【作者】 王海萍；

【导师】 迟兆富；

【作者基本信息】 山东大学 ， 神经病学， 2009， 博士

【摘要】 研究背景:缺血性脑血管病是当今国内外死亡率和致残率较高的疾病之一,是临床常见病多发病,具有高发生率、高致残率、高死亡率的特点,严重危害人类健康。模拟人类缺血性脑血管病的发展过程,建立重复性好、生理指标控制严格的标准化活体脑缺血动物模型,始终是研究者们所追求的目标。随着缺血性神经元损伤与修复机制研究的日趋深入,对脑缺血动物模型的建立提出了更高的要求。如何在现有的条件下建立标准化、规范化的脑缺血动物模型,并合理的选择性的将其用于脑缺血损害的基础研究是值得探索的问题。建立一种廉价、简便、可靠的动物模型,对缺血性脑血管病的防治研究具有重大意义。大脑中动脉是临床缺血性脑血管病的多发部位,故大鼠大脑中动脉闭塞再灌注（middle cerebral artery occlusion reperfusion,MCAO/R）模型已被广泛应用于急性局灶性脑梗死的研究。以往是在颅内阻断大脑中动脉（middle cerebral artery,MCA）,但该手术涉及范围广,损伤严重。自1981年Tamura等采用开颅电凝阻断大脑中动脉建立局灶性脑缺血动物模型以来,经过多次改进,目前认为线栓法是建立脑缺血动物模型的较好途径。目的:建立经典的大鼠大脑中动脉闭塞再灌注模型。方法:选择健康雄性Wistar大鼠200只,体重240-260g,用10%水合氯醛(300mg.kg^-1)麻醉大鼠,把头端涂胶的尼龙线（直径0.28～0.30mm）自左颈外动脉缓慢进入大脑中动脉的起始段,分别阻断血流0h、0.5h、1.0h、1.5h、2.0h后拔除线栓在灌注24h,术中保持室温在26℃。大鼠苏醒后,进行神经行为功能评分;应用干湿重法测定脑组织含水量;应用三苯基四氮唑（TTC）染色对模型进行评价。结果:200只大鼠存活165只,死亡35只。存活大鼠不同程度出现Horner征及自主活动时向右旋转等功能障碍,缺血0h、0.5h、1.0h、1.5h及2.0h组大鼠神经行为功能评分分别为0.00±0.00,2.07±0.51,2.91±0.44,3.24±0.49,3.61±0.39;脑组织含水量分别为78.93±1.22,81.44±1.41,83.63±1.39,84.15±1.44,84.81±1.37;脑梗死体积分别为0%,65.63±5.1%,72.76±4.2%,75.39±4.8%和80.21±4.2%。结论:随着缺血时间延长,大鼠的神经功能行为评分依次升高,脑组织含水量及梗死体积也依次增加;另外,该模型可以控制缺血和再灌注时间,是研究缺血性脑卒中病理生理学的经典动物模型。研究背景基质金属蛋白酶是一类Zn²⁺离子依赖的蛋白水解酶,参与细胞外基质的代谢。近年来,MMPs被认为参与了脑卒中后的病理生理过程,脑缺血时MMPs上调并且可以直接导致神经元死亡,而应用广谱MMPs抑制剂或抗体可减少脑梗死的体积。其中MMP-9起主导作用,研究表明,MMP-9基因敲除鼠可相对的免除脑缺血所致的脑损伤。在短暂局灶性脑缺血鼠模型中MMP-9的过度活跃不仅能降解神经血管的基质,损伤血脑屏障（blood brainbarrier,BBB）引起水肿和出血,而且血脑屏障的开放是脑缺血再灌注后脑水肿、炎性细胞浸润进而导致神经细胞死亡的重要原因。神经调节素（Neuregulins,NRGs）是一个由四种基因编码的多肽家族,由neuregulin-1（nrg-1）基因编码产物的选择性剪接体所组成,包含四种异构体NRG-1、2、3和4。近年来发现,NRGs在体内外和中枢神经系统都是抗炎物质,能抑制脑缺血再灌注损伤诱导的神经元凋亡、胶质反应和炎症反应,诱导有关神经营养因子的表达,具有神经保护作用。但具体机制不明,目前应进一步研究NRG-1β在缺血性脑损伤中的神经保护作用及作用机制,以探讨其临床应用的潜在价值。目的观察大鼠脑缺血再灌注损伤基质金属蛋白酶（matrixmetalloproteinases,MMP-9）表达及神经调节素-1β（neuregulin-1β,NRG-1β）的干预作用。方法成年健康雄性Wister大鼠200只,应用线拴法经颈外-颈内动脉插线建立大脑中动脉闭塞再灌注（models of middle cerebral artery occlusion/reperfusion,MCAO/R）动物模型,治疗组经颈内动脉单剂量注射1.5%NRG-1β5μL干预。氯化三苯基四氮唑（TTC）染色测定脑梗死体积,原位TUNEL方法检测细胞凋亡,免疫组化、免疫荧光双标记法和免疫印迹法（Western-blotting）观察脑组织MMP-9表达。结果脑缺血再灌注损伤可诱导大脑皮质和纹状体区脑组织细胞凋亡和MMP-9表达。随着缺血缺氧时间的延长,对照组皮质区细胞凋亡数量自缺血0h、0.5h、1.0h、1.5h和2.0h逐渐增加,分别为1.78±0.15、5.78±0.51、10.35±0.77、21.50±1.19和32.00±1.78;而纹状体区细胞凋亡数量自缺血0h、0.5h、1.0h、1.5h和2.0h也逐渐增加,分别为1.46±0.21、4.12±0.54、7.33±0.71、16.54±1.63和19.03±1.44（t=5.44～21.80,P＜0.05）;对照组MMP-9蛋白表达（t=3.363～25.523,P＜0.01）逐渐增加。应用NRG-1β后,皮质区细胞凋亡数量自缺血0h、0.5h、1.0h、1.5h和2.0h明显减少,分别为1.66±0.11、4.80±0.61、5.63±0.56、9.75±1.22和13.54±1.26;而纹状体区细胞凋亡数量自缺血0h、0.5h、1.0h、1.5h和2.0h也明显减少,分别为1.34±0.14、3.35±0.32、4.55±0.50、7.63±1.41和10.46±0.98（t=3.68～18.93,P＜0.05）;MMP-9表达水平较对照组同一时间点相应脑区显著降低（t=2.678～8.535,P＜0.05）;脑梗死体积明显缩小（t=4.645～17.837,P＜0.01）。