甘珀酸对兔脑血管痉挛时缝隙连接蛋白43磷酸化表达的影响

目的 探讨甘珀酸对兔实验性蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)时缝隙连接蛋白43(Cx43)磷酸化表达的影响.方法 建立兔二次SAH模型,脑池及静脉分别给予甘珀酸,脑血管造影及光镜观察分析基底动脉直径及形态学变化并应用Western blot检测基底动脉Cx43蛋白磷酸化表达的变化.结果 SAH组与正常组相比,脑血管造影及光镜观察结果 证实基底动脉痉挛明显;痉挛动脉肇磷酸化的Cx43(P-Cx43)蛋白表达显著升高,但去磷酸化的Cx43(NP-Cx43)蛋白表达显著减少.甘珀酸脑池处理组及静脉处理组与SAH组相比,脑血管造影及光镜观察结果 证实基底动脉痉挛显著减轻;痉挛动脉壁P-Cx43蛋白表达显著减少,但NP-Cx43蛋白表达显著升高.结论 SAH后,Cx43蛋白磷酸化表达发生变化,脑池或静脉给予甘珀酸能明显缓解SAH后CVS,其作用机制可能与基底动脉Cx43蛋白磷酸化表达变化有关.     

脑血管痉挛中缝隙连接蛋白Cx43磷酸化位点分析及S368位点与其关系的研究

目的 观察脑血管痉挛中缝隙连接蛋白Cx43的磷酸化位点及其S368位点磷酸化水平的变化,探讨其与脑血管痉挛的关系. 方法 新西兰大白兔78只按随机数字表法分为4组:正常对照组(n=6)、单纯脑池注血组(n=24)、甘珀酸(CBX)脑池处理组(n=24)和溶媒脑池处理组(n=24),后3组应用枕大池二次注血法建立兔蛛网膜下腔出血后脑血管痉挛模型及相应给药,并按1d、3d、7d、14d分成4亚组,每亚组6只.采用磷酸化蛋白富集试剂盒富集各组基底动脉中Cx43磷酸化总蛋白,再利用质谱技术鉴定出其磷酸化位点;应用Western blotting方法分析各组Cx43S368位点磷酸化水平的变化;通过数字减影血管造影技术(DSA)观察各组基底动脉直径变化情况. 结果 (1)质谱技术成功鉴定出Cx43的4个磷酸化位点,分别为Y265、S364、S365、S368.(2)Western blotting结果显示:正常对照组基底动脉Cx43 S368位点磷酸化水平较低(17.0％±2.3％);单纯脑池注血组及溶媒脑池处理组与正常对照组相比,Cx43 S368位点磷酸化水平在1d、3d、7d、14d各时间点均显著升高,差异有统计学意义(P＜0.05),且以7d表达最高,14d开始下降;CBX脑池处理组各时间点基底动脉Cx43 S368位点磷酸化水平显著低于单纯脑池注血组及溶媒脑池处理组,差异有统计学意义(P＜0.05).(3)DSA显示正常对照组第二次与首次造影基底动脉直径的百分比值平均为99.1％±1.3％,单纯脑池注血组、CBX脑池处理组、溶媒脑池处理组分别为66.1％±7.2％、91.3％±5.3％、63.7％±6.6％,CBX脑池处理组基底动脉直径显著狭窄于单纯脑池注血组,差异有统计学意义(P＜0.05). 结论 缝隙连接蛋白Cx43 S368位点磷酸化可能与脑血管痉挛密切相关,且其可能是CBX缓解脑血管痉挛的机制之一.     

