Nimodipine Attenuation of Early Brain Dysfunctions Is Partially Related to its Inverting Acute Vasospasm in a Cisterna Magna Subarachnoid Hemorrhage (SAH) Model in Rats

ABSTRACT Subarachnoid hemorrhage (SAH)-induced brain injury is highly related to neurological deficits and mortality. Regional cerebral blood flow (rCBF) changes and vasoconstriction are two complications that occur soon after SAH experimentally. In this study we investigated the changes in rCBF and vertebro-basilar arterial diameter in a cisterna megna SAH model in Sprague-Dawley rats and intended to explore whether improving early rCBF reduction and cerebral vasospasm could contribute to alleviating blood-brain barrier (BBB) dysfunction. In rats for rCBF, vasospasm and BBB permeability assessments, nimodipine (NDP) or saline was administered intravenously 5 minutes after SAH. rCBF within the first 60 minutes after SAH was measured by laser Doppler flowmetry. BBB permeability indexed by Evans Blue extravasation was assessed 4?hours after SAH. Angiography for the caliber changes of the vertebro-basilar artery were conducted 30 minutes post SAH. Pronounced rCBF reduction and vasospasm were observed soon after SAH, followed by BBB permeability increment. NDP administration could improve rCBF and attenuate vasospasm, followed by the alleviation of BBB permeability. Our results demonstrate that early improvement of cerebral circulation by NDP may contribute to the reduction in brain injury indexed by BBB disruption.     

盐酸法舒地尔对蛛网膜下腔出血后血管痉挛的实验研究

目的通过建立大鼠蛛网膜下腔出血（SAH）模型,探讨盐酸法舒地尔对蛛网膜下腔出血后血管痉挛的缓解作用和神经保护作用,并与尼莫地平对比,观察疗效。方法通过枕大池二次注血法建立大鼠SAH模型,观察基底动脉和海马神经元形态变化,测量基底动脉管径和管壁厚度,计算海马CA1区神经元密度,检测基底动脉内皮型一氧化氮合成酶（eNOS）的表达。结果各组模型大鼠的基底动脉均出现血管痉挛,海马CA1区正常神经元数目明显减少,多数神经元发生变性,基底动脉的eNOS表达明显减弱。但注射法舒地尔组与其他模型组相比能较大程度的缓解以上变化,具有统计学差异（P〈0.05）,且优于尼莫地平。结论法舒地尔可以有效缓解SAH后的迟发性脑血管痉挛,具有神经保护作用,其缓解迟发性脑血管痉挛作用和神经保护作用与动脉壁产生的一氧化氮（NO）有关。     

Mechanism of hemolysate-induced [Ca2+]i elevation in cultured fibroblasts.

Erythrocyte lysate (hemolysate) released from blood clot after subarachnoid hemorrhage is the causative agent for chronic cerebral vasospasm, a prolonged contraction of cerebral arteries. Fibroblasts, the outer layer cells of vessel wall that in contact with blood clot directly, may contribute to cerebral vasospasm. However, the effect of hemolysate on intracellular Ca2+ ([Ca2+]i) mobilization in fibroblasts has not been studied. We investigated hemolysate-induced [Ca2+]i mobilization in cultured neonatal human dermal and canine middle cerebral arterial fibroblasts by using fura-2 microfluorimetry. Hemolysate increased [Ca2+]i by releasing internal Ca2+ stores and promoting Ca2+ entry. Tyrosine kinase inhibitors partially but significantly reduced the effect of hemolysate. The major components of hemolysate, oxyhemoglobin (OxyHb) and adenosine triphosphate (ATP) failed to mimic the effect of hemolysate. In cultured canine middle cerebral arterial fibroblasts, hemolysate produced similar Ca2+ mobilization to that of dermal cells. OxyHb and ATP failed again to reproduce the effect of hemolysate. We conclude that hemolysate increases [Ca2+]i in fibroblasts and this effect of hemolysate is not mediated by OxyHb or ATP but by some unknown factors.     

