人脑挫裂伤早期周围组织AQP4表达及血脑屏障超微结构观察

目的观察人脑挫裂伤后AQP4和血脑屏障超微结构在脑水肿形成中不同时间点的变化特征,探讨脑水肿的形成机制。方法取脑挫裂伤区组织标本60例(观察组),10例非功能区正常脑组织标本(对照组)。采用免疫组化和图像分析技术测定正常组及观察组伤后2～72 h相应时间点水肿区AQP4的表达水平,同时观察脑水肿含水量,血脑屏障指数,血脑屏障超微结构的变化。结果与正常组相比较,脑挫裂伤组在伤后2 h后AQP4表达开始增加(P<0.05),6 h、8 h、12 h明显增加(P<0.01),24～72 h达到最高(P<0.01)。AQP4表达与脑含水量的变化趋于一致(r=0.912,P<0.01);血脑屏障(BBB)指数与脑含水量的变化趋于一致(r=0.877,P<0.01);水通道蛋白4表达与BBB指数呈显著正相关(r=0.908,P<0.01)。伤后早期血脑屏障结构即发生改变,随后血脑屏障结构被明显破坏,24 h、72 h血脑屏障破坏最为严重。结论脑挫裂伤后AQP4表达明显增强,BBB的通透性增加,提示AQP4在损伤后脑水肿的形成过程中起重要作用。     

大鼠重型颅脑损伤急性期水通道蛋白4的表达

目的探讨水通道蛋白（AQP4）在大鼠重型脑外伤急性期的表达变化及其与脑水肿间的关系。方法49只成年雄性SD大鼠,随机分为对照组及实验组（伤后4h、8h、12h、24h、5d共5组）。制作重度冲击加速性损伤模型,分别于伤后4h、8h、12h、24h、72h、5d采用干湿比重法测脑组织含水量,原子吸收分光光度法测定Na^＋、K^＋含量,Evans Blue（EB）测定法观察大鼠血-脑屏障（BBB）通透性变化,半定量逆转录聚合酶链反应（RT-PCR）检测脑组织AQP4 mRNA表达及其变化。结果脑组织AQP4 mRNA在伤后4h开始表达上调,8h、12h依次增高,24h达到峰值（P〈0.05）,3d时仍维持较高水平,伤后5d有所降低。脑含水量、Na^＋含量的变化与AQP4 mRNA表达变化一致。经相关性分析,AQP4 mRNA的表达与脑含水量及脑EB含量均呈正相关（P〈0.05）。结论重型脑损伤急性期,AQP4 mRNA表达的变化与颅脑损伤后BBB的破坏及脑水肿的形成和发展密切相关。AQP4可能参与重型脑损伤后脑水肿的形成并起重要作用。     

线粒体钙单向转运体对脑缺血再灌注大鼠脑水肿的影响

实验探讨线粒体钙单向转运体抑制剂钌红及激动剂精胺对缺血再灌注大鼠脑水肿的影响。采用线栓法建立大鼠左侧大脑中动脉闭塞大鼠模型,缺血再灌注24 h后,脑缺血再灌注模型大鼠、钌红及精胺干预的脑缺血再灌注大鼠神经功能评分均显著低于假手术大鼠,脑组织含水量,水通道蛋白4蛋白表达、IgG渗出含量均显著高于假手术大鼠;与脑缺血再灌注模型大鼠和脑缺血再灌注后精胺干预大鼠比较,钌红干预的脑缺血再灌注大鼠神经功能评分明显升高,脑组织含水量,水通道蛋白4蛋白表达及IgG渗出含量明显减少。提示预防性应用线粒体钙单向转运体抑制剂钌红可显著的降低水通道蛋白4和IgG的表达,影响血脑屏障通透性,进而降低脑水肿的程度。结论 线粒体钙单向转运体可能在大鼠脑缺血再灌注损伤中起重要作用,并能影响AQP4的表达和血脑屏障通透性。     

Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

BACKGROUND: Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE: To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING: The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS: A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 (Santa Cruz, USA) and Evans blue (Sigma, USA) were used to analyze the tissue. METHODS: The rats were randomized into sham-operated (n = 16) and ischemia/reperfusion (n = 96) groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES: Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS: Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, and 72 hours after reperfusion, as well as both hemispheres in the sham-operated group (P<0.05). Evans blue content significantly increased on the ischemic side at 4 hours after ischemi',dreperfusion, and reached a peak at 48 hours. Evans blue content was greater on the ischemic hemispheres, compared with the control hemispheres at various time points, as well as both hemispheres in the sham-operated group (P<0.05). Aquaporin-4-positive cells were detected in the cortex and hippocampus, surrounding the ischemic penumbra focus, at 4-6 hours after ischemia/reperfusion. The number of positive cells significantly increased at 12 hours and reached a peak at 48-72 hours. Aquaporin-4 was, however, weakly expressed in the control hemispheres and the sham-operated group. The absorbance ratio of aquaporin-4 to β-actin was greater at 12, 24, 48, and 72 hours following cerebral ischemia/reperfusion, compared with the sham-operated group (P<0.05). Aquaporin-4 expression positively correlated to brain water content and Evans blue staining following cerebral ischemia/reperfusion (r1 = 0.68, r2= 0.81, P<0.05). CONCLUSION: Aquaporin-4 is highly expressed in brain tissues, participates in the occurrence of ischemic brain edema, and is positively correlated to blood-brain barrier permeability following cerebral ischemia/reperfusion.     

大鼠脑挫裂伤半暗带水肿与血-脑屏障破坏的关系

目的研究大鼠局灶性脑挫裂伤半暗带的水肿变化与血-脑屏障(BBB)破坏的关系。方法将126只雄性SD大鼠随机分为3组:假手术组、挫裂伤组和给药组,采用Feeney法制作脑挫裂伤模型,给药组采用曲克芦丁脑蛋白水解物合剂腹腔给药,分别用伊文思蓝(EB)染色法和干湿法观察BBB的变化和脑组织的水肿情况,同时电镜观察超微结构。结果EB染色显示:挫裂伤组和给药组在伤后1 h EB开始漏出,6 h最严重。水含量测定结果显示:挫裂伤组和给药组伤后6 h水含量开始增加,72 h增至高峰。与挫裂伤组比较,给药组脑组织EB溢出量、水含量降低(P<0.05)。结论大鼠脑挫裂伤半暗带BBB通透性改变早于脑水肿的发生,提示BBB破坏可能是早期创伤性脑水肿的结构基础。曲克芦丁脑蛋白水解物合剂可通过改善BBB等多种途径治疗脑挫裂伤。     

血管内皮生长因子在高原脑水肿形成中作用的实验研究

目的:探讨血管内皮生长因子(VEGF)在高原脑水肿形成中的作用。方法:建立大鼠模拟高原模型,应用脑干湿重比率法定量脑水肿情况、应用荧光素钠透过率测定BBB通透性、应用实时荧光定量RT-PCR法检测脑组织VEGF mRNA含量以及应用蛋白印迹法半定量脑组织VEGF含量。结果:大鼠在高原24 h后脑组织含水率明显增高(P<0.05),荧光素钠透过率显著增加(P<0.01);VEGF mRNA转录及其表达显著增高(P<0.001)。结论:VEGF表达在高原脑水肿形成中起重要作用。     

Protective effects of dl-3n-butylphthalide against diffuse brain injury

Dl-3n-butylphthalide can effectively treat cerebral ischemia; however, the mechanisms underlying the effects of dl-3n-butylphthalide on microcirculation disorders fol owing diffuse brain injury remain ...     

缺氧预处理对创伤性脑损伤大鼠 Claudin5 表达及血脑屏障通透性的影响

目的探讨缺氧预处理对创伤性脑损伤大鼠脑组织紧密连接蛋白Claudin-5的表达及血-脑屏障通透性的影响。方法204只大鼠随机分为创伤性脑损伤组（T组）96例,缺氧预处理后脑损伤组（H组）96例及对照组12例。T组行自由落体撞击法建立大鼠创伤性脑损伤模型,H组给予3d缺氧预处理后,同法致脑损伤,两组大鼠于伤后1h、4h、8h、12h、24h、3d、7d、14d断头处死。采用干湿重法测脑组织含水量：Real—timePCR和Westernblot检测各组大鼠挫伤区周围脑组织Claudin-5mRNA及蛋白表达变化;IgG法检测血一脑屏障通透性变化。结果T组和H组伤后1hClaudin-5mRNA及蛋白表达开始降低,8～12h降至最低点,1d开始上升,直至伤后14d渐趋于对照组水平;其中H组各时间点Claudin-5mRNA及蛋白表达均高于T组。T组各时间点血一脑屏障通透性及脑组织含水量均明显高于H组（P〈0．05）,且两组均高于对照组（P〈0．05）。结论缺氧预处理可在创伤性脑损伤早期上调紧密连接蛋白Claudin-5的表达,维持血-脑屏障完整性,减轻脑水肿。     