缝隙连接阻断剂1-庚醇对脑血管痉挛的抑制作用

目的探讨缝隙连接在脑血管痉挛中的作用及观察其阻断剂在动物实验中的治疗作用。方法建立兔二次蛛网膜下腔出血模型,通过脑血管造影观察经动脉或池内注入缝隙连接阻断剂heptanol,对脑血管痉挛的抑制和治疗作用,并观察基底动脉的形态学变化。结果枕大池注血后血管造影显示基底动脉出现痉挛。在脑血管痉挛后,动脉给予heptanol对急、慢性脑血管痉挛有显著的治疗作用。预先枕大池注入heptanol再注血,造影显示基底动脉痉挛不明显(P>0.05),heptanol枕大池预处理后能显著抑制急、慢性期脑血管痉挛的形成。形态学检查发现,对照组第7d的基底动脉光镜见内皮细胞核染色质聚集,内弹力膜波纹状,平滑肌细胞分布稀疏等。动脉给药组和池内给药组也有类似变化,但范围局限、程度轻。结论缝隙连接阻断剂heptanol能有效抑制兔蛛网膜下腔出血后急性和慢性脑血管痉挛,体内试验表明缝隙连接在脑血管痉挛中可能发挥重要作用,应用其阻断剂heptanol具有显著的治疗作用。     

缝隙连接蛋白43在内皮素诱导的脑基底动脉收缩中的表达变化

目的 探讨Cx43在内皮素诱导的脑基底动脉收缩中的表达变化及其可能的作用.方法 血管环张力实验检测内皮素诱导的脑基底动脉的收缩变化并应用Western blot检测基底动脉Cx43蛋白的表达变化,染料传输实验用来检测脑基底动脉收缩过程中平滑肌细胞间缝隙连接的功能变化.结果 浓度递增的内皮素导致脑基底动脉呈显著浓度依赖性的收缩,一定浓度缝隙连接阻断剂苷珀酸显著缓解该收缩;收缩过程中,Cx43的蛋白表达呈显著时间依赖性的升高,苷珀酸减弱该表达的升高;内皮素刺激下,血管平滑肌细胞间的染料传输呈时间依赖性的升高,苷珀酸显著减少染料在细胞间的传输.结论 脑血管痉挛过程中,通过增加Cx43的表达,血管细胞间缝隙连接的功能被内皮素激活并在血管痉挛病理过程中发挥重要作用;抑制缝隙连接的功能是有效缓解蛛网膜下腔出血后脑血管痉挛的新途径.     

实验性蛛网膜下腔出血后脑血管痉挛兔基底动脉Cx43蛋白表达的时相变化

目的 通过建立兔二次蛛网膜下腔出血实验模型,观察兔蛛网膜下腔出血后基底动脉缝隙连接蛋白Cx43表达的时相变化特点,初步探讨蛛网膜下腔出血后脑血管痉挛的形成机制.方法 选择健康新西兰大白兔30只,随机分为5组:正常对照组(n=6)和蛛网膜下腔出血模型组(1d、3d、7d和14d,n=6):建立兔二次蛛网膜下腔出血后脑血管痉挛实验模型,脑血管造影分析基底动脉的直径变化并应用Western Blot检测基底动脉Cx43蛋白的表达变化.对血管直径与Cx43表达变化情况进行相关分析.结果 成功建立兔二次蛛网膜下腔出血模型;脑血管造影显示注血后1d基底动脉即出现痉挛(85.7%±8.6%,P<0.05);7d时达高峰(66.5%±7.6%,P<0.01);14d时仍有痉挛(78.4%±8.2%,P<0.05)但程度较前缓解.Cx43蛋白表达在建立SAH模型后1d(38.6%±5.6%,P<0.05)、3d(50.2%±5.7%,P<0.05)、7d(57.8%±5.3%,P<0.01)、14d(32.4%±3.6%.P<00.05)均升高,其中7d为高峰,14d开始下降.Cx43蛋白表达的时相性变化与SAH后基底动脉直径的时相性变化相关系数为0.914.结论 实验结果 显示蛛网膜下腔出血后兔基底动脉缝隙连接蛋白Cx43的表达发生了时相性变化,并且Cx43蛋白表达强弱与蛛网膜下腔出血后脑血管痉挛程度在时程上存在正相关关系,表明缝隙连接蛋白Cx43可能参与蛛网膜下腔出血后脑血管痉挛的形成.

