Morphological Analysis Of Rat Hippocampal Neurons Exposed To Ethanol By Atomic Force Microscope

Background and purpose:In today’s society,there are many countless problems caused by drinking,which brings great pressure to social governance.Many civil and criminal cases are caused by excessive or untimely drinking,which not only brings harm to the parties themselves and their families,but also affects the stability of the society.In forensic identification,the role of ethanol in the injury or death and the determination of legal liability are always the main points of social controversy.How to diagnose and identify the effect of ethanol is a big problem that needs to be solved in practice.Accordingly,there is a great significance to make a study on the effects of ethanol,which can provide a theoretical basis for solving the problem of forensic medical identification.Hippocampus is an important part in the limbic system of the brain.On the one hand,hippocampus has a function of memory and spatial localization;on the other hand,it also participates in the processing of sensory information,emotion control,movement,and many other advanced neural activities.Ethanol-exposure may cause the structure of hippocampal neurons abnormal,and disturb their physiological function,as a result,leading to the disorder of memory,learning and social behavior.Currently,researches about the effects of ethanol on the structure and morphology of neuronal bodies and neurites are still insufficient.In this study,the morphological characteristics of primary hippocampal neurons cultured in vitro were observed by atomic force microscope,which has an advantage of high resolution imaging in the area of cell biology.The effects of different concentrations of ethanol on cell morphology were studied that can help further understand the structure of hippocampal nerve cells and the injury mechanism of ethanol to nervous system.Methods:The brain of newborn SD rat within 24 hours was dissected and hippocampal tissue was isolated from the brain.The hippocampal nerve cells were dispersed by trypsin digestion and vitreous tube blowing.After collected by centrifugation,primary hippocampal nerve cells were cultured in CO₂ incubator.On the first day,fourth day and seventh day of the cell culturing,hippocampal neuron samples were fixed with4%paraformaldehyde,and their morphological characteristics and changes were observed by atomic force microscope,collecting the relevant data simultaneously.The hippocampal neurons cultured on the 7th day were randomly divided into four groups:control group,low concentration group,medium concentration group and high concentration group.The cells in the low,medium and high concentration groups were cultured in proper amount of anhydrous ethanol,where the final concentration of ethanol was 0.5 g/L,1.5 g/L and 3.0 g/L.In the control group,the same amount of sterilized PBS solution was added to the culture medium.The cells in the four concentration groups were randomly divided again into four teams according to different period of ethanol exposure,which were 1 h,4 h,8 h and 12 h before fixed with 4%paraformaldehyde.Atomic force microscope was used to observe the morphology of the cells and to collect the image data for analysis.Results:1.After growing for 1 day,hippocampal neurons gradually grew from the original round or ellipse into fusiform or triangular shape,and neurites outgrew from the cell soma.After growing for 4 days,the neurites of hippocampal nerve cells were further extended and branched.The neurites of the cells came into contact with each other,and the network structure was preliminarily formed.At this stage,the size of hippocampal nerve cells increased and hole-like depression could be observed on the soma.After culturing for 7 days,the hippocampal neurons matured basically,with full soma and clear outline.There were circular holes on the surface of the soma,most of which appeared at one end of the cell body,with a pore diameter of 1.60μm approximately and a depth of 80¹70 nm.The elongation of the neurites reached2³ times of the length of the soma.2.The morphology of the hippocampal neurons was influenced by ethanol.The changes in the height of the cell and the surface roughness caused by ethanol at different concentrations were distinguishable.In low concentration ethanol group,the morphology of the cells was basically the same as that of the control group when hippocampal neurons were cultured for 1h,4h,and 8h.The height of the soma and the surface roughness decreased when cells were cultured for 12h.In medium concentration ethanol group,the cell neurites were depressed and the integrity of the cell structure was destroyed.The cell soma was partially collapsed,with the height of the soma and the surface roughness decreased.In high concentration ethanol group,the height of the soma and the surface roughness was smaller than those of the control group,and there was a seriously collapse in the middle part of the cell.The outline of the cell disappeared,and the shape of the cell body was irregular.Conclusion:1.During the growth of hippocampal nerve cells,there were circular holes on the surface of the soma,with a pore diameter of 1²μm and a depth of 80¹70 nm.2.Medium and high concentration of ethanol had an effect on the morphology of the hippocampal neurons,which resulted in the decrease of cell height and the surface roughness,and structure destruction of neural soma and neurite with local collapse and structural loss.