脑缺血炎症反应的分子成像研究进展

中枢神经系统疾病常同时伴炎症反应,小胶质细胞激活是脑缺血后炎症反应的标志事件,其过度活化导致的神经毒性作用备受关注。以活化小胶质细胞作为生物标记实现炎症反应可视化,对诊断疾病、监测病情和个体化治疗有重要意义。本文就小胶质细胞相关的分子影像技术在脑缺血后炎症成像中的研究进展进行综述。     

A1型星形胶质细胞：治疗神经退行性和神经炎性疾病的新靶点

星形胶质细胞是中枢神经系统中数量最多的一类细胞，在生理条件下，它具有多种功能，并在维持中枢神经系统稳态中发挥着重要作用。但在神经系统损伤、炎症、缺氧等情况下，星形胶质细胞在基因表达、形态和功能上发生快速变化，这些反应统称为星形胶质细胞反应性。活化的星形胶质细胞可分为神经毒性表型（A1型）和神经保护表型（A2型）。研究表明，A1型星形胶质细胞广泛参与中枢神经系统疾病的发生和发展。因此，结合多种技术通过靶向调控激活的星形胶质细胞有望成为中枢神经系统疾病的一种潜在治疗方法。本文回顾性综述了近年来有关A1型星形胶质细胞的相关研究进展，包括其表型转换的分子机制及其在神经退行性和神经炎性疾病中的作用。     

视神经脊髓炎IgG刺激原代培养大鼠星形胶质细胞免疫反应

视神经脊髓炎(NMO)主要累及星形胶质细胞,导致中枢神经系统炎症、脱髓鞘和组织损伤。脑内病灶常高表达水通道蛋白4(AQP4),AQP4被认为是NMO血清IgG的抗原目标。根据NMO的可逆性及对NMO病灶演变特点的分析,笔者推测NMO IgG可能诱导了星形胶质细胞的动态免疫过程。培养原代大鼠星形胶质细胞,加入从NMO患者血清中提取或纯化的IgG,星形胶质细胞出现炎性增殖,同时基因表达出现与之匹配的明显变化。且星形胶质细胞反应因子脂质运载蛋白-2、多种趋化因子、细胞因子、应激反应因子的表达均增加。而在培养的原代星形胶质细胞中加入从正常对照者血清中提取的IgG则无上述变化。此星形胶质细胞的反应还与疾病种类相关。从复发缓解型多发硬化、Sj觟gren's或系统性红斑狼疮患者血清中提取的IgG加入培养的原代星形胶质细胞中也不能导致上述变化。笔者推测是NMO IgG与AQP4结合,导致星形胶质细胞出现炎症反应,并出现基因在转录和翻译水平的相应变化。抑制星形胶质细胞的炎症反应可能有助于减少星形胶质细胞增殖,缓解NMO病灶的发展。     

星形胶质细胞与雌激素神经保护作用关系的 研究进展

雌激素对脑损伤具有神经保护作用,而脑内星形胶质细胞在此过程中起重要作用,二者之间存在诸多交叉联系,星形胶质细胞可能是雌激素在中枢神经系统(CNS)中的重要靶细胞之一,除表达雌激素受体(ER)外,自身还分泌内源性雌激素。雌激素不仅能增强星形胶质细胞胶质纤维酸性蛋白(GFAP)的表达,增加反应性星形胶质细胞的数量,促进突触发生;同时还可通过星形胶质细胞表达的ER介导的脑保护作用减轻炎症反应,增加对谷氨酸的摄取能力,减少兴奋性损伤,抑制胶质瘢痕,促进神经功能恢复。因此,深入探究星形胶质细胞如何参与雌激素的脑保护作用将有助于确定雌激素的特定靶点,进而促进其在CNS疾病防控中的临床应用。     

