Distinctive growth patterns between cerebral and cerebellar astrocytomas–a tissue culture study

Astrocytomas have been alternatively considered as a single entity with topographical variabilities or as two different tumours–cerebral astrocytomas and mid line-cerebellar, astrocytomas. Twenty-two astrocytomas, 13 of the cerebral hemispheres, six cerebellar, two brain stem and one of spinal cord, have been studied by short-term tissue cultures. Two distinctive growth patterns have been found. Cerebral astrocytomas grew in the first week as radially arranged bipolar cells and subsequently by multipolar astrocytes in a reticular pattern. On the other hand, astrocytomas of the cerebellum, brain stem and spinal cord showed radial growth of long bipolar cells which persisted during the first 2–3 weeks. Only in the fourth week did a reticular pattern develop, but most of the cells remained bipolar. After the fourth week, a small number of multipolar astrocytes appeared and in two cases Rosenthal fibres were found. It is thus suggested that astrocytomas may be subdivided into two entities, those of the cerebral hemispheres and others in the cerebellum, optic nerve, brain stem and spinal cord which can be grouped as midline astrocytomas.     

Topographic representation of the sciatic nerve motor neurons in the spinal cord of the adult rat correlates to region-specific activation patterns of microglia

The frequent use of the adult rat sciatic nerve as a model to study the neuronal responses to injury, nerve regeneration and in transplantation studies, requires a detailed knowledge of the projection pattern of motor neurons into this nerve. Thus, as a first goal we determined this topographical projection of motor neurons and labelled small contingents by applying the fluorescent dye DiI in localised incisions made in the dorsal, rostral, ventral or caudal quadrants of the nerve. As a second goal we analysed with immunohistochemical methods the response of microglial cells within the topographical area corresponding to the incision and within areas outside this location. Uptake of the dye occurred only within the area confined to the incision, thus allowing the identification of the corresponding motor neuron perikarya within the ventral horn, eight to ten days later. In serial transverse sections of the lumbosacral spinal cord the number of labelled cells, their position within the ventral horn, and their longitudinal extent have been determined. The data suggest that the gross projection of the lumbosacral motor neuron column at the mid-thigh level of the sciatic nerve is topographic. In accordance, microglial cells showed fast activation within the injured topographic area, and a less pronounced and delayed response within the non-injured areas of the ventral horn. The graded response of microglial cells suggests that these cells possess a potential of local activation by sensing whether neurons are axotomised or just irritated by axotomy of their neighbours. The topographic organisation proves to be useful in studies on local injuries to the sciatic nerve and when analysing retrograde responses within the lumbosacral spinal cord.     

Spinal afferents and cortical efferents of the anterior intralaminar nuclei: an anterograde-retrograde tracing study

The topographical relations among the terminal field of spinothalamic fibers and the cells projecting upon areas 4 and 5 were studied in the anterior intralaminar nuclei of the cat. Terminals anterogradely labeled from the spinal cord and cell populations retrogradely labeled from the lateral pericruciate and anterior suprasylvian cortex were simultaneously observed by means of a multiple fluorescent tracing strategy. The present findings confirm that spinal afferents in the central lateral and paracentral nuclei overlap with the cells projecting to area 4. Further, the present data demonstrate that spinal terminals are largely segregated from the intralaminar cell population projecting to area 5.     

Magnetic resonance tracking of magnetically labeled autologous bone marrow CD34+ cells transplanted into the spinal cord via lumbar puncture technique in patients with chronic spinal cord injury: CD34+ cells' migration into the injured site

