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。     

急性运动轴索型神经病患者血清对体外培养感觉和运动神经元的影响

目的观察急性运动轴索型神经病(AMAN)患者血清对体外培养的正常胚胎大鼠背根神经节神经元和脊髓前角运动神经元的影响。方法取胚胎SD大鼠的背根神经节和脊髓腹侧组织体外分离，建立原代单细胞培养体系，并经免疫组织化学染色进行鉴定；应用AMAN患者血清(25％)对培养细胞进行干预，观察神经元胞体和突起的变化，并经Guillery Shirra及Webster变性纤维染色法进行染色，计数发生变性和无变性的神经纤维数目。结果AMAN患者血清对培养的感觉神经元无影响；但可引起培养的运动神经元的突起变性和神经元胞体继发性改变，最终导致细胞死亡。结论AMAN患者血清可特异性的引起运动神经轴索损害。     

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.     

大鼠坐骨神经切断后腰脊髓腹角内胶质细胞和神经元的可塑性变化

目的：探讨大鼠单侧坐骨神经切断后腰脊髓腹角胶质细胞和运动神经元的反应及其相互关系。方法：用免疫组织化学技术、HE染色和Tunnel法，观察坐骨神经切断后1，6，12，24h及3，7和14d腰脊髓腹角胶质原纤维酸性蛋白(GFAP)标记的星形胶质细胞、OX-42标记的小胶质细胞及运动神经元的变化。结果：坐骨神经切断侧腰脊髓腹角可见星形胶质细胞和小胶质细胞活化，星形胶质细胞的活化早于小胶质细胞；后期运动神经元发生凋亡，HE染色显示凋亡细胞周围为反应性OX-42阳性小胶质细胞和GFAP阳性星形胶质细胞包绕。结论：研究结果提示，坐骨神经切断后切断侧腰脊髓腹角活化的胶质细胞与凋亡的运动神经元之间关系密切。     

实验性内囊出血后脊髓前角缝连接对肌痉挛的影响

目的:观察大鼠脑出血后脊髓运动神经无缝隙连接(gap junction,GJ)对肌痉挛的影响。方法:采用自体血脑内注入法制备大鼠内囊出血致肌痉挛模型,检测皮质运动诱发电位(Motor Evoked Potential,MEP),应用免疫组化方法观察脊髓前角运动神经元连接蛋白C_x32的改变。结果:大鼠内囊出血后病灶侧MEP波幅较对侧显著降低(P<0.01),且出血后1周病灶侧波幅较出血前显著降低(P<0.01);MEP潜伏期显著延长(P<0.01)。脑出血后第1周,左侧脊髓灰质前角GJ蛋白C_x32显著升高,同时动物肌痉挛状态明显加重。结论:内囊定向的脑出血模型引起内囊损伤侧MEP波幅明显降低、潜伏时延长,脊髓前角运动神经元GJ数量增多。     

CNTF对大鼠脊髓运动神经元NF积聚影响的研究

目的 观察肌萎缩侧索硬化症（ALS）病人脑脊液和谷氨酸对体外培养SD大鼠脊髓前角运动神经元损伤及神经细丝（Neurofilament,NF）积聚的变化,探讨睫状神经营养因子（Ciliary neurophic　factor,CNTF）保护运动神经元的作用机制。方法 体外培养SD大鼠脊髓前角运动神经元,然后分组观察正常脑脊液、ALS病人脑脊液和谷氨酸对神经元损伤作用及CNTF的保护作用,免疫组化观察各组神经元中神经细丝NF积聚的情况。结果　ALS病人脑脊液和谷氨酸对体外培养的运动神经元有明显损伤作用,神经元数量减少（P<0.05）,免疫组化显示有明显的NF积聚现象,但先加入CNTF后,运动神经元数量无明显变化（P>0.05）,免疫组化显示无明显NF积聚的现象。结论 体外培养SD大鼠脊髓前角运动神经元,在加入CNTF后,可以减少ALS病人脑脊液和谷氨酸对运动神经元的直接损伤作用,并显示有抑制NF积聚的作用,提示CNTF确有保护神经元的作用。     

