| Background and objectiveWith the increase of consumption of alcoholic beverages, more and more person was reported to die after drinking accompany with traumatic subarachnoid haemorrhage (TSAH). Forensic workers has given more attention to theses cases in our country and overseas. Many scholars define TSAH characteristic as caused fatal diffuse subarachnoid hemorrhage by lighter blunt external force, and occur in a large number of drinking. Our team has established TSAH model after drinking,with the result of the incidence rate of TSAH: 82.4%in the chronic group, 28.6 % in the acute group; the mortality rate: 58.8% in the chronic group, 0% in the acute group. That chronic alcoholism rats are more greater mortality with TSAH than acute alcoholism group. In addition, the death rats have demonstrated diffuse subarachnoid hemorrhage. However, high mortality and death mechanism in chronic alcoholism rats of TSAH is not fully clear. Because no reports and system-related research studies in domestic. It is difficult for analysising the cause of death, it is necessary and urgent to solve the problems. Accordingly, we study the high mortality rates and death mechanisms of TSAH in chronic alcoholism group rats.In view of previous studies on the mechanism of death major from chronic alcoholism in neurons of energy metabolism, apoptosis, haemostasis and coagulation, vascular biomechanics and dark neurons. Stress resistance of the body for the Survival of biological is very important. However, from chronic alcoholism changes the body stress to induced the death of the high mortality rate and its mechanism of death is rarely reported. This study was designed on the alcoholism rat model of TSAH, Observing by HSP70 and nNOS expression of chronic alcoholism rat brains. From stress changes and their influence of the body to discuss the further death mechanism. In order to provide science theory for the TSAH medicolegal expertise and clinical prevention and cure.Materials and Methods1 Animals and groups Sixty-one male SPF SD rats, each weighting 300±50g,were randomly divided into the acute and chronic alcoholism groups, and bred in several cages. The acute group was subdivided into 4 groups: acute alcoholism group with or without strike, acute water group with or without strike. For each group, 7 rats were given intragastrically edible spirituous liquor (15 ml/kg) for one time. The chronic group was subdivided into 3 groups: chronic alcoholism groups without strike, chronic alcoholism groups with strike 2 or 12 h after the last alcoholism. Based on the result of beating, the last 2 groups were subdivided into death group and surviving group. Chronic alcoholism group rats were given liquor for 4 weeks, two times for each day, with an interval of 8h and the dosage of 8 ml / kg for the first 2 weeks and 12 ml / kg / time for the last 2 weeks.2 Methods Two or 12 h after alcoholism, the rats were given a concussion strike. For rats that died after strike, they were performed thoracotomy and about 5 ml blood in the left ventricular was collected immediately. Then the rats were decapitated, and their brains were fixated in 4% neutral paraformaldehyde (pH 7.2-7.4). The other survival rats were observed for 4 h, and then the thoracic cavity was exposed, and the right atrial appendage was incised to permit outflow of blood and other liquids. After that, the aortic cannula in left ventricle wad perfused immediately with physiological saline solution until all the blood was cleaned out of the vessels. Then fixative containing 4% neutral paraformaldehyde was perfused to fixate the animal. After opening the skull, the brain was acquired and fixated with 4% paraformaldehyde. Paraffin sections of brain tissue were observed with HE staining, immunohistochemistry of nNOS and HSP70 and SUM IOD and MENA IOD will be got by Image-Pro Plus 6.0, blood alcohol concentration was tested. To observe the pathological changes by patho-true-color image analytical system quantizationly, all datas would be analyzed by SPSS 15.0 (Independent-samples T-test and One-way ANONA) and Excel 2003 software.Results1 General observation1.1 General state of alcoholismRats immediately after acute alcoholism showed increased Excitatory, and were in an alert state. About 0.5h later, Central nervous system depression symptom began to appear to the rats, they side, lethargy, loss of retroflexion, unresponsive to the stimuli, slow breathing, low blood pressure and other clinical symptoms of acute alcoholism. and they gradually returned to normal after 6h later. Chronic alcoholism group rats had chronic malnutrition, gastrointestinal disorders, spiritual malaise.1.2 Weight changesWeight of Chronic alcoholism group rats decreased during the alcoholism periods (P <0.01), and decreased the most fastest in 1st week and 3rd week. Acute group rats'weight showed a slow growth (P> 0.05), until the 4th week, it got a significant difference (P <0.05).1.3 Blood pressure changesAcute and Chronic alcoholism rats'arteria caudilis blood pressure(ACBC) after infusing wine 2h were lower strikingly than before infusing wine 0.5h(P<0.01), but after infusing wine 2h then strike later 4h compared to infusing wine 0.5h was no significant difference(P>0.05). Rats'ACBC was no significant difference before and after irrigatting water in actue water