Functional regeneration of respiratory pathways after spinal cord injury

Spinal cord injuries often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. The effects of impaired breathing are a leading cause of death from spinal cord injuries, underscoring the importance of developing strategies to restore respiratory activity. Here we show that, after cervical spinal cord injury, the expression of chondroitin sulphate proteoglycans (CSPGs) associated with the perineuronal net (PNN) is upregulated around the phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with chondroitinase ABC (denoted ChABC) could, by itself, promote the plasticity of tracts that were spared and restore limited activity to the paralysed diaphragm. However, when combined with a peripheral nerve autograft, ChABC treatment resulted in lengthy regeneration of serotonin-containing axons and other bulbospinal fibres and remarkable recovery of diaphragmatic function. After recovery and initial transection of the graft bridge, there was an unusual, overall increase in tonic electromyographic activity of the diaphragm, suggesting that considerable remodelling of the spinal cord circuitry occurs after regeneration. This increase was followed by complete elimination of the restored activity, proving that regeneration is crucial for the return of function. Overall, these experiments present a way to markedly restore the function of a single muscle after debilitating trauma to the central nervous system, through both promoting the plasticity of spared tracts and regenerating essential pathways.     

Reformation of long axon pathways in adult rat central nervous system by human forebrain neuroblasts.

The failure of lesioned axons to regenerate over long distances in the mammalian central nervous system (CNS) is not due to an inability of central neurons to regenerate, but rather to the non-permissive nature of the CNS tissue environment. Regenerating CNS axons, which grow well within a peripheral nerve, for example, fail to penetrate mature CNS tissue by more than about 1 mm. Recent evidence indicates that this may be due to inhibitory membrane proteins associated with CNS oligodendrocytes and myelin. We report here that human telencephalic neuroblasts implanted into the excitotoxically lesioned striatum of adult rats can escape or neutralize this inhibitory influence of the adult CNS environment and extend axons along major myelinated fibre tracts for distances of up to approximately 20 mm. The axons were seen to elongate along the paths of the striato-nigral and cortico-spinal tracts to reach the substantia nigra, the pontine nuclei and the cervical spinal cord, which are the normal targets for the striatal and cortical projection neurons likely to be present in these implants.     

Regeneration by supernumerary axons with synaptic terminals in spinal motoneurons of cats

Axons in the central nervous system (CNS) of mammals do not normally regrow if they are cut, which severely limits restoration of function after injury. We have studied the reactions of adult cat spinal alpha-motoneurons after chronic transection of their axons in the periphery by labelling single cells with horseradish peroxidase. Twelve weeks after the operation, about a third of the axotomized cells had developed a 'supernumerary' axon originating from the cell-body region. These supernumerary axons had variable trajectories and termination fields in the ipsilateral spinal cord but generally anomalous projections. Ultrastructural examination shows that they give rise to boutons that form morphologically normal synaptic contacts with neuronal profiles, although they contain dense-cored vesicles not normally seen in central terminals of alpha-motor axons. We conclude that axotomized neurons in the mammalian CNS may be able to form new synaptic contacts by means of supernumerary axons in the absence of local damage.     

Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth

The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS). In addition to myelin-associated glycoprotein (MAG) and Nogo-A, available evidence suggests the existence of additional inhibitors in CNS myelin. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR) as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.     

Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein

Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.     

Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors

After lesions in the differentiated central nervous system (CNS) of higher vertebrates, interrupted fibre tracts do not regrow and elongate by more than an initial sprout of approximately 1 mm. Transplantations of pieces of peripheral nerves into various parts of the CNS demonstrate the widespread capability of CNS neurons to regenerate lesioned axons over long distances in a peripheral nerve environment. CNS white matter, cultured oligodendrocytes (the myelin-producing cells of the CNS), and CNS myelin itself, are strong inhibitors of neuron growth in culture, a property associated with defined myelin membrane proteins of relative molecular mass (Mr) 35,000 (NI-35) and 250,000 (NI-250). We have now intracerebrally applied the monoclonal antibody IN-1, which neutralizes the inhibitory effect of both these proteins, to young rats by implanting antibody-producing tumours. In 2-6-week-old rats we made complete transections of the cortico-spinal tract, a major fibre tract of the spinal cord, the axons of which originate in the motor and sensory neocortex. Previous studies have shown a complete absence of cortico-spinal tract regeneration after the first postnatal week in rats, and in adult hamsters and cats. In IN-1-treated rats, massive sprouting occurred at the lesion site, and fine axons and fascicles could be observed up to 7-11 mm caudal to the lesion within 2-3 weeks. In control rats, a similar sprouting reaction occurred, but the maximal distance of elongation rarely exceeded 1 mm. These results demonstrate the capacity for CNS axons to regenerate and elongate within differentiated CNS tissue after the neutralization of myelin-associated neurite growth inhibitors.     

Chemotropic guidance of developing axons in the mammalian central nervous system

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon guidance depends partly on the recognition of cell-surface and extracellular matrix cues derived from cells along the pathways. It has also been proposed that neuronal growth cones are guided by gradients of chemoattractant molecules emanating from their intermediate or final cellular targets. Although there is evidence that the axons of some peripheral neurons in vertebrates are guided by chemotropism and the directed growth of some central axons to their targets is consistent with such a mechanism, it remains to be determined whether chemotropism operates in the central nervous system. During development of the spinal cord, commissural axons are deflected towards a specialized set of midline neural epithelial cells, termed the floor plate, which could reflect guidance by substrate cues or by diffusible chemoattractant molecules. Here we provide evidence in support of chemotropic guidance by demonstrating that the rat floor-plate cells secrete a diffusible factor(s) that influences the pattern and orientation of commissural axon growth in vitro without affecting other embryonic spinal cord axons. These findings support the hypothesis that chemotropic mechanisms guide developing axons to their intermediate targets in the vertebrate CNS.     

Functional regeneration of sensory axons into the adult spinal cord

The arrest of dorsal root axonal regeneration at the transitional zone between the peripheral and central nervous system has been repeatedly described since the early twentieth century. Here we show that, with trophic support to damaged sensory axons, this regenerative barrier is surmountable. In adult rats with injured dorsal roots, treatment with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure. The observed effects of neurotrophic factors corresponded to their known actions on distinct subpopulations of sensory neurons. Neurotrophic factor treatment may thus serve as a viable treatment in promoting recovery from root avulsion injuries. I     

En passant neurotrophic action of an intermediate axonal target in the developing mammalian CNS.

During development, neurons extend axons to their targets, then become dependent for their survival on trophic substances secreted by their target cells. Competition for limiting amounts of these substances is thought to account for much of the extensive naturally-occurring cell death that is seen throughout the nervous system. Here we show that spinal commissural neurons, a group of long projection neurons in the central nervous system (CNS), are also dependent for their survival on trophic support from one of their intermediate targets, the floor plate of the spinal cord. This dependence occurs during a several-day-long period when their axons extend along the floor plate, following which they develop additional trophic requirements. A dependence of neurons on trophic support derived en passant from their intermediate axonal targets provides a mechanism for rapidly eliminating misprojecting neurons, which may help to prevent the formation of aberrant neuronal circuits during the development of the nervous system.     

Neural stem cell transplantation in the repair of spinal cord injury

Neural stem cells are a pronising candidate for neural transplantation aimed at neural cell replacement and repair of the damaged host central nervous system (CNS). Recent studies using neural stem cells have shown that implanted neural stem cells can effectively incorporate into the damaged CNS and differentiate into neurons, astrocytes, and oligodendrocytes. The recent explosion in the field of neural stem cell research has provided insight into the inductive factors influencing neural stem cell differentiation and may yield potential therapies for several neurological disorders, including spinal cord injury. In this review, we summarize recent studies involving neural stem cell biology in both rodents and humans. We also discuss unique advantages and possible mechanisms of using neural stem cell trans plantation in the repair of spinal cord injury.     

