Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Hydralazine inhibits compression and acrolein-mediated injuries in ex vivo spinal cord

We have previously shown that acrolein, a lipid peroxidation byproduct, is significantly increased following spinal cord injury in vivo , and that exposure to neuronal cells results in oxidative stress, mitochondrial dysfunction, increased membrane permeability, impaired axonal conductivity, and eventually cell death. Acrolein thus may be a key player in the pathogenesis of spinal cord injury, where lipid peroxidation is known to be involved. The current study demonstrates that the acrolein scavenger hydralazine protects against not only acrolein-mediated injury, but also compression in guinea pig spinal cord ex vivo . Specifically, hydralazine (500 μmol/L to 1 mmol/L) can significantly alleviate acrolein (100–500 μmol/L)-induced superoxide production, glutathione depletion, mitochondrial dysfunction, loss of membrane integrity, and reduced compound action potential conduction. Additionally, 500 μmol/L hydralazine significantly attenuated compression-mediated membrane disruptions at 2 and 3 h following injury. This was consistent with our findings that acrolein-lys adducts were increased following compression injury ex vivo , an effect that was prevented by hydralazine treatment. These findings provide further evidence for the role of acrolein in spinal cord injury, and suggest that acrolein-scavenging drugs such as hydralazine may represent a novel therapy to effectively reduce oxidative stress in disorders such as spinal cord injury and neurodegenerative diseases, where oxidative stress is known to play a role.     

Role of melatonin in the dynamics of acute spinal cord injury in rats

Melatonin is well-documented to have the ability of reducing nerve inflammation and scavenging free radicals. However, the therapeutic effect of melatonin on spinal cord injury has not been fully described. In this study, we assessed the effect of melatonin on T9 spinal cord injury established by Allen method in rats. Melatonin deficiency significantly delayed the recovery of sensory and motor functions in SCI rats. Treatment with melatonin significantly alleviated neuronal apoptosis and accelerated the recovery of spinal cord function. These results suggest that melatonin is effective to ameliorate spinal cord injury through inhibition of neuronal apoptosis and promotion of neuronal repair.     

大鼠脊髓损伤的弱激光治疗参数选择

弱激光在周围神经损伤治疗中取得了理想效果,但在脊髓损伤修复方面的研究很少.本实验应用Allen＇s造模法构建大鼠急性脊髓损伤模型,通过测量穿透功率及组织温度变化筛选出用于脊髓损伤治疗的弱激光照射参数,照射组按照筛选出的参数连续治疗14 d,于术后1、3、7、14、21 d应用BBB评分法评价大鼠后肢运动功能的恢复情况,苏木精-伊红染色（HE染色）观察脊髓病理变化并测量空洞面积.结果显示应用筛选出的照射参数（810 nm、光斑面积0.2 cm2、500 mW/cm2、510 J/cm2）连续照射14 d,术后21 d照射组的运动功能评分显著高于未照射组（P＜0.05）,术后7、14、21d照射组脊髓空洞面积显著小于未照射组（P＜0.05）.结果表明810 nm、光斑面积0.2 cm2、500 mW/cm2、510 J/cm2的弱激光照射能促进急性脊髓损伤大鼠后期运动功能的恢复.     

BMP inhibition enhances axonal growth and functional recovery after spinal cord injury

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP-2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.     

Large Animal Model for Development of Functional Restoration Paradigms Using Epidural and Intraspinal Stimulation

Restoration of movement following spinal cord injury (SCI) has been achieved using electrical stimulation of peripheral nerves and skeletal muscles. However, practical limitations such as the rapid onset of muscle fatigue hinder clinical application of these technologies. Recently, direct stimulation of alpha motor neurons has shown promise for evoking graded, controlled, and sustained muscle contractions in rodent and feline animal models while overcoming some of these limitations. However, small animal models are not optimal for the development of clinical spinal stimulation techniques for functional restoration of movement. Furthermore, variance in surgical procedure, targeting, and electrode implantation techniques can compromise therapeutic outcomes and impede comparison of results across studies. Herein, we present a protocol and large animal model that allow standardized development, testing, and optimization of novel clinical strategies for restoring motor function following spinal cord injury. We tested this protocol using both epidural and intraspinal stimulation in a porcine model of spinal cord injury, but the protocol is suitable for the development of other novel therapeutic strategies. This protocol will help characterize spinal circuits vital for selective activation of motor neuron pools. In turn, this will expedite the development and validation of high-precision therapeutic targeting strategies and stimulation technologies for optimal restoration of motor function in humans.     