groups. Chronic alcoholism group rats'ACBC increased from the third week and last the end week.1.4 Blood alcohol concentration(BAC)Survival group of chronic alcoholism after strike BAC was 60.08±13.80 mg/dL, Acute alcoholism group with strike BAC was 40.90±13.2 mg/dL, the former was higher (P>0.05). Dead group of chronic alcoholism after strike BAC was 215.63±26.71 mg/dL and Chronic alcoholism groups without strike BAC was 208.50±32.35 mg/dL. Both of them were higher compared to Acute alcoholism groups without strike which BAC was 180.16±16.58 mg/dL (P<0.05). Survival group spacing 12h of chronic alcoholism after strike BAC was 16.43±8.20 mg/dL and Acute water group BAC was 0.2 Morbility and mortality of TSAHTSAH and death didn't occur in Acute water group. However, morbility of TSAH in Acute alcoholism group with strike was 28.6%, no rat died. Morbility of TSAH in Chronic alcoholism group with strike was 84.2%, grade 0-3; mortality was 52.6% and TSAH was found in all dead rats, major in grade 2-3. Morbility of TSAH was 71.4%, grade 0-3, mortality was 28.6% in Spacing 12h of chronic alcoholism with strike. Base on the research group of the previous two experiments, Morbility of TSAH and mortality had significant difference between Chronic alcoholism groups with strike and Acute groups with strike (P<0.05). Morbility in Chronic alcoholism groups with strike was higher compared to Spacing 12h of chronic alcoholism with strike (P<0.05), but morbility of TSAH no significant difference between them (P>0.05).3 Morphologic changes SAH and cerebral contusion no found in Acute water group and brain showed pale. Brain tissue staining with HE observed that no dark neurons, central Nissl body was clearly, and no brain contusion, hemorrhage and so on. Frontal lobe and cerebellum was congestion in Chronic alcoholism groups without strike. SAH was found in Acute alcoholism group with strike of Dorsal and ventral brain stem. Brain staining with HE observed that cerebrovascular was empty or congestion, thinly SAH, neurons and glial cells cell gap expanded slightly; central Nissl body was clearly and no dark neurons.Chronic alcoholism with strike with thick SAH, may involve of the whole brain and its more common saw at the ventra brainstem. HE staining: frontal lobe, brain stem, cerebellum with heavily subarachnoid hemorrhage. Frontal cortex neurons arranged loosely, nuclei condensed slightly, acidophilia increased, more neuronophagia phenomenon; Purkinje cells arranged in loose, shape was irregular like triangular, synapse not clear, some nuclei condensed even dissolved away and structure was not clear. Oblongata showed many nuclear condensed and neurons acidophilia metachromatic named dark neurons.4 The immunohistochemistry expression observation4.1 The immunohistochemistry expression of nNOSThere were no significant difference in SUM IOD and MEAN IOD between the Acute and Chronic groups in mesencephalon (P>0.05). Chronic alcoholism SUM IOD and MEAN IOD value of frontal lobe, oblongata, cerebellum were higher than Acute groups (P<0.05). Survival group of chronic alcoholism after strike SUM IOD and MEAN IOD of frontal lobe, oblongata, cerebellum were higher than Dead group of chronic alcoholism after strike, Survival group spacing 12h of chronic alcoholism after strike, Chronic alcoholism groups without strike (P<0.01). There were no significant difference of SUM IOD and MEAN IOD Among Dead group of chronic alcoholism after strike, Survival group spacing 12h of chronic alcoholism after strike, and Chronic alcoholism groups without strike. Acute alcoholism groups SUM IOD of frontal lobe, oblongata, cerebellum were higher than Acute water groups (P<0.01). MEAN IOD were no significant difference between Acute alcoholism group and Acute water group except in cerebellum (P>0.05).4.2 The immunohistochemistry expression of HSP70HSP70's SUM IOD and MEAN IOD of frontal lobe, oblongata, caudex encephali pontilis and cerebellum in Survival group of chronic alcoholism after strike compared to the other chronic alcoholism groups were higher (P<0.01). There was no significant difference between Dead group of chronic alcoholism after strike and Chronic alcoholism groups without strike (P>0.05), and both of them were lower compared to Survival group spacing 12h of chronic alcoholism after strike (P<0.01). HSP70's SUM IOD and MEAN IOD in oblongata were no significant difference in Acute alcoholism group (P>0.05), but it was higher than Acute water group; frontal lobe, and the HSP70 in caudex encephali pontilis and cerebellum showed negative. HSP70 in Acute water group showed all negative. HSP70's SUM IOD and MEAN IOD in Chronic alcoholism groups were higher than Acute groups(P<0.01).Conclusions1. Chronic alcoholism, blood alcohol concentration, the morbility and mortality of TSAH are significant correlation amomg them. If head was hit by concussion force result of TSAH and death by effects of alcohol, concussion forces and other factors in long-term alcohol abusing. It could be said occasionally case.2. The Expression of nNOS and HSP70 in Chronic alcoholism rats'brain are significantly enhanced. It is may be the important material foundation in nerve center of long-term alcohol abuse, and also it is one of the TSAH high mortality and death mechanisms. |
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