HYPOXIA AND NEUROTROPHINS IN DEVELPOING CNS

BOGETTI, MARÍA EUGENIA; BONAFINA, ANTONELA; FISZER DE PLAZAS, S.

MENDOZA

Congreso; V Neurotoxicity Meeting; 2011

NEUROTOXICITY SOCIETY

HYPOXIA AND NEUROTROPHINS IN DEVELOPING CNS.            Maria Eugenia Bogetti; Antonela Bonafina; Sara Fiszer de Plazas   Hypoxia is an injury that consists in a decreased concentration of O2 in blood and tissues, which leads to cell death. During embryonic development, hypoxia causes irreversible damage in the CNS. Previous work in our model of acute hypoxia (60 min, 8% of O2) in chicken embryos has shown that in the optic tectum (OT), hypoxic cell death (HxCD) presents an apoptotic phenotype. The HxCD is delayed after the end of the hypoxia treatment, and it is not massive, i.e. only a small group of neurons die as consequence of hypoxia. In the OT there are mainly two great waves of NOCD (naturally occurring cell death) that differ in time, the later of which is known for being dependent on neurotrophins (NTF). We hypothesized that a NTF-regulated pathway, involved in neuronal survival/death during development, could be altered by the hypoxic insult. One of the objectives of the present work was to analyze the influence of NTF on NOCD and on HxCD in developing chick OT both in vivo and in vitro. For this purpose we aimed to determine the OT ontogenetic period in which neurotrophic death cells occurs. We have discovered that, in the OT, the second wave of NOCD occurs between embryonic day (ED) 14 and ED16. (Number of TUNEL (+) neurons/ mm2 observed in ED15: 416± 21.4 vs. number of TUNEL(+) neurons/mm2 observed in ED17: 96±9.6 ). For our experiments, we designated   ED15 as a representative day of this period. We demonstrated both in vivo and in vitro that cells in the OT at ED15 are vulnerable to acute hypoxia, showing a decrease in the number of living cells. (50 -70%, ANOVA test, p<0.05) Secondly, by means of the neutralization technique, we studied the effect of endogenous NGF (Nerve Growth Factor) when applying different amounts of anti-NGF antibody on primary cultures of OT neurons at ED 15, showing that neuronal cells seem to require self produced NGF for their survival. Lately, our result suggests a neuroprotective role of NGF when it is administrated to primary neuronal cultures half hour before hypoxia. Otherwise, we characterized the presence of mature astrocytes in different ED. Western Blot studies demonstrated that differential developmental GFAP (glial fibrillary acid protein) levels increase with the embryonic age, first identified at ED14. We also aimed to study the role of astrocytes during the hypoxic insult. For this objective, we first analyzed the levels of GFAP in our model of acute hypoxia. Preliminary results demonstrated an increase in the level of GFAP delayed 6 h after the end of the hypoxia treatment while at 24, 30, 48, 56h post-hypoxia times (p-hx.) the levels of GFAP expression  were similar to the controls. In conclusion, hypoxic cell death during the second wave of NOCD is sensible to the influence of endogenous NTF, and it is prevented when NGF is administrated before the injury. Also, hypoxia would modulate GFAP expression at ED15.