实验性脑出血急性期凝血酶致病机制的探讨

目的通过观察凝血酶特异性抑制剂水蛭素对脑出血后血肿周围组织微管相关蛋白2(MAP2)表达及脑组织含水量变化的影响,探讨凝血酶在脑出血后继发性损伤中的致病机制。方法采用自体未抗凝动脉血注入法,制作实验性脑出血动物模型。将大鼠随机分为正常对照组、单纯脑出血组、水蛭素组。采用干湿比重法定量测定脑组织含水量;免疫组化染色观察脑出血后MAP2的变化。结果脑出血后6h开始MAP2表达逐渐减弱(P<0.05),3d达到最低(P<0.05)。脑出血后脑组织含水量逐渐增加,从1d开始增加显著(P<0.05),3d达高峰(P<0.05)。水蛭素组,均较相应时间点单纯出血组脑组织含水量降低(P<0.05);MAP2阳性细胞数目增多(P<0.05),变形程度减轻,染色加深。直线回归分析表明,脑出血后1周内MAP2的变化与脑组织含水量变化呈负相关关系。结论脑出血急性期凝血酶可能通过MAP2的破坏发挥其致病作用;血肿局部应用水蛭素具有保护作用。     

局部亚低温对脑出血后水肿影响的实验研究

目的探讨局部亚低温对大鼠脑出血后水肿形成的影响及其可能机制。方法雄性Wistar大鼠230只随机分为:对照组;脑出血组;脑出血加局部亚低温组;凝血酶加局部亚低温组。应用Evans-Blue测定血脑屏障(BBB)通透性,应用干湿重法测定脑水含量。结果与对照组相比,大鼠注血后6h开始出现脑组织水含量和BBB通透性的增加,在72h达到高峰,然后逐渐消退。不同时程局部亚低温均可以显著降低脑出血后72h时脑组织水含量及BBB通透性(P<0.01),其中给以4h局部亚低温时,降低最明显。注射凝血酶6h后,脑组织水含量及BBB通透性显著增高(P<0.01),于24~48h达高峰,然后逐渐下降。凝血酶 局部亚低温组在各个时间点与凝血酶组相比,脑组织水含量及BBB通透性明显降低(P<0.01)。结论局部亚低温可能是通过抑制凝血酶的毒性作用来减轻脑出血后水肿的形成及血脑屏障的破坏。     

NMDA受体NR2A亚单位在凝血酶诱导的大鼠脑出血后脑损伤作用机制的研究

目的探讨N-甲基-D天冬氨酸受体(NMDA)受体NR2A亚单位在凝血酶诱导的大鼠脑出血后脑损伤中的作用机制。方法将健康雄性SD大鼠随机分为生理盐水对照组、凝血酶组、凝血酶+阿加曲班组,建立大鼠脑出血模型。造模后48h,采用伊文思蓝法检测血脑屏障(BBB)的通透性,干-湿重法测脑组织的含水量。采用Western-blot方法观察出血后各时间点(0h、0.5h、6h、24h、72h、120h)血肿周围脑组织NR2A分布及动态变化规律。结果 (1)阿加曲班可以明显改善大鼠脑出血BBB通透性(P<0.05),血肿周围脑水肿明显减轻(P<0.05)。(2)Western-blot结果显示,NR2A在6h时开始增加,72h达到高峰,在120h时基本恢复。阿加曲班可以明显减少NR2A在72h时的表达(P<0.05)。结论 (1)NR2A参与了凝血酶诱导的脑出血后脑损伤的病理生理过程。(2)凝血酶可能通过上调NR2A表达引起脑出血后脑组织的损伤。     

大鼠脑出血后血肿周围凝血酶与脑细胞凋亡和脑水肿的关系

目的 研究大鼠脑出血后血肿周围脑组织中凝血酶的变化及其与脑细胞凋亡和脑水肿的关系.方法 105只成年雄性SD大鼠随机均分为脑出血组、生理盐水组和正常对照组.每组再随机均分为7个时相,分别为6 h、12 h、24 h、48 h、3 d、5 d、7 d;用立体定向脑内注射法制备脑出血模型;用ELISA法检测脑组织中凝血酶-抗凝血酶复合物的含量;用流式细胞仪检测脑细胞的凋亡率;用干-湿重法检测脑组织的含水量;用透射电镜观察脑组织的形态学变化.结果 制备动物模型后,脑出血组各时相血肿周围脑组织中凝血酶含量、脑细胞凋亡率、脑组织含水量均明显高于生理盐水组和正常对照组;血肿周围脑组织中凝血酶-抗凝血酶复合物的含量与脑细胞凋亡率和脑组织含水量之间均呈正相关;电镜下,在脑出血组各时相脑组织切片中均可见大量的脑细胞凋亡和毛细血管周围大片组织间隙水肿,以及神经纤维脱髓鞘改变.结论 脑出血后,血肿周围脑组织中凝血酶含量明显升高,可导致脑细胞凋亡和脑水肿,凝血酶的含量与脑细胞凋亡率和脑组织含水量呈正相关;尽早清除血肿有望改善凝血酶引起的继发性脑损伤.     

