【作者】 酉建；

【导师】 徐如祥；

【作者基本信息】 南方医科大学 ， 神经外科学， 2013， 博士

【摘要】 脊髓损伤(spinal cord injury, SCI)这一重大疾病在全球呈现高发生率、高致死率和高致残率的特点,其救治一直是世界性的医学难题之一。相对于周围神经损伤后的自发性修复,成年哺乳动物中枢神经系统(central nervous system, CNS)受损后,被截断的神经元轴突再生能力极其有限,造成的运动感觉等功能障碍难以恢复。因此,脊髓损伤后的神经再生与修复仍是目前研究的热点和难点。上世纪八十年代,Aguayo及其同事突破性的研究颠覆了神经科学界的传统观念,他们发现中枢神经轴突再生困难并非是缺乏再生能力,而是中枢内微环境限制了神经轴突的再生。近年来的研究表明,有两个主要因素形成了不利于轴突再生的微环境。首先是少突胶质细胞及其形成的髓鞘上含有多种轴突生长抑制因子,在CNS受损后被大量释放出来,如Nogo-A、髓鞘相关糖蛋白(MAG)、少突胶质细胞髓鞘糖蛋白(OMgp),三者都是通过与神经元上的NgR受体复合物结合,激活RhoA介导的信号传导通路,调节细胞骨架蛋白,导致生长锥的塌陷而抑制轴突再生。阻碍中枢神经轴突再生的另一重要原因是胶质瘢痕,它主要是由胶质细胞和结缔组织成分构成,被认为是阻碍再生轴突穿越的机械屏障。此外,由瘢痕细胞分泌的细胞外基质成分在损伤区的堆积也是重要的抑制因素,主要代表是硫酸软骨素蛋白多糖(CSPGs)和细胞粘合素-R(Tenascin-R, TN-R)。TN-R是目前已知的一个抑制神经轴突再生的重要因子。它属于细咆粘合素家族,该家族还包括Tenascin-C、-W、-X、-Y。TN-R是一个大的多域糖蛋白,形成二聚体(160kDa亚型)或三聚体(180kDa亚型),它存在于中枢神经系统,主要由少突胶质细胞和某些类型的神经元表达并分泌到细胞外基质中。体外研究发现,TN-R蛋白对神经元有抗细胞粘附的排斥作用,以及阻碍神经突起生长的抑制作用。这些作用是由TN-R蛋白的EGF-L结构域与神经元生长锥上的细胞粘附分子F3/F11结合后介导的。脊髓损伤后,伤灶处TN-R mRNA的表达上调。有学者利用TN-R基因敲除鼠发现脊髓损伤后TN-R可以阻碍运动神经元与其他神经元重建突触联系,从而限制了SCI后运动功能的恢复。对TN-R基因敲除鼠面神经损伤模型的研究也发现TN-R不利于神经损伤后的功能恢复。为了消除轴突再生抑制因子的抑制效应,神经科学家们常常应用抗体阻断抑制因子或其受体,以此达到促进神经元轴突再生及脊髓损伤等CNS损伤后功能恢复的目的。如采用IN-1单克隆抗体中和Nogo-A以封闭其抑制活性的策略就收到了很好的疗效。除了直接应用特异性抗体进行封闭即被动免疫治疗外,还可以通过主动免疫刺激机体产生相应的中和抗体。相关研究表明通过不同主动免疫方式刺激机体产生对抗相关抑制性因子的抗体,能够有效的促进损伤轴突的再生及功能恢复。然而,虽然主动免疫能够起到良好的作用,但是在临床实践中,绝大多数人也许不会考虑在受伤前就进行此种疫苗的接种。再者,损伤后急性期单独采用主动免疫方式接种,机体也需要经历较长的一段时间后才能够产生保护性抗体,而实际上此时早期干预治疗很有必要,有利于伤后的康复。因此,在损伤急性期通过被动免疫治疗应用中和抗体消除或削弱相关抑制性因子可能更具有临床可行性。综上,TN-R已经被证实能抑制神经轴突再生并阻碍脊髓损伤后的功能恢复,因此以TN-R为治疗靶点,开发拮抗TN-R功能的药物,如TN-R的抗体药物等,将为临床上治疗脊髓损伤提供新的选择和手段。本研究拟以人TN-R蛋白EGF-L结构域为靶点,设计并合成多肽片段作为抗原制备兔源性TN-R多克隆抗体,检测其效价和特异性,并在体外观察其对大鼠皮层神经元突起生长的影响；最后.建立大鼠背侧半横断脊髓损伤模型,对其进行TN-R多克隆抗体被动免疫治疗,观察抗体对脊髓损伤大鼠的疗效。第一部分Tenascin-R多克隆抗体的制备及鉴定目的：以人TN-R蛋白EGF-L结构域为靶点制备TN-R多克隆抗体,检测其效价,并初步验证其对靶分子的特异性识别能力。方法：根据人TN-R蛋白EGF-L结构域的氨基酸序列信息设计多肽抗原,序列长度在10-20个氨基酸残基之间,合成后与载体蛋白相偶联,充分乳化后免疫新西兰大白兔,免疫前采集兔血清用作阴性对照。经过一次基础免疫和三次加强免疫,ELISA测定抗体效价达标后放血并分离血清,获得的抗血清采用抗原亲和纯化。间接ELISA法测定纯化后抗体的效价。以人重组TN-R蛋白为上样蛋白,制备的TN-R多克隆抗体为一抗,Western Blot法检验抗体的特异性。结果：查询GenBank数据库,获取人TN-R蛋白EGF-L结构域的氨基酸序列信息(aa199-323, ID:NP003276.3),将肽段CDSEYSGDDCSELRCP确定为多肽抗原,经合成获得多肽10mg,纯度>85%。免疫前获得4mL阴性对照血清。第三次加强免疫后少量采血行ELISA法测定抗血清中多抗效价>1：105,达到预期效价,耳缘静脉放血收获血液共26mL,制备抗血清10mL,经抗原亲和纯化后获得多克隆抗体8mL, BCA法测得浓度为0.36mg/mL。经间接ELISA法检测,TN-R多抗滴度>1：512,000。Western Blot结果显示,在TN-R多抗侧相对分子量约180kDa处出现阳性条带,而对照血清侧为阴性,表明TN-R多抗能与上样的人重组TN-R蛋白结合。结论：通过合成多肽抗原,成功制备了兔源性TN-R多克隆抗体,其效价高、对TN-R蛋白有良好的特异结合性,为进一步研究奠定了基础。第二部分Tenascin-R多克隆抗体对大鼠皮层神经元及其突起生长的影响目的：了解体外培养的大鼠皮层神经元及其突起在TN-R蛋白上的生长情况,以及TN-R多克隆抗体对其有无影响,为TN-R多克隆抗体的应用提供体外实验数据。方法：在西胞多孔培养板上包被TN-R蛋白,经过免疫前血清或者TN-R多克隆抗体处理后,加入原代培养的新生SD大鼠皮层神经元(小组织块、单层细胞),间接免疫荧光细胞化学法对神经元及其突起进行染色。定性观察不同包被基质的边界抑制效应以及不同基质环境中神经元的黏附数量,利用软件对神经元突起进行长度测量和统计学分析。结果：TN-R蛋白存在区域的边界抑制效应十分明显,大鼠皮层神经元的突起不能长入TN-R蛋白的涂布区域,在其边缘停止生长或转向生长,单纯包被PLL则没有此现象,此外TN-R蛋白区域内黏附的神经元数目很少。TN-R多克隆抗体处理后能减弱这些抑制效应,但免疫前血清没有类似作用。对三组神经元突起长度进行测量和统计分析发现,三组数值差异具有显著性(F=58.654,P=0.000),其中正常对照组神经元的平均突起长度为152.35±11.29μm,培养在TN-R蛋白+免疫前血清包被基质上的神经元,突起生长受到显著抑制,平均突起长度为38.79±4.96μm(P<0.001)。当基质经过TN-R多克隆抗体处理后,TN-R蛋白的抑制作用得到部分逆转,神经元突起长度为112.41±8.41μm(P<0.001),但与正常对照组相比仍有统计学差异(P<0.05)。结论：在体外,TN-R蛋白能明显抑制神经元的黏附及突起的生长伸展,TN-R多克隆抗体的应用能有效地部分阻断TN-R蛋白对神经元及其突起的抑制作用。第三部分脊髓半横断模型大鼠Tenascin-R多克隆抗体被动免疫治疗的效果评估目的：建立大鼠背侧半横断脊髓损伤模型,应用TN-R多克隆抗体被动免疫治疗,观察体内治疗效果并对相关机制进行初步探讨。方法：33只成年雌性SD大鼠随机分为假手术组(8只)、对照组(13只)和实验组(12只)。假手术组大鼠仅接受椎板切开术,另外两组均行T8-9脊髓背侧半横断术,并在皮下埋置微量渗透压泵,分别局部给予兔免疫前血清和TN-R多克隆抗体。