The purpose of this study was to demonstrate the possibility of delivering autologous bone marrow precursor cells into the spinal cord via lumbar puncture technique (LP) in patients with spinal cord injury (SCI). Magnetic resonance imaging provides a noninvasive method for studying the fate of transplanted cells in vivo. Considering these propositions, we studied magnetic resonance tracking of autologous bone marrow CD34(+) cells labeled with magnetic nanoparticles delivered into the spinal cord via LP in patients with SCI. Sixteen patients with chronic SCI were enrolled and divided into two groups; one group got their own labeled-CD34(+) cells injected into the spinal cord via LP (n = 10); the others received an injection, but it contained magnetic beads without stem cells (controls, n = 6). CD34(+) cells were magnetically labeled with magnetic beads coated with a monoclonal antibody specific for the CD34 cell membrane antigen. Magnetic resonance images were obtained by a standard turbospin echo-T2 weighted sequences before and 20 and 35 days after post-transplantation. The median number of CD34(+) cells injected via LP was 0.7 x 10(6) (range 0.45 to 1.22 x 10(6)). Magnetically labeled CD34(+) cells were visible at the lesion site as hypointense signals in five patients of the labeled-CD34(+) group 20 and 35 days after transplantation; these signals were not visible in any patient of the control group. We suggested for the first time that autologous bone marrow CD34(+) cells labeled with magnetic nanoparticles delivered into the spinal cord via LP technique migrated into the injured site in patients with chronic SCI.     

培养胚胎神经干细胞移植入纹状体后的形态学观察

本研究的目的是 :将体外培养的小鼠胚胎大脑皮层和脊髓的神经干细胞经单细胞悬液微移植后观察其在大鼠纹状体的存活、迁移和分化的状况。实验在无血清条件下将这些细胞扩增、培养再经活细胞荧光染料 Di I标记后 ,采用微移植的方法 ,通过脑立体定位仪上用微玻璃针将干细胞分别植入成年大鼠双侧纹状体的对称部位。大鼠存活 8周后 ,经灌注固定、恒冷箱切片 ,在荧光显微镜下观察标记的移植细胞的迁移状况 ;用星形胶质细胞特异性抗体观察移植区 GFAP的表达 ,以显示移植细胞分化状况。结果表明 :来源于胚胎小鼠大脑皮层和脊髓的体外培养神经干细胞经微移植后 ,均可在成年大鼠脑内纹状体区域存活 ,移植的神经干细胞可向周围的脑实质内迁移 ,迁移细胞沿特定的纹状体结构分布。神经干细胞主要分化成星形胶质细胞。本实验结果提示 ,移植的神经干细胞可在脑实质内存活、迁移和分化。     

Encephalitogenic T cells use LFA-1 for transendothelial migration but not during capture and initial adhesion strengthening in healthy spinal cord microvessels in vivo

LFA-1 on the surface of encephalitogenic T cells has been suggested to be involved in the pathogenesis of experimental autoimmune encephalomyelitis. By applying a novel technique of intravital fluorescence microscopy that enables us to visualize the interaction of circulating encephalitogenic T lymphoblasts within the healthy spinal cord white matter microvasculature in vivo, we investigated the possible involvement of LFA-1 on circulating encephalitogenic T cells in their multi-step interaction with the blood-brain barrier endothelium in vivo. LFA-1 was found to mediate neither the G-protein-independent capture nor the G-protein-dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessels. In contrast, blocking of LFA-1 on encephalitogenic T lymphoblasts resulted in a significantly reduced number of T cells firmly adhering within spinal cord microvessels 2 h after injection and in a significantly reduced number of T cells subsequently migrating across the vascular wall into the spinal cord parenchyme. Our study provides the first direct evidence that encephalitogenic T cells use LFA-1 for transendothelial migration but not for capture and initial adhesion in spinal cord microvessels in vivo.     

大鼠脊髓神经干细胞的鉴定及光镜和电镜观察

本研究应用无血清培养技术对分离的大鼠胚胎脊髓神经干细胞进行体外培养,在倒置显微镜和电镜下观察细胞形态,应用BrdU标记、免疫荧光染色检测细胞增殖、分化情况。免疫荧光显示nestin、MAP2、GFAP以及BrdU/nestin、BrdU/MAP2、Br-dU/GFAP均有阳性表达,说明从大鼠胚胎脊髓可成功分离出神经干细胞,它们分化后可以表达神经元、星形胶质细胞的特异性抗原。脊髓神经干细胞具有自我更新能力,能分化为神经元和星型胶质细胞。     

Electroporation as a tool to study in vivo spinal cord regeneration.

