The axon reaction in spinal ganglion neurons of acrylamide-treated rats

Summary Rats were given acrylamide in doses of either 30 or 50 mg/kg (5 days each week) for up to 3 weeks and killed at weekly intervals. The right sciatic nerve was tied tightly at the level of the major trochanter 4 days before killing the animals by perfusion fixation when ipsilateral and contralateral sensory ganglia (L5 and L6) were removed. The effects on neuronal perikarya of axotomy alone, of acrylamide alone and of these combined were studied by light and electron microscopy.The responses to axotomy and to acrylamide intoxication shared certain features, namely peripheral Nissl substance and to a lesser degree nuclear eccentricity, nucleolemmal crenation and mitochrondrial enlargement. Neurofilament loss was present only with acrylamide. In combined axotomy and acrylamide all these five features were prominent.These findings indicate firstly that the individual reponses to axotomy and to acrylamide, while sharing several features, are subtly different and secondly that acrylamide appears to impede the vital neuronal responses directed towards repair of the axon.     

高压氧预处理对脊髓损伤后轴突再生影响实验研究

目的探讨高压氧预处理(HBO-PC)对成年大鼠脊髓损伤后轴突再生的影响,以探讨HBO-PC对脊髓损伤后的神经保护作用。方法成年雌性SD大鼠40只,体重250～300 g。随机分为HBO-PC组、非预处理组各20只。大鼠急性脊髓损伤模型制作,采用改良Allen’s法,分别于伤后8w行神经束路示踪(顺行和逆行),以脊髓损伤部位为中心,上下各2 cm取材,免疫组化染色及图像分析检测组织中示踪剂阳性纤维的表达。结果在胶质瘢痕的周围及上下两端,HBO-PC组示踪剂阳性纤维表达数量均明显高于非预处理组。结论 HBO-PC可诱导脊髓损伤后轴突再生,对神经损伤起到保护作用。     

Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey

The sea lamprey recovers normal-appearing locomotion after spinal cord transection and its spinal axons regenerate selectively in their correct paths. However, among identified reticulospinal neurons some are consistently bad regenerators and only about 50% of severed reticulospinal axons regenerate through the site of injury. We previously suggested (Shifman, M. I., and Selzer, M. E., 2000a. Expression of netrin receptor UNC-5 in lamprey brain; modulation by spinal cord transection. Neurorehabilitation and Neural Repair 14, 49–58; Shifman, M. I., and Selzer, M. E., 2000b. In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression. Journal of Neuroscience Methods 104, 19–25) that selective chemorepulsion might explain why some neurons are bad regenerators and others not. To explore the role of additional chemorepulsive axonal guidance molecules during regeneration, we examined the expression of the repulsive guidance molecule (RGM) and its receptor neogenin by in situ hybridization and quantitative PCR. RGM mRNA was expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells. Following spinal cord transection, RGM message was downregulated in neurons close (within 10 mm) to the transection at 2 and 4 weeks, although it was upregulated in reactive microglia at 2 weeks post-transection. Neogenin mRNA expression was unchanged in the brainstem after spinal cord transection, and among the identified reticulospinal neurons, was detected only in “bad regenerators”, neurons that are known to regenerate well never expressed neogenin. The downregulation of RGM expression in neurons near the transection may increase the probability that regenerating axons will regenerate through the site of injury and entered caudal spinal cord.     

The spinobulbar system in lamprey

Locomotor networks in the spinal cord are controlled by descending systems which in turn receive feedback signals from ascending systems about the state of the locomotor networks. In lamprey, the ascending system consists of spinobulbar neurons which convey spinal network signals to the two descending systems, the reticulospinal and vestibulospinal neurons. Previous studies showed that spinobulbar neurons consist of both ipsilaterally and contralaterally projecting cells distributed at all rostrocaudal levels of the spinal cord, though most numerous near the obex. The axons of spinobulbar neurons ascend in the ventrolateral spinal cord and brainstem to the caudal mesencephalon and within the dendritic arbors of reticulospinal and vestibulospinal neurons. Compared to mammals, the ascending system in lampreys is more direct, consisting of excitatory and inhibitory monosynaptic inputs from spinobulbar neurons to reticulospinal neurons. The spinobulbar neurons are rhythmically active during fictive locomotion, representing a wide range of timing relationships with nearby ventral root bursts including those in phase, out of phase, and active during burst transitions between opposite ventral roots. The spinobulbar neurons are not simply relay cells because they can have mutual synaptic interactions with their reticulospinal neuron targets and they can have synaptic outputs to other spinal neurons. Spinobulbar neurons not only receive locomotor inputs but also receive direct inputs from primary mechanosensory neurons. Due to the relative simplicity of the lamprey nervous system and motor control system, the spinobulbar neurons and their interactions with reticulospinal neurons may be advantageous for investigating the general organization of ascending systems in the vertebrate.     

