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 effect of regional hypothermia on mechanical nociceptive thresholds in the equine distal forelimb.

Regional hypothermia has shown promise as analgesic in horses when used to manage painful conditions of the distal limb such as laminitis. In this prospective study, the analgesic effects of regional hypothermia were assessed using mechanical nociceptive thresholds during distal limb cooling. The study population consisted of eight healthy adult Standardbred horses, selected from a teaching herd. A distal forelimb of each horse was cooled using water immersion at the following sequential target water temperatures: 34 °C, 20 °C, 10 °C, 5 °C, 1 °C, 5 °C, 10 °C, 20 °C. Limb surface temperature was measured after 30 min at each target water temperature and the mechanical force required to elicit a response (mechanical nociceptive threshold) was determined using a pneumatic actuator. Both forelimbs of each horse were tested one week apart. At skin surface temperatures above 7 °C, there was little association between skin surface temperature and the mechanical force required to elicit a response. As the skin surface temperature decreased below 7 °C, there was a rapid increase in the force required to elicit a response (P = 0.036). Skin surface temperatures of <7 °C required water temperatures below 2 °C. The results of this study suggest that hypothermia has potential to provide distal limb analgesia in horses at skin surface temperatures below 7 °C. Further evaluation of the technique is warranted.

Pharmacokinetic profile and behavioral effects of gabapentin in the horse.

Gabapentin is being used in horses although its pharmacokinetic (PK) profile, pharmacodynamic (PD) effects and safety in the equine are not fully investigated. Therefore, we characterized PKs and cardiovascular and behavioral effects of gabapentin in horses. Gabapentin (20 mg/kg) was administered i.v. or p.o. to six horses using a randomized crossover design. Plasma gabapentin concentrations were measured in samples collected 0-48 h postadministration employing liquid chromatography-tandem mass spectrometry. Blood pressures, ECG, and sedation scores were recorded before and for 12 h after gabapentin dosage. Nineteen quantitative measures of behaviors were evaluated. After i.v. gabapentin, the decline in plasma drug concentration over time was best described by a 3-compartment mammillary model. Terminal elimination half-life (t(1/2γ) ) was 8.5 (7.1-13.3) h. After p.o. gabapentin terminal elimination half-life () was 7.7 (6.7-11.9) h. The mean oral bioavailability of gabapentin (± SD) was 16.2 ± 2.8% indicating relatively poor absorption of gabapentin following oral administration in horses. Gabapentin caused a significant increase in sedation scores for 1 h after i.v. dose only (P < 0.05). Among behaviors, drinking frequency was greater and standing rest duration was lower with i.v. gabapentin (P < 0.05). Horses tolerated both i.v. and p.o. gabapentin doses well. There were no significant differences in and . Oral administration yielded much lower plasma concentrations because of low bioavailability.

Influence of digital hypothermia on lamellar events related to IL-6/gp130 signalling in equine sepsis-related laminitis.

BACKGROUND: Interleukin-6 (IL-6) is consistently increased in the digital lamellae in different studies of sepsis-related laminitis (SRL). IL-6 signalling through the gp130 receptor activates similar signalling (i.e. mTORC1-related signalling) previously reported to be activated in models of endocrinopathic laminitis. OBJECTIVES: To assess the activation state of signalling proteins downstream of IL-6/gp130 receptor complex activation in an experimental model of SRL. STUDY DESIGN: Randomised experimental study. METHODS: Lamellar phospho-(P) protein concentrations downstream of the IL-6/gp130 receptors were assessed in the oligofructose (OF) model of SRL. Fifteen Standardbred horses were administered water (CON, n = 8) or oligofructose (OF, n = 7) via a nasogastric tube. At 12 h post-OF/water administration, one randomly assigned forelimb was exposed to continuous digital hypothermia (CDH) by placement in ice water (ICE, maintained at <7°C); the other forelimb was maintained at ambient temperature (AMB). Lamellar tissue samples were collected after 24 h of CDH from both ICE and AMB forelimbs and immediately snap-frozen. Lamellar proteins of interest were assessed by immunoblotting and immunofluorescence. RESULTS: Immunoblotting revealed increase (P<0.05) in the phosphorylation states of Akt (Ser 473), RPS6 (Ser235/236), RPS6 (Ser240/244), STAT3 (Ser727) and STAT3 (Tyr705) in lamellar tissue from OF-treated animals (AMB OF vs. AMB CON limbs); CDH resulted in decreased (P<0.05) lamellar concentrations of phosphorylated Akt, p70S6K, RPS6 (235/236), RPS6 (240/244) and STAT3 (S727) in OF-treated animals (AMB OF vs. ICE OF). Immunofluorescence showed that activated/phosphorylated forms of RPS6 and STAT3 were primarily localised to lamellar epithelial cells. MAIN LIMITATIONS: The nature, sequence and timing of sub-cellular events in this experimental model may differ from those that accompany naturally occurring sepsis. CONCLUSIONS: There were increased lamellar concentrations of activated signalling proteins downstream of the IL-6/Gp130 receptor complex in OF-treated horses; CDH inhibited this activation for the majority of the proteins assessed. These results demonstrate similar lamellar signalling (e.g. mTORC1-related signalling) and, therefore, possible therapeutic targets occurring in sepsis-related laminitis as previously reported in models of endocrinopathic laminitis.