Abstract：

Objective The study was designed to explore the change of expression of connexin43(Cx43)protien in the model of subarachnoid hemorrhage(SAH)of rabbits,hoping to provide the basis to study the mechanism of cerebral vasospasm(CVS).Methods 30 New Zealand rabbits were divided into 5 groups:SAH group(1d,3d,7d,14d,n=6)and control group(n=6).The model of CVS following SAH was established.Digital subtraction angiography was performed to detect the change of the basilar arteries diameter.The expression of Cx43 protien in basilar arteries tissue at different time points following experimental SAH was examined by using western blotting analysis.The data were statistically analyzed using the bivariate correlations test.Results The model of SAH in rabbits was successfully established.All 30 rabbits were analyzed.Cerebral angiograms on 1d,3d,7d and 14d showed severe narrowing of the BAs,and on 7d showed the most narrowing and on 14d began to Relieve.Western blotting showed that the expression of Cx43 protein were detected in normal rabbit basilar arteries tissue.However,the expression of Cx43 protein increased gradually and significantly in models compared with that of control(P<0.05),which reached peak on 7d(P<0.01)and then decreased on 14d(P<0.05).There was positive correlation between expression of Cx43 and cerebral vasospasm.Conclusions The above results demonstrates at the first time that the Cx43 protein expression is altered after the SAH,and exhibits a time-dependent change.which might be connected with the development of CVS.In summary,our data demonstrates gap junctions may play an important role in the pathogenesis of cerebral vasospasm after SAH.     

大鼠两种脑血管痉挛模型的早期脑损伤研究

目的研究大鼠蛛网膜下腔出血后脑血管痉挛模型的早期脑损伤程度。方法将Wistar大鼠随机分为假手术组、枕大池一次注血组及二次注血组。制模后24h灌注取脑,采用HE染色观察基底动脉,TUNEL检测细胞凋亡,免疫组化染色检测Bax和Bcl-2的表达水平。结果枕大池二次注血组的基底动脉横截面积明显小于假手术组(P0.01)。电镜下海马组织呈现损伤表现。同假手术组比较,注血组大脑皮层及海马组织中凋亡细胞计数明显增加,Bax的表达增强,而Bcl-2的表达下降(P0.01)。结论枕大池一次注血及二次注血法均可复制蛛网膜下腔出血后脑血管痉挛模型,并造成早期脑损伤。     

Cx43/Cx45异型缝隙连接通道参与实验性蛛网膜下腔出血后脑血管痉挛的实验研究

目的探讨缝隙连接通道参与脑血管痉挛(CVS)的机制。方法选择健康新西兰大白兔36只,随机分为3组:正常对照组(每组n=12)、SAH-7d组(每组n=12)、SAH-7d+甘珀酸(CBX)组(每组n=12);用二次注血法制成兔SAH模型。应用脑血管造影技术观察造影前后各组基底动脉的血管直径,免疫共沉淀技术观察Cx43与Cx45在各实验组中相互作用的变化。结果脑血管造影显示单纯注血7d组较正常组基底动脉明显痉挛(P0.01),正常对照组及SAH-7d+CBX组基底动脉无明显痉挛,免疫共沉淀示Cx43与Cx45在体内的相互作用在SAH-7d组较正常组及SAH-7d+CBX组均明显增加(P0.01)。结论实验结果显示SAH后兔基底动脉较正常组明显痉挛,单纯出血组Cx43与Cx45组成的异型缝隙连接通道较正常组增加,而CBX能缓解SAH后的CVS及抑制Cx43/Cx45缝隙连接蛋白的高表达,即Cx43/Cx45异型通道的增加可能参与SAH后CVS的形成。     

COX-2和NF-κB在兔蛛网膜下腔出血脑血管痉挛模型中的表达

目的 研究COX-2和NF-κB在蛛网膜下腔出血后脑血管痉挛时表达情况及作用.方法 将16只同龄新西兰兔随机分成对照组、血管痉挛两组.血管痉挛组通过枕大池注自身动脉血方法制成蛛网膜下腔出血模型,对照组动物仅在枕大池注入1.5ml生理盐水.3d后处死动物,断头取脑;每个动物基底动脉及其附近结构标本的组织切片至少分成3份,分别检测蛛网膜下腔出血后,脑血管痉挛模型血管内皮细胞和周围组织COX-2和NFKB表达情况,及HE染色,并用计算机图像分析系统分别对两组脑标本基底动脉血管内径进行测定.对血管内径与血管内皮细胞COX-2和NF-KB表达情况进行多元相关分析.结果 在蛛网膜下腔出血后,脑血管痉挛组动物可以观察到COX-2和NF-KB在基底动脉内皮细胞过度表达,两者之间存在着正相关关系,并且两者表达强度与血管痉挛程度呈正相关关系.结论 COX-2和NF-KB在蛛网膜下腔出血后脑血管痉挛形成过程中发挥重要作用,并且两者相互间作用是其发挥作用的基础.     