大鼠脑出血后血肿周围凝血酶与脑细胞凋亡和脑水肿的关系

目的 研究大鼠脑出血后血肿周围脑组织中凝血酶的变化及其与脑细胞凋亡和脑水肿的关系.方法 105只成年雄性SD大鼠随机均分为脑出血组、生理盐水组和正常对照组.每组再随机均分为7个时相,分别为6 h、12 h、24 h、48 h、3 d、5 d、7 d;用立体定向脑内注射法制备脑出血模型;用ELISA法检测脑组织中凝血酶-抗凝血酶复合物的含量;用流式细胞仪检测脑细胞的凋亡率;用干-湿重法检测脑组织的含水量;用透射电镜观察脑组织的形态学变化.结果 制备动物模型后,脑出血组各时相血肿周围脑组织中凝血酶含量、脑细胞凋亡率、脑组织含水量均明显高于生理盐水组和正常对照组;血肿周围脑组织中凝血酶-抗凝血酶复合物的含量与脑细胞凋亡率和脑组织含水量之间均呈正相关;电镜下,在脑出血组各时相脑组织切片中均可见大量的脑细胞凋亡和毛细血管周围大片组织间隙水肿,以及神经纤维脱髓鞘改变.结论 脑出血后,血肿周围脑组织中凝血酶含量明显升高,可导致脑细胞凋亡和脑水肿,凝血酶的含量与脑细胞凋亡率和脑组织含水量呈正相关;尽早清除血肿有望改善凝血酶引起的继发性脑损伤.     

人脑出血后血肿周围缺血半暗带中凝血酶与脑水肿的关系

目的研究人脑出血后,血肿周围缺血半暗带脑组织中凝血酶与脑水肿的关系及二者的变化规律。方法以脑出血患者术中需要切除的缺血半暗带脑组织为实验组,远离血肿需要内减压切除的脑组织为对照组。用ELISA法检测脑组织凝血酶含量;干-湿重法测脑组织含水量;在电镜下,观察脑组织显微结构。比较两组脑组织含水量和凝血酶含量的差异,并分析不同时间窗入院患者这两个指标的变化规律。结果实验组脑组织中凝血酶的含量明显高于对照组。实验组脑组织含水量明显高于对照组。电镜下,实验组脑水肿明显比对照组严重。结论脑出血后,血肿周围缺血半暗带中凝血酶含量明显增高,凝血酶与脑水肿密切相关。     

大鼠脑出血后血肿周围凝血酶与脑细胞凋亡和脑水肿的关系

目的 研究大鼠脑出血后血肿周围脑组织中凝血酶的变化及其与脑细胞凋亡和脑水肿的关系.方法 105只成年雄性SD大鼠随机均分为脑出血组、生理盐水组和正常对照组.每组再随机均分为7个时相,分别为6 h、12 h、24 h、48 h、3 d、5 d、7 d;用立体定向脑内注射法制备脑出血模型;用ELISA法检测脑组织中凝血酶-抗凝血酶复合物的含量;用流式细胞仪检测脑细胞的凋亡率;用干-湿重法检测脑组织的含水量;用透射电镜观察脑组织的形态学变化.结果 制备动物模型后,脑出血组各时相血肿周围脑组织中凝血酶含量、脑细胞凋亡率、脑组织含水量均明显高于生理盐水组和正常对照组;血肿周围脑组织中凝血酶-抗凝血酶复合物的含量与脑细胞凋亡率和脑组织含水量之间均呈正相关;电镜下,在脑出血组各时相脑组织切片中均可见大量的脑细胞凋亡和毛细血管周围大片组织间隙水肿,以及神经纤维脱髓鞘改变.结论 脑出血后,血肿周围脑组织中凝血酶含量明显升高,可导致脑细胞凋亡和脑水肿,凝血酶的含量与脑细胞凋亡率和脑组织含水量呈正相关;尽早清除血肿有望改善凝血酶引起的继发性脑损伤.     

