急慢性酒精中毒大鼠脑组织S-100β、MBP表达与TSAH死亡关系的研究

发布时间：2018-07-08 13:49

本文选题：法医病理学 + TSAH　；参考：《汕头大学》2010年硕士论文

【摘要】： 背景与目的外伤性蛛网膜下腔出血(traumatic subarachnoid haemorrhage,TSAH)是一种不同于脑挫伤所致的蛛网膜下腔出血和病理性蛛网膜下腔出血的独立蛛网膜下腔出血,与外伤性硬脑膜外出血、硬脑膜下腔出血并列为三大外伤性颅内腔隙出血。国内外学者报道的TSAH常与酗酒有关,但其发生及死亡机制的研究性报道较少见。因此,探讨和阐释急慢性酒精中毒相关的TSAH发生及死亡机制,可为其死因分析、司法审判量刑提供科学的理论依据。本课题组前期工作已成功地建立了大鼠急慢性酒精中毒TSAH模型,结果显示,脑震荡性轻微外力打击灌酒后大鼠的TSAH发生率:慢性组82.4㳠、急性组28.6%,死亡率:慢性组58.8㳠、急性组0㳠,慢性灌酒组TSAH发生率及死亡率远大于急性灌酒组。并从MMP-9血管壁毒性物质、血管壁形态结构及其生物力学,Ngb、Hif-1α、EPO、Na~+,K~+-ATPase等脑氧和能量代谢物,tPA、Cyt-C、Caspase-9、Caspase-3、Bcl-2、Bax等细胞凋亡相关物质,证实了酒精的综合性毒理学作用在TSAH发生及死亡机制中具有重要作用。本实验是在此基础上,进一步探讨慢性灌酒大鼠TSAH高死亡率与胶质细胞改变的关系,选择与中枢神经纤维功能结构密切相关的胶质细胞特异性标志物S-100β、MBP,探讨急慢性酒精中毒情况下脑组织S-100β、MBP的表达,及其与大鼠TSAH死亡机制的相关性。材料与方法 1动物模型及分组 SPF级成年雄性SD大鼠61只,体重300±50g,分笼适应性饲养2周,随机分组。 1.1急性灌酒模型 28只大鼠随机分急性单纯灌酒组、急性灌酒打击组、急性单纯灌水组、灌水打击组共4组,每组7只。灌酒组一次性白酒(北京红星酒厂二锅头,52%v/v)灌胃15ml/kg;灌水组同等剂量的食用水灌胃。打击组在灌白酒或食用水后2h,给予脑震荡性打击。 1.2慢性灌酒模型 33只大鼠随机分为慢性单纯灌酒组7只、慢性灌酒停酒打击组7只、慢性灌酒打击组19只(其中死亡10只归为死亡组,存活9只归为存活组)共4组。各组大鼠给予白酒灌胃(北京红星酒厂二锅头,52%v/v),一天两次,间隔8小时,前2周每次予灌胃剂量8ml/kg、后2周每次灌胃剂量予12ml/kg,最后一次灌酒后2h,慢性灌酒停酒打击组于灌酒后12h,给予脑震荡性打击。 2方法各灌酒打击和灌水打击组大鼠予脑震荡打击后,放置4h予乙醚麻醉,开胸左心室取血5ml,死亡的大鼠立即开胸取血5ml,经左心室-主动脉插管,灌注4%中性多聚甲醛液处死兼固定脑组织,开颅取全脑,置于4%中性多聚甲醛固定、取材,常规石蜡包埋、HE切片染色,固绿(Fast green)髓鞘染色观察脑组织及髓鞘组织形态学,S-100β、MBP免疫组化染色,Image-Pro Plus 6.0图像分析软件体视学量化组织病理学观测,BAC检测。所有数据均应用SPSS15.0统计软件,t检验和单因素方差、卡方检验统计分析。结果 1急慢性酒精中毒大鼠行为学表现和BAC变化急性灌酒组大鼠灌酒后0.5h内活动增多、不停张望,呈兴奋状态,之后活动减少、步态不稳、动作笨拙,呈昏睡、呼吸缓慢,反应迟钝等醉酒表现。灌酒后2h血酒精浓度(BAC)180.16±16.58 mg/dL、血压14.13±0.34 Kpa,较灌酒前血压17.02±0.88 Kpa显著下降(P0.01);灌水组灌胃后行为及血压均无明显变化(P0.05)。慢性灌酒组大鼠灌酒期间,逐渐出现消瘦、营养不良、进食减少,精神萎靡、部分一侧肢体偏瘫等慢性酒精中毒改变,4w后体重251.8±19.6g较灌胃前307.2±28.9g明显下降(P0.01)、血压18.16±0.82Kpa较灌胃前16.60±0.76Kpa明显增高(P0.01)。灌酒后0.5h内呈侧卧、昏睡、翻正反射消失等急性酒精中毒表现,最后一次灌酒2h后BAC 208.50±32.35 mg/dL高于急性组。 