蛛网膜下腔出血大鼠基底动脉血小板源性生长因子的表达变化

目的探讨血小板源性生长因子(PDGF)在大鼠蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)血管壁的表达及关系。方法将30只大鼠按照枕大池二次注血的方法建立模型,然后分别于建立模型后的1 d、3 d、5 d、7 d、14 d将大鼠处死,取出基底动脉制作石蜡切片在光镜下观察。采用免疫组化法检测大鼠基底动脉血管壁PDGF的表达水平。结果模型组中PDGF在基底动脉血管壁上的表达,3 d和5 d组最明显,与脑血管痉挛程度的变化是一致的。结论通过枕大池二次注血能够成功的模拟SAH后CVS的发生。PDGF参与了SAH后CVS的过程,并可能起了重要的作用。     

实验性蛛网膜下腔出血后兔脑血管病理结构的动态变化

目的观察蛛网膜下腔出血后脑血管病理结构的动态变化,以建立可靠的脑血管痉挛模型。方法实验分正常组、对照组(枕大池注入等量生理盐水)、SAH3d组、SAH5d组、SAH7d组、SAH10d组和SAH14d组,枕大池二次注血法建立兔蛛网膜下腔出血模型,应用脑血管造影、光镜和透射电镜检查等方法观察SAH后基底动脉形态改变。结果脑血管造影发现SAH后第3d基底动脉狭窄,第7d达高峰。光镜和透射电镜检查显示出血后第3d开始出现血管管腔狭窄、管壁增厚、内皮细胞和平滑肌细胞变性,并随时间推移加重,第5d和第7d病理改变更明显,尤以第7d为著,10d后缓解。结论兔枕大池二次注血法是可靠的SAH后脑血管痉挛模型制作方法。     

Potential role of Ras in cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits

Previous studies have demonstrated that mitogen-activated protein kinase (MAPK) is involved in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). Ras, an upstream regulator of MAPK, may be activated following SAH. The aim of this study was to investigate the role of Ras in cerebral vasospasm in a rabbit model of SAH. We first investigated the time course of Ras and ERK1/2 activation in the basilar artery after SAH. Next, for the time point at which Ras was maximally activated, we assessed the effect of FTI-277 (a Ras farnesyltransferase inhibitor) on cerebral vasospasm. SAH was induced by injecting autologous blood into the cisterna magna on both day 0 and day 2. FTI-277 was injected into the cisterna magna every 24 hours, beginning 30 minutes after blood injection to the last day of the experiment. Elevated expression of Ras-GTP and phosphorylated ERK1/2 was detected in the basilar artery after SAH and expression peaked on day 3. FTI-277 administration resulted in lower Ras-GTP and phosphorylated ERK1/2 levels and markedly attenuated vasospasm in the basilar arteries relative to animals that did not receive FTI-277. Our results suggest that Ras protein is activated in the arterial wall after SAH and contributes to vasospasm development.     

2-methoxyestradiol reduces cerebral vasospasm after 48 hours of experimental subarachnoid hemorrhage in rats