Topical application of dynorphin A (1-17) antiserum attenuates trauma induced alterations in spinal cord evoked potentials,microvascular permeability disturbances,edema formation and cell injury: an experimental study in the rat using electrophysiological and morphological approaches

Summary.  Dynorphin is a neuropeptide that is present in high quantities in the dorsal horn of the spinal cord. The peptide is actively involved in pain processing pathways. However, its involvement in spinal cord injury is not well known. Alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. Infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. Antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improve motor recovery, reduce edema and cell changes. However, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. Spinal cord evoked potentials (SCEP) are good indicator of spinal cord pathology following trauma. Therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in SCEP were examined in our rat model. In addition, spinal cord edema formation, microvascular permeability disturbances and cell injury were also investigated. Our results show that topical application of dynorphin antiserum (1 : 200) two min before injury markedly attenuated the SCEP changes immediately after injury. In the antiserum treated animals, a significant reduction in the microvascular permeability, edema formation and cell injury was observed in the traumatised spinal cord. These observations suggest that (i) dynorphin is involved in the altered bioelectrical activity of the spinal cord following trauma, (ii) the peptide actively participates in the pathophysiological processes of cell injury in the spinal cord trauma, and (iii) the dynorphin antiserum has potential therapeutic value for the treatment of spinal cord injuries. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

肠道菌群影响脊髓损伤后焦虑情绪的研究进展

脊髓损伤作为一种严重的创伤性应激可以引发焦虑情绪,对患者心理健康造成极大影响。研究发现,脊髓损伤后肠道菌群失调与焦虑情绪的发生存在密切联系,因此本文从5-羟色胺系统失调、多巴胺系统失调、脑源性神经营养因子缺乏及炎症反应4个方面,探讨脊髓损伤后肠道菌群改变影响焦虑情绪发生的机制,为今后治疗脊髓损伤后焦虑情绪的深入研究和药物开发提供理论依据。     

Expression of neural cell adhesion molecule in spinal cords following a complete transection

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

神经生长因子对兔坐骨神经损伤后脊髓前角运动神经元乙酰胆碱酯酶的影响

钳夹损伤兔右坐骨神经,于损伤处注射蛇毒ＮＧＦ４００Ｂｕ／ｋｇ／日,损伤术后１,３,７天和２,３,４,６,８周动态观察脊髓腰段伤侧第Ⅸ板层外侧群的大型运动神经元的ＡＣｈＥ活性改变。结果表明术后１,３天实验组（指损伤给药组）和对照组（指损伤对照组）ＡＣｈＥ活性均下降（Ｐ＞００５）;术后１,２,３周对照组ＡＣｈＥ活性明显下降,而实验组ＡＣｈＥ活性逐渐趋于恢复（Ｐ＜００１）;术后６周实验组ＡＣｈＥ活性恢复至正常水平（Ｐ＜００１）。本研究显示蛇毒ＮＧＦ对坐骨神经损伤后脊髓前角运动神经元ＡＣｈＥ活性恢复有促进作用,从而对运动神经元可起一定的保护作用和促进恢复的作用     

