免疫介导的脊髓前角运动神经元损伤过程中神经丝异常的观察

目的 研究免疫介导的脊髓前角运动神经元损伤过程中神经丝(NF)磷酸化及超微结构的特征,探讨免疫与肌萎缩侧索硬化发病之间的关系.方法 通过透射电镜技术及免疫组化方法,对免疫介导的脊髓前角运动神经元损伤过程中NF异常聚集的超微结构特征及异常磷酸化状态进行研究.结果 电镜观察发现免疫后动物脊髓前角运动神经元胞质及轴索近端有神经丝异常聚集;免疫组化证实抗非磷酸化神经丝(SMI-32)抗体阳性的脊髓前角运动神经元(个/张脊髓切片)数量(12.00±1.05)与对照组(18.00±1.83)相比,明显减少(P＜0.05),而抗磷酸化NF抗体(SMI-31)阳性的脊髓前角运动神经元数量(13.00±1.60)与对照相比(3.23±1.33)明显增加(P＜0.01).结论 在免疫介导的运动神经元损伤过程中存在类似于肌萎缩侧索硬化的神经丝结构及代谢异常特征,两者之间可能存在共同的发病机制.     

实验性自身免疫性灰质病大鼠脊髓病理学、Bcl-2和Bax蛋白表达的研究

目的:观察实验性自身免疫性灰质病大鼠脊髓的病理改变及Bcl-2和Bax蛋白的表达。方法:利用猪脊髓前角匀浆作为抗原,免疫Lewis大鼠,观察大鼠行为学、肌电图、脊髓前角运动神经元的组织学改变,应用免疫组化方法,研究免疫介导的神经元损伤过程中Bcl-2和Bax表达的变化。结果:所有被免疫的大鼠均未出现肢体无力、明显的肌肉萎缩,肌电图表现有失神经改变,组织学证实脊髓运动神经元有不同程度的变性丢失,有卫星、噬节及墓穴现象形成。在实验性自身免疫性灰质病(experi mental autoi mmune gray matter disease EAGMD)大鼠的脊髓前角运动神经元中抗凋亡基因Bal-2的表达减少;促凋亡基因Bax的表达增加。结论:免疫可导致Lewis大鼠脊髓前角运动神经元的变性丢失,与细胞凋亡密切相关的基因蛋白Bcl-2和Bax参与了EAGMD的发病过程。     

p38MAPK信号转导通路参与免疫介导的豚鼠脊髓运动神经元损伤

目的明确p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路是否参与免疫介导的豚鼠脊髓运动神经元损伤。方法免疫组织化学检测磷酸化p38MAPK(p-p38MAPK)在豚鼠脊髓内的表达与分布。结果免疫后瘫痪豚鼠及免疫后未瘫痪豚鼠的颈膨大p-p38MAPK免疫反应阳性神经元数目较正常对照组、免疫对照组均有显著性增加(P<0.01);免疫后瘫痪豚鼠的腰膨大p-p38MAPK免疫反应阳性神经元数目较免疫后未瘫痪豚鼠、正常对照组、免疫对照组均有显著性增加(P<0.01)。结论p38MAPK信号转导通路在实验性自身免疫性灰质病动物模型脊髓运动神经元变性中发挥了一定的作用。     

实验性自身免疫灰质病模型IgG的相关研究

目的:建立实验性自身免疫性灰质病(EAGMD)模型,通过观察IgG在神经肌肉接头(NMJ)处和运动神经元(MN)中的沉积情况,探讨IgG损伤MN的机制。方法:用牛脊髓免疫豚鼠建立EAGMD模型,对动物的临床表现,病理改变进行观察。结果:EAGMD动物临床表现为肌肉无力和萎缩;病理上出现腰髓运动神经元数量明显减少;IgG在NMJ处的沉积率明显增高(P<0.01),并且NMJ处IgG沉积率与动物病情进展程度有相关性,r=0.61。结论:EAGMD动物IgG可能通过NMJ参与MN的损伤。     