2 TSAH发生率、死亡率及病理学观察急性灌酒打击组大鼠TSAH发生率为28.6%,无死亡,灌水打击组未见TSAH发生及死亡;慢性灌酒打击组的TSAH发生率为84.2%、死亡率52.6%,慢性灌酒停酒打击组的TSAH发生率为71.4%、死亡率28.6%,死亡率较慢性灌酒打击组有显著差异(P0.01)。急性单纯灌水组大脑表面散在局限性淤血;急性灌水打击组大鼠脑表面光滑,呈蜡样苍白;急性单纯灌酒组大鼠额叶及小脑表面大片淤血;急性灌酒打击组发生TSAH的大鼠脑表面及脑干背侧、腹侧斑片状薄层SAH;慢性灌酒打击组发生TSAH的大鼠全脑弥漫厚层SAH,以脑干腹侧面为重。组织学检查,急性灌酒组脑神经元及神经胶质细胞轻度水变性、细胞周隙轻度扩大,打击组脑干及小脑薄层SAH。慢性灌酒组脑皮质各层神经元排列紊乱、数目减少、核固缩,多见噬神经现象;小脑蒲肯野细胞疏松较少、胞体三角形固缩变小、突起减少、核固缩深染,部分溶解消失;延脑多见均质化胞浆深染固缩、核固缩的暗神经元(Dark cell);打击组脑干、额顶、小脑等处大片厚层SAH。固绿髓鞘染色(髓鞘呈深绿色、细胞核呈红色),急性单纯灌水组脑干、胼胝体及小脑神经纤维髓鞘平直、排列紧密;急性灌酒打击组的髓鞘排列稍疏松、间隙大,着色稍淡;慢性打击组的髓鞘疏松、间隙增大,着色浅淡不均,脑干可见明显不规则增粗扭曲、断裂神经纤维,周隙明显扩大。 3 S-100β蛋白、MBP的表达 3.1 S-100β蛋白阳性细胞数急性灌酒打击组和灌水打击组大鼠各脑区S-100β阳性细胞数均显著多于急性灌酒和急性单纯灌水组(P0.05);打击组之间、单纯组之间均无统计学差异(P0.05)。慢性灌酒打击存活组和慢性灌酒停酒打击存活组大鼠各脑区阳性细胞数均显著多于慢性单纯灌酒组和慢性灌酒打击死亡组(P0.05);慢性灌酒打击存活组和慢性灌酒停酒打击存活组之间、慢性单纯灌酒组和慢性灌酒打击死亡组之间均无显著差异(P0.05)。慢性灌酒打击存活组和慢性灌酒停酒打击存活组大鼠各脑区阳性细胞数均显著多于各急性组(P0.05);慢性单纯灌酒组和慢性灌酒打击死亡组各脑区阳性细胞数均多于各急性单纯灌酒和急性单纯灌水组(P0.05),与急性灌酒打击组和灌水打击组无显著差异(P0.05)。 3.2 S-100β蛋白IOD值急性灌酒打击组和灌水打击组各脑区S-100βIOD值均高于急性单纯灌酒组和急性单纯灌水组(P0.05),打击组之间、单纯组之间无统计学差异(P0.05)。慢性单纯灌酒组的IOD值最高,均显著高于其它慢性灌酒组(P0.05);慢性灌酒打击死亡组的IOD值最低,均显著低于其它慢性灌酒组(P0.05);慢性灌酒打击存活组与慢性灌酒停酒打击存活组的IOD值无显著差异(P0.05)。除慢性灌酒打击死亡组以外,其它慢性灌酒组的IOD值均高于急性组;慢性灌酒打击死亡组的IOD值与急性灌酒打击和灌水打击组无显著差异(P0.05),高于急性灌酒和急性单纯灌水组(P0.05)。 3.3 MBPIOD值急性灌酒打击组和急性灌水打击组脑干和胼胝体MBP IOD值均显著低于单纯灌酒和单纯灌水组(P0.05),打击组之间、单纯组之间无统计学差异(P0.05);小脑各组间均无显著差异(P0.05)。慢性单纯灌酒组各脑区MBP IOD值最高,高于其它各慢性灌酒打击组(P0.05);各慢性灌酒打击组之间胼胝体及小脑MBP IOD值无显著差异(P0.05);慢性灌酒打击死亡组脑干MBP IOD值高于慢性灌酒打击存活组及慢性灌酒停酒打击存活组(P0.05),存活组之间无显著差异(P0.05)。除慢性单纯灌酒组与急性打击组大鼠各脑区MBP IOD值无显著差异外(P0.05),慢性灌酒组各脑区MBPIOD值均低于急性组(P0.05)。结论 1、急慢性酗酒均可协同脑震荡性打击促发TSAH,慢性酗酒是TSAH死亡重要因素。 2、慢性酒精中毒脑组织胶质细胞S-100β蛋白表达增强、神经纤维髓鞘MBP表达减弱,可能参与慢性酗酒的酒精性脑萎缩和酒精性脑病发生机制,并且构成慢性酗酒的TSAH死亡机制的物质基础。
[Abstract]:Background and Purpose