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and antiproapoptotic activities. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF-1alpha). We hypothesized that hypoxia in the major cerebral arteries might activate a unique signaling pathway, hypoxia-inducible factor-1alpha (HIF-1alpha), to produce or enhance cerebral vasospasm after subarachnoid hemorrhage (SAH). Sprague-Dawley male rats (n = 70) were randomly divided into 5 groups: Sham operated, SAH without treatment, SAH treated with vehicle (DMSO), SAH treated with two HIF-1alpha inhibitors, 2ME2, and D609 (positive control of 2ME2). SAH model was produced by middle cerebral artery perforation. 2ME2 and D609 were administered intraperitoneal at 1 h after SAH; rats were sacrificed after 48 h of SAH. Thick blood clot was observed around basilar artery under arachnoids in all animals except Sham group; severe morphological vasospasm was observed in basilar arteries in SAH and SAH+DMSO rats, and the mild vasospasm in rats treated with 2ME2 and D609; 2ME2 and D609 reduced the activity of HIF-1alpha in the basilar arteries by HIF-1alpha DuoSet ELISA; reduce the expression of HIF-1alpha, VEGF, BNIP3 and PCNA in basilar arteries by Western blotting and immunohistochemical staining. In addition, it decreased the mortality and improved the neurological deficits. In conclusion, 2ME2 is a powerful agent to reduce cerebral vasospasm by inhibiting HIF-1alpha activity and the expression of VEGF as its downstream, suppressing endothelium and VSMCs apoptosis via BNIP3 pathway, and attenuating vasoproliferation.     

蛛网膜下腔出血后白细胞介素-8基因在兔脑基底动脉中表达的变化

目的探讨实验性蛛网膜下腔出血（SAH）诱发脑血管痉挛时,白细胞介素-8（IL-8）基因在兔脑基底动脉中表达的变化及在诱发脑血管痉挛中的作用。方法35只健康日本大耳白兔随机分为生理盐水组、SAH组。SAH组根据第一次注血时间又分为四组,分别为第一次注血后第1、4、7、14天。以枕大池二次注血法构建迟发性脑血管痉挛模型,采用RT—PCR法观察兔基底动脉中细胞因子IL-8mRNA表达的变化。结果IL-8mRNA在SAH组第一次注血后第4—7天升高,14天趋于正常。SAH组IL-8的表达水平与基底动脉的狭窄程度呈正相关（r=0．642,P〈0．01）。结论IL．8在基底动脉中的表达水平与脑血管痉挛的程度紧密相关,提示IL-8可能作为免疫／炎症因素因素参与了SAH后迟发性脑血管痉挛的发生。     

蛛网膜下腔出血后白细胞介素-8基因在兔脑基底动脉中表达的变化(英文)

目的探讨实验性蛛网膜下腔出血(SAH)诱发脑血管痉挛时,白细胞介素-8(IL-8)基因在兔脑基底动脉中表达的变化及在诱发脑血管痉挛中的作用。方法 35只健康日本大耳白兔随机分为生理盐水组、SAH组。SAH组根据第一次注血时间又分为四组,分别为第一次注血后第1、4、7、14 天。以枕大池二次注血法构建迟发性脑血管痉挛模型,采用RT-PCR法观察兔基底动脉中细胞因子IL-8 mRNA表达的变化。结果 IL-8mRNA在SAH组第一次注血后第4- 7天升高,14 天趋于正常。SAH组IL-8 的表达水平与基底动脉的狭窄程度呈正相关(r = 0.642,P < 0.01)。结论 IL-8在基底动脉中的表达水平与脑血管痉挛的程度紧密相关,提示IL-8可能作为免疫/炎症因素因素参与了SAH 后迟发性脑血管痉挛的发生。     

17β-雌二醇对蛛网膜下腔出血脑血管痉挛的疗效与其可能机制

性别的差异是否影响颅内动脉瘤破裂所引发蛛网膜下腔出血后的预后,仍未有定论,雌性素对于血管扩张的可能作用也尚未确定。本研究评估17β-雌二醇（estradiol,E2）在大鼠两次出血的蛛网膜下腔出血动物模型中,对于蛛网膜下腔出血引发的脑血管痉挛的治疗效果与可能机制。方法 以0.3 mg/ml E2混合玉米油填充于30 mm长的硅胶管（Silastic tube）,于雄性大鼠引发蛛网膜下腔出血后1 h,包埋于动物皮下。测量包埋的第0、1、2、3、4及7天大鼠血中E2浓度。脑血管痉挛的程度以第一次出血后7天的基底动脉横切面平均面积来评估。同时检查基底动脉内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）及诱导型一氧化氮合酶（Inducible nitric oxide synthase,iNOS）的表现。结果 以E2治疗大鼠的血中浓度维持在生理浓度（56～92 pg/ml）,与给予赋形药物的溶剂对照组相比较,研究组E2浓度的增加有统计学上的意义。E2治疗能有意义的减少蛛网膜下腔出血引发的脑血管痉挛（P <0.01）。E2治疗能有意义的减少脑血管痉挛后基底动脉iNOS-mRNA及蛋白质的表达增加,而对照组无此作用。脑血管痉挛后eNOS-mRNA及蛋白质的表达受压抑,但可经E2治疗而减缓。结论 建议持续性给予E2,维持其于生理浓度,可防止蛛网膜下腔出血后的脑血管痉挛,E2防止蛛网膜下腔出血后脑血管痉挛的效益,部分与E2可防止蛛网膜下腔出血后iNOS的表达及保留eNOS的表达有关。因此,E2对于治疗蛛网膜下腔出血后的脑血管痉挛是一种值得进一步探讨的方法。     