抗氧化反应元件活化剂对选择性运动神经元损伤的保护作用

目的 观察抗氧化反应元件(ARE)活化剂5,6-二氢环戊烯并1,2-二硫杂环戊烯-3-硫酮(CPDT)对肌萎缩侧索硬化(ALS)体外器官培养模型的影响.探讨激活ARE途径后,在氧化应激状态下对运动神经元的保护作用.方法 取出生8 d乳鼠的脊髓腰段组织切片做脊髓器官培养,在培养液中加入谷氨酸转运体抑制剂苏-羟天冬氨酸(THA),升高细胞间隙谷氨酸浓度,造成脊髓前角运动神经元特异性损伤,制备成ALS体外器官培养模型.在此模型的基础上,将不同浓度的CPDT(分别为15、30 μmol/L)加入培养液中进行干预.用神经元的特异性免疫组织化学染色剂非磷酸化神经丝单克隆抗体(SMI-32)对脊髓前角运动神经元进行鉴定,电镜观察运动神经元超微结构.结果 用不同浓度的CPDT提前干预组运动神经元数目(个/张脊髓片,15 μmol/L组15.81±6.97,30 μmol/L组16.25±6.74)均较THA模型组(5.31±5.76)增多(n=15,P＜0.05),且神经元周围存在着丰富的突起.而CPDT非提前干预组运动神经元数目分别为6.00±6.01、6.42±4.88,与模型组的运动神经元数目及形态差异无统计学意义.电镜观察CPDT提前干预组运动神经元超微结构保存较完整,与正常对照组比较无明显差异.结论 活化ARE途径可以在一定程度上保护THA引起的运动神经元损伤.     

先天性巨脑回畸形

目的报告1例(右侧颞顶枕叶局限性)先天性巨脑回畸形患儿的影像学和临床病理学特征,以及诊断与治疗要点,以期提高对该病的认识。方法与结果男性患儿,2岁,临床表现为发作性意识丧失伴肢体抽搐18个月并进行性加重,MRI以右侧颞顶枕叶皮质发育畸形为特征,手术切除右侧颞顶枕区致灶。术后组织学形态呈现灰质不均匀增厚、白质减少,皮质分层结构消失,代之以大量异常神经元和"气球"样细胞,以及白质内散在"气球"样细胞;免疫组织化学染色,异常神经元表达非磷酸化神经丝重链SMI-32、神经元微管相关蛋白-2和波形蛋白或神经微丝蛋白,异常神经元和"气球"样细胞同时表达磷酸化S6核糖体蛋白(前者强于后者),但"气球"样细胞不表达神经元微管相关蛋白-2和波形蛋白。随访1年无意识障碍、肢体抽搐发作。结论先天性巨脑回畸形为脑发育早期增殖、移行障碍所致大脑皮质发育不良,应注意与局灶性皮质发育不良Ⅱb型和结节性硬化症相鉴别,综合临床病史、影像学和组织学特征明确诊断。     

携带cdc2-siRNA重组腺相关病毒对C型尼曼-皮克病小鼠神经元细胞骨架损伤的保护作用

目的 评估经小脑注射携带cdc2-siRNA的重组腺相关病毒(rAAV)对C型尼曼-皮克病(NPC)小鼠神经元细胞骨架损伤是否具有保护作用.方法 将携带cdc2-siRNA重组腺相关病毒(rAAV) 注射入 2周龄npc-/-小鼠的小脑,采用免疫组织化学及免疫印迹方法观察该病毒对 npc-/-小鼠神经元细胞骨架损伤的保护作用.结果 (1)携带cdc2-siRNA的rAAV明显减少npc-/-小鼠轴突球状体的数量;(2)携带cdc2-siRNA的rAAV有效抑制磷酸化的神经丝(由SMI31识别)及磷酸化的Tau蛋白(由PHF-1识别)的表达. 结论小脑注射携带cdc2-siRNA的rAAV对npc-/-小鼠神经元细胞骨架损害具有保护作用.     

重症肌无力动物模型的建立及应用

<正>重症肌无力(myasthenia gravis,MG)是一种T细胞依赖B细胞介导的自身免疫病,主要以乙酰胆碱受体介导的自身免疫性神经肌肉接头传递障碍为特点[1]。实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)代表一种研究人类疾病发病机制与开发疾病相关免疫治疗方法的优良模型。首次关于重症肌无力模型的报道要追溯至30年前,当时Patrick等[2]提出由电鳗电器官纯化得到的     

重组Bcl-2腺病毒对损伤运动神经元的保护作用

目的:观察携带Bcl-2基因的重组腺病毒(Ad/s-Bcl-2)对损伤运动神经元的保护作用.方法:采用培养脊髓运动神经元的谷氨酸损伤模型,评价重组Bcl-2腺病毒对损伤运动神经元的影响.指标是Bcl-2原位杂交和Bcl-2免疫组化染色、TUNEL阳性神经元计数、运动神经元[Ga2+]i的检测以及台盼蓝拒染法检测培养神经元存活.结果:①Ad/s-Bcl-2可转染原代培养的脊髓运动神经元并使其过表达Bcl-2.②过表达Bcl-2可延长培养神经元的生存时间.③过表达Bcl-2可显著减少谷氨酸诱导的原代培养脊髓运动神经元的凋亡,并显著降低谷氨酸诱导的神经元[ca2+]i的增高.结论:腺病毒中介Bcl-2基因过表达对神经毒性损伤的运动神经元具有保护作用.     

