Development of vulnerability to hypoxic damage in in vitro hippocampal neurons

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Abstract
We investigated the relationship between sensitivity to hypoxia and culture age in in vitro hippocampal neurons. Hypoxia was induced by 24 hr incubation in an oxygen‐free environment. Up to 6 days in vitro (DIV) mortality was very low or negligible, with few exceptions. Starting at 7 DIV, significant mortality began to be observed: in the age range 7–10 DIV, mortality of 50% or more was observed in five out of 11 experiments (45%) and average mortality was 51 ± 15% (mean standard deviation, N = 11). In older (12–18 DIV) cultures, mortality of 50% or more was the rule (13 out of 13 experiments) and average mortality was 83±16% (mean±standard deviation, N = 13). The data could be fitted by a sigmoid line (r = 0.87, P < 10−6) in which 50% mortality corresponds to 8.6 DIV. The N‐methyl‐d‐aspartate antagonist amino‐phosphono‐valerate and the nitric oxide synthase inhibitor nitroarginine both provided protection. Degree of protection was comparable for the two compounds, but was not observed in cultures younger than approximately 7 DIV. By contrast, exogenous creatine was not protective, at variance with findings from other models. The data represent the first description of how sensitivity to hypoxic damage varies during the lifetime of an in vitro neuronal hippocampal culture. Moreover, they suggest the hypothesis that some maturational changes occurring at 7–9 days in vitro may make previously resistant in vitro neurons significantly sensitive to hypoxic damage, and that at least some of these changes may reflect the development of N‐methyl‐d‐aspartate‐mediated glutamatergic transmission.