灌酒大鼠脑组织tPA、MMP-9表达变化与外伤性蛛网膜下腔出血的关系研究

发布时间：2018-07-07 16:37

  本文选题：外伤性蛛网膜下腔出血(TSAH) + 酗酒　； 参考：《汕头大学》2007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)约占无颅骨骨折时颅内出血的90％。早在1963年国外学者报道了酗酒后TSAH死亡案例中，多是颜面或颈部遭受拳击等轻微暴力，导致致死性TSAH，而不合并其他颅脑损伤，或仅伴有轻微脑挫伤。但是，关于TSAH发生机制及其与酗酒关系和参与度，国内外尚无统一认识，亦未见系统研究报道，一定程度上影响了此类案件的法医学死因分析鉴定和审判量刑。 本文目的是在本课题组已经建立的大鼠灌酒后TSAH模型基础上进一步实验，拟观察急慢性酗酒时脑组织tPA及MMP-9表达情况，以期从毒理学、组织病理学及分子生物学角度阐述TSAH发生和死亡的相关机制。 材料与方法 健康雄性SD大鼠，体重250～350g，随机分为急性单纯灌酒组和急性灌酒后打击组，每组7只；慢性单纯灌酒组和慢性灌酒后打击组，每组17只；对照为灌水打击组，每组5只。实验组予食用白酒北京二锅头(56％v／v)灌胃，急性灌酒组一次性灌酒剂量为15ml／Kg；慢性灌酒组连续灌酒1个月、每天两次间隔8h，，前2周灌酒量8ml／kg／次、后2周12ml／kg／次。对照组灌同剂量纯净水。急性组灌酒后2h、慢性组最后一次灌酒后2h，用自制单摆式定位定量脑损伤打击装置复制大鼠脑震荡性损伤模型，急性灌酒打击组分别于打击后0.5h、2h、4h、6h、12h断头处死取血，慢性灌酒组打击后2h断头处死，对照组处死时间比照实验组相应时间段。 观察灌酒后大鼠生命体征变化，检测血乙醇浓度，观察脑组织形态学变化及tPA、MMP-9表达情况。采用SPSS统计软件和Excel数据库处理和统计分析实验数据。 结果 1动物醉酒行为观察 大鼠灌酒后短时间内兴奋性较强、不停走动，步态不稳，约0.5h后逐渐出现急性酒精中毒临床表现：侧卧、昏睡、翻正反射消失，呼吸缓慢，血压降低(P＜0.05)。灌酒组与灌水组大鼠血压在打击后呈现一过性短暂性血压升高(P＜0.05)，呼吸未见改变。慢性组大鼠灌酒2周后，毛发粗糙暗淡、精神萎靡，进食量减少；3周后体重较对照组增长缓慢(P＜0.05)，4周后基础血压升高(P＜0.05)。 2酒精浓度检测 急性组大鼠灌酒后2h血乙醇浓度达最高峰值188.8±5.8 mg／dl，之后逐渐降低，12h降至1.7±0.5 mg／dl。 3 TSAH发生率及死亡率 急性灌酒组TSAH发生率为28.6％，死亡率为0，自发性SAH发生率为0。慢性灌酒组TSAH发生率为82.4％，死亡率为58.8％；自发性SAH发生率为5.9％，死亡率为0。 4 HE染色观察 急性灌酒组大鼠伤后各时间段大鼠脑组织形态学表现为不同程度轻度淤血、肿胀。组织学观察，脑蛛网膜疏松淤血水肿，薄层片状蛛网膜下腔出血，多见于脑干腹侧区，神经元及小胶质细胞轻度水变性(颗粒性肿胀)。 慢性灌酒组大鼠脑组织形态学表现为不同程度中至重度淤血、肿胀，不同程度蛛网膜下腔出血。组织学观察，脑蛛网膜疏松淤血水肿，大片蛛网膜下腔出血，神经元中重度水变性，细胞周隙扩大，可见神经元核固缩、数量减少，胶质细胞增生明显。 5免疫组织化学染色观察 正常大鼠脑组织tPA表达微弱，急性灌酒组及打击后各时间段脑海马、小脑、枕叶tPA阳性细胞数表达高于灌水打击对照组，tPA和MMP-9阳性细胞表达的光密度值亦均高于对照组(P＜0.05)。其中，6h组表达高于0.5h组(P＜0.05)。但是，额叶与对照组无统计学差异。 慢性灌酒组脑组织各区域tPA阳性细胞数及光密度值表达均强于对照组(P＜0.01)，MMP-9光密度值高于对照组(P＜0.01)。急慢性灌酒组及其相应打击组大鼠脑组织tPA、MMP-9表达无明显差异(P＞0.05)。 结论 1建立了稳定的大鼠急慢性灌酒后外伤性蛛网膜下腔出血模型，证实了灌酒后轻微头部暴力打击与TSAH高发生率及死亡率相关，首次实验证实了慢性灌酒大鼠TSAH、SAH发生率及死亡率均明显高。 2证实了长期慢性灌酒大鼠基础血压升高，灌酒后血压照灌酒前降低，打击后血压呈一过性升高，容易发生血管破裂出血。 3证实了急性和慢性灌酒组大鼠脑组织海马、小脑、枕叶中tPA与MMP-9阳性细胞数和光密度值均较对照组表达增强，提示损伤早期脑组织tPA可大量释放，继而通过MMP-9造成对血管壁结构破坏，促进出血。大量长期饮酒后tPA、MMP-9过度表达对神经元具有致坏死作用。尚未见同类研究报道。 此外，tPA一直被临床用作SAH和脑血栓急性期治疗药物。本实验结果提示在酒后TSAH的救治时，应尽量减少tPA的释放并对其活性加以抑制，降低再次出血的危险。同时，注意减少和避免神经元不可逆损伤的发生。
[Abstract]:Background and Purpose






