Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study

BACKGROUND: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis. HYPOTHESIS: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis. ANIMALS: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5). METHODS: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2. RESULTS: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.     

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.     

Evaluation of equine digital Starling forces and hemodynamics during early laminitis

A carbohydrate overload model was used in 8 horses to evaluate Starling forces and hemodynamics of the digit during the prodromal stage of acute laminitis. A pump-perfused extracorporeal digital preparation was used to evaluate blood flow, arterial pressure, venous pressure, capillary pressure, isogravimetric capillary filtration coefficient, osmotic reflection coefficient, and vascular compliance. From these data, pre- and postcapillary resistances and pre- to postcapillary resistance ratios were determined. Vascular and tissue oncotic pressures were estimated from plasma and lymph protein concentrations, respectively. The osmotic reflection coefficient, an estimation of capillary permeability, was determined by means of the lymph protein wash-down technique. Using the collected data, tissue pressure in the digit was calculated by use of the Starling equation. In the isolated digit, mean isogravimetric capillary pressure was 55.13 mm of Hg, mean plasma and lymph oncotic pressures were 22.29 mm of Hg and 7.2 mm of Hg, respectively, the mean osmotic reflection coefficient was 0.66, the mean capillary filtration coefficient was 0.003 ml/min/mm of Hg/100 g, and mean interstitial fluid pressure was 44.82 mm of Hg. The high capillary pressure appeared to be caused by high vascular resistance from the venous side, predisposing to enhanced capillary filtration and interstitial fluid accumulation.     

Nuclear magnetic resonance-based metabonomic study of early time point laminitis in an oligofructose-overload model

Reasons for performing study: NMR‐metabonomics is an unbiased evaluation method, which allows to comprehensively study changes of the equine metabolic profile in early time point laminitis. This might give insight into the early stages of disease development. Objectives: To detect hitherto unknown changes in blood metabolites during the development of oligofructose‐induced laminitis by comparing pre‐ and post induction blood samples. Methods: Prior to laminitis induction blood was sampled to establish control values. Post oligofructose administration (POA) blood was collected every 3 h for 24 h. One‐dimensional ¹H‐NMR spectra of the blood plasma were statistically analysed. Results: NMR resonances of >20 metabolites were identified using this technique. Already known changes (e.g. lactate concentrations) were confirmed using this method. Interestingly, oligofructose, a carbohydrate usually considered indigestible in the small intestine, or derivatives of oligofructose, was detected in plasma. Horses also showed increased phosphatidylcholine and/or low density lipoprotein levels POA, indicating a change in blood lipid composition. An increase in phosphatidylcholine is consistent with the breakdown of the mucosal layer of the large intestine and increased permeability of the gut. Conclusion: Due to the nontargeted approach of metabonomics, new unexpected changes can be identified, in this case the hitherto unknown oligofructose uptake through the mucosal wall and the phospholipid changes. Potential relevance: Metabolic changes in disease can be observed using NMR metabonomics. Oligofructose is used in feedstuffs and transport mechanisms through the mucosa should be studied. Phospholipids could point to a compromise of the intestinal wall during laminitis development.     

Pain management for laminitis in the horse

The inability to control pain is the most common reason for cessation of treatment and euthanasia in cases of laminitis, yet pain also serves a unique protective function in these cases, particularly in the acute phase when lamellar integrity is weakened. Successful analgesia requires an understanding of the disease pathophysiology, the sources of pain in laminitis, methods of serial pain evaluation, and methods of analgesia including systemic and regional techniques. This review discusses the approach to analgesia including detail of traditional and novel analgesic medications and techniques with specific reference to the laminitis case.     

Pathophysiology of acute laminitis

This article reviews research findings relating to the pathophysiology of acute laminitis in horses. The data presently available suggest that the onset of the condition may be due to constriction of the postcapillary vessels in the digit, leading to increased capillary hydrostatic pressure and movement of fluid into the interstitial space.     

Histopathology of insulin-induced laminitis in ponies

Reasons for performing study: Ponies with laminitis associated with insulin resistance and hyperinsulinaemia lack systemic and/or intestinal inflammatory signs, suggesting a different pathogenesis potentially reflected in differing histopathology. Objectives: To describe the histological appearance and quantify morphological changes in primary and secondary epidermal lamellae (PEL and SEL) of laminitis lesions from ponies with insulin‐induced laminitis. Methods: Equine hoof lamellar tissue was obtained from 4 control ponies and 5 ponies with laminitis induced following infusion of insulin (1036 ± 55 µU/ml) while maintaining euglycaemia for 55.4 ± 5.5 h. Sections from all 4 hooves were stained and examined by a veterinary pathologist. Measurements of lamellar length (PEL and SEL) were made in mid‐dorsal sections of the right forefeet by 2 blinded observers. Immunolabelling for calprotectin was performed using a monoclonal antibody. Results: No lesions were detected in normal ponies. Lesions detected in ponies with laminitis were variable in severity between ponies. Within ponies, SEL lesions were more severe along the axial region of PEL. Lesions included swelling, disorganisation and abnormal keratinisation of epidermal cells, increased mitotic activity and apoptosis. Separation of basement membranes was minimal. Immunostaining revealed inflammatory cells within the lamellar dermis. SEL were significantly elongated in laminitic hooves relative to controls, with the greatest elongation in those attached to abaxial and middle regions of PEL. Conclusions: Laminitis induced by prolonged infusion of insulin lacked widespread basement membrane disintegration, and increases in epidermal cellular proliferation at axial aspects were marked for this acute stage of disease. Potential relevance: Defining equine laminitis entirely in terms of separation of the basement membrane may not be appropriate for laminitis associated with hyperinsulinaemia.     

Effects of an endothelin receptor antagonist and nitroglycerin on digital vascular function in horses during the prodromal stages of carbohydrate overload-induced laminitis

OBJECTIVE: To evaluate changes in digital vascular function in horses with carbohydrate overload (CHO)-induced laminitis and determine the effects of an endothelin (ET) receptor antagonist and nitroglycerin on laminitis-associated vascular dysfunction. ANIMALS: 20 adult horses without abnormalities of the digit. PROCEDURES: Hemodynamic variables were recorded before (baseline) and hourly after all horses were administered a CHO ration via nasogastric tube. In 4 groups of 5 horses each, saline (0.9% NaCl) solution or ET receptor antagonist (10(5)M in digital blood) was administered into the digital arterial circulation according to 1 of 2 schedules. During anesthesia, blood flow; arterial, venous, and capillary pressures; and total, precapillary, and postcapillary resistances were measured in an isolated perfused digit of each horse. In all groups, nitroglycerin was infused (10(5)M in digital blood), and digital microvascular assessments were repeated. RESULTS: The CHO caused a significant decrease in right atrial pressure by 14 hours that was not affected by administration of saline solution or ET receptor antagonist. In isolated digits of anesthetized horses, CHO resulted in a significant decrease in digital blood flow associated with a significant increase in total and postcapillary resistances. Treatment with the ET receptor antagonist and nitroglycerin caused a significant decrease in total resistance. Postcapillary resistance was significantly decreased following treatment with the ET receptor antagonist but was not altered by treatment with nitroglycerin. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with an ET receptor antagonist and nitroglycerin resulted in significant improvement in vascular resistance in isolated perfused digits of anesthetized horses with CHO-induced laminitis.     

Glucocorticoids and laminitis in the horse.

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