【作者】 崔向宁；

【导师】 王玉来； 尹岭；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2005， 博士

【摘要】 1 目 的 本研究根据祖国医学血水相关理论,结合现代医学知识和实验方法,对创伤性脑水肿主要病理生理改变进行研究,探讨创伤性脑水肿发病机理和证治规律及治疗的新途径。利用创伤性脑水肿动物模型,以颅脑损伤后脑微循环改变为切入点,从微血管结构和功能的改变,探讨颅脑损伤后脑微循环紊乱的特点及其对脑血流灌注、血脑屏障通透性及脑水肿等的影响,进一步探讨活血利水法对颅脑损伤后脑微循环的改善及脑水肿的防治作用,为活血利水法治疗创伤性脑水肿提供理论依据及实验基础。通过脑损伤后脑微血管基底膜基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)及星形胶质细胞足突水通道蛋白 4(aquaporin,AQP4)的动态变化探讨脑微血管损伤的机制及活血利水中药脑疏宁对微血管损伤的保护机制。 2 方 法 2.1 按 Feeney 自由落体撞击法建立大鼠创伤性脑损伤(traumatic brain injury,TBI)模型,设假手术组、创伤组、脑疏宁组、甘露醇组。术前 3d 开始连续给予脑疏宁水煎液灌胃,第 4d 给药后 1h 造模;甘露醇组给予 20%甘露醇 1g/kg 股静脉注射。术后 7d 连续动态观察体重及神经功能评分的变化。 2.2 用干/湿重法和 Evans 蓝测定法观察大鼠 TBI 后 6h、24h、3d 及 5d 脑组织含水量和血脑屏障通透性的变化。 2.3 采用血管组化染色及图像分析方法,对大鼠TBI后不同时相的脑微血管分布密度进行动态观察和定量分析,并结合HE染色、透射电镜及MSB染色等技术对脑损伤后脑微血管形态学改变及凝血功能等进行研究。 2.4 采用免疫组化方法观察大鼠 TBI 后不同时相脑组织 MMP-2、MMP-9 及 AQP4的表达。 2.5 运用磁共振成像(magnetic resonance imaging,MRI)技术连续动态观察脑挫伤区信号与体积变化。 3 结 果 3.1 创伤性脑损伤后大鼠出现明显行走功能障碍伴体重减轻。伤后 7d 创伤组大鼠完成木条行走作业的时间为 16.46±3.27s,体重较术前减轻 17.75±5.09g;脑疏宁组行走时间(10.24±1.38s)缩短,与创伤组比较有显著性差异(P<0.01)。体重变化(11.75±4.27g)较小,与创伤组比较有显著性差异(P<0.05);甘露醇组伤后 7d 大鼠完成木条行走作业的时间及体重变化与创伤组比较无显著性差异(P>0.05)。 - 2 - 活血利水法治疗创伤性脑水肿的理论与实验研究3.2 创伤性脑损伤后 6h 伤侧脑组织含水量(78.76±0.49%)及 EB 含量(10.41±1.50μg/g)较假手术组(77.20±0.45%,3.27±1.05μg/g)增高,24h 达高峰,分别为79.63±0.45%和 11.76±2.22μg/g,伤后 5d 未恢复正常。脑疏宁组伤后 6h、24h、3d、5d 脑组织含水量分别为 78.14±0.37%、78.93±0.47%、78.23±0.63%、77.70±0.33%,与创伤组同一时间点相比,脑组织含水量降低,有显著性差异(P<0.05);甘露醇组脑组织含水量分别为 78.01±0.82%、78.80±0.57%、78.26±0.82%、77.81±0.48%,6h、24h、3d 时间点较创伤组降低,有显著性差异(P<0.05),伤后 5d 两组比较无显著性差异(P>0.05)。脑疏宁组与甘露醇组同一时间点脑组织含水量比较无显著性差异(P>0.05)。脑疏宁组脑组织 EB 含量分别为 8.50±1.33μg/g、7.97±1.08μg/g、6.57±1.93μg/g、5.31±1.19μg/g,与创伤组同一时间点相比,EB 含量明显降低,有显著性差异(P<0.01);甘露醇组伤后 24h、3d、5d 脑组织 EB 含量与创伤组比较无显著性差异(P>0.05)。3.3 应用 DAB 组织化学染色,大鼠脑微血管呈棕黄至棕黑色。颅脑损伤后 24h,脑微血管面积密度(0.021±0.0032%)下降至假手术组(0.051±0.0022%)的 41%,血管染色浅淡,部分血管有扭曲、断裂现象,血管粗细不均匀;脑疏宁组伤后 24h 开始微血管面积密度(0.027±0.0056%)明显增加,与创伤组比较有显著性差异(P<0.05)。伤后 24h 大鼠血浆纤维蛋白原(Fib)及 D-二聚体(D-dimer)含量(3.05±0.71g/L,1.04±0.32μg/ml)较假手术组增高(2.34±0.23g/L,0.31±0.084μg/ml),提示机体处于高凝状态并继发纤溶亢进;脑疏宁组 Fib 及 D-dimer 均显著降低(1.98±0.39 g/L,0.52±0.30μg/ml),与创伤组比较有显著性差异(P<0.05)。3.4 正常大鼠大脑半球 MMP-2 有少量表达,主要见于脑微血管周围;创伤组大鼠伤后 6h 伤灶周围 MMP-2 表达开始增高,至伤后 24h,其表达继续增加,5d 时达到高峰;脑疏宁组与创伤组比较,伤后 24h 开始 MMP-2 表达降低,有显著性差异(P<0.01)。正常大鼠脑组织未发现 MMP-9 免疫阳性;创伤组大鼠于术后 6h 伤灶周围可见棕黄色MMP-9 阳性染色细胞,24h 达到高峰,5d 时仍维持较高水平;脑疏宁治疗组与创伤组同一时间点相比,MMP-9 表达下降,有显著性差异(P<0.01)。3.5 AQP4 在各组大鼠脑组织中均有表达,阳性表达主要见于脑室和导水管系统的室管膜、软脑膜及血管周围的星形胶质细胞足突膜上,假手术组呈低表达;TBI 后 6h 伤灶周围水肿区星形胶质细胞足突 AQP4 表达开始增高,24h 表达继续增加,3d 后逐渐降低;脑疏宁组大鼠各时间点伤灶周围 AQP4 的表达与创伤组相比明显减低,具有显著性差异(P<0.05)。3.6 T2WI 检测显示创伤组大鼠 1d、3d、5d 时间点,右脑额顶叶出现高信号区。伤后5d更多还原