大鼠脊髓损伤后感觉、运动及电生理变化

在应用磁控机械夹断法复制的大鼠脊髓损伤模型上,动态地观察了脊髓损伤后的感觉及运动机能变化,并进行了电生理学研究。结果表明,0.3A电流未能导致永久性瘫痪。术后2周,后肢的感觉及运动功能逐渐恢复;可记录到体感诱发电位(SEP)。0.4,0.5和0.8A电流均能导致大鼠永久性瘫痪;倾斜板及开阔场地行走分数均显著低于0.3A组;术后4周这些大鼠可产生行走样动作,于损伤部位再次切断脊髓后仍能出现这些动作;0.4A组可记录到早期SEP,再次切断脊髓后SEP消失。结果提示:(1)脊髓不全横断后,由于残留纤维活动,可在相当程度上导致大鼠感觉和运动机能的恢复;(2)脊髓完全横断后,后肢的上行冲动可能经再生的神经纤维向中枢端传导至脑;(3)大鼠脊髓内可能存在行走中枢模式发生器(CPG),适当刺激可激发其活动,并产生行走样运动。     

bFGF通过抑制Nogo-A信号减少脊髓损伤后胶质瘢痕形成

脊髓损伤后胶质瘢痕的形成是阻碍神经恢复的关键原因之一。碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）具有良好的神经保护及促进脊髓损伤的修复作用,然而其对于胶质瘢痕的影响及其机制仍不清楚。本研究通过采用血管动脉夹（30 g）夹闭雌性SD大鼠脊髓2 min造成急性脊髓损伤模型并予以每天皮下注射bFGF（80 μg/kg）,探讨bFGF促进脊髓损伤的恢复作用是否涉及到胶质瘢痕调控和Nogo-A/NgR信号的相关机制。通过检测损伤后28 d,各组BBB评分和斜板试验,发现bFGF显著促进脊髓损伤后大鼠运动功能的恢复。HE及尼氏染色显示,bFGF处理组相对于生理盐水处理组,其神经元明显增多,空洞面积减少。同时,星形胶质细胞标记物GFAP免疫荧光结果表明,bFGF减少胶质瘢痕形成,抑制星形胶质细胞过度激活。同样,通过Western 印迹检测发现,bFGF处理后,胶质瘢痕相关蛋白（如GFAP, neurocan）以及神经突生长抑制蛋白（Nogo-A）信号通路相关蛋白质表达量下降。上述结果表明,bFGF可能通过抑制Nogo-A信号蛋白的表达,从而抑制胶质瘢痕的形成,促进脊髓损伤的恢复。此机制研究为脊髓损伤的治疗和恢复提供全新的思路和药物靶点。     

FGF22 signaling regulates synapse formation during post‐injury remodeling of the spinal cord

The remodeling of axonal circuits after injury requires the formation of new synaptic contacts to enable functional recovery. Which molecular signals initiate such axonal and synaptic reorganisation in the adult central nervous system is currently unknown. Here, we identify FGF22 as a key regulator of circuit remodeling in the injured spinal cord. We show that FGF22 is produced by spinal relay neurons, while its main receptors FGFR1 and FGFR2 are expressed by cortical projection neurons. FGF22 deficiency or the targeted deletion of FGFR1 and FGFR2 in the hindlimb motor cortex limits the formation of new synapses between corticospinal collaterals and relay neurons, delays their molecular maturation, and impedes functional recovery in a mouse model of spinal cord injury. These results establish FGF22 as a synaptogenic mediator in the adult nervous system and a crucial regulator of synapse formation and maturation during post‐injury remodeling in the spinal cord.     

Stress-induced phosphoprotein 1 restrains spinal cord ischaemia-reperfusion injury by modulating NF-κB signalling

Spinal cord injury (SCI), a major cause of disability, causes high global disease and economic burdens. Stress-induced phosphoprotein 1 (STIP1) has been identified to be involved in spinal cord ischaemia-reperfusion injury (SCII); however, the effect of STIP1 on SCII remains unclear until now. This study aimed to examine the role of STIP1 in SCII and unravel the possible mechanisms. Western blotting and immunohistochemical staining showed that STIP1 expression rapidly increased and then decreased in rat spinal cord following SCII treatment. Neurological function scoring, HE staining, immunohistochemical staining and Western blotting revealed that STIP1 overexpression alleviated SCII-induced motor dysfunction of hind limbs, neuronal loss and inflammation in spinal cord, and inhibited activity of nuclear factor kappa B (NF-κB) signalling in rats. Immunoprecipitation identified that STIP1 was co-located with Iba-1. In addition, STIP1 was found to ameliorate oxygen and glucose deprivation (OGD)-induced inflammation and activation of NF-κB signalling in mouse microglia BV2 cells, and STIP1 resulted in decrease of heat shock protein family A member 8 (HSPA8), increase of IκBβ expression and reduced binding of IκBβ to HSPA8 in BV2 cells. The results of the present study demonstrate that STIP1 alleviates ischaemia/reperfusion-induced neuronal injury and inflammation in rat spinal cord and mouse microglial cells by deactivating NF-κB signalling. These findings may provide novel insights for the clinical diagnosis and treatment of SCI.     

