Cystatin C对SAH后早期脑血管痉挛防治及与自噬相关性的实验研究

目的 观察SD大鼠蛛网膜下腔出血(subarachnoid hemor rhage,SAH)后自噬在基底动脉壁的表达,探讨半胱氨酸蛋白酶抑制剂C(Cystatin C)对SD大鼠SAH后早期脑血管痉挛(cerebral vasospasm,CVS)的干预作用,并分析其与自噬(autophagy)的相关性.方法 40只SD大鼠随机分为对照组(A组)、蛛网膜下腔出血组(B组)、安慰剂组(C组)、Cystatin C治疗组(D组),每组10只.对照组大鼠在枕大池单纯注入生理盐水,其余三组大鼠在枕大池一次性注血制作SAH模型,治疗组在注血前30min经枕大池注入Cystatin C水溶剂0.1ml(10μg/0.1ml),安慰剂组在注血前30min经枕大池注入等量生理盐水.均于术后48h处死大鼠,取脑基底动脉部位标本作病理检查,测定基底动脉内径周长和血管壁厚度,以此评价有无脑血管痉挛及程度;应用自噬标记抗体LC3和Beclin-1对各组大鼠基底动脉行Western blot分析.结果 SAH组和安慰剂组大鼠基底动脉内径周长较对照组明显缩短(P<0.01),血管壁厚度明显增加(P<0.01).Cystatin C治疗组基底动脉内径周长增大,管壁增厚程度减轻以及CVS值下降,与SAH组及安慰剂组比较差异有统计学意义(P<0.01).Western blot分析显示自噬相关指标LC3和Beclin-1在对照组呈低表达,在SAH组及安慰剂组表达明显增高,与对照组比较差异有统计学意义(P<0.05).在Cystatin C治疗组,LC3 和Beclin-1的表达进一步增高,与SAH组及安慰剂组比较差异有统计学意义(P<0.01).结论 SAH后自噬在基底动脉壁被激活,提示自噬可能作为一种保护机制参与了CVS的病理及生理过程,经枕大池注入Cystatin C能诱导自噬在基底动脉壁的高表达,对CVS有一定的防治作用.     

大鼠蛛网膜下腔出血后额叶脑组织转化生长因子β_1的表达

目的探讨蛛网膜下腔出血后大鼠额叶脑组织中转化生长因子β1表达的变化及其意义。方法枕大池2次注血法制备蛛网膜下腔出血模型。30只成年健康雄性SD大鼠,随机分为对照组(仅在蛛网膜下腔注入等量生理盐水,n=6)和SAH组(n=24),其中SAH组分别于第1、3、5、7天随机均分为4组(n=6)。通过HE染色光镜下观察基底动脉的病理学变化,测定血管腔内径,免疫组化法检测各组大鼠额叶脑组织中TGF-β1在不同时间点的表达。结果与对照组比较,SAH各组基底动脉内径明显减小(P<0.01);SAH1 d时额叶脑组织TGF-β1表达增加,3 d时达高峰,而5、7 d时低于1、3 d(P<0.01),但仍高于对照组(P<0.01)。结论SAH后额叶脑组织中TGF-β1的表达发生了变化,并与CVS有关,提示TGF-β1参与了CVS的病理过程。     