以天麻和钩藤为主的中药制剂干预体外模拟脑缺血再灌注损伤大鼠碱性成纤维细胞生长因子及其受体的表达

目的：观察碱性成纤维细胞生长因子及其受体在体外模拟脑缺血再灌注损伤中的表达变化及以天麻和钩藤为主的中药制剂——抗呆Ⅰ号对其的影响。 方法：实验于2002-03／2004-04在科学实验中心完成。先分别进行大鼠大脑皮质星形胶质细胞和神经元的分离纯化培养及体外模拟脑缺血再灌注损伤模型的建立，然后分别进行下列实验：①按随机数字表法将传代后培养5d的星形胶质细胞分为正常对照组、缺血再灌注模型组和缺血用药组（应用抗呆Ⅰ号），在体外模拟脑缺血4h和再灌注3h，18h，24h，36h，48h和72h后行碱性成纤维细胞生长因子的免疫细胞化学染色。②用再灌注18h后收集的星形胶质细胞条件培养液、经抗呆Ⅰ号作用的星形胶质细胞条件培养液及星形胶质细胞条件培养液与抗呆Ⅰ号联合应用以1：5的浓度来培养损伤后的神经元。再对其培养的神经元行碱性成纤维细胞生长因子受体的免疫组化染色。 结果：①体外培养大鼠的大脑皮质星形胶质细胞在模拟脑缺血再灌注损伤后各时相点分泌碱性成纤维细胞生长因子的能力均显高于正常对照组（P〈0．05—0．01）；除再灌注72h外，其余各时相点缺血用药组碱性成纤维细胞生长因子的表达水平均显高于缺血再灌注模型组（P〈0．05—0．01）。②星形胶质细胞条件培养液组、经抗呆Ⅰ号作用的星形胶质细胞条件培养液组和联合应用组脑缺血再灌注损伤后各时相点的碱性成纤维细胞生长因子受体表达水平大多显高于缺血再灌注模型组（P〈0．05-0．01），均在再灌注18h时达到高峰，其作用强度为经抗呆Ⅰ号作用的星形胶质细胞条件培养液〉联合应用〉星形胶质细胞条件培养液。 结论：体外模拟脑缺血再灌注损伤使星形胶质细胞表达碱性成纤维细胞生长因子的能力增强，星形胶质细胞条件培养液能促进受损神经元碱性成纤维细胞生长因子受体的表达。抗呆Ⅰ号可增强受损神经组织的分泌功能，促进其损伤后的修复。     

大鼠局灶性脑缺血后星形胶质细胞的活化及桂哌齐特对神经功能的影响

目的：观察大鼠局灶性脑缺血再灌注后脑星形胶质细胞的不同活化状态的动态变化及桂哌齐特对鼠神经功能的影响，探讨星形胶质细胞在脑缺血损伤中的作用机制。方法：健康成年雄性SD大鼠24只，单纯随机分为3组：假手术组(n=6)、模型组(n=8)和桂哌齐特组(n=8)。采用免疫组织化学法检测脑缺血再灌注后22h和70h梗死灶周边区星形胶质细胞胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)的表达及药物的影响，并进行神经功能评分。结果：大鼠再灌注后22h皮质及纹状体梗死周边区的星形胶质细胞大量增生，细胞突起缩短增粗，染色增强；桂哌齐特组GFAP表达为8484．46&;#177;787．45，与模型组9565．17&;#177;1105．52比较显著减少(P&;lt;0．05)。再灌注后70h相应脑区域内星形胶质细胞GFAP的表达较22h有所减弱，桂哌齐特组GFAP表达显著增强(P&;lt;0.05)，神经功能评分均明显减少(P&;lt;0．05)。结论：脑缺血损伤后GFAP的活化反应在不同的时间具有双向作用，桂哌齐特显著改善神经功能，可能与其调节反应性星形胶质细胞的活性有关。     

小胶质细胞对星形胶质细胞的免疫和神经保护功能的调节作用(英文)

在中枢神经系统中,小胶质细胞和星形胶质细胞对神经元的发育、功能维持及细胞存活具有重要的作用。但在炎症反应过程中,星形胶质细胞增生的作用及其对神经元的影响目前并不十分清楚。因此,本研究对不同种类的细胞进行培养,给予细菌脂多糖(lipopolysaccharide,LPS)刺激,观察各种细胞的反应。结果显示,星形胶质细胞可以减轻炎症反应的神经元毒性,提示星形胶质细胞增生主要具有神经保护作用。如果去掉上清液中由星形胶质细胞分泌的胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF),这种神经保护作用显著降低。为了研究星形胶质细胞的免疫功能,本研究培养了高浓度的星形胶质细胞,并发现LPS不能诱导肿瘤坏死因子(tumor necrosis factor,TNF)-α及诱导型一氧化氮合酶(inducible nitric oxide synthase,iN OS)/一氧化氮(nitric oxide,NO)(iN OS/NO)释放。但如果在培养的高浓度星形胶质细胞中增加0.5%~1%的小胶质细胞,则可以诱导TNF-α及iN OS/NO的释放。这提示小胶质细胞和星形胶质细胞间的相互作用对于LPS诱导星形胶质细胞释放前炎症因子及GDNF是必须的。我们还发现,小胶质细胞释放的TNF-α可以通过旁分泌作用调节星形胶质细胞的神经保护功能。综上所述,本研究结果提示星形胶质细胞可能不会直接被LPS激活,而是通过与小胶质细胞相互作用被激活,释放神经营养因子,减少因炎症反应激活的小胶质细胞释放的毒性物质对神经元的损害作用。     