结论脑缺血后MMP-9表达增强,参与了脑缺血再灌注损伤后的炎症反应过程。NRG-1β可能通过下调MMP-9的表达而抑制脑缺血再灌注损伤后炎症反应诱发的细胞凋亡。研究背景核转录因子（nuclear factorκB,NF-κB）作为一种多向性转录调节蛋白,能调节多种炎症因子的表达和细胞凋亡,在缺血再灌注中起了重要作用。NF-κB是Rel蛋白家族中的一组转录因子,是由多肽链p50和p65二个蛋白亚基组成同源或异源性二聚体。NF-κB被激活后,启动一系列免疫和炎症反应相关基因的转录程序,诱导众多炎症因子的高表达。炎症因子通过促进花生四烯酸代谢产物的释放及氧自由基、脂质过氧化物的生成,引起细胞毒性脑水肿、血管内皮细胞肿胀坏死及内皮细胞间隙紧密连接的破坏,通透性增加,或通过抑制谷氨酸的吸收造成血管源性脑水肿、血脑屏障破坏,进一步加重脑组织损伤。胶质纤维酸性蛋白（gelatinous fiber acid protein,GFAP）是脑内成熟星型细胞的主要中间丝,是星型胶质细胞的一种标志蛋白,在星型细胞中有丰富、唯一的表达。胶质细胞浸润和活化,是缺血性脑卒中诱导的炎症反应的特征反应。MCAO的鼠中,梗塞周围区域24h后GFAP免疫反应明显增强,提示星形胶质细胞的存在。有研究显示,MCAO/再灌注24h后结果相同,在对照组梗塞周围发现了重GFAP着色斑。然而,NRG-1β治疗组半暗带区GFAP的表达则大大降低。神经调节素（Neuregulins,NRGs）是一个由四种基因编码的多肽家族,包含四种异构体NRG-1、2、3和4,NRG-1β在体内外和中枢神经系统都是抗炎物质,可下调白细胞介素-1（interleukin-1,IL-1）、核转录因子-κB（neuclear factor-κB,NF-κB）等炎性细胞因子的表达。近年来发现,NRGs在脑缺血再灌注损伤的病理生理过程中发挥神经保护作用。其中,NRG-1β被认为与神经元的生存和功能有关,但具体机制不明,目前应进一步研究NRG-1β在缺血性脑损伤中的神经保护作用及作用机制,以探讨其临床应用的潜在价值。目的观察大鼠脑缺血再灌注损伤核转录因子（nuclear factorκB,NF-κB）、胶质纤维酸性蛋白（gelatinous fiber acid protein,GFAP）的表达及神经调节素-1β（neuregulin-1β,NRG-1β）的干预作用。方法成年健康雄性Wister大鼠200只,应用线拴法经颈外-颈内动脉插线建立大脑中动脉闭塞再灌注（models of middle cerebral artery occlusion/reperfusion,MCAO/R）动物模型,治疗组经颈内动脉单剂量注射1.5%NRG-1β5μL干预。氯化三苯基四氮唑（TTC）染色测定脑梗死体积,原位TUNEL方法检测细胞凋亡,免疫组化、免疫荧光双标记法和免疫印迹法（Western-blotting）观察脑组织NF-κB和GFAP表达。结果脑缺血再灌注损伤可诱导大脑皮质和纹状体区脑组织细胞凋亡、NF-κB和GFAP表达。随着缺血缺氧时间的延长,对照组细胞凋亡数量（t=5.44～21.80,P＜0.05）、NF-κB（t=3.363～25.523,P＜0.01）和GFAP蛋白表达（t=3.16～21.38,P＜0.01）逐渐增加。应用NRG-1β后,NF-κB（t=2.46～5.46,P＜0.05）、GFAP（t=3.03～5.89,P＜0.01）表达水平较同一时间点对照组相应脑区显著降低,细胞凋亡数量明显减少（t=3.68～18.93,P＜0.05）,脑梗死体积明显缩小（t=4.645～17.837,P＜0.01）。结论脑缺血后NF-κB和GFAP表达增强,参与了脑缺血再灌注损伤后的炎症反应过程。NRG-1β可能通过下调NF-κB和GFAP的表达而抑制脑缺血再灌注损伤后炎症反应诱发的细胞凋亡。更多还原

【Abstract】 Background Ischemic stroke is one of the leading causes of death in the world,and continues to be the leading causes of long-term disability.The research for a good repeating.reliable animal model which is similiar to the pathophysiology of human ischemic stroke has been prompted by many scholars. With the development of the neuron injuring and repairment,a better ischemic animal model is wanted.How to develop a standarized ischemic animal model under the condition and choose the proper one for the research of the ischemic stroke is a good question to us now.To development a cheaper.simple and reliable animal model is very important for the research of the ischemic stroke. Many ischemic stroke happen owing to the occlusion of middle cerebral artery(MCA).So middle cerebral artery occlusion/reperfusion(MCAO/R)model has been widely used today.A direct microsurgical technique for permanently occlusion the MCA through a craniectomy had also