慢性渐进性压迫对大鼠行为功能及脊髓运动神经递质表达的影响

目的：观察脊髓慢性受压后实验动物的行为功能与运动神经递质表达的变化情况。方法：采用大鼠后路渐进性脊髓压迫动物模型，观察联合行为评分（CBS），常规病理及免疫组化检测胆碱乙酰转移酶（ChAT)的变化。结果：免疫组织化学染色显示，在正常大鼠脊髓前角运动神经元及大小神经元ChAT均表达阳性，脊髓损伤后ChAT阳性细胞数减少，CBS升高，二者具有相关关系。结论：脊髓受压抑制大鼠脊髓神经元合成ChAT，从而影响实验动物的行为功能。     

氧乐果致运动神经元agrin基因mRNA水平改变

目的分析不同浓度氧乐果对运动神经元agrin基因mRNA转录水平的影响,探讨有机磷中毒突触前损害的发病机制。方法体外培养大鼠脊髓运动神经元,氧乐果染毒,检测不同浓度氧乐果对运动神经元agrin基因mRNA转录水平的影响,检测有机磷中毒是否对运动神经元的agrin基因表达产生影响。结果(1)正常组大鼠脊髓运动神经元agrin基因mRNA水平明显高于氧乐果染毒组大鼠脊髓运动神经元agrin基因mRNA水平;(2)不同浓度氧乐果染毒组(0.1mmol/L,0.01mmol/L)大鼠脊髓运动神经元agrin基因mRNA水平,各组间存在显著差异,损害程度与有机磷杀虫剂剂量存在正相关,即浓度大,agrin基因转录水平低;(3)0.1mmol/L浓度氧乐果染毒组大鼠脊髓运动神经元存活时间小于12h,0.01mmol/L浓度氧乐果染毒组大鼠脊髓运动神经元可较长时间存活。结论有机磷杀虫剂影响脊髓运动神经元agrin基因表达,且损害程度与有机磷杀虫剂剂量存在正相关,即浓度大,agrin基因转录水平低,神经肌肉接头损害严重,存在突触前损害。     

GDNF对培养脊髓运动神经元的影响

目的　研究不同浓度胶质细胞源性神经营养因子 (GDNF)对大鼠胚胎脊髓运动神经元生长活性的作用。方法　取大鼠胚胎脊髓腹侧组织进行原代体外分离培养 ,从细胞形态学及应用MTT法观察GDNF对大鼠脊髓运动神经元的影响。结果　GDNF能明显促进体外培养的大鼠脊髓运动神经元存活及突起的生长 ,并且有剂量依赖的趋势。结论　不同浓度GDNF对体外培养大鼠胚胎脊髓运动神经元有不同程度的促生长作用。     

慢性渐进性压迫对大鼠行为功能及脊髓运动神经递质表达的影响

目的 :观察脊髓慢性受压后实验动物的行为功能与运动神经递质表达的变化情况。方法 :采用大鼠后路渐进性脊髓压迫动物模型 ,观察联合行为评分 (CBS) ,常规病理及免疫组化检测胆碱乙酰转移酶 (ChAT)的变化。结果 :免疫组织化学染色显示 ,在正常大鼠脊髓前角运动神经元及大小神经元ChAT均表达阳性 ,脊髓损伤后ChAT阳性细胞数减少 ,CBS升高 ,二者具有相关关系。结论 :脊髓受压抑制大鼠脊髓神经元合成ChAT ,从而影响实验动物的行为功能     

Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene

Immunohistochemical study was performed to examine if calcium-binding proteins are involved in the degeneration of motor neurons in the brain stems and the spinal cords of transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn neurons in the spinal cord. On the other hand, calbindin D-28k immunoreactivity was much less apparent than that observed with parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k, whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn neurons were severely reduced, even at the presymptomatic stage, whereas calbindin-positive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1-transgenic mouse model.     

Potential roles of Alzheimer precursor protein A4 and beta-amyloid in survival and function of aged spinal motor neurons after axonal injury

To study the potential role of Alzheimer precursor protein A4 (APP) and beta-amyloid (A/beta) on aging motor neuron survival, expression of APP, A/beta, and choline acetyltransferase (ChaT) were investigated in aged rats after either distal axotomy or root avulsion injury. Approximately 45% in number of total aged spinal motor neuron were normally APP-positive. A/beta-positive neurites were observed normally in the spinal ventral horn of aged rats. After distal axotomy, without apparent neurodegeneration such as cell loss and decreased ChaT-immunoreactivity, increased levels of APP expression were observed in the spinal cords of aged rats post-injury. In contrast, after avulsion, expression of APP and A/beta were downregulated in the spinal ventral horn of aged rats, and marked loss of spinal motor neurons and downregulated ChaT expression were observed. Our data indicate that APP and A/beta might play beneficial roles in neuronal survival of aged spinal motor neurons after axonal injury.     