术后3天和28天取各组脊髓作冰冻切片,免疫组织化学染色检测渗透入脊髓损伤灶处的兔源性抗体。对术后3天的组织切片行间接免疫荧光染色检测损伤灶及其周围组织TN-R蛋白表达情况。术后3天,提取各组脊髓组织蛋白并测定蛋白浓度,利用蛋白质沉淀技术及Western Blot检测方法对各组脊髓损伤处组织行RhoA活性分析。术后1天及每周,对各组动物盲法行BBB行为学评分以评价大鼠运动功能的恢复情况。结果：兔源性抗体渗透性分析：术后3天,假手术组脊髓切片兔源性抗体免疫组化染色阴性,对照组及实验组脊髓损伤处免疫组化染色均为阳性；术后28天,对照组及实验组脊髓伤灶及其周围组织仍可检测到兔源性抗体。TN-R多抗结合特异性分析：实验组切片TN-R染色强度明显弱于对照组,但与假手术组相比免疫强度稍强；对各组TN-R免疫荧光染色的IOD值进行定量统计分析发现,三组IOD值差异具有显著性(F=24.197,P=0.000),对照组IOD值显著高于假手术组(P<0.01),实验组IOD值比对照组显著降低(P<0.05),但仍高于假手术组(P<0.01)。脊髓组织活化RhoA信号检测：各组RhoA蛋白总表达量无差异,但活化RhoA蛋白量以对照组最高,实验组次之,假手术组最少。以RhoA总蛋白为校正标准,获取活化RhoA蛋白光密度指数为统计量进行统计学分析发现,三组光密度指数值差异具有显著性(F=168.224,P=0.000),对照组的活性RhoA显著高于假手术组(P<0.001),实验组的活性RhoA比对照组显著降低(P<0.001),但仍高于假手术组(P<0.001)。BBB评分结果：在4周的观察期内,大鼠后肢运动功能逐渐恢复,但从7天开始一直到观察终点28天,在各固定时间点,实验组大鼠的BBB评分始终要高于对照组(P<0.01),总体来说,两组BBB评分也存在统计学差异(F=16.279,P=0.001)。21天时,BBB评分达到或超过14分的比率,实验组要高于对照组(62.5%vs.11.1%),统计学上有显著性差异(P=0.0498);28天时,实验组动物的这一比率也要高于对照组(87.5%vs.22.2%),差异具有显著性(P=0.0152)。结论：通过微量渗透压泵的局部给药,TN-R多克隆抗体能在不少于4周的时间窗内持续渗透入损伤脊髓组织,并且能够与组织中大部分上调的TN-R蛋白特异性结合。TN-R多抗可显著削弱胞内轴突再生抑制信号的激活,从而促进脊髓损伤后神经元轴突的再生,以及损伤脊髓的功能恢复。总结本研究制备了一种针对轴突再生抑制因子TN-R的多克隆抗体,该抗体是以人TN-R蛋白EGF-L结构域为靶点,通过合成多肽抗原制备的,经鉴定具有高效价与良好的特异性,对其在体外和体内的功能进行检测和评估发现,该抗体能显著削弱TN-R蛋白对神经元及其突起的抑制作用,阻碍中枢神经系统内的神经抑制信号传递,促进受损脊髓的功能恢复。但是,这一中和抗体的生物安全性还需要进一步评估,其相关的作用机制也有待于阐明。本研究提供的证据表明：TN-R拮抗剂的被动免疫具有治疗脊髓损伤的潜在应用价值,为治疗中枢神经系统损伤提供了一种新的、有前途的手段和策略。更多还原

【Abstract】 Spinal cord injury （SCI）, one most serious trauma in the world with high incidence, results in damage to axonal tracts that control motor and sensory function, leading to high morbidity and disability rate. The treatment for SCI has been one of worldwide medical problem. Relative to the spontaneous repairing after peripheral nerve injury, injured axons have weak regenerative capacity in adult mammalian central nervous system （CNS）, limiting functional recovery. Therefore, neural regeneration and repairing after SCI is still an active area of current research with difficulty.In the1980s, breakthrough research of Aguayo and his colleagues overturned the traditional concept in neuroscience, they found that an important barrier against regeneration is not the intrinsic property of neurons, but the microenvironment encountered by the sprouting fibers. In recent years, accumulating evidences have demonstrated that there are two main factors in the formation of inhibitive microenvironment, which is firstly associated with the neurite outgrowth inhibitors that lie in myelin and oligodendrocytes, such as Nogo-A, myelin-associated glycoprotein （MAG） and oligodendrocyte myelin glycoprotein （OMgp）. A large number of these molecules are released post CNS injury, combining with a complex composed of NgR and its co-receptors on neurons, activating RhoA-mediated inhibitory signaling pathway, regulating cytoskeleton proteins, resulting in the growth cone collapse and axonal elongation failure. Another important reason for the inhibition of axonal regeneration is glial scar, mainly consisted of glial cells and connective tissue components, which is considered as a mechanical barrier impeding the regenerating axons to cross. Moreover, the inhibitory property of glial scar is also related to the accumulation of extracellular matrix molecules secreted by scar cells in the lesion site, mainly including chondroitin sulfate proteoglycans （CSPGs） and