Laminitis in the perfused isolated equine foot

Equine laminitis is a common, severe and often life threatening disease in horses, the pathogenesis of which is not yet fully understood. Several mechanisms for the development of laminitis have been proposed, and there are two main theories currently being investigated, one focussing on the changes in the blood flow in the distal limb, and the other focussing on the presence and tissue activity of trigger factors such as metalloproteinases and lipopolysaccharides. Most studies into equine laminitis have been either in-vivo studies, where the horses showed clinical signs and pain, or studies in isolated small tissue specimens, such as single vessels or hoof wall explants. In order to increase our understanding of this disease, which is necessary for the development of better prophylactic and therapeutic measures, the investigation of the equine distal limb in toto will offer a complex inter- active system, without the necessity of animal experiments. The possibility of blood perfusion of equine distal limbs has been investigated by our research group and was found to be allowing the establishment of physiological conditions within isolated the distal limb for up to 10 hours. In the study presented in this proposal, we intend to investigate several mechanisms that have been shown to induce laminitis in live horses in isolated, extracorporeal perfused distal limbs of horses. Specifically, we propose to compare the laminitic changes caused by endotoxine addition to the perfusate, MMP-activation and as a consequence of a reperfusion injury with and without cycling loading in a material testing machine, simulating loading of the limb in vivo. In these samples histological and electromicroscopical changes will be analysed. In additional limbs, laminitis will be induced and the effect of two temperatures (hyperthermia and crotherapy) changes will be evaluated. For the comparison of the groups, statistical analysis will be carried out using ANOVA and Friedman tests for parametric and non-parametric data respectively. From the three laminitis induction methods, we expect to be able to select a suitably naturalistic laminitis model that can be the basis for future investigations that allow in-depth analysis of the effect of drug applications and biomechanical treatments of the hoof, while following the 3R guidelines to reduce, replace and refine animal experiments. The research group into extracorporeal perfusions is the only one in Austria, and is keen to develop this model for other organs and other species for the veterinary and human medicine sectors. The proposed project is an essential step towards achieving this goal.

This study used limbs of horses slaughtered for human consumption in a regular abattoir together with the same horses blood for a ten hour perfusion of these limbs. The aim of this study was to investigate the development of equine laminitis, an excruciatingly painful disease that carries a poor prognosis. Laminitis is an inflammation of the suspensory apparatus of the pedal bone within the hoof capsule, that in the worst case can lead to the loss of the hoof and therefore necessitate euthanasia of the horse. Even in the less severe cases the pain caused by this disease is so painful, that comparisons to bone metastases have been made.For the present study the following factors that are known risk factors for equine laminitis were examined: 1.) endotoxemia that is the accumulation of fragments of bacterial walls that are being absorbed into the blood stream from the intestines during colic or from the uterus during retention of fetal membranes. In this part of the study we could show, that - within only a few hours - endotoxins damage the suspensory apparatus of the pedal bone and lead to such an accumulation of leukocytes that endotoxins should not be regarded as a risk factor only but as causative agents in their own right. 2.) hyperinsulinaemia, an increased concentration of insulin that is commonly found in horses with metabolic dyndrome and insuline resistance. Here we could show that a few hours of increased insulin concentration increase the vascular resistance and create edema in the suspensory apparatus of the pedal bone, mediated by endothelin receptors. This is an explanation for the increased risk of horss with naturally occurring hyperinsulinaemia for the development of laminitis.Summarising the results of this study it can be said that the research into equine laminitis on the isolated equine limb is in accordance with the 3R criteria aiming at a more critical use of in vivo experiments, and that a considerable contribution to the understanding of the development of laminitis was achieved; such contributions are also expected in the future with the application of this methodology.