Retinoic acid synthesis by a population of NG2-positive cells in the injured spinal cord

Retinoic acid (RA) promotes growth and differentiation in many developing tissues but less is known about its influence on CNS regeneration. We investigated the possible involvement of RA in rat spinal cord injury (SCI) using the New York University (NYU) impactor to induce mild or moderate spinal cord contusion injury. Changes in RA at the lesion site were determined by measuring the activity of the enzymes for its synthesis, the retinaldehyde dehydrogenases (RALDHs). A marked increase in enzyme activity occurred by day 4 and peaked at days 8-14 following the injuries. RALDH2 was the only detectable RALDH present in the control or injured spinal cord. The cellular localization of RALDH2 was identified by immunostaining. In the noninjured spinal cord, RALDH2 was detected in oligodendroglia positive for the markers RIP and CNPase. Expression was also intense in the arachnoid membrane surrounding the spinal cord. After SCI the increase in RALDH2 was independent of the RIP- and CNPase-positive cells, which were severely depleted. Instead, RALDH2 was present in a cell type not previously identified as capable of synthesizing RA, that expressed NG2 and that was negative for markers of astrocytes, oligodendroglia, microglia, neurons, Schwann cells and immature lymphocytes. We postulate that the RALDH2- and NG2-positive cells migrate into the injured sites from the adjacent arachnoid membrane, where the RALDH2-positive cells proliferate substantially following SCI. These findings indicate that close correlations exist between RA synthesis and SCI and that RA may play a role in the secondary events that follow acute SCI.     

Schwann cell remyelination of demyelinated axons in spinal cord multiple sclerosis lesions

To investigate remyelination in multiple sclerosis lesions, we immunostained spinal cord sections from patients with multiple sclerosis and neurological normal (control) patients with antisera to P0 protein, a major constituent of peripheral nervous system myelin, and myelin basic protein, which is found in both central and peripheral nervous system myelin. In sections from five of the eight patients with no clinical or pathological evidence of neurological disease, P0 immunostaining was confined to peripheral myelin sheaths in dorsal and ventral roots. They were intensely stained, and peripheral--central nervous system transition zones were clearly demarcated. Sections from the other three control patients contained a few P0-stained sheaths in the central nervous system near root entry zones or among marginal glia near the dorsal sulcus. Spinal cord sections from six of the ten patients with multiple sclerosis contained clusters of myelin sheaths immunostained by P0 antiserum. These regenerating sheaths of peripheral nervous system origin were most numerous in large lesions and were commonly located in central areas or peripherally near root entry zones. The sheaths were observed frequently in areas of active demyelination and appeared morphologically normal even when surrounded by debris-filled macrophages. Near margins of small inactive plaques were a few basic protein--stained oligodendroglia extending processes to thin basic protein--stained sheaths.(ABSTRACT TRUNCATED AT 250 WORDS)     

The guidance molecule Semaphorin III is expressed in regions of spinal cord and periphery avoided by growing sensory axons

Previous studies in rat, showing a transient pattern of expression of the α7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of α7 mRNA and α-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels Of α7 mRNA and α-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both α7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, α7 mRNA and α-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3–5. Quantitative analysis reveals a dramatic decrease in the levels of expression of α7 mRNA and α-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of α7 mRNA or α-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thulamocurticul development of rat and mouse taken together with the present results suggest that α7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development. © 1995 Wiley-Liss, Inc.     

Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti-NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody-induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX-42, and Rip antibodies did not co-localise with NG2+ cells in double immunostained tissue sections. NG2+ cells incorporated BrdU 48–72 h following the onset of demyelination. After the onset of remyelination (10–14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x-irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x-irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself. GLIA 22:161–170, 1998. © 1998 Wiley-Liss, Inc.     

