家兔自发性蛛网膜下腔出血早期脑损伤模型的初步研究

目的：建立一种可重复的、有典型神经损伤症状的自发性蛛网膜下腔出血（sAH）动物模型,为研究蛛网膜下腔出血后早期脑损伤（EBI）的机制提供可靠的动物模型。方法：选用新西兰大耳白兔60只,随机分为实验组、对照组、空白组。采用枕大池穿刺一次注入自体动脉血法建立SAH模型。在4h、8h、12h、24h、48h、72h时间点,观察比较行为学与脑组织形态学的变化。结果：（1）实验组枕大池和蛛网膜下腔内发现大量的血凝块和血液。（2）实验组光镜下可观察到蛛网膜下腔大量红细胞,神经元细胞水肿,电镜下可见胶质细胞空泡样改变,神经元细胞线粒体肿胀,髓鞘内存在空泡和板层分离现象。（3）实验组造模后均出现明显的神经系统损伤,对照组4h出现典型神经系统损伤表现,12h后恢复正常,空白组未见神经系统损伤表现。结论：枕大池一次注血法是一种简便、可重复的症状性自发性蛛网膜下腔出血早期脑损伤模型,适宜于研究早期脑损伤的致病机制。     

褪黑素对蛛网膜下腔出血后继发性脑损伤及认知功能的影响及其机制研究

目的:探讨褪黑素对蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后神经元细胞凋亡、坏死及继发性认知功能障碍的影响。方法:选择80只成年健康雄性SD大鼠,随机分为四组:正常组(n=20)、单纯SAH组(n=20)、SAH+安慰剂治疗组(n=20)和SAH+褪黑素治疗组(n=20),经大鼠自体尾动脉(股动脉)非肝素化动脉血在20 s内注入视交叉池建立蛛网膜下腔出血模型,褪黑素注射剂量为150 mg/kg,1次/12 h,在蛛网膜下腔出血建模后48h处死各组部分大鼠,取血凝块周围的皮层脑组织(额颞底)做标本,通过TUNEL荧光染色及Fluoro-Jade B荧光染色测定神经元凋亡及坏死的情况,各组剩余大鼠在SAH后48小时开始通过Morris水迷宫试验测试其认知功能。结果:SAH组大鼠的活动功能评分、Morris水迷宫试验的逃避潜伏期及总路程、神经元细胞凋亡和坏死的百分比均较正常对照组大鼠显著升高(P0.01),而褪黑素治疗组以上指标均显著低于安慰剂治疗组(P0.05),但SAH组和安慰剂组之间以上指标比较均无统计学意义(P0.05)。结论:褪黑素可能通过减少神经元细胞的凋亡和坏死改善蛛网膜下腔出血后大鼠的认知功能障碍。     

去铁敏干预蛛网膜下腔出血后溶酶体组织蛋白酶表达研究

目的:探讨大鼠蛛网膜下腔出血(SAH,Subarachnoid Hemorrhage)早期脑损伤中溶酶体组织蛋白酶B(CathepsinB)、溶酶体组织蛋白酶D(Cathepsin D)的表达变化及去铁敏(DFO)对其的影响.方法:将24只SD大鼠随机分为四组:正常对照组(6只),SAH模型组(6只),安慰剂组(6只),DFO药物组(6只),视交叉前池注血法(APC)制作大鼠SAH模型,免疫组化分别检测CathepsinD、CathepsinB的蛋白表达,干湿法测定48 h脑含水量.结果:与正常组比较,SAH模型组大鼠48 h后CathepsinD、Cathepsin B的蛋白表达明显增强,水肿指数增高,DFO药物组大鼠48h后Cathepsin D、Cathepsin B的蛋白表达较SAH组明显减低,水肿指数减低.结论:Cathepsin D、Cathepsin B在大鼠蛛网膜下腔出血后表达增强,DFO能减少其表达并对早期脑损伤有保护作用,溶酶体可能参与了蛛网膜下腔出血早期脑损伤的过程,稳定溶酶体膜,减少Cathepsin B/D的释放可能为SAH后早期脑损伤提供新的治疗途径.     

