中枢神经系统突触发生研究方法概述

突触是神经元进行信息交换的特殊部位。突触发生中神经元突起通过延伸、辨认相应的靶细胞并与其接触形成稳定的突触。晚近的一些实验方法对突触发生做了研究，认为电镜、电生理、免疫组织化学及实时荧光成像等多种方法相结合进行研究更可信，研究突触发生可能有助于进一步探讨中枢神经系统疾病的康复机制。     

吗啡对大鼠海马神经元突触传递的作用及机制

目的从离子通道角度研究吗啡对中枢神经系统兴奋性及抑制性突触传递的作用,以探讨吗啡镇痛机制.方法原代培养新生Wistar大鼠的海马神经元.采用膜片钳技术研究吗啡对其兴奋性及抑制性突触后电流及谷氨酸诱发电流的影响.结果①吗啡可明显增强海马神经元兴奋性突触传递,加吗啡后自发兴奋性突触后电流发放频率增加了207.8%(t=42.182 8,P＜0.01).此作用可被阿片受体阻断剂纳洛酮阻断;②吗啡对微小兴奋性突触后电流的发放频率及谷氨酸诱发电流的幅度没有明显影响(t=0.962,t=0.791,P＞0.05);③吗啡可明显抑制神经元自发抑制性突触后电流,纳洛酮可拮抗吗啡作用(P＜0.01).结论吗啡对海马神经元的兴奋作用不是由于吗啡直接作用于兴奋性氨基酸-谷氨酸突触传递过程,而是可能由于抑制了抑制性中间神经元,间接产生的兴奋达到镇痛作用.     

黄芪益母草注射液对脑缺血大鼠神经元突触结构的保护作用

目的：观察脑缺血-再灌注后大鼠皮质神经元突触结构的变化，并试图探讨黄芪和益母草发挥脑保护作用的可能机制，方法：采用四血管结扎法制作再灌注模型。运用电子显微镜摄像和微观计量方法，观察假手术组，模型组和黄芪益母草注射液组大鼠皮质神经元GrayⅠ型突触的结构和突触活性区长度，突触致密物质厚度，突触小泡数量的变化。结果：模型组突触结构模糊，线粒体肿胀，突触活性区长度，突触致密物质厚度，突触小泡数量与假手术组比较均有显著增加；黄芪益母草注射液组突触结构较清晰，结构相对完整，3项突触微观指标与模型组比较有显著下降。结论：缺血后大鼠皮质神经元GrayⅠ型突触结构遭破坏，黄芪益母草可能是通过保护神经元突触结构的基本完整，抑制脑缺血后神经毒性作用，从而起到脑保护作用。     

突触形成调控蛋白的研究与进展：

背景：干细胞在体外也被证实可以分化为神经元细胞,然而干细胞分化成神经细胞后如何形成突触连接而实现信息传递功能,以及突触形成的调控机制是什么,这些尚未可知。目的：通过对2000年以来影响突触形成调控蛋白的实验研究检索,总结这些蛋白在突触研究中的作用。方法：以“synapse、synaptogenesis”为检索词,应用计算机检索中国知网和pubmed数据库相关文章,排除蘑复性研究,保留39篇文章做进‘步分析。结果与结论：突触形成的过程主要包括3个方面的相关内容：①突触结构的形成。②些突触由无活性剑有活性的转换。③非必要突触的消除。而与之相关的蛋白及其功能得以肯定并得到初步研究的主要育血小板反应蛋白、突触分化诱导基凶产物、突触细胞黏附分子、主要组织相容性复合物I型、肌细胞增强因子2、脆性X智力低下蛋白等。这些蛋白在突触的形成,发育和成熟过程中发挥着重要作用。     

吗啡对大鼠海马神经元突触传递的作用及机制

目的：从离子通道角度研究吗啡对中枢神经系统兴奋性及抑制性突触传递的作用，以探讨吗啡镇痛机制。方法：原代培养新生Wistar大鼠的海马神经元。采用膜片钳技术研究吗啡对其兴奋性及抑制性突触后电流及谷氨酸诱发电流的影响。结果：①吗啡可明显增强海马神经元兴奋性突触传递，加吗啡后自发兴奋性突触后电流发放频率增加了207．8％(t=42．1828，P&;lt;0．01)。此作用可被阿片受体阻断剂纳洛酮阻断；②吗啡对微小兴奋性突触后电流的发放频率及谷氨酸诱发电流的幅度没有明显影响(t=0．962，t=0．791，P&;gt;0．05)；③吗啡可明显抑制神经元自发抑制性突触后电流，纳洛酮可拮抗吗啡作用(P&;lt;0．01)。结论：吗啡对海马神经元的兴奋作用不是由于吗啡直接作用于兴奋性氨基酸-谷氨酸突触传递过程，而是可能由于抑制了抑制性中间神经元，间接产生的兴奋达到镇痛作用。     

