Equine Laminitis: a Tissue Microdialysis Study of Lamellar Bioenergetics Carlos Eduardo Medina Torres DVM, MSc, DVSc, DipACVIM A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 School of Veterinary Science Abstract Laminitis is a debilitating and painful ailment of the equine foot. The pathophysiological mechanisms are diverse and still poorly understood. Disruption of cellular energy pathways may play a role in the different forms of laminitis including sepsis- and endocrine- associated laminitis and particularly supporting limb laminitis (SLL) where decreased perfusion of the lamellar tissue is suspected. The aim of this thesis was to characterize the normal bioenergetic profile of the lamellar tissue, and to establish if changes in metabolite patterns and microvascular perfusion occur as a result of modifications in limb load cycling activity and during the development of experimentally induced sepsis-associated laminitis. A tissue microdialysis technique was developed for serial measurement of lamellar energy metabolites and perfusion over a 24 hour period. Microdialysis probes were placed in the lamellar, sublamellar, and skin dermis of 14 healthy horses and glucose, lactate, pyruvate, urea and glycerol concentrations, lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios were determined in microdialysis samples (dialysate). Dialysate volume and metabolite concentrations were stable for 24 hours and the technique was clinically well tolerated and resulted in minimal histological reaction at the probe site. There were key significant differences in dialysate composition between lamellar and skin dialysate (lower glucose and urea concentraitons and higher L:G in lamellar dialysates). Lamellar probe position (sublamellar dermis vs lamellar dermis) significantly affected the dialysate composition. In two subsequent studies, 9 horses instrumented with lamellar and skin microdialysis probes were subjected to sequential interventions designed to modify tissue perfusion (4 interventions) and limb load cycling activity and weight bearing (4 interventions). Urea (20 mmol/L) was added to the perfusion fluid to investigate if urea clearance (a microdialysis- based method used to assess local blood flow) could identify lamellar perfusion changes. Energy metabolite concentrations (glucose, lactate, pyruvate), L:G, L:P and urea clearance were determined during each intervention and compared to baseline. A following study investigated changes in lamellar bioenergetic composition and perfusion during the development of experimentally induced, sepsis-associated laminitis. Microdialysis probes were placed in the lamellar tissue of one forelimb in 12 horses. Treatment (n=6) and control (n=6) horses received oligofructose (OF) and water via nasogastric tube. Bihourly sampling was performed. Lamellar metabolite composition, L:G, ii L:P and urea clearance were determined and compared. Plasma glucose, lactate, pyruvate and urea were measured and compared; their correlation with dialysate composition was investigated. In another study using lamellar and skin dialysate and tissue sections from healthy horses (n=7), and lamellar dialysate and plasma samples from OFT (n=4) and CON (n=4) horses, targeted metabolomic analysis (i.e. 44 central carbon metabolites) was performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The differences between the lamellae and skin in healthy horses and the differences between OFT and CON horses were investigated. In the intervention studies, urea clearance was capable of detecting profound changes in lamellar perfusion, and, in combination with fluctuations in energy metabolite patterns, mild perfusion changes were also detected. Significant changes in lamellar energy metabolite composition and urea clearance were observed when limb load cycling activity was modified: significant increases in glucose and urea clearance, compatible with an increase in lamellar perfusion, were observed when limb activity increased. In the OF study, glucose decreased significantly in lamellar dialysate in the OFT group (OFT-L), and was consistently lower in OFT than in CON horses. Lactate was higher in OFT skin (OFT-S) compared with OFT-L and pyruvate decreased significantly from baseline in OFT-L and was also significantly less than CON-L. L:G and L:P increased significantly compared with baseline in OFT-L and OFT-S, but not CON-L or plasma from either group. Urea concentration in OFT-L decreased significantly compared with baseline (increased urea clearance); urea remained stable in CON-L. This study demonstrated that ischaemia does not occur during the developmental phase of laminitis in the OF model. There was evidence of lamellar hyperaemia, together with some bioenergetic disturbances. Despite increased perfusion, glucose concentrations in the lamellar interstitium were markedly reduced. There was no definitive evidence of lamellar bioenergetic failure, however it is still possible that reduced glucose availability could have compromised lamellar basal epithelial cell metabolism in particular. Metabolomic analysis of lamellar and skin dialysate showed no difference in metabolite composition in these two tissues in healthy horses, and the metabolome of dialysate and tissue samples was also found to be similar. A difference in central carbon metabolism (CCM) between plasma and lamellar dialysate, and between OFT and CON horses was observed. iii Metabolomic analysis of lamellar CCM was capable of differentiating horses developing experimental laminitis from controls. Lamellar malate, pyruvate, aconitate and glycolate, and plasma malate alone, were identified as the source of differentiation between OFT and CON groups. The novel research described in this thesis demonstrated that lamellar microdialysis is well tolerated, and together with urea clearance it can be used for assessment of lamellar bioenergetic and perfusion changes. Microdialysis urea clearance demonstrated an increase in lamellar perfusion rather than ischaemia during the development of sepsis-associated laminitis (OF study). Results also suggest that lamellar perfusion and energy balance may be related to limb load cycling and are particularly affected by ambulation. The findings support the hypothesis that reduced limb load cycling activity, but not increased weight bearing, may be associated with reduced lamellar perfusion. Though further studies are required to determine if lamellar hypoperfusion and energy failure are key contributors to SLL, the development of strategies to increase limb load cycling in patients at risk of SLL may be important for prevention in horses at risk. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Carlos E. Medina-Torres v Publications during candidature Peer-reviewed papers C.E. Medina-Torres, C.C. Pollitt, C. Underwood, S. Collins, R.E. Allavena, D.W. Richardson, A.W. van Eps. (2014) Equine lamellar energy metabolism studied using tissue microdialysis. The Veterinary Journal 201, 275 – 282. C.E. Medina-Torres, C. Underwood, C.C. Pollitt, E.M. Castro-Olivera, M.P. Hodson, D.W. Richardson, A.W. van Eps. (2015) Microdialysis measurements of equine lamellar perfusion and energy metabolism in response to physical and pharmacological manipulations of blood flow. Submitted to The Equine Veterinary Journal. C.E. Medina-Torres, C. Underwood, C.C. Pollitt, E.M. Castro-Olivera, M.P. Hodson, D.W. Richardson, A.W. van Eps. (2014) The effect of weight bearing and limb load cycling on equine lamellar perfusion and energy metabolism measured using tissue microdialysis. The Equine Veterinary Journal, [epub ahead of print]: doi: 10.1111/evj.12377 C.E. Medina-Torres, C. Underwood, C.C. Pollitt, E.M. Castro-Olivera, M.P. Hodson, D.W. Richardson, A.W. van Eps. (2015) Microdialysis measurements of lamellar perfusion and energy metabolism during the development of laminitis in the oligofructose model. The Equine Veterinary Journal, [epub ahead of print]: doi: 10.1111/evj.12417 C.E. Medina-Torres, A.W. van Eps, L.K. Nielsen, M.P. Hodson. (2015) A liquid