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、72 h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。     

单唾液酸神经节苷脂对大鼠体外循环脑损伤的影响

目的：单唾液酸神经节苷脂（GM-1）是神经细胞膜上的一种鞘糖脂,参与多种生理功能,具有抗氧化和神经保护作用,本课题主要探讨单唾液酸神经节苷脂对大鼠体外循环（CPB）脑损伤的影响。方法：成年雄性SD大鼠30只,14月龄,体重300-400g,采用随机数字表法,将大鼠随机分成3组（n=10）：假手术组（s组）、CPB组和GM-1组。采用右颈静脉腔房引流,右颈动脉灌注法建立大鼠CPB模型。CPB组和GM-1组行CPB1h,其中CPB组转流液中加入GM-120mg／kg,GM-1组给予等容量的生理盐水。CPB结束后3h和s组机械通气结束后3h时,断头取左侧脑组织,透射电镜下观察海马超微结构变化;TUNEL法检测海马神经元调亡情况。结果：电镜下CPB组海马可见异染色质明显边集、凝聚,线粒体嵴减少或空泡变性,细胞器消失等不可逆性的损伤改变;GM-1组神经元细胞核圆形,线粒体嵴少量减少,细胞器仍可见。与S组比较,CPB组和GM-1组海马神经元病理损伤严重,且凋亡神经元明显增多（P〈0．05）。与CPB组相比,GM-I组海马神经元病理损伤减轻,且凋亡神经元有所减少（P〈0．05）。结论：体外循环可导致以大鼠海马神经元超微结构发生改变为标志的脑损伤,GM-1可减轻CPB诱发的脑损伤,其机制可能与其抑制神经元凋亡有关。     

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.     

血红素氧合酶-1在脑损伤过程中的作用的研究进展

来源于出血后血红蛋白或衰老细胞释放的血红素能够诱导血红素氧合酶-1（HO-1,HSP-1）的表达。血红素氧合酶-1催化血红素生成气体介质一氧化碳,铁和胆绿素。胆绿素和它的代谢产物胆红素都是有效的抗氧化剂;同时铁诱导的铁蛋白和CO也发挥着各自的保护作用。因此,HO-1的表达被看作一种重要的保护机制。在各种不同的脑病理改变发生后,如蛛网膜下腔出血,脑梗死,创伤性脑损伤及神经变性疾病,HO-1明显表达于小胶质细胞,星形细胞和神经元细胞,从而发挥其重要脑保护作用。     

辛伐他汀对大鼠蛛网膜下腔出血后脑血管痉挛的治疗效果研究

目的:观察辛伐他汀对大鼠蛛网膜下腔出血后脑血管痉挛的治疗效果并探讨其可能机制。方法:将48只健康Wistar大鼠随机分为正常对照组、模型组、辛伐他汀组,每组各16只。模型组以及辛伐他汀组给予枕大池注入自体动脉血法以建立大鼠蛛网膜下腔出血后脑血管痉挛的动物模型,辛伐他汀组于第1次注血后开始给予辛伐他汀皮下注射,其余两组给予等体积生理盐水皮下注射。比较各组大鼠情况、组织学检查结果、蛛网膜下腔出血量评分、神经功能缺损评分、血管内径、血管舒张度、血管痉挛程度、eNOS以及TNF-α蛋白表达水平。结果:实验过程中,正常对照组无大鼠死亡,模型组大鼠死亡率为33.33%,辛伐他汀死亡率为6.67%,较模型组显著降低(P0.05)。治疗后,与正常对照组相比,模型组及辛伐他汀组大鼠蛛网膜下腔出血量评分及TNF-α阳性表达率较高(P0.05),神经功能缺损评分、血管内径、D/T及eNOS阳性表达率较低(P0.05);与模型组相比,辛伐他汀组蛛网膜下腔出血量评分及TNF-α阳性表达率较低(P0.05),神经功能缺损评分、血管内径、D/T及eNOS阳性表达率较高(P0.05)。结论:辛伐他汀可有效改善蛛网膜下腔出血后脑血管痉挛,这可能与其上调eNOS蛋白表达并下调TNF-α蛋白表达有关。     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

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。     

Enhanced Therapeutic Potential of Nano-Curcumin Against Subarachnoid Hemorrhage-Induced Blood–Brain Barrier Disruption Through Inhibition of Inflammatory Response and Oxidative Stress