Cystatin C对大鼠蛛网膜下腔出血后的自噬的影响

目的:探讨CystatinC对大鼠蛛网膜下腔出血后自噬的影响.方法:成年雄性SD大鼠48只,随机分为假手术组(n＝8)、SAH组(n=8)、安慰剂组(n=8)、Cystatin C组(分为低浓度(2μg/ml)、中浓度(5μg/ml)、高浓度(10 μg/ml),每个亚组(n=8)).采用视交叉池注血技术建立大鼠蛛网膜下腔出血模型.各组于建模后48 h后取颞底脑皮层,分别用western blot技术和免疫组化技术测定脑皮层组织中自噬标志物LC3和Beclin-1的变化,透射电镜观察脑皮层组织中自噬体和自噬溶酶体.结果:与假手术组相比,SAH组、安慰剂组大鼠脑皮层组织中自噬标志物Beclin-1及LC3于蛛网膜下腔出血后48 h后明显升高;与SAH组、安慰剂组相比,Cystatin C干预后大鼠脑皮层中Beclin-1及LC3表达水平进一步升高,且与浓度呈正相关,透射电镜下并可见Cystatin C组大鼠脑皮层组织中自噬体和自噬溶酶体数量增加.结论:Cystatin C可以进一步诱导激活大鼠蛛网膜下腔出血后脑组织的自噬,可能对蛛网膜下腔出血后的早期脑损伤起保护作用.     

二次造影对首次造影阴性的自发性蛛网膜下腔出血的价值

目的:探讨二次造影对首次血管造影阴性的自发性蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)的价值.方法:回顾性分析2009年7月至2011年7月我院共收治的自发性蛛网膜下腔出血患者115例,在确诊蛛网膜下腔出血后1-3天行股动脉插管全脑血管造影并3D重建和颅脑CT平扫.首次DSA结果阴性者于2-8周行二次全脑血管造影.结果:115例中83例共发现颅内动脉瘤87个,9例为脑血管畸形、Moyamoya病等,23例首次造影阴性.但23颅脑CT显示弥漫性蛛网膜下腔出血16例,中脑周围蛛网膜下腔出血5例,局限于一个脑池或脑叶内蛛网膜下腔出血1例,颅脑CT阴性1例.二次造影后在16例弥漫性蛛网膜下腔出血患者中共发现动脉瘤3例.结论:二次造影对首次血管造影阴性的自发性蛛网膜下腔出血患者具有较高的临床意义,尤其是对于首次造影高度怀疑动脉瘤的弥漫性蛛网膜下腔出血患者,进行二次造影是必要的.     

动脉瘤性蛛网膜下腔出血后早期脑损伤的研究现状

蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是临床上最具破坏性和难以治疗的神经疾病之一,尤其是动脉瘤性蛛网膜下腔出血(aneurysmal subarachnoid hemorrhage,aSAH)。尽管经过数十年的研究,我们对aSAH病理生理学的认识和动脉瘤破裂的处理(包括手术夹闭或血管内治疗)有所改善,但a SAH仍然是中国和世界范围内严重和显著的健康问题。aSAH后造成脑损伤的机制至今仍不清楚。传统上,在动脉瘤破裂后3～7天内出现的脑动脉痉挛被认为是脑损伤的最重要决定因素,所以大多数的体内研究都着重强调了迟发性脑血管痉挛在aSAH后的病理生理学或形态学上变化的基本机制。然而,最近的研究表明,预防迟发性脑血管痉挛的发生并不能改善患者的预后。这一发现最终将重点转向了早期脑损伤对aSAH患者预后的影响上。大量的证据表明,脑损伤从动脉瘤破裂开始,随着时间的推移而发生变化,在患者的预后中起着重要的作用。因此,文章将从a SAH后脑损伤机制开始,回顾aSAH的研究历史,以及早期脑损伤的当前研究现状和未来发展。     