Tailed amphibians such as axolotls and newts have the unique ability to fully regenerate a functional spinal cord throughout life. Where the cells come from and how they form the new structure is still poorly understood. Here, we describe the development of a technique that allows the visualization of cells in the living animal during spinal cord regeneration. A microelectrode needle is inserted into the lumen of the spinal cord and short rapid pulses are applied to transfer the plasmids encoding the green or red fluorescent proteins into ependymal cells close to the plane of amputation. The use of small, transparent axolotls permits imaging with epifluorescence and differential interference contrast microscopy to track the transfected cells as they contribute to the spinal cord. This technique promises to be useful in understanding how neural progenitors are recruited to the regenerating spinal cord and opens up the possibility of testing gene function during this process.     

少突胶质前体细胞修复损伤脊髓的组织学变化

背景：脊髓损伤的发病率呈上升趋势,而脊髓损伤后的修复机制尚不完全清楚。 目的：探讨少突胶质前体细胞在脊髓损伤修复过程中的作用。 方法：采用Allen's重物撞击法建立小鼠脊髓损伤模型。病理学检测脊髓损伤程度,体外分离、纯化和诱导分化绿色荧光蛋白转基因鼠的少突胶质前体细胞并移植到脊髓损伤模型鼠体内。按照不同的治疗方式分为模型组、假手术组、治疗组和对照组。 结果与结论：小鼠脊髓损伤模型建模成功率100%。培养的少突胶质前体细胞具有自我增殖能力,并且可以分化为少突胶质细胞。移植后的少突胶质前体细胞不仅可以与宿主脊髓组织较好的整合,并可迁移到损伤部位,替代损伤组织。说明外源性少突胶质前体细胞可以在脊髓损伤小鼠受损部位存活并与宿主细胞较好的整合。     

维甲酸、音猬因子对大鼠脊髓神经干细胞向运动神经元样细胞分化的影响

目的:分离大鼠胚胎的脊髓神经干细胞进行体外培养,探讨维甲酸(RA)、音猬因子(Shh)诱导其向运动神经元样细胞分化. 方法:应用无血清培养技术分离培养脊髓神经干细胞,通过5-溴-2-脱氧尿苷标记、免疫荧光显色检测细胞增殖、分化情况;培养液分组添加Shh、 RA或Shh+RA,观察神经干细胞向运动神经元样细胞的分化情况. 结果:大鼠胚胎脊髓可成功分离神经干细胞,分化后可表达神经元、星形胶质细胞的特异性抗原;诱导分化后结果显示Shh组未检测到胆碱乙酰转移酶阳性细胞,RA组为20.63%, Shh+RA组为66.84%,其差异具统计学意义.结论:从大鼠胚胎脊髓可成功分离神经干细胞,Shh、 RA可不同程度地诱导脊髓神经干细胞分化为运动神经元样细胞.     

Electroporation loading of calcium-sensitive dyes into the CNS

Calcium imaging of neural network function has been limited by the extent of tissue labeled or the time taken for labeling. We now describe the use of electroporation-an established technique for transfecting cells with genes-to load neurons with calcium-sensitive dyes in the isolated spinal cord of the neonatal mouse in vitro. The dyes were injected subdurally, intravascularly, or into the central canal. This technique results in rapid and extensive labeling of neurons and their processes at all depths of the spinal cord, over a rostrocaudal extent determined by the position and size of the electrodes. Our results suggest that vascular distribution of the dye is involved in all three types of injections. Electroporation disrupts local reflex and network function only transiently (approximately 1 h), after which time they recover. We describe applications of the method to image activity of neuronal populations and individual neurons during antidromic, reflex, and locomotor-like behaviors. We show that these different motor behaviors are characterized by distinct patterns of activation among the labeled populations of cells.     

