| Background and Objective:Ethanol has neurotoxic effect to the brain. When ethanol gets into the nervous system, it can generate toxic effect directly to the cells and induces apoptosis. Ethanol makes the neurobehavior changed, it can damage the association, attention, judgement and discrimination along with the blood ethanol concentration increased; thus it can cause criminal and civil case. So it is necessary to research the effect of ethanol with the neuron. In the study, we treated with different concentration of ethanol to the primary cultures of rat cortical neurons, used inverted phase contrast microscopy, H.E. Staining and the scanning electron microscopy to observe the changes of the neuronal morphology. We study the effect of different level of ethanol on the expression of CYP2E1, iNOS and NSE within the cortical neurons by immunohistochemistry staining; in order to investigate the interrelationship among them and the mechanism of them participate to the brain injuries.Methods:1. Cortical neurons of Sprague-Dawley neonatal rats (24 h) were cultured in vitro, and then were identified by immunohistochemistry staining with anti-NSE. The cultured cortical neurons with growth in good condition were randomLy divided into control group (C), low-dose group (L), moderate-dose group (M) and high-dose group (H). Cells of control group were fed with medium that did not contain ethanol, other group were treated with different concentration of ethanol (0.5 g/L,1.5 g/L or 3.0 g/L). The four groups were treated for different time intervals (0.5 h,1h,2h,4h,8h,12h or 24 h).2. The changes of the cortical neuronal morphology were observed by H.E. staining.3. The changes of the cortical neuronal ultrastructure were observed by the scanning electron microscopy.4. The changes of the cortical neuronal expression of CYP2E1, iNOS and NSE were studied by immunohistochemistry staining.5. Statistical analysis:all the data were dealt with SPSS 12.0 statistical software package and the measurement data were indicated with mean±standard deviation (x±s). Comparing groups used ANOVA, LSD test and a value of .P<0.05 was considered as statistical significant.Result:1. The cortical neurons'morphological findings:After cultured 9 days, mature cortical neurons showed abundant cytoplasm, good refraction, and the nuclei were located in the center of the cell or leaned to one side, the nucleus and nucleolus could be seen clear, the neurite and dendrite of the cortical neurons interlaced into reticulate.2. Indentification of cortical neurons:Immunohistochemistry staining with anti-NSE demonstrated that 90% of the cultured cells were cortical neurons.3. The cortical neurons'morphological findings observed by the inverted phase contrast microscopy:There were no significant differences of morphological alteration between L and C groups. In the M group, after 8 h, the morphological characteristics of the neurons altered, the neurons became clostridial form or long clostridial form and could be observed round vacuoles in the cytolymph. In the H group, the neuronal morphology were changed obviously along with the concentration of ethanol and action time increased, the neurons were swollen and round, the round vacuoles in the cytolymph increased, the neuronal apophysises became shorter obviously, the nucleus were swollen or disappeared and the cell-cell junction disappeared.4. The results of H.E. Staining:The neuronal structure in the L group was similar to that of the control group. In the M group, after 8 h, the morphology of neurons were changed, such as the neurons were swollen and became round, the round vacuoles could be observed in the cytoplasm and the nucleus were light stained irregularly. In the H group, the morphology of neurons were changed obviously, such as the neurons were swollen and became round; the round vacuoles which in the cytoplasm increased; some cell body became small, deep stained or became triangle; the cytoplasm of deep red colour, the nucleolus became small and deep stained, the nucleoli wasn't clear; at last, some cell-cell junction disappeared.5. The results that observed by scanning electron microscopy:There were no significant differences of morphological alteration between L and C groups. In the M group, after 8 h, the morphological characteristics of the neurons were changed, some neurons became clostridial form, long clostridial form or irregular form; the neuronal apophysises became shorter obviously; the morphology of the dendritic spine from the surface of a small proportional neuronal cell bodies altered, meanwhile, the neuronal cell bodies lost the linkage with other neurons partly. In the H group, the neuronal morphology were changed obviously along with the concentration of ethanol and action time increased, the neuronal cell bodies were swollen and became round or irregular form, the neuronal apophysises became shorter, the morphology of the dendritic spine altered; along with the action time increased, the surface of the neurons of pounch form and irregular form increased, the morphology of the dendritic spine altered obviously, the neurons of tile form increased; at last, most neurons collapsed to tile form, the neuronal apophysises disappeared and the cell-cell junction disappeared.6. The results of immunohistochemistry staining:(1) The expression of CYP2E1:There is only a little expression in the control group. At 2 h after treated with ethanol 1.5 g/L, the expression of CYP2E1 increased, which was highest at 12 h, and remained higher than control group. At 1 h after treated with ethanol 3.0 g/L, the expression of CYP2E1 increased, which was highest at 8 h, and remained higher than control group. (2) The expression of iNOS:There is only a little expression in the control group. At 2 h after treated with ethanol 1.5 g/L, the expression of iNOS increased, which was highest at 12 h, and remained higher than control group. At 1 h after treated with ethanol 3.0 g/L, the expression of iNOS increased, which was highest at 8 h, and remained higher than control group. (3) The expression of NSE: There is a great positive expression in the control group. At 2 h after treated with ethanol 1.5 g/L, the expression of NSE decreased, which was lowest at 12 h, and remained lower than control group. At 1 h after treated with ethanol 3.0 g/L, the expression of NSE decreased, which was lowest at 8 h, and remained lower than control group.Conclusion:1. After treated with different concentration of ethanol to the primary cultures of rat cortical neurons, observed by the inverted phase contrast microscopy and H.E. staining, showed that the moderate and high concentration of ethanol could cause the morphology of neurons changed in a short time.2. Observed by the scanning electron microscopy, showed that moderate and high concentration of ethanol could make the primary cultures of rat cortical neurons damaged, cause the ultrastructure of cortical neurons changed and was closely associated with the action time.3. Ethanol could induce the expression of CYP2E1 and iNOS increased along with the concentration of ethanol and action time increased. Meanwhile, made the expression of NSE decreased. It can associated them to conclude the action time of ethanol in forensic medicine analysis and assessment. |
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