Influence Of Astrocyte-conditioned Medium On Anoxia Damaged Neurons

ObjectiveTo analyse the protective effect of astrocytes and adenosine on the hypoxia/reoxygenation damaged neurons and explore the nutritional role of the culture medium of astrocytes after adenosine-preconditioning for further revealing the neuroprotective mechanisms of adenosine. That can make foundation for the clinical treatment of ischemic cerebrovascular disease.MethodsCultured SD rat cerebral cortical astrocytes, neurons and hippocampal neurons were purified to establish models of simulated cerebral ischemia-reperfusion injury. After reperfusion for18h, astrocyte condition medium (ACM) and adenosine-preconditioning astrocyte conditioned medium (ACMa) were collected. Then damaged neurons, from models of simulated cerebral ischemia-reperfusion injury were cultured in ACM, ACMa and ACM+a (ACM mixed with DMEM medium containing100μmol/L adenosine). The concentration is1:5. Light microscopy was used to observe the morphological changes of neurons and determine the neuronal activity. Immunofluorescence and immunocyto-chemical technique were used to detect the expression level of NSE and Bcl-2positive cells.ResultsObservation by light microscopy:In normal group, neuron grew well whose morphology was regular, cell body oval, refraction good, synaptic connection closely reticulate. In model group, neuronal cell body became larger and irregular. Refraction was poor. Synapse was significantly reduced or disappeared. Neuronal condition in ACMa group, ACM+a group and ACM group was between the above two. By overall visual observation, their roles presumed to be:ACMa> ACM+a> ACM. Detection of the neuronal activity by XTT:In model group, the neuronal activity decreased, OD value was significantly lower than that in normal control group. The cell viability in ACMa group, ACM+a group and ACM was all higher than that in model group. According to the OD value, their roles presumed to be:ACMa> ACM+a> ACM. Determination of NSE and Bcl-2:The expression ability of NSE and Bcl-2determination in ACMa group, ACM+a group and ACM group improved, compared with model group. Took the positive staining of cell culture-chip for image analysis, their roles presumed to be:ACMa> ACM+a> ACM.Conclusions1. Detection of the cell activity and NSE expression of damaged neurons reflect that ACM plays an important role in protecting and restoring damaged neurons.2. Changes about bcl-2expression of damaged neurons show that the ACM has anti-apoptotic effect.3. Adenosine can indirectly protect and repair damaged nerves through astrocytes, which plays key roles in cerebral ischemia-reperfusion injury.