Tenascin-R （TN-R）.TN-R is now known as a crucial molecule involved in inhibition of axonal regrowth in impaired CNS, which is belong to the tenascin family including tenascin-C,-W,-X,-Y. TN-R is a neural specific multidomain glycoprotein presenting as dimer （160kDa） or trimer isoform （180kDa）, which is an extracellular matrix component expressed by oligodendrocytes and subpopulations of neurons in CNS. Previous works have shown that neurites of neurons are repelled by a substrate border of TN-R in vitro. TN-R also has inhibitory functions in the outgrowth and guidance of optic axons in vivo. The inhibitory effect of TN-R on neurite outgrowth is mediated by the interaction of TN-R EGF-L domain with the cell adhesion molecule F3/F11on neuronal growth core. It is reported that TN-R mRNA expression is up-regulated in the lesion area after SCI. Better functional outcome of facial nerve repair is observed in TN-R null mutant mice, suggesting that TN-R impedes recovery after nerve lesion. In addition, a study on TN-R-deficient mice shows that TN-R restricts functional recovery from SCI by limiting posttraumatic remodeling of synapses around motor neurons. In order to neutralize the inhibitory activity, antibodies against inhibitors or their receptors are usually applied by many neuroscientists, aiming at promoting axonal regeneration and functional recovery from SCI and other CNS injury. For example, Blocking Nogo-A with infusion of monoclonal antibodies1N-1promotes long distance axonal regeneration after SCI. It has been reported that rats immunized with recombinant inhibitory protein or DNA vaccine encoding multiple inhibitors, show functional improvements and axonal regeneration in histomorphology with polyclonal antibodies detected in vivo. These findings suggest that active immunity could stimulate the generation of antibodies which effectively promote structural and functional recovery following SCI by blocking inhibitors. However, in clinical practice, no one would like to receive vaccine immunization before SCI occurs. Even if the subject is immunized immediately after injury, it will take several weeks at least to generate sufficient antibodies. In fact, the intervention at early stage of injury is especially important and beneficial for rehabilitation. To achieve this goal, it could be more practical for clinical application to apply passive immunization and administer neutralizing antibodies.In summary, it is confirmed that TN-R is involved in the inhibition of axonal regeneration and functional recovery from SCI. Therefore, TN-R could be a novel target for the treatment of SCI, and specific TN-R antagonist may represent a useful therapeutic approach for promoting neural repair after SCI. In this study, we attempted to develop a TN-R antagonist, the rabbit-derived TN-R polyclonal antibody, which was induced by a fragment of peptide designed and synthesized according to the amino acid residues of EGF-L domain of human TN-R. Firstly, the titer and specificity of this prepared antibody was detected; Then, we tested if TN-R polyclonal antibody could promote neurite outgrowth and adhesive number of rat cortical neurons cultured on TN-R protein in vitro; Finally, we investigated in vivo efficacy of passive immunotherapy with TN-R polyclonal antibody on spinal