Glycine-like immunoreactivity in synaptic boutons of identified inhibitory interneurons in the mammalian spinal cord

Glycine is thought ti be a major inhibitory neurotransmitter in the mammalian CNS. Two types of physiologically identified interneurons, Renshaw cells and Ia inhibitory interneurons, were intracellularly staind with horseradish peroxidase, and their axon terminals were studied at the electron microscopic level. Post-embedding immunogold procedures weer used to reveal the presence of glycine-like immunoreactivity. The synaptic terminals of both types of interneuron were significantly enriched with glycine-like immunoreactivity, providing support for the idea that glycine is a mediator of synaptic transmission in the recurrent and reciprocal inhibitory pathways to motoneurons.     

Axonal regeneration of descending and ascending spinal projection neurons in spinal cord-transected larval lamprey

The distributions of descending and ascending spinal projection neurons (i.e., spinal neurons with moderate to long axons) were compared in normal larval lamprey and in animals that had recovered for 8 weeks following a complete spinal cord transection at 50% body length (BL, normalized distance from the anterior head). In normal animals, application of HRP to the spinal cord at 60% BL (40% BL) labeled an average of 713.8 +/- 143.2 descending spinal projection neurons (718.4 +/- 108.0 ascending spinal projection neurons) along the rostral (caudal) spinal cord, most of which were unidentified neurons. Some of these neurons project for at least approximately 50-60 spinal cord segments (approximately 36-47 mm in animals with an average length of approximately 90 mm used in the present study). At 8 weeks posttransection, the numbers of HRP-labeled descending or ascending spinal neurons that extended their axons through the transection were about 40% of those in similar areas of the spinal cord in normal animals. Thus, in larval lamprey, axonal regeneration of descending and ascending spinal projection neurons is incomplete, similar to that found for descending brain neurons. The majority of restored projections were from unidentified spinal neurons that have not been documented previously. In contrast to results from several other lower vertebrates, in the lamprey ascending spinal neurons exhibited substantial axonal regeneration. Identified descending spinal neurons, such as lateral interneurons and crossed contralateral interneurons, and identified ascending spinal neurons, such as giant interneurons and edge cells, regenerated their axons at least 9 mm beyond the transection site in animals with an average length of approximately 90 mm, which is appreciably farther than previously reported. In contrast, most dorsal cells, which are centrally located sensory neurons, exhibited very little axonal regeneration.     

以NgR为靶点治疗脊髓损伤的研究进展

NgR是最近发现并克隆的一种糖基磷脂酰肌醇（GPI）锚定膜蛋白，因其在髓磷脂抑制轴突再生信号转导过程中特殊的靶分子效应，日益受到重视。以NgR为靶点促进脊髓轴突再生的尝试为中枢神经系统（CNS）损伤的治疗提供了有益的启示。     

大鼠脊髓损伤后轴突病理变化与胶质瘢痕的关系

目的 观察脊髓损伤(SCI)后轴突变化及其与胶质瘢痕的关系.方法 应用Allen's法建立大鼠脊髓损伤模型,通过行为学评分、免疫荧光及神经束路示踪等观察SCI后轴突的病理变化,及其与胶质瘢痕的关系,并测量胶质瘢痕的厚度.结果 SCI后损伤处的轴突呈断裂、扭曲状,SCI后1 周损伤轴突呈再生趋势,2周时再生明显,与此相应动物运动功能逐渐恢复,4周时胶质瘢痕形成,再生的轴突被瘢痕阻挡.头尾侧胶质瘢痕厚度(107.00±20.12)μm大于两侧边厚度(69.92±24.37)μm.结论 SCI后轴突仍具有再生能力,但被胶质瘢痕所阻挡,瘢痕厚度的测量为将来去除胶质瘢痕提供了实验依据.     