诱导多功能干细胞治疗脊髓损伤的现状及思考

背景：目前对脊髓损伤的治疗方法有激素冲击治疗,急诊手术减压及最新发展的干细胞治疗等方法。已有大量工作聚焦于干细胞治疗脊髓损伤的研究方面,并已成为热点。 目的：综述诱导多功能干细胞治疗脊髓损伤的相关研究现状及未来发展趋势。 方法：应用计算机检索PubMed 数据库1980年1月至2012年12 月期间的相关文章,检索关键词为“spine injury,induced pluripotent stem cells,IPS”。 结果与结论：共纳入35篇文章,均就诱导多能干细胞治疗脊髓损伤持赞同或支持态度。结果表明,诱导多功能干细胞直接来源于自体分离得到,解决了干细胞移植中伦理以及道德等问题,同时避免了异体排斥反应也为移植提供了大量的细胞来源。对于脊髓损伤的治疗方面目前只有动物实验及少量临床实验支持,但其在动物实验中效果良好,安全性较高。诱导多功能干细胞治疗脊髓损伤现存的问题主要是诱导多功能干细胞的诱导分化方法不成熟,并发症也较多。因此,诱导多功能干细胞的诱导分化及使用安全性将成为研究关键。     

基于鸡胚电转技术研究脊髓神经干细胞相关基因功能方法的建立

目的建立一种基于鸡胚电转技术研究脊髓神经干细胞(NSCs)相关基因功能的方法。方法 RT-PCR检测鸡胚发育不同时期脊髓NSCs表面标志物;在鸡胚胚龄(E)E2.5~E3时,利用活体电转基因技术将p CAGGSGFP质粒转染到鸡胚脊髓,E6时体视荧光显微镜下筛选绿色荧光蛋白(GFP)阳性胚胎,每组至少取材5个;通过脊髓横切及open-book技术观察神经纤维投射情况;普通光学显微镜下剥离出3~5条脊髓,经胰蛋白酶消化、离心后,无血清NSCs培养基重悬获得细胞铺板,于37℃、5%CO_2细胞培养箱内培养,定时观察GFP阳性脊髓NSCs的形态变化。结果 RT-PCR结果表明,鸡胚脊髓中阳性表达NSCs表面标志物;随后的脊髓横切及open-book结果表明,GFP阳性转染侧的神经纤维能穿过底板,投射到脊髓对侧;而脊髓NSCs体外培养结果显示,GFP标记的脊髓细胞具有典型的NSCs形态,继续培养后有明显突起产生。结论本实验成功建立了一种基于鸡胚电转技术研究脊髓神经干细胞相关基因功能的方法。     

Long-term Fate of Allogeneic Neural Stem Cells Following Transplantation into Injured Spinal Cord

To characterize the fate of allogeneic neural stem cells (NSCs) following transplantation into injured spinal cord, green fluorescent protein (GFP)-NSCs isolated from GFP transgenic Sprague–Dawley rat embryos were transplanted into contused spinal cords of Wistar rats. The GFP-NSCs survived for at least 6 months in injured spinal cord; most of them differentiated rapidly into astrocytes, and a few were able to undergo proliferation. After transplantation, the GFP-NSCs remained in the transplantation site at the early stage, and then migrated along white-matter, and gathered around the injured cavity. At 6 months post-transplantation, CD8 T-lymphocytes infiltrated the spinal cord, and mixed lymphocyte culture from host and donor showed that lymphocytes from the host spleen were primed by allogeneic GFP-NSCs. At 12 months post-transplantation, most GFP cells in the spinal cord lost their morphology and disintegrated. The Basso, Beattie and Bresnahan score and footprint analysis indicated that the improvement of locomotor function in transplanted rats appeared only at the early stage, and was not seen even at 6 months after transplantation All these results suggest that the allogeneic NSCs, after transplantation into injured spinal cord, activate the host immune system. Therefore, if immunosuppressive agents are not used, the grafted allogeneic NSCs, although they can survive for a long time, are subjected to host immune rejection, and the effect of NSCs on functional recovery is limited.     