A new population of neurons with crossed axons in the lamprey spinal cord

Neurons with contralateral, rostrally and caudally projecting axons were studied in whole mounts of lamprey spinal cord using retrograde labelling techniques with fluorescent dextran-amines, cobalt-lysine or horseradish peroxidase. A previously unknown large population (180-300 cells per hemisegment) of small (less than 25 microns) cells with contralateral projecting axons is described. Their axons extend over less than 5 segments rostrally or caudally. The number of these cells per segment was relatively constant in the rostral half of the spinal cord, but increased significantly in the caudal half. In comparison, medium-sized cells with contralateral axons corresponding to previously identified premotor interneurons were far less numerous (14-21 per hemisegment) and their axons extended more than 5 segments. Contralaterally projecting edge cells (intraspinal stretch receptor neurons) with principal rostral or caudal axons plus short collaterals in the other direction were distributed throughout the length of the spinal cord, whereas large and giant cells with a varied morphology were found in the caudal half.     

NG2 expression in rats with acute T10 spinal cord injury

Rat models of T10 spinal cord injury were established with a clamp method.NG2 expression was detected with immunohistochemical staining and western blot.Ten days after spinal cord injury,the number of NG2-positive cells in the damaged areas and NG2 absorbance were both significantly increased.The findings indicate that acute T10 spinal cord injury in rats can lead to upregulation of NG2 protein expression in damaged areas.     

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.     

Spontaneous regeneration of intrinsic spinal cord axons in a novel spinal cord slice culture model

A substantial problem in research concerned with axonal repair is the use of a wide variety of lesion models and the complexity associated with the respective in vivo lesion paradigms. Organotypic slice cultures are a potential in vitro alternative because the cytoarchitectonic tissue organization is well preserved and the slices can be maintained in culture for several weeks. Until now no spinal cord slice culture model for the study of axonal growth has been available. Here we present a spinal cord slice culture model that is well suited for the study of axonal growth. The spinal cord slices were cut not in the transverse but in the sagittal longitudinal plane such that several spinal cord segments were included in the slice culture. In these cultures the typical ventro-dorsal polarity of the spinal cord was maintained and intrinsic spinal cord axons formed a strong fibre tract extending along the longitudinal axis of the slice. The axons became myelinated during the culture period and synaptic contacts were present in these cultures. After mechanical lesions the intrinsic spinal cord axons had a substantial potential for axonal growth and regeneration. The number of regenerating axons crossing the lesion site decreased with increasing maturation of the culture, but even in mature cultures a small number of crossing fibres were present. This slice culture model could provide an important tool for several aspects of spinal cord research in the fields of axonal growth and regeneration, synapse formation, formation of intrinsic spinal cord circuits and myelination.     

L1 and GAD65 are expressed on dorsal commissural axons in embryonic rat spinal cord

Using immunocytochemical methods, the cell adhesion molecule L1 was detected on axons crossing in the dorsal commissure of developing rat spinal cord. Immunoreactive axons were found in locations similar to fiber bundles illustrated by Ramón y Cajal and designated the anterior, middle and posterior bundles of the dorsal commissure. L1-immunoreactive dorsal commissural axons were first observed on embryonic day 17 (E17), appeared more numerous by E19, and remained detectable in early postnatal ages. The massive middle axon bundles extended bilaterally from the dorsolateral funiculi towards the midline and crossed in the central part of the commissure. In horizontal sections, bundles of L1-labeled middle axons were observed to traverse the dorsal commissure in a periodic pattern along the entire rostrocaudal extent of the spinal cord. Bundles of glutamic acid decarboxylase (GAD65)-positive axons were detected crossing in the middle and posterior regions of the dorsal commissure between E17 and E20. Results from double-labeling experiments demonstrated that GAD65-positive fibers were embedded in larger bundles of L1-labeled axons and that some dorsal commissural axons were double-labeled. To determine if there were axons crossing in the dorsal commissure that did not express L1, double-labeling experiments were conducted using neurofilament and L1 antibodies. Results indicated that bundles of axons identified with anti-neurofilament antibodies were also L1-positive, whereas individually coursing axons within the commissure were L1-negative. The predominance of L1 on fiber bundles traversing the dorsal commissure adds to the growing evidence that this molecule may play a role in axon outgrowth and fasciculation.     

