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。     

甘珀酸治疗蛛网膜下腔出血后脑血管痉挛的体内实验研究

目的 探讨缝隙连接抑制剂甘珀酸对实验性蛛网膜下腔出血后脑血管痉挛的治疗作用.方法 建立兔蛛网膜下腔二次出血模型,脑池及静脉分别给与缝隙连接抑制剂甘珀酸,脑血管造影及光镜观察分析基底动脉的直径及形态学变化,并应用Western blotting检测基底动脉Cx43蛋白的表达变化.结果 给与甘珀酸后,基底动脉狭窄程度及光镜下形态学变化显著减轻:单纯注血组(65.7±10.3)%,脑池处理组(91.2±6.4)%,静脉处理组(96.4±11.0)%,腑池预处理组(89.7±12.8)%;同时也显著抑制了痉孪后Cx43蛋白表达水平的升高:单纯注血组(57.2±2.8)%,脑池处理组(10.0±5.3)%,静脉处理组(15.2±1.7)%.结论 缝隙连接抑制剂甘珀酸可能对蛛网膜下腔出血后脑血管痉挛起到预防和治疗作用.     

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.     

大鼠蛛网膜下隙出血脑血管痉挛模型的建立

目的 采用两种注血方法建立大鼠蛛网膜下隙出血早期脑血管痉挛模型,比较两种制备方法对脑血管痉挛程度的影响.方法 分别采用枕大池一次注血和二次注血法制备蛛网膜下隙出血早期基底动脉痉挛动物模型,HE染色观察基底动脉形态变化,计算基底动脉血管横截面积;透射电子显微镜观察基底动脉超微结构变化.结果 光学显微镜观察,枕大池一次注血组和二次注血组大鼠颅底有凝血块沉积;一次注血组大鼠基底动脉血管横截面积略有缩小[(2.68±0.48)×10-2mm2],但与假手术组比较差异无统计学意义(q=2.630,P=0.070),二次注血组大鼠基底动脉血管横截面积[(2.41±0.24)×10-2mm2]显著小于假手术组(q=4.660,P=0.003),但与一次注血组之间差异无统计学意义(q=2.031,P=0.166).电子显微镜观察,一次注血组大鼠基底动脉内弹力层局部变薄,内皮细胞胞质少量空泡形成,胞膜损伤崩解,中膜平滑肌细胞空泡样变性;二次注血组大鼠中膜平滑肌细胞和内皮细胞胞质空泡样变性,内皮细胞胞膜受损.结论 经枕大池注血法可复制蛛网膜下隙出血早期脑血管痉挛动物模型,以二次注血法所复制的脑血管痉挛模型更为稳定.     

兔脑血管痉挛模型的建立及DSA和TCD对其的评价比较

目的 建立简便可靠的兔蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后脑血管痉挛的模型,并运用数字减影血管造影(digital subtraction angiography,DSA)、经颅多普勒(transcranial doppler,TCD)对基底动脉痉挛进行评价比较.方法 新西兰纯种家兔36只,随机分为第一次注血后3 d,5 d,7 d,10 d,14 组(n=6),经颈枕部切开行枕大池二次注血来制作SAH模型,另6只为假注血组,并用DSA及TCD对基底动脉的管径及血流速率进行检测.结果 兔二次注血SAH模型很好地模拟了人类SAH后迟发性脑血管痉挛的病理过程,DSA和TCD的结果 郁显示基底动脉狭窄随时间逐渐加重,7 d左右到达高峰,而后义逐渐减轻,两者显示了很好的一致性.结论 经颈枕部切升枕大池二次注血兔脑血管痉挛模型的制作简便、易行、可靠,TCD对兔基底动脉痉挛程度的评价比DSA更简便易行.     

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.     

蛛网膜下腔出血后基底动脉和软脑膜动脉缝隙连接蛋白表达与血管痉挛的关系

目的探讨蛛网膜下腔出血(SAH)后颅内基底动脉和软脑膜动脉痉挛与缝隙连接蛋白的相关性。方法选取健康成年雄性SD大鼠90只,并随机分为对照组、假手术组、SAH模型组、18β-甘草次酸(18β-GA)干预组(以下简称干预组)。采用改良版颅内视交叉前池二次注血法建立SAH模型。于造模后7 d对各组SD大鼠进行Garcia神经行为学评分;对全脑切片行HE染色法后运用软件系统测量大鼠软脑膜动脉直径;运用压力肌动图检测大鼠基底动脉直径;采用Western-blot法测量软脑膜动脉和基底动脉缝隙连接蛋白40(Connexin 40, Cx40)、Cx43和Cx45的表达水平,采用Pearson相关性分析血管痉挛与缝隙连接蛋白之间的相关性。结果与假手术组比较,SAH模型组大鼠神经行为学评分明显降低(P0.05),软脑膜动脉直径和基底动脉直径及其Cx40表达均降低(P0.05),而软脑膜动脉和基底动脉Cx43和Cx45表达升高(P0.05),与SAH模型组比较,干预组大鼠神经行为学评分高(P0.05),软脑膜动脉直径和基底动脉直径及Cx40明显增加(P0.05),而软脑膜动脉和基底动脉Cx43和Cx45表达均明显降低(P0.05)。软脑膜血管直径和基底动脉直径与Cx40表达水平均呈正相关(r=0.749,P0.05;r=0.866,P0.05),而与Cx43和Cx45均呈负相关(r=-0.836),P0.05;r=-0.697,P0.05;r=-0.411,P0.05;r=-0.979,P0.05)。结论 SAH后软脑膜动脉和基底动脉发生血管痉挛,且其血管直径与Cx40呈正相关,与Cx43、Cx45呈负相关。     