Peripheral thermal injury causes early blood-brain barrier dysfunction and matrix metalloproteinase expression in rat

High mortality incidence after serious systemic thermal injury is believed to be linked to significant increases in cerebral permeability, ultimately leading to irreversible blood-brain barrier (BBB) breakdown. The aim of this study was to investigate whether disruption of microvascular integrity in a rat thermal injury model is associated with early matrix metalloproteinase (MMP) expression. A total of 35 Sprague-Dawley rats were studied in thermal injury and control groups, each group containing two subgroups, one for brain edema and Evans blue analysis and another for MMP mRNA analysis. Thermally injured animals were anesthetized and submerged vertically in 85 degrees C water to the neck for 6 seconds producing a third degree burn affecting 70% of the total body surface area. BBB integrity was determined by measuring amount of Evans blue after 7 hours of injury with a spectrophotometer. Brain edema was detected by calculating water content. Brain mRNA levels were determined with real-time PCR 3 and 7 hours post-injury. Brain water content was significantly increased after peripheral injury at hour 7. Evans blue leakage was also significantly increased at the same time, suggesting an impaired BBB function after injury. Expressions of MMP-2 and MMP-9 mRNA in brain were increased as early as 3 hours after injury and remained at hour 7. Our study demonstrated a significant increase in cerebral permeability that occurs after serious systemic thermal injury. The underlying mechanisms could be related to early expression of MMPs.     

家兔上矢状窦中1/3及其回流静脉结扎后脑水肿变化和水通道蛋白-4表达的研究

目的探讨家兔上矢状窦中1/3及其回流静脉结扎后窦旁皮层组织水肿变化、水通道蛋白-4(AQP4) 的表达及其变化。方法建立家兔上矢状窦中1/3及其回流静脉结扎的动物模型,用干湿重法、免疫组化方法和半定量逆转录聚合酶链反应(RT-PCR)分别检测窦旁皮层组织脑水肿、AQP4蛋白和AQP4mRNA的表达及其变化。结果家兔上矢状窦中1/3及其回流静脉结扎后,家兔可长期存活而无死亡;窦旁皮层组织4 h出现脑水肿,8 h到达高峰,以后随时间延长而逐渐减轻,长期组脑水肿接近于假手术对照组。家兔上矢状窦中1/3及其回流静脉结扎后窦旁皮层组织存在AQP4蛋白的表达,结扎后4 h AQP4表达显著高于正常水平,8 h达到峰值,其表达水平以后随时间延长而逐渐回落。AQP4的表达与脑含水量呈显著正相关(r=0．778,P<0．01);而窦旁皮层组织也存在 AQP4mRNA的表达,其结果与AQP4蛋白的表达相似。AQP4mRNA的表达与脑含水量呈显著正相关(r=0．885,P <0．01)。AQP4mRNA与AQP4蛋白的表达呈显著正相关(r=0．753,P<0．01)。结论家兔上矢状窦中1/3及其回流静脉结扎后窦旁皮层组织存在脑水肿,AQP4蛋白与AQP4mRNA在兔脑中有表达。两者的表达增强可能与上矢状窦中1/3及其回流静脉结扎后脑水肿的形成和发展密切相关。AQP4可能参与结扎后脑水肿的形成。     