Curcumin and nano-curcumin both exhibit neuroprotective effects in early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH). However, the mechanism that whether curcumin and its nanoparticles affect the blood–brain barrier (BBB) following SAH remains unclear. This study investigated the effect of curcumin and the poly(lactide-co-glycolide) (PLGA)-encapsulated curcumin nanoparticles (Cur-NPs) on BBB disruption and evaluated the possible mechanism underlying BBB dysfunction in EBI using the endovascular perforation rat SAH model. The results indicated that Cur-NPs showed enhanced therapeutic effects than that of curcumin in improving neurological function, reducing brain water content, and Evans blue dye extravasation after SAH. Mechanically, Cur-NPs attenuated BBB dysfunction after SAH by preventing the disruption of tight junction protein (ZO-1, occludin, and claudin-5). Cur-NPs also up-regulated glutamate transporter-1 and attenuated glutamate concentration of cerebrospinal fluid following SAH. Moreover, inhibition of inflammatory response and microglia activation both contributed to Cur-NPs’ protective effects. Additionally, Cur-NPs markedly suppressed SAH-mediated oxidative stress and eventually reversed SAH-induced cell apoptosis in rats. Our findings revealed that the strategy of using Cur-NPs could be a promising way in improving neurological function in EBI after experimental rat SAH.     

老年蛛网膜下腔出血的治疗方法和预后分析

目的:探讨70岁以上老年蛛网膜下腔出血患者的治疗方法和预后。方法:回顾性分析我院2006-2012年收治的49例老年蛛网膜下腔出血患者的临床资料及其预后。结果:49例患者入院时Hunt-Hess分级:Ⅰ级14例,Ⅱ级8例,Ⅲ级6例,Ⅳ级6例,Ⅴ级15例,其中14例采用开颅动脉瘤夹闭术,10例采用血管内介入栓塞术,25例采用内科保守治疗。治疗后6月,根据ADL量表评价预后(ADL≥60分为预后良好):内科保守治疗者仅3例预后良好,手术及栓塞术者17例预后良好。结论:对于老年蛛网膜下腔出血患者而言,影响患者预后的原因主要是出血导致的脑损伤和症状性脑血管痉挛,通过手术或栓塞术积极治疗破裂动脉瘤是预后良好的重要因素。     

Role of autophagy in early brain injury after subarachnoid hemorrhage in rats

Early brain injury (EBI) occurred after aneurismal subarachnoid hemorrhage (SAH) strongly determined the patients’ prognosis. Autophagy was activated in neurons in the acute phase after SAH, while its role in EBI has not been examined. This study was designed to explore the effects of autophagy on EBI post-SAH in rats. A modified endovascular perforating SAH model was established under monitoring of intracranial pressure. Extent of autophagy was regulated by injecting autophagy-regulating drugs (3-methyladenine, wortmannin and rapamycin) 30 min pre-SAH intraventricularly. Simvastatin (20 mg/kg) was prophylactically orally given 14 days before SAH induction. Mortality, neurological scores, brain water content and blood–brain barrier (BBB) permeability were evaluated at 24 h post-SAH. Microtubule-associated protein light chain-3 (LC3 II/I) and beclin-1 were detected for monitoring of autophagy flux. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, expression of cleaved caspase-3 and cytoplasmic histone-associated DNA fragments were used to detect apoptosis. The results showed that mortality was reduced in rapamycin and simvastatin treated animals. When autophagy was inhibited by 3-methyladenine and wortmannin, the neurological scores were decreased, brain water content and BBB permeability were further aggravated and neuronal apoptosis was increased when compared with the SAH animals. Autophagy was further activated by rapamycin and simvastatin, and apoptosis was inhibited and EBI was ameliorated. The present results indicated that activation of autophagy decreased neuronal apoptosis and ameliorated EBI after SAH. Aiming at autophagy may be a potential effective target for preventing EBI after SAH.     