been done.Since Traura invented the MCAO model through a craniectomy,it has been studied and modified extensively.Now the model of reversible MCAO without craniectomy is accepted widely.Objective To establish a classical model with MCAO/R in ratMethods We choosed 200 healthy male Wister rats,weighing between240-260g.All rats were anesthetized with hydrochloride ketemine.The nylon sutures were coated with silica gel at one end.Then we introduced the nylon sutures into the cervical internal carotid artery and advanced them intracranically to block blood flow into the MCA at 0h.0.5h,1.0h,1.5h and 2.0h.then drawed out the suture.So the occlused MCA were reperfused.Neurologic deficit,morality rate,brain wet content and infarct volume were evaluated in all surving rats. Results 165 rats survived and 35 rats were dead.Of all surving rats,the grades of neurologic defict were 0.00±0.00,2.07±0.51,2.91±0.44,3.24±0.49and 3.61±0.39 at the different groups which were divided according the ischemic time from 0h to 2h one by one.The brain wet contents were 78.93±1.22,81.44±1.41,83.63±1.39,84.15±1.44,84.81±1.37 and the infarct volumes were 0%,65.63±5.1%,72.76±4.2%,75.39±4.8%and 80.21±4.2%at different groups.Conclusion The MCAO/R model is stable with accurate timing control of occlusion and reperfusion.It is the classical one for researching of pathophysiology in ischemic stroke. Background The matrix metalloproteinases(MMPs),may degrade extracellular matrix and trigger anoikis-like cell death in neurons.MMPs comprise an important family of proteases associated with basement membrane and extracellular matrix remodeling and are involved in both physiological and pathological CNS processes.In particular,the gelatinases(MMP-2 and MMP-9) have been implicated specifically in cerebral ischemia.Knock-out mice deficient in MMP-9 are protected against brain trauma and focal cerebral ischemia.The mechanisms of injury may involve MMP-9-mediated disruption of blood-brain barrier integrity,edema,and hemorrhagic conversion.Neuregulins are signalling proteins and the NRG1 family encodes at least 15 different splice variants,classified into four isoforms.They play an important role in cell differentiation,migration, myelination and proliferation of oligodendrocytes and neurons.In animal models of ischemia/hypoxia,NRG1 has been shown to act as a therapeutic,neuroprotective agent and should be investigated in more detail in transgenic animal models.A recent study demonstrated that NRG-1 blocked delayed neuronal death and pro-inflammatory responses following focal ischemic stroke in the