Oxyhemoglobin produces apoptosis and necrosis in cultured smooth muscle cells

Confluent rat aortic smooth muscle cells were treated with OxyHb in a concentration- and time-dependent manner. A high concentration of OxyHb (100 microM) within 24 h decreased cell density. DNA analysis showed a smear pattern characteristic of cell necrosis. Transmission electron microscopy demonstrated disintegration of the cell membrane and destruction of cell organelles. Western blotting using PARP antibody revealed that 116 kDa PARP was not cleaved to 85 kDa, an apoptosis-related fragment. On the contrary, a low concentration of OxyHb (10 microM) produced apoptotic cell death at 72 h that was supported by DNA analysis and TUNEL staining. These results demonstrated that a high level of OxyHb induced necrosis within 24 h and a low concentration of OxyHb produced apoptosis after 72 h in cultured smooth muscle cells. Morphological alterations induced by OxyHb might contribute to the vascular wall changes in the cerebral arteries following subarachnoid hemorrhage (SAH).     

尼莫地平不同给药途径治疗蛛网膜下腔出血后脑血管痉挛

脑血管痉挛(CVS)一直被认为是蛛网膜下腔出血(SAH)后严重的并发症.本文回顾了尼莫地平不同给药途径治疗SAH后CVS的研究进展.尼莫地平作为第二代双氢吡啶类钙离子拮抗剂能有效降低SAH后的全脑梗塞和不良预后.尼莫地平局部应用可增加药物浓度,减少全身应用引起的副作用,有效扩张血管管腔,改善术中CVS,减少手术后症状性CVS的发生,改善动脉瘤SAH患者的预后.尼莫地平口服、静脉应用对于治疗SAH后CVS已取得肯定的效果,更加深入研究其局部应用治疗SAH后CVS的作用及其作用机理,可进一步提高尼莫地平的疗效.     

Cisternal sustained release dihydropyridines for subarachnoid hemorrhage

Nimodipine improved outcome in patients with subarachnoid hemorrhage (SAH) although hypotension limited the dose that could be administered systemically. Subarachnoid delivery of nicardipine or nimodipine may be more efficacious. We tested the efficacy of cisternal application of sustained release nicardipine and nimodipine in SAH in monkeys and dogs, respectively. SAH was created in 13 cynomolgus macaques by placement of autologous blood clot around right middle cerebral, anterior cerebral, and internal carotid arteries. Placebo poly-D,L-lactide coglycolide (PLGA), nicardipine PLGA or mibefradil PLGA was inserted in the clots. Catheter and computed tomography angiography (CTA) were performed at baseline and 7 days later (day 7). Cerebral infarction was assessed on day 7 by magnetic resonance imaging. Six dogs underwent baseline angiography and injection of autologous blood plus PLGA or nimodipine-loaded PLGA microparticles into the cisterna magna. Blood injection was repeated 2 days later and angiography 7 and 14 days later. Animals were euthanized and brains were examined histologically. Cerebrospinal fluid and serum nimodipine concentrations were measured. Nicardipine, but not mibefradil PLGA decreased vasospasm in monkeys (paired t-tests) although there was no significant effect on infarctions see on MRI. In dogs, nimodipine-PLGA produced high local concentrations of nimodipine that were associated with reduced basilar artery vasospasm. No untoward histological effects were observed. There was no reduction in microthrombi in animals treated with nimodipine PLGA compared to placebo PLGA. Site-specific, sustained release formulations of dihydropyridines can deliver high concentrations to the cerebrospinal fluid without causing systemic side effects, and may reduce angiographic vasospasm after SAH. Since nimodipine improves outcome in patients with SAH without necessarily preventing vasospasm, further studies are warranted.     

