The Effect Of Ethanol On The Differentiation Of Cortical Neurons In Newborn Rats

Objective: Ethanol(alcohol)is abused seriously today, leading to varioussocial problems. The current studies have shown that ethanol can causedegeneration, atrophy and apoptosis of nerve cells in cerebral cortex,hippocampus and cerebellum and inhibit the differentiation of nerve cells.Fetal alcohol syndrome caused by maternal alcoholism is one of seriousconsequences by ethanol on the central nervous system. It would be showed asneurodegeneration, apoptosis, degeneration necrosis in neuronal and disorderof differentiation and migration in neuronal systems.During the development of the central nervous system, almost all of thenerve cells would experience the process of differentiation and migration toplay their role. If this process is broken, kinds of primary neurologicaldiseases will appear. So far, studies have showed that ethanol may affect themigration and differentiation of nerve cells, while it is unclear about themechanisms and related genes protein. This study is designed tosystematically observe the change of expression in microtubeasso ciatedprotein-2(MAP2), topoisomereⅡβ(TopⅡβ) and nuclear receptor-relatedfactor1(NURR1) with in vivo and vitro cell experiments. The proteins areclosely related to the differentiation and migration in nerve cells. The resultswill reveal the relation between MAP2, TopⅡβ, NURR1and differentiationof nerve cells caused by abnormal ethanol and provide the scientific basis forthe prevention and treatment to pathological state induced by anomalousdifferentiation in nerve cells.Methods:1The newborn mice (born within24h) were disinfected with75%v/v ethanoland decapitated. Isolated cortex neurons were planted on culture plates, andtreated with75mM ethanol for3days,5days and7days.Immunohistochemical double staining was used to observe the changes of MAP2expression, and Fluoro-Jade B fluorescence staining to detect neuronaldegeneration and necrosis.2The whole animal models: Healthy pregnant Wistar rats, weighed260±10gwere used. The rats were randomly divided into the control group andalcoholism group. Rats were treated with wine of Beijing Hongxing(5g/kg)orally through biunt tipped needle, two times each day at interval of over12hours. Water instead of wine was given to control group. Water was givenorally through blunt tipped needle to adapt the process in advance. Thepregnant rats were gavaged of wine from the6thday to the end of pregnancy.After models were established, the brain tissue was taken to dehydration,paraffined and sliced. We used immunohistochemistry to observe the changesof of cortical neurons.3The data were presented as Mean±SD, analyzed with ANOVA and LSDusing SPSS13.0statistical program. A level of p<0.05was considered asstatistical significance.Results:1In vitro experiments(The culture of primary cortical cells in newborn rat)1.1The effect of ethanol on expression of MAP2, NURR1and TopⅡ β. As theethanol treatment progressed, the expression of MAP2decreased. It was mostsignificant after7day treatment. Compared with the normal control group, thedistribution of cell was relatively sparse and the synaptic became shorter andfiner. The red fluorescent signal in the cytoplasmic weakened significantly andthe positive expression reduced apparently. With the extension of exposuretime,the expression of topoisomerase Ⅱβ and Nurr1also reduced.1.2Fluoro-Jade B staining marking the situation of degeneration and necrosisin nerve cells.The primary nerve cells in the normal control group was not seen positivecells of degeneration and necrosis. The5d-treatment group had occasionalpositive cells, while the7d-treatment group had some positive cells.2In vivo experiment(Newborn rats of the pregnancy alcoholism group)2.1results of MAP2-immunohistochemical staining The cytoplasm and synaptic of cortical neurons in newborn rats had theMAP2expression showing as brown granular. Compared with the normalgroup,the expression at different time points all reduced in the cerebral cortexof newborn rats of alcoholism group.2.2results of TopⅡ β and NURR1-immunohistochemical stainingThere were obvious brown expression in the nucleus of cortical neuronsin newborn rats. Compared with the normal group,the expression at differenttime points all reduced in the cerebral cortex of newborn rats of alcoholismgroup.Conclusion:Neurotoxic effects of ethanol decrease the expression of TopⅡβ, NURR1and MAP2, suggesting that ethanol can affect the differentiationof cortical neuronal,reduce the activity of the immune in synapse, accumulatemicrotubules and damages the integrity of cytoskeleton. All of these lead todegeneration and necrosis of cortical neuronal.