Effects of hypoxia on neuronal phenotype and viability in organotypic cultures.

KRUSE MS, BARUTTA J, BOTI V, REY M, SARACENO E, CAPANI F, COIRINI H

Buzios, Brasil

Congreso; I IBRO/LARC Congress of Neuroscience of Latin America, Caribbean and Iberian Peninsula.; 2008

IBRO/LARC

Perinatal asphyxia occurs in approximately 0.3% full-term newborn babies and this percentage has not decreased despite medical advances. There are evidences indicating that neurosteroids are important in neurodevelopment showing neuroprotective effects. We studied the potential protective effect of  allopregnanolone (AP) in vitro using organotypic cultures from neocortex, striatum and hippocampus. Organotypic cultures were prepared from P3–P5 rats following Stoppini protocol with modifications (Cereb cortex 13:11, 2003). After 6 days in vitro cultures were exposed to 5μM AP or vehicle for 24h then subjected to hypoxia for 1h in an anaerobic chamber (Billups-Rothenberg, Inc.) and incubated at 5% CO2 and 95% air at 37ºC for 24h. Results: First, the changes previously observed in our animal model were evaluated. Immunocytochemistry and confocal microscopy showed an increase of GFAP signal in the studied brain areas after hypoxia. Western blot studies supported these results (striatum 116%; hippocampus 101%; cortex 82%, n=3 p<0.05). The presence of the NOS enzyme was studied by NADPH diaphorase technique. After hypoxia we observed an increase in NADPH positive neurons in the neocortex (66%) and the striatum (40%) but not in the hippocampus (n=3 p<0.05). Cell survival was analyzed by measuring LDH. After 1h of hypoxia, all cultures showed a non-significant increase of LDH which was greater after 24h of hypoxia (striatum-cortex co-cultures 140%, hippocampus 111% n=3 p<0.05). LDH levels have no changes after AP pretreatment before hypoxia. However tissues AP treated showed a non significant slight decrease of the GFAP signal compared to control cultures. Conclusion: Organotypic culture is a suitable method to study the effects of hypoxia in vitro. 24h pretreatment with 5μM of AP does not change neuronal viability and it does not prevent astroglyosis induced by hypoxia. 2 and 95% air at 37ºC for 24h. Results: First, the changes previously observed in our animal model were evaluated. Immunocytochemistry and confocal microscopy showed an increase of GFAP signal in the studied brain areas after hypoxia. Western blot studies supported these results (striatum 116%; hippocampus 101%; cortex 82%, n=3 p<0.05). The presence of the NOS enzyme was studied by NADPH diaphorase technique. After hypoxia we observed an increase in NADPH positive neurons in the neocortex (66%) and the striatum (40%) but not in the hippocampus (n=3 p<0.05). Cell survival was analyzed by measuring LDH. After 1h of hypoxia, all cultures showed a non-significant increase of LDH which was greater after 24h of hypoxia (striatum-cortex co-cultures 140%, hippocampus 111% n=3 p<0.05). LDH levels have no changes after AP pretreatment before hypoxia. However tissues AP treated showed a non significant slight decrease of the GFAP signal compared to control cultures. Conclusion: Organotypic culture is a suitable method to study the effects of hypoxia in vitro. 24h pretreatment with 5μM of AP does not change neuronal viability and it does not prevent astroglyosis induced by hypoxia.