睫状神经营养因子对坐骨神经切断吻合后大鼠脊髓前角胶质纤维酸性蛋白

背景：睫状神经营养因子具有多种生物活性,在神经系统发育、分化和损伤修复中具有重要意义。 目的：观察睫状神经营养因子对坐骨神经切断吻合后大鼠相应脊髓节段前角星形胶质细胞的特异标记物胶质纤维酸性蛋白表达的影响。 方法：将SD大鼠随机分为对照组、模型组、生理盐水组及药物组。除对照组外,对所有大鼠实施双侧坐骨神经切断吻合术,药物组手术区局部注射睫状神经营养因子100 ng/kg,1次/d,生理盐水组局部注射等量生理盐水。术后1,3,7,14,21,28 d取相应脊髓节段,免疫组织化学染色观察胶质纤维酸性蛋白的表达,苏木精-伊红染色、TUNEL染色对脊髓前角神经元进行计数。 结果与结论：大鼠坐骨神经切断吻合后相应脊髓节段星形胶质细胞胞体大,突起分枝多且粗大,神经元数目逐渐减少,凋亡神经元增多,胶质纤维酸性蛋白表达增高。与模型组和生理盐水组比较,药物组神经元存活数目增多,凋亡减少,胶质纤维酸性蛋白表达明显增加(P < 0.05或P < 0.01)。同时,药物组大鼠的运动功能障碍较轻,恢复较快。说明睫状神经营养因子可以通过促进大鼠脊髓前角胶质纤维酸性蛋白的表达起到神经保护作用。 关键词：胶质纤维酸性蛋白;睫状神经营养因子;星形胶质细胞;神经元凋亡;周围神经损伤     

Midkine expression in rat spinal motor neurons following sciatic nerve injury

Midkine (MK), a heparin-binding growth factor, is produced in the developing and damaged nervous system. However, the role of MK in peripheral nerve injury has not been clarified. Here, we investigated MK expression in lumbar spinal motor neurons after rat sciatic nerve injury by immunohistochemical, in situ hybridization, and Western blot analyses. The rat sciatic nerve showed complete degeneration after local freezing. Numerous regenerated myelinated and thin nerve fibers were observed 3 weeks after injury. Intense MK immunoreactivity was detected in the ipsilateral spinal motor neurons of the anterior horn of the lumbar spinal cord after 1 day and in ipsilateral and contralateral spinal motor neurons from 4 days to 1 week after injury. It decreased after 2 weeks and again transiently increased in spinal motor neurons after 3 weeks. MK was found in the motor neurons and axon of the sciatic nerve. However, it was not detected in normal neurons and axon. In situ hybridization showed the expression of MK mRNA in lumbar spinal motor neurons of the anterior horn, but it was not present in Schwann cells or non-neuronal cells. Low-density lipoprotein receptor-related protein (LRP) immunoreactivity, a cell membrane receptor of MK, was observed in anterior horn motor neurons, but receptor-type protein tyrosine phosphatase zeta (PTPzeta) immunoreactivity as a signaling receptor complex of MK was not observed. LRP and PTPzeta immunoreactivities were observed in Schwann cells of the injured and uninjured sciatic nerve. Our findings suggest that MK is synthesized, released, and taken up in anterior horn motor neurons in an autocrine fashion with LRP. MK may have a role in degeneration and regeneration after peripheral nerve injury.     

Fragmentation of the Golgi apparatus of the anterior horn cells in patients with familial amyotrophic lateral sclerosis with SOD1 mutations and posterior column involvement

The Golgi apparatus (GA) of the anterior horn cells in the spinal cord was examined by immunohistological methods with an antibody against the MG-160 protein, a conserved intrinsic membrane sialoglycoprotein of the medial cisternae of the GA, in three patients with familial amyotrophic lateral sclerosis (FALS) with posterior column involvement. Large motor neurons in the anterior horns were markedly reduced in number and 10 of total 14 remaining large motor neurons showed fragmentation and a reduction in the number of the elements of the GA. The fragmentation of the GA was identical to that previously reported in motor neurons of the spinal cord and motor cortex from patients with sporadic ALS and in transgenic mice expressing the G93A mutation of the gene encoding the Cu/Zn superoxide dismutase months before the onset of paralysis. This is the first report of fragmented GA of the anterior horn cells in patients with FALS with posterior column involvement. The findings suggest that the GA is a common target in the neuronal degeneration in sporadic and FALS.     