血管性痴呆的发病与突触可塑性

目的：血管性痴呆是主要由脑血管疾病引起的获得性、慢性、进行性认知障碍，是老年期痴呆的主要类型。其发病机制尚不明确。突触损伤可能是其早期的病理改变，与其认知功能障碍关系密切。 资料来源：应用计算机检索Medline1990—01／2004—12关于血管性痴呆、脑缺血及认知障碍与突触改变的文章，检索词包括“cerebral is-chemia and synapse；cognitive impairment and synapse；vascular demen-tia and synapse；demetian and synapse，long-termp rotection”等，并限定文章语言种类为English。应用计算机检索维普全文数据库1990—01／2004-12关于血管性痴呆、脑缺血及认知障碍与突触改变的文章，检索词为“突触结构，可塑性，血管性痴呆、突触功能，突触蛋白”。并限定语言种类为中文。 资料选择：对资料进行初审，选择脑缺血时突触改变、认知障碍时突触改变、血管性痴呆时突触改变的文章，然后筛除与以上要求无明显联系的文章。排除重复性研究。 资料提炼：共收集到78篇相关的中英文文献，排除43篇，纳入35篇，其中涉及血管性痴呆与突触13篇，脑缺血与突触17篇，认知障碍与突触22篇。资料综合：突触是神经元之间的结构和功能的接触点，突触的可塑性是学习记忆的神经生物学基础，血管性痴呆的发病过程中突触改变可能是其重要机制。突触形态结构的可塑性和传递效能的可塑性的物质基础都涉及神经元和突触部位的某些蛋白质、受体、神经递质、离子及信使分子的物理化学变化。目前研究较多的突触蛋白是突触膨体素、突触素、突触后致密结构-95等。 结论：突触损伤在血管性痴呆发病的早期即存在，且与其认知功能障碍密切相关。对血管性痴呆发病中突触可塑性的研究有利于进一步阐明其发病机制，从而更有效地对其进行防治。     

重组α-突触核蛋白对SD大鼠黑质多巴胺能神经元的作用

目的：研究重组人全长α-突触核蛋白对大鼠黑质多巴胺能神经元的霉性作用。方法：原核表达、鉴定α-突触核蛋白；雄性SD大鼠30只，随机分为α-突触核蛋白注射组、六羟多巴注射组及α-突触核蛋白和六羟多巴联合注射组。用立体定位的方法分别右侧黑质注射α-突触核蛋白，六羟多巴及α-突触核蛋白 六羟多巴，左侧黑质注射等量的生理盐水，测定大鼠纹状体多巴胺含量和黑质酪氨酸羟化酶阳性神经元数目。结果：六羟多巴和α-突触核蛋白 六羟多巴注射使注射侧多巴胺分别减少到对照侧的36．98％和21．79％，多巴胺能神经元数减少到30．81％和28．05％。而α-突触核蛋白注射对纹状体多巴胺和黑质多巴胺能神经元数目无显著影响。结论：没有发现重组α-突触核蛋白对大鼠黑质多巴胺能神经元有明显的毒性作用。     

电针促进阿尔茨海默病模型小鼠（SAMP8）海马神经元突触可塑性的神经细胞黏附机制

目的:研究电针对快速老化痴呆模型小鼠(SAMP8)海马神经元突触超微结构、神经细胞黏附分子(NCAM)、多聚唾液酸转移酶ST8SiaII/IV的影响,从神经细胞黏附角度探讨电针治疗阿尔茨海默病(AD)的可塑性作用机制。方法:以SAMP8小鼠作为AD动物模型,电针"百会"、"涌泉"穴,1次/d,连续刺激21d。以Morris水迷宫测试小鼠学习记忆能力的变化;电镜观察海马神经元突触界面曲率、突触后致密物(PSD)厚度和突触间隙宽度的变化;以免疫组织化学、原位杂交方法检测海马神经元NCAM及其mRNA、ST8SiaII/IVmRNA的表达。结果:①模针组小鼠平均逃避潜伏期较模型组小鼠短(P<0.05);模针组小鼠在平台所在象限停留的时间(39.55±5.47s)较模型组小鼠(30.27±6.12s)长(P<0.05);②模针组突触后致密物厚度(76.928±25.236nm)较模型组(65.371±24.219nm)增厚(P<0.05);突触间隙宽度(25.941±6.217nm)较模型组(29.161±7.830nm)减小(P<0.05);突触界面曲率(1.083±0.049)较模型组(1.062±0.048)变化不明显(P>0.05);③与模型组比较,模针组小鼠NCAM及其mRNA、ST8SiaII/IVmRNA阳性表达明显增强(P<0.05)。结论:①电针能有效改善SAMP8小鼠学习记忆能力;②电针能有效调整海马神经元突触形态,使PSD增厚,突触间隙宽度变窄,促进突触形态可塑性的发挥;③电针改善SAMP8小鼠学习记忆能力的效应可能是通过诱导NCAM及ST8SiaII/IVmRNA的合成,促进神经细胞黏附,促进神经元突触形态可塑性来实现的。     

