【作者】 齐倩；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 法医学， 2010， 硕士

【摘要】 目的:随着经济的发展及国民生活水平的不断提高,各种酒类开始大量涌现。嗜酒、酗酒、酒依赖逐渐形成普遍性,随之滋生的社会问题日益严重。大量研究表明长期大量饮酒可通过调节脑血流、脑血管舒缩功能,影响血压及血管通透性,改变血液粘稠度及凝血功能等多种途径促进脑血管意外的发生,但其具体机制尚未明了,成为法医学、临床医学及基础医学的研究焦点。在法医学检案中,饮酒后轻度外力作用导致蛛网膜下腔出血的案件较为常见,饮酒的参与度问题成为此类案例鉴定的难点。本实验拟以整体动物模型为研究对象,应用HE染色、特殊染色、透射电子显微镜(TEM)技术与免疫组织化学方法,深入观察急慢性饮酒大鼠脑基底动脉的组织结构改变,并对其分子机制进行探讨,进一步阐明饮酒对脑血管的影响及作用机制,为饮酒后轻度外力作用导致蛛网膜下腔出血案件的饮酒参与度鉴定提供理论基础。方法:健康雄性Sprague—Dawley(SD)大鼠,体重220g±10g,随机分成对照组、急性组、慢性低剂量组、慢性中剂量组、慢性高剂量组。用市售白酒红星二锅头(56%v/v)制备模型,采用大鼠灌胃器进行灌胃,每天2次,间隔10小时。实验前给予适应性蒸馏水灌胃1周。急性组给酒量为56%v/v 1.2mL/100g/次,分别于实验1天、1周、2周、3周末次灌胃24小时后处死大鼠;慢性各组自实验开始至第3周给酒量、给酒浓度逐渐递增,至第4周达到足量(慢性低剂量组:56%v/v 0.4 mL/100g/次;慢性中剂量组:56%v/v 0.8 mL/100g/次;慢性高剂量组:56%v/v 1.2 mL/100g/次),维持至第12周末。实验期间观察大鼠行为学改变,测量并记录大鼠体重和收缩压,并分别于第4周、8周、12周末次灌胃24小时后处死大鼠。对照组用蒸馏水代替白酒进行灌胃。取脑后全脑固定制作石蜡切片,进行Weigert弹力纤维染色观察大鼠脑基底动脉内弹力膜改变;在体视显微镜下剥离大鼠脑基底动脉,观察基底动脉超微结构改变;进一步应用免疫组织化学方法观察大鼠脑血管基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)蛋白的表达变化。数据采用均数士标准差(Mean士SD)表示,用SPSS 16.0统计分析软件进行统计学分析,各组均数的比较行单因素方差分析(ANOVA),用最小显著差法(LSD)作两两比较,以P<0.05为有显著性差异。结果:1行为学改变及体重变化:单次给酒数分钟后大鼠即出现不同程度的酒后表现,包括:兴奋性增加,平衡不稳,反应迟钝,嗜睡甚至瘫软等。随实验时间的延长,各慢性饮酒组大鼠进食量减少,倦怠少动,反应性差,体重增长缓慢,实验后期甚至不增反降,慢性中剂量组、高剂量组大鼠体重在实验第4周末与对照组相比有显著性差异(P<0.05);慢性低剂量组大鼠体重在实验第12周末与对照组相比有显著性差异(P<0.05)。2收缩压变化:对照组大鼠收缩压波动平稳,各慢性组大鼠收缩压随实验时间的延长呈上升趋势,慢性高剂量组大鼠收缩压到实验第10周末与对照组相比有显著性差异(P<0.05);慢性低剂量、中剂量组到第12周末与对照组相比有显著性差异(P<0.05)。3组织病理学改变:3.1肝脏病理学改变:急性组肝脏主要表现为肝细胞水肿;慢性低剂量组第4周末可见肝细胞水肿,随实验时间延长肝细胞水肿弥漫,且出现脂肪变性;慢性中剂量组较早可见肝细胞水肿伴部分肝细胞轻度脂肪变性,后期出现肝细胞嗜酸性变,炎细胞聚集;慢性高剂量组病变程度较低、中剂量组严重,第8周末可见弥漫性脂肪变性,进而又出现多发片状坏死。3.2中枢神经系统病理学改变:急性组可见大脑皮层神经元水肿,毛细血管周围间隙扩大;慢性组大鼠在实验过程中大脑皮层神经元水肿,排列紊乱,神经元细胞、小脑蒲肯野细胞核固缩浓染,可见嗜神经元现象和毛细血管周围间隙扩大等现象。3.3脑基底动脉内弹力膜形态改变:急性组内弹力膜(IEL)未见明显改变,慢性各组内弹力膜出现质地松散、局部紊乱,甚至分层、断裂。3.4脑基底动脉超微结构改变:实验第4周末慢性高剂量组出现内弹力膜质地不均,薄厚不一;第8周末慢性各组普遍可见内皮细胞形态不规则,表面微绒毛减少,内皮细胞内及内皮细胞与内弹力膜之间出现空泡,内弹力膜质地不均,厚薄不一,甚至出现分叉、分层、断裂。4 MMP-2、MMP-9免疫组织化学结果:对照组脑实质毛细血管、小血管和基底动脉血管壁在各观察点均无明显MMP-2、MMP-9蛋白表达;急性组1周、2周末脑实质内毛细血管、小血管血管壁可见MMP-9阳性表达;而各时间点脑实质内毛细血管、小血管血管壁均未出现MMP-2阳性表达;急、慢性各组大鼠脑基底动脉动脉壁未见明显MMP-2,MMP-9阳性表达。结论:1本实验中慢性中剂量组大鼠饮酒8周后(第1周:56%V/V白酒用蒸馏水1:1稀释,大鼠单次饮酒0.4mL/100g;第2周:56%V/V白酒用蒸馏水2:1稀释,大鼠单次饮酒0.6mL/100g;第3周:56%V/V白酒,大鼠单次饮酒0.6mL/100g;第4~8周,56%V/V白酒,大鼠单次饮酒0.8mL/100g)脑血管损伤明显,可作为探讨饮酒对脑血管影响的较为理想的实验模型。2慢性饮酒对大鼠脑基底动脉壁具有损伤作用,使该血管可能成为在机械外力、各种应激、疾病等因素刺激下容易发生破裂出血的部位。更多还原

【Abstract】 Objective: With development of economy and improvement of living standards, all kinds of alcoholic drinks became popular. The phenomenon of drinking, alcohol abuse and alcohol dependence was widespread. These social problems should not be ignored. Long-term drinking promoted the occurrence of cerebrovascular disease by regulating the blood flow and vasomotor function of cerebral vessels, raising blood pressure and vascular