A. W. van Eps

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.

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.

Equine laminitis: cryotherapy reduces the severity of the acute lesion

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

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: comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in standardbred horses.

Laminitis has many triggers and comparing the histopathology of lesions induced by different causes may help to establish whether a common mechanism or multiple pathologies are involved. The aim of this study was to describe the microscopical lesions and to quantify morphometric changes in the lamellae of horses with insulin-induced (n=4) and oligofructose (OF)-induced laminitis (n=4) compared with normal controls (n=4). Archived lamellar samples collected during two previous studies were used. Laminitis was induced within 48 h in standardbred horses with either a euglycaemic, hyperinsulinaemic clamp (EHC) technique or, in a separate experiment, with an overdose of alimentary OF. Normal tissue was obtained from control horses in the EHC experiment that received a balanced electrolyte solution intravenously for 48 h. Six measurements of lamellar length and width were recorded for each hoof. Leucocyte infiltration was assessed by immunolocalization of calprotectin. All control horses exhibited normal lamellar architecture, whereas treated horses developed clinical and histopathological changes consistent with laminitis. Laminitic samples displayed lengthening and narrowing of secondary epidermal lamellae (SELs), rounded epidermal basal cell (EBC) nuclei, mitosis and apoptosis. In the fore feet of laminitic horses, the length from the end of the keratinized axis to the axial tip of the primary epidermal lamellae (PELs) was increased (P<0.05). SELs were significantly longer (P<0.05) and narrower (P<0.05) in the treated horses compared with controls. The two treated groups did not differ from each other in SEL length or width. Calprotectin expression was absent in control horses, moderate in hyperinsulinaemic horses and marked in OF-treated horses. Laminitis induced experimentally with insulin or OF results in comparable lengthening and narrowing of the SELs and elongation of the axial end of the PELs at 48 h. Immunolocalization of calprotectin indicated that hyperinsulinaemia induces less leucocyte emigration than carbohydrate overload at 48 h. The microscopical lesion of laminitis is similar, but not identical in different forms of the disease.

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.

Digital hypothermia inhibits early lamellar inflammatory signalling in the oligofructose laminitis model.

REASONS FOR PERFORMING STUDY The pathophysiological events inhibited by prophylactic digital hypothermia that result in reduction of the severity of acute laminitis are unknown. OBJECTIVES To determine if digital hypothermia inhibits lamellar inflammatory signalling during development of oligofructose (OF) induced laminitis. METHODS Fourteen Standardbred horses were given 10 g/kg bwt OF by nasogastric tube with one forelimb (CRYO) continuously cooled by immersion in ice and water and one forelimb (NON-RX) at ambient temperature. Lamellae were harvested prior to the onset of lameness (24 h post OF administration, DEV group, n = 7) or at the onset of lameness (OG1 group, n = 7). Lamellar mRNA was purified and cDNA produced for real time-quantitative PCR analysis of mRNA concentrations of cytokines (IL-6, IL-1β, IL-10), chemokines (CXCL1, CXCL6, CXCL8/IL-8, MCP-1, MCP-2), cell adhesion molecules (ICAM-1, E-selectin), COX-2 and 3 housekeeping genes. Data were analysed (NON-RX vs. CRYO, NON-RX vs. archived control [CON, n = 7] lamellar tissue) using nonparametric tests. RESULTS Compared with CON, the OG1 NON-RX had increased (P<0.05) lamellar mRNA concentrations of all measured mediators except IL-10, IL-1β and MCP-1/2, whereas only CXCL8 was increased (P<0.05) in DEV NON-RX. Within the OG1 group, CRYO limbs (compared with NON-RX) had decreased (P<0.05) mRNA concentrations of the majority of measured inflammatory mediators (no change in MCP-1 and IL-10). Within the DEV group, mRNA concentrations of CXCL-1, ICAM-1, IL-1β, CXCL8 and MCP-2 were decreased (P<0.05) and the anti-inflammatory cytokine IL-10 was increased (compared with NON-RX limbs; P<0.05). CONCLUSIONS Digital hypothermia effectively blocked early lamellar inflammatory events likely to play an important role in lamellar injury including the expression of chemokines, proinflammatory cytokines, COX-2 and endothelial adhesion molecules. POTENTIAL RELEVANCE This study demonstrates a potential mechanism by which hypothermia reduces the severity of acute laminitis, and may help identify molecular targets for future laminitis intervention.

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.

Prolonged, continuous distal limb cryotherapy in the horse

REASONS FOR PERFORMING STUDY The recommended duration of cryotherapy in horses is currently extrapolated from human medicine. Prolonged, continuous cryotherapy (days rather than minutes) may be of therapeutic value if it is safe and well tolerated. OBJECTIVES To evaluate the effect of prolonged, continuous application of ice and water to the equine distal limb. METHODS A slurry of ice and water was applied to the right forelimb of 4 Standardbred horses for 48 h. Hoof temperature, ambient temperature and ice boot temperature were logged continuously and clinical observations recorded every 2 h. Lameness examinations were performed prior to application and 1 week, 6 months and 1 year after removal of the ice boot. RESULTS Continuous cryotherapy was well tolerated and resulted in marked cooling of the treated foot. No significant variation in clinical parameters was noted, and no lameness or gross pathology noted in the treated limbs at any examination period. CONCLUSIONS The continuous application of ice and water is a safe and effective means of cooling the equine distal limb. The extremely low, constant temperatures achieved may be of benefit in the management of various distal limb conditions. POTENTIAL RELEVANCE Providing clinicians and research workers with a potent therapeutic/prophylactic modality for disorders of the equine distal limb.

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.