中枢髓鞘源性抑制蛋白与中枢神经再生

Nogo是一类中枢髓鞘源性抑制蛋白,主要由少突胶质细胞表达,是抑制中枢神经元轴突再生的抑制因子。这些研究成果为探讨CNS损伤的治疗提供了新思路。论文综述了Nogo的结构及在CNS中对神经元轴突再生的抑制作用。     

体育功能锻炼对截瘫患者康复影响及评价分析

目的:探讨体育功能锻炼对截瘫患者后期康复治疗的影响及康复评价.方法:76例在脊髓损伤后出现截瘫患者,进行体育功能锻炼的康复治疗,追踪记录,采用美国脊髓损伤学会(ASIA)脊髓损伤神经功能分类国际标准评价康复效果.结果:经统计,胸腰髓不完全损害者康复锻炼前后ASIA损伤评分运动、感觉评分均有显著意义(P<0.05).胸腰髓完全损伤者运动、感觉ASIA评分好转较明显(P<0.05).结论:体育功能锻炼能帮助截瘫患者后期恢复,显著改善和提高患者的综合功能.     

Preliminary animal studies on observation of injured spinal cord with intraoperative ultrasound backscatter microscopy

Many studies have shown that strategies of nerve regeneration and cell-based transplantation are valid based on animal models of spinal cord injury (SCI).To apply these strategies and bridge spinal cord defects,the identification and precise localization of lesions during spinal cord surgery is necessary.The aim of the present experiment was to evaluate the capabilities of ultrasound backscatter microscopy (UBM) in identifying morphologic changes after SCI.After laminectomy,high-resolution ultrasound images of the spinal cord were obtained in one normal and seven spinal cord-injured adult Wistar rats using a UBM system with a 55-MHz center frequency scanner.Comparison between histoanatomic and UBM images was also performed.The results showed that UBM can identify cysts after the experimental SCI is removed in adult rats.In addition,the glial scar formed in secondary injury showed obvious hyperechoic speckle in the UBM image and correlated with the histoanatomic image.UBM has obvious clinical value in nerve regeneration and cell-based transplantation strategies in injured spinal cords.     

Studies on repairing of hemisected thoracic spinal cord of adult rats by using a chitosan tube filled with alginate fibers

A chitosan tube filled with alginate fibers was implanted into the injured spinal cord of a rat for repairing the damaged tissue. Twelve months after the operation, the morphological observation demonstrated that this chitosan tube could induce regeneration of myelinated and non-myelinated axons and blood vessels. The Basso-Beattie-Bresnahan (BBB) behavioral evaluation confirmed that the implants played a key role in the long-term restoration of rats motor functions. It is a promising start in the treatment of the patients with the injury of the spinal cord.     

Monoclonal antibodies against carbohydrate differentiation antigens identify subsets of primary sensory neurones

Dorsal root ganglion (DRG) neurones transmit cutaneous sensory information from the periphery to the spinal cord. Within the dorsal horn of the spinal cord, classes of sensory fibres that are activated by different cutaneous stimuli terminate in separate and highly restricted laminae. Although the developmental events resulting in the laminar organization of sensory afferent terminals have not been defined, it is likely that interactions between surface molecules on DRG and dorsal horn neurones are involved in the generation of afferent synaptic connections. The identification of surface antigens that distinguish functional subclasses of DRG neurones would represent a first step in establishing the existence and nature of such molecules. We report here that monoclonal antibodies directed against carbohydrate differentiation antigens identify cytoplasmic and cell surface molecules expressed selectively by functional subsets of DRG neurons.     