Differential effect of aging on axon sprouting and regenerative growth in spinal cord injury

The demographics of acute spinal cord injury (SCI) are changing with an increased incidence in older age. However, the influence of aging on the regenerative growth potential of central nervous system (CNS) axons following SCI is not known. We investigated axonal sprouting along with the efficiency of the infusion of the stromal cell-derived growth factor-1 (SDF-1/CXCL12) and regenerative growth along with the anti-scarring treatment (AST) in young (2–3 months) and geriatric (22–28 months) female rats following SCI. AST included local injection of iron chelator (2,2′-dipyridine-5,5′-dicarboxylic acid) and 8-bromo-cyclic adenosine monophosphate solution into the lesion core. Axon outgrowth was investigated by immunohistological methods at 5 weeks after a partial dorsal hemisection at thoracic level T8. We found that aging significantly reduces spontaneous axon sprouting of corticospinal (CST), serotonergic (5-HT) raphespinal and catecholaminergic (TH) coerulospinal tracts in distinct regions of the spinal cord rostral to the lesion. However, impairment of axon sprouting could be markedly attenuated in geriatric animals by local infusion of SDF-1. Unexpectedly and in contrast to rostral sprouting, aging does not diminish the regenerative growth capacity of 5-HT-, TH- and calcitonin gene-related peptide (CGRP)-immunoreactive axons at 5 weeks after SCI. Moreover, 5-HT and TH axons maintain the ability to react upon AST with significantly enhanced regeneration in aged animals. These data are the first to demonstrate, that old age compromises axonal plasticity, but not regenerative growth, after SCI in a fiber tract-specific manner. Furthermore, AST and SDF-1 infusions remain efficient, which implicates that therapy in elderly patients is still feasible.     

Axon reaction in the red nucleus of the rat

Summary Two groups of 60 day old male Wistar Rats were subjected to right-sided rubro-spinal tractotomy at the fourth cervical and thirteenth thoracic vertebral levels respectively. Four animals in each group were sacrificed at each of the time intervals 1, 3, 4, 7, 14, 21 and 55 days. Counts of chromatolytic neurons from both groups of animals at time intervals ranging from 1–21 days established a time course for the chromatolytic response. Morphological observations showed a more severe central chromatolysis in the cervical group commencing at Day 3, and a much less severe central chromatolysis in the thoracic group starting at Day 4. By Day 21 the majority of the neurons of the cervical group were atrophic and by 55 days all were atrophic. The reaction following thoracic lesions was less intense and the maximum number of reacting neurons was observed at Day 7. Following this stage the neurons gradually returned to a normal morphological state which was complete by Day 55. Measurements of neuronal diameters from semi-thin Epon sections in the cervical group yielded statistically significant alterations in the perikaryal diameters of experimental neurons, demonstrating swelling at Days 1, 3 and 4 followed by shrinkage at Days 7, 14 and 21.The findings of this study suggest that the severity of the chromatolytic reaction in intrinsic neurons is intimately related to the distance of the lesion from the neuronal soma, while the time of onset of chromatolysis varies with lesion distance but is not directly proportional to it.This investigation was supported by Medical Research Council of Canada Grants MA-3417 and MA-4471.     

小发夹RNA抑制Nogo基因表达促进脊髓损伤修复的实验研究

目的 利用RNA干涉（RNAi）技术使特定的基因（Nogo）沉默，探索脊髓损伤的治疗方法。方法 设计有小发夹结构的两条DNA序列，PCR扩增带有U6启动子的小发夹，腺病毒包装重组体，转染少突胶质细胞，采用Westernblot法分析Nogo-66的蛋白表达水平。结果成功地构建了靶向Nogo基因RNAi的带有U6启动子的小发夹重组体，Nogo-66蛋白表达水平明显下降。结论 靶向RNAi小发夹重组体经腺病毒包装后，成功转染少突胶质细胞并能有效抑制Nogo-66基因的表达。     

Mouse olfactory ensheathing glia enhance axon outgrowth on a myelin substrate in vitro

Olfactory ensheathing glia (OEG) express cell adhesion molecules and secrete growth factors that support newly generated olfactory axons and are a promising therapeutic treatment to facilitate axonal regeneration after spinal cord injury (SCI). To study the molecular mechanisms underlying the ability of OEG to enhance axonal outgrowth, we designed an outgrowth assay using spinal cord myelin as a substrate to mimic an injury environment. We asked if olfactory bulb-derived OEG could enhance outgrowth of dorsal root ganglion (DRG) axons on myelin. When grown on myelin alone DRG axons have limited outgrowth potential. However, when OEG are co-cultured with DRG on myelin, twice as many neurons generate axons and their average length is almost twice that grown on myelin alone. We used this OEG/DRG co-culture to determine if a cell adhesion molecule expressed by OEG, L1, and a factor secreted by OEG, brain-derived neurotrophic factor (BDNF), contribute to the ability of OEG to enhance axonal outgrowth on myelin. Using OEG and DRG from L1 mutant mice we found that L1 expression does not contribute to OEG growth promotion. However, both BDNF and its receptor, TrkB, contribute to OEG-enhanced axon regeneration as function-blocking antisera against either component significantly decreased outgrowth of DRG axons. Additional BDNF further enhanced DRG axon growth on myelin alone and on myelin co-cultured with OEG. This simple mouse outgrowth model can be used to determine the molecules that contribute to OEG-enhancement of axonal outgrowth, test therapeutic compounds, and compare the outgrowth potential of other treatments for SCI.     