凝血酶在自发性脑出血后脑水肿形成中的作用

脑出血后脑水肿形成是导致继发性神经损害的一个重要因素。近年来，有关脑出血后脑水肿产生机制的研究表明，凝血时释放的凝血酶可能是引起脑出血后脑水肿的重要物质之一，凝血酶在脑出血后脑水肿形成、炎症反应等方面起着重要作用。本文对近年来凝血酶在自发性脑出血后脑水肿形成中的作用研究进展作一综述。     

凝血酶对大鼠脑内MMP-9、MMP-2表达的影响

目的探讨凝血酶对大鼠脑内MMP-9、MMP-2表达的影响。方法Wistar大鼠随机分为假手术组及实验组。实验组脑内注入凝血酶,在不同时间点采用干湿重法测脑水含量,免疫组化方法检测脑内MMP-9、MMP-2的表达。假手术组注入等量生理盐水。结果脑组织水含量在凝血酶注入后6h开始增加,3d达高峰。MMP-9阳性细胞在注射凝血酶后6h即开始表达增加,与对照组相比差异显著(P<0.01),3d达高峰,随后持续下降。阳性微血管数也有相似的变化趋势。MMP-2阳性细胞在注射凝血酶后1d才开始有少量表达,后持续增多,5d达高峰,随后有所下降,但14d仍有明显表达,与对照组相比差异显著(P<0.01)。阳性微血管数也有相似的变化趋势。结论凝血酶在脑出血后脑水肿及脑组织损伤中起了关键的作用,MMP-9、MMP-2参与了急性期脑水肿、炎症反应等过程,MMP-2在损伤修复中可能有重要作用。     

凝血酶对脑组织毒性损伤机制及水蛭素、尼莫地平干预作用的研究

目的 探讨凝血酶对脑组织损害的机制,及凝血酶抑制剂水蛭素和尼莫地平对其的影响。方法 将不同剂量的凝血酶或／和水蛭素注入SD大鼠尾状核,尼莫地平腹腔注射。采用干湿重法、免疫组化法、原位末端缺口标记法及镜下观察在不同时点大鼠脑含水量、细胞骨架蛋白、神经元凋亡数及组织学改变。结果 （1）大剂量凝血酶组出现以下改变：早期（4h）明显味水肿,其高峰时间为24～48h,3d后水肿逐渐消退,7d趋于正常;细胞骨架蛋门出现不同程度变形甚至崩解,24h损伤进一步加重,导致不可逆损伤,3～7d神经细胞坏死;神经细胞凋亡,其高峰时间为24～48h,持续到7d;小剂量凝血酶组及生理盐水组无以上作用。（2）水蛭素能特异性抑制凝血酶的作用,而钙离子拈抗剂可减轻或延迟细胞的损伤。结论 大剂量凝血酶可导致脑水肿、脑细胞不同逆损伤及脑细胞凋亡,其高峰时间均在24～48h。早期干预可改善脑出血（ICH）后血肿周围脑组织水肿和继发性神经几损伤,改善ICH的预后。     

Plasminogen potentiates thrombin cytotoxicity and contributes to pathology of intracerebral hemorrhage in rats.

Thrombin and plasmin are serine proteases involved in blood coagulation and fibrinolysis, whose precursors are circulating in blood stream. These blood-derived proteases might play important roles in the pathogenesis of intracerebral hemorrhage by acting on brain parenchymal cells. We previously reported that thrombin induced delayed neuronal injury through extracellular signal-regulated kinase (ERK)-dependent pathways. Here, we investigated potential cytotoxic actions of plasminogen, a precursor protein of plasmin, using slice cultures prepared from neonatal rat brain and intracortical microinjection model in adult rats. Although plasminogen alone did not evoke prominent neuronal injury, plasminogen caused significant neuronal injury when combined with a moderate concentration of thrombin (30 U/mL) in the cerebral cortex of slice cultures. The cortical injury was prevented by tranexamic acid and aprotinin. The combined neurotoxicity of thrombin and plasminogen was also prevented by PD98059, an inhibitor of ERK pathway, as well as by other agents that have been shown to prevent cortical injury induced by a higher concentration (100 U/mL) of thrombin alone. Extracellular signal-regulated kinase phosphorylation after plasminogen exposure was localized in cortical astrocytes. Moreover, microinjection of plasminogen in vivo potentiated thrombin-induced cortical injury, and inhibition of plasmin ameliorated hemorrhage-induced neuronal loss in the cerebral cortex. These results suggest that plasminogen/plasmin system augmenting thrombin neurotoxicity participates in hemorrhagic cortical injury.     

