依达拉奉改善大鼠血脑屏障损伤的AQP-4机制研究

目的研究大鼠脑出血后血脑屏障(BBB)通透性与水通道蛋白4(aquaporin-4,AQP-4)的关系及依达拉奉的干预作用。方法采用自体动脉血注入尾状核法制成大鼠脑出血模型,免疫组化法观察AQP-4的表达,伊文思蓝法测BBB的通透性,干湿重法计算脑含水量表示脑水肿。结果与对照组相比,模型组及依达拉奉组BBB通透性均在出血后6h开始升高,1~3d最高,依达拉奉组明显小于模型组(P0.01)。2组AQP-4也于6h开始升高,1~3d达到高峰依达拉奉组明显小于模型组(P0.05)。BBB通透性与AQP-4表达呈显著正相关(r=0.880,P0.01),与脑水肿变化趋势一致。结论脑出血后产生的AQP-4增多,从而增加BBB通透性,参与脑水肿形成和发展,依达拉奉可抑制AQP-4的表达,从而有减轻脑水肿的作用。     

H-7在脑出血后脑损伤中作用机制的研究

目的探讨蛋白激酶C(PKC)抑制剂异喹啉磺酰类(H-7)在脑出血后脑损伤中的作用机制。方法SD雄性大鼠随机分为对照组、假手术组、脑出血组、H-7治疗组,采用自体血注入法建立大鼠脑出血模型,治疗组应用H-7每12 h腹腔注射一次。分别用伊文思蓝(EB)测血脑屏障(BBB)通透性,干-湿重法测脑组织含水量,同时进行组织病理学观察。结果H-7治疗组可以明显改善大鼠脑出血后BBB通透性(P<0.05),血肿周围脑水肿明显减轻(P<0.05),并且减少脑出血后血肿周围炎症细胞浸润。结论H-7通过抑制PKC,减轻脑出血后脑水肿?血脑屏障的开放和炎症细胞浸润,揭示PKC参与了脑出血后脑损伤。     

高同型半胱氨酸血症与脑出血后迟发性脑水肿的临床观察

目的 探讨高同型半胱氨酸血症(Hcy)对脑出血后迟发性脑水肿发生的影响.方法 将本院收治的伴高同型半胱氨酸血症的脑出血患者87例随机分成治疗组(44例)和对照组(43例),对照组常规治疗,治疗组在常规治疗的基础上补充叶酸,测定入院和一月时空腹血浆同型半胱氨酸血水平,定期行头颅CT动态观察脑水肿情况.结果 治疗组同型半胱氨酸水平下降显著,治疗组迟发型脑水肿发生5例,对照组发生13例迟发型脑水肿,两组差异均明显.结论 叶酸治疗可能降低伴高同型半胱氨酸血症的脑出血患者迟发性脑水肿的发生.     

尼膜同对大鼠脑出血后血脑屏障通透性及水通道蛋白4mRNA表达的影响

目的研究大鼠脑出血后血脑屏障(BBB)通透性与水通道蛋白4(AQP 4)的关系及尼膜同的干预作用。方法采用自体动脉血注入尾状核法制成大鼠脑出血模型,RT-PCR法观察AQP 4 mRNA的表达,伊文思兰法测量BBB通透性,干湿重法计算脑含水量表示脑水肿。结果与对照组相比,脑出血组及尼膜同组BBB通透性均在出血后6h开始升高(0.5955±0.0956、0.5092±0.0309),1d~3d最高(0.8889±0.0968、0.7826±0.0339和0.7914±0.0520、0.7442±0.0753),尼膜同组低于脑出血组(P<0.05);两组AQP 4 mRNA表达也于6h即开始升高(1.06±0.12、0.90±0.15),3d时达到高峰(1.34±0.14对1.27±0.14),尼膜同组低于脑出血组(P<0.05);BBB通透性与AQP 4 mRNA表达呈显著正相关(r=0.686,P<0.01),与脑水肿变化趋势一致。结论脑出血后可能通过上调APQ 4 mRNA表达,增加BBB通透性,参与脑水肿形成;尼膜同可抑制此过程。     