Membrane currents and morphological properties of neurons and glial cells in the spinal cord and filum terminale of the frog

Using the patch-clamp technique in the whole-cell configuration combined with intracellular dialysis of the fluorescent dye Lucifer yellow (LY), the membrane properties of cells in slices of the lumbar portion of the frog spinal cord (n=64) and the filum terminale (FT, n=48) have been characterized and correlated with their morphology. Four types of cells were found in lumbar spinal cord and FT with membrane and morphological properties similar to those of cells that were previously identified in the rat spinal cord (Chvátal, A., Pastor, A., Mauch, M., Syková, E., Kettenmann, H., 1995. Distinct populations of identified glial cells in the developing rat spinal cord: Ion channel properties and cell morphology. Eur. J. Neurosci. 7, 129-142). Neurons, in response to a series of symmetrical voltage steps, displayed large repetitive voltage-dependent Na(+) inward currents and K(+) delayed rectifying outward currents. Three distinct types of non-neuronal cells were found. First, cells that exhibited passive symmetrical non-decaying currents were identified as astrocytes. These cells immunostained for GFAP and typically had at least one thick process and a number of fine processes. Second, cells with the characteristic properties of rat spinal cord oligodendrocytes, with passive symmetrical decaying currents and large tail currents after the end of the voltage step. These cells exhibited either long parallel or short hairy processes. Third, cells that expressed small brief inward currents in response to depolarizing steps, delayed rectifier outward currents and small sustained inward currents identical to rat glial precursor cells. Morphologically, they were characterized by round cell bodies with a number of finely branched processes. LY dye-coupling in the frog spinal cord gray matter and FT was observed in neurons and in all glial populations. All four cell types were found in both the spinal cord gray matter and FT. The glia/neuron ratio in the spinal cord was 0.78, while in FT it was 2.0. Moreover, the overall cell density was less in the FT than in the spinal cord. The present study shows that the membrane and morphological properties of glial cells in the frog and rat spinal cords are similar. Such striking phylogenetic similarity suggests a significant contribution from distinct glial cell populations to various spinal cord functions, particularly ionic and volume homeostasis in both mammals and amphibians.     

绿色荧光蛋白转基因小鼠CD34+/CD45+细胞在脊髓全横断大鼠模型中的存活及迁移

目的:探讨CD34⁺/CD45⁺细胞移植入脊髓全横断大鼠模型后的存活、迁移及分布情况。方法:体外培养绿色荧光蛋白（GFP）转基因小鼠骨髓细胞，经CD34、CD45单克隆抗体鉴定后移植入脊髓全横断大鼠模型脊髓横断处尾侧，分别在术后24 h、48 h、1周、2周、4周和8周行左心腔内灌注，取出脊髓，连续切片（片厚 10 μm），置于荧光显微镜下观察切片中有无绿色荧光细胞，并观察荧光细胞的分布范围；用免疫组化法检测CD34⁺/CD45⁺细胞的存活。 结果:脊髓横断处头尾两侧均可见绿色荧光细胞，且多分布于灰质中，散在或聚集成片；免疫组化法可见切片中有CD34⁺/CD45⁺细胞散在。 结论: CD34⁺/CD45⁺细胞可在脊髓全横断大鼠模型的脊髓中存活，并可迁移至脊髓横断处头侧，且随时间的延长迁移距离有所增加。     

The effects of dorsal rhizotomy and spinal cord isolation on calcitonin gene-related peptide-labeled terminals in the rat lumbar dorsal horn

In the present study, the origin of calcitonin gene-related peptide (CGRP) to the dorsal horn in the rat lumbar spinal cord is investigated. CGRP immunoreactivity is examined following multiple unilateral and bilateral dorsal rhizotomies and isolated cord preparations (spinal cords are isolated by transecting the cord in two places and cutting all dorsal roots between the transections). Seven to 11 days after surgery, unilateral multiple dorsal rhizotomies result in a drastic decrease in CGRP-stained terminals on the operated side; following bilateral dorsal rhizotomies and isolated cord preparations, one or two CGRP varicosities remain in the dorsal horn in each section. The numbers of CGRP-immunostained varicosities observed in the latter two preparations are not significantly different, suggesting that few if any axons descending from the brain contribute to the CGRP terminal population in the spinal cord dorsal horn. Based on these data, we hypothesize that dorsal root ganglion cells are the only source of CGRP to the rat lumbar dorsal horn.     

