DF-521巴曲酶对大鼠脑出血后脑水肿区补体表达的影响

目的 :观察降纤酶制剂对大鼠脑出血后脑水肿形成及血肿周边脑组织补体C3d和C9表达的影响。方法 :一侧基底节区注射自体血 10 0 μL制作大鼠脑出血 2 4、72h模型 ,一组给予腹腔注射降纤酶制剂DF 5 2 1巴曲酶处理 ,一组不给 ,另设一组空白对照脑内注射生理盐水 10 0 μL。用干湿重法 ,测定大鼠脑组织的含水量 ;免疫组化法检测C3d的表达 ,免疫Westernblot方法研究脑组织补体C9表达情况。结果 :大鼠脑出血后 ,血肿侧脑组织含水量上升 ,同时出血侧脑组织内C3d和C9的表达量增加 ;给予DF 5 2 1巴曲酶制剂处理后 ,血肿侧脑水肿减轻〔2 4h由 (81 8± 1 14 ) %降至 (79 46± 0 68% ) ,P <0 0 1;72h由 (83 14± 0 3 6) %降至 (81 0 9±0 3 4) % ,P <0 0 1〕 ;并且脑组织C3d和C9的表达较对照组有所下降。结论 :降纤酶制剂能下调脑出血后血肿周边脑组织补体C3d和C9的表达并减轻大鼠脑出血后脑水肿的程度。     

Df—521巴曲酶对大鼠脑出血后脑水肿形成的影响

目的:观察Df-521巴曲酶对大鼠脑出血后脑水肿对血肿周边区脑组织ICAM-1表达的影响。方法:一侧基底节注射自体血制作大鼠脑出血模型,用干湿重法,测定脑组织含水量;用免疫组化法检测血肿周边水肿带ICAM-1的表达情况。结果:大鼠脑出血后,血肿侧脑组织含水量上升,同时水肿区ICAM-1的表达量增加;给予Df-521巴曲酶制剂处理后,血肿侧脑水肿减轻,脑组织ICAM-1的表达有所下降。结论:Df-521巴曲酶能下调脑出血后血肿周边组织ICAM-1的表达并减轻脑水肿的程度。     

MMP-2/9与脑出血后脑水肿的关系探讨

目的 探讨MMP-2／9与脑出血后脑水肿的关系。方法 采用胶原酶复制大鼠脑出血模型．通过免疫组化和原位杂交技术测定血肿周围脑组织中MMP-2／9的mRNA和蛋白表达，同时进行脑水含量和渗出脑血管外EB量的测定。结果 大鼠脑出血后血肿周围有明显的脑水肿发生和EB含量显著增加．均于24～48h达高峰。正常和假手术组可见极少量MMP-2蛋白和mRNA阳性染色细胞；而无MMP-9的表达。模型组出现大量MMP-2／9蛋白和mRNA阳性染色细胞；且MMP-9蛋白和mRNA表达在24～48h达高峰，与脑水含量和EB含量呈正相关。结论 大鼠脑血肿周围早期存在MMP-2／9的表达上调，其可能促进了出血后血管源性脑水肿的形成。     

实验性脑出血大鼠脑水肿的动态变化

目的研究大鼠脑出血后脑水肿的动态变化。方法应用立体定向技术,用大鼠自体股动脉血液100μl缓慢注入大鼠尾状核,制成脑出血模型,动态观察脑水肿的时空变化。脑水肿的测定应用干/湿重法。结果脑出血血肿周围组织含水量与对照组间差别有显著性意义,出血侧脑组织含水量与出血对侧脑组织含水量间差别有显著性意义,以出血后24-72h最明显, 至脑出血后第7d大致恢复正常。24h出血侧基底节脑组织含水量为(77．80±0．53)％,出血对侧为(77．53±0．43)％;72h出血侧基底节为(79.42±0．89)％,出血对侧为(77．64±0．34)％;假手术组手术侧基底节为(76．86±0．88)％,手术对侧为(76．89± 0．87)％。结论脑出血后脑水肿的变化是导致疾病恶化的主要原因之一。     

