Culture Of Cerebral Cortex Astrocytes And The Effects Of Ciliary Neurotrophic Factor On Activation Of Astrocytes

Objective: To obtain high purity of astrocytes and identification of them through thecell isolation, primary culture, purified cells and subsequent subculture. To investigatethe activation effect of ciliary neurotrophic factor (CNTF) on astrocyte (Ast) in vitro,explore the best activation state of Ast after treatment of subculture Ast with CNTF fordifferent time periods and provide a new way to explore mechanism of epilepsy.Methods:(1) Newborn SD rat pups within24h of age were decapitated and theforebrains were removed. The meninges and blood capillaries were removed carefullyfrom the forebrains. Brain tissues were minced into0.5~1mm3pieces by blade,incubated in0.25%trypsin solution for15min, then used the medium with serum toterminate digestion and preparation it into single cells suspension. Then, plated the cellsinto25cm2culture flasks at a density of5×105/ml in DMEM-F12medium for7~10d(37oC,5%CO2), after being counted and being treated with the method of differentialvelocity adherent. After cells full of the bottom of the culture flasks, then culture flaskswere shaken (240rpm,37oC) on thermostat shaker for18h. Removed suspended cellsand medium immediately, cleaned culture flasks for2~3times. The non-adherentastrocyte cells were trypsinized and collected, and then subcultured into new cultureflasks. Circulated the process of subculture cells3times conventionally, and then usedthe double labeled immunofluorescence method to detect the purity of astrocytes.(2) Blade method and scissors method were used respectively to mince the cerebralcortex tissue (any other conditions are the same). The cerebral cortex tissue wasprepared into single cell suspension, and then cell counter was used to count cells. Both methods were applied to count cells randomly10times in each experiment and wererepeated for3times. Last, statistical methods were used to analyze the results.(3) Activation of CNTF on Ast: the astrocytes were randomly divided intoexperimental group and control group. In the experimental group, the SABC methodcombine with DAB chromogenic method were used to stain astrocytes respectively at12h,24h,36h and48h. In the control group, astrocytes were put into the sameconditions without any treatment. After each task was completed, put them under themicroscope and observed the cell color depth change at each time point in each group,then calculated the percentage of positive cells. Last, statistical methods were used toanalyze the results.Results:(1) Comparison of blade method and scissors method for preparing to obtainthe number of single cells from the suspension, the t-test was used for statistical analysis.The results of the blade method was14.611.84, scissors method was5.382.01,compared the two groups was P<0.001, the differences were statistically significant.Thus, we could think of the blade method to obtain the number of single cells from thesuspension was obviously more than scissors method.(2) After the cell isolation, primary culture, purified cells and subsequentsubculture, and through the GFAP double immunofluorescence identification, the resultsshowed that almost all the cells were glial fibrillary acidic protein (GFAP)-positive cells.The purity of astrocytes was99.890.04%, showing that almost all of the cells wereastrocytes and can be used in subsequent experiments.(3) Activation of CNTF on Ast:1) According to the degree of GFAP-positive cellscoloration: each section of the control group was light yellow(+); in the experimentalgroup,12h、36h、48h group was brown yellow (++),24h group was tan (+++), theoverall trend of the degree of coloration with time changed was: color from light to deepto light, the deepest one was24h group. 2) According to the percentage of GFAP-positive cells. From the time effect, theoverall trend of GFAP-positive expression rate with the time changed in12h,24h,36h,48h group was: no significant changed in the control group; in the experimental group,the GFAP-positive expression rate first increased and then decreased, the highest onewas the24h group. Statistical analysis: a. comparison of the experimental group andcontrol group in the each same time points used the F-test, all the results of P values<0.001, the differences were statistically significant; b. in the control group, comparisonbetween the different time points used the F-test and the pairwise comparison used theq-test, all the results of P values>0.05, the differences had no statistical significance; c.in the experimental group, it also used the F-test to different time points and used theq-test to pairwise, all the results of P values <0.01, the differences were statisticallysignificant.3) Integral comprehensive metrological analysis results: the control group wasweak positive (+), the12h,36h,48h group were positive (++), the24h group wasstrongly positive (+++). The results of our research showed: it presented a bestactivation state at24h, after activated by CNTF.Conclusions:(1) Compared with the scissors method, in the preparation of the primarysingle cells suspension, blade method could significantly increase the number of singlecells in suspension.(2) Through the cell isolation, primary culture, purified cells and subsequentsubculture, we could obtain high purity of astrocytes.(3) CNTF could activate Ast and significantly upregulate the expression of GFAP.It may present a best activation state at24h, after activated by CNTF.