軸索再生、組織修復 Axonal Regeneration and Tissue Repair
P3-1-92 損傷後の脳における線維性瘢痕の形成と軸索再生の阻害について On the formation and axonal growth-inhibiting property of the fibrotic scar which occurs after traumatic brain injury ○川野仁1, 木村-黒田純子1, 小牟田縁1, 吉岡望2, 武内恒成2 ○Hitoshi Kawano1, Junko Kimura-Kuroda1, Yukari Komuta1, Nozomu Yoshioka2, Kosei Takeuchi2 東京都医学研・脳発達再生1, 新潟大学医学部生化学2 Dept Brain Dev Neural Regen, Tokyo Mtropol Inst Med Sci, Tokyo1, Dept Biochem, Niigata Univ, Niigata2 Following traumatic injury of the central nervous system of adult mammals, transected axons do not regenerate beyond the fibrotic scar which is formed in the lesion center. After the injury, meningeal fibroblasts migrate to the lesion site, proliferate and secrete type IV collagen (Col IV) to form the fibrotic scar. We have demonstrated that suppression of the fibrotic scar formation is required for axonal regeneration in the damaged brain in a variety of animal models, such as (1) suppression of Col IV synthesis (Kawano et al., 2005), (2) newborn mouse (Kawano et al., Ibid), (3) the mouse hypothalamic arcuate nucleus (Homma et al., 2006), (4) degradation of glycoaminoglycan side chains of chondroitin sulfate proteoglycans with chondroitinase ABC (Li et al., 2007), (5) transplantation of olfactory ensheathing cells (Teng et al., 2008) and (6) suppression of transforming growth factor-β (TGF-β) function (Yoshioka et al., 2011). Addition of TGF-β1 to the coculture of meningeal fibroblasts and cerebral astrocytes induced a fibrotic scar-like cell cluster which repels neurites of cerebellar neurons (Kimura-Kuroda et al., 2010). The fibrotic scar and TGF-β1-induced cell cluster intensely expressed both dermatan sulfate (DS) and condroitin sulfate (CS). Administration of enzymes specifically degrading DS or CS in injured brains and in cell culture demonstrated that DS is involved in the fibrotic scar formation and CS inhibits axonal regeneration (Li, Komuta et al., 2013 in press).	P3-1-93 ラット脊髄完全損傷に対するsemaphorin3A阻害剤とtreadmill訓練の併用療法の検討 Rewiring of Regenerated Axons by Combining Treadmill Training with Semaphorin3A Inhibition ○張亮1,2,3,5, 金子慎二郎3,5, 菊池薫4, 佐野明彦4, 前田美穂4, 岸野晶祥4, 芝田晋介2, 向野雅彦1, 戸山芳昭3, 里宇明元1, 木村徹4, 岡野栄之2, 中村雅也3 ○Liang Zhang1,2,3,5, Shinjiro Kaneko3,5, Kaoru Kikuchi4, Akihiko Sano4, Miho Maeda4, Akiyoshi Kishino4, Shinsuke Shibata2, Masahiko Mukaino1, Yoshiaki Toyama3, Meigen Liu1, Toru Kimura4, Hideyuki Okano22, Masaya Nakamura3 慶應大院・医・リハビリ1, 慶應大院・医・生理2, 慶應大院・医・整形3, 大日本住友製薬4, 国立医療機構村山医療センター5 Dept Rehab, Univ of Keio, Tokyo1, Dept Physiol, Univ of Keio, Tokyo2, Dept Orthopedic, Univ of Keio, Tokyo3, Dainippon Sumitomo Pharma Co. Ltd.4, Clinical Research Center, National Hospital Organization, Murayama Medical Center5 Objective: To examine the effect of functional recovery after combining treadmill training with Semaphorin3A inhibitionMethod: Female SD adult rats were suffered total spinal cord transection(SCT) and a selective semaphorin3A inhibitor(SM-345431) or placebo was administered at the lesion site by a newly drug delivery system(DDS). After surgery, rat were divided into three groups randomly(Placebo, SM-345431, combinatorial group) and treadmill training started at 1 week after SCT in combinatorial group. Detailed motor function recovery and tissue analysis was performed at 12 weeks after SCT.Result: Control SCT rats show almost no axonal regeneration and functional recovery. Treatment with SM-345431 by this newly DDS enhanced axon regeneration with significant but limited hindlimb motor functional recovery. Although extensive treadmill training with SM-345431 administration did not further improve axon regeneration, significant functional recovery, even the plantar step with body support, was observed. Further detailed analyses indicate SM-345431 improves axonal regeneration, remyelination and angiogenesis, forming a plasticity environment around lesion site after SCT. Extensive treadmill training could remodels extensor lumbar spinal circuitries, and therefore improves the ability of body support after spinal cord injury. Furthermore, it rewires regenerated axons from supraspinal or propriospinal neuron after SM-345431 administration at/around lesion site. Rewiring of regenerated axons by treadmill training in plasticity environment improves connectivity between supraspinal and lumbar spinal circuitries, together with remodeling of the extensor lumbar spinal circuitries, rehabilitate the capability of plantar step with body support.Conclusion: This study highlights the importance of combining treatments that yield axon regeneration with specific and appropriate rehabilitations, aim for rewiring, for treatment of spinal cord injury.
