The Protective Effect Of Astrocyte-conditioned Medium On The Hypoxia/Reoxygenation Damaged Neurons And Approaching To Its Mechanism

Background:As the life span of general population has been greatly improved, the risk forcerebrovascular disease has rapidly increased and become the first cause of cripplingand fatal. In thecerebral vascular diseases.ischemic stroke accounts80%~85%Ischemic stroke is not only causing a huge threat to human’s health, but also bringserious negative impact to the patient’s family and society. However, there is no exactmethod of treatment for ischemic stroke so far, therefore it’s very necessary to find anew method for improving the treatment of ischemic stroke.During the study of transient ischemic attack, we found the ischemic preconditioningof the brain tissue. Ischemic preconditioning had protective effect on brain cells undercertain conditions and reduced the degree of ischemic tissue damage.Induced brainischemic tolerance, resistanced long-term sustainability of cerebral ischemia. After all,the ischemic preconditioning is unrealistic in the clinical treatment and suggesting thatthe drug pretreatment in order to eliminate the trauma by ischemia, excited by drugs orsimulator endogenous substances to play a protective role, such as adenosine,bradykinin and nitric oxide (nitric oxide, NO).Adenosine (adenosine, Ado) and its receptor has important role in the protection oforgan ischemia-reperfusion injury. Under normal circumstances, the levels of adenosine was30~300nmol/L in brain extracellular fluid, but in pathological statessuch as cerebral ischemia, can increasing to5-40umol/L,200times of the normalrange. Thus, the sharp increase of adenosine may be play an important role inendogenous neuroprotective responsing.Astrocytes (astrocyte, AS) is mainly present in neurons of the central nervoussystem,which has the support and nutritional effects on nervous. Activated astrocytescan release a variety of neurotransmitters.When body experience tissue ischemiapathological state, astrocytes release substances can significantly increase to protectthe damaged neurons and accelerate lesions neuron function recovery. A largenumber of cell culture experiments in vitro also confirmed a variety of nutrients in theliquid (astrocyte-conditioned medium, ACM), suggesting ACM can improve thesurvival of damaged neurons and accelerate its functional recovery. Although theprotective effect on neurons of astrocytes has been recognized by the scholars, itsmechanism is not very clear. And need further investigate to the nutrition function ofdamaged neurons of ACM.This issue got astrocytes and neurons by using primary cell culture technology,created a simulated cerebral ischemia and reperfusion injury model, interventedhypoxic injury of neurons with the ACM, ACMa and ACM+a. observed morphologicalchanges of glial cells and neurons under an inverted microscope, assayed activity byXTT method, measured expression levels of neuron-specific enolase(neuron-specificenolase, NSE) strongly positive cells by immunofluorescence andimmune cells chemical technology. Analyzed caspase-3activity in groups of neuronsby Western blot. So explore the nutritional role of astrocyte culture medium afteradenosine preconditioning and analyze the protective effect of Astrocytes andadenosine to hypoxia/reoxygenation damage neurons while further reveals theprotection mechanisms to nervous system of adenosine, and play the theoreticalfoundation for the adenosine used in clinical treatment of cerebral vascular diseases.ObjectiveAnalysis of the protection mechanisms and Ado in hypoxia/reoxygenation injury of neurons andastrocytes after exploring medium of nutritional role adenosine preconditioning-further explorethe specific protective mechanism of adenosine. Can be better used for clinical treatment ofischemic cerebrovascular disease to provide theoretical support. Material and MethodsSD rats were cultured in vitro cerebral cortex and hippocampus cortical neurons AS;Through purification, to build a simulation model of cerebral ischemia-reperfusioninjury; Collected18h after reperfusion by Ado pretreatment ACM and18h ofreperfusion ACMa (ACM+containing100μmol/L Ado in DMEM solution); Thenuse the ACM, ACMa and ACM+a concentration of1:5culture hypoxia/reoxygenationneurons. AS using the inverted microscope and neuronal morphological changes in themicroscope. AS and neuronal activity were determined using the XTT method.Immunocyto-chemical technology and the expression level of immunofluorescence detectionresearch of NSE positive cells and glial fibrillaryacidic protein(GFAP). Analyzedastrocyte lactate dehydrogenase (lactate dehydrogenase, LDH) enzyme-linkedimmunosorbent by ELISA kit. used of flow cytometry to detect apoptosis. Followedthe instructions of Colorimetric kit of the CaspACETM Assay System of PromegaCorporation to measure each group neurons caspase-3activity and analyzed byWestern blot.ResultsLight microscopy: Normal Group: neurons refraction good oval cell body, Good growth,synaptic connections, mesh. Model group: neurons have poor refraction, and irregular cellbody, synapse significantly reduced or disappeared. ACM group, ACM+a group andACMa cell state between normal group and simulation, the overall observation,neurons form the role change: ACMa> ACM+a> ACM.Apoptosis by flow cytometry results: Compared with normal group, model group,ACMa group, ACM+a group and ACM group were significantly increased apoptosis.And ACMa group, ACM+a group of apoptosis and ACM group was significantly lowerthan the model group, ACMa apoptotic cells was significantly lower than ACM+agroup and ACM group, ACM+a group and no difference in cell apoptosis ACM.Detection of the neuronal activity by XTT: Model group: neuronal activity decreased,the absorbance OD value was significantly lower than the control group OD value.Intervention group: ACMa group, ACM+a group and the group of neurons ACMactivity were higher than model group, according to the absorbance OD values, and itsintensity ACMa group> ACM+a group> ACM groups.Determination of NSE: Intervention group: ACMa group, ACM+a group and the group of neurons expressing ACM NSE high capacity compared with the model group.Image analysis staining positive cell culture chip, and its role: ACMa group> ACM+agroup> ACM group.The caspase-3activity: the caspase-3activity in ACMa group, ACM+a group andACM group were decreased,compared with the model group. their roles presumed tobe: ACMa> ACM+a> ACM.The results of caspase-3activity measurement: Caspase-3activity in ACMa group,ACM+a group and ACM group were significantly reduced,compared with the modelgroup. Caspase-3activity in ACMa group is less than the ACM+a group and ACMgroup, apoptosis between the ACM+a and ACM group were no significant difference.Conclusions1. Through the cell activity and impaired neuronal expression of NSE, reflectingACM hypoxia/reoxygenation injury of neurons that support the role of protectionand restoration.2. According to the detection of neuronal apoptosis and changes about caspase-3expression of damaged neurons show that the ACM has anti-apoptotic effect.3. AS ischemia-reperfusion injury in the brain plays an important role, Adopretreatment enhances AS damaged neurons support the protection and repair.