Equine laminitis model: lamellar histopathology seven days after induction with oligofructose.

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

Equine laminitis model: cryotherapy reduces the severity of lesions evaluated seven days after induction with oligofructose.

REASONS FOR PERFORMING STUDY: A previous preliminary study demonstrated the potential of distal limb cryotherapy (DLC) for preventing laminitis. Clinically, DLC must be effective for periods longer than 48 h and the preventive effect must extend beyond its discontinuation. OBJECTIVES: To evaluate the effect of DLC, applied during the developmental phase of induced laminitis, on the severity of clinical laminitis and lamellar histopathology 7 days after dosing. METHODS: Eighteen normal Standardbred horses were divided into 3 groups of 6. Continuous cryotherapy was applied for 72 h to the distal limbs of the first group. The second and third groups were administered laminitis inducing doses of oligofructose and 72 h of cryotherapy applied (immediately after dosing) to the second group. After clinical assessment all horses were subjected to euthanasia 7 days after dosing and hoof lamellar tissues were harvested and analysed. RESULTS: In the laminitis induced horses clinical lameness and laminitis histopathology was significantly reduced in horses that underwent 72 h of DLC compared with untreated controls. Cryotherapy alone produced no significant lameness or other ill effect. CONCLUSIONS: Continuous, medium- to long-term (72 h) cryotherapy applied to the distal limbs of horses safely and effectively ameliorates the clinical signs and pathology of acute laminitis. POTENTIAL RELEVANCE: Pre-emptive distal limb cryotherapy is a practical method of ameliorating laminitis in ill horses at risk of developing the disease.

The relationship between bronchoalveolar lavage fluid cytology and airway hyper-reactivity in a population of Australian horses presented for poor performance.

OBJECTIVE: To investigate the relationship between bronchoalveolar lavage fluid cytology, particularly mast cells, and airway hyper-reactivity in athletic horses presented for poor performance that included a respiratory tract evaluation in two disparate locations in Australia. DESIGN: Multi-centre, retrospective and prospective cross-sectional study METHODS: Eighty four adult horses underwent both pulmonary function testing and histamine bronchoprovocation with a commercial flowmetric plethysmography system. A bronchoalveolar lavage was performed four to twelve hours later. Bronchoalveolar lavage fluid cytology was categorised using two differing classification systems to define mild equine asthma. Statistical analysis was used to assess associations between bronchoalveolar lavage fluid relative inflammatory cell percentages, and airway hyper-reactivity and their associated categorisations. RESULTS: Sixty four percent (54/84) of horses displayed airway hyper-reactivity, as defined by PC35 < 6 mg/ml of histamine. A relative mastocytosis was the most common bronchoalveolar lavage fluid cytological abnormality. Horses with a sole mast cell response of ≥ 5% within their bronchoalveolar lavage fluid displayed airway hyper-reactivity at a lower dose of nebulized histamine than horses with normal bronchoalveolar lavage fluid cytology. Horses with mixed cell responses (relative mast cell percentage > 2% and/or relative neutrophil percentage > 5% and/or eosinophil relative cell percentage ≥ 1%) displayed airway hyper-reactivity at a lower dose of nebulized histamine than horses with normal bronchoalveolar lavage fluid cytology. CONCLUSION: In the Australian context, recently revised increased bronchoalveolar lavage fluid cytology relative cell percentage cut offs appear appropriate for sole mast cell responses. The historical lower cut offs appear to be appropriate for mixed inflammatory cell responses.

