【作者】 刁云锋；

【导师】 孙晓枫；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:弥漫性轴索损伤(diffuse axonal injury,DAI)是一种常见的颅脑损伤类型,其特点是症状危重、昏迷时间长、伤残率及死亡率高。DAI的形成包括一系列病理生理学变化过程,除了剪应力直接作用形成的原发性损伤外,脑损伤后的继发性改变如缺血、缺氧、水肿等引起的损伤更为严重。伴随DAI产生的血脑屏障(blood-brain barrier,BBB)破坏,及由之引起的脑水肿等继发性损伤是导致本病预后较差的主要原因。近年来发现的水通道蛋白4(Aquaporin-4,AQP4)与DAI后脑水肿的形成关系密切,但AQP4是否引起BBB通透性的改变,以及Ca2+是否与二者有关联尚不清楚。研究证明在大鼠脑出血模型中,Ca2+拮抗剂尼膜同能够下调脑组织AQP4 mRNA表达,降低BBB通透性,从而抑制脑水肿。本实验拟通过动态观察大鼠DAI后脑组织含水量、BBB通透性和AQP4的表达,以及使用尼膜同后的变化,探讨DAI后脑水肿的发生机制,从而为DAI防治提供理论依据。方法:健康雄性成年SD大鼠110只,体重350-375g,由河北医科大学实验动物中心提供。按完全随机分组原则分为3组:对照组、DAI组和尼膜同治疗组。对照组10只,DAI组和尼膜同治疗组各50只。后两组再各随机分为6h、1d、3d、5d、7d组5个亚组,每个亚组10只大鼠。DAI组按照Marmarou等的方法制作大鼠DAI模型;尼膜同治疗组腹腔注射尼膜同(0.5mg/kg体重)20min后,按照Marmarou等的方法制作大鼠DAI模型,以后每24h注射1次尼膜同(剂量同前);对照组大鼠切开头皮暴露颅骨后直接缝合。造模后大鼠常规进食。从DAI组和尼膜同治疗组每个亚组中随机抽取五只大鼠,连同从对照组中随机抽取的五只大鼠,按预定时间,4%多聚甲醛溶液灌注后断头取脑,沿正中矢状线将脑组织分为左右两部分。左半脑组织立即进行脑组织含水量的测定;右半脑组织常规固定、脱水、透明、石蜡包埋后连续切片厚度4μm,进行相应染色、观察。取上述各时间点的左半脑组织,采用干湿重法计算脑组织含水量,以此代表脑水肿的程度。各组中剩余的大鼠均采用分光光度法检测各时间点的脑组织中伊文思兰(evens blue,EB)的含量,以定量评价BBB功能损害的情况。在光镜下,通过对标本的HE染色观察DAI后脑水肿的变化,如细胞水肿、细胞间隙和血管周围间隙增宽程度等。通过对标本的免疫组化染色观察细胞胞膜呈棕黄色或褐色深染的AQP4蛋白阳性表达。采用Motic6.0数码医学图像分析系统定量分析每个视野阳性目标平均吸光度值(OD),代表AQP4的表达强度。应用SPSS13.0统计学软件,所得数据均以均数±标准差( x±s)表示。两组间比较采用两样本t检验,多组间比较采用单因素方差分析,当方差分析有显著性差异时,进一步用q检验作两两比较;以及根据实验要求作直线相关性分析。以P<0.05作为判断差异有无统计学意义的标准。结果:1肉眼及光镜观察结果肉眼观察:DAI组和尼膜同治疗组可见软脑膜血管扩张淤血,散在的点灶状蛛网膜下腔出血,未见明显的脑挫伤及脑实质内出血;HE染色:DAI组可见脑组织疏松水肿,细胞空泡样变,细胞间隙和血管周围间隙增宽。尼膜同治疗组病理变化较DAI组轻。对照组无上述病理变化;免疫组化染色:DAI组可见室管膜、软脑膜和血管周围的星形胶质细胞胞膜呈棕黄色或褐色深染的AQP4阳性表达。尼膜同治疗组AQP4表达较DAI组稍弱,但强于对照组。对照组AQP4呈弱阳性表达。2脑组织含水量的变化DAI组和尼膜同治疗组脑组织含水量均呈现一个由低向高,到达顶峰后逐渐降低的过程。从致伤后6h开始增加,1d达到高峰,之后开始下降,7d脑组织含水量仍高于正常。与对照组比较,DAI组和尼膜同治疗组都具有统计学差别(P<0.05)。尼膜同治疗组达到高峰时间与DAI组相同,但6h、1d、3d、5d脑组织含水量均低于DAI组(P<0.05),7d脑组织含水量无明显差异(P>0.05)。3 BBB通透性的变化(EB含量的变化)DAI组和尼膜同治疗组BBB通透性均呈现一个逐渐上升,到达顶峰后缓慢降低的过程。从致伤后6h开始升高,1d~3d时最高(1d与3d比较无明显差异),之后逐渐降低,7天BBB通透性仍高于正常。与对照组比较,DAI组和尼膜同治疗组都具有统计学差别(P<0.05)。尼膜同治疗组的变化与DAI组相似,但各对应时间点BBB通透性均低于DAI组(P<0.05)。4脑组织AQP4表达的变化DAI组和尼膜同治疗组脑组织AQP4表达均呈现一个由低向高,到达顶峰后逐渐降低的过程。从致伤后6h开始增加,3d达到高峰,之后开始下降,7d脑组织AQP4表达仍高于正常。与对照组比较,DAI组和尼膜同治疗组都具有统计学差别(P<0.05)。尼膜同治疗组达到高峰时间与DAI组相同,但各对应时间点AQP4表达均低于DAI组(P<0.05)。5脑含水量、EB含量、AQP4表达的相关分析大鼠DAI后脑含水量、EB含量与AQP4表达的变化规律一致,相关分析显示脑含水量与AQP4表达正相关(r=0.824,P<0.01);EB含量与AQP4表达也呈正相关(r=0.871,P<0.01)。结论:1大鼠DAI后脑组织含水量、BBB通透性、AQP4表达均呈现一个由低向高,到达顶峰后开始逐渐降低的过程。AQP4蛋白的变化规律,与脑组织含水量、BBB通透性呈正相关,证实AQP4与DAI后脑水肿的形成,BBB损伤之间存在密切正相关关系。2给予尼膜同治疗后,与DAI组相比,脑组织含水量、BBB通透性、AQP4表达在各对应时间点均有明显的差异(P <0.05)。表明尼膜同通过降低DAI后AQP4的表达,降低BBB通透性,减轻脑水肿,在DAI后BBB的修复过程中发挥一定的作用。更多还原

【Abstract】 Objective: Diffuse axonal injury (DAI) is a common type of brain injury, characterized by severe symptoms, a long time coma and high rate of disability and mortality. The formation of DAI included a series of pathophysiological changes. Except the primary injury caused by shear stress directly, the secondary brain injury caused by changes such as ischemia, hypoxia, edema were more serious. The blood-brain barrier (BBB) damage Accompanied with DAI led to cerebral edema and other secondary nerve damage, which were the main reason of the poor prognosis. The Aquaporin-4(AQP4) discovered In recent years had close relations with the formation of brain edema after DAI, but it is still unclear that if AQP4 caused the changes of the permeability of BBB, and Ca2+ have relations with them. The study shows that in the model cerebral hemorrhage rat, Ca2+ antagonist nimoton can decrease the expression of AQP4 mRNA in brain tissue, lower the BBB permeability, so as to inhibit brain edema. Though dynamically observing the brain water content, BBB