| Background and purpose:In China,drinking is a very common social behavior,which will cause a series of social and medical problems.The former includes traffic accidents,fights,domestic violence,child abuse,etc.While the latter includes mental illness,cancer,etc.In the practice of forensic medicine,there are many cases related to ethanol.The interaction mechanism,causality and identification of ethanol,external force and disease in death or injury cases are often the focus of debate.Ethanol has obvious neurotoxicity,which will lead to learning and memory dysfunction.As one of the structural bases of learning and memory,hippocampus is highly sensitive to ethanol.The research is dedicated to the study of the effect of ethanol on hippocampal neurons in rats.In the early stage,scanning electron microscopy,immunohistochemistry,atomic force microscopy and other techniques were used to observe the morphological changes of hippocampal neurons exposed to ethanol.And the external morphological changes are closely related to the changes of ultrastructure and physiological process inside the cells.Mitochondrion is the most important organelle,whose basic function is to provide energy for all kinds of cell activities and functions.And calcium ion is important second messenger in the cell signal transmission process.Therefore,we study the changes of these two aspects to further explore the damage caused by ethanol to hippocampal neurons from the changes of ultrastructure and internal mechanism.Methods:1.At the ultra-clean working table,the brain of SD rat within 24 hours after birth was dissected and brain was taken.Then hippocampal tissue was isolated from the brain to prepare cell suspension.Primary culture in vitro lasted for 7-8 days.Hippocampal neurons with good growth and high purity were randomly divided into control group and groups with low,medium and high concentration of ethanol.Anhydrous ethanol was added into the culture medium to make the final concentration 0.5g/L,1.5g/L and 3.0g/L,respectively.The control group was added with the same amount of PBS solution.And each group lasted for 4h,8h,12h and 24h,respectively.Each group repeated 3 times.2.After treatment,taking 24-well plates cultured with hippocampal neurons and the culture medium was discarded.Incubating hippocampal neurons with 600?L Jc-1 fluorescent probe and 600?L fresh culture medium for 20min and washing with Jc-1(1x).Then adding with fresh culture medium.Some areas were randomly selected and photographed under an inverted fluorescence microscope to collect image data.Average fluorescence intensity was calculated by Image-J software,and the ratio of average red-green fluorescence intensity could represent the change of mitochondrial membrane potential.3.After treatment,taking special dishes for a laser scanning confocal microscope and the culture medium was discarded.Incubating hippocampal neurons with Fluo-3 AM fluorescent probe for 30min and washing with PBS.Then adding with fresh culture medium.Some areas were randomly selected and photographed under a laser scanning confocal microscope to collect image data.Average fluorescence intensity was calculated by Image-J software,and the value of average fluorescence intensity could represent the relative value of calcium ion concentration.4.SPSS 21.0 software was used for statistical analysis of the obtained data.Results1.The hippocampal neurons were incubated with JC-1 fluorescence probe.Observing the hippocampal neurons in the control groups after the action of PBS solution.The red fluorescence was brighter and the green fluorescence was darker.After ethanol treatment,compared with the control groups,the red fluorescence became darker and the green fluorescence became brighter.The results indicated that the mitochondrial membrane potential of hippocampal neurons decreased after the effect of ethanol.The ratio of the average red-green fluorescence intensity was calculated by Image-J software,which represented the mitochondrial membrane potential of hippocampal neurons.Compared with the control group,the mitochondrial membrane potential of hippocampal neurons all decreased when they were cultured with low,medium and high concentration ethanol for 4h,8h,12h and 24h(P<0.05).Compared with the low concentration ethanol group,the mitochondrial membrane potential decreased after the action of medium concentration ethanol for 8h and 24h(P<0.05),and decreased after the action of high concentration ethanol for 4h and 8h(P<0.05).In the 4h group,the mitochondrial membrane potential decreased gradually with the increase of ethanol concentration.Compared with the 4h group,the mitochondrial membrane potential increased after the action of low and high concentration ethanol for 8h,12h and 24h(P<0.05),while the mitochondrial membrane potential increased after the action of medium concentration ethanol for 12h(P<0.05).2.The hippocampal neurons were incubated with Fluo-3 AM fluorescence probe.Observing the hippocampal neurons in the control groups after the action of PBS solution,whose fluorescence is dark.After ethanol treatment,compared with the control groups,the fluorescence of hippocampal neurons became brighter.Especially the changes in the cell body were more obvious.The neurites were also clear and visible compared with the control groups,and its fluorescence became brighter.This indicated that the concentration of calcium ions in hippocampal neurons increased after the effect of ethanol.The average fluorescence intensity value was calculated by Image-J software to represent the calcium concentration in hippocampal neurons.Compared with the control group,the calcium concentration in hippocampal neurons did not increase significantly after the action of low concentration ethanol for 4h,while the calcium concentration increased after the action of medium and high concentration ethanol for 4h(P<0.05).And in the 4h group,the calcium concentration increased gradually with the increase of ethanol concentration.Compared with the control group,the calcium concentration increased after the action of low,medium and high concentration ethanol for 8h,12h and 24h(P<0.05).Compared with the 4h group,the calcium concentration increased after the action of low and medium concentration ethanol for 8h,12h and 24h(P<0.05),and increased after the action of high concentration ethanol for 8h and 24h(P<0.05).Conclusion:Ethanol can reduce the mitochondrial membrane potential and increase the concentration of calcium ions in hippocampal neurons,which suggests that ethanol could lead to the inj ury of hippocampal neurons through this mechanism. |
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