Conduction of impulses by axons regenerated in a Schwann cell graft in the transected adult rat thoracic spinal cord

Central nervous system axons regenerate into a Schwann cell implant placed in the transected thoracic spinal cord of an adult rat. The present study was designed to test whether these regenerated axons are capable of conducting action potentials. Following the transection and removal of a 4- to 5-mm segment of the thoracic spinal cord (T8-T9), a polymer guidance channel filled with a mixture of adult rat Schwann cells and Matrigel was grafted into a 4- to 5-mm-long gap in the transected thoracic spinal cord. The two cut ends of the spinal cord were eased into the guidance channel openings. Transected control animals received a channel containing Matrigel only. Three months after implantation, electrophysiological studies were performed. Tungsten microelectrodes were used for monopolar stimulation of regenerated axons within the Schwann cell graft. Glass microelectrodes were used to record responses in the spinal cord rostral to the stimulation site. Evoked responses to electrical stimulation of the axon cable were found in two out of nine Schwann cell-grafted animals. These responses had approximate latencies in the range of those of myelinated axons. No responses were seen in any of the Matrigel-grafted animals. Histological analysis revealed that the two cases that showed evoked potentials had the largest number of myelinated axons present in the cable. This study demonstrates that axons regenerating through Schwann cell grafts in the complete transected spinal cord can produce measurable evoked responses following electrical stimulation.     

Olig genes are expressed in a heterogeneous population of precursor cells in the developing spinal cord

Recent results from multiple laboratories have identified Olig genes as important in regulating glial differentiation. Here we show that Olig2 expression at early stages of development (prior to E16.5) identifies a domain in the developing spinal cord, which contains a heterogeneous population of progenitors that includes stem cells and glial progenitors. We show that Nkx2.2 and Olig2, which are present initially in nonoverlapping domains, are coexpressed at later stages, likely due to a second wave of Olig expression. We find that Olig1, like Olig2, is present in cells that coexpress astrocytic and radial glial markers and that Olig1/2 double knockouts lead to a loss of oligodendrocytes with preservation of NG2 expression. These results coupled with previously published data indicate that Olig1/2 and Nkx2.2, while clearly important in regulating early progenitor cell differentiation, do not unambiguously demonstrate the existence of an oligodendrocyte-neuron precursor or negate the existing retroviral lineage and clonal analysis data that suggest the existence of other types of precursors such as oligodendrocyte-astrocyte precursors or neuronal precursors.     

Pregnancy and autonomic hyperreflexia in patients with spinal cord lesions

Symptoms of autonomic hyperreflexia in patients with complete and incomplete paraplegia above D 7 can be caused by almost any stimulus in the abdominal area or in the lower extremities, specifically during parturition by the uterine contractions. The symptoms vary from pilo-erection and outbreaks of sweating to serious blood pressure crises and cerebrovascular accidents. Epidural anaesthesia and general anaesthesia are effective as therapy and also as prophylaxis. Frequent complications are anemia and urinary tract infections. Changes in bladder function as a result of pregnancy and childbirth were observed. Paraplegic expectant mothers experience premature labour pains more frequently than do others, and this implies the necessity for earlier clinical surveillance up to the time of delivery. The secondary uterine inertia frequently requires an operative termination of the birth. The perception of labour pains is clearly possible also with lesions above D 10.     