克林澳注射液对大鼠脑缺血后神经细胞凋亡及磷酸化Akt蛋白表达的影响

目的动态观察SD大鼠局灶性脑缺血再灌后神经细胞凋亡的变化及磷酸化Akt蛋白的表达并探讨克林澳注射液（简称克林澳）对其保护作用。方法用线栓法制备局灶性大脑中动脉缺血再灌注模型,以尼氏染色观察大鼠脑缺血再灌后皮质存活神经元,末端转移酶介导的缺口末端标记（TUNEL）方法检测神经细胞凋亡,免疫组化（SABC）方法检测磷酸化Akt蛋白表达。结果克林澳组再灌12h后各时间点半暗带区神经元较模型组多（P〈0.01）,TUNEL阳性细胞数显著减少（P〈0.01）,磷酸化Akt阳性细胞数明显增加（P〈0.01）。结论减少神经元凋亡,上调磷酸化Akt蛋白表达可能是克林澳保护脑缺血损伤的机制之一。     

Motor neuron reactivity of sera from animals with autoimmune models of motor neuron destruction.

To determine whether mammalian upper and lower motoneurons share common epitopes, we assessed the immunohistochemical reactivity of serum IgG of two animal models, experimental autoimmune motor neuron disease with destruction of lower motoneurons (EAMND) and experimental autoimmune gray matter disease with destruction of both lower and upper motoneurons (EAGMD). EAMND serum IgG reacted with the cytoplasm of guinea pig spinal motoneurons in a patchy granular pattern paralleling the in vivo localization in the guinea pig model. The EAMND serum IgG also reacted with human upper and lower motoneurons. EAGMD serum IgG reacted with the cytoplasm and subsequently the external membranes of upper and lower motoneurons of guinea pig and human tissues, also paralleling the in vivo localization. IgG was detected in ipsilateral ventral horn following hind limb injections of EAMND and EAGMD serum, thereby suggesting retrograde transport to the motoneuron cell body. The presence of shared epitopes in upper and lower human motoneurons, and the ability of IgG to reach the motoneuron cell body by retrograde transport from the periphery provides evidence for the potential role of autoimmune mechanisms in motoneuron diseases.     

Motor neurons are rich in non-phosphorylated neurofilaments: cross-species comparison and alterations in ALS

The localization and distribution of non-phosphorylated neurofilaments (NP-NF) in the upper and lower motor neurons was investigated in the rat, the common marmoset, the rhesus monkey and man using the SMI-32 antibody. Within the spinal cord of all species studied, the most intense NP-NF immunoreactivity was observed within the ventral horn alpha-motor neurons. Concurrent staining for the cholinergic marker choline acetyltransferase (ChAT) demonstrated that virtually all of the ChAT-positive alpha-motor neurons contain NP-NF immunoreactivity. Although NP-NF staining was also observed in other neurons within the ventral and intermediate horns, these neurons were loosely scattered and contained a considerably lower staining intensity. The only other prominent NP-NF staining in the spinal cord occurred within the neurons of the dorsal nucleus of Clark and the intermediolateral cell column. Phosphorylated neurofilament (P-NF) immunoreactivity was found primarily in neuronal processes. Occasionally, a solitary motor neuron contained weak P-NF immunoreactivity. Within the brainstem, neurons in all cranial nerve motor nuclei contained intense NP-NF immunoreactivity. The distribution and apparent density of NP-NF immunoreactive neurons in these nuclei was virtually identical to that observed for neurons immunoreactive for ChAT. NP-NF immunoreactive neurons of relatively lower intensity were found in many other regions of the brainstem. All of the giant Betz cells of layer (L) V in the motor cortex contained dark NP-NF immunoreactivity. Within the spinal cord of amyotrophic lateral sclerosis (ALS) patients, both Nissl and NP-NF staining demonstrated the dramatic loss of alpha-motor neurons characteristic of this disorder. Some of the remaining motor neurons contained intense P-NF immunoreactivity. These observations suggest that NP-NF immunoreactivity is a good marker for motor neurons in health and disease and may be a useful tool for studies of motor neuron degeneration (MND).     

Cyclophosphamide alters the clinical and pathological expression of experimental autoimmune gray matter disease

Guinea pigs inoculated with bovine spinal cord ventral horn homogenate develop a syndrome termed experimental autoimmune gray matter disease (EAGMD) characterized by extremity weakness, bulbar signs, and a loss of lower and upper motoneurons. To provide evidence for the role of autoimmune mechanisms, we have administered the immunosuppressant cyclophosphamide prior to and after gray matter immunization. Pretreatment with cyclophosphamide prevented the appearance of clinical signs of disease and decreased the loss of spinal cord motoneurons, the appearance of damaged motoneurons, and the antibody titer to motoneurons. Treatment 7 days after immunization attenuated the expression of disease. Treatment immediately after signs also improved the clinical and pathological findings. In all cyclophosphamide-treated animals there was less IgG within motoneurons and less inflammation. These results support the role for autoimmune mechanisms in motoneuron loss and degeneration in EAGMD.     