White matter inhibitors in CNS axon regeneration failure

Multiple lines of evidence have indicated that the inability of adult mammalian central nervous system (CNS) axons to regenerate after injury is partly due to the growth inhibitory property of central myelin. Three prototypical myelin-associated inhibitors of neurite outgrowth have been identified, including Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp). These inhibitory ligands, their receptors and signaling pathways are being intensively investigated for their roles in CNS axon regeneration failure. In addition, several members of the axon guidance molecules have been implicated in restricting CNS axon regeneration, some of which are expressed by mature oligodendrocytes. Here we review in vitro and in vivo studies of these molecules in neurite growth and in axon regeneration failure and discuss the implications of these studies. While the increasing number of potential axon regeneration inhibitors highlights the complexity of the restrictive CNS environment, it provides new windows of opportunity as well as new challenges for therapeutic development for spinal cord injury and related neurological conditions.     

Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats

Nogo-66 receptor (NgR), a common receptor for the three known myelin-associated inhibitors, i.e., Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), plays a key role in the failure of axonal regeneration in the adult mammalian central nervous system (CNS). Here we report a novel vaccine approach that stimulates the production of anti-NgR antibody to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). We showed that adult rats immunized with recombinant NgR produced high titers of the anti-NgR antibody and that antisera obtained from the immunized rats promoted neurite outgrowth of rat cerebellar neurons on the inhibitory MAG substrate in vitro. In a spinal cord dorsal hemisection model, NgR immunization promoted regeneration of lesioned corticospinal tract (CST) axons, anterogradely labeled with biotin dextran amine (BDA), beyond the lesion site. In a contusive SCI model, NgR immunization markedly reduced the total lesion volume and improved Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking performance. Thus, the NgR vaccine approach may represent a promising repair strategy to promote structural and functional recovery following SCI.     

Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord

Olfactory ensheathing cells (OECs) or Schwann cells were transplanted into the transected dorsal columns of the rat spinal cord to induce axonal regeneration. Electrophysiological recordings were obtained in an isolated spinal cord preparation. Without transplantation of cells, no impulse conduction was observed across the transection site; but following cell transplantation, impulse conduction was observed for over a centimeter beyond the lesion. Cell labelling indicated that the regenerated axons were derived from the appropriate neuronal source, and that donor cells migrated into the denervated host tract. As reported in previous studies, the number of regenerated axons was limited. Conduction velocity measurements and morphology indicated that the regenerated axons were myelinated, but conducted faster and had larger axon areas than normal axons. These results indicate that the regenerated spinal cord axons induced by cell transplantation provide a quantitatively limited but rapidly conducting new pathway across the transection site.     

慢性压迫性脊髓损伤后周围神经退变与MAP1B表达变化的实验研究

目的 观察慢性压迫性脊髓损伤大鼠运动功能、周围神经形态学改变及微管相关蛋白1B(MAP1B)的表达变化情况. 方法 50只Wistar雌性大鼠按照随机数字表法分为正常组、假手术组、慢性压迫20％组、慢性压迫40％组、慢性压迫60％组,每组10只.后3组置入平头塑料螺钉对大鼠脊髓进行后路渐进性压迫,2个月后分别压迫到20％、40％、60％左右程度.正常组不做任何处理,假手术组只做L5棘突和部分椎板咬除术,不实施压迫.造模后60 d处死大鼠取坐骨神经作为标本,行HE染色,于光镜及电镜下观察,同时行MAP1B的免疫组化染色. 结果 各压迫组大鼠运动功能减退;Tarlov评分、斜板实验评分、BBB-21评分均明显低于正常组大鼠,差异有统计学意义(P＜0.05).各压迫组大鼠均出现明显的周围神经退变现象,并且压迫程度越重变化越显著.各压迫组周围神经轴突中MAP1B的表达均降低,与正常组比较差异有统计学意义(P＜0.05). 结论 脊髓损伤后周围神经发生退变,且因为神经元凋亡、坏死或受抑制使得轴突的再生不明显,造成脊髓损伤后功能恢复不良.     