二硫代氨基甲酸吡咯烷对实验性脑出血血肿周围脑组织含水量及水通道蛋白-9表达的影响

目的探讨二硫代氨基甲酸吡咯烷(PDTC)对实验性脑出血血肿周围脑组织含水量和水通道蛋白9(AQP9)表达的影响。方法将128只SD大鼠随机分成正常组、对照组、脑出血组及PDTC组,建立脑出血模型;制模2 h予PDTC组腹腔注射PDTC;通过干湿法和SABC法检测各组大鼠各时点脑组织含水量、AQP9表达。结果(1)脑出血组脑组织含水量出血侧在制模后4 h开始升高,72 h达高峰,持续到120 h后下降;出血对侧在4 h也升高,48 h达高峰;脑组织含水量较对照组明显增加,出血侧更明显(均P<0.01)。(2)脑出血组AQP9表达制模后4 h开始升高,72 h表达最强,120 h降低,较对照组明显增加,出血侧较出血对侧增加更明显(均P<0.01)。(3)PDTC组脑组织含水量和AQP9表达在制模后24 h开始各时间点较脑出血组明显降低(均P<0.01)。结论脑出血后脑组织含水量和AQP9表达均呈平行增加,PDTC干预对出血后脑组织含水量和AQP9表达有平行抑制作用,提示AQP9参与了脑出血后脑水肿的形成。     

磁共振成像评价去铁酮、氯碘羟喹对大鼠脑出血后铁超载的干预效果

目的比较去铁酮、氯碘羟喹对大鼠脑出血模型铁含量及脑水肿的干预效果。方法将24只Wistar大鼠随机分为假手术组、脑出血(ICH)组、去铁酮组、氯碘羟喹组,大鼠右侧纹状体区注入胶原酶IV 0.4 U、肝素钠4 U制作脑出血模型。术后6 h开始灌胃干预组分别给予去铁酮[125 mg/(kg·次),1次/12 h]、氯碘羟喹[50 mg/(kg·次),1次/12 h],假手术组和脑出血组给予等量溶剂。并于术后1 d、3 d、7 d、14 d各时间点行神经功能学行为评分,通过ESWAN评估病灶铁含量、T2*WI评估血肿体积、T_2WI评估血肿周围水肿体积,14 d处死大鼠行脑组织铁染色。结果脑出血1 d后即出现神经功能缺损以1~3 d最重。14 d与假手术组无明显差别。与此相似,MRI显示血肿周围脑水肿体积以1~3 d最明显,7~14 d与假手术组无显著差别。MRI同时显示出血后脑铁含量增加,以7~14 d最明显,其后逐渐消退。氯碘羟喹可改善大鼠神经功能缺损,以第3~7 d最明显,可减少出血后3 d血肿周围水肿及3~7 d血肿周围脑组织铁含量,而去铁酮干预显著减轻脑出血后3~7 d铁含量,血肿周围水肿体积未见明显减少、神经功能缺损未见明显改善。结论 MRI显示脑出血后1~3 d血肿周围水肿体积、7~14 d脑铁含量较对照组显著增加,去铁酮、氯碘羟喹均可降低脑铁含量,氯碘羟喹可降低出血后3d血肿周围水肿体积,改善出血后3~7 d神经功能缺损。     

实验性脑出血后脑水肿的动态变化及其与MMP-9的关系

目的探讨脑出血后脑水肿和MMP-9表达的动态变化,并初步探讨其关系。方法健康雄性Wistar大鼠56只,将动物随机分成假手术组和脑出血组,大鼠尾壳核区注入自体非肝素抗凝动脉血建立脑出血模型,采用干湿重法测量脑组织水含量;免疫组织化学方法观察术后不同时间点MMP-9的动态变化。结果血肿形成后脑水肿产生迅速,从脑出血后3h开始增加,48~72h达高峰;脑出血后12h时,血肿周围组织中开始出现大量的深棕黄色阳性染色细胞,术后72h达高峰,以后逐渐下降,至120h时仍维持在较高水平。结论脑出血后脑水肿可能与MMP-9的活化有关。     

