Direct evidence of primary afferent sprouting in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and CGRP

Mechanical and thermal allodynia develops after spinal cord injury in three areas relative to the lesion: below level, at level, and above level. The present study tests colocalization of CGRP, associated with nociceptive neurons, with growth-associated protein (GAP-43), expressed in growing neurites, to test for neurite sprouting as a mechanism for reorganization of pain pathways at the level of the lesion and distant segments. Male Sprague-Dawley rats were divided into three groups: sham control (N = 10), hemisected at T13 and sacrificed at 3 days (N = 5) and at 30 days (N = 5) following surgery, the spinal cord tissue was prepared for standard fluorescent immunocytochemistry using mouse monoclonal anti-GAP-43 (1:200) and/or rabbit polyclonal anti-CGRP (1:200), density of immunoreaction product (IR) was quantified using the Bioquant software and values from the hemisected group were compared to similar regions from the sham control. We report significant increases at C8 and L5, in CGRP-IR in lamina III compared to control tissue (P < 0.05). We report significant bilateral increases in GAP-43-IR at C8, T13, and L5 segments in lamina I through IV, at 3 days post hemisection, compared to control tissue (P < 0.05), some of which is colocalized with alpha-CGRP. The increased area and density of GAP-43-IR is consistent with neurite sprouting, and the colocalization with alpha-CGRP indicates that some of the sprouting neurites are nociceptive primary afferents. These data are consistent with endogenous regenerative neurite growth mechanisms that occur near and several segments from a spinal lesion, that provide one of many substrates for the development and maintenance of the dysfunctional state of allodynia after spinal cord injury.     

The development of the relationship between dorsal root afferents and motoneurons in the larval bullfrog spinal cord

The relationship of dorsal root afferents to motoneuron somata and dendrites was studied by labelling dorsal and ventral roots of the tadpole lumbar enlargement with HRP at different stages of hindlimb development. Procedures were used which allowed for sequential light and electron microscopic analysis to determine whether close appositions between labelled elements represented synaptic contacts. Lateral motor column (LMC) motoneuron dendrites grow first into the lateral funiculus, and later begin arborizing within the spinal gray, concurrent with the arrival of developing dorsal root afferent fibers. Mature-appearing synaptic contacts between dorsal root afferents and motoneuron dendrites are established first on distal dendrites, and are observed on progressively more proximal dendrites as hindlimb development proceeds. Migrating motoneurons were also labelled in some animals. Distinct dorsal and ventral migratory pathways were noted; cells migrating dorsally were contacted by developing dorsal root afferents. Migrating motoneurons were associated with radially oriented processes, and were often closely apposed to other cells. The coincident development of dorsal root projections and the motoneuron dendrites which these fibers innervate in the adult, as well as the interaction between these two systems during cell migration, suggest that these two systems may be interdependent in establishing their normal relationship during development.     

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。     

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。     

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.     

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.     

Induction of c-fos gene expression by spinal cord transection in the rat

Sympathetic nerve activity is maintained after high spinal injury through circuits that remain in question. We evaluated patterns of c-fos gene induction as a monitor of spinal neurons responding to high spinal cord transection in the rat. Rats were anesthetized with isofluorane. Lower cervical or upper thoracic spinal segments were exposed, immersed in warm mineral oil and transected. Spinal cords were exposed but not transected in anesthetized controls. After 2.5 h, spinalized and control rats were perfused for immunocytochemistry. Cervical and thoracolumbar spinal segments and dorsal root ganglia were sectioned coronally. Tissues were incubated in primary, polyclonal antisera raised in rabbit or sheep against a peptide sequence unique to the N-terminal domain of Fos, and processed immunocytochemically. Neurons were induced to express Fos-like immunoreactivity (FLI), bilaterally, in the spinal gray, but not in primary sensory ganglia. Spinal cord transection induced neurons to express FLI in thoracic laminae I, IIo (outer substantia gelatinosa). Vre (lateral reticulated division), VII (lamina intermedia) and X, and the intermediolateral cell column. Lamina VIII was also labeled in spinal-injured but not in control animals. Immunolabeled nuclei were prominent in lumbar segments and were concentrated in the medial third of laminae I and IIo, and in laminae VII and X. Few cells were labeled in upper cervical or sacral segments. FLI was sparse in the spinal gray of controls and expressed mainly within the dorsal root entry zone of upper thoracic segments. Patterns of c-fos gene expression were site-specific and correlated with laminae that respond predominantly to noxious stimulation and that contain sympathetic interneurons. Laminae that are responsive to non-noxious stimuli and activated by walking, IIi, nucleus proprius, medial V and layer VI were not induced to express FLI. We conclude that neurons in specific spinal laminae that process high threshold afferents and that harbor neurons with sympathetic nerve-related activity are activated selectively by spinal cord transections. We hypothesize that peripheral afferents processed by spinal-sympathetic circuit neurons may regulate sympathetic discharge in the absence of supraspinal drive.     

