Chronic Alcohol Intoxication Induced Cerebral Cortex Neuronal Cell Apoptosis In Mice

ObjectiveChronic alcohol intoxication is a chornic progressing lesion, which is characterized of the loss of control alcohol consumption repeatedly long-term, and often lead to the psychical, body variation, and social, legal problems. Chronic alcohol intoxication lead to the simpairment of many systems and organs of body, such as nervous system, cardiovascular system, digestive system, immune system, muscle and so on. caspase-3 is a cysteinyl aspartate-specific proteinase, and to be exist in cytoplasm by means of precursor incompetence which the molecular weight 32kD approximately. After the Dead singal transmitted into intra-cellular, acvitvated caspase protein family by a series of cascade reaction and further activated caspase-3 (splited into p11, p17, p19 and p20 fragment).further transmitted the successor signal into endonuclear, induced cell apoptosis. In the present study, based on the establishment of the animal models of chronic alcohol intoxication in mice, the level of caspase-3 and neuron-specific enolase(NSE) would be detected by immunohistochemical technique and Western blot, and observed the cerebral cortex nerve cells apoptosis by terminal deoxynucleotide transferase-mediated deoxyuridine-biotin nick end labeling(TUNEL). The investigation is aimed to explore the level of NSE in the cerebral cortex nerve cells, and the correlation between the cerebral cortex nerve cell apoptosis and caspasep-3.Materials and Methods1. Establishment of animal model and packetA total of 48 adult, healthy kunming mice of male(8-9weeks), each weighting 30-42g, were divided into eight groups randomly. one control group is ninety days group, another control is one hundred eighty days group; experimental groups are ninety days groups with 10%,20%,30% ethanol(v/v) and one hundred eighty days groups with 10%,20%,30% ethanol(v/v). After normal feeding five days, the contral group was supplied with purified water freely and others with 10%,20%,30% ethanol(v/v) differently as the drinking water which were dispensed with purified water and ethanol. Ninety days groups would been feed ninety days, and one hundred eighty days groups would been feed one hundred eighty days.At the end of animal model of chronic alcohol intoxication, to draw blood from vena caudalis of the mice,all of animals were killed directly with decapitation after anesthetized by inhalating diethyl ether, took out of the whole brain tissue, to cut the cerebellum and brain stem and remain the cerebrum for HE staining, immunohistochemical staining, TUNEL and Western blot.2. MethodsTo make use of gas chromatography(GC) to detect the concentration of ethanol in mice, The level of procaspase-3 and active caspase-3 in cerebral cortex neuronal cell of mice wound be detected by immunohistochemical staining and Western blot. The level of NSE by immunohistochemical staining. And the mouse drinking purified water freely was used as the control. The number and ratio of procaspasep-3, NSE and TUNEL positive cells were counted and analyzed by microscope. The average integrated optical density of were calculated by by software of Motic Image Advanced 3.2 image analysis system. The band AverGray was analyzed by software of Fluochem V2.0. The data were analyzed comparatively by the software of SPSS13.0 for Windows.ResultsAll kinds of the cerebral cortex of mice had displayed apparently abnormal behave of chronic ethanol poision, such as lethargy, irritability, loss of weight, and the fur become tarnished, tangle sparseness chronic alcohol intoxication mice compared to the contral. To accompany with the augmentation of alcohol consumption and the time of alcohol consumption to extend, There were significant differences in the weight between chronic alcohol intoxication groups and the contral (P<0.05). The ethanol was detected in blood of all kinds of chronic alcohol intoxication mice except the control, and there were significant differences in blood ethanol level between alcoholism groups and the contral (P<0.05). And The brain tissue in chronic alcohol intoxication mice is tender, swelling, and the sulcus is superficial. HE staining of cerebrum slice in chronic alcohol intoxication mice showed reinforcement of eosinophilic staining, Nissl body gathered, nucleolus anachromasis, neuronophagia. There were significant differences in ratios and average integral optical density of NSE-positive cells between alcoholism groups and the contral (P<0.05). And there were significant differences in ratios and average integral optical density of TUNEL-positive cells between alcoholism groups and the contral (P<0.05). As well, there were significant differences in ratios and average integral optical density of procaspase-3-positive cells between alcoholism groups and the contral (P<0.05) except 10% ethanol group(P>0.05). The obvious bands of 32kD and 17kD were noticed in all kinds of groups except the controls in which there are only bands of 32kD. And there were significant differences in average integral optical density of peocaspase-3-positive and active caspase-3 bands between alcoholism groups and the contral (P<0.05).Conclusions1. The express of the cerebral cortex of chronic alcohol intoxication mice is decrease compared to the contral, and the nerve cells function is abnormality.2. The chronic alcohol intoxication induce cerebral cortex nerve cells apoptosis in mice.3. The chronic alcohol intoxication lead to activation of caspase-3 of the cerebral cortex nerve cells in mice. 4. The apoptosis level of cerebral cortex neuron cells are time-correlated and concentration-correlated of alcohol consumption, the more alcohol consumption and the high concentration, the more apoptosis.