Effect of 18β-glycyrrhetinic acid on cerebral vasospasm caused by asymmetric dimethylarginine after experimental subarachnoid hemorrhage in rats

Abstract

Objectives:

Cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) is characterized by the severe constriction of an artery, which often leads to unfavorable outcomes. CVS after SAH is closely associated with asymmetric dimethylarginine (ADMA) and connexin. The effect of 18β-glycyrrhetinic acid (18β-GA), an inhibitor of gap junction, on ADMA, connexin, and CVS after SAH were investigated.

Methods:

Sprague–Dawley rats (n = 120), weighing 300–350 g, were divided into the control group, sham, SAH, and SAH + 18β-GA groups. In the SAH group, blood was injected into the prechiasmatic cistern of the rats, and 18β-GA (10 mg/kg) was intraperitoneally injected. The neurological score, basilar artery diameter, ADMA, and connexin protein contents (Cx40, Cx43, and Cx45) were measured using Kaoutzanis scoring system, pressure myograph, enzyme linked immunosorbent assay kit, and Western blot, respectively, 1, 3, 5, 7, and 14 days after SAH.

Results:

The neurological score significantly decreased 3, 5, 7, and 14 days after SAH. The basilar artery diameter significantly decreased, and the ADMA level in the cerebrospinal fluid (CSF) significantly increased at all time points. The level of Cx40 significantly decreased on days 3, 5, 7, and 14, and the level of Cx43 and Cx45 significantly increased at all time points. ADMA and Cx43 are positively correlated. However, the upregulated level of ADMA, Cx43, and Cx45 were attenuated. The neurology result significantly improved in the SAH + 18β-GA group.

Conclusions:

Treatment with 18β-GA in SAH rats decreases Cx43 and Cx45 in basilar artery and ADMA in CSF. ADMA is probably involved in the pathophysiological events of CVS after SAH by altering connexin proteins. The mechanism of connexin protein changes caused by ADMA needs to be further studied.     

Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage.

Our previous studies indicate that apoptosis in endothelial cells of major cerebral arteries contributes to cerebral vasospasm after subarachnoid hemorrhage (SAH). This study examined the pathologic roles of tumor suppressor p-53 in cerebral vasospasm using an established dog double-hemorrhage model. Twenty mongrel dogs were divided into four groups: (1) control, (2) SAH, (3) SAH+DMSO (vehicle), and (4) SAH+pifithrin-alpha (PFT) (p53 inhibitor). The p53 inhibitor (200 nmol/L) was injected into the cisterna magna daily from Day 0 through Day 3. Angiogram was performed on Day 0 and Day 7. Western blot, cell proliferation assay, histology, and TUNEL staining were conducted on the basilar arteries collected on Day 7 after SAH. The arterial diameter on Day 7 was 42%+/-4%, 40%+/-5%, and 59%+/-4% for SAH, SAH+DMSO, and SAH+PFT, respectively. In addition, positive staining of TUNEL and increased protein expression of p53, Bax, and PCNA in the basilar artery were observed on Day 7. PFT suppressed apoptosis in endothelial cells and proliferation in smooth muscle cells, and attenuated angiographic vasospasm. In conclusion, p53 may be a key factor in endothelial apoptosis and smooth muscle proliferation after SAH. Inhibition of p53 may potentially reduce or even prevent cerebral vasospasm.