Effects Of Ethanol On The Morphology And Cell Activity Of Primary Cultured Rat Hippocampal Neurons

Background and purpose:Now, "wine" has become a necessary "drink" in our daily life, and even develops into a kind of wine culture. The main ingredient, ethanol, is a substance that can freely diffuse and slowly be oxidized and metabolized. It makes damages to many organs and systems of the human body, and by easily passing through the blood-brain barrier, causes injuries of central nervous system. In China, due to the continuous improvement of living standards, the impact of China’s traditional culture of drinking, and the social role of "wine" in interpersonal communication, alcohol abuse and alcohol poisoning are increasing and drinkers are becoming younger in age. In the central nervous system, nerve cells in the hippocampus are highly sensitive to ethanol injury relatively, causing structural and functional changes of hippocampal neuronal cell which results in memory damage. By culturing primary rat hippocampal neural cells, this study observed the changes in morphology and cell activity under different concentration of ethanol, providing certain basis for the further study on the ethanol’s mechanism on neural cell injury. Methods:Using SD rat infants reared 24 h in the laboratory, isolate hippocampus neural cells and culture in serum to seventh days in vitro. Identify the purity of hippocampus nerve cells by NSE immunocytochemical staining. Divide the cultured hippocampal neurons into low dose group, middle dose group, high dose group and control group randomly, and add ethanol solution making the final concentration of ethanol in the medium 11 mM, 33 mM, 66 mM. Add equal volume of saline into control group. Culturing in CO2 incubator for 1h, 12 h and 24 h, observe the morphological changes of hippocampal neurons under different action time and ethanol concentration by immunocytochemical staining, and measure cell activity of hippocampal neurons by CCK-8 assay. Results:1. The morphological changes of hippocampal neural cells under different ethanol concentration and different action time: in low dose group with 11 mM ethanol, the cell morphology did not change significantly at each period. Cytoplasm and process staining did not change significantly, and the characteristics of nerve cells were basically the same with normal control groups. In middle dose group with 33 mM ethanol, the edge of few cells blurred after cultured 1h and significant cell aggregation did not appear. Cell neurite’s reduction was not obvious. After 12 h cells began to gather into clusters and visible glial cells began to proliferate. After 24 h cells, cell density decreased significantly. The number of process and synaptic connections reduced. Neural network was sparse but still visible. In high dose group with 66 mM ethanol, length of cell process became shorter and the number of process reduced after 1h. After 12 h, cells tufted aggregation was obvious and glial cells proliferated largely. After 24 h, a large number of cells died and the cell density was very low. Length of process became shorter, and cell synaptic connection was few, hardly forming neural network.2. The cell activity changes of hippocampal neural cells under different ethanol concentration and different action time: there were obvious changes on the viability of hippocampal neural cells between different concentration of ethanol and control group. The cell activity decreased in different time, and the extent of activity changes depended on ethanol concentration. Middle dose group and high dose group showed more obvious changes. And the same concentration of ethanol in different time to the influence of the activity of hippocampal neuron was statistically significant compared with control group. Conclusion:1. Combining vaginal smear and circulate mating method successfully obtains accurate pregnancy pregnant mice and P0 of suckling mice, with simplifying animal breeding process, ensuring the supply of suckling mice experiments.2. Middle dose and high doses of ethanol can cause damages to the cell bodies decrease the amount of synapses and reduce the process’ s length and amount of primary cultured rat hippocampal neurons, and it is associated with the time of action.3. The cell activity of rat hippocampal neurons in primary culture is decreased by ethanol and it has dose depend relationship with ethanol concentration, and it is related to the action time.