Masahiko Mukaino

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. “Safe” iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as “unsafe” showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

Roles of ES Cell-Derived Gliogenic Neural Stem/ Progenitor Cells in Functional Recovery after Spinal Cord Injury

Transplantation of neural stem/progenitor cells (NS/PCs) following the sub-acute phase of spinal cord injury (SCI) has been shown to promote functional recovery in rodent models. However, the types of cells most effective for treating SCI have not been clarified. Taking advantage of our recently established neurosphere-based culture system of ES cell-derived NS/PCs, in which primary neurospheres (PNS) and passaged secondary neurospheres (SNS) exhibit neurogenic and gliogenic potentials, respectively, here we examined the distinct effects of transplanting neurogenic and gliogenic NS/PCs on the functional recovery of a mouse model of SCI. ES cell-derived PNS and SNS transplanted 9 days after contusive injury at the Th10 level exhibited neurogenic and gliogenic differentiation tendencies, respectively, similar to those seen in vitro. Interestingly, transplantation of the gliogenic SNS, but not the neurogenic PNS, promoted axonal growth, remyelination, and angiogenesis, and resulted in significant locomotor functional recovery after SCI. These findings suggest that gliogenic NS/PCs are effective for promoting the recovery from SCI, and provide essential insight into the mechanisms through which cellular transplantation leads to functional improvement after SCI.

Anti-IL-6-receptor antibody promotes repair of spinal cord injury by inducing microglia-dominant inflammation

We previously reported the beneficial effect of administering an anti-mouse IL-6 receptor antibody (MR16-1) immediately after spinal cord injury (SCI). The purpose of our present study was to clarify the mechanism underlying how MR16-1 improves motor function after SCI. Quantitative analyses of inflammatory cells using flow cytometry, and immunohistochemistry with bone marrow-chimeric mice generated by transplanting genetically marked purified hematopoietic stem cells, revealed that MR16-1 dramatically switched the central player in the post-traumatic inflammation, from hematogenous macrophages to resident microglia. This change was accompanied by alterations in the expression of relevant cytokines within the injured spinal cord; the expression of recruiting chemokines including CCL2, CCL5, and CXCL10 was decreased, while that of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF), a known mitogen for microglia, was increased. We also showed that the resident microglia expressed higher levels of phagocytic markers than the hematogenous macrophages. Consistent with these findings, we observed significantly decreased tissue damage and reduced levels of myelin debris and Nogo-A, the axonal growth inhibitor, by MR16-1 treatment. Moreover, we observed increased axonal regeneration and/or sprouting in the MR16-1-treated mice. Our findings indicate that the functional improvement elicited by MR16-1 involves microglial functions, and provide new insights into the role of IL-6 signaling in the pathology of SCI.

Beneficial compaction of spinal cord lesion by migrating astrocytes through glycogen synthase kinase-3 inhibition

The migratory response of astrocytes is essential for restricting inflammation and preserving tissue function after spinal cord injury (SCI), but the mechanisms involved are poorly understood. Here, we observed stimulation of in vitro astrocyte migration by the new potent glycogen synthase kinase‐3 (GSK‐3) inhibitor Ro3303544 and investigated the effect of Ro3303544 administration for 5 days following SCI in mice. This treatment resulted in accelerated migration of reactive astrocytes to sequester inflammatory cells that spared myelinated fibres and significantly promoted functional recovery. Moreover, the decreased extent of chondroitin sulphate proteoglycans and collagen IV demonstrated that scarring was reduced in Ro3303544‐treated mice. A variety of in vitro and in vivo experiments further suggested that GSK‐3 inhibition stimulated astrocyte migration by decreasing adhesive activity via reduced surface expression of β1‐integrin. Our results reveal a novel benefit of GSK‐3 inhibition for SCI and suggest that the stimulation of astrocyte migration is a feasible therapeutic strategy for traumatic injury in the central nervous system.

Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition

BackgroundRats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the “rewiring” of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment.ResultsTreatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles).ConclusionsThis study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

Regulation of RhoA by STAT3 coordinates glial scar formation

Understanding how the transcription factor signal transducer and activator of transcription–3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3+/− mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.

Novel concept of motor functional analysis for spinal cord injury in adult mice.