cord dorsal hemisection model in rats.The study includes three chapters:Chapter I Preparation and identification of Tenascin-R polyclonal antibodyObjective:To prepare rabbit-derived polyclonal antibody targeting on EGF-L domain of human TN-R, and identify titer and specificity of the prepared antibody.Methods:A fragment of peptide （10-20aa） was designed and synthesized according to the amino acid residues of EGF-L domain of human TN-R, coupled to keyhole limpet hemocyanin, and used to immunize rabbit as antigen. The rabbit-derived antiserum was collected after the4th immunization and purified by antigen coupling sepharose chromatography. And rabbit serum before immunization was collected for control. The titer of polyclonal antibody was assessed by indirect enzyme-linked immunosorbent assay （ELISA）. Western blotting was performed to detect antibody specificity:0.75μg recombinant human TN-R protein （-180kDa） per lane was loaded on a5%stacking gel and an8%separating gel, and the prepared antibody was diluted to1:1,000in PBS as the primary antibody to sample, while pre-immune serum was used as control.Results:10mg peptide antigen （CDSEYSGDDCSELRCP） with>85%purity was designed and synthesized according to the amino acid residues of EGF-L domain （aa199-323） of human TN-R provided by GenBank （ID:NP₀03276.3）.4mL control serum was harvested before the immunization with coupled peptide. After the4th immunization and antigen affinity purification,8mL rabbit-derived TN-R polyclonal antibody （0.36mg/mL） was prepared and antibody response specific to antigen （TN-R peptide） was assessed by ELISA. The results showed that high level of antibody against TN-R peptide was detected and the average antibody titer was>1:512,000. While in rabbit pre-immune serum, anti-TN-R antibody was not detected. And notably, Western blotting showed that the prepared antibody could specifically recognize a band of～180kDa, the loaded recombinant human TN-R protein, and pre-immune serum did not react with the TN-R protein. These results indicate that the TN-R polyclonal antibody is successfully prepared with a high titer and high specificity to TN-R protein.Conclusion:The rabbit-derived TN-R polyclonal antibody is successfully prepared with a high titer and high specificity to TN-R protein, which is induced by a fragment of synthesized peptide antigen, establishing the foundation of further research on TN-R function.Chapter Ⅱ Effect of Tenascin-R polyclonal antibody on adhesion and neurite outgrowth of rat cortical neuronsObjective:To examine neurite outgrowth and adhesive number of rat cortical neurons cultured on TN-R protein, as well as the effect of TN-R polyclonal antibody, provide in vitro data for the application of the TN-R polyclonal antibody.Methods:Cortical neurons isolated from neonatal Sprague-Dawley rats （small blocks of tissue or cells in single layer） were primarily cultured on cell plates coated with TN-R protein in the presence of pre-immune serum or TN-R polyclonal antibody. Neurons and their neurites were stained by indirect immunofluorescence cytochemistry. We qualitatively observed boundary inhibitory effects of different immobilized substrates and adhesive number of neurons in different substrates, and measured neurite lengths of neurons in different