Identification of CD4- CD8- alpha beta T cells in the subarachnoid space of rats with experimental autoimmune encephalomyelitis. A possible route by which effector cells invade the lesions.

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats to elucidate the origin of effector T cells and the route by which they invade lesions. Since mouse studies have suggested that some autoimmune diseases are induced by extrathymic T cells in the liver, we focused our attention on the properties of mononuclear cells (MNC) isolated from the liver and other organs in rats with EAE. A small but significant proportion of LFA-1+ alpha beta T cells was identified in the liver as early as day 7 after immunization with myelin basic protein (MBP). Such LFA-1+ alpha beta T cells were also abundant among MNC attached to the spinal cord (i.e. subarachnoid space), and MNC infiltrated the spinal cord in rats with EAE (day 12). In electron microscopy, MNC attached to the spinal cord were found to be quite unique in terms of their large cell size with well-developed microvilli. More importantly, they were comprised of a considerably large proportion of double-negative CD4- CD8- T cells as well as single-positive CD4+ T cells. However, the cells which infiltrated the spinal cord were mainly CD4+. The present results raise the possibility that the subarachnoid space might be a major site for the expansion of extrathymic T cells in rats with EAE, and that only a limited population of CD4+ T cells invade the spinal cord directly through the outer layer and elicit EAE.     

大鼠脊髓半横断损伤后硫酸软骨素蛋白多糖Versican的变化

目的观察大鼠脊髓半横断损伤(hSCI)位点头尾段硫酸软骨素蛋白多糖Versican的表达变化,探讨其与脊髓损伤修复的关系。方法20只成年健康SD大鼠随机分为假手术对照组和脊髓半横断损伤3d、7d和14d组。建立成年SD大鼠脊髓半横断(T9～T10)模型。取损伤位点头尾段T9、T10节段制作冰冻切片,运用Versican抗血清进行免疫组化ABC法染色。分别观察并计数各组前角中Versican阳性细胞数,采用计算机图像分析技术测量免疫阳性产物的平均光密度(A)值。结果Versican阳性产物主要分布于细胞外基质中,前角神经元和胶质细胞的胞浆中也有分布;损伤后前角中Versican阳性神经元和胶质细胞数较对照组明显减少(P<0.05),免疫阳性产物平均A值较对照组明显增加(P<0.05)。结论脊髓半横断损伤后,表达增加的Versican很可能参与损伤后轴突生长被抑制的过程。     

Longitudinal extent of dorsal root fibres in the spinal cord and brain stem of the frog

The longitudinal arrangement of dorsal root fibres was investigated with a modified cobalt labelling technique in the spinal cord and brain stem of frogs. The topographical order of dorsal root fibres in the dorsal white column closely resembles the well-known scheme of the mammalian spinal cord. A significant difference between frogs and mammals is the extension of fibres up to the cerebellar plate. The ascending fibres of different origin are organized in concentric rings in the medulla. An oval-shaped area and a triangular area in the dorsal horn, and the motor horn, receive fibre collaterals in the spinal cord. Thoracic dorsal root fibres terminate exclusively in the oval-shaped area. Fibre terminations clearly outline the dorsal column nuclei which begin in the obex region and end at the level of the glossopharyngeal nucleus. The spinal nucleus of the trigeminus is richly supplied by both thin and thick calibre dorsal root fibres in its entire rostrocaudal extension. Two parts of the reticular formation receive dorsal root fibres; the first is in the dorsal gray matter ventral and lateral to the solitary fascicle in the medulla, the second is the lateral reticular zone. In the vestibular region, the medial, lateral and superior vestibular nuclei are innervated by dorsal root fibres. The granular layer of the cerebellum receives a significant contingent of dorsal root fibres. Fibres terminating in the vestibular region and in the cerebellum arise from limb-innervating spinal ganglia.The results indicate a close similarity in the longitudinal arrangement of dorsal root fibres in frogs and in higher vertebrates. The several collaterals that terminate in the hindbrain may modulate the function of the receiving structures. On the basis of present and previous findings the aggregation of primary sensory fibres and the convergence of their terminations are surveyed in the hindbrain.