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。     

eNOS与蛛网膜下腔出血后脑血管痉挛的关系

目的 探讨内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)在蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后不同时间点基底动脉表达情况及其与脑血管痉挛的关系.方法 新西兰大白兔72只,随机分成两组:(1)对照组(12只);(2)SAH组(60只).SAH组进一步按时间随机分为术后1d、3d、5d、7d、10d5个亚组,每组12只.采用枕大池二次注血法建立兔SAH模型.二次注血后在各个时间点采用灌流固定法处死动物,测定基底动脉横截面积判断有无脑血管痉挛.应用Western blotting分别观察eNOS在基底动脉中的表达情况.结果 枕大池二次注血法成功制作SAH模型,基底动脉发生了不同程度的血管痉挛.Western blotting观察到eNOS在对照组大量表达,实验组第1天表达开始减少,第5天表达水平最低,之后表达逐渐增加.结论 SAH后eNOS表达水平的变化与脑血管痉挛发展的时相性具有一定的一致性,eNOS蛋白减少可能参与蛛网膜下腔出血后脑血管痉挛的发展.     

SAH后迟发性脑血管痉挛中ICAM-1的表达变化及ICAM-1单克隆抗体和甲基强的松龙的防治作用

目的检测自发性蛛网膜下腔出血(SAH)后迟发性脑血管痉挛(DCVS)动脉中内皮细胞间粘附分子-1(ICAM-1)表达变化,同时评价ICAM-1单克隆抗体和甲基强的松龙的防治效果。方法中国白兔48只,体重2～2.5kg,随机分为四组。A组:正常对照组(n=8),不做枕大池穿刺注血,Day0处死;B组:SAH组(n=24),采用枕大池二次注血SAH模型,即在Day0和Day2第一、二次枕大池注血,并分别在Day4、Day7和Day14分三批处死,每批8只;C组:SAH+ICAM-1单克隆抗体和甲基强的松龙组(n=8),枕大池注射ICAM-1单克隆抗体10μg,耳缘静脉注射甲基强的松龙30mg/kg,并在Day7处死;D组:SAH+生理盐水组(n=8),用等量37℃生理盐水代替ICAM-1单克隆抗体和甲基强的松龙,第一次注血后Day7处死。结果A组动物基底动脉组织结构正常。B组动物基底动脉在Day4、Day7时出现血管痉挛,Day7时最严重,Day14时血管痉挛缓解。C组动物基底动脉痉挛明显缓解(P<0.05)。而D组动物基底动脉脑血管痉挛无缓解。B组动物基底动脉壁ICAM-1表达于day4开始增加,day7达到高峰,day14降至正常水平。C组ICAM-1表达明显减弱,与B组(day7)和D组比较有显著差异(P<0.01)。结论在兔SAH后迟发性脑血管痉挛中ICAM-1表达变化与DCVS炎症反应时相一致。应用ICAM-1单克隆抗体和甲基强的松龙,可抑制炎症反应的启动和进展,减轻炎症反应从而缓解DCVS。     

Academic Enhancement site

This off-reservation boarding school serves over 600 students in grades 4-12; approximately 85% of the students reside in campus dormitories. After having documented significant improvement on a number of outcomes during a previous High Risk Youth Prevention demonstration grant, the site submitted a Therapeutic Residential Model proposal, requesting funding to continue successful elements developed under the demonstration grant and to expand mental health services. The site received Therapeutic Residential Model funding for school year 2001-2002. Once funds were received, the site chose to shift Therapeutic Residential Model funds to an intensive academic enhancement effort. While not in compliance with the Therapeutic Residential Model initiative and therefore not funded in subsequent years, this site created the opportunity to enhance the research design by providing a naturally occurring placebo condition at a site with extensive cross-sectional data baselines that addressed issues related to current federal educational policies.     

Recording electrode site nomenclature

Several additions and extensions to the 10-20 System have been proposed. The major sets of proposals are reviewed here. The principal uses of these additional scalp recording sites are for clinical evoked potential testing and for acquisition of EEGs from very large numbers of scalp sites. Three separate systematic nomenclatures have arisen for filling in the half-step gaps in the 10-20 placements. These are compared and contrasted here.     