rat.However,the mechanisms that underlie the neuroprotective effects of NRG-1 are unclear.Objective To observe the expression of matrix metalloproteinase-9 (MMP-9) and the influence of neuregulin-1β(NRG-1β) on brain tissue of rats following cerebral ischemia/reperfusion.Methods The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by a monofilament method from left external-internal carotid artery in 200 adult healthy male Wister rats.The rat models in the treatment group were injected 1.5%NRG-1β5μL from internal carotid artery (ICA).The cerebral infarct volume was measured by TTC stain,the apoptosis was determined wit in situ TUNEL method,the expressions of MMP-9 were determined by immunohistochemical and immumofluorescent double staining and western-blotting assay.Results Cerebral ischemia reperfusion can induce apoptosis and expression of MMP-9 in cerebral cortex and striatum.With the ischemic time prolonging,the number of apoptotic cells in cortex from ischemic 0h、0.5h、1.0h、1.5h to 2.0h increased from 1.78±0.15,5.78±0.51,10.35±0.77,21.50±1.19 to 32.00±1.78,while the number of apoptotic cells in striatum from ischemic 0h、0.5h、1.0h、1.5h to 2.0h also increased successfully from 1.46±0.21、4.12±0.54、7.33±0.71、16.54±1.63 to 19.03±1.44(t=5.44～21.80,P＜0.05) and the expression of MMP-9 increased successfully(t=3.363～25.523,P＜0.01) in the control group. With NRG-1βtreatment,the number of apoptotic cells incortex from ischemic 0h、0.5h、1.0h、1.5h to 2.0h reduced from 1.66±0.11、4.80±0.61、5.63±0.56、9.75±1.22 to 13.54±1.26;while the number of apoptotic cells in striatum from ischemic 0h、0.5h、1.0h、1.5h to 2.0h also decreased successfully from 1.34±0.14、3.35±0.32、4.55±0.50、7.63±1.41 to 10.46±0.98(t=3.68～18.93,P＜0.05),the expression of MMP-9 decreased(t=2.678～8.535,P＜0.05),and the infarct volume decreased significantly(t=4.645～17.837,P＜0.01 ) compared with that in the control group at the same timepoint and the corresponding region.Conclusion The expression of MMP-9 is high after cerebral ischemia/reperfusion,that contribute to the inflammatory reaction.NRG-1βmight down-regulate the expression of MMP-9 to inhibit apoptosis inducing by inflammatory reaction in cerebral ischemic reperfusion. Background Nuclear factor-kappaB(NF-κB) is a transcription factor that is activated after cerebral ischemia.NF-κB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke,causing enhanced release of proinflammatory cytokines,which are known to be associated with delayed neuronal death following ischemia.Gelatinous fiber acid protein (GFAP) is the main fiber of astrocytes in brain.It