神经营养因子联合治疗促进脊髓轴索再生的研究现状

神经营养因子（neurotrophic factors,NTFs）在急性脊髓损伤（spinal cord injury,SCI）后神经细胞的生长发育、保护和修复的过程中发挥重要作用。然而,单一的治疗尚不足以激活神经元内源性的再生程序;其次,再生抑制因子限制了NTFs对SCI后结构和功能恢复。因此,越来越多的学者选择以联合的方式探索NTFs对再生的促进作用。本文归纳了NTFs以联合的方式治疗轴索再生的基本原理和最新进展,旨在为联合治疗的进一步研究提供科学指导。     

Nogo-66 receptor antagonist peptide promotes axonal regeneration

Myelin-derived axon outgrowth inhibitors, such as Nogo, may account for the lack of axonal regeneration in the central nervous system (CNS) after trauma in adult mammals. A 66-residue domain of Nogo (Nogo-66) is expressed on the surface of oligodendrocytes and can inhibit axonal outgrowth through an axonal Nogo-66 receptor (NgR). The IN-1 monoclonal antibody recognizes Nogo-A and promotes corticospinal tract regeneration and locomotor recovery; however, the undefined nature of the IN-1 epitope in Nogo, the limited specificity of IN-1 for Nogo, and nonspecific anti-myelin effects have prevented a firm conclusion about the role of Nogo-66 or NgR. Here, we identify competitive antagonists of NgR derived from amino-terminal peptide fragments of Nogo-66. The Nogo-66(1 40) antagonist peptide (NEP1 40) blocks Nogo-66 or CNS myelin inhibition of axonal outgrowth in vitro, demonstrating that NgR mediates a significant portion of axonal outgrowth inhibition by myelin. Intrathecal administration of NEP1 40 to rats with mid-thoracic spinal cord hemisection results in significant axon growth of the corticospinal tract, and improves functional recovery. Thus, Nogo-66 and NgR have central roles in limiting axonal regeneration after CNS injury, and NEP1-40 provides a potential therapeutic agent.     

大鼠坐骨神经切断后经皮低频高强度电刺激相应脊髓节段中GAP-43表达的变化

目的探讨成年Wister大鼠在坐骨神经切断后GAP-43于相应脊髓节段前角运动神经元内的表达变化.方法选取健康成年雄性Wister大鼠60只,将坐骨神经切断,随机分为实验组和对照组,实验组给予经皮低频高强度电刺激,分别于术后1,2,4,8,12,16周处死,取其L4~L6脊髓,利用免疫组织化学技术检测GAP-43在相应脊髓节段中的表达变化,并利用影像分析系统进行统计学分析.结果对照组:1周时前角细胞胞体内GAP-43有明显表达,4周时达到高峰,5~8周时逐渐下调,9~16周时GAP-43在前角细胞胞体内中仍有少量表达,并呈弱阳性.实验组:1周时前角细胞胞体内GAP-43有明显表达,2周时达到高峰,且在4~16周时在神经元中仍有表达,并呈阳性.结论坐骨神经切断可导致成年大鼠相应脊髓节段中前角运动神经元GAP-43表达明显增加,可证明在周围神经损伤后神经元的再生能力增强,但时效很短.而给予低频高强度电刺激疗法后,GAP-43的表达在时长和量上都有明显增加.     

Ependymal cells of chordate larvae are stem-like cells that form the adult nervous system

In ascidian tunicates, the metamorphic transition from larva to adult is accompanied by dynamic changes in the body plan. For instance, the central nervous system (CNS) is subjected to extensive rearrangement because its regulating larval organs are lost and new adult organs are created. To understand how the adult CNS is reconstructed, we traced the fate of larval CNS cells during ascidian metamorphosis by using transgenic animals and imaging technologies with photoconvertible fluorescent proteins. Here we show that most parts of the ascidian larval CNS, except for the tail nerve cord, are maintained during metamorphosis and recruited to form the adult CNS. We also show that most of the larval neurons disappear and only a subset of cholinergic motor neurons and glutamatergic neurons are retained. Finally, we demonstrate that ependymal cells of the larval CNS contribute to the construction of the adult CNS and that some differentiate into neurons in the adult CNS. An unexpected role of ependymal cells highlighted by this study is that they serve as neural stem-like cells to reconstruct the adult nervous network during chordate metamorphosis. Consequently, the plasticity of non-neuronal ependymal cells and neuronal cells in chordates should be re-examined by future studies.