permeability, AQP4 expression after DAI, and the changes after the use of Nimoton, this research discussed the mechanism of cerebral edema after DAI, so as to provide a theoretical basis for prevention and cure of DAI.Methods: 110 adult male SD rats, each animal weight 350-375g, afforded by the Hebei Medical University Laboratory Animal Centre. These animals were divided into three groups according to the principle of completely randomized: the control group, the DAI Group and the nimoton treatment group. The control group had 10 rats, while the DAI group and the nimoton treatment group each 50. The DAI group and the nimoton treatment group were randomly divided into 6 h, 1d, 3d, 5d, 7d five sub-groups, each sub-groups had 10 rats. The DAI Group, in accordance with the Marmarou’s production method to cause DAI model; The nimoton treatment group were intraperitoneal injected of nimoton (0.5mg/kg body weight), after 20 min, according to Marmarou’s production method to cause DAI model, then injected nimoton once every 24 h (the same dose as before); The control rats sutured directly after incising scalp and exposing skull. The rats ate normaly after having been injured. Selected five rats from each sub-groups and the control group randomly. According to the scheduled time, after being reperfused by 4% paraformaldehyde solution decapitated and took out brain, The brain tissue will be divided into two parts along the median sagittal line. The left brain tissue was used to determinate the brain water content immediately. The right brain tissue were fixed, dewatered, transparented, paraffin imbeded for staining and observing.Using the wet-dry weighting method to measure the water content of the left brain tissue, as the representative of the extent of brain edema.Using spectrophotometric to measure the EB content in brain of the remaining rats at every time point in order to quantitatively evaluate the functional damage of BBB.In light microscopy, specimens after HE staining was to detect the changes of brain edema, such as cell edema, the extent of cell gap and vascular peripheral gap widened. specimens after immunohistochemical staining was to detect the AQP4 protein expression which the cell membrane was yellow or brown. Using Motic 6.0 digital medical image analysis system for quantitative analysis of each field of the positive reaction target’s average optical density (OD), on behalf of the intensity of AQP4 expression.Applying SPSS 13.0 statistical software, the data are mean±standard deviation ( x±s). Between the two groups using the two-sample t-test, multiple comparisons using single-factor analysis of variance, when there are significant differences using analysis of variance, for further testing by q comparison. Based on the experimental requirements, as well as for linear correlation analysis. To P<0.05 as a judge if there was significant difference between the standards.Results: 1. Naked eye and light microscopy observationNaked eye: We can see leptomeningeal blood vessel dilated and congested, scattered subarachnoid hemorrhage point in the DAI group and the nimoton treatment group, but there was no obvious brain contusion and brain hemorrhage; HE staining: In the DAI group, we can see brain tissue osteoporosis and edema, cell vacuolar changes, cell clearance and the gap around vascular widened. The nimoton treatment group’s pathological changes was lighter than the DAI group; The control group did not have these pathological changes; Immunohistochemical staining: In the