P3-1-94 顔面神経損傷後のマウスにおけるGABAシグナルの変化 GABAergic signaling during degeneration and regeneration of mouse facial nerves ○金正泰1, 高山千利1 ○Jeongtae Kim1, Chitoshi Takayama1 琉球大学大学院　医学研究科　分子解剖学講座1 Department of Molecular Anatomy, School of Medicine, University of the Ryukyus, Okinawa, Japan1 In the present study, we focused on the GABAergic role in neuronal regeneration in adult mice. The damage of facial nerve caused varying degrees of facial muscle paralysis, sensory and autonomic nervous functional abnormality. To reveal how GABAergic signals take part in regeneration on facial motor neuron, we performed facial nerve transection injury. Under deeply anesthesia, the right facial nerves were cut completely transected and sutured end-to-end. To examine the time-course of degeneration and regeneration of facial motor neurons and their axons, we performed Nissl staining and immunohistochemistry for choline acetyltransferase (ChAT) using frozen sections of pons. After axons were sutured, no significant differences in the number and shape of large motor neurons were detected between sutured and intact side. Facial motor neurons of intact side continued to be occupied by ChAT-positive neurons after operation. In contrast, immunolabeling of ChAT in large motor neurons decreased in intensity on Day 14 in the sutured side. Next, to examine the changes in expression of GABAergic signaling, we performed immunohistochemistry for glutamic acid decarboxylase (GAD), GABA transporter 1 and 3 (GAT1 and GAT3), vesicular GABA tansporter (VGAT) and potassium sodium chloride co-transporter 2 (KCC2). We used GAD, GAT1and VGAT for detection of changes in presynapses and GAT3 was examined for the detection of the changes in the astroglia. And we could not detect any changes in their expression on the sutured side comparing the intact side. However, KCC2 as a marker for GABAergic inhibition, reduced in intensity at the sutured side on Day 7 and Day 14. These results suggested that there is no significant change of GABAergic network on facial motor neurons after transection. And the downregulation of KCC2 in the GABAergic postsynapses may contribute to switch the GABAergic role from inhibition to excitation by elevation of intracellular Cl-.	P3-1-95 Phosphacanが中枢神経損傷後の軸索再生阻害因子の本体である Phosphacan is the major inhibitor for axonal regeneration after injury ○坂元一真1, 門松健治1 ○Kazuma Sakamoto1, Kenji Kadomatsu1 名古屋大学大学院　医学系研究科　生物化学1 Dept Biochem, Nagoya University, Nagoya1 Proteoglycans (PGs) are glycoproteins in which unique sugar chains called as glycosaminoglycans (GAGs) covalently attach to their core proteins. PGs are components of extracellular matrix (ECM) in our central nervous system (CNS). However, once the CNS is damaged, overproduced PGs act as inhibitors for axonal regeneration. It has been well known that inhibitory activity of PG depends on chondroitin sulfate (CS) chain on PGs. CS is reported to be recognized by neuronal receptors such as PTPσ and LAR, but the detailed mechanisms are still unclear. In a series of our study, we had asked whether keratan sulfate (KS), another class of GAGs, also works as an inhibitor for axonal regeneration. Here, we show that KS also inhibits axonal regeneration after spinal cord injury (SCI). Null mouse for GlcNAc6ST-1, an essential enzyme for KS biosynthesis, showed better functional recovery after SCI compared to wild type mouse. Moreover, infusion of Keratanase II, a degrading enzyme for KS, promotes axonal regeneration and functional recovery in rat model of SCI. PGs which contain KS strongly inhibit neurite outgrowth from cerebellar granule neurons in a KS-dependent manner in vitro. Surprisingly, KS and CS seemed to work in the same axis both in vitro and in vivo. Thus, we hypothesized that a KS/CS-chimeric PG which has both KS and CS simultaneously is a major inhibitor for axonal regeneration. In good accordance with this idea, KS/CS-chimeric PG is significantly up-regulated in primary cultured reactive astrocytes and inhibits neurite outgrowth in both KS and CS dependent manners. Using proteomics technique and RNA interference method, we had found out that phosphacan was responsible for this phenomena. Phosphacan was modified with bot KS and CS, and recombinant phosphacan expressed in COS-1 cells inhibits neurite outgrowth in both KS and CS dependent manners. Taken together, our findings will provide new structural basis of PG-mediated inhibition of axonal regeneration.