甲状腺素对动脉瘤性蛛网膜下腔出血大鼠脑缺氧诱导因子-1α表达的影响及其机制*

目的:探讨甲状腺素(T4)对动脉瘤性蛛网膜下腔出血后大鼠脑缺氧诱导因子-1α(HIF-1α)表达的调节及其机制。方法:72只雄性成年SD大鼠随机分为以下4组:蛛网膜下腔出血模型组(SAH)(n=18)、蛛网膜下腔出血+甲状腺素组(SAH+T4)(n=18)、蛛网膜下腔出血+溶剂组(SAH+溶剂组)(n=18)、假手术组(n=18)。颈内动脉穿刺法建立蛛网膜下腔出血的模型,术后行颅脑CT平扫,建模后立即开始给药,按3 μg/100 g腹腔注射,每隔24 h一次,连续3 d,SAH+T4组予甲状腺素干预,SAH+溶剂组予等体积溶剂干预,均在建模后72 h处死;各组6只大鼠经多聚甲醛灌注处死后石蜡包埋切片行免疫组化染色检测HIF-1α及p-Akt蛋白、6只用TUNEL法检测凋亡,6只用干湿重法做脑水肿含量检测。结果:建模成功后SAH组及SAH+T4组、SAH+溶剂组大鼠的脑组织肿胀明显,蛛网膜下腔可见暗红色血凝块。SAH组神经行为学评分、脑含水量、凋亡率、HIF-1α蛋白、p-Akt蛋白均较假手术组明显增高(P＜0.05);SAH+T4组神经行为学评分、HIF-1α蛋白、p-Akt蛋白均较SAH组明显增高,其脑含水量、凋亡均较SAH组明显减少(P＜0.05)。结论:使用T4替代治疗可以上调动脉瘤性蛛网膜下腔出血后大鼠脑HIF-1α蛋白表达水平,可能是通过激活三磷酸肌醇激酶／蛋白激酶B(PI3K/Akt)信号通路,使凋亡率减小,最终大鼠行为学得以改善,对大鼠脑产生保护作用。     

新生儿缺氧缺血性脑病MRI表现及病理基础与预后评估

目的:探讨新生儿缺氧缺血性脑病(HIE)的MRI表现及病理基础与预后评估。方法:回顾性分析42例HIE患儿初期的MRI表现。结果:MRI能显示HIE患儿的9种早期异常征象,其中T1WI皮层高信号,侧脑室周围深部白质高信号以及蛛网膜下腔出血(SAH),硬膜下出血(SDH)弥漫性脑水肿,T1WI双侧基底节高信号伴内囊后肢高信号消失,弥漫性脑实质出血,灰白质分界不清,大面积脑梗死,后五种征象提示预后不良,常会留下严重神经系统的后遗症;侧脑室周围病变一般会影响小儿运动功能。结论:MRI可以客观反映新生儿HIE早期脑损伤的严重程度,并可早期评估预后。     

表没食子儿茶素没食子酸酯对蛛网膜下腔出血大鼠脑损伤的保护作用研究

目的:探讨表没食子儿茶素没食子酸酯(EGCG)对蛛网膜下腔出血(SAH)脑损伤的影响及其可能机制。方法:选取健康雄性Sprague-Dawley(SD)大鼠80只,按照随机数字表法分为假手术组(Sham组)、SAH组、SAH+EGCG组、蛛网膜下出血+生理盐水组(SAH+NS组),每组各20只。SAH造模成功后1d、3d,检测大鼠神经功能评分及脑组织含水量;造模成功3d后,脑组织石蜡切片HE染色观察脑细胞形态,并采用超敏C-反应蛋白(hs CRP)试剂盒检测血清hs CRP水平,通过Western blotting检测脑组织脑源性神经生长因子(brain derived neurotrophic factor,BDNF)及与其高度亲和的酪氨酸激酶受体B(tropomyosin-related kinase B,TrkB)的表达。结果:(1)SAH+EGCG组神经功能评分较Sham组明显降低,但较同期SAH组增高明显(P0.05);(2)与Sham组比较,SAH+EGCG组脑组织含水量明显增加,但与同期SAH组比较,SAH+EGCG组脑组织含水量明显降低(P0.05);(3)与SAH组比较,SAH+EGCG组血清hs CRP水平明显降低(P0.05);(4)与SAH组比较,SAH+EGCG组脑组织石蜡切片HE染色神经细胞水肿明显减轻,且坏死神经元减少,细胞核固缩明显减轻;(5)与SAH组比较,SAH+EGCG组脑组织BDNF、TrkB的表达量明显升高(P0.05)。结论:EGCG能够有效的保护大鼠SAH后的脑损伤,促进神经功能修复,减轻预后神经功能障碍,可能与其促进脑组织BDNF、TrkB的表达有关。     

