The Neuroprotective Effects Of Danhong Injection On NMDA Induced Neuronal Injury In Cultured Cortical Neurons Of Embryonic Mice In Vitro

Backgrounds The neuroprotection after acute cerebral ischemia is a hot topic in stroke research. As a common therapy for cerebral ischemia, traditional Chinese medicine has tremendous potential. Clinical trials and researches on animal models have shown that Danhong injection is effective in treating acute cerebral ischemia. Up till now, most researches in vitro have studied effects of Danhong injection on vascular endothelial cells, it remains to be explored whether Danhong injection can protect neurons from damage induced by NMDA, which mediates excitotoxicity as one of the main sources of damage after cerebral ischemia and can result in cell apoptosis.Objective To provide cultured embryonic mice neurons with exogenous NMDA in vitro. To study whether Danhong injection can protect neurons from excitotoxicity induced by NMDA. To make the basis for further analysis of effective components of Danhong injection and its protective mechanisms.Methods Primary cortical cultures were prepared from 17 or 18-day-old embryos obtained from pregnant KM mice. Neuronal cell populations are determined by immunocytochemistry against microtubule-associated protein (MAP-2). 10 days later, cultures were exposed to exogenous NMDA (50,150 and 300umol/l) randomly. Cell survival was evaluated by MTT assay and LDH release. The concentration of NMDA leading to the most marked cell damage was chosen to establish the model of NMDA induced neuronal injury. Cultured neurons were divided into 3 groups: normal control group, NMDA injury group and Danhong treatment group. Danhong injection(0.005μl/ml and 0.01μl/ml) was added to the culture medium of the treatment group simultaneously with NMDA. Cell survival was evaluated by MTT assay. Cell injury was determined by LDH release. Apoptosis was estimated by Hoechst staining and TUNEL.Results A dose-response curve of NMDA neurotoxicity after 24 h of exposure to cultured neurons was performed and revealed the most significant reduction in MTT activity and the highest LDH release at a concentration of 300umol/l. Therefore, this concentration was chosen to perform all the following experiments. Under microscopy, after NMDA incubation, cultured neurons were damaged as evidenced by the presence of swelling and fragmented neuritis. When cortical cells were coincubated with NMDA and Danhong injection, cultures were better preserved and more intact neurons were visible. Danhong treatment group had both increased MTT activity and decreased LDH release and no statistical difference was observed between the 2 treatment groups(0.005μl/ml and 0.01μl/ml). The percentage of apoptotic neurons decreased as was labeled by Hoechst staining and TUNEL.Conclusions Danhong injection can relieve NMDA induced neuronal injury in cultured cortical neurons of embryonic mice and inhibit NMDA induced neuronal apotosis.