GAP-43 expression in the developing rat lumbar spinal cord.

The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period E11 (embryonic day 11), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (E11-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (E14-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During the period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At E17-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At E19-P2, GAP-43 is concentrated in axons of substantia gelatinosa. Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.     

The distribution of GAP-43 in normal rat spinal cord

Summary We have studied the distribution of the growth-associated protein GAP-43 in the spinal cord of adult rats by light and electron microscopy, using a new antiserum raised against GAP-43/-galactosidase fusion protein. We show that GAP-43 is present at all vertebral levels but is more concentrated in cervical and thoracic regions. In addition to heavy staining in the corticospinal tracts of the white matter, staining can be seen at the light microscopic level throughout the grey matter and is particularly heavy around the central canal and in the superficial dorsal horn. Electron microscopic examination revealed that GAP-43 immunostaining is confined to a subpopulation of axons and axon terminals. Staining occurs in small myelinated and unmyelinated fibres and in terminals which are mainly small and make single axodendritic or axosomatic synapses. Staining in such terminals occurs in the axoplasm but is heaviest immediately adjoining the axolema. Staining was not observed in dendrites, nor in large myelinated axons or large axon terminals. Our results indicate that GAP-43 is expressed in adult rat spinal cord in a subpopulation of small diameter fibres and axon terminals. The distribution and morphology of these terminals is consistent with several different possible origins including corticospinal projection neurons, small diameter primary afferent neurons, and descending raphe-spinal serotonin containing neurons.     

Up-regulation of GAP-43 and growth of axons in rat spinal cord after compression injury

Summary The growth-associated protein-43 (GAP-43) is an axonal phosphoprotein which is expressed at high levels during development and is reinduced by regeneration in the PNS. Consequently it is believed to be a key molecule in the regulation of axonal growth. However, injury to the CNS does not result in significant regeneration and this has been suggested to correlate with a failure of central neurons to up-regulate GAP-43 after axotomy. We have examined a model of spinal cord injury which is unique in two respects; first dural integrity is maintained by compression of the cord with smooth forceps (thus excluding connective tissue elements) and, secondly, considerable axonal growth has been reported through the resulting lesion. Our previous studies have shown that GAP-43 is extensively distributed in the rat spinal cord (see accompanying paper), but here we have used anti-GAP-43 antiserum at a dilution which did not yield any immunostaining in normal cord. However, supranormal levels of GAP-43 were detected in cell bodies and axons around the lesion within four days of compression injury. Double immunostaining with the RT97 monoclonal antibody indicated that a small subpopulation of neurons local to the site of compression were axotomized and expressed GAP-43 and phosphorylated neurofilament epitopes in their cell bodies. Although damage to long axon tracts was extensive, there was no evidence of regeneration in white matter. On the other hand cavities which formed in grey matter provided an environment for axonal elongation. Immunolabelling with markers for astrocytes and endothelial cells was used to evaluate the interaction of elongating axons with endogenous CNS cell types. Sprouting axons, identified by the presence of elevated levels of GAP-43, did not appear to grow in contact with astrocytes but preliminary evidence suggested that newly formed capillaries provided an appropriate substrate.     

α-硫辛酸对大鼠全横断脊髓损伤后GAP-43和Caspase-3表达的影响

目的:研究α-硫辛酸对脊髓全横断损伤(SCI)大鼠损伤部位神经生长相关蛋白(GAP-43)、神经细胞凋亡相关蛋白(Caspase-3)的表达,探讨α-硫辛酸对大鼠脊髓全横断损伤功能恢复的作用。方法:制作并评价SD大鼠SCI模型后,64只大鼠随机分为假手术组、脊髓损伤组(SCI组)、SCI+游泳训练组(游泳组)和SCI+α-硫辛酸组(硫辛酸组)。各组按手术后7、14、21、28 d等4个时间点收集标本,每个时间点大鼠均为4只。各组大鼠进行BBB评分。各组4个时间点的GAP-43和Caspase-3表达用免疫组化测定,同时用Western Blot检测GAP-43表达。结果:游泳训练和用硫辛酸均能提高脊髓损伤大鼠的BBB评分(P0.05)。与SCI组比较,游泳组和硫辛酸组GAP-43表达显著增加,Caspase-3表达则明显降低(P0.05),且硫辛酸组比游泳组的GAP-43表达增加和Caspase-3表达降低更为明显(P0.05)。结论:游泳训练和α-硫辛酸可以上调脊髓损伤大鼠GAP-43表达和抑制Caspase-3表达促进损伤神经修复,对改善大鼠运动功能有一定疗效。     