substrates.Results:Boundary inhibitory effect of TN-R protein substrate was most obvious, and few neurons adhered within the TN-R region. Treatment with TN-R polyclonal antibody, but not pre-immune serum. could attenuate these inhibitory effects. Neurite lengths in three groups were measured and statistical analyzed. There is a statistically significant difference between the three groups （F=58.654,.P=0.000）. The average neurite length of neurons was152.35±11.29μm in normal control group. Neurite outgrowth was significantly inhibited in neurons cultured on TN-R+pre-immune serum, and the average neurite length was only38.79±4.96μm （P<0.001）. When substrate had been treated with the TN-R polycional antibody, the inhibitory effect of TN-R protein could be partially reversed, the average length of neurites in neurons reached to112.41±8.41μm （P<0.001）, but compared with normal control group, there is still a statistically significant difference between the two groups（P<0.05）.Conclusion:TN-R proteins significantly inhibit neuronal adhesion and neurite outgrowth, application of TN-R polyclonal antibody can effectively and partly block the inhibitory action of TN-R protein on neurons and their neurites.Chapter Ⅲ Application and efficacy assessment of passive immunotherapy with Tenascin-R polyclonal antibody on spinal cord hemisection model in ratsObjective:To investigate the in vivo efficacy of passive immunotherapy with TN-R polyclonal antibody on spinal cord dorsal hemisection model in rats and to discuss the possible mechanism of passive immunotherapy, to provide experimental basis for clinical application.Methods:33Adult female Sprague-Dawley rats were randomly divided into three groups:sham-operated group （N=8）, control group （N=13）, experimental group （N=12）. Rats in sham-operated group received laminectomy only, and spinal cord dorsal hemisection at the level of T8-9was performed on animals in the other two groups. Immediately after corticospinal tract （CST） transection, the lesion site was adjacent to the open of a fixed catheter connected to a primed mini-osmotic pump inserted into subcutaneous space. Each pump held and locally delivered rabbit pre-immune serum or TN-R polyclonal antibody. Spinal cord cryo-sections from each group at3d and4wk post-injury were stained using immunohistochemistry to detect the rabbit-derived immunoglobulin infiltrating into the lesioned spinal cord. For TN-R staining, indirect immunofluorescence staining was performed on sections from each group at3d post-surgery. Two rats in each group were sacrificed3days after SCI and injured spinal cords were rapidly removed to quantify RhoA activation. Total protein of spinal cord tissue was extracted and the protein concentration was determined. Following the pulldown assay, GTP bound and total RhoA proteins were detected by Western blotting. Gross functional recovery was evaluated according to the BBB locomotor rating scale, this testing was performed at24h and once weekly thereafter up to4weeks post-injury with observers blinded to the study groups.Results:Immunoglobulins detection:The rabbit-derived immunoglobulins were detected in injured spinal cords from both control and experimental groups3days after SCI. Moreover, the staining for immunoglobulins at lesion sites4weeks after SCI