植入异物法构建自发性脑出血实验动物模型

目前对于自发性脑出血的病理生理机制及治疗方法的研究大多基于动物试验。国内外不同实验室已经针对多种动物制作了实验性脑出血模型。根据其脑内移植的组织不同，分为缺血诱发脑出血、外伤导致脑出血、自发性脑出血和颅内植入异物导致脑出血4类。颅内植入异物导致脑出血根据植入异物的不同，又分为植入惰性物质导致的脑出血、植入生物制剂诱导脑出血、植入自体动脉血模拟脑出血3种。文章归纳总结实验性脑出血动物模型总类、制作方法和特点。     

Stem cell-based therapies for intracerebral hemorrhage in animal model: a meta-analysis

Stem cell to be a new intervention for treating intracerebral hemorrhage (ICH) might benefit humans. Therefore, we collected animal studies to find the effect of this innovative treatment. In July 2014, we searched Medline (from 1950), Embase (from 1980), China Biology Medicine disk (from 1978) for studies on stem cells used for treating experimental ICH in animal models that reported neurobehavioral and structural outcome. We evaluated the quality of these studies and used a weighted mean difference random affects model for the meta-analysis. We have collected 30 studies from 650 publications identified through systematic review describing the effects of 5 different type of stem cells on 12 different neurobehavioral scales with 1101 rodents or monkeys. Although there is lack of uniformity of the evaluation methods, these researches showed consistent improvements both in neurobehavioral function and structural outcomes. Besides, the quality of these studies needs to be raised. In conclusion, stem cells hold extensive potential in treating ICH, which should be further evaluated with more evidence-based, high-quality animal studies.     

幼猪脑出血模型的建立与评价

目的探索建立稳定、可靠、重复性好的幼猪脑内血肿模型。方法利用未肝素化自体血制作幼猪脑基底节区血肿。实验分缓慢注射组(n=12)和快速注射组(n=12),通过取脑切片,采用计算机图像分析系统计算各组脑内血肿体积,同时观察血肿周围组织病理变化,从而评价两种注射法的差异。结果缓慢注射法(二次注入法)可形成较稳定、形态规则的血肿,无蛛网膜下腔和脑室积血表现,脑内血肿体积(1.84±0.28)cm3。快速注射组血肿易破入蛛网膜下腔和脑室内,脑内血肿体积(1.13±0.48)cm3,两种注射法所形成的脑内血肿大小比较差异有统计学意义(P<0.05)。而两组血肿周围脑组织病理形态学表现均为血肿周围白质疏松水肿与炎性细胞侵润。结论采用缓慢注射法(二次注入法),将未肝素化自体血缓慢匀速注入幼猪脑基底节区,可形成稳定、可靠、重复性好的脑内血肿模型。     