维生素C在急性大剂量乙醇摄入后颅脑损伤中作用的研究

目的探讨维生素C（VitC）在急性大剂量乙醇摄入后颅脑损伤（TBI）后继发性脑损伤的作用及其可能机制。 方法将实验大鼠随机分为打击+生理盐水组（TBI+only）;打击+乙醇+生理盐水组（TBI+EtOH）;打击+VitC组（TBI+VitC）;打击+乙醇+VitC组（TBI+EtOH+VitC）,每组10只。建立中度TBI模型,TBI后24 h进行神经功能损伤评分、脑含水量及伊文氏蓝含量测定;采用Western blot法测定TBI后24 h的损伤侧脑组织中细胞色素P450（CYP2E1）,水通道蛋白-4（AQP-4）,基质金属蛋白酶-9（MMP-9）和血脑屏障紧密连结蛋白（Occludin）的表达情况。 结果打击24 h后,TBI+EtOH组的神经功能损伤评分、脑含水量和伊文氏蓝含量明显增加,VitC干预后,各项指标明显减少,组间差异有统计学意义（P<0.05）;与TBI+EtOH组相比,TBI+EtOH+VitC组的CYP2E1表达减少,AQP-4和MMP-9的含量降低,Occludin的含量增高,组间差异有统计学意义（P<0.05）。 结论急性乙醇摄入加剧了TBI后细胞毒性脑水肿,而VitC可通过CYP2E1减少AQP-4和MMP-9的激活,增加Occludin蛋白的表达,减少血脑屏障紧密连接的分解及完整性的破坏,降低血脑屏障的开放并减轻脑水肿,最终改善神经功能。     

Apelin-13 as a novel target for intervention in secondar y injur y after traumatic brain injur y

The adipocytokine, apelin-13, is an abundantly expressed peptide in the nervous system. Apelin-13 protects the brain against ischemia/reperfusion injury and attenuates traumatic brain injury by suppressing autophagy. However, secondary apelin-13 effects on traumatic brain injury-induced neural cell death and blood-brain barrier integrity are still not clear. Here, we found that apelin-13 signiifcantly de-creases cerebral water content, mitigates blood-brain barrier destruction, reduces aquaporin-4 expression, diminishes caspase-3 and Bax expression in the cerebral cortex and hippocampus, and reduces apoptosis. These results show that apelin-13 attenuates secondary injury after traumatic brain injury and exerts a neuroprotective effect.     

大鼠弥漫性颅脑损伤后脑水肿和病理学变化

目的探讨大鼠弥漫性颅脑损伤后病理学变化规律及意义。方法成年健康SD雄性大鼠100只，以自由落体法建立大鼠弥漫性颅脑损伤模型，随机平均分为对照组及外伤后1h、3h、6h、12h、24h、48h、72h、7d和14d等10组．检测大鼠脑组织含水量并观察脑组织中炎症反应、毛细血管和神经元的结构变化。结果外伤后脑皮层组织含水量呈上升趋势．3h含水量有较大幅度升高，24h达高峰，其后下降，14d时仍处于较高水平，伤后6、12、24、48和72h组与对照组相比．脑组织含水量明显高于对照组（P〈0．05）。脑组织炎症反应、毛细血管和神经元结构变化与脑水肿变化相平行。结论弥漫性颅脑损伤后发生缺血、缺氧．导致微循环障碍，最终血-脑屏障结构破坏，神经细胞的代谢活动紊乱，使脑组织发生继发性脑损伤。弥漫性颅脑损伤后脑水肿是造成继发性脑损伤病理学变化的基础。     

4000/预防性应用克罗卡林对大鼠脑缺血后AQP-4和血脑屏障通透性的影响

Cromakalim,an adenosine triphosphate-sensitive potassium channel opener,exhibits protective effects on cerebral ischemia/reperfusion injury.However,there is controversy as to whether this effect is associated with aquaporin-4 and blood-brain barrier permeability.Immunohistochemistry results show that preventive administration of cromakalim decreased aquaporin-4 and IgG protein expression in rats with ischemia/reperfusion injury;it also reduced blood-brain barrier permeability,and alleviated brain edema,ultimately providing neuroprotection.     

Cerebral microembolization in the rat: Changes in blood-brain barrier permeability and cerebral blood flow as related to the degree of ischemia

Unilateral cerebral microembolism was performed in the rat by injecting calibrated, 50 micrometers in diameter, carbonized microspheres into the internal carotid artery. The events that follow brain ischemia due to cerebral embolization were studied by the analysis of the blood-brain barrier (BBB) function, the degree of regional cerebral blood flow (CBF) and the development of brain edema. Two hours after embolization there was no change in the brain water content. The local CBF (14C-ethanol technique) was only reduced in the ipsilateral hemisphere. Twenty-four hours after embolization the brain water content was increased significantly in the ipsilateral, but not in the contralateral hemisphere. Local CBF further decreased in the ipsilateral hemisphere and a reduction in flow was also observed in the contralateral hemisphere. Embolization led to an increase in the BBB permeability, analysed as regional penetrability of 3H-dextran and of Evans blue-albumin complexes, which was restricted to the side of the injection of the microspheres.     