Tetrodotoxin-resistant sodium channels Na(v)1.8/SNS and Na(v)1.9/NaN in afferent neurons innervating urinary bladder in control and spinal cord injured rats

Tetrodotoxin-resistant (TTX-R) sodium channels Na(v)1.8/SNS and Na(v)1.9/NaN are preferentially expressed in small diameter dorsal root ganglia (DRG) neurons. The urinary bladder is innervated by small afferent neurons from L6/S1 DRG, of which approximately 75% exhibit high-threshold action potentials that are mediated by TTX-R sodium channels. Following transection of the spinal cord at T8, the bladder becomes areflexic and then gradually hyper-reflexic, and there is an attenuation of the TTX-R sodium currents in bladder afferent neurons. In the present study, we demonstrate that Na(v)1.8 is expressed in both bladder and non-bladder afferent neurons, while Na(v)1.9 is expressed in non-bladder afferent neurons but is rarely observed in bladder afferent neurons. In spinal cord transected rats 28-32 days following transection, there is a decreased expression of Na(v)1.8 sodium channels in bladder afferents, but no change in the expression of Na(v)1.8 in non-bladder afferent neurons. Both bladder and non-bladder afferent neurons exhibit limited increases in Na(v)1.9 expression following spinal cord transection. These results demonstrate that the expression of TTX-R channels in bladder afferent neurons changes after spinal cord transection, and these changes may contribute to the increased excitability of these neurons following spinal cord injury.     

Distribution of B-50(GAP-43) mRNA and protein in the normal adult human spinal cord

B-50(GAP-43) is a phosphoprotein mainly found in the nervous system which plays a major role in neurite growth during development and regeneration as well as in synaptic remodelling. In the mature intact central nervous system, intense B-50 immunoreactivity (B-50-IR) can still be detected in regions which maintain residual capacity for structural re-organization. B-50 expression has been studied extensively in laboratory animals; however, its distribution and regulation in the human spinal cord is largely unknown. As a first step to analyze lesion-induced structural alterations, we investigated the distribution of B-50 protein and mRNA in the normal adult human spinal cord and dorsal root ganglia. Intense B-50-IR was localized to the superficial laminae of the dorsal horn at all segmental levels, the intermediolateral nucleus at thoracic levels and Onuf’s nucleus at sacral levels. Scattered neurons, particularly in the ventral horn of lumbar and sacral segmental levels (and occasionally also in Clarke’s nucleus) displayed intense B-50-IR in close apposition to the perikaryal and proximal dendritic surfaces. Nonradioactive in situ hybridization indicated that B-50 mRNA could also be detected in neurons of the ventral horn and also in the intermediolateral nucleus. The distribution of B-50 mRNA and protein in the normal human spinal cord shows a marked similarity to that reported in experimental animals, including the selective labelling of Onuf’s nucleus. However, the strong B-50-IR on the surface of some large anterior horn motor neurons has not been observed in other mammals. This finding might reflect a particular state of readiness for synaptic plasticity. Received: 4 August 1997 / Accepted: 27 October 1997     

Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: Effects of olfactory ensheathing glia

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.     