经腹腔应用阿加曲班对脑出血后凝血酶神经毒性的抑制作用

目的:探讨经腹腔应用阿加曲班治疗脑出血（ICH）后凝血酶神经毒性损伤的可能性。方法:①研究阿加曲班对ICH及凝血酶注入后脑水肿、细胞损伤的影响。Wistar大鼠60只,随机分为假手术对照组（只进针不注血）;ICH组（50μL自体尾血注入右尾状核）;ICH+阿加曲班干预组(50μL自体尾血注入大鼠右侧尾状核,分别于术后3和12h经腹腔给予阿加曲班0.9mg·kg^-1,总量0.6mL);凝血酶组(10U·2μL^-1凝血酶注入大鼠右侧尾状核);凝血酶+阿加曲班干预组(10U·2μL^-1凝血酶注入大鼠右侧尾状核,分别于术后3和12h经腹腔给予阿加曲班0.9mg·kg^-1,总量为0.6mL)。各组均n=12,术后24h处死各组大鼠,每组中6只用于检测脑组织水含量,6只检测caspase-3免疫反应细胞及TUNEL阳性细胞数。②经腹腔注射阿加曲班对血肿容积的影响:建立胶原酶ICH大鼠模型组:注入Ⅰ型胶原酶+肝素;阿加曲班组:胶原酶ICH模型成功后3及12h分别经腹腔注入阿加曲班溶液0.9mg·kg^-1,每次注入液体量为0.6mL,测定两组大鼠脑组织血肿血红蛋白的含量(测定血红蛋白A₅₅₀值)以评价阿加曲班对血肿容量的影响。结果:自体血ICH及凝血酶模型大鼠在阿加曲班干预后,ICH组及凝血酶组的TUNEL阳性细胞数、caspase-3阳性细胞数明显下降（P<0.01或P<0.05）,脑组织水肿含量百分比明显降低（P<0.05）。胶原酶ICH血红蛋白A值为（0.45±0.12）,阿加曲班干预组为（0.46±0.09）,差异无统计学意义（P>0.05）。结论:ICH后3～12h经腹腔注入阿加曲班可减轻ICH后的脑水肿及细胞凋亡性损伤,且没有血肿增大的不良反应。     

高血压脑出血病理生理机制研究进展

高血压脑出血具有发病率高、致残率高、死亡率高的特点。其治疗近年来虽无突破性进展,但对血肿扩大、脑水肿等方面的研究较多。现就脑出血后脑组织的损伤机制、血肿扩大原因及其毒性作用以及出血半暗带、局部脑血流改变、炎症反应等在脑水肿形成中的作用等方面对脑出血的病理生理学机制研究的新进展作一综述。     

动脉瘤早期手术夹闭后术区继发性脑内血肿的分析

目的 探讨早期手术成功夹闭动脉瘤之后,术区形成继发性脑内血肿的原因.方法 134例颅内动脉瘤开颅夹闭的患者中5例(3.7%)形成术后继发脑内血肿需二次开颅处理,分析术中(包括减压后)额叶挫裂伤程度、创面渗血和术中止血情况、脑压板对脑组织造成的损伤程度.1例难以止血者先留取部分脑挫裂伤组织提前送病理检查;4例第二次开颅清除血肿同时留取出血脑组织送病理检查,综合了解出血的原因.结果 5例手术中均发现蛛网膜下腔出血,脑肿胀明显,脑组织外观呈"紫红色";术中均有不同程度脑压板造成的损伤;病理主要表现为局部出血.出血周围可见"异常血管集中区",主要为细小静脉及毛细血管,血管呈弥漫扩张,部分血栓形成,一些血管壁明显厚薄不一,壁内见有少量中性白细胞浸润.结论 这种脑内血肿的发生与动脉瘤出血所致的早期脑肿胀、患者本身存在脑组织内小血管畸形及术中脑压板牵拉伤有关,术后严密观察能够早期发现,及时处理,效果良好.     

