations (n = 100) were investigated. Populations were selected on the basis of substrate hardness and the amount of travel typical for the population. Feet were radiographed and photographed, and digital images were surveyed by two experienced assessors blinded to each other's assessment and to the population origin. Lamellar samples from 15 feet from three populations were investigated histologically for evidence of laminitis. RESULTS: There was a total of 377 gross foot abnormalities identified in 100 left forefeet. There were no abnormalities detected in three of the feet surveyed. Each population had a comparable prevalence of foot abnormalities, although the type and severity of abnormality varied among populations. Of the three populations surveyed by histopathology, the prevalence of chronic laminitis ranged between 40% and 93%. CONCLUSIONS: Foot health appeared to be affected by the environment inhabited by the horses. The observed chronic laminitis may be attributable to either nutritional or traumatic causes. Given the overwhelming evidence of suboptimal foot health, it may not be appropriate for the feral horse foot to be the benchmark model for equine foot health.

The feral horse foot. Part A: observational study of the effect of environment on the morphometrics of the feet of 100 Australian feral horses.

AIM: To better understand the morphology of, and the effect of different travel patterns and varying substrate environments on, the feral horse foot to better manage the feet of domestic horses. METHODS: The left forefeet of 20 adult feral horses from each of five geographically separated populations in Australia (n = 100) were investigated. Populations were selected on the basis of substrate hardness under foot and the amount of travel typical for the population. Feet were radiographed and photographed and 40 morphometric measurements of each foot were obtained. RESULTS: Of the 40 parameters, 37 differed significantly (P < 0.05) among the populations, which suggested that substrate hardness and travel distance have an effect on foot morphology. Harder substrates and longer travel distances were associated with short hoof walls and minimal hoof wall flaring. Softer substrates and moderate travel distances were associated with long flared walls, similar to that of typical untrimmed feet of domestic horses. CONCLUSIONS: The morphology of the feral horse foot appeared to be affected by the distance travelled and by the abrasive qualities and mechanical properties of the substrate under foot. There were marked differences in some conformation parameters between the feral horses in the current study and domestic horses in previous studies. Although the conformation of the feral horse foot may have some prescriptive value, concerns regarding abnormal foot anatomy warrant further investigation.

Population dynamics of feral horses (Equus caballus) following above-average rainfall in a semi-arid environment of Australia.

BACKGROUND: Recent record rainfall in much of semi-arid Central Australia is the most likely reason for a feral horse population increase in excess of normal. Uncontrolled numbers of feral horses have habitat degradation and animal welfare implications. OBJECTIVES: The aims of this study were to investigate the social structure of feral horses and assess their population growth rate following unseasonably high rainfall. METHODS: The study area was 4000 km(2) of unmanaged, semi-arid country in Central Australia (latitude 24.50°S, longitude 132.10°E). Horses were identified by descriptive features from ground searches, movement-activated cameras and 'hides' positioned at key water holes. Wherever possible, sex and age categories were documented. Population growth rate was estimated by the number of foals divided by the number of horses older than 1 year in the observed population. RESULTS: A total of 1424 horses were identified and categorised, of which 335 were foals born within the current year. Only 123 juveniles were identified. Of the adult horses, 53.4% were male and 46.6% were female and this differed from parity (P = 0.04). Of the mares, 71.9% had a foal at foot and the population growth rate was 29.5%. CONCLUSIONS: With a sustained population growth rate of 29.5%, this population of feral horses will more than double within 3 years. The high population increase will likely have a detrimental effect on native fauna and flora and the fragile, semi-arid ecosystems of Central Australia. After a period of high rainfall and plentiful resources, 'normal' drought conditions will return and many feral horses will starve and die as they compete for limited resources.

Adaption of horses to a novel dynamic feeding system: movement and behavioural responses.

REASONS FOR PERFORMING STUDY: Many domestic horses and ponies are sedentary and obese due to confinement to small paddocks and stables and a diet of infrequent, high-energy rations. Severe health consequences can be associated with this altered lifestyle. OBJECTIVES: The aims of this study were to investigate the ability of horses to learn to use a dynamic feeder system and determine the movement and behavioural responses of horses to the novel system. METHODS: A dynamic feed station was developed to encourage horses to exercise in order to access ad libitum hay. Five pairs of horses (n = 10) were studied using a randomised crossover design with each pair studied in a control paddock containing a standard hay feeder and an experimental paddock containing the novel hay feeder. Horse movement was monitored by a global positioning system (GPS) and horses observed and their ability to learn to use the system and the behavioural responses to its use assessed. RESULTS: With initial human intervention all horses used the novel feeder within 1 h. Some aggressive behaviour was observed between horses not well matched in dominance behaviour. The median distance walked by the horses was less (P = 0.002) during a 4 h period (117 [57-185] m) in the control paddock than in the experimental paddock (630 [509-719] m). CONCLUSIONS: The use of an automated feeding system promotes increased activity levels in horses housed in small paddocks, compared with a stationary feeder. POTENTIAL RELEVANCE: The novel feeder system may have application in the husbandry of horses and ponies kept in small paddocks by encouraging a natural pattern of exercise without human intervention and an ad libitum diet of hay. This may improve the health and welfare of horses.