大鼠脊髓损伤后挫伤部位的X线照射治疗对运动功能恢复的作用研究

目的 探讨脊髓挫伤部位的X线照射治疗对脊髓损伤后运动功能恢复的作用.方法 将70只雌性Wistar大鼠按照纽约大学的重力冲击方法建立大鼠脊髓(T10)损伤动物模型,按照随机数字表法分为7组,每组10只,其中6组大鼠在损伤后不同时间(损伤后20 min、1 d、2 d、4 d、7 d、17 d)对挫伤部位进行X线(20 Gy)照射,第7组则不予照射(对照组).然后根据Basso、Beattie、Bresnahan(BBB)评分标准评测各组大鼠运动功能恢复情况,并进行统计学比较.采用快蓝染色对存活6周以上的大鼠进行挫伤脊髓的组织形态学观察.结果脊髓挫伤后20 min、1 d、2 d行X线照射组BBB评分明显高于对照组,差异均有统计学意义(P<0.05),且在脊髓损伤后的2～3周进展较快,后期恢复缓慢.组织形态学观察可见应用X线治疗组周边组织残存区面积大于对照组.结论 脊髓挫伤部位伤后早期行X线照射治疗可保护脊髓残存的神经组织,改善运动功能的恢复.     

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     

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.     

Spontaneous longitudinally orientated axonal regeneration is associated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat

Spontaneous cellular reorganisation at the lesion site has been investigated following massive spinal cord compression injury in adult rats. By 2 days post operation (p.o.), haemorrhagic necrosis, widespread axonal degeneration, and infiltration by polymorphnuclear granulocytes and OX42-positive macrophages were observed in the lesion site. By 7 days p.o., low affinity nerve growth factor receptor-positive Schwann cells, from activated spinal roots, were identified as they migrated far into the lesion. Between 7 and 14 days p.o., the overlapping processes of Schwann cells within the macrophage-filled lesion formed a glial framework which was associated with extensive longitudinally orientated ingrowth by many neurofilament-positive axons. Relatively few of these axons were calcitonin gene-related peptide (CGRP)-, substance P (SP)-, or serotonin (5HT)-positive; however, many were glycinergic or gamma aminobutyric acid (GABA)ergic. At 21 and 28 days p.o. (the longest survival times studied), a reduced but still substantial amount of orientated Schwann cells and axons could be detected at distances of up to 5 mm within the lesion. Glial fibrillary acidic protein (GFAP) immunoreactivity demonstrated the slow formation of astrocytic scarring which only became apparent at the lesion interface between 21 and 28 days p.o. The current data suggest the possibility of developing future therapeutic strategies designed to maintain or even enhance these spontaneous and orientated regenerative events. J. Neurosci. Res. 53:51–65, 1998. © 1998 Wiley-Liss, Inc.     

Resistance to anoxic injury in the dorsal columns of adult rat spinal cord following demyelination

In order to examine the relationship between myelination and sensitivity to anoxia in adult white matter, we studied action potential conduction in the spinal cord dorsal column of adult rats in which focal demyelinating lesions had been produced using ethidium bromide/X-irradiation. Acutely isolated spinal cords from control rats and following demyelination were maintained in vitro at 36°C and compound action potentials were studied following supramaximal stimulation. The compound action potential was totally abolished within 12 min of the onset of anoxia in normal dorsal columns, but was not abolished until 50 min following the onset of anoxia in demyelinated dorsal columns. Compound action potentials showed significantly greater recovery (to 58.1±12.2% of control amplitude) in demyelinated dorsal columns compared to controls (30.8±5.3%) following 120 min of reoxygenation. These results show that focal demyelination is associated with reduced sensitivity to anoxia within white matter of the adult spinal cord.     

Inflammation is increased with anxiety- and depression-like signs in a rat model of spinal cord injury

Spinal cord injury (SCI) leads to increased anxiety and depression in as many as 60% of patients. Yet, despite extensive clinical research focused on understanding the variables influencing psychological well-being following SCI, risk factors that decrease it remain unclear. We hypothesized that excitation of the immune system, inherent to SCI, may contribute to the decrease in psychological well-being. To test this hypothesis, we used a battery of established behavioral tests to assess depression and anxiety in spinally contused rats. The behavioral tests, and subsequent statistical analyses, revealed three cohorts of subjects that displayed behavioral characteristics of (1) depression, (2) depression and anxiety, or (3) no signs of decreased psychological well-being. Subsequent molecular analyses demonstrated that the psychological cohorts differed not only in behavioral symptoms, but also in peripheral (serum) and central (hippocampi and spinal cord) levels of pro-inflammatory cytokines. Subjects exhibiting a purely depression-like profile showed higher levels of pro-inflammatory cytokines peripherally, whereas subjects exhibiting a depression- and anxiety-like profile showed higher levels of pro-inflammatory cytokines centrally (hippocampi and spinal cord). These changes in inflammation were not associated with injury severity; suggesting that the association between inflammation and the expression of behaviors characteristic of decreased psychological well-being was not confounded by differential impairments in motor ability. These data support the hypothesis that inflammatory changes are associated with decreased psychological well-being following SCI.     

