Anoxia / Reoxygenation of equine endothelial cells produces reactive oxygen species: an oxymetric and electron paramagnetic resonance investigation G. de la Rebière de Pouyade, A. Mouithys-Mickalad, A. Salciccia, J. Ceusters, G. Deby-Dupont, D. Serteyn Equine Clinic, Large Animal Surgery, Faculty of Veterinary Medicine and Center for Oxygen Research and Development, University of Liège, Sart Tilman, 4000 Liège, Belgium Take Home Message: Equine endothelial cells (EC) submitted to anoxia/reoxygenation (A/R) produce ROS (free radicals) and show an altered respiratory function (oxygen consumption by mitochondria). Introduction: Endothelium participates in the regulation of the inflammatory response.1 In laminitis, ischemia/reperfusion-induced injuries and oxidative damages have been suspected.2,3 Herein, we investigated the effect of anoxia/reoxygenation on ROS production by EC and mitochondrial respiratory function. Materials and Methods: EC cultures were obtained from equine carotids. Detached cells (107) were submitted either to 3 cycles of 20min anoxia followed by reoxygenation in the presence of POBN (50mM)/ethanol (2%) mixture as spin trap (Fig.1) or to 1h of anoxia followed by reoxygenation and immediate addition of 100mM DMPO as spin trap. Normoxic cells were used as control (n=3). Anoxia and reoxygenation were monitored by high-resolution respirometry and free radical production was evidenced by electron paramagnetic resonance (EPR). After each A/R, the respiratory slopes were measured and compared to the first pre-anoxia slope taken as 100% (n=10). O2 100% 89.05% 74.24% 60.06% * Fig.1: Typical graph of A/R cycles followed by oxymetry. *p<0.05 compared to slope1 Results: After A/R cycles, high intensity EPR spectra, assigned to the POBN/•CH(OH)CH3 adducts were observed (Fig 2B), compared to normoxic cells (control, Fig. 2A). Respiratory slopes 3 and 4 were significantly decreased (p<0.05) compared to the slope 1 without significant cell viability changes between the onset and the end of the experiment (Fig.1). After 1h of anoxia followed by reoxygenation, an enhanced production of DMPO-OH adducts was observed (Fig. 3B) compared to control (Fig. 3A). EPR device Oxygraph Discussion: The EPR spectra were consistent with the trapping of superoxide (DMPO adduct) and hydroxyl radical (POBN adduct) produced by EC. Such a production could derive from either xanthine oxidase activity, NADPH oxidase activation or mitochondrial dysfunction.4 Significant reduction of the respiratory rate over A/R cycles supports the mitochondrial dysfunction theory. Production of ROS by EC could be involved in the disruption of the endothelial barrier, the recruitment of neutrophils and participate to the development of laminitis. Control Anoxia Fig. 2 Control Anoxia Fig. 3 Conclusion: ROS from EC submitted to A/R might play a crucial role in the oxidative aspect of laminitis likely by the pathway of mitochondrial dysfunction. The use of drugs (e.g: LMWH) able to protect endethelium may have beneficial effects in the therapeutic approach of laminitis Duffy et al., 2004. J. Vet. Em. Crit. Care. 14, 84-99 Hood et al., 1993. J. Vet. Int. Med. 7, 228-234 Yin et al., 2009. Vet. Immunol. Immunopathol. 129, 211-215 Ray and Shah, 2005. Clin. Sci. 109, 217-226 Acknowledgment: This work was supported by a FNRS-FRIA scholarship