Neuronal nitric oxide synthase (nNOS) immunoreactivity in the spinal cord of transgenic mice with G93A mutant SOD1 gene

Immunohistochemical and quantitative analyses were used to examine the evolution of neuronal nitric oxide synthase (nNOS) with time in spinal motor neurons of transgenic mice with a G93A mutant Cu/Zn superoxide dismutase (SOD1) gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the controls, the anterior horn including the anterior horn neurons was not immunostained for nNOS. In the transgenic mice, at the age of 24 weeks (early presymptomatic), when no pathological change was observed in the spinal cord, anterior horn neurons were only occasionally immunostained for nNOS (0.3%). At the age of 28 weeks (late presymptomatic), nNOS-positive anterior horn neurons and their neuronal processes were occasionally observed (7.6%), and at the age of 32 weeks (early symptomatic), nNOS-positive anterior horn cells, including degenerated ones showing central chromatolysis, were frequently demonstrated (27.6%) and nNOS-positive cord-like swollen proximal axons were also observed in the anterior horns. nNOS expression in the anterior horn neurons was almost always observed in the somata. At the age of 35 weeks (end stage), neuronal loss of the anterior horn cells was severe, and nNOS-positive anterior horn neurons and cord-like swollen axons in the anterior horns were less prominent compared to those at the age of 32 weeks (33.8%), but many reactive astrocytes were immunostained for nNOS. Thus, nNOS immunoreactivity in the anterior horn neurons is observed as early as the presymptomatic stage and varies with the progression of the disease. The selective localization of positive nNOS immunoreactivity in the anterior horn neurons and degenerated ones in particular, and swollen proximal axons suggests that nNOS immunoreactivity may be involved in the degeneration of anterior horn neurons in this SOD1 transgenic mouse model.     

Basic fibroblast growth factor attenuates the degeneration of injured spinal cord motor endplates**

The distal end of the spinal cord and neuromuscular junction may develop secondary degeneration and damage following spinal cord injury because of the loss of neural connections. In this study, a rat model of spinal cord injury, established using a modified Allen’s method, was injected with basic fibroblast growth factor solution via subarachnoid catheter. After injection, rats with spinal cord injury displayed higher scores on the Basso, Beattie and Bresnahan locomotor scale. Motor function was also well recovered and hematoxylin-eosin staining showed that spinal glial scar hyperplasia was not apparent. Additionally, anterior tibial muscle fibers slowly, but progressively, atrophied. Immunohistochemical staining showed that the absorbance values of calcitonin gene related peptide and acetylcholinesterase in anterior tibial muscle and spinal cord were similar, and injection of basic fibroblast growth factor increased this absorbance. Results showed that after spinal cord injury, the distal motor neurons and motor endplate degenerated. Changes in calcitonin gene related peptide and acetylcholinesterase in the spinal cord anterior horn motor neurons and motor endplate then occurred that were consistent with this regeneration. Our findings indicate that basic fibroblast growth factor can protect the endplate through attenuating the decreased expression of calcitonin gene related peptide and acetylcholinesterase in anterior horn motor neurons of the injured spinal cord.     

Frontotemporal dementia with cerebral intraneuronal ubiquitin-positive inclusions but lacking lower motor neuron involvement

A case of frontotemporal dementia with cerebral intraneuronal ubiquitin-positive, tau- and alpha-synuclein-negative inclusions is reported. A 50-year-old female patient exhibited mental changes; however, no clinical evidence of motor neuron disease was detected in her 11-year history. Neuronal loss and spongiform changes were mainly found in the frontotemporal cortices. Degeneration of the pyramidal tract was observed. Depletion of Betz cells was observed, whereas motor neurons of the hypoglossal nuclei and spinal anterior horn were well preserved. Immunohistochemically, intraneuronal ubiquitin-positive, tau and alpha-synuclein -negative inclusions were present in the small neurons of the dentate gyrus, frontal cortices and putamen. Neither Betz cells nor the anterior horn cells contained any inclusions. Fragmentation of the Golgi apparatus was visible only in 2.2% of anterior horn cells. A large number of tau-positive glial structures lacking argyrophilia were seen in the area of the frontopontine tract in the cerebral peduncle. The pyramidal tract lesions of the present case may be based on frontal lobe degeneration with spread of lesions to the motor cortex. Except for the pyramidal tract lesions, our case is similar to cases of motor neuron disease-inclusion dementia.     

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.