| 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. |
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