尼莫地平对蛛网膜下腔出血大鼠额叶转化生长因子-β1表达的影响

目的：探讨尼莫地平对蛛网膜下腔出血（SAH）大鼠额叶转化生长因子-β1（TGF—β1）表达和血管痉挛的影响。方法：枕大池二次注血法制作SAH模型。54只成年健康雄性SD大鼠随机分为对照组（n=6）、SAH组（n=24）和尼莫地平处理组（n=24），其中SAH组和尼莫地平处理组随机均分为1、3、5和7d等4组（n=6）。尼莫地平处理组于二次注血后30min时经股静脉注入尼莫地平2mg／kg，此后每天经腹腔注射尼莫地平2mg／kg。HE染色光镜下观察基底动脉病理学变化，测定内径；免疫组化法检测各组大鼠额叶TGF—β1表达。结果：SAH组和尼莫地平处理组基底动脉内径显著小于对照组（P〈0．01），而SAH组与尼莫地平处理组无显著差异。SAH1d时，TGF—β1表达增加，3d时达高峰，5d和7d时显著低于1d和3d时（P〈0．01），但仍照著高于对照组（P〈0．01）。与SAH组相比，尼莫地平处理组1d和3d时TGF-β1表达无显著差异，5d和7d时显著增加。结论：SAH后额叶TGF—β1表达发生改变，与脑血管痉挛有关，提示TGF-β1参与了脑血管痉挛的病理学过程。尼莫地平可能通过增加SAH后啮组织中TGF-β1表达对缺血脑组织起着保护作用。     

外膜成纤维细胞在实验性脑血管痉挛中的作用

目的 在体外模拟血管壁外层结构,建立外膜成纤维细胞(AF)和平滑肌细胞(SMC)共培养,研究血管AF在蛛网膜下腔出血后脑血管痉挛中的作用.方法 (1)以多聚碳酸酯膜(PET膜)作为载体,将AF和SMC进行共培养.(2)模拟体内出血环境,在PET膜一侧接种AF细胞并加入含10-6 mol/L OxyHb的培养液,分别共培养24h、48h、72h,应用电镜观察SMC的长度.(3)提取各组AF缝隙连接蛋白Cx43进行RT-PCR半定量分析.结果 (1)扫描电镜测量发现,加入OxyHb处理的AF能使PET膜对侧未经OxyHb处理的SMC产生收缩(P＜0.001),且收缩程度与OxyHb处理AF细胞时间成正比.(2)AF缝隙连接蛋白Cx43半定量RT-PCR分析显示:24h、48h、72h组中Cx43mRNA平均相对含量较正常组上调(P＜0.01).结论 血管壁外层的AF受到OxyHb作用后可以引起未直接接触OxyHb的SMC发生持续收缩且缝隙连接蛋白Cx43mRNA表达上调,提示AF在蛛网膜下腔出血引起的脑血管痉挛中可能发挥重要作用.     

Angiographic Features and Clinical Outcomes of Intra-Arterial Nimodipine Injection in Patients with Subarachnoid Hemorrhage-Induced Vasospasm

Objective

The aim of this study was to determine the role of intra-arterial (IA) nimodipine injections for cerebral vasospasm secondary to ruptured subarachnoid hemorrhage (SAH) and to investigate the factors that influence vasodilation and clinical outcomes.

Methods

We enrolled 29 patients who underwent aneurysm clipping for ruptured cerebral aneurysms between 2009 and 2011, and who received IA nimodipine after subsequently presenting with symptomatic vasospasm. The degree of vasodilation shown in angiography was measured, and the correlation between the degree of vasodilation and both the interval from SAH to cerebral vasospasm and the interval from clipping to cerebral vasospasm was determined. The change in blood flow rate after IA injection was assessed by transcranial Doppler ultrasound. Multiple clinical parameters were completed before and after IA nimodipine injection to evaluate any improvements in clinical symptoms.

Results

For eight patients, Glasgow Coma Scale (GCS) scores increased by two or more points. The regression analysis demonstrated a positive correlation between the change in GCS scores after IA nimodipine injection and the change in blood vessel diameter (p=0.025). A positive correlation was also observed between the interval from SAH to vasospasm and the change in diameter (p=0.040); and the interval from clipping to vasospasm and the change in diameter (p=0.022).