血管性痴呆的发病与突触可塑性

目的:血管性痴呆是主要由脑血管疾病引起的获得性、慢性、进行性认知障碍,是老年期痴呆的主要类型。其发病机制尚不明确。突触损伤可能是其早期的病理改变,与其认知功能障碍关系密切。资料来源:应用计算机检索Medline1990-01/2004-12关于血管性痴呆、脑缺血及认知障碍与突触改变的文章,检索词包括“cerebralis-chemiaandsynapse;cognitiveimpairmentandsynapse;vasculardemen-tiaandsynapse;demetianandsynapse,long-termprotection”等,并限定文章语言种类为English。应用计算机检索维普全文数据库1990-01/2004-12关于血管性痴呆、脑缺血及认知障碍与突触改变的文章,检索词为“突触结构,可塑性,血管性痴呆、突触功能,突触蛋白”。并限定语言种类为中文。资料选择:对资料进行初审,选择脑缺血时突触改变、认知障碍时突触改变、血管性痴呆时突触改变的文章,然后筛除与以上要求无明显联系的文章。排除重复性研究。资料提炼:共收集到78篇相关的中英文文献,排除43篇,纳入35篇,其中涉及血管性痴呆与突触13篇,脑缺血与突触17篇,认知障碍与突触22篇。资料综合:突触是神经元之间的结构和功能的接触点,突触的可塑性是学习记忆的神经生物学基础,血管性痴呆的发病过程中突触改变可能是其重要机制。突触形态结构的可塑性和传递效能的可塑性的物质基础都涉及神经元和突触部位的某些蛋白质、受体、神经递质、离子及信使分子的物理化学变化。目前研究较多的突触蛋白是突触膨体素、突触素、突触后致密结构-95等。结论:突触损伤在血管性痴呆发病的早期即存在,且与其认知功能障碍密切相关。对血管性痴呆发病中突触可塑性的研究有利于进一步阐明其发病机制,从而更有效地对其进行防治。     

突触改变在病理性神经痛中的作用

传统观点为病理性神经痛的产生机制提供了一个单纯“神经元模式”的解释，但是仍存在着一些疑问是这个模式所不能解释的。作为神经元传递过程中的重要结构，近年来突触的病理改变在病理性神经痛发病中的作用日益受到重视，下面就病理性神经痛突触研究的进展做一综述。     

Cell-based therapy for ischemic stroke

Introduction: Stroke is a major cause of mortality and disability in adults worldwide. Unfortunately, current therapy which targets vessel recanalization has a narrow treatment window, and at this time neuroprotective approaches are not effective for stroke treatment. However, after stroke the parenchymal and endothelial cells in the central nervous system (CNS) respond in concert to ischemic stressors and create a microenvironment in which successful recovery may ensue. Neurogenesis, synaptogenesis, axonal sprouting, glial cell activation, angiogenesis and vascular remodeling within the brain and the spinal cord are stimulated post stroke. Cell based-therapy amplifies these endogenous restorative effects within the CNS to promote functional outcome.

Areas covered: This article reviews current knowledge of cell-based therapy in the adult brain after stroke, including transplanted cell type, benefits and risks, with an emphasis on mechanisms of action.

Expert opinion: Experimental studies and clinical trials with cell-based therapy in stroke appear promising. Cell-based therapy is not intended for the replacement of damaged cells, but for the remodeling of the CNS by promoting neuroplasticity, angiogenesis and immunomodulation. However, there are risks associated with the use of cell-based therapy, and adequate evaluation of these potential risks is a prerequisite before clinical application for stroke patients.     