permeability, disordering blood viscosity and coagulation and so on. But the mechanisms were not clear, and were brought to the researchers’notice in forensic medicine field, clinical medicine field and preclinical medicine field. The cases of subarachnoid hemorrhage(SAH) due to slight violence after drinking were frequently reported. It was troublesome problem to assess the relationship between drinking and SAH.Our experiment intended to establish the model of alcohol-treated rats and observe pathological changes of cerebral blood vessels in acute and chronic groups with HE stain, Weigert elastic fiber staining, transmission electron microscopy(TEM) and immunohistochemical staining, explore the mechanisms, and provide a reliable theoretical basis to assess the relationship between drinking and SAH.Methods: Sprague—Dawley(SD) rats (220g±10g) were randomly divided into following groups:control group, acute group, chronic low-dose group, chronic medium-dose group and chronic high-dose group. 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 were given orally through blunt tipped needle to adapt all rats to the process in advance. The acute group was treated with 56%v/v 1.2 mL·100g-1 dose each time and rats were respectively sacrificed after 1-day, 1-week, 2-week and 3-week treatment. The concentration and volume given to chronic groups increased gradually until the 4th week(chronic low-dose group: 56%v/v 0.4mL·100g-1 each time, chronic medium-dose group: 56%v/v 0.8mL·100g-1 each time, chronic high-dose group: 56%v/v 1.2mL·100g-1 each time), and then the full dose was supplied until the end of the 12th week. Observe behavior changes and record the weights and the systolic blood pressure using the tail-cuff method. Rats from each chronic group were respectively sacrificed after 4-week, 8-week and 12-week treatment. Water instead of wine was given to control group. Brains fixed were used for Weigert elastic fiber staining to observe the morphological changes of rat basilar artery walls. Basilar arteries dissected with stereomicroscope were observed by TEM. And expression of metrix metalloproteinase-2(MMP-2) and metrix metalloproteinase-9(MMP-9) was detected by immunohistochemical stain.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: 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. A significant weight lose in chronic high-dose group and chronic medium-dose group was observed at the 4th week compared to control group(P<0.05). And then significant lose in chronic low-dose group was also observed at the 12th week compared to control group(P<0.05).2. Systolic blood pressure changes: Systolic BP fluctuated smoothly in control group, but rose in each chronic group with the prolonged treatment. Systolic BP in chronic high-dose group rose most apparently, and it had increased at the 10th week significantly(P<0.05). There was also a significant increase in chronic medium-dose and low-dose groups at the 12th week(P<0.05) . 