尼膜同对大鼠脑出血后血脑屏障通透性及水通道蛋白4mRNA表达的影响

目的研究大鼠脑出血后血脑屏障(BBB)通透性与水通道蛋白4(AQP4)的关系及尼膜同的干预作用。方法采用自体动脉血注入尾状核法制成大鼠脑出血模型,RT-PCR法观察AQP4mRNA的表达,伊文思兰法测量BBB通透性,干湿重法计算脑含水量表示脑水肿。结果与对照组相比,脑出血组及尼膜同组BBB通透性均在出血后6h开始升高(0.5955±0.0956、0.5092±0.0309),1d~3d最高(0.8889±0.0968、0.7826±0.0339和0.7914±0.0520、0.7442±0.0753),尼膜同组低于脑出血组(P<0.05);两组AQP4mRNA表达也于6h即开始升高(1.06±0.12、0.90±0.15),3d时达到高峰(1.34±0.14对1.27±0.14),尼膜同组低于脑出血组(P<0.05);BBB通透性与AQP4mRNA表达呈显著正相关(r=0.686,P<0.01),与脑水肿变化趋势一致。结论脑出血后可能通过上调APQ4mRNA表达,增加BBB通透性,参与脑水肿形成,尼膜同可抑制此过程。     

MK-801通过抑制RhoA在出血性脑损伤机制的研究

目的观察大鼠脑出血后血肿周围Rho GTP酶(RhoA)的表达变化及地佐环平(dizo-cilpine,MK-801)对其表达的影响,探讨在脑出血后脑损伤中N-甲基-D-门冬氨酸(N-methyl-D-aspartate,NMDA)与RhoA的相关性。方法 SD雄性大鼠随机分为生理盐水组、出血组和治疗组,采用鼠尾自体血注入尾状核法建立大鼠脑出血3d模型,治疗组用MK-801在脑出血模型前1h腹腔注射,分别用伊文思蓝(Evans blue,EB)测血脑屏障(blood brain barrier,BBB)通透性,干-湿重法测血肿周围脑组织含水量,Western blot法测定患侧RhoA在各组的表达量。结果 MK-801治疗组较脑出血BBB通透性明显降低(P<0.05),血肿周围脑水肿明显减轻(P<0.05),RhoA表达量明显较少(P<0.05)。结论 MK-801能减轻脑出血后脑水肿、血脑屏障的开放,揭示MK-801通过抑制RhoA的表达可能参与出血性脑损伤。     

脑出血后发生迟发性脑水肿的相关危险因素分析

目的 探讨脑出血后发生迟发性脑水肿的相关危险因素。方法 回顾性分析本院神经内科2012年7月～2014年6月158例非手术治疗的脑出血患者的临床资料,根据患者脑水肿情况分为迟发性脑水肿组(观察组73例)和非迟发性脑水肿组(对照组85例),对可能影响脑出血后发生迟发性脑水肿的因素危险因素进行单因素和多因素Logistical回归分析。结果 单因素分析表明2组患者年龄、高血压和吸烟史、美国国立卫生研究卒中量表(National Institutes of Health Stroke Scale,NIHSS)评分、血糖值、纤维蛋白原、基质金属蛋白酶9(Matrix Metalloproteinases 9,MMP-9)、出血量有明显的差异(P<0.05); 多因素Logistical回归分析表明纤维蛋白原,MMP-9,NIHSS评分、血糖水平及出血量是脑出血后发生迟发性脑水肿的独立危险因素。结论 纤维蛋白原,MMP-9,NIHSS评分,血糖水平及出血量是脑出血后发生迟发性脑水肿的独立危险因素。     