实验性脑出血急性期灶周组织水肿及炎性细胞因子的表达

目的 研究脑出血血肿周围脑组织炎性细胞因子的表达,探索其动态变化规律及与脑水肿的关系.方法 采用Wistar大鼠脑内缓慢注入自体血的方法建立实验性脑出血动物模型;随机分为6组,测定实验性脑出血6h、12h、24h、3d、5d、7d时间点血肿周围脑组织的含水量;应用免疫组化法及免疫印迹法(Western Blot)观察血肿周围脑组织中细胞因子IL-6、TNFα的表达.结果 脑出血后血肿周围脑组织的含水量逐渐增加,于3d达高峰;免疫组化染色显示血肿周围组织中神经细胞、血管内皮细胞均有IL-6及TNFa的阳性表达.免疫印迹半定量分析显示脑出血后6h血肿周围脑组织中即可见IL-6、TNFα较高水平的表达,24h达高峰,此后逐渐下降.结论脑出血急性期(6h)II-6及TNFα参与了血肿周围脑组织的损伤过程.IL-6变化曲线与脑水肿曲线呈现出一致性,推测其参与了血肿周围水肿的形成.     

一种大鼠脑内出血模型的建立与评价

目的建立稳定、制作过程创伤小的大鼠脑出血模型,研究其行为、脑组织水肿及组织结构的变化。方法取大鼠自体动脉血注入尾壳核不同时间对行为评分、测定脑含水量并观察脑组织病理变化结果出血后,。6h出现行为异常,2周行为异常好转脑含水量;24h增加(P<0.05),48～72h达高峰(P<0.01);1周血肿开始吸收,4周血肿大部分吸收镜下,血肿侧早期脑室受压、细胞肿胀晚期胶质增生结论此模型制作方便创伤小血肿形成稳定脑出血病理生理变化接近临床患者表现。     

实验性大鼠脑出血后TNF-α、ICAM-1的表达和脑水肿的研究

目的:探讨TNF-α、ICAM-1在大鼠脑出血后脑水肿中的表达及意义。方法:利用立体定向技术建立中等 量大鼠自体尾动脉血脑出血模型(50μl),于脑出血后6h、24h、48h、72h处死大鼠,进行脑含水量测定,并于脑出血后3h、 6h、12h、24h、48h、72h、7d处死大鼠,进行TNF-α、IGAM-1的免疫组化染色,并对结果进行统计处理。结果:大鼠脑出血 后脑水肿于48h达到高峰;大鼠脑组织表达TNF-α也于48h达到高峰,高于假手术组,分别为58．4±6．19和2±1．12,P <0．01;ICAM-1表达72h达高峰,与假手术组比较存在显著性差异,分别为7．8±0．84和0．8±0．84,P<0．01。结论: TNF-α、ICAM-1在大鼠脑出血后血肿周围表达的高峰与脑水肿高峰存在时间上的相关性,提示高表达的TNF-α、ICAM- 1可能参与了脑水肿形成。     

Activation of p44/42 mitogen activated protein kinases in thrombin-induced brain tolerance

BACKGROUND: Our recent studies have shown that prior intracerebral injection of a low dose of thrombin attenuates the brain edema formation that results from either an intracerebral hematoma, an intracerebral injection of a large dose of thrombin or cerebral ischemia. The aim of the current study is to investigate whether thrombin-induced tolerance (thrombin preconditioning; TPC) is associated with activation of p44/42 mitogen activated protein (MAP) kinases. METHODS: This study contained three parts. In the first, rats received an intracerebral infusion of either saline or one unit thrombin (the TPC dose) into the right caudate nucleus. After 1, 3 and 7 days, the rats will be killed and brains used to detect p44/42 MAP kinases activation using Western blot analysis and immunohistochemistry. In the second and third parts, rats received intracerebral infusions of either vehicle, one unit thrombin (TPC) or one unit thrombin and 5 nmol PD 098059. These rats were either killed to detect kinases activation after 24 h or received a second intracerebral infusion of five-unit thrombin 7 days later with brain edema being assessed after a further 24 h. RESULTS: Western blot analysis demonstrated that p44/42 MAP kinases were activated in the ipsilateral basal ganglia after the intracerebral infusion of thrombin one unit. Cells immunoreactive for activated p44/42 MAP kinases were found in the ipsilateral basal ganglia and ipsilateral cortex. PD 098059, a MAP kinase kinase inhibitor, abolished thrombin-induced activation of p44/42 MAP kinases. TPC suppressed thrombin-induced brain edema while PD 098059 blocked this protective effect. The water contents in the ipsilateral basal ganglia 24 h after infusion of thrombin five units were 82.6+/-0.8%, 79.2+/-0.4% and 81.8+/-1.9% in the control, TPC alone and TPC plus PD 098059 groups, respectively. CONCLUSION: Thrombin can activate p44/42 MAP kinases within the brain and the protective effects of thrombin preconditioning on brain edema formation are related to this activation.     