Chronic transplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord transection in the rat

The goal of this study was to ascertain whether olfactory ensheathing cells (OECs) were able to promote axonal regeneration and functional recovery when transplanted 45 days after complete transection of the thoracic spinal cord in adult rats. OECs promoted partial restitution of supraspinal pathways evaluated by motor evoked potentials and modest recovery of hindlimb movements. In addition, OEC grafts reduced lumbar reflex hyperexcitability from the first month after transplantation. Histological results revealed that OECs facilitated corticospinal and raphespinal axons regrowth through the injury site and into the caudal spinal cord segments. Interestingly, raphespinal but not corticospinal fibers regenerated long distances through the gray matter and reached the lower lumbar segments (L5) of the spinal cord. However, delayed OEC grafts failed to reduce posttraumatic astrogliosis. In conclusion, the beneficial effects found in the present study further support the use of OECs for treating chronic spinal cord injuries.     

Modulation of spinal reflex by assisted locomotion in humans with chronic complete spinal cord injury

Objective

In healthy subjects, spinal reflexes (SR) evoked by non-noxious tibial nerve stimulation consist of an early (60–120 ms latency) and an occasional late-appearing (120–450 ms latency) component in the ipsilateral tibialis anterior. In chronic (>1 year) complete spinal cord injured (cSCI) subjects early components are small or lacking while late components are dominant. Here we report on the modulation of SR by assisted locomotion in healthy and chronic motor cSCI subjects.

Methods

SR was evoked by tibial nerve stimulation at the terminal stance phase during assisted locomotion and was compared to SR recorded during upright stance.

Results

In chronic cSCI subjects only a late SR component was consistently present during upright stance. However during assisted locomotion, an early SR component appeared, while amplitude of the late SR component became small. In contrast, in healthy subjects the early SR component dominated in all conditions, but a small late component appeared during assisted locomotion.

Conclusion

A more balanced activity of early and late SR components occurred in both subject groups if an appropriate proprioceptive input was provided.

Significance

Early and late SR components are assumed to reflect the activity of separate neuronal circuits, which are associated with the locomotor circuitry possibly by shaping the pattern.     

慢性压迫性脊髓损伤后巢蛋白mRNA表达的观察

目的观察成年大鼠慢性压迫性脊髓损伤后及减压后早期巢蛋白与巢蛋白mRNA的相关性表达。方法选用健康wistar大鼠50只，体重280～320g，制备慢性压迫性脊髓损伤中度、重度及重度损伤减压后3d、10d模型，取自距胜迫边缘5mm段脊髓组织切片。正常成年大鼠作为对照组。行巢蛋白免疫组织化学染色，巢蛋白mRNA原位杂交实验，计算机罔像分析仪定量分析，观察巢蛋白、巢蛋白mRNA在脊髓中央管、灰质和白质中表达的变化，探讨巢蛋白与巢蛋白mRNA表达的相关性。结果成年大鼠慢性膻迫性脊髓损伤中、重度及重度压迫损伤减压后3d，巢蛋白在自、灰质及脊髓中央管室管膜细胞中均有明显表达（P〈0．05），以重度压迫组最为显著（P〈0．01）。减压后10d组灰质与正常对照组比较，差异无显著性意义（P=0．483）。重度压迫组及减压后3d组，巢蛋白mRNA在脊髓灰质、白质及中央管室管膜细胞中均有显著性表达（P〈0．05），以灰质前角第Ⅸ板层、后角和室管膜下区最为显著。中度压迫组，巢蛋白mRNA在灰质前角第Ⅸ板层及中央管室管膜细胞中有显著性表达（P〈0．05），其余区域仅有微弱表达，而白质内巢蛋白mRNA表达于软脊膜下星形胶质细胞的足突中。减压后10d组灰质内巢蛋白mRNA的表达与正常对照组比较，无显著性差异（P=0．375）。正常对照组中无表达。结论成年大鼠慢性压迫性脊髓损伤及减压后早期存在神经前体细胞的增殖。增殖的神经前体细胞巢蛋白与巢蛋白mRNA表达的相关性具有与胚胎发育期脊髓相似的特征。