| Excessive drinking leads to tremendous damage on health and numerous negative consequences on societies, and it is estimated that 25 million death and huge number of sickness and harm are caused by drinking every year, especially among younger generations. According to "Global Status Report on Alcohol and Health" published by WHO, alcohol is the third risk factor of diseases around the world. Researches nationally and internationally suggest that large amount of drinking may trigger cerebrovascular diseases in various ways, however, the mechanism is not clear. In forensic practice, subarachnoid hemorrhage(SAH) caused by mild external forces after massive drinking is relatively common, so cerebral damage due to drinking and participation of alcohol in such cases become a research focus of forensic medicine field, clinical medicine field and preclinical medicine field.Objective:The purpose of this experiment was to observe the influence of different time length of massive drinking on rats with reference to previous experiment of acute and chronic alcohol-treated rat carried out by our team. The experiment utilized headspace gas chromatograph to determine changes of blood alcohol concentration(BAC) after consumption of large amount of alcoholic drink. Then series of indexes of coagulation function were determined by automated coagulation analyzer, with connection between series of indexes were observed. Changes of cerebrovascular structure and vascular endothelial cells(VEC) were observed through HE dyeing, immunohistochemical staining, and transmission electron microscopy(TEM). Also, expression of CD146 in vascular endothelial cells and molecular mechanism were discussed, the influence and mechanism of massive drinking towards cerebrovascular were explained, and theoretic foundation of SAH caused by mild external forces and participation of alcohol in such cases were reinforced.Methods: Healthy male Sprague-Dawley rats (SD) which weighted 220g±10g were used. The rats were treated with wine of Beijing Hongxing (56%v/v) orally through blunt tipped needle, two times each day at interval of over 10 hours. Water was given orally through blunt tipped needle to adapt all rats to the process in advance.1 BAC determination in timeline: 6 SD rats were treated with 56% v/v 1.2 mL/100g, and then blood samples were collected at 0.5h, 1h, 2h, 3h, 6h, 12h respectively after treated. Blood was collected from orbital venous plexus inner canthus of left and right eyes by turns. About 200-300μl blood was taken and sealed each time in each rat. BAC at each time point was measured by headspace gas chromatograph to determine the time of peak for each.2 Sixty rats were randomly assigned into control group, spirit filling for 1 time group, filling for 7 days group, 14 days group, 21 days group and 28 days group. Rats in each group were filled with 56% v/v 1.2 mL/100g for twice a day with interval of 10 hours except the 1 time group. Controls were filled with distilled water instead of spirit. Behavioral changes of rats were observed during this period. Weight changes, survival status of the rats were measured and recorded. Rats were sacrificed when the peak BAC were recorded after the last filling. Heart-blood, brain and liver tissue were obtained. PT, TT, APTT and FBG were analyzed by automated coagulation analyzer. Liver tissues were fixed in paraffin for observing histopathologic changes after HE dyeing; brain tissues were also prepared in paraffin for examining cerebral pathologic changes after HE dyeing; expression changes of CD146 in cerebrovascular were analyzed using immunohistochemical stain. Post perfusion basilar arteries and middle cerebral artery dissected with stereomicroscope were observed by TEM.Data were presented as Mean±SD and analyzed with one way ANOVA and least significant difference test(LSD) by SPSS 16.0 statistical program. A level of P<0.05 was considered statistically significant.Results: 1 Behavior changes and weight changes: Experimental groups were treated harder than the control group with distilled water. Excitation, unsteadiness on feet, torpidity to stimulus, lethargy and debilitation came out a few minutes after treated with alcohol each time. With prolonged alcohol-treatment, rats appeared anepithymia, torpidity to stimulus, weight-decrease and so on. Comparing 21 days group and 28 days group to control group, there were significant differences (21 days P<0.05, 28 days P<0.01).2 BAC: Treated with 56% v/v 1.2 mL/100g, BAC of rats raised to the maximum 225.06±43.10mg/dL 1h after intragastric administration, and BAC were 25.37±18.21mg/dL after 12h.3. Correlation analysis of blood coagulation function: TT was significantly shorter in the group of 14 days, compared to others groups(P<0.05); PT significantly longer in the group of 14 days, compared to others groups(P<0.01); Comparing APTT in each experimental group to control, there were not significant differences; FBG was significantly higher in the group of 14 days, compared to others groups(P<0.05).4. Pathological changes4.1 Pathological changes of liver: In 1 day group cellularedema of liver was observed. Microvesicular adipose degeneration appeared at 7 days, mild microvesicular adipose degeneration at 14 days, diffuse microvesicular adipose degeneration, necrosis and inflammatory infiltration at 21days and 28 days.4.2 Expression of CD146: Immunohistochemical stain images of CD146 were observed in small veins, arteries and capillaries. Positive expression was observed in control group(+); weak positive expression in 1 day group rats(±);positive expression in 7 days group, 14 days group and 21 days group rats(++~++++); weak positive expression in 28 day group rats(±). In the same experimental group, expression changes in veins and capillaries of brain parenchyma were more obvious than in arterial.4.3 Ultra-structure changes of rat basilar artery and middle cerebral artery: 2/3 fusion occurred in 7 days group; irregularly shaped EC, curly microvilli and vacuolation in EC, EC cell junction 1/2 fusion in 14 days group; the formation of a large number of pinocytotic vesicles, EC cell junction 4/5 fusion and irregularly shaped EC in 21 days group; broken mitochondrial crista, membrane fusion, rough endoplasmic reticulum degranulation, almost all fusion, nucleoplasm edema and irregularly shaped EC in 28 days group.Conclusions:1 Drinking rats were killed in different periods (the time were killed 2 hours after drinking alcohol), test results showed that abnormal coagulation function in rats.2 The different time length of heavy drinking lead to varying degree of CD146 protein expression in cerebral vascular, and varying degree of damage in brain vascular endothelial connection, which affects the brain vascular permeability, results in an increase of subarachnoid hemorrhage risk after drinking.
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