Resumo

Introduction: Ischemia induces neuronal death through a series of events that involve multiple interdependent molecular pathways. It is well known that, in the process of ischemia, a pivotal change is the increase in extracellular levels of glutamate, the main excitatory neurotransmitter in the central nervous system and the retina (Osborne et al., 2004). Spider toxins PhTx3, Tx3–3 or Tx3–4 are calcium channel blockers (Prado et al., 1996; Guatimosim et al., 1997). The aim of this study was to investigate the effect of spider toxins on retinal slices injury induced by oxygen deprivation and low glucose (ODLG) insult on slices of rat retina. Methods: Experimental Animal Ethics Committee: 4201092005-6. The retinal slices that were to receive ODLG insult were transferred to small pre-incubation chambers (25 mL). After 30 min, the slices and their floating nylon platforms were transferred to insult chambers (25 mL) containing KRB supplemented with 4 mmol/L glucose and bubbled with 95% N2/5% CO2 for 45 min at 36.5 °C. Test compounds were present (or absent thus forming the control) during both the 30 min pre-incubation period and the 45 min ODLG insult. After ODLG insult cell viability in retinal slices was assessed by confocal microscopy and epifluorescence using the live/dead kit containing calcein-AM and ethidium homodimer. The glutamate content on the control or ischemic crushed retinal slices was determined in the presence of glutamate dehydrogenase. The method was carried out by monitoring the increase in NADPH+. Results: Confocal imaging of retinal slices subject to ischemic insult treated with PhTx3, Tx3–3 and Tx3–4 showed a decrease in cell death that amounted to 79.5 ± 3.1, 75.5 ± 5.8 and 61 ± 3.8, respectively. The neuroprotection promoted on retinal slices by PhTx3, Tx3–3 or Tx3–4 was also observed when the toxins were applied 15, 30, 60 or 90min after induction of the ODLG injury. During the ischemic insult, glutamate release from retinal slices was increased 53% % (from 6.2 ± 1.0 nM/mg of protein control retinal slices not subjected to ischemia to 13.3 ± 0,9 nM/mg of protein in slices exposed to ischemia. PhTx3, Tx3–3 and Tx3–4 toxins inhibited the ischemia-induced increase on glutamate release by 38.5%, 34.6% and 44.2% respectively. Discussion and Conclusion: Thus, PhTx3, Tx3-3 and Tx3-4 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of retinal ischemia. REFERENCES: Guatimosim C et al. Br J Pharmacol. (1997) 122(3):591-7 Osborne NN et al., Prog Retinal Eye Res (2004) 23:91–147 Prado MA et al. Biochem J. (1996) 15(314) :145-50 SUPPORTED BY: FAPEMIG, CNPq, Instituto Milênio, Grupo Santa Casa