补阳还五汤对脑出血大鼠PI3K/AKT信号通路的影响及其神经保护作用的机制

目的观察并探究补阳还五汤对脑出血大鼠脑组织磷脂酰肌醇3激酶/丝氨酸/苏氨酸蛋白激酶(PI3K/AKT)信号转导通路的影响及其神经保护作用的可能机制。方法 72只SD大鼠随机分为四组:假手术组、模型组、补阳还五汤组、银杏叶片组,每组18只。其中模型组、补阳还五汤组、银杏叶片组采用Rosenberg法制作脑出血大鼠模型。Garcia法检测大鼠神经功能评分,电镜观察神经元线粒体超微结构,western blot法检测磷酸化蛋白激酶(p-AKT)蛋白表达,TUNEL法检测细胞凋亡变化,免疫组化法检测B细胞淋巴瘤基因-2(bcl-2)蛋白、Bcl-2相关X蛋白(bax)的表达,甲酰胺法检测血脑屏障(BBB)通透性,干湿重法检测脑组织含水量。结果与脑出血模型组相比,补阳还五汤治疗明显提高神经功能学评分(P0.05),降低BBB通透性,减少脑组织水含量(P0.05),上调p-AKT、bcl-2蛋白表达,下调bax蛋白表达,减轻线粒体损伤,抑制神经元凋亡。结论补阳还五汤抗脑出血引起脑损伤的作用机制可能与其激活PI3K/AKT信号途径,抑制神经元凋亡,降低BBB通透性,减轻脑水肿有关。     

脑出血后迟发性脑水肿与甘露醇治疗的临床观察

目的 分析和探讨高血压性脑出血后迟发性脑水肿与甘露醇的关系.方法 对我院收治的56例高血压脑出血患者随机分成混合组和甘露醇组,混合组给予甘油果糖和呋塞米,甘露醇组给予20%甘露醇治疗15天.结果 治疗后甘露醇组的迟发性脑水肿明显重于混合组,有显著统计学差异性(P＜0.05).结论 高血压性脑出血后迟发性水肿的发生可能与脑出血后甘露醇的大量、长时程应用有关.     

局部亚低温对大鼠脑出血后基质金属蛋白酶-9及血脑屏障通透性的影响

目的探讨局部亚低温对大鼠自体血注入法脑出血模型基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)mRNA及蛋白表达的影响以及局部亚低温减轻脑出血后水肿的可能机制。方法雄性Wistar大鼠240只,随机分为脑出血(ICH)组和脑出血加局部亚低温(ICH H)组。每组分为对照、脑出血后6h、24h、72h、5d、7d共6个亚组,ICH H组于注血后立即给以4h的局部亚低温治疗,各亚组分别进行血脑屏障(BBB)通透性、脑水含量的检测以及应用RT-PCR及Western印记对MMP-9进行测定。结果ICH组大鼠脑内注血后6h开始出现脑组织水含量(P<0.01)及BBB通透性(P<0.05)的显著增加,二者在72h达到高峰,然后逐渐消退,ICH组MMP-9蛋白表达量与脑含水量和血脑屏障通透性呈正相关(r=0.88和r=0.96),ICH组MMP-9 mRNA表达量也与脑含水量和血脑屏障通透性呈正相关(r=0.78和r=0.85)。ICH H组大鼠脑组织水含量、BBB通透性以及MMP-9蛋白的表达与ICH组各时间点相比较,明显降低,而MMP-9 mRNA的表达与ICH组相比仅有轻度下降。结论脑出血后MMP-9的变化与BBB通透性和脑水肿密切相关,局部亚低温可以抑制脑出血后MMP-9蛋白表达的增加以及脑水肿的形成。提示局部亚低温可能通过影响MMP-9的变化来抑制脑出血后的水肿形成。     