蛛网膜下腔出血后血脑屏障通透性的实验研究

目的观察蛛网膜下腔出血后脑血管痉挛后血管通透性改变以及探讨其发生机制。方法将36只新西兰大白兔随机平分为6组,各手术组通过枕大池注血建立兔蛛网膜下腔出血后脑血管痉挛的模型,假手术组枕大池注入生理盐水。假手术1组和手术1组用于通过免疫组化方法检测血管内皮细胞的caspase-3表达情况,作为细胞凋亡的标志;假手术2组和手术2组用于检测注血后3d处死动物,比较手术组与对照组脑含水量。假手术3组和手术3组用于通过Evan's蓝荧光染色方法检测手术组和非手术组血脑屏障的通透性。结果手术1组的基底血管内皮细胞caspase-3高度表达,与假手术1组相比有显著统计学差异,反映了蛛网膜下腔出血后基底血管内皮细胞发生了细胞凋亡;手术2组与假手术2组脑含水量相比增加,有显著统计学差异;手术3组脑组织Evan,s蓝含量明显增加,与假手术3组相比有显著统计学差异,反映了蛛网膜下腔出血后血脑屏障的通透性也发生了明显改变。结论血管内皮细胞凋亡可能是蛛网膜下腔出血后脑血管痉挛引发血脑屏障损伤的重要原因。     

阿托伐他汀对大鼠脑缺血再灌注后血脑屏障通透性的影响

目的 评估阿托伐他汀对大鼠脑缺血再灌注后血脑屏障通透性的影响。方法 采用常规尼龙线栓法制备SD大鼠脑缺血再灌注模型,并将大鼠随机分为假手术组、大脑中动脉阻断再灌注(Middle cerebral artery occlusion/reperfusion,MCAO/R)(对照)组和MCAO/R阿托伐他汀(治疗)组; 对照组和治疗组分别于脑缺血2 h再灌注24 h处死; 标准湿干法测定脑组织含水量; 实时聚合酶链反应(Real-time polymerase chain reaction,RT-PCR)检测基质金属蛋白酶-2(Matrix metalloproteinases-2,MMP-2)和基质金属蛋白酶-9(Matrix metalloproteinases-9,MMP-9)的mRNA表达水平; 应用免疫组化法测定Ⅳ型胶原蛋白(Ⅳ type collagen,CoⅣ)水平; 电镜观察显示血脑屏障超微结构的改变。结果 治疗组与对照组比较,脑组织含水量减少(P<0.01); 阿托伐他汀治疗显著降低了MMP-2和MMP-9的mRNA表达水平; 治疗组脑组织CoⅣ水平高于对照组(P<0.01); 电镜观察显示治疗组血脑屏障超微结构的改变明显好于对照组。结论 阿托伐他汀可以降低脑缺血再灌注大鼠血脑屏障的通透性,从而减轻脑水肿。     

白果内酯抑制持续性脑栓塞大鼠水通道蛋白1，4及胶质纤维酸性蛋白的表达

实验结果发现白果内酯预处理可以降低永久性大脑中动脉闭塞大鼠脑组织含水量和梗死面积;下调水通道蛋白1,4 mRNA在水肿脑组织中的表达,继而抑制其合成,特别是在缺血的早期阶段（8 h）;抑制胶质纤维酸性蛋白的表达,减轻反应性胶质增生。说明白果内酯可通过抑制水通道蛋白的表达减轻脑水肿。     

Brain injury, edema, and vascular permeability changes induced by oxygen-derived free radicals

We studied the cerebral effects of oxygen-derived free radicals generated from the xanthine oxidase/hypoxanthine/ADP-Fe3+ system. Xanthine oxidase/hypoxanthine/ADP-Fe3+ solution (0.1 ml) was infused into caudate putamen, and brain was frozen rapidly in situ. Brain water and sodium content increased concomitant with decreased potassium content at 24 hours and 48 hours after the infusion. The degree of brain edema and injury depended on the dose of xanthine oxidase. Spongy neuropil and neuronal cytoplasmic vacuoles were seen at 2 hours, with an infiltration by polymorphonuclear leukocytes at 24 hours, followed by lipid-laden macrophages and reactive astrocytes. Leakage of fluorescent dye into neuropil was seen at 2 hours, but not later. These data suggest that oxygen-derived free radicals damage endothelial cells of the blood-brain barrier; the brain injury is characterized by edema and by structural damage of neurons and glia.