Continuous fluid infusion per rectum compared with intravenous and nasogastric fluid administration in horses.

BACKGROUND: Rectal fluid administration may offer a simple, safe and inexpensive alternative to intravenous or nasogastric fluid therapy in equine clinical cases. OBJECTIVES: To evaluate the tolerance and effects of rectally administered fluid and compare the measurements of haemodilution and intravascular volume with those during nasogastric and intravenous fluid administration. STUDY DESIGN: Randomised controlled experimental trial. METHODS: Six clinically normal Standardbred geldings were used in a 4-way crossover study: each received three different fluid treatments (intravenous, nasogastric and rectal) at 5 mL/kg/h for 6 h and underwent a control (no treatment) with water and feed withheld. Bodyweight was measured at baseline and 6 h. Packed cell volume (PCV), total solids (TS), albumin, electrolytes, lactate, urine specific gravity, vital parameters, gastrointestinal borborygmi and central venous pressure were measured every 2 h. RESULTS: Rectal fluid administration with plain water was well tolerated and caused clinical chemistry changes consistent with haemodilution, indicating absorption. Mean (95% confidence interval) PCV decreased from 40% [40-42] at 0 h to 35% [34-36] at 6 h during rectal fluid treatment (P<0.001), similar to decreases in PCV occurring also with i.v. and nasogastric (NGT) treatment (P<0.001). The TS also decreased with i.v. and rectal fluid (P<0.001). There was a decrease in bodyweight in the control (P<0.001) but not with any of the fluid treatments. MAIN LIMITATIONS: A small sample size of healthy, euhydrated horses and a relatively short duration of fluid administration was used. CONCLUSIONS: Rectal fluid administration requires clinical evaluation, but may offer an inexpensive, safe alternative or adjunct to i.v. fluid administration, particularly when administration via NGT is not possible or contraindicated. The Summary is available in Portuguese - see Supporting Information.

Continuous digital hypothermia prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Continuous digital hypothermia can prevent the development and progression of laminitis associated with sepsis but its effects on laminitis due to hyperinsulinaemia are unknown. OBJECTIVES: To determine the effects of continuous digital hypothermia on laminitis development in the euglycaemic hyperinsulinaemic clamp model. STUDY DESIGN: Randomised, controlled (within subject), blinded, experiment. METHODS: Eight clinically normal Standardbred horses underwent laminitis induction using the euglycaemic hyperinsulinaemic clamp model (EHC). At initiation of the EHC, one forelimb was continuously cooled (ICE), with the other maintained at ambient temperature (AMB). Dorsal lamellar sections (proximal, middle, distal) were harvested 48 h after initiation of the EHC and were analysed using histological scoring (0-3) and histomorphometry. Cellular proliferation was quantified by counting epidermal cell nuclei staining positive with an immunohistochemical proliferation marker (TPX2). RESULTS: Severe elongation and disruption of SEL with dermo-epidermal separation (score of 3) was observed in all AMB feet at one or more section locations, but was not observed in any ICE sections. Overall 92% of the AMB sections received the most severe histological score (grade 3) and 8% were grade 2, whereas ICE sections were classified as either grade 1 (50%) or grade 2 (50%). Relative to AMB feet, ICE sections were 98% less likely to exhibit grades 2 or 3 (OR: 0.02, 95% CI 0.001, 0.365; P<0.01). Histomorphometry measurements of total and nonkeratinised primary epidermal lamellar length were significantly increased (P<0.01) in AMB limbs compared with ICE. TPX2 positive cell counts were significantly increased (P<0.01) in AMB limbs compared with ICE. MAIN LIMITATIONS: Continuous digital hypothermia was initiated before recognition of laminitis and therefore the clinical applicability requires further investigation. CONCLUSIONS: Continuous digital hypothermia reduced the severity of laminitis in the EHC model and prevented histological lesions compatible with lamellar structural failure.

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.

Elapid snake envenomation in horses: 52 cases (2006-2016).