补体在脑出血后脑组织损伤机制中的作用

目的研究补体C9在大鼠实验性脑出血(ICH)后血肿周围组织中的表达情况,探讨补体C9在ICH后脑水肿中的作用以及应用眼镜蛇毒因子(CVF)干预后对血肿周围组织C9表达及脑组织含水量变化的影响。方法采用立体定向技术,将自体不凝血注入大鼠尾状核制备ICH模型,将动物分为假手术组、出血组和CVF干预组,分别在不同时间断头取脑,连续切片分别作补体C9免疫组化染色和HE染色,并进行脑组织含水量测定(干湿重法)。结果ICH后2h血肿周围脑组织开始表达C9,24h达高峰。血肿周围脑组织含水量在ICH后2h开始增加(P<0.05),6h明显增加,24～72h达高峰(P<0.01),此后逐渐回落,1周基本恢复正常水平,脑组织含水量与C9的表达呈正相关关系(r=0.938,P<0.01);对侧半球相应部位及假手术对照组脑组织含水量没有明显变化;经CVF干预后,血肿周围组织C9表达明显下降,干预组与出血组之间比较有显著差异(P<0.01)。CVF干预组脑组织含水量明显低于常规ICH组(P<0.01)。结论脑出血后补体级联激活C9表达明显增加,并证明通过CVF干预后,C9表达下降,脑水肿减轻,能达到神经保护作用。     

The effects of thrombin preconditioning on focal cerebral ischemia in rats

Our previous studies have shown that prior intracerebral infusion of a low dose of thrombin (thrombin preconditioning; TPC) reduces the brain edema that follows a subsequent intracerebral infusion of a high dose of thrombin or an intracerebral hemorrhage. In vitro studies have also demonstrated that low concentrations of thrombin protect neurons and astrocytes from hypoglycemia and oxidative stress-induced damage. This study, therefore, examines the hypothesis that TPC would offer protection from ischemic brain damage in vivo. This was a blinded design study. The rat brain was preconditioned with 1 U thrombin by direct infusion into the left caudate nucleus. Seven days after thrombin pretreatment, permanent middle cerebral artery occlusion (MCAO) was induced. Twenty-four hours post-ischemia, neurological deficit was evaluated and infarction volume, brain water and ion contents were measured. Compared to saline-treated rats, thrombin pretreatment significantly attenuated brain infarction in cortex (90+/-33 vs. 273+/-22 mm(3); P<0.05) and basal ganglia (56+/-17 vs. 119+/-12 mm(3); P<0.05) that followed 24 h of permanent MCAO. TPC also reduced the brain edema in cortex and basal ganglia by 50 and 53% (P<0.05). Neurological deficit was improved in thrombin pretreatment group (P<0.05). These effects of TPC were, in part, prevented by co-injection of hirudin, a thrombin inhibitor, indicating that the protection was indeed thrombin mediated. Cerebral TPC significantly reduces ischemic brain damage, perhaps by activation of the thrombin receptor. This finding provides a new mechanism by which to study ischemic tolerance.     

强啡肽A1—13对大鼠脑出血后脑水肿的作用

在建立稳定的大臣脑出血模型的基础上，用放免法测定不同脑区和垂体中DynA1-13含量，干湿重法测定含水量，旨在观察DynA1-13对脑出血后脑组织合水量的影响。结果发现：大鼠尾核注射胶原酶，可造成稳定的脑实质出血；注射胶原酶后4，24，48h脑组织含水量均明显升高（P＜0．01）；在脑出血后4，24，48h，Dy－nA1-13含量，在皮质、海马与尾壳核均明显降低（P＜0．05，P＜0．01），而在下丘脑及垂体则明显升高（P＜0．05，P＜0．01）；DynA1-13可减轻胶原酶诱导的脑出血后脑水肿（P＜0．01），并呈剂量依赖效应。本研究提示：脑组织DynA1-13含量降低，可能是造成脑出血后脑水肿的一个重要原因，DynA1-13对脑出血后脑水肿具有潜在的治疗价值。     