GAP-43治疗大鼠脊髓横断后神经中丝NF200表达的变化

通过制备成年SD大鼠完全性脊髓损伤模型,研究生长相关蛋白(GAP-43)治疗大鼠脊髓损伤后神经中丝(NF200)表达的变化,探讨GAP-43在再生修复中的作用,为临床治疗提供实验依据。实验采用雌性8周龄SD大鼠75只,制成脊髓损伤模型后随机分为三组:GAP-43抗体组、GAP-43抗原组和对照组,每组25只。使用直接注射法将GAP-43抗原和GAP-43多克隆抗体分别注入抗原组和抗体组的大鼠脊髓的断端,对照组仅切断脊髓而不给药,最后观察各组大鼠肢体功能的恢复情况。用BBB评分法对不同时段大鼠的行为学表现进行评分,用HE染色及免疫组化染色观察NF200的表达,并对其进行相关性分析。结果发现对照组在不同的时间段行为学评分最低和抗原组评分最高,脊髓损伤区病理改变明显好转,NF200的表达呈进行性增高,且前角神经元NF200的表达早于后角神经元。抗体组早期恢复出现明显的停滞状态,但停药后能很快恢复。说明GAP-43能促进损伤脊髓的恢复,而抗体对损伤脊髓恢复的影响是可逆的,这对于脊髓再生的研究是一种值得探讨的新方法,对进一步探索脊髓损伤的治疗具有重要的意义。     

Continuous brain-derived neurotrophic factor (BDNF) infusion after methylprednisolone treatment in severe spinal cord injury

Although methylprednisolone (MP) is the standard of care in acute spinal cord injury (SCI), its functional outcome varies in clinical situation. Recent report demonstrated that MP depresses the expression of growth-promoting neurotrophic factors after acute SCI. The present study was designed to investigate whether continuous infusion of brain-derived neurotrophic factor (BDNF) after MP treatment promotes functional recovery in severe SCI. Contusion injury was produced at the T10 vertebral level of the spinal cord in adult rats. The rats received MP intravenously immediately after the injury and BDNF was infused intrathecally using an osmotic mini-pump for six weeks. Immunohistochemical methods were used to detect ED-1, Growth associated protein-43 (GAP-43), neurofilament (NF), and choline acethyl transferase (ChAT) levels. BDNF did not alter the effect of MP on hematogenous inflammatory cellular infiltration. MP treatment with BDNF infusion resulted in greater axonal survival and regeneration compared to MP treatment alone, as indicated by increases in NF and GAP-43 gene expression. Adjunctive BDNF infusion resulted in better locomotor test scores using the Basso-Beattie-Bresnahan (BBB) test. This study demonstrated that continuous infusion of BDNF after initial MP treatment improved functional recovery after severe spinal cord injury without dampening the acute effect of MP.     

The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels

Appropriate target reinnervation and functional recovery after spinal cord injury depend on longitudinally directed regrowth of transected axons. To assess the capacity to promote directed axon regeneration, alginate-based highly anisotropic capillary hydrogels (ACH) were introduced into an axon outgrowth assay in vitro and adult rat spinal cord lesions in vivo. In an entorhino-hippocampal slice culture model, alginate-based scaffolds elicit highly oriented linear axon regrowth and appropriate target neuron reinnervation. Coating of alginate-based ACH with the extracellular matrix components collagen, fibronectin, poly L-ornithine and laminin did not alter the axon regrowth response as compared to uncoated alginate-based ACH. After implantation into acute cervical spinal cord lesions in adult rats, alginate-based ACH integrate into the spinal cord parenchyma without major inflammatory responses, maintain their anisotropic structure and in parallel to findings in vitro induce directed axon regeneration across the artificial scaffold. Furthermore, adult neural progenitor cells (NPC), which have been shown to promote cell-contact-mediated axon regeneration, can be seeded into alginate-based ACH as a prerequisite to further improve the regenerative capacity of these artificial growth supportive matrices. Thus, alginate-based ACH represent a promising strategy to induce directed nerve regrowth following spinal cord injury.     