Conclusion

IA nimodipine injection for SAH-induced vasospasm led to significant vasodilation in angiography and improvement in clinical symptoms without significant complications. Our findings suggest that IA nimodipine injection should be utilized when intractable vasospasm develops despite rigorous conservative management.     

颅内动脉瘤性蛛网膜下腔出血血管痉挛治疗的临床探讨

目的探讨颅内动脉瘤性蛛网膜下腔出血脑血管痉挛的治疗方法。方法回顾性分析颅内动脉瘤性蛛网膜下腔出血合并脑血管痉挛患者临床资料67例。结果 23例并发脑血管痉挛,6例发生一侧肢体功能障碍,3例发生脑血栓形成。经治疗后61例C-反应蛋白恢复正常,6例明显下降,4例肢体功能恢复正常,1例恢复到4级,1例死亡。结论炎症因子在脑血管痉挛中明显升高;钙离子拮抗剂尼莫地平加抗氧化剂依达拉奉中药川芎嗪联合治疗动脉瘤性蛛网膜下腔出血所致的脑血管痉挛有较好疗效。     

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.     

鼠脑血管痉挛时尼莫地平对体感诱发电位的影响

目的 探讨蛛网膜下腔出血（SAH）后脑血管痉挛（CVS）对体感诱发电位（SEP）的影响,及尼莫地平（ND）的保护作用。方法 对单纯SAH组和ND处理组大鼠观察手术前后基底动脉管径,并检测24h内局部脑血流量（rCBF)、SEP潜伏期及脑组织内皮素-1（ET-1）含量的动态变化。结果 SAH组大鼠在诱导SAH后rCBF立即降低,并持续24h,同时有基底动脉痉挛;SAH后1h开始至24hSEP潜伏期逐渐延长,脑组织ET-1含量显著增加,ND处理组大鼠上述变化均较轻。结论 SAH后CVS可通过脑血流的降低,脑组织ET-1增加而导致SEP潜伏期延长,ND通过拮抗脑组织ET-1变化而对之具有保护作用。     

Caspase inhibitors prevent endothelial apoptosis and cerebral vasospasm in dog model of experimental subarachnoid hemorrhage.

Apoptosis in the endothelium of major cerebral arteries may play a role in the initiation and maintenance of cerebral vasospasm after subarachnoid hemorrhage (SAH). We tested the therapeutic effect of caspase inhibitors on endothelial apoptosis and on cerebral vasospasm in an established dog double-hemorrhage model. Thirty-one mongrel dogs were divided into five groups: control; SAH; SAH treated with vehicle [DMSO]; SAH treated with Ac-DEVD-CHO [a specific caspase-3 inhibitor]; and SAH treated with Z-VAD-FMK [a broad caspase inhibitor]. The inhibitors (100 microM) were injected into the cisterna magna daily from Day 0 through Day 3. Angiography was performed on Day 0 and Day 7. Histology, TUNEL staining, and immunohistochemistry were conducted on basilar arteries collected on Day 7 after SAH. Positive staining of TUNEL, poly(ADP)-ribose polymerase (PARP), caspase-3, and caspase-8 was observed in the endothelial cells of the spastic arteries. Double fluorescence labeling demonstrated co-localization of TUNEL with caspase-3 and TNFalpha receptor-1 (TNFR1). Ac-DEVD-CHO and Z-VAD-FMK prevented endothelial apoptosis and reduced angiographic vasospasm. The mechanism of apoptosis in endothelial cells involves TNFR1 and the caspase-8 and caspase-3 pathways. Caspase inhibitors may have potential in the treatment of cerebral vasospasm.     