BACKGROUND: Snake envenomation is a cause of morbidity and mortality in domestic animals worldwide. The clinical features of crotalid snake (pit viper) envenomation are widely reported and well described in horses but elapid snake envenomation is poorly characterised. OBJECTIVES: To describe the presentation, clinical and laboratory findings, treatment and outcome of horses with a diagnosis of elapid snake envenomation in Australia. STUDY DESIGN: Retrospective case series. METHODS: Medical records of horses with a diagnosis of elapid snake envenomation (2006-2016) at several university and private veterinary practices were reviewed. Inclusion criteria comprised one or more of the following: 1) observed snakebite, 2) positive snake venom detection kit (SVDK) result, 3) appropriate clinical response to treatment with antivenom or 4) supportive post-mortem findings. RESULTS: Fifty-two cases met the inclusion criteria. Most cases (94%) demonstrated clinical signs of neurotoxicity, characterised by generalised neuromuscular weakness. Associated neurologic signs included staggering gait, muscle fasciculations, recumbency, mydriasis, ptosis and tongue paresis. Concurrent clinically important conditions included rhabdomyolysis (50%) and haemolysis (19%). Of 18 urine samples evaluated with a SVDK, only three (17%) were positive. Overall survival was favourable (86%) among 49 horses who received antivenom. Eighteen surviving horses (43%) required more than one vial of antivenom. MAIN LIMITATIONS: Possible cases within the searchable database were not included if horses died acutely or responded to symptomatic treatment without receiving antivenom. CONCLUSIONS: Elapid snake envenomation is primarily a syndrome of neuromuscular weakness. Supportive anamnesis or an obvious bite site is rarely encountered. In endemic areas, this diagnosis should be considered for horses with generalised neuromuscular weakness, altered mentation, rhabdomyolysis and/or haemolysis; especially during spring and summer months. Diagnostic suspicion is best confirmed by response to treatment with antivenom.

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

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

Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response.

REASONS FOR PERFORMING STUDY: Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. OBJECTIVES: To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the black walnut extract (BWE) and oligofructose (OF) models of laminitis. METHODS: Real-time qPCR was used to assess lamellar mRNA expression of interleukins-1beta, 2, 4, 6, 8, 10, 12 and 18, and tumour necrosis factor alpha and interferon gamma at the developmental stage and at the onset of lameness. RESULTS: Significantly increased lamellar mRNA expression of cytokines important in the innate immune response were present at the developmental stage of the BWE model, and at the onset of acute lameness in both the BWE model and OF model. Of the cytokines characteristic of the Th1 and Th2 arms of the adaptive immune response, a mixed response was noted at the onset of acute lameness in the BWE model, whereas the response was skewed towards a Th1 response at the onset of lameness in the OF model. CONCLUSIONS: Lamellar inflammation is characterised by strong innate immune response in the developmental stages of laminitis; and a mixture of innate and adaptive immune responses at the onset of lameness. POTENTIAL RELEVANCE: These results indicate that anti-inflammatory treatment of early stage laminitis (and the horse at risk of laminitis) should include not only therapeutic drugs that address prostanoid activity, but should also address the marked increases in lamellar cytokine expression.

A novel model to assess lamellar signaling relevant to preferential weight bearing in the horse.

Supporting limb laminitis (SLL) is a devastating sequela to severe unilateral lameness in equine patients. The manifestation of SLL, which usually only affects one limb, is unpredictable and the etiology is unknown. A novel, non-painful preferential weight bearing model designed to mimic the effects of severe unilateral forelimb lameness was developed to assess lamellar signaling events in the supporting limb (SL). A custom v-shaped insert was attached to the shoe of one forelimb to prevent normal weight bearing and redistribute weight onto the SL. Testing of the insert using a custom scale platform built into the floor of stocks confirmed increased distribution of weight on the SL compared with the unloaded forelimb (UL) and the contralateral (CH) and ipsilateral (IH) hind limbs in six Standardbred horses. In a second part of the study, eight healthy Standardbred horses were fitted with the insert and tied with consistent monitoring and free access to hay and water for 48 h, after which the lamellae were harvested. Real-time qPCR was performed to assess lamellar mRNA concentrations of inflammatory genes and immunoblotting and immunofluorescence were performed to assess lamellar protein concentration and cellular localization of hypoxia-related proteins, respectively. Lamellar mRNA concentrations of inflammatory signaling proteins did not differ between SL and either CH or IH samples. HIF-1α concentrations were greater (P < 0.05) in the SL compared to the CH. This work establishes an experimental model to study preferential weight bearing and initial results suggest that lamellar hypoxia may occur in the SL.