Traumatic subarachnoid hemorrhage ( TSAH ) is a kind of independent subarachnoid hemorrhage which is different from the subarachnoid hemorrhage and pathological subarachnoid hemorrhage due to brain contusion . It is related to the traumatic epidural hemorrhage and the inferior vena cava and is classified as three major traumatic intracranial lacunar hemorrhage . The research of the occurrence and death mechanism of TSAH is seldom seen at home and abroad . Therefore , the research and interpretation of the occurrence and death mechanism of TSAH related to acute and chronic alcoholism can provide scientific basis for the death cause analysis and the sentencing of judicial trial .

The TSAH model of acute and chronic alcoholism in rats was successfully established in the previous work of the study group . The results showed that the incidence and mortality of TSAH in rats with chronic cerebral concussion were 82.4 ? , 28.6 % in the acute group and 58.8 ? in the acute group .

On the basis of this , the relationship between the high mortality of TSAH and the changes of glial cells in rats with chronic alcohol intoxication was studied . The glial cell specific marker S - 100尾 , MBP , which was closely related to the functional structure of the central nervous fibers , was selected to investigate the expression of S - 100尾and MBP in brain tissue under acute and chronic alcoholism , and its correlation with the death mechanism of TSAH in rats .

Materials and Methods

1 Animal model and grouping

Sixty - one SPF adult male SD rats weighing 300 卤 50g were randomly divided into two weeks .

1.1 Model of acute alcohol filling

28 rats were randomly divided into 4 groups randomly divided into acute pure alcohol group , acute alcohol infusion group , acute simple irrigation group and irrigation control group .

1.2 Model of chronic drinking wine

Thirty - three rats were randomly divided into four groups : chronic simple alcohol - infused group ( 7 ) , chronic alcohol ( n = 19 ) , group 4 ( death group , survival group 9 , survival group ) . Each group was given a dose of 8 ml / kg twice a day . After 2 weeks of administration , the rats were given a dose of 8 ml / kg twice a day . After the last two weeks , the rats were given a dose of 8 ml / kg . After the last two weeks , the rats were given a dose of 12 ml / kg . After the last time , the rats were given a dose of 12 ml / kg . After the last time , the rats were given a concussion . After the last time , the rats were given a concussion .

2 Method

All the data were analyzed by SPSS 15.0 statistical software , t - test and single - factor variance , chi - square test and statistical analysis .

Results

Behavior and BAC changes in rats with acute and chronic alcoholism

After drinking , the activity of the rats was increased , and the activity decreased , the activity decreased , the gait was unstable , the action was awkward , the blood pressure was 14.13 卤 0.34 Kpa , and the blood pressure before the infusion was 17.02 卤 0.88 Kpa ( P0.01 ) .

