Studies on the pathogenesis of insulin-induced laminitis in horses

Abstract

Laminitis is a common condition of the horse’s foot and is a major source of decreased welfare and economic loss. Laminitis can be categorised into three main forms: endocrinopathic laminitis, sepsis-associated laminitis and supporting limb laminitis. Endocrinopathic laminitis is the most common form of laminitis and develops in association with insulin dysregulation, particularly hyperinsulinaemia. While it is recognised that hyperinsulinaemia is an important component of the pathogenesis, there is still uncertainty as to how hyperinsulinaemia induces laminitis. In addition, there is limited evidence to support a particular therapeutic intervention to limit lesion progression in acute cases.Continuous digital hypothermia (CDH) has been shown to protect the lamellar tissue in sepsis-associated laminitis and is widely used in clinical practice. Furthermore, evaluation of how CDH affects specific pathophysiological events in experimentally-induced sepsis-associated laminitis has led to a better understanding of the pathogenesis of that form of laminitis. Therefore, studies aimed at investigating the effects of CDH during the development of insulin-induced laminitis could provide insight into the pathogenesis and facilitate the development of targeted therapeutics. The purpose of this thesis was to investigate the pathogenesis of insulin-induced laminitis in horses. The studies were conducted in four parts; firstly, the effect of CDH on the development of acute insulin-induced laminitis was investigated; secondly, lamellar energy metabolism and perfusion were investigated during the development of insulin-induced laminitis; thirdly, the effects of CDH on lamellar energy metabolism and perfusion were investigated in healthy horses and during the development of insulin-induced laminitis, and lastly, the effect of CDH on lamellar inflammatory signaling was investigated in insulin-induced laminitis. Eight Standardbred horses underwent laminitis induction using the euglycaemic hyperinsulinaemia clamp (EHC) model of endocrinopathic laminitis with one forelimb subjected to CDH and the other maintained at ambient temperature. Laminitis was induced in all horses and dorsal lamellar sections were harvested and analysed. Based on histological analysis, it was found that CDH reduced the severity of laminitis and prevented histological lesions compatible with lamellar failure. The findings of this first study established CDH as a research tool to investigate the pathogenesis of insulin-induced laminitis, and support the use of CDH as a first aid therapy for acute laminitis associated with hyperinsulinemia. In a subsequent study, lamellar energy metabolism and perfusion were investigated during the development of laminitis in the EHC model using lamellar microdialysis. Lamellar microdialysate samples collected every 6 hours were analysed for glucose, lactate and pyruvate concentrations and lactate to pyruvate ratio (L:P) to investigate lamellar energy metabolism. Microdialysis urea clearance was used to estimate lamellar tissue perfusion. The findings of the study indicated that insulin-induced laminitis develops in the absence of ischaemia or cellular energy deprivation and therefore therapies aimed at improving lamellar perfusion are unlikely to affect the initial development of insulin-induced laminitis.The third and fourth part of this project aimed to understand the protective effects of CDH during laminitis development in order to provide insight into the pathogenesis. Lamellar microdialysate samples from horses that underwent the EHC model were analysed together with archived samples from healthy Standardbred horses and horses that underwent the oligofructose (OF) laminitis model. In each of the induced and control horses one forelimb had been treated with CDH and the other maintained at ambient temperature. The findings of the study showed that a profound decrease in the rate of glucose metabolism occurs in CDH-treated limbs. In addition, evidence of lamellar hypoperfusion was observed in CDH-treated limbs of healthy horses and in horses that underwent the EHC model; however, despite the evidence of lamellar hypoperfusion, lamellar glucose availability was maintained. The findings of this study suggest that CDH may act by limiting energy supply to pathologic cellular processes whilst preserving those critical to lamellar homeostasis, and have identified that the effect of hyperinsulinaemia on lamellar metabolism (particularly protein metabolism) warrants further investigation.The effect of CDH on lamellar inflammatory signalling was investigated by analysing lamellar tissue in ambient and CDH-treated limbs from the EHC model study using polymerase chain reaction for pro-inflammatory cytokine and chemokine genes (CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1β, IL11, COX1 and COX2, TNF-α, E-selectin, and ICAM-1) and immunoblotting for inflammation-associated proteins (STAT1 and STAT3). The findings of this study showed that CDH inhibited lamellar inflammatory signalling in the EHC model and therefore support further studies that investigate the cause, timeline and role of lamellar inflammation in the pathogenesis of insulin-induced laminitis.The novel research described in this thesis expands our understanding of the role of lamellar energy metabolism, lamellar perfusion and lamellar inflammatory signaling in the pathogenesis of insulin-induced laminitis. Importantly, these studies provide support for the use of CDH as a first aid therapy for acute laminitis associated with hyperinsulinaemia. Further work needs to be directed at the systemic alterations induced by hyperinsulinaemia in the EHC model, in particular, hypoaminoacidaemia and its effect on lamellar structural protein homeostasis.