大鼠局灶性脑缺血后星形胶质细胞的活化及桂哌齐特对神经功能的影响

目的:观察大鼠局灶性脑缺血再灌注后脑星形胶质细胞的不同活化状态的动态变化及桂哌齐特对鼠神经功能的影响,探讨星形胶质细胞在脑缺血损伤中的作用机制。方法:健康成年雄性SD大鼠24只,单纯随机分为3组:假手术组(n=6)、模型组(n=8)和桂哌齐特组(n=8)。采用免疫组织化学法检测脑缺血再灌注后22h和70h梗死灶周边区星形胶质细胞胶质纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)的表达及药物的影响,并进行神经功能评分。结果:大鼠再灌注后22h皮质及纹状体梗死周边区的星形胶质细胞大量增生,细胞突起缩短增粗,染色增强;桂哌齐特组GFAP表达为8484.46±787.45,与模型组9565.17±1105.52比较显著减少(P<0.05)。再灌注后70h相应脑区域内星形胶质细胞GFAP的表达较22h有所减弱,桂哌齐特组GFAP表达显著增强(P<0.05),神经功能评分均明显减少(P<0.05)。结论:脑缺血损伤后GFAP的活化反应在不同的时间具有双向作用,桂哌齐特显著改善神经功能,可能与其调节反应性星形胶质细胞的活性有关。     

脑缺血小胶质细胞的表型转化与靶向治疗研究

炎症反应在脑缺血的发病机制中起重要作用。小胶质细胞(MG)是中枢神经系统的固有免疫细胞,脑缺血发生后MG可被迅速活化为M1、M2不同表型,参与炎症反应的发生及后期的组织修复等过程。MG的过度活化可导致一系列细胞毒性因子的累积,对参与MG激活过程中的受体、信号通路及分泌物等的调节都可能是一个新的治疗策略。临床前研究表明几类药物具有上述作用,本文主要从脑缺血损伤后MG的活化表型、作用及临床前研究中对靶向抑制MG激活的药物进行综述。     

大鼠局灶性脑缺血后星形胶质细胞Cyclin D1及Cyclin E的表达

目的:研究大鼠局灶性脑缺血后星形胶质细胞细胞周期蛋白Cyclin D1及Cyclin E的表达差异及变化。方法:建立大鼠大脑中动脉闭塞模型,双光源流式细胞术检测假手术组及缺血再灌注后1d、3d、7d、15d组大鼠大脑皮质星形胶质细胞Cyclin D1及Cyclin E的表达。结果:假手术组大鼠星形胶质细胞表达Cyclin D1及Cyc-lin E,Cyclin E的表达高于Cyclin D1(P0.05);局灶性脑缺血后,星形胶质细胞Cyclin D1的表达逐渐增加,7d及15d时高于假手术组(P0.05);局灶性脑缺血后,星形胶质细胞Cyclin E的表达无明显变化,各组间比较差异无统计学意义。结论:局灶性脑缺血后星形胶质细胞Cyclin D1表达增强,提示Cyclin D1可能参与脑缺血后星形胶质细胞的活化增殖。     

家兔脑干机械性损伤后S100β表达与损伤时间的关系

目的：研究家兔脑干机械性损伤后S100β含量随时间变化规律。方法健康家兔24只,经自制打击装置打击脑干部位,分别于损伤后0、2、4、8、16、24、48、72h解剖取脑干,甲醛固定,免疫组织化学和图像分析技术检测。结果损伤后0h家兔脑干中可见S100β阳性染色均匀分布在星形胶质细胞胞体及突起中。脑干机械性损伤后2h ,星形胶质细胞结构破坏,大量的S100β从中漏出,突起消失;之后随着时间的延长S100β逐步升高,24h达到高峰,之后下降,但仍高于0h （LSD检验,P＜0．05）。结论 S100β主要存在于神经胶质细胞,是中枢神经损伤的敏感生化指标,根据S100β的含量变化可作检测脑早期缺血缺氧性损伤和预后的黄金指标;同时可以应用于法医学中枢神经系统损伤时间的推断,为法医学鉴定提供一定的实验依据。     

In vivo visualization of reactive gliosis using manganese-enhanced magnetic resonance imaging

Reactive astrogliosis occurs after diverse central nervous system (CNS) insults. While astrogliosis provides protection against inflammation, it is also obstructive in the progress of neuranagenesis after CNS insults. Thus, a method that enables in vivo visualization and tissue characterization for gliosis would be invaluable for studies of CNS insults and corresponding treatments. Manganese has proven to be a useful MRI contrast agent that enters cells via Ca²⁺ channels and has been applied to manganese-enhanced MRI (MEMRI) for neuronal functional mapping. This study investigated whether MEMRI can detect astrogliosis after focal ischemia in vivo. Rats were divided into groups according to the number of days after either transient middle cerebral artery occlusion or a sham. Ring- or crescent-shaped enhancement of MEMRI corresponded to the GFAP-positive astroglia observed in the peripheral region of the ischemic core 11 days after middle cerebral artery occlusion. This indicates that MEMRI enhancement predominantly reflects reactive astrogliosis after stroke.     