脑淋巴引流阻滞加重蛛网膜下腔出血后大鼠皮层神经细胞的凋亡

本文旨在探讨在体脑淋巴引流阻滞(cerebral lymphatic blockage,CLB)对蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后大鼠皮层神经细胞凋亡的影响及机制。选用健康成年Wistar大鼠,随机分为正常对照组、SAH组、SAH+CLB组。采用枕大池二次注血法建立SAH模型。于第二次注血3d后,采用HE染色法及碘化丙啶(PI)染色法观察各组大鼠皮层神经细胞形态结构变化;TUNEL荧光标记法检测原位凋亡情况;免疫组织化学激光共聚焦检测大鼠皮层神经细胞Caspase-3和Bcl-2的蛋白表达。结果显示:(1)HE染色和PI染色可见SAH组大鼠部分皮层神经细胞皱缩,细胞核呈波纹状或折缝样,部分呈新月形;SAH+CLB组神经细胞分布稀疏,核碎裂,可见凋亡小体,周围有空泡形成;(2)SAH组和SAH+CLB组TUNEL阳性细胞的数量均高于正常对照组,而SAH+CLB组又显著高于SAH组;(3)SAH组和SAH+CLB组Caspase-3的表达量均高于正常对照组,而SAH+CLB组又显著高于SAH组;(4)SAH组和SAH+CLB组Bcl-2蛋白表达量均高于正常对照组,而SAH+CLB组显著低于SAH组。以上结果表明,CLB可以通过Caspase-3高表达和Bcl-2低表达加重SAH后大鼠皮层神经细胞的凋亡。     

GPR18 Agonist Resolvin D2 Reduces Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage by Multiple Protective Mechanisms

Early brain injury (EBI) is the early phase of secondary complications arising from subarachnoid hemorrhage (SAH). G protein-coupled receptor 18 (GPR18) can exert neuroprotective effects during ischemia. In this study, we investigated the roles of GPR18 in different brain regions during EBI using a GPR18 agonist, resolvin D2 (RvD2). Location and dynamics of GPR18 expression were assessed by immunohistochemistry and western blotting in a rat model of SAH based on endovascular perforation. RvD2 was given intranasally at 1 h after SAH, and SAH grade, brain water content and behavior were assayed before sacrifice. TUNEL and dihydroethidium staining of the cortex were performed at 24 h after SAH. Selected brain regions were also examined for pathway related proteins using immunofluorescence and Western blotting. We found that GPR18 was expressed in meninges, hypothalamus, cortex and white matter before EBI. After SAH, GPR18 expression was increased in meninges and hypothalamus but decreased in cortex and white matter. RvD2 improved neurological scores and brain edema after SAH. RvD2 attenuated mast cell degranulation and reduced expression of chymase and tryptase expression in the meninges. In the hypothalamus, RvD2 attenuated inflammation, increased expression of proopiomelanocortin and interleukin-10, as well as decreased expression of nerve peptide Y and tumor necrosis factor-α. In cortex, RvD2 alleviated oxidative stress and apoptosis, and protected the blood–brain barrier. RvD2 also ameliorated white matter injury by elevating myelin basic protein and suppressing amyloid precursor protein. Our results suggest that GPR18 may help protect multiple brain regions during EBI, particularly in the cortex and hypothalamus. Upregulating GPR18 by RvD2 may improve neurological functions in different brain regions via multiple mechanisms.

     

Apelin-13 attenuates early brain injury through inhibiting inflammation and apoptosis in rats after experimental subarachnoid hemorrhage

Molecular Biology Reports - Early brain injury (EBI) has been considered as the major contributor to the neurological dysfunction and poor clinical outcomes of subarachnoid hemorrhage (SAH)....     

Attenuation of Early Brain Injury and Learning Deficits Following Experimental Subarachnoid Hemorrhage Secondary to Cystatin C: Possible Involvement of the Autophagy Pathway

Cystatin C (CysC) is a cysteine protease inhibitor and previous studies have demonstrated that increasing endogenous CysC expression has therapeutic implications on brain ischemia, Alzheimer’s disease, and other neurodegenerative disorders. Our previous reports have demonstrated that the autophagy pathway was activated in the brain after experimental subarachnoid hemorrhage (SAH), and it may play a beneficial role in early brain injury (EBI). This study investigated the effects of exogenous CysC on EBI, cognitive dysfunction, and the autophagy pathway following experimental SAH. All SAH animals were subjected to injections of 0.3 ml fresh arterial, nonheparinized blood into the prechiasmatic cistern in 20 s. As a result, treatment with CysC with low and medial concentrations significantly ameliorated the degree of EBI when compared with vehicle-treated SAH rats. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosomes, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased in the cortex 48 h after SAH and were further up-regulated after CysC therapy. By ultrastructural observation, there was a marked increase in autophagosomes and autolysosomes in neurons of CysC-treated rats. Learning deficits induced by SAH were markedly alleviated after CysC treatment with medial doses. In conclusion, pre-SAH CysC administration may attenuate EBI and neurobehavioral dysfunction in this SAH model, possibly through activating autophagy pathway.     

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。