3. Pathological changes3.1 Pathological changes of liver: In acute group cellularedema of liver was observed. In chronic low-dose group, cellularedema was observed and aggravated since the 4th week, and then mild microvesicular adipose degeneration emerged. In chronic medium-dose group, there was mild microvesicular adipose degeneration, and acidophilic degeneration and inflammatory infiltration were observed later. In chronic high-dose group, the pathological changes were more serious: diffuse microvesicular adipose degeneration was obvious at the 8th week, and then multiple necrosis emerged.3.2 Pathological changes of central nervous system: In acute group, neuron edema and expansion of the space surrounding capillaries were seen. In chronic groups, there were neuron edema, disorder of cell-scatterring, karyopyknosis and nuclear condensation of neurons or purkinje cells, neuronophagia and expansion of the space surrounding capillaries.3.3 Changes of internal elastic lamina(IEL) by Weigert elastic fiber staining: There was no significant change of IEL in acute group. IEL in chronic groups had loose texture, delamination and disruption.3.4 Changes of rat basilar artery ultra-structure: The chronic hige-dose group began to appear uneven texture and unhomogeneous thickness of IEL at the 4th week. Each chronic group at the 8th week showed reduction of endothelial cell microvillus, vacuolation in or under endothelial cells(EC), uneven texture, unhomogeneous thickness, bifurcation, delamination and even fragmentation of IEL.4. Expression of MMP-2 and MMP-9: Negative expression of MMP-2 or MMP-9 was observed in walls of capillaries, small vessels or basilar arteries of control group. There were positive expression of MMP-9 in walls of capillaries and small vessels at the 1st week and 2nd week of acute treatment. Negative expression of MMP-2 was observed in capillaries or small vessels. And there were negative of MMP-2 or MMP-9 in basilar artery walls in each group. Conclusions:1. There is obvious impairment of rat cerebral vessels in medium-dose group for 8 weeks(At 1st week: 28%V/V wine, 0.4mL/100g each time; At 2nd week: 37.3%V/V wine, 0.6 mL/100g each time; At 3rd week: 56%V/V wine, 0.6 mL/100g each time; At 4th ~8th week: 56%V/V wine, 0.8mL/100g each time). These rats are ideal models to study the alcohol effect on cerebral vessels.2. Chronic alcohol treatment brings impairment in basilar artery, which makes the basilar artery fragile to external force, various stress and diseases.更多还原