, 2004, Kolko et al., 2007 and Bazan et al., 1986). Under abnormal conditions PLA2 and its products are involved in neuroinflammation processes, oxidative stress and neural cell injury. Furthermore, PLA2 and metabolites in excess are also related with Alzheimer’s and Parkinson’s diseases, ischemia and in multiple sclerosis (Farooqui and Horrocks, 2006). In brain, PLA2s mediate several

physiological responses: neurotransmitters release, neurite outgrowth, cellular membrane repair and cellular growth and differentiation (Farooqui and Horrocks, 2004). Based on these last results, the aim of this work was to analyze the effect of a PLA2 isolated from Lachesis muta snake venom (named LM-PLA2-I) on axotomized retinal ganglion cell survival kept in culture and also to investigate the mechanism of action of this PLA2 in cell survival. Medium 199 and fetal calf serum (FCS) were purchased from see more Gibco (Gaithersburg, USA), L. muta snake HSP inhibitor venom, 1.2-bis (2-aminophenexy) ethane-N,N,NX,NX-tetraaceticacid (BAPTA-AM), glutamine,

penicillin, lysophosphatidylcholine from egg yolk (L 4129), fatty acids mixture (1892-1AMP and 49441-U), streptomycin, p-bromophenacyl bromide (p-BPB), poly-l-ornithine and horseradish peroxidase (HRP) were obtained from Sigma (St. Louis, USA). Chelerythrine chloride came from BIOMOL (Plymouth Meeting, USA). Dimethyl sulphoxide (DMSO) was obtained from Mallinckrodt Baker (New Jersey, USA). The inhibitor of JNK V (1,3-Benzothiazol-2-yl-(2-((2-(3-Pyridinil)ethyl)amino)-4-pyrimidinyl)acetonitrile) and Rottlerin were purchased from Calbiochem (California, USA). Trypsin was obtained from Worthington Biochemical (New Jersey, USA). The phospholipase A2 enzyme (LM-LPA2-I) was purified by two chromatographies steps; a gel filtration on a Sephacryl S-200HR followed by a reverse-phase C2/C18 using FPLC apparatus as previously described (Fuly et al., 1997). Lipids (LPC as well as fatty acids) were dissolved in small volumes of chloroform, dried to a thin film under a gentle nitrogen

flow and Arachidonate 15-lipoxygenase vacuum pumped to remove the organic solvent. After, dried lipid was suspended in 150 mM NaCl, extensively vortexed and then ultrasonically dispersed in a bath sonicator for 5 min at room temperature. Then, lipids were kept at 4 °C until experiments. The enzymatic activity of LM-LPA2-I was measured by the indirect hemolysis method, by using washed rabbit erythrocytes and hen’s egg yolk as substrate, as previously described (Fuly et al., 1997 and Fuly et al., 2002). Modification on histidine residues of LM-LPA2-I was carried out by incubating PLA2 with p-bromophenacyl bromide (10 μM, final concentration) dissolved in DMSO. Incubation was carried out overnight at 4 °C, and then the excess of reagent was removed by dialysis, when necessary. Immediately after, samples were assayed for the indirect hemolytic test and the effect on retinal ganglion cell survival.