Inducible cyclooxygenase-2 expression after experimental intracerebral hemorrhage

Cyclooxygenase-2 (COX-2) is an inducible isoform of cyclooxygenase, which catalyzes the conversion of arachidonic acid to prostaglandins and thromboxane. Recent evidence suggests it has a pathological role in cerebral insults, but its involvement in intracerebral hemorrhage (ICH) is unknown. The present study investigates the temporal and anatomic distribution of COX-2 as well as the effect of the selective COX-2 inhibitor NS-398 on brain edema formation and cerebral blood flow in a rat model of ICH. Immunohistochemistry for COX-2 was performed in control rats and 6 h, as well as 1, 3, 7 and 10 days after the injection of 100 microl autologous blood into the right basal ganglia. Double-labeling immunohistochemistry was used to determine the type of COX-2 immunoreactive microvascular-associated cells. Western blot analysis was used to quantify COX-2 protein. The effect of NS-398 on brain water content, ion concentration and cerebral blood flow were assessed 24 h after ICH. The results demonstrated that COX-2 protein was expressed in control brain tissue and induced significantly in the ipsilateral hemisphere at 6 h, as well as 1 and 3 days after ICH. Increased staining of COX-2 in neurons was observed around the blood clot with a peak at 6 h. COX-2 was induced in endothelial cells, perivascular cells as well as infiltrating leukocytes 1 day after ICH. Brain water and ion contents and cerebral blood flow were unaffected by NS-398 administration. Thus, although COX-2 expression was increased in the ipsilateral hemisphere after an autologous blood injection, its products do not appear to be major regulators of blood flow or edema formation following ICH.     

Mild hypothermia effects on matrix metalloproteinase-9 expression in the perihematomal region of rats following experimental intracerebral hemorrhage**☆

BACKGROUND： Matrix metalloproteinase-9 （MMP-9） expression increases with intracerebral hemorrhage, and participates in the pathophysiological processes of secondary brain injury after intracerebral hemorrhage.
OBJECTIVE： To investigate the effects of mild hypothermia on MMP-9 expression and brain edema in the perihematomal region of experimental intracerebral hemorrhage rats.
DESIGN, TIME AND SETTING： The randomized, controlled experiment was performed at the Central Laboratory of Shandong Provincial Hospital between May and September 2007.
MATERIALS： Seventy-two, Wistar, male rats, 12-weeks old, were used for this study. Rabbit anti-MMP-9 primary antibody was purchased from Boster, China.
METHODS： Wistar rats were equally and randomly divided into normothermia and mild hypothermia groups. The two groups each comprised control, 6-hour intracerebral hemorrhage, 24-hour intracerebral hemorrhage, 48-hour intracerebral hemorrhage, 72-hour intracerebral hemorrhage, and l-week intracerebral hemorrhage subgroups, with six rats in each subgroup. Rat models of intracerebral hemorrhage were established by injecting 100 μL of autologous blood into the rat caudate nucleus. Rats in the mild hypothermia group received four hours of local mild hypothermia immediately following the injection. lntracerebral temperature was maintained at （33 ± 0.5） ℃. Subsequently, intracerebral temperature was spontaneously recovered at 25 ℃. Rats in the control subgroup were not injected with autologous blood and received only with intracerebral hemorrhage.
MAIN OUTCOME MEASURES： Brain water content and MMP-9 expression surrounding the hematoma region. RESULTS： MMP-9 expression increased at 6 hours, and brain edema reached a peak at 48 hours after intracerebral hemorrhage. MMP-9 expression was significantly decreased in the mild hypothermia group compared with the normothermia group at each time point （P 〈 0.05）.
CONCLUSION： Mild hypothermia can significantly inhibit MMP-9 overexpression and reliev     

Mild hypothermia effects on matrix metalloproteinase-9 expression in the perihematomal region of rats following experimental intracerebral hemorrhage