Absence of endogenous interleukin-10 enhances secondary inflammatory process after spinal cord compression injury in mice

Interleukin-10 (IL-10) exerts a wide spectrum of regulatory activities in the immune and inflammatory response. The aim of this study was to investigate the role of endogenous IL-10 on the modulation of the secondary events in mice subjected to spinal cord injury induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5–T8 laminectomy. IL-10 wild-type mice developed severe spinal cord damage characterized by oedema, tissue damage and apoptosis (measured by Annexin-V, terminal deoxynucleotidyltransferase-mediated UTP end labeling staining, Bax, Bcl-2, and Fas-L expression). Immunohistochemistry demonstrated a marked increase of localization of TNF-α, IL-1β and S100β, while western blot analysis shown an increased immunoreactivity of inducible nitric oxide synthase in the spinal cord tissues. The absence of IL-10 in IL-10 KO mice resulted in a significant augmentation of all the above described parameters. We have also demonstrated that the genetic absence of IL-10 worsened the recovery of limb function when compared with IL-10 wild-type mice group (evaluated by motor recovery score). Taken together, our results clearly demonstrate that the presence of IL-10 reduces the development of inflammation and tissue injury events associated with spinal cord trauma.     

铁死亡参与脊髓损伤调控的研究进展

铁死亡是一种铁依赖性的,以细胞内脂质活性氧堆积为特征的细胞程序性死亡方式。广泛存在于肿瘤、癌症、急性肾损伤等多种疾病当中。脊髓损伤(spinal cord injury, SCI) 是一种严重的创伤性神经系统疾病,具有高发病率、高死亡率、高致残率的特点。目前,脊髓损伤的具体发生机制及高效治疗方法仍在探索当中,这也是亟待解决的世界性难题。研究表明,脊髓损伤后调控神经细胞的程序性死亡是治疗SCI的重点。然而,对于铁死亡参与脊髓损伤的分子生物学机制尚缺乏系统和深入的认识。收集和整理了近几年国内外有关脊髓损伤后铁死亡方面的相关文献,针对铁死亡参与脊髓损伤的调控机制和研究进展进行了综述,以期为治疗脊髓损伤带来新的思路。     

Acrolein scavenging: a potential novel mechanism of attenuating oxidative stress following spinal cord injury

It has long been established that oxidative stress plays a critical role in the pathophysiology of spinal cord injury, and represents an important target of therapeutic intervention following the initial trauma. However, free radical scavengers have been largely ineffective in clinical trials, and as such a novel target to attenuate oxidative stress is highly warranted. In addition to free radicals, peroxidation of lipid membranes following spinal cord injury (SCI) produces reactive aldehydes such as acrolein. Acrolein is capable of depleting endogenous antioxidants such as glutathione, generating free radicals, promoting oxidative stress, and damaging proteins and DNA. Acrolein has a significantly longer half‐life than the transient free radicals, and thus may represent a potentially better target of therapeutic intervention to attenuate oxidative stress. There is growing evidence, from our lab and others, to suggest that reactive aldehydes such as acrolein play a critical role in oxidative stress and SCI. The focus of this review is to summarize the cellular and biochemical mechanisms of acrolein‐induced membrane damage, mitochondrial injury, oxidative stress, cell death, and functional loss. Evidence will also be presented to suggest that acrolein scavenging may be a novel means of therapeutic intervention to attenuate oxidative stress and improve recovery following traumatic SCI.