大鼠蛛网膜下腔出血后NLK在基底动脉壁的表达变化

目的 探讨大鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后Nemo样激酶(nemo-like kinase,NLK)在基底动脉壁中的表达变化及其与脑血管痉挛(cerebral vasospasm,CVS)的关系.方法 60只健康成年SD大鼠随机分为对照组(NS组)和SAH组,SAH组又分为1d、3d、5d、7d和14d5个亚组,各10只大鼠.应用枕大池一次注血法制成SAH动物模型;通过HE染色光镜下观察基底动脉的病理学变化,测定血管壁厚度及血管腔内径,计算出不同时期的基底动脉血管腔横截面积,以此评价脑血管痉挛;并应用免疫组化法及Western bolt评估大鼠基底动脉管壁NLK在不同时间点的表达.结果 HE染色显示SAH组大鼠基底动脉管腔狭窄,内膜皱褶,平滑肌细胞肥大,3d时病理学变化最显著,管腔狭窄明显,血管腔横截面积约为对照组的43.5%,且管壁明显增厚,约为对照组的1.7倍,之后狭窄程度逐渐减轻,14d时接近正常.SAH后基底动脉壁NLK逐渐减少,表达低谷为注血后3d,随后开始表达增加,14 d基本恢复至正常水平.结论 通过构建大鼠枕大池一次注血模型,能够观察到基底动脉发生明显的细胞形态改变、管壁的增厚及管腔的狭窄,提示SAH后存在CVS;SAH后NLK在基底动脉壁的表达先下降后上调,并与CVS的时相基本一致,提示NLK可能参与CVS的病理过程.     

大鼠蛛网膜下腔出血后额叶脑组织转化生长因子β1的表达

目的探讨蛛网膜下腔出血后大鼠额叶脑组织中转化生长因子β1表达的变化及其意义。方法枕大池2次注血法制备蛛网膜下腔出血模型。30只成年健康雄性SD大鼠，随机分为对照组（仅在蛛网膜下腔注入等量生理盐水，n=6）和SAH组（n=24），其中SAH组分别于第1、3、5、7天随机均分为4组（n=6）。通过HE染色光镜下观察基底动脉的病理学变化，测定血管腔内径，免疫组化法检测各组大鼠额叶脑组织中TGF-β1在不同时间点的表达。结果与对照组比较，SAH各组基底动脉内径明显减小（P〈0．01）；SAH1d时额叶脑组织TGF-β1表达增加，3d时达高峰，而5、7d时低于1、3d（P〈0．01），但仍高于对照组（P〈0．01）。结论SAH后额叶脑组织中TGF-β1，的表达发生了变化，并与CVS有关，提示TGF—β1参与了CVS的病理过程。     

兔脑血管痉挛后大脑皮质神经胶质原纤维酸性蛋白表达及尼莫地平的神经保护作用

目的:研究脑血管痉挛(cerebralvasospasm,CVS)后兔大脑皮质神经胶质原纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)表达和尼莫地平的神经保护作用。方法:实验于2004-02/07在唐都医院中心实验室进行。枕大池二次注血法制备兔CVS模型,28只新西兰兔随机分为3组:假手术组4只,蛛网膜下腔出血(subarachnoidhemorrhage,SAH)组和尼莫地平组各12只,后两组按照2,24h,3,7d各时相点均分为4组(n=3)。全部动物于首次注血(或生理盐水)前及处死前行脑血管造影(DSA),测量基底动脉直径。二次注血后处死,苏木精-伊红染色观察兔基底动脉及大脑皮质形态学改变,免疫组织化学染色检测皮质GFAP的表达。结果:与假手术组相比,SAH组和尼莫地平组基底动脉直径明显减小(P=0.036),苏木精-伊红染色示SAH组、尼莫地平组基底动脉出现不同程度的管壁痉挛或增厚;3,7d时大脑皮质神经元明显缺血性改变;GFAP二次注血后2h大量表达,24h达高峰,3d仍有表达,7d时明显下降;与SAH组同时点比较,尼莫地平组血管痉挛减轻,皮质神经元缺血明显改善,GFAP表达明显减少(P<0.001)。结论:星形胶质细胞在SAH后脑血管痉挛所致迟发性脑缺血发挥重要作用,尼莫地平对其缺血损伤有神经保护作用,并能够减少GFAP蛋白表达。     