Compared with the acute group , the average weight was 251.8 卤 19.6g , which was significantly higher than that in the acute group ( P 0.01 ) , the blood pressure was 18 . 16 卤 0 . 82Kpa , the average age of the last time was 2 h , the BAC was 201.50 卤 32.35 mg / dL above the acute group .

2 . Incidence , mortality and pathology of TSAH

The incidence of TSAH in the control group was 28.6 % , there was no death . The incidence of TSAH in the treatment group was 84.2 % , the mortality rate was 52.6 % , the incidence of TSAH in the treatment group was 71.4 % , the mortality rate was 28.6 % , and the mortality rate was significantly different from the control group ( P0.01 ) .

In the acute irrigation group , the surface of the brain was smooth , the surface of the brain was pale , the frontal lobe and the cerebellum surface of the acute pure irrigation group had a large amount of blood stasis , the brain surface of the rats with TSAH in the acute irrigation group and the back side of the brain stem and the lamellar thin layer SAH on the ventral side of the brain , and the SAH of the whole brain of the rats with the TSAH in the treatment group of the chronic alcohol infusion set the weight of the ventral surface of the brain stem .

histological examination , mild water degeneration of cranial nerve element and glial cell in acute alcohol infusion group , mild enlargement of that peripheral space of the cell , the decrease of the number of neurons in the brain cortex , the reduction of the numb of neurons in the brain cortex , the reduction of the number of nuclei , the reduction of the nucleus , the deep dyeing of the nucleus , the partial dissolution and disappearance of the nucleus , the deep - staining of the cytoplasm of the cytoplasm , the dark cell of the nuclear fixation , and the SAH of the thick layer of the brain stem , forehead , cerebellum and the like .

The myelin sheath of the acute irrigation group was slightly loose , the gap was enlarged , the color was slightly different , the brain stem was obviously irregular , thickened and twisted , and the broken nerve fibers were obviously enlarged .

Expression of 3 S - 100 尾 protein and MBP

3.1 S - 100尾protein positive cell count

The number of S - 100尾 - positive cells in each brain area was significantly higher than that in the control group ( P0.05 ) , but there was no significant difference between the two groups ( P0.05 ) .

The number of positive cells in each brain area was significantly higher than that in the control group ( P0.05 ) , and the number of positive cells in each brain area was more than that of the acute pure alcohol and acute pure irrigation group ( P0.05 ) . There was no significant difference between the treatment group and the acute irrigation group ( P0.05 ) .

3.2 S - 100尾 Protein IOD Value

The values of S - 100 尾 IOD in the brain areas of the acute irrigation group and the control group were higher than those in the acute pure irrigation group and the acute pure irrigation group ( P0.05 ) . There was no statistical difference between the control group and the simple group ( P0.05 ) .

The IOD value of the chronic alcohol - infused group was higher than that in other chronic alcohol groups ( P0.05 ) , and the IOD value was significantly lower in the treatment group than in the other groups ( P0.05 ) , and there was no significant difference between the survival group and the survival group ( P0.05 ) .

There was no significant difference between the values of IOD in the group of acute alcohol and the control group ( P0.05 ) , which was higher than that in the acute and acute irrigation groups ( P0.05 ) .

3.3 MBF D value

The values of MBP IOD in brain stem and corpus callosal were significantly lower than those in the control group ( P0.05 ) , but there was no significant difference between the groups ( P0.05 ) .

There was no significant difference in MBP IOD ( P0.05 ) between the two groups ( P0.05 ) . There was no significant difference between the survival group and the survival group ( P0.05 ) . Except for the chronic simple alcohol infusion group and the acute attack group , the MBP IOD value of the brain regions was lower than that in the acute group ( P0.05 ) .

Conclusion

1 . The acute and chronic alcoholism can be combined with cerebral concussion to blow up TSAH , and chronic alcoholism is an important factor in TSAH death .

2 . The expression of S - 100尾 protein in the glial cells of chronic alcoholism brain increased , the expression of MBP in nerve fibers decreased , the mechanism of alcoholic brain atrophy and alcoholic encephalopathy could be involved in chronic alcoholism , and the material basis of the death mechanism of TSAH of chronic alcoholism .
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919.1

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