MicroRNA-181c provides neuroprotection in an intracerebral hemorrhage model

Apoptosis is an important factor during the early stage of intracerebral hemorrhage.MiR-181 c plays a key regulatory role in apoptosis.However,whether miR-181 c is involved in apoptosis of prophase cells after intracerebral hemorrhage remains unclear.Therefore,in vitro and in vivo experiments were conducted to test this hypothesis.In vivo experiments:collagenase type VII was injected into the basal ganglia of adult Sprague-Dawley rats to establish an intracerebral hemorrhage model.MiR-181 c mimic or inhibitor was injected in situ 4 hours after intracerebral hemorrhage.Neurological functional defects(neurological severity scores)were assessed 1,7,and 14 days after model establishment.Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and western blot assay were conducted 14 days after model establishment.In vitro experiments:PC12 cells were cultured under oxygen-glucose deprivation,and hemins were added to simulate intracerebral hemorrhage in vitro.MiR-181 c mimic or inhibitor was added to regulate miR-181 c expression.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,luciferase reporter system,and western blot assay were performed.Experimental results revealed differences in miR-181 c expression in brain tissues of both patients and rats with cerebral hemorrhage.In addition,in vitro experiments found that miR-181 c overexpression could upregulate the Bcl-2/Bax ratio to inhibit apoptosis,while inhibition of miR-181 c expression could reduce the Bcl-2/Bax ratio and aggravate apoptosis of cells.Regulation of apoptosis occurred through the phosphoinositide 3 kinase(PI3 K)/Akt pathway by targeting of phosphatase and tensin homolog deleted on chromosome ten(PTEN).Higher miR-181 c overexpression correlated with lower neurological severity scores,indicating better recovery of neurological function.In conclusion,miR-181 c affects the prognosis of intracerebral hemorrhage by regulating apoptosis,and these effects might be directly mediated and regulated by targeting of the PTEN\PI3 K/Akt pathway and Bcl-2/Bax ratio.Furthermore,these results indicated that miR-181 c played a neuroprotective role in intracerebral hemorrhage by regulating apoptosis of nerve cells,thus providing a potential target for the prevention and treatment of intracerebral hemorrhage.Testing of human serum was authorized by the Ethics Committee of China Medical University(No.2012-38-1)on February 20,2012.The protocol was registered with the Chinese Clinical Trial Registry(Registration No.ChiCTR-COC-17013559).The animal study was approved by the Institutional Animal Care and Use Committee of China Medical University(approval No.2017008)on March 8,2017.     

Nec-1在小鼠脑出血中通过抑制程序性坏死发挥神经保护作用

目的研究程序性坏死的特异性抑制剂Nec-1对小鼠脑出血神经功能的保护作用,并探讨其机制。方法选取健康雄性ICR小鼠,体重25~30 g,采用在纹状体部位注射胶原酶Ⅳ或生理盐水的方法建立小鼠脑出血组或者脑出血对照组,在脑出血前15 min分别在侧脑室注射Nec-1溶液或vehicle溶液。将实验随机分为:对照组、脑出血+vehicle处理组、脑出血+Nec-1处理组。分别利用干湿重法测定脑水肿程度,神经功能评分检测神经运动功能,Western blotting检测cleaved caspase-3,Bcl-2蛋白表达情况。结果脑出血加剧脑水肿程度,而Nec-1处理组减轻脑水肿程度(P0.05)。经Nec-1处理后,可以提高小鼠脑出血神经运动功能(P0.05)。脑出血可以增加cleaved caspase-3蛋白表达,抑制Bcl-2蛋白表达,经Nec-1处理后,抑制cleaved caspase-3的表达水平(P0.05),增加Bcl-2的表达(P0.05)。结论 Nec-1对脑出血发挥重要神经保护作用,这种保护作用可能是通过抑制凋亡通路实现的,提示程序性坏死在脑出血中具有重要作用,将为脑出血的治疗提供新的思路。     

实验性脑出血动物模型

脑出血的发病率和致残（死）率很高，是威胁人类生命健康的主要疾病之一。脑出血临床救治主要包括内科保守和外科手术两种方法，但均有尚需改进和提高的方面。目前，有关脑出血的实验研究仍然是脑科学研究的重点之一，而建立稳定性高、重复性好的脑出血动物模型是该研究的前提，现将常用的脑出血动物模型及其建立方法归纳如下：     

细胞移植对脑出血脑损伤的保护作用

目前用于脑出血治疗研究的主要细胞有：神经干细胞、遗传工程神经干细胞、骨髓间质干细胞、脐血细胞、胚胎干细胞、重组细胞、微囊化人工细胞等，本文就前5种细胞移植在出血性脑损伤中的保护作用进行综述。干细胞移植在脑出血性动物身上的实验所取得的良好效果，显示细胞移植重建损伤的脑组织、改善脑功能成为治疗脑出血疾病的必然途径。临床实验也取得了一定效果，但要真正应用于临床目前还有许多问题要解决：要想准确的定向诱导细胞分化，需要更深入的研究局部微环境，细胞因子及基因对干细胞分化的作用；细胞移植促进脑出血功能的恢复，其确切的机制还需要进一步研究 ；诱导分化的神经干细胞是否能象正常神经细胞一样能分泌神经递质，是否具有复杂的电生理等。     