奥拉西坦联合尼莫地平对自发性蛛网膜下腔出血患者血清IGF-1, sICAM-1 及sVCAM-1 水平的影响

目的:探讨奥拉西坦联合尼莫地平对自发性蛛网膜下腔出血患者血清胰岛素样生长因子-1(Insulin like growth factor-1,IGF-1)、可溶性细胞间黏附分子-1(Soluble intercellular adhesion molecule-1,s ICAM-1)及可溶性血管间内皮细胞黏附分子-1(Soluble vascular adhesion molecule-1,s VCAM-1)水平的影响。方法:选取我院收治的自发性蛛网膜下腔出血患者46例,随机分配为实验组与对照组,每组23例。对照组患者给予尼莫地平治疗,实验组在对照组的治疗基础上加用奥拉西坦。比较治疗前后两组患者血清IGF-1、s ICAM-1及s VCAM-1水平,同时比较治疗结束后两组患者的临床总有效率。结果:与治疗前相比,两组患者治疗后血清IGF-1水平升高,s ICAM-1及s VCAM-1水平降低(P0.05);且与对照组相比,实验组患者血清IGF-1水平较高,s ICAM-1及s VCAM-1水平较低,临床总有效率较高(P0.05)。结论:奥拉西坦联合尼莫地平能够提高自发性蛛网膜下腔出血患者的临床疗效,其机制与提高患者血清IGF-1水平,降低s ICAM-1及s VCAM-1水平有关。     

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.     

蛛网膜下腔出血对大鼠脑血流量和体感诱发电位的影响

目的：探讨蛛网膜下腔出血（ＳＡＨ）后脑血流量、体感诱发电位（ＳＥＰ）潜伏期的改变及其与一氧化氮（ＮＯ）的关系。方法：对假手术对照组和ＳＡＨ模型组大鼠检测２４ｈ局部脑血流量（ｒＣＢＦ）、ＳＥＰ潜伏期和血清及脑组织ＮＯ含量动态变化。结果：非开颅刺破Ｗｉｌｌｉｓ环的方法可成功地诱发ＳＡＨ。ＳＡＨ后ｒＣＢＦ立即降低,在２４ｈ内无恢复趋势。ＳＥＰ潜伏期于ＳＡＨ后１ｈ开始至２４ｈ明显延长。血清和脑组织ＮＯ含量     

Tert-Butylhydroquinone Alleviates Early Brain Injury and Cognitive Dysfunction after Experimental Subarachnoid Hemorrhage: Role of Keap1/Nrf2/ARE Pathway

Tert-butylhydroquinone (tBHQ), an Nrf2 activator, has demonstrated neuroprotection against brain trauma and ischemic stroke in vivo. However, little work has been done with respect to its effect on early brain injury (EBI) after subarachnoid hemorrhage (SAH). At the same time, as an oral medication, it may have extensive clinical applications for the treatment of SAH-induced cognitive dysfunction. This study was undertaken to evaluate the influence of tBHQ on EBI, secondary deficits of learning and memory, and the Keap1/Nrf2/ARE pathway in a rat SAH model. SD rats were divided into four groups: (1) Control group (n = 40); (2) SAH group (n = 40); (3) SAH+vehicle group (n = 40); and (4) SAH+tBHQ group (n = 40). All SAH animals were subjected to injection of autologous blood into the prechiasmatic cistern once in 20 s. In SAH+tBHQ group, tBHQ was administered via oral gavage at a dose of 12.5 mg/kg at 2 h, 12 h, 24 h, and 36 h after SAH. In the first set of experiments, brain samples were extracted and evaluated 48 h after SAH. In the second set of experiments, changes in cognition and memory were investigated in a Morris water maze. Results shows that administration of tBHQ after SAH significantly ameliorated EBI-related problems, such as brain edema, blood-brain barrier (BBB) impairment, clinical behavior deficits, cortical apoptosis, and neurodegeneration. Learning deficits induced by SAH was markedly alleviated after tBHQ therapy. Treatment with tBHQ markedly up-regulated the expression of Keap1, Nrf2, HO-1, NQO1, and GSTα1 after SAH. In conclusion, the administration of tBHQ abated the development of EBI and cognitive dysfunction in this SAH model. Its action was probably mediated by activation of the Keap1/Nrf2/ARE pathway.     