Cytokines as mediators in the central nervous system

Cytokines are soluble mediators involved in cell-cell regulations in the immunological and the hematopoietic system. We review various cytokine effects on the central nervous system, including growth-promoting activity, neuromodulatory action, fever induction, sleep and decreased food intake. In addition, cytokines, neuropeptides, neurotransmitters and hormones all participate in an intricate inter-relationship to contribute to the development and maintenance of brain homeostasis. Cytokines are also involved in the wounding responses of injured brain after trauma, infection or neurodegenerative processes. Pharmacological modulation of the expression and/or actions of cytokines in the brain may represent a new field of research of therapeutic benefit in the treatment of central disorders.     

Neuronal damage in rat brain and spinal cord after cardiac arrest and massive hemorrhagic shock

OBJECTIVE: Severe global ischemia often results in severe damage to the central nervous system of survivors. Hind-limb paralysis is a common deficit caused by global ischemia. Until recently, most studies of global ischemia of the central nervous system have examined either the brain or spinal cord, but not both. Spinal cord damage specifically after global ischemia has not been studied in detail. Because the exact nature of the neuronal damage to the spinal cord and the differences in neuronal damage between the brain and spinal cord after global ischemia are poorly understood, we developed a new global ischemia model in the rat and specifically studied spinal cord damage after global ischemia. Further, we compared the different forms of neuronal damage between the brain and spinal cord after global ischemia. DESIGN: Randomized, controlled study using three different global ischemia models in the rat. SETTING: University research laboratory. SUBJECTS: Male, adult Sprague-Dawley rats (300 g). INTERVENTIONS: Animals were divided into three experimental groups, group A (n = 6, survived for 7 days), 12 mins of hemorrhagic shock; group B (n = 6, survived for 7 days), 5 mins of cardiac arrest; or group C (n = 6, each for 6 hrs, 12 hrs, 1 day, 3 days, and 7 days), 7 mins of hemorrhagic shock and 5 mins of cardiac arrest. Motor deficit of the hind limbs was studied 6 hrs to 7 days after resuscitation. Also, nonoperated animals (n = 6) were used as the control. Histologic analysis (hematoxylin and eosin, Fluoro-Jade B, terminal deoxynucleotidyl transferase- mediated dUTP end-labeling [TUNEL], Klüver-Barrera) and ultrastructural analysis using electron microscopy were performed on samples from the CA1 region of the hippocampus and lumbar spinal cord. Demyelination of the white matter of the lumbar spinal cord was analyzed semiquantitatively using Scion Image software. MAIN RESULTS: No paraplegic animals were observed in either group A or B. All group C animals showed severe hind-limb paralysis. Severe neuronal damage was found in the CA1 region of the hippocampus in all groups, and the state of delayed neuronal cell death was similar among the three groups. Neuronal damage in the lumbar spinal cord was detected only in group C animals, mainly in the dorsal horn and intermediate gray matter. Demyelination was prominent in the ventral and ventrolateral white matter in group C. A significant difference was observed between control and group C rats with Scion Image software. Ultrastructural analysis revealed extensive necrotic cell death in the intermediate gray matter in the lumbar spinal cord in group C rats. CONCLUSION: The combination in the global ischemia model (i.e., hemorrhagic shock followed by cardiac arrest) caused severe neuronal damage in the central nervous system. Thereby, hind-limb paralysis after global ischemia might result from spinal cord damage. These results suggest that therapeutic strategies for preventing spinal cord injury are necessary when treating patients with severe global ischemia.     