Remote site extradural haematoma

Postoperative extradural haematoma is a well described and serious complication of intracranial procedures that usually occurs at the site of the operation. Remote site extradural haematoma (that occurring distant to the site of the craniotomy) is relatively rare and may occasionally cause significant neurological morbidity or even death. We report this rare complication in an 18-year-old male who underwent craniotomy for excision of recurrent left temporal ganglioglioma.     

L3 Therapeutic Model site

L3 is an intertribal residential school enrolling approximately 200 students in grades 5-8 from tribes in the northern Midwest. As a result of successful grant-writing which espoused Circle of Courage and Asset-Building, the school built up an impressive configuration of programs funded by a variety of sources, including a cadre of mental health professionals, and began increasingly to rely on their assessments and services. First funded by the Therapeutic Residential Model program in the 2002-2003 school year, L3 used the funding that year primarily to increase professional-level services of a psychiatrist and psychologist, and to maintain or expand programs which would otherwise have been terminated as funding from short-term sources was running out. Evaluation of this project began in January 2003, when the site was assessed and determined to be strongly oriented toward provision of Level Three, or professional-level, psychiatric and medical mental health services. The initial evaluation report identified a low retention rate and raised concerns that the presence of more than 200 staff on campus had resulted in a diffusion of responsibility, lack of consistency, and duplication or redundancy of services; that elements of the environment appeared to be detrimental to social development and emotional stability; and that an unusually high proportion of students were receiving psychiatric diagnoses and medication. The site was asked to address these issues, and additional funding was provided to bolster lower levels of triage by adding paraprofessional case managers to advocate or students and coordinate provision of services for them. Retention remained low at this site throughout the course of funding, and there were a high number of assaults and psychiatric hospitalizations compared with other sites.     

Level 2 Therapeutic Model site

L2, one of the original sites first funded under the Therapeutic Residential Model Initiative in 2001-2002, is operated as a peripheral dormitory. This dormitory cares for 185 boys and girls in grades 1-12 who attend local public schools. L2 presented an outstanding proposal which identified gaps in services and presented a reasonable budget to address those gaps by adding additional mental health services and increasing the number of residential and recreation staff. With only minor modifications to this budget, the site efficiently and effectively implemented the strategies it had proposed and utilized evaluation feedback to fine-tune systems and maximize positive outcomes. The Therapeutic Residential Model funds enabled the site to move from a functional dormitory to a therapeutic residential situation where the needs of students are assessed and addressed. Outcome indicators in spring 2002, 2003, 2004, and 2005 showed impacts in a number of areas when compared with the baseline year of 2000-2001: Retention of students steadily increased going from 40.7% in 2000-2001 to 68.4% in 2004-2005; 75 students graduated from high school during the four Therapeutic Residential Model years, compared with 41 in the preceding four years; Academic Proficiency and ACT scores improved significantly; Thirty-day cigarette use dropped from 62% in spring 2001 to 38% in spring 2005 among 7th and 8th graders, from 58% to 33% among 9th and 10th graders, and from 72% to 29% among 11th and 12th graders; Alienation indices showed an increase in feelings of inclusion and a decrease in lack of meaning. This site is an outstanding example of what can be done with a well-designed and responsibly implemented Therapeutic Model Program, and the measurable impacts which can result from such strategic use of resources.     

Level 1 Therapeutic Model site

This site is an intertribal residential grant school annually enrolling over 250 students in grades 1-8 from tribes located in three states on the Northern Great Plains. From its inception in 1890, the boarding school's mission has been to provide services for young children in need of a safe and supportive living and learning environment. For over a decade, this site has used strategies centered on respecting children, structuring students' time, and providing the therapeutic benefits of a well-maintained campus. This site also has a long history of believing in each child's inherent value and potential. When Therapeutic Residential Model funding commenced at the midpoint of the 2002-2003 school year, L1 focused these new resources on strengthening and refining its program. The number of personnel positions increased from 98 to 135, with new positions principally going to dormitory staff and four Masters-level counselor positions. This increase in staff allowed L1 to proactively address the children's developmental needs. The site also adopted and implemented the Applied Humanism caregiving model. In accordance with Applied Humanism, an interview was utilized that allowed the site to identify and hire applicants possessing the attitudes and skills necessary to be good caregivers, existing staff were trained so that they understood the kind of caregiving that would be expected of them, supervision procedures and practices were implemented that supported and encouraged good caregivers and provided time-limited assistance to those who were not, and relevant agency policies and procedures were revised as needed to align with the Applied Humanism philosophy. In addition, the Morningside program was brought in to systematically address the students' academic lags in reading. The results of implementing the Therapeutic Residential Model were a reduction in behavioral incidents, a decrease in the amount of money spent on external mental health services, an increase in the retention rate, an increase in academic skills in selected areas, and higher scores on pre-post measures of adjustment, interpersonal relationships, and adaptability.