In situ tolerance within the central nervous system as a mechanism for preventing autoimmunity

Multiple sclerosis (MS) is believed to be an autoimmune disease in which autoreactive T cells infiltrate the central nervous system (CNS). Animal models of MS have shown that CNS-specific T cells are present in the peripheral T cell repertoire of healthy mice and cause autoimmune disease only when they are activated by immunization. T cell entry into the CNS is thought to require some form of peripheral activation because the blood-brain barrier prohibits trafficking of this tissue by naive cells. We report here that naive T cells can traffic to the CNS without prior activation. Comparable numbers of T cells are found in the CNS of both healthy recombinase activating gene (Rag)(-/)- T cell receptor (TCR) transgenic mice and nontransgenic mice even when the transgenic TCR is specific for a CNS antigen. Transgenic T cells isolated from the CNS that are specific for non-CNS antigens are phenotypically naive and proliferate robustly to antigenic stimulation in vitro. Strikingly, transgenic T cells isolated from the CNS that are specific for myelin basic protein (MBP) are also primarily phenotypically naive but are unresponsive to antigenic stimulation in vitro. Mononuclear cells from the CNS of MBP TCR transgenic but not nontransgenic mice can suppress the response of peripheral MBP-specific T cells in vitro. These results indicate that naive MBP-specific T cells can traffic to the CNS but do not trigger autoimmunity because they undergo tolerance induction in situ.     

成功治疗儿童罕见髓系/自然杀伤细胞祖细胞急性白血病1例

本文总结儿童髓系/自然杀伤细胞祖细胞急性白血病(M/NKPAL)的治疗经验以提高对该病的认识。对1例罕见的3岁8个月女童M/NKPAL合并中枢神经系统白血病进行了确诊分析,并对其治疗经过及长期随访结果进行了总结。结果表明,女童M/NKPAL合并中枢神经系统浸润得到了确诊,其免疫表型特征为CD7,CD33,CD34,CD56和HLA-DR共表达,MPO阴性,其他NK细胞和T、B细胞分化抗原阴性,染色体核型有+8和12p-。采用柔红霉素+阿糖胞苷化疗后达完全缓解,随后应用急性髓系白血病的化疗方案巩固强化治疗5个疗程,化疗方案中均含有大剂量阿糖胞苷,期间共行腰穿及鞘内注射治疗10次。停止治疗后中枢神经系统白血病复发,腰穿及鞘内注射治疗9次后行颅脑放疗36 Gy,取得了长期生存。结论:M/NKPAL是一种罕见的白血病,有特异的免疫表型特征,应用含有大剂量阿糖胞苷的急性髓系白血病化疗方案可能取得较好疗效。     

Labelling of olfactory ensheathing cells with micron‐sized particles of iron oxide and detection by MRI

A crucial issue in transplant‐mediated repair of the damaged central nervous system (CNS) is serial non‐invasive imaging of the transplanted cells, which has led to interest in the application of magnetic resonance imaging (MRI) combined with designated intracellular magnetic labels for cell tracking. Micron‐sized particles of iron oxide (MPIO) have been successfully used to track cells by MRI, yet there is relatively little known about either their suitability for efficient labelling of specific cell types, or their effects on cell viability. The purpose of this study was to develop a suitable MPIO labelling protocol for olfactory ensheathing cells (OECs), a type of glia used to promote the regeneration of CNS axons after transplantation into the injured CNS. Here, we demonstrate an OEC labelling efficiency of >90% with an MPIO incubation time as short as 6 h, enabling intracellular particle uptake for single‐cell detection by MRI without affecting cell proliferation, migration and viability. Moreover, MPIO are resolvable in OECs transplanted into the vitreous body of adult rat eyes, providing the first detailed protocol for efficient and safe MPIO labelling of OECs for non‐invasive MRI tracking of transplanted OECs in real time for use in studies of CNS repair and axon regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Experimental therapies for repair of the central nervous system: stem cells and tissue engineering

Several stem cell‐based therapeutic tools are currently being investigated for the regeneration of central nervous system (CNS) injuries. This review focuses on innovative approaches for CNS tissue repair via the use of implantable cellular devices. These devices are supported by biopharmaceuticals and conventional physiotherapy for the restoration of lost neuronal circuits and CNS function. This paper further reviews new and promising tools currently in pre‐clinical and clinical tests for the treatment of CNS diseases where substantial loss of cellular and extracellular components of neural tissue has occurred such as stroke, encephalopathy and traumatic neural injuries. We also discuss selected 3D bioscaffolds co‐cultured with clinically applicable human mesenchymal stem cells. Recent advances in neural tissue engineering and stem cell differentiation methods have shown promise for their clinical application in treating yet incurable CNS deficits. Copyright © 2012 John Wiley & Sons, Ltd.     