Mechanisms of ischemic cerebral injury

Normal compensatory mechanisms protect the central nervous system (CNS) from moderate hypoxia and ischemia; however, after more severe ischemia progressive brain hypoperfusion ensues and irreversible damage occurs. Ischemic brain injury remains greatly significant clinically and elucidating the determinants of ischemic neuronal injury and death continues to challenge researchers. Although altered perfusion and decreased energy charge may contribute to the production of irreversible damage, the distribution of lesions seen after insult does not correspond with the degree of ischemic blood flow impairment, nor can neuronal energy deprivation explain the cell damage. Other factors, such as derangements in astrocyte function, calcium homeostasis, free radical metabolism, acid-base regulation and excitatory neurotransmitters also probably mediate ischemic neuronal death. Continued investigation to establish the cellular pathophysiology of cerebral ischemia can guide rational research and therapeutic strategies.     

The role of cytokines in the generation of inflammation and tissue damage in experimental gram-positive meningitis

Cytokines mediate many host responses to bacterial infections. We determined the inflammatory activities of five cytokines in the central nervous system: TNF-alpha, IL-1 alpha, IL-1 beta, macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). Using a rabbit model of meningeal inflammation, each cytokine (except IL-1 beta) induced enhanced blood brain barrier permeability, leukocytosis in cerebrospinal fluid, and brain edema. Homologous antibodies to each mediator inhibited leukocytosis and brain edema, and moderately decreased blood brain barrier permeability. In rabbits treated with anti-CD-18 antibody to render neutrophils dysfunctional for adhesion, each cytokine studied lost the ability to cause leukocytosis and brain edema. After intracisternal challenge with pneumococci, antibodies to TNF or IL-1 prevented inflammation, while anti-MIP-1 or anti-MIP-2 caused only a 2-h delay in the onset of inflammation. We suggest these cytokines have multiple inflammatory activities in the central nervous system and contribute to tissue damage during pneumococcal meningitis.     

神经干细胞对缺血性脑损伤的反应能力

背景:当中枢神经系统受损伤时,Nestin蛋白的重新表达可能增加细胞抗损伤的能力,有利于损伤灶的修复。目的:通过永久性脑缺血神经干细胞迁移及Nestin蛋白表达的变化,探讨永久性脑缺血情况下神经干细胞的反应性。设计:以实验动物为研究对象,随机对照的验证性研究。单位:一所专科学校的解剖学教研室和一所大学的解剖学教研室。材料:实验于1999-10/2001-01在西安交通大学医学院人体解剖学教研室进行,选择健康SD大鼠75只。随机分为正常对照组、实验组和假手术组,每组25只。各组动物均在术后1,3,7,14和28d,断头取脑,每个时间点5只大鼠。方法:以永久性大鼠脑缺血为模型,采用免疫组化染色方法,观察脑缺血1,3,7,14和28d时,神经干细胞的迁移及其神经干细胞标记物Nestin蛋白的变化。主要观察指标:①免疫组化染色结果。②SZa区Nestin阳性细胞在正常和缺血后不同时间点脑组织中的迁移距离。③缺血后不同时间点缺血区附近Nestin阳性细胞数的变化。结果:通过Nestin免疫组化染色,发现正常脑组织中神经干细胞主要位于室管膜下区。室管膜下区的神经干细胞在缺血后沿胼胝体腹侧向缺血区方向发生了迁移。其中,缺血7d时达到最远。缺血灶附近在缺血1d时就出现较多的Nestin阳性细胞,缺血3d以后逐渐减少。结论:神经干细胞对缺血性脑损伤有一定的反应能力,Nestin蛋白表达于缺血附近可能是一种对损伤的保护机制。     

脑缺血再灌注海马齿状回神经干细胞Mash-1的表达

背景:Mash-1在中枢神经系统和周围神经系统发育中都有表达,对神经元和神经胶质细胞的分化都有很重要的作用。目的:检测Mash-1在大鼠脑缺血再灌注海马组织神经干细胞中的表达及其变化。方法:建立大脑中动脉栓塞制作大鼠脑缺血再灌注模型。实验分为假手术组、缺血再灌注3,7,14,21,28d组。结果与结论:免疫组织化学检测结果,随着脑缺血再灌注第3天海马神经元BrdU阳性细胞明显增多,第7天达高峰,然后逐渐减少。Mash-1反应产物随脑缺血再灌注时间延长逐渐增多,第21天表达最强,以后表达逐渐减少。提示Mash-1呈时间依赖性表达,与神经干细胞增殖分化进程相吻合,说明其在神经干细胞晚期分化中起重要调控作用。     

Ischemia as a possible effect of increased intra-abdominal pressure on central nervous system cytokines,lactate and perfusion pressures

Introduction  

The aims of our study were to evaluate the impact of increased intra-abdominal pressure (IAP) on central nervous system (CNS) cytokines (Interleukin 6 and tumor necrosis factor), lactate and perfusion pressures, testing the hypothesis that intra-abdominal hypertension (IAH) may possibly lead to CNS ischemia.