Protective effects of free radical inhibitors in intracerebral hemorrhage in rat

Iron compounds formed in the degradation of a hematoma can accelerate the formation of free radicals in adjacent ischemic or hypoperfused tissue. The purpose of this study was to examine the efficacy of compounds that quench free radicals in improving the outcome in rats with experimental intracerebral hemorrhage. Intracerebral hemorrhage was induced in rats by injection of bacterial collagenase and heparin into the caudate nucleus. Rats were treated with α-tocopherol plus ascorbic acid starting before hemorrhage, or with dimethylthiourea or α-phenyl-N-tert-butyl nitrone starting 2 h after hemorrhage, with treatment continued for 10 days after induction of hemorrhage. Outcome was assessed by behavioral analyses, magnetic resonance imaging, and histopathology. A trend towards behavioral improvement was found for rats treated with α-tocopherol/ascorbic acid, while behavior was significantly improved following intracerebral hemorrhage in rats treated with dimethylthiourea or α-phenyl-N-tert-butyl nitrone. These results suggest that free radicals may play a role in the development of brain injury following intracerebral hemorrhage, and that compounds that interrupt the free radical cascade may improve outcome. However, treatment did not significantly affect edema, resolution of the hematoma, or neuronal injury in tissue adjacent to the hemorrhage.     

A double-injection model of intracerebral hemorrhage in rabbits

We aimed to develop a double-injection model of intracerebral hemorrhage (ICH) in rabbits and to evaluate it as a tool for investigating post-ICH brain injury. Rabbits were injected with 300 μL fresh autologous whole blood into the right basal ganglia. Behavioral changes were rated, brain water content (BWC) was measured and brain tissue morphology was also examined. ICH was established in 93.5% of the blood injection group. At 1, 3 and 7 days after ICH, there were significant differences in the total neurological scores (p < 0.01) and BWC (p < 0.01) between a sham-operated group and the ICH group. These findings suggest that the model produces a persistent neurological deficit, hematoma volume and perihematomal edema and closely mimics human hypertensive basal ganglia ICH; it is a controllable and reproducible hematoma that lends itself to quantitative investigation.     

Toxic role of prostaglandin E2 receptor EP1 after intracerebral hemorrhage in mice

Inflammatory mechanisms mediated by prostaglandins may contribute to the progression of intracerebral hemorrhage (ICH)-induced brain injury, but they are not fully understood. In this study, we examined the effect of prostaglandin E₂ receptor EP1 (EP1R) activation and inhibition on brain injury in mouse models of ICH and investigated the underlying mechanism of action. ICH was induced by injecting collagenase, autologous blood, or thrombin into the striatum of middle-aged male and female mice and aged male mice. Effects of selective EP1R agonist ONO-DI-004, antagonist SC51089, and nonspecific Src family kinase inhibitor PP2 were evaluated by a combination of histologic, magnetic resonance imaging (MRI), immunofluorescence, molecular, cellular, and behavioral assessments. EP1R was expressed primarily in neurons and axons but not in astrocytes or microglia after ICH induced by collagenase. In middle-aged male mice subjected to collagenase-induced ICH, EP1R inhibition mitigated brain injury, brain edema, cell death, neuronal degeneration, neuroinflammation, and neurobehavioral deficits, whereas its activation exacerbated these outcomes. EP1R inhibition also was protective in middle-aged female mice and aged male mice after collagenase-induced ICH and in middle-aged male mice after blood- or thrombin-induced ICH. EP1R inhibition also reduced oxidative stress, white matter injury, and brain atrophy and improved functional outcomes. Histologic results were confirmed by MRI. Src kinase phosphorylation and matrix metalloproteinase-9 activity were increased by EP1R activation and decreased by EP1R inhibition. EP1R regulated matrix metalloproteinase-9 activity through Src kinase signaling, which mediated EP1R toxicity after collagenase-induced ICH. We conclude that prostaglandin E₂ EP1R activation plays a toxic role after ICH through mechanisms that involve the Src kinases and the matrix metalloproteinase-9 signaling pathway. EP1R inhibition could be a novel therapeutic strategy to improve outcomes after ICH.     