Repetitive intrathecal injections of poliovirus replicons result in gene expression in neurons of the central nervous system without pathogenesis

Poliovirus-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. In the current study, a replicon encoding green fluorescent protein (GFP) was encapsidated into authentic poliovirions, using established procedures. Intrathecal delivery of encapsidated replicons encoding GFP to the CNS of mice transgenic for the human poliovirus receptor did not result in any functional deficits as judged by behavioral testing. Histological analysis of the CNS of mice given a single intrathecal injection of poliovirus replicons encoding GFP revealed no obvious pathogenesis in neurons (or other cell types) within the CNS. The expression of GFP was confined to motor neurons throughout the neuroaxis; a time course of expression of GFP revealed that expression was detectable 24 hr postinoculation and returned to background levels by 120 hr postinoculation. A procedure was devised to allow repetitive inoculation of replicons within the same animal. Behavioral testing of animals that had received 6 to 13 independent inoculations of replicons revealed no functional deficits. Histological analysis of the CNS from animals that had received 6 to 13 sequential inoculations of replicons revealed no obvious abnormalities in neurons or other cell types in the CNS; expression of GFP was demonstrated in neurons 24 to 72 hr after the final inoculation of the replicon. Furthermore, there was no obvious inflammatory response in the CNS after the multiple inoculations. These studies establish the safety and efficacy of replicons for gene delivery to the CNS and are discussed with respect to use of replicons as new therapeutic strategies for spinal cord injuries and/or neurological diseases.     

Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression

EAE is a mouse T cell–mediated autoimmune disease of the CNS used to model the human condition MS. The contributions of B cells to EAE initiation and progression are unclear. In this study, we have shown that EAE disease initiation and progression are differentially influenced by the depletion of B cells from mice with otherwise intact immune systems. CD20 antibody–mediated B cell depletion before EAE induction substantially exacerbated disease symptoms and increased encephalitogenic T cell influx into the CNS. Increased symptom severity resulted from the depletion of a rare IL-10–producing CD1d^hiCD5⁺ regulatory B cell subset (B10 cells), since the adoptive transfer of splenic B10 cells before EAE induction normalized EAE in B cell–depleted mice. While transfer of regulatory B10 cells was maximally effective during early EAE initiation, they had no obvious role during disease progression. Rather, B cell depletion during EAE disease progression dramatically suppressed symptoms. Specifically, B cells were required for the generation of CD4⁺ T cells specific for CNS autoantigen and the entry of encephalitogenic T cells into the CNS during disease progression. These results demonstrate reciprocal regulatory roles for B cells during EAE immunopathogenesis. The therapeutic effect of B cell depletion for the treatment of autoimmunity may therefore depend on the relative contributions and the timing of these opposing B cell activities during the course of disease initiation and pathogenesis.     

Ultra-sensitive molecular MRI of cerebrovascular cell activation enables early detection of chronic central nervous system disorders

Since endothelial cells can be targeted by large contrast-carrying particles, molecular imaging of cerebrovascular cell activation is highly promising to evaluate the underlying inflammation of the central nervous system (CNS). In this study, we aimed to demonstrate that molecular magnetic resonance imaging (MRI) of cerebrovascular cell activation can reveal CNS disorders in the absence of visible lesions and symptoms. To this aim, we optimized contrast carrying particles targeting vascular cell adhesion molecule-1 and MRI protocols through both in vitro and in vivo experiments. Although, pre-contrast MRI images failed to reveal the ongoing pathology, contrast-enhanced MRI revealed hypoperfusion-triggered CNS injury in vascular dementia, unmasked amyloid-induced cerebrovascular activation in Alzheimer's disease and allowed monitoring of disease activity during experimental autoimmune encephalomyelitis. Moreover, contrast-enhanced MRI revealed the cerebrovascular cell activation associated with known risk factors of CNS disorders such as peripheral inflammation, ethanol consumption, hyperglycemia and aging. By providing a dramatically higher sensitivity than previously reported methods and molecular contrast agents, the technology described in the present study opens new avenues of investigation in the field of neuroinflammation.     

Another VCP interactor: NF is enough

Inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD) is a multisystem degenerative disorder caused by mutations in the valosin-containing protein (VCP) gene. How missense mutations in this abundant, ubiquitously expressed, multifunctional protein lead to the degeneration of disparate tissues is unclear. VCP participates in diverse cellular functions by associating with an expanding collection of substrates and cofactors that dictate its functionality. In this issue of the JCI, Wang and colleagues have further expanded the VCP interactome by identifying neurofibromin-1 (NF1) as a novel VCP interactor in the CNS. IBMPFD-associated mutations disrupt binding of VCP to NF1, resulting in reduced synaptogenesis. Thus, aberrant interactions between VCP and NF1 may explain the dementia phenotype and cognitive delay observed in patients with IBMPFD and neurofibromatosis type 1.