Traumatic subarachnoid hemorrhage ( TSAH ) accounts for 90 % of the intracranial hemorrhage in the absence of skull fracture . In 1963 , foreign scholars reported that there were mild violence such as boxing and other minor violence , leading to fatal TSAH without merging other brain injury , or with mild brain contusion . However , there was no uniform understanding at home and abroad about the mechanism of TSAH and its relationship with alcohol and alcohol .






The aim of this paper is to further experiment on the model of TSAH , which has been established in the study group , and to observe the expression of the tissue and MMP - 9 in brain tissue during acute and chronic alcoholism , with a view to elaborating the mechanism of TSAH ' s occurrence and death from toxicology , histopathological and molecular biology .






Materials and Methods






Male SD rats weighing 250 - 350g were randomly divided into acute pure alcohol group and acute alcohol - infused group , each group was 7 rats .
17 rats in each group ;
In the control group , 5 rats in each group were divided into two groups : the experimental group was given edible white spirit Beijing two - pot head ( 56 % v / v ) , and the dose of one - time infusion was 15ml / Kg .
In the control group , the rats were killed at 0.5 h , 2 h , 4 h , 6 h , 12 h , and the control group was killed for 2 h after the last time . The control group was killed for 2 h and the control group was killed for a period of time compared with the experimental group .






The changes of vital signs in rats were observed , the concentration of ethanol was detected , the morphological changes of brain tissues and the expression of tissue morphology , and the expression of MMP - 9 were observed . SPSS statistical software and Excel database were used to analyze and analyze the experimental data .






Results






An Observation on the Behavior of Drinking Wine in 1 Animal






The results showed that the blood pressure of the rats was decreased ( P & lt ; 0 . 05 ) . After 2 weeks , the hair was rough , the spirit was depressed , and the consumption of food was decreased .
After 3 weeks , the body weight was slower than that in the control group ( P & lt ; 0.05 ) , and the basic blood pressure increased after 4 weeks ( P & lt ; 0.05 ) .






2 . Determination of alcohol concentration






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