A value of P < 0.001 was considered significant. To investigate

the effect of LM-PLA2-I on retinal ganglion cell survival, we added increasing concentrations of the enzyme to culture medium. Fig. 1A reports the influence of LM-PLA2-I (2.5–12.5 μg/mL) on ganglion cell survival. Addition of LM-PLA2-I (5.0 μg/mL) to cell culture resulted in a 50% SRT1720 manufacturer increase on retinal ganglion cell survival. As also observed in Fig. 1A, at higher concentrations of LM-PLA2-I (12.5 μg/mL), the effect upon ganglion cells survival was less pronounced, but surprisingly a neuronal outgrowth was observed (data not shown). The effect of LM-PLA2-I upon ganglion cells was a bell-shaped curve with a maximum survival effect at 5.0 μg/mL (Fig. 1A). Accordingly, we use 5.0 μg/mL of LM-PLA2-I in further experiments to investigate the

mechanism of action of the enzyme upon retinal ganglion cell survival. This survival effect of LM-PLA2-I upon ganglion cells was dependent of its enzymatic activity, since when LM-PLA2-I was chemically modified with p-BPB (10 μM), both activities (named survival and hemolysis) were abolished with this treatment (data not shown), clearly showing a parallelism between them, and suggesting learn more the need of generation of LPC by the PLA2 enzyme to express the observed effect on the retina. Indeed, Fig. 1B shows that commercial LPC, at 10 μM also much protected retinal ganglion cells from death. On the other hand, higher concentrations of LPC (up to 25 μM) led cells to death, being considered toxic on such concentrations; while at lower concentrations (5 μM), LPC was ineffective upon ganglion cells (Fig. 1B). It is worthwhile emphasizing

that a synergic effect between LPC (5 μM or 10 μM) and fatty acids (10 μM) upon ganglion cells survival was not observed (data not shown). Moreover, fatty acids alone (5–50 μM) also did not interfere on ganglion cell survival; neither stimulated nor inhibited (data not shown). The mechanism of action of LM-PLA2-I on the survival effect of ganglion cells was investigated (Fig. 2). When cultures were treated with 1.25 μM chelerythrine chloride (a PKC enzyme inhibitor) or the inhibitor of JNK (iJNK), the survival effect of LM-PLA2-I upon retinal ganglion cells was completely abolished (Fig. 2A and B, respectively), suggesting that PKC and JNK enzyme activities are important steps on LM-PLA2-I-induced ganglion cells survival. In contrast, when cells were treated with BAPTA-AM (10 μM), that is an intracellular calcium chelator, the ganglion cells survival induced by LM-PLA2-I was not abolished (Fig. 2C). It is important to emphasize that chelerythrine chloride, iJNK or BAPTA-AM alone did not interfere on ganglion cell survival (Fig. 2A–C). Later, the participation of PKCδ (novel class of PKC isoform) was investigated.