Intracisternal administration of SB203580, a p38 mitogen-activated protein kinase inhibitor,attenuates cerebral vasospasm via inhibition of tumor-necrosis factor-α

Tumor-necrosis factor-α (TNF-α) is critical to the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Hence, therapeutic strategies targeting TNF-α can attenuate cerebral vasospasm. This study investigated the effects of SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, on TNF-α concentration in the cerebral arteries and the cerebrospinal fluid (CSF) after SAH and on subsequent cerebral vasospasm. Twenty-three rabbits were divided into four groups: (i) control (without SAH), (ii) SAH (SAH only), (iii) dimethylsulfoxide (DMSO, vehicle), and (iv) SB203580. The severity of vasospasm and the immunoreactivities of TNF-α and phosphorylated p38 MAPK in the brain vessels were determined in all animals, and the concentrations of TNF-α in the CSF were also assessed. Severe vasospasm was observed in the rabbits from the SAH and DMSO groups. SB203580 reversed vasospasm after SAH. Lower immunoreactivities of TNF-α and phosphorylated p38 MAPK were found in the basilar artery in the SB203580 group than in the DMSO group. The concentration of TNF-α in the CSF increased after SAH, but treatment with SB203080 after SAH suppressed this increase. Our data show that SB203580 reversed cerebral vasospasm by inhibiting the phosphorylation of p38 MAPK in the basilar artery and by suppressing the increase in TNF-α in the basilar artery and CSF after SAH. SB203580 could therefore potentially be used for the treatment of cerebral vasospasm after SAH.     

Rho激酶、氧合血红蛋白与蛛网膜下腔出血后脑血管痉挛的关系

目的探讨Rho激酶、氧合血红蛋白在蛛网膜下腔出血后患者脑脊液中的表达与浓度及其在脑血管痉挛的调控作用。方法采集62例蛛网膜下腔出血患者入院后第1、3、7、10、14 d脑脊液标本,RT-PCR方法检测Rho激酶mRNA的表达,采用比色法测定脑脊液中氧合血红蛋白的浓度,采用TCD检测颅内主要动脉血流速度。结果出血后第3 d脑脊液中Rho激酶表达及氧合血红蛋白浓度增高,在出血后第7 d达到高峰,而后逐渐下降。结论蛛网膜下腔出血患者脑脊液中Rho激酶的表达与氧合血红蛋白浓度有关,参与蛛网膜下腔出血后脑血管痉挛的发生,其含量与脑血管痉挛程度相关。     

非开颅蛛网膜下腔出血大鼠脑血流量和水、电解质含量研究

利用非开颅大鼠模型观察蛛网膜下腔出血（ＳＡＨ）后２４ｈ内脑血流量和水电解质含量的动态变化和尼莫地平对其影响。结果发现ＳＡＨ后脑血迅速降低，１ｈ了低值，２４ｈ内持于低水平状态。ＳＡＨ后１ｈ开始脑组织Ｃａ＾２＋答聚，６ｈ后出现脑水肿。尼莫地平对上述指标均有改善作用。提示脑血管痉挛及微循环异常所致脑血流减少在ＳＡＨ继发性的损害中起重要作用，尼莫地平可减轻上述病理改变。     

Haemoglobin and ATP levels in CSF from a dog model of vasospasm.

Haemoglobin and adenosine 5'-triposphate (ATP) released from lysed erythrocytes have been postulated to be responsible for delayed cerebral vasospasm after subarachnoid haemorrhage (SAH). However, the concentrations of haemoglobin and ATP in cerebrospinal fluid (CSF) in patients or in an animal model of vasospasm have not been reported. In this study, 12 mongrel dogs underwent a double blood injection via the cisterna magna on day 0 and 2, after an initial collection of CSF. On day 3, 5 or 7, the dogs were sacrificed after a second collection of CSF. An angiogram was recorded on day 0 and on the day of sacrifice. Results showed that the diameter of the dog's basilar artery was reduced 20% on day 3 (P > 0.05), 35% on day 5 (P < 0.05) and 45% on day 7 (P < 0.05). The concentrations of OxyHb, deOxyHb and MetHb in CSF were increased (P < 0.05), and all peaked on day 3. OxyHb and MetHb remained significantly higher than control (day 0) from day 3 to day 7, while deOxyHb remained at a high level on day 5 but returned to normal on day 7. In contrast, ATP was decreased (P < 0.05) on days 5 and 7 after SAH compared with day 0. The results indicate that haemoglobin might be involved in the development of cerebral vasospasm. The possible role of ATP in vasospasm remains unclear.     