P3-1-96 骨髄間質細胞の培養メディウムによる脊髄損傷ラットに対する行動回復効果 Effects of the conditioned medium of cultured BMSCs on the locomotor recovery in spinal cord injured rat ○本間玲実1, 中野法彦1, 井出千束1 ○Tamami Homma1, Norihiko Nakano1, Chizuka Ide1 藍野大学　医療保健学部・再生医療研究所1 Institute of Regeneration and Rehabilitation, Faculty of Nursing and Rehabilitation, Aino Univ. Osaka, Japan1 Our previous study showed that the transplantation of bone marrow stromal cells (BMSCs) into the the cerebrospinal fluid (CSF) had beneficial effects on locomotor improvement as well as on tissue repair, including axonal regeneration, in the spinal cord injury (SCI) rats. This study suggested that BMSCs might secret some neurotrophic factors into the CSF. To elucidate this hypothesis, the conditioned medium (CM) of cultured BMSCs was administrated intracerebroventricularly or intravenously in SCI rats. CM was applied intracerebroventricularly three times everyday for two weeks, beginning immediately after SCI (n=4), or intravenously beginning at 1 week following SCI (n=4). The spinal cord was contused by dropping a weight at the thoracic 8-9 level. Injection of Dulbecco's modified Eagle's medium (DMEM) alone was used as control (n=4). The functional recovery was evaluated by the Basso, Beattie and Bresnahan (BBB) scale. BBB test was performed for 4 weeks before the SCI, and every week after CM administration. In the intracerebroventricular administration group, BBB score was significantly higher in CM than in DMEM rats 4 weeks after the administration. However, there was no significant difference in BBB score between CM and DMEM rats in the vascular administration group. These results show that the intravenous administration of CM of BMSCs has benefical effects on locomotor recovery following SCI in rat, and support the hypothesis that BMSCs secret some trophic factors beneficial for locomotor improvement into the CSF.	P3-1-97 末梢知覚神経傷害時におけるsyndecan-1ヘパラン硫酸プロテオグリカンの発現 Expression and distribution of syndecan-1 heparan sulfate proteoglycan in injured sensory neurons ○村上公一1, 吉田成孝1 ○Koichi Murakami1, Shigetaka Yoshida1 旭川医科大学　解剖学講座　機能形態学分野1 Department of Functional Anatomy and Neuroscience, Asahikawa Medical University, Asahikawa1 Heparan sulfate proteoglycans, which bear long chains of heparan sulfate glycosaminoglycan, play significant roles during embryogenesis, including the formation of the CNS. However, their involvement in nerve inuries and regeneration have not yet been clarified. Previously, we reported that the injuries of cranial motor nerves, sciatic nerves and infraorbital nerves induce the expression of syndecan-1 heparan sulfate proteoglycan in the injured neurons. In this study, we examined the expression and distribution of syndecan-1 in sciatic nerve injuries. Sciatic nerve injuries induced the expression of syndecan-1 in dorsal root ganglion (DRG) neurons, but not in injured spinal motor neurons. DRG neurons expressing syndecan-1 were smaller in size, and syndecan-1 was localized with CGRP and isolectin B4, markers for C-fibers, in the injured nerve fibers. Syndecan-1 was also distributed in lamina II of the dorsal horn of the spinal cord and localized with presynaptic structures. These results suggest that syndecan-1 is specifically expressed in the injured C-fibers in the sensory nervous systems, and syndecan-1 transported to the presynaptic structures may be related to synaptic plasticity after sciatic nerve injuries.

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