Local Delivery of Nimodipine by Prolonged-Release Microparticles—Feasibility,Effectiveness and Dose-Finding in Experimental Subarachnoid Hemorrhage

Background and Purpose

To investigate the effect of locally applied nimodipine prolonged-release microparticles on angiographic vasospasm and secondary brain injury after experimental subarachnoid hemorrhage (SAH).

Methods

70 male Wistar rats were categorized into three groups: 1) sham operated animals (control), 2) animals with SAH only (control) and the 3) treatment group. SAH was induced using the double hemorrhage model. The treatment group received different concentrations (20%, 30% or 40%) of nimodipine microparticles. Angiographic vasospasm was assessed 5 days later using digital subtraction angiography (DSA). Histological analysis of frozen sections was performed using H&E-staining as well as Iba1 and MAP2 immunohistochemistry.

Results

DSA images were sufficient for assessment in 42 animals. Severe angiographic vasospasm was present in group 2 (SAH only), as compared to the sham operated group (p<0.001). Only animals within group 3 and the highest nimodipine microparticles concentration (40%) as well as group 1 (sham) demonstrated the largest intracranial artery diameters. Variation in vessel calibers, however, did not result in differences in Iba-1 or MAP2 expression, i.e. in histological findings for secondary brain injury.

Conclusions

Local delivery of high-dose nimodipine prolonged-release microparticles at high concentration resulted in significant reduction in angiographic vasospasm after experimental SAH and with no histological signs for matrix toxicity.     

p53、BCL-2、Caspase－3等凋亡相关蛋白在大鼠脑损伤中的表达及意义

目的：检测脑外伤大鼠中p53、bcl-2及caspase-3表达,并分析其与脑外伤之间的关系,为脑损伤患者预后提供部分参数依据。方法：建立脑外伤大鼠实验动物模型,用免疫组化方法检测p53、Bcl-2和Caspase-3的表达。结果：脑创伤后在创伤周围区的神经元会发生凋亡,凋亡的发生可能与p53、bcl-2、caspase-3等基因及蛋白的调节有关。结论：脑创伤后在创伤周围区的神经元会发生凋亡,凋亡的发生可能与p53、bcl-2、caspase-3等基因及蛋白的调节有关。     

A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer''s statistical analysis.Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 μm ± 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 ± 3; p=0.004). There were no mortalities from cerebral vasospasm.Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated ^{7, 8}. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.     

大鼠脑血管痉挛时NO和ET—1变化及尼莫地平的影响

目的探讨蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)时脑组织一氧化氮(NO)和内皮素-1(ET-1)含量变化及尼莫地平(ND)对其影响。方法将135只Wistar大鼠随机均分为SAH组、ND处理组和假手术组,观察手术前后基底动脉管径,及24h内局部脑血流量(rCBF)、脑组织NO和ET-1含量动态改变,并行海马病理检查。结果SAH后rCBF明显而持续降低,基底动脉管径显著缩小;海马CAl区锥体细胞严重受损;脑组织NO和ET-1含量均在SAH后1～24h显著增加(P＜0．05～0．01)。ND处理后使上述异常变化均减轻。结论SAH后脑组织NO、ET-1增多可能参与了CVS所致脑损害过程,ND通过减轻CVS和拮抗脑组织NO及ET-1的病理性改变而发挥脑保护作用。