BACKGROUND:Matrix metalloproteinase-9(MMP-9)expression increases with intracerebral hemorrhage,and participates in the pathophysiological processes of secondary brain injury after intracerebral hemorrhage.OBJECTIVE:To investigate the effects of mild hypothermia on MMP-9 expression and brain edema in the perihematomal region of experimental intracerebral hemorrhage rats.DESIGN,TIME AND SETTING:The randomized,controlled experiment was performed at the Central Laboratory of Shandong Provincial Hospital between May and September 2007.MATERIALS:Seventy-two,Wistar,male rats,12-weeks old,were used for this study.Rabbit anti-MMP-9 primary antibody was purchased from Boster,China.METHODS:Wistar rats were equally and randomly divided into normothermia and mild hypothermia groups.The two groups each comprised control,6-hour intracerebral hemorrhage,24-hour intracerebral hemorrhage,48-hour intracerebral hemorrhage,72-hour intracerebral hemorrhage,and 1-week intracerebral hemorrhage subgroups,with six rats in each subgroup.Rat models of intracerebral hemorrhage were established by irtjecting 100 μL of autologous blood into the rat caudate nucleus.Rats in the mild hypothermia group received four hours of local mild hypothermia immediately following the injection.Intracerebral temperature was maintained at(33±0.5)℃.Subsequently,intracerebral temperature was spontaneously recovered at 25℃.Rats in the control subgroup were not injected with autologous blood and received only with intracerebral hemorrhage.MAIN OUTCOME MEASURES:Brain water content and MMP-9 expression surrounding the hematoma region.RESULTS:MMP-9 expression increased at 6 hours,and brain edema reached a peak at 48 hours after intracerebral hemorrhage.MMP-9 expression was significantly decreased in the mild hypothermia group compared with the normothermia group at each time point (P<0.05).CONCLUSION:Mild hypothermia can significantly inhibit MMP-9 overexpression and relieve brain edema following intracerebral hemorrhage.     

大鼠实验性脑出血后脑组织中c-fos基因的表达与局部脑血流的改变

目的 探讨大鼠实验性脑出血后脑组织中即刻早期基因c-fos的表达和局部脑血流的变化。方法 采用Nath改良法建立大鼠脑出血模型；免疫组化法及RT－PCR法测定其脑组织中fos蛋白和c-fos mRNA的表达；氢清除法测定其局部脑血流。结果 大鼠血肿周围区（基底节）在脑出血后1小时即出现fos蛋白的表达，至3小时达高峰；c-fos mRNA于出血后1小时达表达高峰,至3小时后仍有较高水平的表达；出血后1小时全脑的的血流量均下降，4小时恢复至对照组水平，并维持至出血后24小时，随着的24小时内再次出现脑血流下降。结论 大鼠脑出血后，血肿周围区和双侧皮质区的脑组织中存在着c-fos基因的快速而长久的诱导表达。局部脑血流的下降相对短暂，且脑血流的下降在时程上与c-fos基因的表达不相一致。     

Effects of hypothermia on thrombin-induced brain edema formation

Recent studies have shown that thrombin plays an important role in brain edema formation after intracerebral hemorrhage (ICH). The possible mechanisms of thrombin-induced brain edema formation include blood-brain barrier (BBB) disruption and inflammatory response involving polymorphonuclear (PMN) leukocyte. Animal experiments have revealed that moderate therapeutic hypothermia improves pathological and functional outcome in various models of brain injury. In this study, we examined the effect of hypothermia on thrombin-induced brain edema formation. Effects of hypothermia on BBB permeability and the accumulation of PMN leukocytes were also determined to clarify the protective mechanism of hypothermia in this model. Anesthetized adult rats received an injection of 10 Units of thrombin into the basal ganglia. Animals were separated into the normothermic and hypothermic groups, which were housed in a room maintained at 25 degrees C and in a cold room maintained at 5 degrees C, respectively, for 24 h after the thrombin injection. The brain temperature in rats housed in a cold room reduced temporarily to approximately 30 degrees C and then gradually recovered to 35 degrees C by the end of the observation. Brain water content in the basal ganglia was significantly reduced in rats treated with hypothermia compared to the normothermic rats (84.3+/-0.2 vs. 82.4+/-0.1%; P<0.01). The decrease of brain water content was accompanied with a significant reduction in BBB permeability to Evan's blue dye and in accumulation of PMN leukocytes. This study indicates that hypothermic treatment significantly reduces thrombin-induced brain edema formation in the rat. Inhibition of thrombin-induced BBB breakdown and inflammatory response by hypothermia appear to contribute to brain protection in this model. Hypothermic treatment may provide an approach to potentially reduce ongoing edema after ICH.