褪黑素对脑出血大鼠的脑保护作用及机制

目的探讨褪黑素对脑出血(ICH)后大鼠脑组织神经细胞超微结构、氧化应激及促炎症因子表达的影响及机制。方法 SD大鼠130只随机分为正常组、假手术组、ICH模型组(模型组)、褪黑素干预组(褪黑素组),除正常组外,各组随机分为12 h、1 d、2 d、4 d、7 d共5个时间点亚组;采用透射电镜技术观察神经细胞超微结构;黄嘌呤氧化法和硫代巴比妥酸比色法测定脑组织超氧化物岐化酶(SOD)活性、丙二醛(MDA)含量;免疫组化法检测核转录因子-κB(NF-κB)p65阳性细胞表达;Western blot法检测肿瘤坏死因子-α(TNF-α)蛋白表达。结果 ICH后模型组神经元肿胀、核膜结构不清、细胞器减少,线粒体肿胀、空泡化,可见类似凋亡的细胞核,同一时间点褪黑素组神经元及线粒体超微结构病理改变轻微。与正常组和假手术组比较,模型组ICH后12 h~4 d时SOD活性明显降低,MDA含量明显升高(均P0.05);与模型组比较,褪黑素组MDA含量显著降低,SOD活性升高(均P0.05);ICH后模型组NF-κBp65阳性细胞表达和TNF-α蛋白表达高于正常组(均P0.05)。ICH后不同时间点褪黑素组NF-κB p65阳性细胞及TNF-α蛋白表达水平显著低于模型组(均P0.05)。结论褪黑素可减轻ICH大鼠神经细胞超微结构损伤及氧化应激水平,褪黑素的神经保护机制可能与其抑制ICH后炎症反应相关。     

补体在脑出血后脑组织损伤机制中的作用

目的研究补体C9在大鼠实验性脑出血(ICH)后血肿周围组织中的表达情况,探讨补体C9在ICH后脑水肿中的作用以及应用眼镜蛇毒因子(CVF)干预后对血肿周围组织C9表达及脑组织含水量变化的影响。方法采用立体定向技术,将自体不凝血注入大鼠尾状核制备ICH模型,将动物分为假手术组、出血组和CVF干预组,分别在不同时间断头取脑,连续切片分别作补体C9免疫组化染色和HE染色,并进行脑组织含水量测定(干湿重法)。结果ICH后2h血肿周围脑组织开始表达C9,24h达高峰。血肿周围脑组织含水量在ICH后2h开始增加(P<0.05),6h明显增加,24～72h达高峰(P<0.01),此后逐渐回落,1周基本恢复正常水平,脑组织含水量与C9的表达呈正相关关系(r=0.938,P<0.01);对侧半球相应部位及假手术对照组脑组织含水量没有明显变化;经CVF干预后,血肿周围组织C9表达明显下降,干预组与出血组之间比较有显著差异(P<0.01)。CVF干预组脑组织含水量明显低于常规ICH组(P<0.01)。结论脑出血后补体级联激活C9表达明显增加,并证明通过CVF干预后,C9表达下降,脑水肿减轻,能达到神经保护作用。     