The activity-dependent plasticity of segmental and intersegmental synaptic connections in the lamprey spinal cord

Activity-dependent synaptic plasticity has been proposed as a contributory factor in the patterning of rhythmic network activity. However, its role has not been examined in detail. Here, paired or triple intracellular recordings have been made from identified neurons in the lamprey locomotor network to examine the potential relevance of activity-dependent synaptic plasticity in segmental and intersegmental spinal networks. Segmental inputs from glutamatergic excitatory interneurons (EIN) to ipsilateral glycinergic crossed caudal (CC) interneurons were facilitated or depressed during spike trains at 5-20 Hz. Connections between EINs were depressed. Glycinergic inputs from small ipsilateral inhibitory interneurons were depressed in motor neurons, but were facilitated in CC interneurons. Excitatory inputs from small crossing interneurons to motor neurons were depressed, whereas inhibitory inputs were unaffected. With the exception of connections between EINs, significant effects occurred with stimulation that mimicked interneuron spiking during network activity. Intersegmental EIN synaptic properties were also investigated. EIN inputs did not differ significantly when examined from zero to four segments rostral to motor neurons or CC interneurons. However, caudally located EINs evoked greater activity-dependent facilitation than did rostral EINs. Whilst the amplitude or plasticity of EIN inputs in the rostral or mid-trunk regions of the spinal cord did not differ, EINs in the caudal trunk region evoked greater facilitation. Synaptic transmission between locomotor network neurons thus exhibits activity-dependent plasticity in response to physiologically relevant stimulation. Activity-dependent plasticity could thus contribute to the patterning of the rhythmic network output.     

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.     

The expression of nerve growth factor receptor on Schwann cells and the effect of these cells on the regeneration of axons in traumatically injured human spinal cord

To investigate the effects of Schwann cells and nerve growth factor receptor (NGFR) on the regeneration of axons, autopsy specimens of spinal cord from 21 patients with a survival time of 2 h to 54 years after spinal cord trauma were studied using immunohistochemistry and electron microscopy. Regenerating sprouts of axons could be observed as early as 4 days after trauma. At 4.5 months after trauma, many regenerating nests of axons appeared in the injured spinal cord. The regeneration nests contained directionally arranged axons and Schwann cells. Some axons were myelinated. In injured levels of the spinal cord, the Schwann cells exhibited an increased expression of NGFR within spinal roots. These results show that an active regeneration process occurs in traumatically injured human spinal cord. The NGFR expressed on Schwann cells could mediate NGF to support and induce the axon regeneration in the central nervous system. Received: 20 June 1995 / Revised, accepted: 18 September 1995     

香芹酚对大鼠急性脊髓损伤的保护作用

目的 探讨香芹酚对大鼠脊髓损伤（SCI）后神经功能的影响及其机制。方法 将60只雄性SD大鼠（200~250 g）随机分为5组:假手术组（n=12）、SCI组（n=12）、香芹酚组（n=36）,香芹酚组根据香芹酚剂量分为低、中、高剂量3个亚组,每亚组12只。低、中、高剂量香芹酚组SCI后30 min腹腔注射香芹酚,剂量分别为10、20、40 mg/kg,每日一次;假手术组和SCI组每日腹腔注射等量生理盐水。采用Allen法建立大鼠SCI模型;假手术组只行椎板切除手术。SCI后24、48、72 h,采用BBB评分评估大鼠神经功能;SCI后72 h,采用ELISA法检测损伤脊髓组织丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、过氧化氢酶（CAT）、caspase-3活性;Western-blot法检测损伤脊髓组织Bax,Bcl-2蛋白表达水平。结果 SCI后,大鼠BBB评分均明显降低（P<0.05）,损伤脊髓组织水肿指数以及MDA、caspase-3和Bax水平均明显增高（P<0.05）,而SOD、GSH、CAT、Bcl-2水平均明显降低（P<0.05）;香芹酚能明显改善大鼠BBB评分（P<0.05）,明显降低水肿指数以及MDA、caspase-3和Bax水平（P<0.05）,而显著增加CAT、SOD、GSH、Bcl-2水平（P<0.05）。结论 香芹酚可通过减轻脊髓水肿、抑制氧化应激反应以及抗凋亡作用而对SCI大鼠发挥神经保护作用。