一氧化碳及一氧化氮对局灶性缺血脑组织的影响

目的 研究一氧化碳与一氧化氮对局灶性缺血脑组织脑含水量及血脑屏障通透性的影响。方法 将SD大鼠随机分为4组：为生理盐水组、Hemin组、ZnPP组及Hemin ZnPP组，分别用等量生理盐水、Hemin、ZnPP、Hemin ZnPP腹腔注射，1h后制成MCAO模型。栓塞后24h检测血浆CO及NO浓度、脑组织含水量、血脑屏障通透性。结果 与生理盐水组相比，Hemin组血浆CO浓度明显升高，NO浓度无变化，脑组织含水量下降，血脑屏障通透性减低；ZnPP组血浆CO浓度明显减低，NO浓度上升，脑组织含水量上升，血脑屏障通透性增高；同时注射Hemin ZnPP时CO浓度无变化，NO浓度升高，脑组织含水量上升，血脑屏障通透性增高。结论 NO及CO在缺血早期对脑组织均具有保护作用，两者之间存在着相互作用，CO通过HO影响NOS及NO。     

脑出血后脑水肿经时变化机制的探讨

目的 研究脑出血(mtracerebral hemorrhage，ICH)后血肿周围脑水肿与血脑屏障(blood-brain barrier，BBB)随时间变化的机制，从而为预防脑水肿提供依据。方法90只大耳白兔随机分为3组。1组：在立体定向仪下将300μl生理盐水注入兔左侧基底节；2组：注入200μl自身动脉血与100μl生理盐水；3组：注入200μl自身动脉血与100μl水蛭素。每组每时相(6h、12h、24h、48h、72h)各6只兔。脑组织含水量采用干湿重法测量，血脑屏障的通透性测定采用伊文思兰法。结果动脉血组及水蛭素干预组血肿周围脑组织含水量均在48h达到高峰．此后逐步降低。动脉血组伊文思兰(EB)于24h到达高峰，水蛭素干预组伊文思兰(EB)于48h达高峰。结论脑出血后脑水肿是多种因素综合作用的结果。早期可能和血块凝缩、流体静力压有关；至中期时凝血酶是主导因素；后期则主要由于红细胞裂解物的损害。     

Hemoglobin-Induced Nitric Oxide Synthase Overexpression and Nitric Oxide Production Contribute to Blood–Brain Barrier Disruption in the Rat

Hemoglobin (Hb) released from extravasated erythrocytes may have a critical role in the process of blood–brain barrier (BBB) disruption and subsequent edema formation after intracerebral hemorrhage (ICH). Excessive nitric oxide (NO) production synthesized by nitric oxide synthase (NOS) has been well documented to contribute to BBB disruption. However, considerably less attention has been focused on the role of NO in Hb-induced BBB disruption. This study was designed to examine the hypothesis that Hb-induced NOS overexpression and excessive NO production may contribute to the changes of tight junction (TJ) proteins and subsequent BBB dysfunction. Hemoglobin was infused with stereotactic guidance into the right caudate nucleus of male Sprague Dawley rats. Then, we investigated the effect of Hb on the BBB permeability, changes of TJ proteins (claudin-5, occludin, zonula occludens-1 (ZO-1), and junctional adhesion molecule-1 (JAM-1)), iron deposition, expression of inducible NOS (iNOS) and endothelial NOS (eNOS), as well as NO production. Hb injection caused a significant increase in BBB permeability. Significant reduction of claudin-5, ZO-1, and JAM-1 was observed after Hb injection as evidenced by PCR and immunofluorescence. After a decrease at early stage, occludin showed a fivefold increase in mRNA level at 7 days. Significant iron deposition was detectable from 48 h to 7 days in a time-dependent manner. The iNOS and eNOS levels dramatically increased after Hb injection concomitantly with large quantities of NO released. Furthermore, enhanced iNOS or eNOS immunoreactivity was co-localized with diffused or diminished claudin-5 staining. We concluded that overexpressed NOS and excessive NO production induced by Hb may contribute to BBB disruption, which may provide an important potential therapeutic target in the treatment of ICH.     

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.     

Signaling pathways for early brain injury after subarachnoid hemorrhage.