小胶质细胞在出血性脑卒中发病机制中的研究进展

出血性脑卒中(intracerebral haemorrhage,ICH)是脑卒中致死率最高的类型,目前临床对其仍缺乏有效的治疗方法。小胶质细胞是ICH后第一个产生免疫应答的中枢神经系统细胞。ICH急性期脑损伤后,小胶质细胞可被诱导为经典的M1型(促炎作用)或补充替代的M2型(抗炎作用),其中,M1型抑制中枢神经系统的修复,M2型通过分泌抗炎因子和神经营养因子来促进组织的再生和修复。同时,小胶质细胞与星形胶质细胞、神经元、少突胶质细胞以及T淋巴细胞生理病理上具有紧密联系,其M1型和M2型极化与其他神经细胞产生不同的交互作用,这些均在ICH的病理过程中具有至关重要的作用。基于此,本文对ICH后小胶质细胞和其他神经细胞的相互作用的关键分子机制进行综述。     

蛛网膜下腔出血性脑血管痉挛动物模型的制作

蛛网膜下腔出血引起的脑血管痉挛是导致蛛网膜下腔出血患者不良预后的主要原因之一,其发生机制至今尚未完全明了。因此,建立一种理想的蛛网膜下腔出血性脑血管痉挛动物模型将对脑血管痉挛发生机制及临床防治的研究起到巨大的推动作用。文章介绍了各种蛛网膜下腔出血性脑血管痉挛动物模型的制作方法、优缺点及应用范围,但目前尚无一种用于研究蛛网膜下腔出血后症状性脑血管痉挛的理想模型。     

Protective role of TLR9‐induced macrophage/microglia phagocytosis after experimental intracerebral hemorrhage in mice

IntroductionIntracerebral hemorrhage (ICH) causes devastating morbidity and mortality, and studies have shown that the toxic components of hematomas play key roles in brain damage after ICH. Recent studies have found that TLR9 participates in regulating the phagocytosis of peripheral macrophages. The current study examined the role of TLR9 in macrophage/microglial (M/M) function after ICH.MethodsRAW264.7 (macrophage), BV2 (microglia), and HT22# (neurons) cell lines were transfected with lentivirus for TLR9 overexpression. Whole blood from C57BL/6 or EGFP^Tg/+ mice was infused for phagocytosis and injury experiments, and brusatol was used for the experiments. Intraperitoneal injection of the TLR9 agonist ODN1826 or control ODN2138 was performed on days 1, 3, 5, 7, and 28 after ICH to study the effects of TLR9 in mice. In addition, clodronate was coinjected in M/M elimination experiments. The brains were collected for histological and protein experiments at different time points after ICH induction. Cellular and histological methods were used to measure hematoma/iron residual, M/Ms variation, neural injury, and brain tissue loss. Behavioral tests were performed premodeling and on days 1, 3, 7, and 28 post‐ICH.ResultsOverexpression of TLR9 facilitated M/M phagocytosis and protected neurons from blood‐derived hazards in vitro. Furthermore, ODN1826 boosted M/M activation and phagocytic function, facilitated hematoma/iron resolution, reduced brain injury, and improved neurological function recovery in ICH mice, which were abolished by clodronate injection. The experimental results indicated that the Nrf2/CD204 pathway participated in TLR9‐induced M/M phagocytosis after ICH.ConclusionOur study suggests a protective role for TLR9‐enhanced M/M phagocytosis via the Nrf2/CD204 pathway after ICH. Our findings may serve as potential targets for ICH treatment.     

脑出血灶周组织ICAM-1与MMP-9表达的实验研究

目的研究大鼠实验性脑出血后血肿周围脑组织细胞间粘附分子-1（ICAM-1），基质金属蛋白酶-9（MMP-9）的表达。方法采用立体定向技术将自体不凝血注入大鼠尾状核区制备脑出血模型，免疫组化染色法检测血肿周围脑组织ICAM-1，MMP-9的表达。结果脑出血后6h血肿旁有少量ICAM-1表达阳性细胞，12h开始增多，3d达高峰；脑出血后6h血肿周围就有较多MMP-9表达阳性细胞，2d时阳性细胞最多；脑出血后ICAM-1与MMP-9的表达呈正相关（y=0．768，P〈0．05）。结论血肿周围组织ICAM-1、MMP-9表达上调提示两者可能参与了脑出血后继发性脑损伤。