Motor neuron destruction in guinea pigs immunized with bovine spinal cord ventral horn homogenate: experimental autoimmune gray matter disease

Guinea pigs immunized with bovine spinal cord ventral horn homogenate develop muscle weakness with electromyographic and morphologic evidence of denervation. Pathological examination demonstrates a loss of motoneurons and scattered inflammatory foci primarily localized to the spinal cord. Immunohistochemical techniques document the presence of immunoglobulin G at the motor end plate and around the external membrane and within the cytoplasm of motoneurons. This syndrome of experimental autoimmune gray matter disease (EAGMD) differs from experimental autoimmune motor neuron disease induced by inoculation with purified motoneurons and also differs from experimental autoimmune encephalomyelitis. The existence of two different forms of immune-mediated motoneuron destruction suggests that a number of cytoplasmic and membrane antigens may give rise to an immunologically based attack on the motor system.     

MR-pathologic comparison of the upper spinal cord in different motor neuron diseases

This MRI study was performed to evaluate in vivo alterations of the spinal cord in defined subgroups of motor neuron diseases. Standard MRI examinations of the cervical and thoracic spinal cord in sporadic amyotrophic lateral sclerosis (ALS; n = 39), sporadic lower motor neuron disease (LMND; n = 19), Kennedy's disease (KD; n = 19) and a control group (n = 96) were analyzed with respect to spinal cord signal changes and the thickness of the spinal cord. No significant changes in thickness or signal alterations were observed when comparing ALS, LMND and control groups with one another. However, in KD patients significant upper spinal cord atrophy was detected at the cervical level as compared with all other groups. At the thoracic level, KD patients had significant upper cord atrophy as compared with controls and LMND. Marked atrophy of the upper spinal cord seems to be a feature of the KD-associated central-peripheral distal axonopathy.     

Ubiquitin-only intraneuronal inclusion in the substantia nigra is a characteristic feature of motor neurone disease with dementia

Two types of ubiquitinated inclusions have been described in motor neurone disease (MND). (1) Skein or globular ubiquitinated inclusions in the motor neurones (more frequently in the lower motor neurones). This is a characteristic feature of all motor neurone disease categories. (2) Dot-shape or crescentric ubiquitinated inclusions in the upper layers of cortex and dentate gyrus described in cases of motor neurone disease with dementia (DMND). We investigated the substantia nigra (SN) in MND cases; two cases of motor neurone disease inclusion body (MND-IB) dementia, six cases of DMND, 14 cases of MND (including one case from Guam and two cases of familial SOD1 mutation), four cases of Parkinson's disease (PD), and 10 cases of age-matched normal controls. SN and spinal cord sections were stained with ubiquitin (alpha-synuclein, tau, PGM1, SMI-31 and SOD1 antibodies). The neuronal density in SN was quantified by using a computer-based image analysis system. Four out of six DMND cases showed rounded ubiquitin positive inclusions with irregular frayed edges, associated with neuronal loss, reactive astrocytosis and a large number of activated microglia cells. These inclusions are negative with antibodies to (alpha-synuclein, tau, SMI-31 and SOD1). The SN in cases from MND-IB dementia and MND showed occasional neuronal loss and no inclusions. The ubiquitin-only inclusions in SN of DMND cases are similar (but not identical) to the ubiquitinated inclusions described previously in the spinal cord of MND cases and are distinct from Lewy bodies (LBs). The degeneration of SN is most likely a primary neurodegenerative process of motor neurone disease type frequently involving the DMND cases. MND disease is a spectrum and multisystem disorder with DMND located at the extreme end of a spectrum affecting the CNS more widely than just the motor system.     

胰岛素样生长因子(IGF-1)对体外培养的脊髓和皮层运动神经元的保护作用

目的研究胰岛素样生长因子(IGF-1)对THA诱导的运动神经元损伤的保护作用。方法选用生后7d和1d的SD乳鼠,7d龄乳鼠用于脊髓片培养,1d龄乳鼠用于脑片培养。在无菌条件下断头取脊髓腰膨大部分或脑组织,将脊髓腰膨大部分和包含运动皮层的脑组织切成薄片进行体外培养,对照组加入正常培养基,模型组给予谷氨酸转运体抑制剂-THA进行干预,IGF-1组于培养液中同时加入THA和不同浓度的IGF-1。药物干预3w后,应用免疫组织化学方法显示运动神经元并计数。结果 THA能够选择性诱导脊髓前角运动神经元和皮层运动神经元死亡,IGF-1能阻止THA诱导的运动神经元的死亡。结论 IGF-1对THA诱导的慢性运动神经元损伤具有保护作用,IGF-1可能有益于ALS的治疗。