Few studies have examined the signaling pathways that contribute to early brain injury after subarachnoid hemorrhage (SAH). Using a rat SAH model, the authors explored the role of vascular endothelial growth factor (VEGF) and mitogen-activation protein kinase (MAPK) in early brain injury. Male Sprague-Dawley rats (n = 172) weighing 300 to 350 g were used for the experimental SAH model, which was induced by puncturing the bifurcation of the left anterior cerebral and middle cerebral arteries. The blood-brain barrier (BBB), brain edema, intracranial pressure, and mortality were evaluated at 24 hours after SAH. The phosphorylation of VEGF and different MAPK subgroups (ERK1/2, p38, and JNK) were examined in both the cortex and the major cerebral arteries. Experimental SAH increased intracranial pressure, BBB permeability, and brain edema and produced high mortality. SAH induced phosphorylation of VEGF and MAPKs in the cerebral arteries and, to a lesser degree, in the cortex. PP1, an Src-family kinase inhibitor, reduced BBB permeability, brain edema, and mortality and decreased the phosphorylation of VEGF and MAPKs. The authors conclude that VEGF contributes to early brain injury after SAH by enhancing the activation of the MAPK pathways, and that the inhibition of these pathways might offer new treatment strategies for SAH.     

AQP-9在大鼠感染性脑水肿脑组织中的表达及意义

目的 探讨水通道蛋白-9(AQP-9)在内毒素脂多糖(LPS)致大鼠感染性脑水肿脑组织中的表达及意义. 方法 1月龄普通级SD大鼠128只采用随机数字表法分为生理盐水(NS)组(64只)和LPS组(64只),采用颈内动脉注射LPS制作大鼠感染性脑水肿模型,模型成功后每组均选取6h、12h、24h和48 h4个时间点,在不同时间点采用HE染色观察脑组织形态学改变;干湿重法测定脑组织含水量(BWC);甲酰胺法测定脑组织伊文思蓝(EB)含量;免疫组织化学法检测脑组织AQP-9蛋白的表达量:采用逆转录多聚酶链反应(RT-PCR)方法 检测AQP-9 mRNA的表达水平并对结果 进行相关性分析. 结果 HE染色结果 显示LPS组血管周围间隙增宽、炎性细胞浸润、胶质细胞体积增大肿胀、神经元空泡变性、细胞核固缩等.与NS组相比,LPS组6 h、12 h、24 h和48 hBWC、EB含量、AQP.9蛋白及AQP.9mRNA表达水平均增高.差异具有统计学意义(P<0.05).同时LPS组BWC和EB含量、AQP-9蛋白、AQP.9 mRNA表达量、AQP-9 mRNA表达量与EB含量、AQP-9蛋白与mRNA表达量均呈正相关. 结论 AQP-9可能参与感染性脑水肿的发生和发展.     

Contribution of poly(ADP-ribose) polymerase to postischemic blood-brain barrier damage in rats.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood-brain barrier (BBB) disruption and edema formation after ischemia-reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia-reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170+/-9, saline: 760+/-95, PJ34: 472+/-61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction     

Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage

Granulocyte colony-stimulating factor (G-CSF) has been used in the treatment of neutropenia in hematologic disorders. The neuroprotective and anti-inflammatory effects of G-CSF were reported in various neurological disease models. In this study, we examined whether G-CSF induces functional recovery after intracerebral hemorrhage (ICH). ICH was induced using collagenase injection in adult rats. Either G-CSF (50 microg/kg, i.p.) or saline was given from 2 h after ICH and every 24 h for 3 days. 72 h after ICH induction, the rats were sacrificed for histological analysis and measurement of brain edema. Behavioral tests were performed before and 1, 7, 14, 21, 28, and 35 days after ICH. We also measured the blood-brain barrier (BBB) permeability using Evans blue dye injection method. G-CSF-treated rats recovered better on rotarod and limb placing tests, starting from 14 days throughout 5 weeks after ICH. The brain water content and BBB permeability of G-CSF-treated group decreased in the lesioned hemispheres compared with those of ICH-only group. In G-CSF-treated group, the number of TUNEL+, myeloperoxidase+, and OX42+ cells was smaller than that of ICH-only group in the periphery of hematoma. These findings suggest that G-CSF induces long-term sensorimotor recovery after ICH with reduction of brain edema, inflammation, and perihematomal cell death.