【Abstract】 1 Objective According to the theory of TCM about the relation of blood and water, the knowledge of mordern medicine and mordern experimental method, the present study tried to investigate the pathological and physiological changes of traumatic brain edema in order to identify the pathogenesis, diagnosis and treatment of brain edema. In order to provide theoretical and experimental evidence for promoting blood flow and alleviating water retention to treat brain edema, TBI animal model was used to study the feature of microcirculation disturbance after TBI and its impact on cerebral blood flow, permeability of blood-brain barrier and brain edema. We also tried to investigate the protective effects of promoting blood flow and alleviating water retention on microcirculation and brain edema after TBI. In addition, through observing the change of MMP-2 and MMP-9 on basement membrane and AQP4 on foot process of astrocyte, the mechanism of microvessel injury and the protective mechanism of chinese medicine Naoshuning to microvessel were investigated. 2 Method 2.1 The model of TBI was established by bumpiness of free falling body according to Feeney’s, all experimental rats were devided into pseudoperarion group, TBI group, Naoshuning group and mannitol group. In Naoshuning group, we successively poured into rats’ stomache with Naoshuning for 3 days before operation. 1 hour after the last administration on the 4th day, TBI models were induced. Rats’ weight and neurological function score were detected each day after opertion within one week. Rats in mannitol group were intravenous injected 20% mannitol after operation. 2.2 We observed the variance of permeability of BBB by the method of Evans blue fluorometry. Water content in brain tissue were also evaluated. 2.3 We observed and analyzed microvessel area density by the method of blood vessel histochemical stain and image analysis. We observed the structural and functional change of microvessels with light and eletron microscope. We also detected fibrinogen and D-dimer in order to observe the change of coagulation-fibrinolusis of the blood system. 2.4 Immunohistochemistry was used to observe the brain protein expression of MMP-2,MMP-9 and AQP4 in rats with TBI. 2.5 MRI was successivly used to observe the the change of signal intensity and volume of abnormal high signal region on T2WI after TBI. 3 Result 3.1 After TBI, the beam-walking ability of rats was seriously damaged and the weight of rats was lost obviously. 7 days after TBI, the beam-walking time of rats in TBI group was 16.46±3.27s, the rats’ weight was lost 17.75±5.09g. The beam-walking time of rats in Naoshuning group (10.24±1.38s) significantly decreased compared with TBI group (P<0.01); The change of rats’ weight (11.75±4.27g) was less compared with TBI group (P<0.05). Compared with TBI group, the beam-walking time of rats and the loss of rats’ weight in mannitol group had no significantly difference (P>0.05). 3.2 6 hours after operation, water content of brain tissue (78.76±0.49%) and EB content (10.41±1.50μg/g) in the contusion side significantly increased in TBI group compared with pseudoperarion group (77.20±0.45%, 3.27±1.05μg/g) (P<0.01). They reached summit (79.63±0.45%, 11.76±2.22μg/g) 24 hours after operation and did not come back normal level 5 days after operation. 6h, 24h, 3d, 5d after operation, water content of brain tissue was respectively 78.14±0.37%, 78.93±0.47%, 78.23±0.63%, 77.70±0.33% in Naoshuning group. There was significant difference between Naoshuning group and TBI group (P<0.05 ). In mannitol group, water content of brain tissue was respectively 78.01±0.82%, 78.80±0.57%, 78.26±0.82%, 77.81±0.48%. There was significant difference between mannitol group and TBI group 6h, 24h and 3d after operation (P<0.05). There was no significant difference between Naoshuning group and mannitol group (P>0.05); In Naoshuning group, EB content in contusion brain tissue was respectively 8.50±1.33μg/g, 7.97±1.08μg/g, 6.57±1.93μg/g, 5.31±1.19μg/g. There was signi更多还原