The Role Of Autophagy-Lysosome Pathway In Primary Cortical Neuron Ischemia Model Of Oxygen And Glucose Deprivation

Aim: To study the role of an autophagy-lysosomal pathway in primary cortical neurons deprived of oxygen and glucose.Methods: The OGD model of primary cortical neuron was established by deprivation of oxygen and glucose. The injury of neurons induced by OGD was detected with MTT test and immunofluorescence. The activation of autophagy in neurons after OGD was observed using MDC staining. Protein levels of LC3, Beclin-1, and Lamp-2 were determined by Western Blot analysis and immunefluorescence. The neuroprotective effects of the autophagy inhibitor 3-methyladenine (3-MA) was assessed with LDH. The apopsis related protein p53 and PUMA as well as the anti-apoptotic protein Bcl-2 were assessed with Western Blot analysis. In order to evaluate the relationship between the autophagy and apoptosis in the OGD model, we assessed the expression level of DRAM by Western Blot analysis.Results: We have successfully established the primary cortical neuron ischemia model by depriving of oxygen and glucose. Immunofluorecsence and MTT test showed that neurons were severly injured 3h after OGD. MDC staining revealed that autophagy was activated after OGD, and the expression of protein LC3 and Beclin-1, which were the markers for autophagy, reached to a peak at 1 h and decreased after 2 h. From LDH test, we observed that the damage of neurons induced by OGD was significantly aggravated after the treatment with 3-MA (10 mM). Western blot analysis showed that p53, PUMA and DRAM rapidly up-regulated after OGD 1 h while Bcl-2 down-regulated, which proved that the apoptosis was triggered after OGD.Conclusions: The primary cortical neurons were damaged by OGD. Autophagy was activated at the early stage of OGD. From this study, we learned that the autophagy played neuroproductive function in the early phase of OGD, but promoted cell to death in the later phase through apoptotic mechanisms.