蛛网膜下腔出血患者继发症状性脑血管痉挛的相关危险因素分析

目的 研究探讨动脉瘤性蛛网膜下腔出血（subarachnoid hemorrhage,SAH）继发症状性脑血管痉挛 （symptomatic cerebral vasospasm,SCVS）的相关危险因素,为SCVS的防治提供参考。 方法 回顾性分析96例SAH患者临床资料,对性别、年龄、高血压史、糖尿病史、Fisher分级等影响因 素进行统计学分析。 结果 96例患者中发生SCVS的患者共39例,单因素分析结果显示SCVS组与非SCVS组在年龄、高血 压、吸烟、脑室内积血、Hunt-Hess分级、Fisher分级、数字减影血管造影（digital subtraction angiography, DSA）显示血管痉挛程度、尼莫地平使用等方面差异有显著性;多因素Logistic回归分析结果显示：低 龄、高血压史、Fisher分级是发生SCVS的危险因素,其比值比（odds ratio,OR）分别为0.567、1.982和 2.713;而尼莫地平的使用是SCVS发生的保护因素,OR为0.799。 结论 SAH后SCVS是多种因素共同作用的结果,其中低年龄、高血压史、Fisher分级是SCVS的独立危 险因素,尼莫地平使用为保护因素。     

脑血管痉挛血管壁损害的发病机制研究

迟发性脑血管痉挛是蛛网膜下隙出血的一种常见并发症。目前,其确切发生机制还不完全清楚。近年来,一些学者发现,蛛网膜下隙出血后血管壁发生了器质性病理变化,伴随蛛网膜下隙出血的免疫炎性反应、血管壁细胞增殖与凋亡导致的血管壁的损害可能是脑血管痉挛发生的关键病理途径。本文就其研究的相关进展进行总结。     

蛛网膜下腔出血大鼠脑干组织叉头框蛋白的表达及尼莫地平的影响

目的 研究丝氨酸/苏氨酸蛋白激酶B[protein Kinase B,PKB(AKT)]、叉头框蛋白[forkhead box(FOXO),FKHR]在大鼠蛛网膜下腔出血脑血管痉挛动物模型中早期(1d)的表达、活化情况及应用尼莫地平的影响.方法 将Wistar大鼠分为假手术组(对照组)、SAH模型组、SAH后给尼莫地平治疗组(治疗组).经颅多普勒超声(TCD)检测大鼠基底动脉的最大血流速度(Maximum velocity of Basilar artery blood flow,Vmba).脑干组织进行免疫组化染色.结果 SAH模型组与对照组相比基底动脉血流加快(P＜0.05).治疗组比SAH模型组基底动脉血流速度慢(P＜0.05).在对照组AKT、P-AKT(磷酸化-AKT)、FKHR、P-FKHR(磷酸化-FKHR)均可见阳性细胞表达.在SAH模型组:AKT表达增多,但与对照组比较差异无显著性(P＞0.05);P-AKT表达减少,与对照组比较差异有显著性(P＜0.05).在治疗组AKT、P-AKT表达较对照组和SAH模型组均增加,差异具有显著性(P＜0.05).在SAH模型组FKHR表达增加,P-FKHR表达减少,与对照组比较差异有显著性(P＜0.05);在治疗组FKHR表达较对照组和SAH模型组均减少,P-FKHR表达较对照组和SAH模型组均增加,差异具有显著性(P＜0.05).结论 PKB/AKT介导的磷酸化能够诱导FOXO蛋白从细胞核到细胞质的重新定位,诱导FOXO蛋白的活性发生改变,并参与了大鼠SAH后早期(1d)CVS的形成.尼莫地平具有缓解SAH后早期(1d)CVS的作用.