兔脑血管痉挛后大脑皮质神经胶质原纤维酸性蛋白表达及尼莫地平的神经保护作用

目的：研究脑血管痉挛(cerebral vasospasm，CVS)后兔大脑皮质神经胶质原纤维酸性蛋白(glial fibrillary acidic protein，GFAP)表达和尼莫地平的神经保护作用。方法：实验于2004—02／07在唐都医院中心实验室进行。枕大池二次注血法制备兔CVS模型，28只新西兰兔随机分为3组：假手术组4只，蛛网膜下腔出血(subarachnoid hemorrhage，SAH)组和尼莫地平组各12只，后两组按照2，24h，3，7d各时相点均分为4组(n=3)。全部动物于首次注血(或生理盐水)前及处死前行脑血管造影(DSA)，测量基底动脉直径。二次注血后处死，苏木精-伊红染色观察兔基底动脉及大脑皮质形态学改变，免疫组织化学染色检测皮质GFAP的表达。结果：与假手术组相比，SAH组和尼莫地平组基底动脉直径明显减小(P=0．036)，苏木精一伊红染色示SAH组、尼莫地平组基底动脉出现不同程度的管壁痉挛或增厚；3，7d时大脑皮质神经元明显缺血性改变；GFAP二次注血后2h大量表达，24h达高峰，3d仍有表达，7d时明显下降；与SAH组同时点比较，尼莫地平组血管痉挛减轻，皮质神经元缺血明显改善．GFAP表达明显减少(P&;lt;0．001)。结论：星形胶质细胞在SAH后脑血管痉挛所致迟发性脑缺血发挥重要作用，尼莫地平对其缺血损伤有神经保护作用，并能够减少GFAP蛋白表达。     

TCD在兔蛛网膜下出血后脑血管痉挛中与病理对照的实验研究

为评价经颅多普勒(TCD)在蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)中的作用,本文在兔枕大池注血建立SAH模型的基础上进行研究,结果表明TCD检测CVS平均血流速度变化与脑血管造影、脑脊液内皮素-1水平变化相关性良好,可早期发现.动态观察CVS的发生、发展过程,认为TCD对CVS的诊断确有实用价值.     

上胸段硬膜外阻滞对兔基底动脉痉挛后内皮素-1和内皮型一氧化氮合酶基因表达的影响

目的:建立兔上胸段硬膜外阻滞(HTEB)模型和蛛网膜下腔出血(SAH)模型,探讨预先HTEB对兔基底动脉痉挛后内皮素-1(ET-1)和内皮型一氧化氮合酶(eNOS)基因表达的影响.方法:切开置管法制备HTEB模型的新西兰免30只,随机分为正常组(N组)、单纯SAH组(S组)和HTEB+SAH组(HS组)3组,每组10只.HS组硬膜外以0.1%罗哌卡因1 mL/h持续给药至实验完毕.用枕大池穿刺一次注血法(0.5 mL/kg)制成兔SAH模型.饲养7 d,用经颅多普勒(TCD)监测第7天兔基底动脉平均血流速度(Vm);用RT-PCR方法测定基底动脉preproET-1 mRNA和eNOS mRNA的表达.结果:实验第7天TCD显示,S组的Vm较N组明显升高(P<0.05),HS组与N组组间无明显变化(P>0.05).preproET-1 mRNA的表达,与N组相比,S组(P<0.01)和HS组(P<0.05)均增高,S组高于HS组(P<0.05);eNOSmRNA的表达,与N组相比,S纽降低(P<0.01),HS组差异无显著性(P>0.05),S组低于HS组(P<0.01).结论:预先HTEB可减轻SAH后脑血管痉挛的程度,并可引起兔基底动脉内皮细胞下调preproET-1 mRNA和上调eNOS mRNA的表达水平.     

