Isolation of Equine Hoof Epidermal Cells for in Vitro Studies

s Vol 30, No 2 (2010) 107 CHO administration or upon reaching Obel grade 3 lameness whichever occurred first. Horses in the treatment group received 8.5 mg/kg PTX in 1 L saline IV over 30 minutes beginning 12 hours prior to CHO administration, then every 12 hours thereafter until the completion of the study. MMP activity in the digital plasma will be evaluated using gelatin zymography. PRELIMINARY RESULTS All control horses (5/5) developed Obel grade 3 lameness, four of them within 32 hours post-CHO. However, only one PTX horse reached Obel grade 3. Of the remaining four PTX horses, two never became lame, one reached Obel grade 2 and then returned to almost complete soundness by 60 hours, and one fluctuated between Obel grades 1 and 2 during the entire study. MMP analysis is being performed at this time. DISCUSSION The horses appear to have responded favorably to PTX treatment with significantly decreased lameness. This response to PTX could result from 1) MMP inhibition, 2) anti-inflammatory effects of PTX, or 3) individual response to CHO. CLINICAL RELEVANCE If future data confirms our preliminary findings, PTX could be used as a preventative/treatment for cases of laminitis associated with carbohydrate overload. CONCLUSION PTX appears to decrease lameness associated with CHOinduced laminitis. Isolation of Equine Hoof Epidermal Cells for in Vitro Studies Hannah Galantino-Homer, Susan Megee, and Makoto Senoo, Laminitis Institute, Department of Clinical Studies/New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA TAKE HOME MESSAGE We present a method for aseptic retrieval of equine hoof epidermal cells. INTRODUCTION Disruption of basal epidermal cell-basement membrane adhesion is a key event during the developmental and acute phases of laminitis and disorganized proliferation of basal cells may contribute to the pathology of chronic laminitis. The isolation of these cells would facilitate in vitro studies of normal and disrupted basal cell-extracellular matrix adhesion and proliferation and could provide basal epidermal progenitor cells (BEPCs) for autografting following hoof wall resection. MATERIALS AND METHODS Aseptic surgical techniques are used to remove a 5 cm by 4 cm section of the dorsal hoof wall of equine cadavers up to 3 hr following euthanasia. Coronary and lamellar epidermal tissue and underlying dermal tissue are dissected by scalpel and washed in DPBS containing antibiotic/antimycotic and held in DMEM, 10% Fetal Calf Sera and antibiotic/antimycotic. Mechanical disruption by dicing, scraping, and electric homogenizer in combination with enzymatic digestion is used to generate a single cell suspension of mixed epidermal and dermal cells that are then frozen or used immediately for in vitro studies. RESULTS Mixed cells and selective culture have been used successfully in a preliminary experiment to generate BEPC holoclones. Non-selective in vitro culture of mixed cells results in non-uniform cell proliferation and differentiation. DISCUSSION AND CLINICAL RELEVANCE Selection of BEPCs is essential for repeatable culture of uniform populations of basal cells for laminitis pathophysiology studies as well as for successful therapeutic epidermal tissue regeneration. CONCLUSION Equine hoof BEPC selective culture from isolated mixed epidermal and dermal cells is currently under development. Chemical and Biological Properties of Aqueous Black Walnut Extracts That Contribute to the Pathogenesis of Laminitis D.J. Hurley, J.N. Moore, K.A. Hurley, L.J. Berghaus, K.L. Galland, and R.S.K. Majerle, Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, the University of Georgia, Athens, GA, Department of Large Animal Medicine, College of Veterinary Medicine, the University of Georgia, Athens, GA, Department of Chemistry, Hamline University, St. Paul, MN 108 Abstracts Vol 30, No 2 (2010) TAKE HOME MESSAGE Black walnut extracts (BWE) generate radicals in aqueous medium, activate leukocytes, alter cell membranes and induce apoptosis in vitro, activities that may account for its rapid effect in horses. INTRODUCTION BWE induces systemic inflammatory activation and laminitis faster than carbohydrate overload. In this study, we examined the capacity of BWE to produce radicals and activate leukocytes. MATERIALS AND METHODS Aqueous and organic BWE were tested for radical-generating capacity in several media, and aqueous BWE were tested for endotoxin. Mononuclear cells treated with BWE (+/polymyxin B, PMB) were assessed for induction of inflammatory products, genes and apoptosis, and neutrophils for changes in membrane fluidity, potential and apoptosis. RESULTS BWE induced radicals in aqueous media, the organic extract was 10x more efficient, radical production was prolonged by lipid binding proteins in the medium, and BWE contained endotoxin. BWE induced production of tissue factor and TNFa by mononuclear cells was reduced 50% by PMB. BWE and LPS induced most inflammatory genes equivalently, but BWE did not induce IL-10, TRAF-1, RANTES, or L-selectin, and LPS did not induce IL-12. Only BWE increased caspase 3/7 activity, membrane fluidity, and Annexin Vbinding, anddecreased membranepotential inneutrophils. DISCUSSION Production of radicals and activation of leukocytes suggests that components of BWE are important in the inflammatory activation associated with the pathogenesis of laminitis. Plant-derived radical generators, irritants and LPS in BWE may be responsible for rapid activation of inflammation in horses. CLINICAL RELEVANCE BWE is used to induce laminits because its actions are rapid and symptoms are transient. Results of this study suggest that BWE contains components that generate radicals and induce inflammatory responses, as well as LPS. The limited lifetime of the plant-derived radical generators and irritants may be responsible for the limited nature of the disease. CONCLUSION BWE contains plant-derived radical generators and irritants, as well as LPS that may result in rapid development of laminitis. Microarray Analysis of Gene Expression in Laminar Tissue From Horses After Carbohydrate Overload So Young Kwon, Michel L. Vandenplas, David J. Hurley, E. Noschka, Marco A. Lopes, Thomas P. Robertson, and James N. Moore, Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, Dept. Pharmacology and Physiology, College of Veterinary Medicine, University of Georgia, Athens, GA TAKE HOME MESSAGE Microarrays can identify gene products that may contribute to the progression of laminitis. INTRODUCTION A 3,000 gene equine cDNA microarray was used to examine gene expression in laminar tissues from horses after carbohydrate (CHO) overload. MATERIALS AND METHODS Horses (6/group) were administered CHO via a nasogastric tube and tissues were collected either 17-18 hours (pre-clinical group) or 22-24 hours later (Obel grade 1 group); control horses received water (24 hours; control group). After euthanasia, high quality RNA was extracted from laminar tissue and used to generate cDNA targets that were hybridized to the mirrored microarrays. Each slide was hybridized with a common reference target prepared from pooled RNA from 6 control horses and an experimental target prepared from each individual CHOtreated horse. Scanned images were normalized and analyzed. RT-qPCR analysis for gene expression of three genes (IL-6, SOD-2 and MCP-3) was preformed on the individual RNA samples. Statistics: modified t-test with significance set at P < 0.05. RESULTS Compared to the control horse group, expression of 241 genes was significantly up-regulated whereas expression of 440 genes was down-regulated in the pre-clinical group. In the Obel grade 1 group, expression of 430 genes was upregulated and expression of 198 genes was downregulated. RT-qPCR data for IL-6, MCP-3 and SOD-2 expression were similar to those identified in laminar tissue of horses with black walnut extract-induced laminitis, with greater expression occurring at Obel grade 1. DISCUSSION CONCLUSIONS AND CLINICAL RELEVANCE Microarray analysis of gene expression in laminitis can determine changes in gene expression that may contribute to