Schwann cell and oligodendrocyte remyelination in lysolecithin-induced lesions in irradiated rat spinal cord

Localised irradiation of adult rat spinal cord was achieved by implanting for 2 weeks a 192Ir pin alongside vertebral segments in the thoraco-lumbar region of the spinal column. Following removal of the implant, lysolecithin (LPC) was injected directly into the dorsal columns in order to induce demyelination in the most intensely irradiated segments of spinal cord. Eight weeks after LPC injection, remyelination was much less extensive in dorsal columns which absorbed more than 40Gy than in LPC lesions in less intensely irradiated spinal cords or in unirradiated animals. No oligodendrocytes, few astrocyte processes and little myelin debris lay among the demyelinated axons. However, capillary vessels were surrounded by astroglial end-feet so that the glial-limiting membrane remained intact in the demyelinated regions. There were some oligodendrocyte remyelinated fibres around the edges of the demyelinated zones but none among the naked axons. Schwann cells, which probably migrated into the lesions from the proximal segments of the dorsal roots, provided some fibres with myelin sheaths. These remyelinated fibres abutted demyelinated axons without an intervening glial limiting membrane or astrocyte process. Oligodendrocytes may fail to migrate into the demyelinated regions because of the scarcity of astrocyte processes. A possible explanation for the limited Schwann cell remyelination may be that the presence of astroglial end-feet around capillaries deprived Schwann cells of ready access to the demyelinated regions.     

Long spinal cord lesions in a patient with pathologically proven multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. We report the case of a 50-year-old man who presented with progressive gait ataxia. Brain magnetic resonance imaging (MRI) on fluid-attenuated inversion recovery revealed a hyperintense lesion in the right temporal white matter. The spinal cord showed a long hyperintense lesion between the vertebral levels C6 and L1 on T2-weighted MRI. Biopsied tissues from the brain lesion demonstrated features of inflammatory demyelination with preservation of astrocytes, consistent with typical MS. This is the first reported case of pathologically proven MS with longitudinally extensive spinal cord lesions.     

Visualization of individual axons in excised lamprey spinal cord by magnetic resonance microscopy

The direct visualization of axons within their native tissue environment by magnetic resonance (MR) microscopy is presented for the first time in the excised larval sea lamprey spinal cord. A home-built transverse radio frequency coil of 1.5 mm diameter was used in conjunction with a commercial 400 MHz MR microscopy system, implementing both 2-D and 3-D imaging pulse sequences. Images having nominal voxel sizes of 9x9x500 and 9x9x125 microm(3), respectively, in the lamprey spinal cord were obtained, resolving individual Mauthner and Müller axons. Furthermore, architectural changes associated with axonal degeneration were visualized in the spinal cord of one animal, excised 8 weeks after hemisection of the cord. Although the lamprey previously has not been the subject of MR microscopy investigations, these results demonstrate the method's potential for imaging this axon system, which is an important model of spinal cord injury and regeneration.     

人胚神经干细胞条件化培养基促进脊髓损伤大鼠皮质脊髓束再生

目的 研究神经干细胞条件化培养基对脊髓损伤大鼠（SCI）皮质脊髓束（CST）再生的促进作用。方法 成年雌性Wistar大鼠30只随机分为两组，神经干细胞条件化培养基治疗组和培养基对照组各15只，所有大鼠于T11水平横切脊髓。治疗组15只大鼠从固定在大鼠皮下Ommaya囊内注入神经干细胞条件化培养基，每周1次，每次注入5μl，对照组从Ommaya囊内注入同等量未培养过干细胞的培养基。利用Basso-Beattie—Bresnahan（BBB）评分客观评价后肢运动功能的恢复，检测生物素葡聚糖胺（Biotin dextran amine，BDA）示踪标记CST的再生，检测损伤部位远端的突触素表达情况。结果 所有大鼠在脊髓损伤后出现下肢截瘫，神经干细胞条件化培养基治疗组的大鼠表现为后肢运动功能的逐渐恢复，BDA标记的再生的轴突穿过了损伤处到达了脊髓的远端；对照组仅表现为轻微的组织和功能变化。治疗组大鼠脊髓在脊髓损伤处远端的神经元和BDA标记的轴突位置有突触素表达，对照组没有突触素表达。结论 神经干细胞条件化培养基可以促进SCI大鼠CST的再生以及CST和神经元之间的解剖学重建；神经于细胞治疗SCI可能通过支持治疗而非替代治疗。