蛛网膜下腔出血后早期基底动脉ADAMTS-1表达与急性脑血管痉挛的相关性研究

目的:检测大鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后早期海马组织中含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶-1(a disintegrin-like and metalloproteinase with thrombospondin type l motifs,ADAMTS-1)在基底动脉中的表达,分析其与大鼠SAH后急性脑血管痉挛(cerebral vasospasm,CVS)的相关性,探讨其在SAH后早期脑损伤(earlybrain injury,EBI)中的作用。方法:健康雄性成年SD大鼠108只,体质量250～300g,随机分为SAH组(n=90)和假手术组(sham组,n=18)。SAH组取大鼠自体动脉血,用视交叉池注血法建立大鼠SAH模型(sham组注入等量0.9%氯化钠液)。将SAH组随机分为6h、12h、24h、48h、72h5个亚组,每个亚组18只,分别在相应时间点处死。用蛋白质印迹(Western-blot)方法及实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测SAH各亚组及sham组基底动脉ADAMTS-1的表达,同时用HE染色法对基底动脉进行形态学观察。探讨ADAMTS-1与实验性大鼠SAH后急性CVS的相关性。结果:与sham组相比,SAH 6h亚组基底动脉ADAMTS-1表达无显著差异;12h亚组基底动脉ADAMTS-1蛋白表达显著增高,24h亚组最高,48h亚组、72h亚组逐渐下降,但仍维持在较高水平。与sham组相比,SAH 6h亚组大鼠基底动脉变化不明显;12h亚组大鼠基底动脉管腔缩小,管壁增厚;24h亚组基底动脉痉挛最为明显;48h亚组基底动脉痉挛较24h亚组减轻;与24h亚组、48h亚组相比,72h亚组基底动脉管腔直径更大、管壁厚度更薄。ADAMTS-1蛋白表达与SAH后急性CVS呈正相关(r=0.916,P=0.003)。结论:大鼠SAH后早期基底动脉ADAMTS-1表达与急性CVS正相关,提示ADAMTS-1可能参与大鼠SAH后EBI的病理过程。     

释放脑脊液对治疗家兔蛛网膜下腔出血后脑血管痉挛的作用

目的探讨家兔蛛网膜下腔出血后释放脑脊液在治疗迟发型脑血管痉挛（DCVS）中的作用及其机制。方法采用枕大池二次注血法制作家兔蛛网膜下腔出血（SAH）模型,动物随机分为SAH组、治疗组和盐水对照组,各组分别于建模后1 d、3 d、5 d、7 d、10 d处死固定,取基底动脉。应用Le ica-Q550CW图像分析系统及软件测量基底动脉周长。数据结果应用统计软件SPSS 13.0进行处理。结果第3天,SAH组和治疗组的血管周长均小于盐水组,第5天治疗组的血管周长大于SAH组,并与盐水组无统计学差异。第7天治疗组血管周长大于SAH组及盐水组。结论 SAH后通过释放脑脊液对DCVS有治疗作用。     

氧化苦参碱对蛛网膜下腔出血大鼠脑血管痉挛的作用及机制研究

目的探讨氧化苦参碱（OMT）对蛛网膜下腔出血（SAH）大鼠脑血管痉挛的作用及机制的研究。 方法将200只雄性大鼠分成假手术组、SAH组、小剂量组和大剂量组,每组50只。采用枕大池二次注血法制作SAH大鼠模型,假手术组大鼠两次均注入等量生理盐水,其余三组在模型建立后分别给予1 ml的0.9%氯化钠注射液,60和120 mg/kg的OMT腹腔注射。在术后1、3、5、7和10 d每组分别处死10只大鼠,测量四组大鼠基底动脉血管直径,检测基底动脉内皮细胞核因子κB（NF-κB）、胞外信号调节激酶（ERK）1/2、p38丝裂原活化蛋白激酶（MAPK）、c-Jun氨基末端激酶（JNK）的蛋白相对表达量,以及基底动脉内皮细胞白细胞介素1β（IL-1β）、IL-6和肿瘤坏死因子α（TNF-α）的mRNA相对表达水平。 结果术后3、5、7和10 d,四组大鼠基底动脉血管直径（F = 11.897、28.957、14.785、8.381,P均< 0.05）,基底动脉内皮细胞NF-κB（F = 17.289、65.602、43.881、26.998,P均< 0.05）、ERK1/2（F = 204.331、145.948、107.442、30.332,P均< 0.05）、p38MAPK（F = 84.908、116.677、83.735、28.338,P均< 0.05）和JNK（F = 26.809、83.419、56.465、27.756,P均< 0.05）蛋白表达,以及基底动脉内皮细胞IL-1β（F = 66.625、262.620、283.499、218.081,P均< 0.05）、IL-6（F = 38.580、170.657、136.253、58.068,P均< 0.05）和TNF-α（F = 31.290、361.661、101.109、23.940,P均< 0.05）mRNA水平的比较,差异均有统计学意义（P均< 0.05）。其中术后3、5、7和10 d时间点上,假手术组和大剂量组大鼠基底动脉血管直径均较SAH组显著增大（P均< 0.05）,而这两组的基底动脉内皮细胞NF-κB、ERK1/2、p38MAPK和JNK的蛋白表达,以及IL-1β、IL-6和TNF-α的mRNA表达均显著少于SAH组（P均< 0.05）。但小剂量组的以上指标和SAH组比较差异均无统计学意义（P均> 0.05）。 结论OMT可改善SAH大鼠脑血管痉挛,其机制可能与OMT抑制脑血管内皮细胞NF-κB、ERK1/2、p38MAPK和JNK等信号通路,降低大鼠基底动脉血管炎症反应有关。     