GAP-43治疗大鼠完全性脊髓损伤后GFAP表达的变化及意义

目的:通过制备完全性脊髓损伤(SCI)成年SD大鼠模型,研究生长相关蛋白(GAP-43)治疗大鼠SCI后胶质原纤维酸性蛋白(GFAP)的变化,探讨GAP-43在再生修复中的作用,为临床治疗提供实验参考。方法:咬除T7-T8棘突及相应椎板,用剪刀将脊髓完全横断,制成SCI模型。雌性8周龄SD大鼠75只,随机分为三组:GAP-43抗体组、GAP-43抗原组、对照组,每组25只。使用直接注射法将GAP-43抗原和GAP-43多克隆抗体分别注入抗原组和抗体组的大鼠脊髓的断端,观察各组大鼠肢体功能的恢复情况,用BBB评分法进行不同时段的行为学评分、免疫组化染色及图像分析方法观察GFAP的表达变化,并对其进行相关性分析。结果:对照组大鼠在不同时间段的行为学评分最低,抗原组评分最高;抗原组GFAP阳性细胞显著增多,而抗体组晚期则显著减少。结论:GAP-43可促进星形胶质细胞增生,而GAP-43抗体对星形胶质细胞的增生则表现为抑制作用。本实验结果表明GAP-43对脊髓损伤具有较好的治疗作用。     

Spinal axon regeneration evoked by replacing two growth cone proteins in adult neurons

In contrast to peripheral nerves, damaged axons in the mammalian brain and spinal cord rarely regenerate. Peripheral nerve injury stimulates neuronal expression of many genes that are not generally induced by CNS lesions, but it is not known which of these genes are required for regeneration. Here we show that co-expressing two major growth cone proteins, GAP-43 and CAP-23, can elicit long axon extension by adult dorsal root ganglion (DRG) neurons in vitro. Moreover, this expression triggers a 60-fold increase in regeneration of DRG axons in adult mice after spinal cord injury in vivo. Replacing key growth cone components, therefore, could be an effective way to stimulate regeneration of CNS axons.     

Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions

While most approaches to repair spinal cord injury (SCI) rely on promoting axon outgrowth, the extensive distance that axons would have to grow to bridge SCI lesions remains an enormous challenge. In this study, we used a new tissue-engineering technique to create long nervous tissue constructs spanned by living axon tracts to repair long SCI lesions. Exploiting the newfound process of extreme axon stretch growth, integrated axon tracts from dorsal root ganglia (DRG) neurons were mechanically elongated in vitro to 10 mm over 7 days and encased in a collagen hydrogel to form a nervous tissue construct. In addition, a modified lateral hemisection SCI model in the rat was developed to create a 1 cm long cavity in the spinal cord. Ten days following SCI, constructs were transplanted into the lesion and the animals were euthanized 4 weeks post-transplantation for histological analyses. Through cell tracking methods and immunohistochemistry, the transplanted elongated cultures were consistently found to survive 4 weeks in the injured spinal cord. In addition, DRG axons were observed extending out of the transplanted construct into the host spinal cord tissue. These results demonstrate the promise of nervous tissue constructs consisting of stretch-grown axons to bridge even extensive spinal cord lesions.     

神经生长相关蛋白GAP-43在脊髓损伤后不同时段的表达

目的探讨成年大鼠脊髓损伤后不同时段神经生长相关蛋白GAP-43表达的改变及其在脊髓损伤修复中的意义。方法应用改良的Allen’s法和数字化脊髓损伤模型制备仪建立大鼠脊髓损伤模型,用免疫组织化学方法检测脊髓在损伤后不同时段GAP-43的表达,分析免疫阳性细胞数和细胞的积分光密度值,资料用q检验进行统计分析。结果GAP-43表达于脊髓神经元胞浆及突起中,在前角运动神经元中更为明显,损伤后一周内免疫反应逐渐增强,损伤后第5天积分光密度值（10D）达到高峰（P〈0．01）,2周后明显下调（P〈0．05）。结论脊髓损伤可能诱导损伤区GAP-43表达,在损伤后7d左右表达高峰期出现,提示其可能参与了脊髓损伤神经的生长修复过程。     

Effect of cervical spinal cord hemisection on the expression of axon growth markers

To evaluate the plasticity processes occurring in the spared and injured tissue after partial spinal cord injury, we have compared the level of axon growth markers after a C2 cervical hemisection in rats between the contralateral (spared) and ipsilateral (injured) cervical cord using western blotting and immunohistochemical techniques. In the ipsilateral spinal cord 7 days after injury, although GAP-43 levels were increased in the ventral horn caudal to the injury, they were globally decreased in the whole structure (C1–C6). By contrast, in the contralateral intact side 7 days and 1 month after injury, we have found an increase of GAP-43 and βIII tubulin levels, suggesting that processes of axonal sprouting may occur in the spinal region contralateral to the injury. This increase of GAP-43 in the contralateral spinal cord after cervical hemisection may account, at least partially, to the spontaneous ipsilateral recovery observed after a cervical hemisection.     