can be expressed only by astrocytes.Astrocytes can be actived after ischemia which is regard as character of the inflammatory response after ischemia.In MACO rats,GFAP was found around the infarct tissue while its expression can decrease after treatment of NRG-1β. Neuregulins are signalling proteins and the NRG1 family encodes at least 15 different splice variants,classified into four isoforms.They play an important role in cell differentiation,migration,myelination and proliferation of oligodendrocytes and neurons.In animal models of ischemia/hypoxia,NRG1 has been shown to act as a therapeutic,neuroprotective agent and should be investigated in more detail in transgenic animal models.A recent study demonstrated that NRG-1 blocked delayed neuronal death and pro-inflammatory responses following focal ischemic stroke in the rat.However,the mechanisms that underlie the neuroprotective effects of NRG-1 are unclear.Objective To observe the expression of nuclear factorκB,NF-κB)、gelatinous fiber acid protein(GFAP) and the influence of neuregulin-1β(NRG-1β) on brain tissue of rats following cerebral ischemia/reperfusion.Methods The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by a monofilament method from left external-internal carotid artery in 200 adult healthy male Wister rats.The rat models in the treatment group were injected 1.5%NRG-1β5μL from internal carotid artery (ICA).The cerebral infarct volume was measured by TTC stain,the apoptosis was determined wit in situ TUNEL method,the expressions of NF-κB and GFAPwere determined by immtmohistochemical and immumofluorescent double staining and western-blotting assay.Results Cerebral ischemia reperfusion can induce apoptosis and expression of NF-κB and GFAP in cerebral cortex and striatum.With the ischemic time prolonging,the number of apoptotic cells(t=5.44～21.80,P＜0.05) and the expression of NF-κB(t=3.363～25.523,P＜0.01) and GFAP(t=3.16～21.38, P＜0.01) increased successfully in the control group.With NRG-1βtreatment, the expression of NF-κB(t=2.46～5.46,P＜0.05) and GFAP(t=3.03～5.89, P＜0.01) decreased,the number of apoptotic cells reduced(t=3.68～18.93,P＜0.05), and the infarct volume decreased significantly(t=4.645～17.837,P＜0.01) compared with that in the control group at the same timepoint and the corresponding region.Conclusion The expression of NF-κB and GFAP is high after cerebral ischemia/reperfusion,that contribute to the inflammatory reaction.NRG-1βmight down-regulate the expression of NF-κB and GFAP to inhibit apoptosis inducing by inflammatory reaction in cerebral ischemic reperfusion.更多还原