Prevention of delayed cerebral vasospasm by continuous intrathecal infusion of glyceroltrinitrate and nimodipine in the rabbit model in vivo

Objective Intrathecal bolus administration of nitric oxide donors and calcium channel antagonists has been proposed to reduce cerebral vasospasm (CVS) in animal subarachnoid hemorrhage (SAH) models. Intrathecal continuous administration of these substances for CVS prevention has not been extensively evaluated. This study compared the efficacy of continuous intrathecal infusions of the NO donor glyceroltrinitrate and nimodipine in preventing delayed CVS associated with SAH in an animal model in vivo. Methods New Zealand White rabbits were randomly assigned to six groups: no SAH/NaCl, no SAH/NO, no SAH/nimodipine, SAH/NaCl, SAH/NO, or SAH/nimodipine. Glyceroltrinitrate (GTN) at 0.5 μg/μl (0.5 μl/h) or nimodipine at 0.2 μg/μl (10 μl/h) or NaCl was continuously infused into the cisterna magna via an Alzet osmotic pump from day 0 to day 5 after injection of 1.0 ml autologous blood. The magnitude of spasm in the basilar artery was determined by comparison of pre- and posttreatment angiography and was calculated as proportional change in intraluminal diameter based on automatic measurements. Results A total of 55 experiments and 110 angiograms were performed. SAH was associated with vasoconstriction of the basilar artery (SAH/NaCl group 19.85 ± 2.94%). Continuous intrathecal injection of GTN and nimodipine prevented SAH-induced CVS. There was significant prevention of CVS in animals treated with GTN (SAH/NO group 5.93 ± 5.2%, n = 11) and nimodipine (SAH/nimodipine group: 0.55 ± 2.66%, n = 9). There was no significant difference between the treatment groups and controls in prevention of CVS. Conclusions This study demonstrates that prophylactic continuous intrathecal administration of either GTN or nimodipine equally prevents SAH-associated CVS in an animal model. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the Cerebrovascular Research Fund from the Departments of Neurosurgery and Intensive Care Medicine (account no. 34-160), University of Berne, Switzerland.     

Origin of pretruncal nonaneurysmal subarachnoid hemorrhage: ruptured vein, perforating artery, or intramural hematoma?

Pretruncal (perimesencephalic) nonaneurysmal subarachnoid hemorrhage (SAH) is a benign variant of SAH. Although angiography fails to show a source of the hemorrhage, mild basilar artery narrowing may be observed. The cause of pretruncal nonaneurysmal SAH has not been established. Recent imaging studies have demonstrated that the center of this type of SAH is not around the mesencephalon but is in the prepontine or interpeduncular cistern with the hemorrhage closely associated with the basilar artery. We review the possible sources of hemorrhage in these cisterns and hypothesize that pretruncal nonaneurysmal SAH is caused by a primary intramural hematoma of the basilar artery. Such an intramural hematoma would explain bleeding under low pressure, the location of the hemorrhage anterior to the brainstem, and the typical findings of hemorrhage adjacent to the basilar artery lumen on magnetic resonance imaging and mild basilar artery narrowing on angiography. Although an intramural hematoma of the basilar artery would be easily identified at surgical exploration, such surgeries have never included the extensive base-of-the-skull approaches that are necessary to visualize the artery in the prepontine cistern.     