高血压脑出血患者血肿周围水肿组织中水通道蛋白-4表达及意义

目的研究高血压脑出血患者血肿周围水肿组织中水通道蛋白-4(aquaporin 4,AQP4)表达及其意义。方法收集2009～2010年高血压脑出血患者血肿周围水肿组织28例,按照脑出血时间分为四组,如<12h,≥12h且<24h,≥24h且<48h和≥48h且<72h,以脑外伤患者术中急性脑膨出行内减压而切除的正常脑组织作为对照,应用Western-Blot方法,以GAPDH为内参,分别检测血肿周围水肿组织及正常脑组织中AQP4的表达水平。结果脑出<12h,≥12h且<24h,≥24h且<48h和≥48h且<72h患者脑内血肿周围水肿组织AQP-4的表达水平明显高于对照组(P<0.05),72h内AQP-4的表达逐渐升高。结论高血压脑出血患者脑内血肿周围水肿组织AQP4的表达增多与脑水肿的形成和发展密切相关。     

Dabigatran abrogates brain endothelial cell permeability in response to thrombin

Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell β1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.     

Plasminogen activator inhibitor-1 induction after experimental intracerebral hemorrhage.

Serine proteases, such as thrombin and tissue-type plasminogen activator, play an important role in brain injury after intracerebral hemorrhage and other neurologic disorders. Plasminogen activator inhibitor-1 is one of the serine protease inhibitors, or serpins. The balance between serine proteases and serpins may affect the outcome of intracerebral hemorrhage. The purpose of this study was to determine whether plasminogen activator inhibitor-1 and tissue-type plasminogen activator are upregulated after intracerebral hemorrhage and the role that thrombin plays in that induction. Plasminogen activator inhibitor-1 protein levels were upregulated after intracerebral hemorrhage. Brain plasminogen activator inhibitor-1 content also increased after thrombin infusion in a dose-dependent manner. Hirudin, a specific thrombin inhibitor, blocked the upregulation of plasminogen activator inhibitor-1 after intracerebral hemorrhage. Time courses showed that plasminogen activator inhibitor-1 levels around the hematoma peaked at the first day. Plasminogen activator inhibitor-1-positive cells were detected in the perihematomal area and the ipsilateral basal ganglia after thrombin infusion, but not in the contralateral hemisphere. Plasminogen activator inhibitor-1 messenger RNA levels were increased at 24 hours after intracerebral hemorrhage and after thrombin infusion. However, tissue-type plasminogen activator protein levels were the same in the control, whole-blood, and thrombin-infusion groups. In conclusion, intracerebral hemorrhage and thrombin infusion stimulate plasminogen activator inhibitor-1 but not tissue-type plasminogen activator production in the brain. The upregulation of plasminogen activator inhibitor-1 may be neuroprotective by limiting thrombin or other serine protease-induced toxicity.     

Reduction of tissue plasminogen activator-induced hemorrhage and brain injury by free radical spin trapping after embolic focal cerebral ischemia in rats.

Thrombolytic stroke therapy with tissue plasminogen activator (tPA) remains complicated by serious risks of cerebral hemorrhage and brain injury. In this study, a novel model of tPA-induced hemorrhage was used in spontaneously hypertensive rats to examine the correlates of hemorrhage, and test methods of reducing hemorrhage and brain injury. Homologous blood clot emboli were used to occlude the middle cerebral artery in spontaneously hypertensive rats, and delayed administration of tPA (6 hours postischemia) resulted in high rates of cerebral hemorrhage 24 hours later. Compared with untreated rats, tPA significantly increased hemorrhage volumes by almost 85%. Concomitantly, infarction and neurological deficits were worsened by tPA. A parallel experiment in normotensive Wistar-Kyoto rats showed markedly reduced rates of hemorrhage, and tPA did not significantly increase hemorrhage volumes. To examine whether tPA-induced hemorrhage was caused by the delayed onset of reperfusion per se, another group of spontaneously hypertensive rats was subjected to focal ischemia using a mechanical method of arterial occlusion. Delayed (6 hours) reperfusion via mechanical means did not induce hemorrhage. However, administration of tPA plus delayed mechanical reperfusion significantly increased hemorrhage volumes. Since reperfusion injury was implicated, a final experiment compared outcomes in spontaneously hypertensive rats treated with tPA plus the free radical spin trap alpha-phenyl tert butyl nitrone (alpha-PBN) versus tPA alone. tPA-induced hemorrhage volumes were reduced by 40% with alpha-PBN, and infarction and neurological deficits were also decreased. These results indicate that (1) blood pressure is an important correlate of tPA-induced hemorrhage, (2) tPA interacts negatively with reperfusion injury to promote hemorrhage, and (3) combination therapies with anti-free radical treatments may reduce the severity of tPA-induced hemorrhage and brain injury after cerebral ischemia.