Effect of anoxia and hypoglycaemia on the expression of neuronal cell markers in the rat retina and hippocampus: Development of a rat ex vivo ischemia model

Abstract

Neuronal ischemia leads to neuronal cells death due to glucose and oxygen deprivation. The goal of this study was the development of a rat ex vivo ischemia model for the deferential identification of anoxic and hypoglycaemic actions of ischemia on neuronal tissues which could subsequently used for the screening of possible neuroprotective agents. Methods: The protocol used was a slight modification of one previously reported. In brief, the retina and 500pm thick hippocampal sections were preincubated for 30min in artificial cerebrospinal fluid pH 7.3 (artiCSF) previously buffered with 95%O 2/5%CO2. Tissues were subsequently incubated in artiCSF previously buffered with 95%O2/5%CO2 (control and hypoglycaema) or 95%N2/5%CO2 (anoxia) in the presence (control and anoxic conditions) or absence of glucose (hypoglycaemia: replacement with sucrose). The effect of anoxia and hypoglycaemia on neuronal cell markers' expression was studied immunohistochemically using antibodies against choline acetyl-transferase (ChAT), γ-amino-butyric acid (GABA), neuronal nitric oxide synthase (bNOS) and neurofilament light (NF-L). Results/Conclusions: In the retina, ex vivo hypoglycemic treatment led to the decrease of ChAT and GABA immunoreactivity in amacrine cells. This decrease was more profound under anoxic conditions. bNOS and NF-L retinal expression as well as GABA hippocampal expression did not seem to be affected by either treatment. As far as ChAT, GABA and bNOS immunoreactivity are concerned our results were in agreement with previous studies of in vivo pressure-induced and in vitro chemical-induced retinal ischemia as well as transient cerebral ischemia. On the other hand, NF-L expression failed to correlate to the decrease of NF-L mRNA levels observed after pressure-induced retinal ischemia/reperfusion. Although further studies are necessary for the assessment of the hypoglycaemia/hypoxia effects on the hippocampal neuronal marker expression this study led to the development of a convenient ex vivo retinal model for the screening of possible neuroprotective agents. ©ΦApmakon-Túttoς.