Potassium-channel openers KMUP-1 and pinacidil prevent subarachnoid hemorrhage-induced vasospasm by restoring the BKCa-channel activity

Alterations in the activity of vascular K channels are commonly associated with abnormalities in cerebral vascular function after subarachnoid hemorrhage (SAH). Subarachnoid hemorrhage-induced vasospasm remains incompletely understood; nevertheless, activation of K channels may be of benefit in relieving spastic constriction. This study was to examine whether the vasodilators KMUP-1 and pinacidil, a KATP-channel opener, have the ability to prevent SAH-induced vasospasm via the large-conductance Ca-activated K (BKCa) channels in cerebral arteries. Rats were divided into four groups including sham-operated, SAH, KMUP-1 treated, and pinacidil treated. Subarachnoid hemorrhage rats were induced by injecting autologous blood into cisterna magna, and then KMUP-1 or pinacidil (1 mg/kg) was injected intraperitoneally 1 and 24 h after SAH. Behavioral tests were assessed on day 2 after SAH before the rats were killed. Cerebral myocytes were enzymatically isolated from rat basilar arteries and used to monitor BKCa-channel activities. In isolated basilar arteries, KMUP-1-treated and pinacidil-treated rats showed normalized vascular reactivity. In whole-cell recordings, BKCa currents were attenuated in SAH rats compared with sham-operated rats. In inside-out patches, the conductance and voltage sensitivity of single BKCa-channels were unchanged among the four groups. In contrast, SAH rats showed markedly decreased BKCa-channel activity and β1-subunit expression associated Ca sensitivity that was further confirmed by immunofluorescence staining and Western blotting. Subarachnoid hemorrhage-induced deficits in motor function and BKCa-channel inhibition were improved by KMUP-1-treated and pinacidil-treated rats. In addition, SAH appears to modify BKCa-channel calcium sensitivity. KMUP-1 and pinacidil prevent SAH-induced vasospasm at least in part by the restoration of BKCa-channel activities.     

尼莫地平联合脑脊液置换治疗蛛网膜下腔出血疗效研究

目的：探讨尼莫地平联合脑脊液置换治疗蛛网膜下腔出血（SAH）的疗效。方法将122例SAH患者分成观察组和对照组,每组各61例。对照组采用尼莫地平常规治疗,观察组患者在对照组基础上加以脑脊液置换治疗。结果观察组左大脑前动脉（ACA）、右ACA、左大脑中动脉（MCA）、右MCA收缩峰流速值均高于对照组（P＜0．05）;观察组脑膜刺激征缓解率优于对照组（P＜0．05）;观察组血管痉挛、梗阻性脑积液、再出血发生率低于对照组（P＜0．05）。结论采用尼莫地平联合脑脊液置换治疗SAH ,能及时清除蛛网膜下腔积血,改善脑部血液循环,降低不良反应发生率,提高患者生存质量。     

Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) causes cerebral ischemia and infarction. To date, the pathogenesis and gene expression associated with vasospasm remain poorly understood. The present study used fluorescent differential display to identify differentially expressed genes in a rat model of SAH. By using quantitative RT-PCR, we found that heme oxygenase-1 (HO-1) mRNA was prominently induced in the basilar artery and modestly in brain tissue in a rat vasospasm model. A significant correlation was observed between the degree of vasospasm and HO-1 mRNA levels in the basilar arteries exhibiting vasospasm. Intracisternal injection of antisense HO-1 oligodeoxynucleotide (ODN) significantly delayed the clearance of oxyhemoglobin and deoxyhemoglobin from the subarachnoid space and aggravated angiographic vasospasm. Antisense HO-1 ODN inhibited HO-1 induction in the basilar arteries but not in the whole brain tissue. This phenomenon was not observed in the nontreated, sense HO-1 ODN-treated, or scrambled ODN-treated arteries. We report the protective effects of HO-1 gene induction in cerebral vasospasm after SAH, a finding that should provide a novel therapeutic approach for cerebral vasospasm.