The investigation of insulin-induced laminitis in horses

Melody De Laat (2011). The investigation of insulin-induced laminitis in horses PhD Thesis, School of Veterinary Science, The University of Queensland.

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Author Melody De Laat
Thesis Title The investigation of insulin-induced laminitis in horses
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
Publication date 2011-07
Thesis type PhD Thesis
Supervisor Professor Christopher Pollitt
Professor Martin Sillence
Dr Catherine McGowan
Total pages 280
Total colour pages 38
Total black and white pages 242
Language eng
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary Equine laminitis is a common, complex and insidious disease affecting the lamellar structures of the horse’s foot. The lamellae are located between the inner hoof wall and the distal phalanx and damage results in lameness and loss of function. Many disparate, inciting factors are responsible for lamellar failure, including hyperinsulinaemia. The pathophysiology of the condition has been pursued by researchers but remains largely undescribed. The pathogenesis of hyperinsulinaemic laminitis has proved difficult to study. However, the discovery of an experimental model that induced laminitis with a prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC) within 72 h in ponies, has enabled research on the ‘laminitogenic’ potential of insulin. This thesis aimed to investigate the mechanism of insulin-induced laminitis in horses by: 1) determining the role of increased (or decreased) blood flow to the hoof in the developmental and acute stages of the disease, 2) determining whether hyperinsulinaemic laminitis occurs due to a whole body or local digital mechanism, 3) describing the lamellar lesion caused by hyperinsulinaemia and comparing this to laminitis caused by other factors and 4) examining the role of degradative enzymes, glucose, advanced glycation end products, reactive oxygen species and the insulin-like growth factor (IGF-1) receptor in insulin-induced laminitis. Initially, the p-EHC was examined in healthy, Standardbred horses, with laminitis occurring within 48 h in the treatment group (n = 4), but not in controls (n = 4). Based on hoof wall surface temperatures, persistent vasodilation appeared to be a feature of the developmental and acute stages of the disease. Insulin is a vasodilator, so to test the hypothesis that persistent digital vasodilation is the principal mechanism of insulin-induced laminitis, a separate study was performed. A regional intra-osseous infusion of the potent vasodilator ATP-MgCl2 induced digital vasodilation in the absence of hyperinsulinaemia. Persistent digital vasodilation was achieved for 48 h, but this did not induce laminitis. Thus, insulin does not cause laminitis solely through a vascular mechanism. To determine whether insulin acts directly in the lamellar microenvironment, or whether a systemic mechanism is required, insulin was administered intra-osseously into the lamellar region without concurrent glucose. Unfortunately, significant digital hyperinsulinaemia did not result and the opportunity to determine whether insulin acts at a local level was thwarted. Further work on local induction techniques for the study of laminitis pathophysiology is required. Adaption of the whole body p-EHC model, to allow examination of lamellar tissue obtained during the developmental (6 h, 12 h, 24 h) phase of insulin-induced laminitis, enabled temporal analysis of laminitis pathology. Samples from the acute and developmental stages of the disease were examined with histology, morphometry, immunohistochemistry and transmission electron microscopy (TEM). The hyperinsulinaemic lamellar lesion was described, and compared to the alimentary carbohydrate overload (ACO) model of laminitis histopathology. Structurally, the lesions of insulin-induced laminitis did not differ significantly from ACO. However, insulin-induced lesions were characterised by less lamellar inflammation, as evidenced by more subdued calprotectin immunoreactivity. Significant lamellar pathology, including secondary epidermal lamellar (SEL) lengthening and narrowing and epidermal cell apoptosis, commenced early in the developmental phase (6 h). Cell proliferation was increased late in the developmental (24 h), and in the acute (48 h) phase. Increased cell proliferation and changes in SEL cell morphology may contribute to significant lamellar lengthening by 48 h, leading to lamellar dysfunction and lameness. Lamellar basement membrane (BM) damage commenced late in the developmental phase (24 h) and became more generalised by the acute phase (48 h), suggesting that it was a downstream event. Ultrastructural analysis revealed decreased hemidesmosome density and significant widening of the BM zone. Gelatin zymography and quantitative real time-PCR studies of lamellar tissue demonstrated that minimal metalloproteinase activity occurred in the lamellae during the developmental and acute stages of insulin-induced laminitis, thus discounting their having a primary pathogenic role in BM proteolysis. Advanced glycation end products (AGEs) were up-regulated in the acute phase of insulin-induced laminitis, compared with control horses, but their receptor, RAGE, was not. However, the AGE increase occurred after significant lamellar pathology was underway. Thus, while a pathogenic role for AGEs is unlikely, they may be an important factor in chronic disease. Reactive oxygen species were not up-regulated in laminitic horses, indicating that oxidative stress is not a feature of acute, hyperinsulinaemic laminitis. However, it may occur in chronic disease secondary to AGE formation. The role of glucose in the pathogenesis of the disease was further investigated. A final experiment determined the endogenous pancreatic response to, and the effect on the lamellae of, a 48 h infusion that delivered an equivalent quantity of glucose to that administered during a p-EHC, but without exogenous insulin. The endogenous insulin response to glucose overload was 5-fold lower (~ 200 µIU/mL) than the serum insulin concentration achieved during the p-EHC. Clinical laminitis did not occur, discounting a primary role for glucose in disease pathogenesis. The resultant endogenous hyperinsulinaemia did induce histological lamellar pathology similar to lesions seen during the developmental stages of the disease. Thus, a possible threshold for disease onset was identified. This suggests that hyperinsulinaemic equids are at considerable risk of subclinical disease in the field, especially during periods of prolonged hyperinsulinaemia. Down-regulation of both the insulin and IGF-1 receptors was observed during the developmental and acute phases of insulin-induced laminitis, suggesting that both receptors were activated. Overstimulation of the IGF-1 system leads to cell proliferation and survival, and this may be the mechanism responsible for the increased cell proliferation and decreased apoptosis occurring in the late developmental (24 h) and acute phases of the disease. Overall, this thesis has contributed significant knowledge about hyperinsulinaemic laminitis and provides clear directions for future research. The findings have largely demonstrated which factors are not involved in the pathogenesis of insulin-induced laminitis, and this work has discounted several theories about the disease mechanism. Further work needs to be directed towards the cellular and intra-cellular consequences of insulin and, in particular, how the interaction between insulin and the IGF-1 system may lead to lamellar failure.
Keyword Equine
Matrix Metalloproteinase
Advanced glycation end product
Insulin-like growth factor 1
Additional Notes 14, 20, 22, 23, 29, 32, 33, 38, 41, 46, 53, 61, 81, 82, 94, 106, 111, 120, 121, 129, 132, 133, 143, 144, 148, 163, 164, 169, 177, 185, 187, 188, 227, 242, 245, 255, 277, 280

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Created: Mon, 27 Feb 2012, 11:40:24 EST by Ms Melody De Laat on behalf of Library - Information Access Service