臂丛损伤再生中GAP-43 mRNA及其蛋白的表达

目的：分析臂丛损伤后脊髓前角运动神经元表达GAP-43 mRNA及其蛋白的变化规律，探讨神经损伤再生的机制。方法：建立3种臂丛损伤模型：右C₇前根撕脱（A组）；右C₇前根撕脱＋同侧C5-T₁后根断离（B组）；右C₇前根撕脱+右C₅C₆间脊髓半横断（C组）。用荧光定量RT-PCR方法检测术后14 d时 C₇前角GAP-43 mRNA的表达量。用免疫组化方法检测术后1、 3、 7、14 d脊髓前角GAP-43免疫阳性运动神经元的表达。结果：对照组C₇前角GAP-43 mRNA呈低表达，损伤组GAP-43 mRNA表达显著上调。损伤组术后1 d、3 d时均未见C₇前角 GAP-43免疫阳性神经元，术后7 d各损伤组GAP-43免疫阳性神经元开始出现，14 d时免疫阳性神经元数目达到高峰。3组间比较，C组表达量最高，B组最低，A组居中。结论：臂丛损伤诱导运动神经元GAP-43 mRNA及其蛋白表达上调，GAP-43合成增加是神经元蛋白重组所致，与轴索再生和神经功能重建有关。     

GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb.

The expression of the growth associated protein, GAP-43, in developing rat hindlimb peripheral nerves has been studied using immunocytochemistry. GAP-43, is first detected in lumbar spinal nerves at embryonic day (E)12 as the axons grow to the base of the hindlimb. It is expressed along the whole length of the nerves as well as in the growth cones. GAP-43 staining becomes very intense over the next 36 h while the axons remain in the plexus region at the base of the limb bud before forming peripheral nerves at E14. It remains intense along the length of the growing peripheral nerves, the first of which are cutaneous, branching away from the plexus and growing specifically to the skin, their axon tips penetrating the epidermis of the proximal skin at E15 and the toes at E19. GAP-43-containing terminals form a dense plexus throughout the epidermis which subsequently withdraws subepidermally in the postnatal period. GAP-43 staining is also evident along the growing muscle nerves during muscle innervation, which follows behind that of skin. Axons branch over the surface of proximal muscles at E15 but do not form terminals until E17. As target innervation proceeds, GAP-43 staining declines in the proximal part of the nerve but remains intense in the distal portions. Overall GAP-43 expression in the hindlimb decreases in the second postnatal week as axon growth and peripheral terminal formation decline.     

Freeze-dried poly(D,L-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-fibroblast growth factor. One group of animals received fibrin glue with acidic-fibroblast growth factor only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord-implant interfaces. With BDNF foam, up to 20% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord-implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly(D,L-lactic acid) foams, with or without axonal growth-promoting factors, seeded with Schwann cells to enhance the axonal regeneration and myelination response.     

人骨髓间质干细胞和嗅鞘细胞移植对大鼠急性脊髓损伤功能修复的比较研究

目的对比人骨髓基质细胞(BMSCs)与人胚嗅鞘细胞(OECs)移植对大鼠脊髓损伤功能修复的影响。方法将45只SD大鼠分成脊髓损伤后BMSCs移植组(BMSCs组)、OECs移植组(OECs组)和PBS对照组(PBS组),通过BBB评分、运动诱发电位(MEP)评估脊髓传导功能的改善状况,免疫组织化学方法检测移植细胞存活和分化情况,病理形态学方法观察组织结构修复情况。结果BMSCs组BBB评分高于OECs组(P<0.05);两细胞治疗组MEP潜伏期明显缩短(P<0.05);BMSCs组嗜银染色可见脊髓损伤近端有较多再生纤维向远端延伸,形成神经纤维束,而OECs组再生纤维较少;两种移植细胞均可在损伤处部分存活,BMSCs组可见BMSCs来源的细胞Nestin、NF、GFAP的阳性表达。结论BMSCs移植比OECs移植能更有效促进大鼠急性脊髓损伤修复。     