remained strong and extended rostrally and caudally for a considerable distance. All sections from sham group had negative staining.TN-R staining:There is a statistically significant difference between the three groups for IOD value （F=24.197, P=0.000）. The immunoreactivity density in control group was much greater than that of the sham （P<0.01）. The TN-R staining in experimental group was much weaker than that of control group. There is a statistically significant difference in labeling density between the two groups（P<0.05）. But compared to the sham, an increase in TN-R immunoreactivity was detected in the experimental group（P<0.01）.RhoA activation assay:There is a statistically significant difference between the three groups for relative RhoA-GTP level （F=168.224, P=0.000）. The RhoA total expression in each group had no difference between each other. The active RhoA level in control group was significantly higher than that of sham group （P<0.001）. Spinal cord tissue homogenates from TN-R antibody-treated rats had lower RhoA-GTP levels than control （P<0.001）, but still higher than the sham （P<0.001）.BBB score:Rats receiving continuous local infusion of pre-immune serum recovered partial function over the4-week duration of the experiment, attaining a mean BBB score of8.1±1.5. Delivery of TN-R antibody resulted in significantly improved BBB scores at the last4time-points （P<0.01）, reaching a mean BBB score of14.6±0.6. And there is a statistically significant difference between the two groups for BBB score （F=16.279, P=0.001）. The proportion of TN-R antibody-treated animals attained a score of14or more was higher than that in control animals,3weeks （62.5%vs.11.1%, P=0.0498） and4weeks （87.5%vs.22.2%,P=0.0152） after SCI.Conclusion:Due to the breakdown of spinal cord blood barrier, the TN-R antibodies could persistently penetrate into the injured spinal cord via mini-osmotic pump for4weeks. TN-R expression is up-regulated in the spinal cord following injury, after passive immunization, most of the endogenous TN-R molecules are blocked by prepared anti-TN-R antibodies. Administration of the TN-R antibody into the injury site could significantly attenuate RhoA activation after SCI, promote neurite outgrowth, culminating in improved functional recovery from CST transection. SummaryTenascin-R （TN-R） is a neural specific protein and an important molecule involved in inhibition of axonal regeneration after spinal cord injury （SCI）. Here we report on rabbit-derived TN-R polyclonal antibody induced by a fragment of synthesized peptide antigen, which acts as a TN-R EGF-L domains antagonist with high titer and high specificity, promoted neurite outgrowth and sprouting of rat cortical neurons cultured on the inhibitory TN-R substrate in vitro. When locally administered into the lesion sites of rats received spinal cord dorsal hemisection, these TN-R antibodies could significantly decrease RhoA activation and improve functional recovery from corticospinal tract （CST） transection. However, the mechanism underlying such neuroprotection remains to be determined and further improvement will entail evaluating the bio-safety of TN-R antagonist. In conclusion, passive immunotherapy with specific TN-R antagonist may represent a promising repair strategy following acute SCI.更多还原