DAI group, we can see AQP4 expression which astrocytes membrane were yellow or deep brown in ependymal, leptomeningeal and the vessels surrounding; The nimoton treatment group’s AQP4 expression was lighter than the DAI group, but stronger than the control group. The control group showed weak AQP4 positive expression.2. The changes of brain water contentThe brain water content of the DAI group and the nimoton treatment group has a tendency from low to high, and reached its peak before gradually reducing process. The brain water content began to increase from 6h and reach a peak 1d, then started to decline, 7d brain water content is still higher than normal. Compared with the control group, The DAI group and the nimoton treatment group have significant differences (P <0.05). The nimoton treatment group reached its peak the same time as the DAI Group, but at 6h, 1d, 3d, 5d time point the brain water content were lower than the DAI group (P<0.05). The 7d brain water content had no significant difference (P>0.05).3. The changes of BBB permeability (The changes of brain EB content)The BBB permeability of the DAI group and the nimoton treatment group has a tendency from low to high, and reached its peak before gradually reducing process. The BBB permeability began to increase from 6h and reach its peak 1d-3d( 1d compared with 3d had no significant differences), then started to decline, 7d BBB permeability is still higher than normal. Compared with the control group, The DAI group and the nimoton treatment group have significant differences (P <0.05). The nimoton group reached its peak the same time as the DAI Group, but each time point BBB permeability were lower than the DAI group(P<0.05).4. The changes of brain AQP4 expressionThe brain AQP4 expression of the DAI group and the nimoton treatment group has a tendency from low to high, and reached its peak before gradually reducing process. The brain AQP4 expression began to increase from 6h and reach its peak 3d, then started to decline, 7d brain AQP4 expression is still higher than normal. Compared with the control group, the DAI group and the nimoton treatment group have significant differences (P<0.05). The nimoton treatment group reached its peak the same time as the DAI Group, but each time point brain AQP4 expression were lower than the DAI group (P<0.05).5. the correlation analysis of brain water content, EB content and AQP4 expressionThe brain water content, EB content and AQP4 expression had the same rule, correlation analysis showed that brain water content and AQP4 expression was positively correlated (r= 0.824,P<0.01); EB content and AQP4 expression was positively correlated (r=0.871,P<0.01).Conclusion: 1. the brain water content, BBB permeability, AQP4 expression has a tendency from low to high, and reached its peak before gradually reducing process in rats after DAI. The AQP4 expression have a positive correlation with the brain water content and the BBB permeability. It was confirmed that the AQP4 changes in the level of protein have a positive correlation with the changes of brain water content and the extent of damage on BBB.2. Given nimoton treatment, compared with the DAI group, the brain water content, BBB permeability, AQP4 expression in the corresponding time points were significant differences (P<0.05). It was confirmed that nimoton can lower BBB permeability and reduce cerebral edema by reducing the AQP4 expression after DAI. It was certain that Nimoton play a role in the process of BBB restoration after DAI.更多还原