腓肠神经移植修复大鼠陈旧性脊髓损伤的作用

目的:探讨利用自体周围神经组织移植修复大鼠陈旧性脊髓损伤病理机制,为临床应用提供实验依据。方法:利用改良Allens撞击方法建立脊髓打击损伤模型,12周后,将大鼠分为2组,实验组切取后肢腓肠神经,利用显微外科技术去除神经外膜,将其修剪成小段,游离移植于脊髓损伤处,对照组不作处理。分别于术后2、4、12周,在光镜及电镜下观察脊髓损伤段及移植周围神经再生情况。术后4、8及12周分别对两组动物进行脊髓诱发电位检查。结果:对照组脊髓变性,可见瘢痕和空洞,实验组术后12周,损伤区脊髓与周围神经融合良好。脊髓诱发电位检查神经移植组优于对照组。结论:周围神经组织游离移植修复大鼠陈旧性脊髓损伤后,存活良好,为再生轴突跨越损伤段脊髓提供通道。     

大鼠坐骨神经损伤与再生中GAP－43表达的实验研究

用免疫组化技术对６０只大鼠坐骨神经切断、卡压和再卡压损伤后不同时期的ＧＡＰ－４３表达作了观察．观察结果：（１）大鼠坐骨神经卡压或切断后，前角运动神经元、后根神经节细胞和坐骨神经纤维均产生免疫反应阳性，其中７ｄ组近段和１４ｄ组远段坐骨神经纤维为强阳性，（２）３０ｄ组各部免疫反应减弱，６０ｄ组基本恢复正常；（３）３０和６０ｄ组的再卡压损伤的各部均较同期其他损伤组免疫反应为强；（４）伤侧腰骶髓前角运动神经元染色逐渐减弱．ＧＡＰ－４３免疫反应结果显示，神经元胞体部分随再生期延长而逐渐减弱，周围部由近至远逐渐增强，又逐渐恢复至正常水平．结果提示ＧＡＰ－４３主要由神经元胞体所产生，随轴突逐渐转运至损伤和再生处，表明了此种蛋白参与神经再生，在神经再生过程中一直起着重要的作用．     

The impact of myelination on axon sparing and locomotor function recovery in spinal cord injury assessed using diffusion tensor imaging

The dysmyelinated axons of shiverer mice exhibit impaired conduction characteristics, similar to early postnatal axons before myelination, whereas the patterns of neuronal activity and connectivity are relatively comparable with those of wild‐type myelinated axons. This unique dysmyelination pattern is exploited in the present study to determine the role of compact myelin in the loss and recovery of function following traumatic spinal cord injury (SCI). We applied in vivo diffusion tensor imaging (DTI) and post‐mortem immunohistochemistry analysis to examine changes in myelin and axonal integrity, and evaluated these changes in concert with the analysis of locomotor function from 1 to 4 weeks following a mid‐thoracic contusion injury in homozygous shiverer and heterozygous littermate mice. The DTI biomarkers, axial and radial diffusivities, are noninvasive indicators of axon and myelin integrity in response to SCI of both myelinated and dysmyelinated spinal cord. We show that myelin is critical for normal hind limb function in open field locomotion. However, when the functional outcome is limited during chronic SCI, the extent of recovery is associated with residual axonal integrity and independent of the extent of intact myelin at the lesion epicenter. Copyright © 2013 John Wiley & Sons, Ltd.     

Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury

Although several approaches to stimulate axonal regeneration after spinal cord injury have succeeded in stimulating robust growth of axons into a lesion site, the growth is generally highly disorganized, losing the distinct arrangement of axonal tracts within the spinal cord. Previously described freeze-dried agarose scaffolds, composed of individual, uniaxial channels extending through their entire length, were prepared with and without recombinant Brain-Derived Neurotrophic Factor (BDNF) protein and tested in an adult rat model of spinal cord injury to determine whether regenerating axons could be guided across a site of injury in an organized fashion. After 1 month, both the cellular and axonal responses within and around scaffolds were evaluated. Scaffolds were found to be well integrated with host tissue, individual channels were penetrated by cells, and axons grew through scaffolds in a strikingly linear fashion. Furthermore, the regeneration was significantly augmented by the incorporation of BDNF protein into the walls and lumen of the scaffold. These findings clearly demonstrate that axonal regeneration can be organized and guided across a site of injury.