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.

A comparison of seven methods for continuous therapeutic cooling of the equine digit.

REASONS FOR PERFORMING STUDY: Digital hypothermia may be effective for laminitis prophylaxis and therapy, but the efficacy of cooling methods used in clinical practice requires evaluation. OBJECTIVES: To use hoof wall surface temperature (HWST) to compare several cooling methods used in clinical practice. STUDY DESIGN: Experimental crossover design with a minimum washout period of 72 h. METHODS: Seven cooling methods (commercially available ice packs, wraps and boots) and one prototypical dry-sleeve device were applied to a single forelimb in 4 horses for 8 h, during which HWST of the cooled forelimb and the uncooled (control) forelimb was recorded hourly. Results were analysed descriptively. RESULTS: The median (range) HWST from 2-8 h was lowest for the ice and water immersion methods that included the foot and extended proximally to at least include the pastern: 5.2°C (range: 4.8-7.8°C) for the fluid bag and 2.7°C (2.4-3.4°C) for the ice boot. An ice boot that included the distal limb but not the foot resulted in a median HWST of 25.7°C (20.6-27.2°C). Dry interface applications (ice packs) confined to the foot only resulted in a median HWST of 21.5°C (19.5-25.5°C) for the coronet sleeve and 19.8°C (17.6-23°C) for a commercial ice pack. For the dry interface applications that included the foot and distal limb, the median HWST was much higher for the ice pack device, 19.9°C (18.7-23.1°C), compared with the perfused cuff prototype of 5.4°C (4.2-7°C). CONCLUSIONS: Immersion of the foot and at least the pastern region in ice and water achieved sustained HWST <10°C as did a prototype perfused cuff device with a dry interface. Variation between cooling methods may have a profound effect on HWST and therefore efficacy in clinical cases where laminitis prophylaxis or therapy is the goal.

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.

Mitogen-activated kinase pathway activation in epidermal lamellae in the acute stages of carbohydrate overload laminitis models and the effect of regional deep hypothermia on signalling pathways.

REASONS FOR PERFORMING STUDY: In sepsis models, mitogen-activated protein kinases (MAPKs) are reported to incite inflammatory injury to tissues and are purported to be a therapeutic target. OBJECTIVES: To assess MAPK signalling in lamellae in sepsis-related laminitis (SRL) at different time points after induction of laminitis via carbohydrate overload, and to determine the effect of regional deep hypothermia (RDH) on MAPK signalling. STUDY DESIGN: In vitro study using archived tissue samples. METHODS: Lamellar concentrations of MAPKs were assessed in archived lamellar samples from 2 studies: 1) the starch gruel model of SRL with 3 groups (n = 6/group) of horses (control, onset of fever [DEV] Obel Grade 1 lameness [OG1]); and 2) from limbs maintained at ambient (AMB) and hypothermic (ICE) temperatures (n = 6/group) in animals given a bolus of oligofructose. Immunoblotting and immunolocalisation were used to assess lamellar concentrations and cellular localisation of total and activated (phosphorylated) forms of p38 MAPK, extracellular-regulated kinase (ERK) 1/2, and stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK) 1/2. RESULTS: Lamellar samples had statistically significant increased concentrations of activated ERK 1/2 at the onset of OG1 laminitis (vs. control) in the starch gruel model, but showed no significant change between ICE and AMB limbs in the RDH model. Phospho-SAPK/JNK 1/2 exhibited a similar significant increase in the OG1 samples, but was also increased in ICE (vs. AMB) limbs. No statistically significant changes in lamellar p38 MAPK concentrations were noted. CONCLUSIONS: Increased concentrations of activated ERK 1/2 and SAPK/JNK in the acute stages of SRL indicate a possible role of these signalling proteins in lamellar injury. Signalling related to ERK 1/2 and SAPK/JNK 1/2 pathways should be further investigated to determine if these play a detrimental role in laminitis and may be therapeutic targets to be manipulated independently of RDH.

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.