In basic research on spinal cord injury (SCI), behavioral evaluation of the SCI animal model is critical. However, it is difficult to accurately evaluate function in the mouse SCI model due to the small size of mice. Although the open-field scoring scale is an outstanding appraisal method, supplementary objective tests are required. Using a compact SCANET system, in which a mouse carries out free movement for 5 min, we developed a novel method to detect locomotor ability. A SCANET system samples the horizontal coordinates of a mouse every 0.1 s, and both the speed and acceleration of its motion are calculated at each moment. It was found that the maximum speed and acceleration of motion over 5 min varied by injury severity. Moreover, these values were significantly correlated with open-field scores. The maximum speed and acceleration of SCI model mice using a SCANET system are objective, easy to obtain, and reproducible for evaluating locomotive function.

After-effects of pedaling exercise on spinal excitability and spinal reciprocal inhibition in patients with chronic stroke.

Purpose of the study: To evaluate the after-effects of pedaling on spinal excitability and spinal reciprocal inhibition in patients with post-stroke spastic hemiparesis. Materials and methods: Twenty stroke patients with severe hemiparesis participated in this study and were instructed to perform 7 min of active pedaling and 7 min of passive pedaling with a recumbent ergometer at a comfortable speed. H reflexes and M waves of paretic soleus muscles were recorded at rest before, immediately after and 30 min after active and passive pedaling. The Hmax/Mmax ratio and H recruitment curve were measured. Reciprocal inhibition was assessed using the soleus H reflex conditioning test paradigm. Results: The Hmax/Mmax ratio was significantly decreased after active and passive pedaling exercise. The decreased Hmax/Mmax ratio after active pedaling lasted at least for 30 min. The H recruitment curve and reciprocal inhibition did not change significantly after active or passive pedaling exercise. Conclusions: Pedaling exercise decreased spinal excitability in patients with severe hemiparesis. Pedaling may be effective in rehabilitation following stroke.

S11-2. The effects of walking exercises on spinal cord injury

The aim of this study was to compare the functional effects of early and delayed interventions involving walking exercises with treadmills, using animal subjects. Contusive spinal cord injury (SCI) was made at the Th10 level in adult Sprague–Dawley rats, which were then assigned to one of the following conditions: control, early training (days 7 to 20), and delayed training (days 21 to 34). A significant increase in step height and step length was observed in the early-training group, while the delayed-training group showed no significant improvement. In addition, a reduction in synergic contraction was observed in electromyographic activity of the hind limb muscles, as well as a reduction in spastic muscle activities, but this was observed only in the early-training group. Immunohistochemical analysis of the lumbar spinal cord using synaptic markers showed a significant decrease in the inhibitory synapses and an increase in the excitatory synapses which were caused by an earlier start to training. Further studies are necessary to determine underlying mechanisms involved the above process.

Combining extensive treadmill training with a selective semaphorin3A inhibitor treatment enhances locomotor functional recovery by wiring regenerated axons in adult spinal cord-transected rats

s / Neuroscience Research 68S (2010) e223–e334 e259 P2-f06 Analysis of IP3 signaling for alpha-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) induced-nuclear Ca2+ increase and the appearance of nuclear granulation in hippocampal neurons Kyoko Ibaraki 1 , Seiji Yamamoto 1, Takashi Tsuboi 2, Susumu Terakawa 1 1 Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan 2 Laboratory of Cell Imaging, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan Under the video enhanced contrast-differential interference contrast microscope, continuous exposure to AMPA dose-dependently induced the nuclear granulation and the cellular swelling in hippocampal neurons. In this process, AMPA significantly increased the percentage of severer DNA damage after nuclear Ca2+ increase and the appearance of nuclear granulation. In this study, we investigated whether IP3 signaling is involved in the nuclear Ca2+ increase and the appearance of the nuclear granulation in hippocampal neuron. AMPA induced IP3 production in hippocampal neuron, since green fluorescent protein-tagged pleckstrin homology domain of PLC1 (GFP-PHD) was translocated from the plasmamembrane to the cytoplasm in response to increased concentration of IP3. A blocker of IP3-induced Ca2+ release, Xestospongin C (1 M), partially blocked the increase of Ca2+ concentration in the nucleus, however, it did not block the appearance of nuclear granulation. These results suggested that IP3-induced Ca2+ increase in the nucleus may not contribute to the induction of nuclear granulation although AMPA induced IP3 production in hippocampal neuron. doi:10.1016/j.neures.2010.07.1148 P2-f07 Apoptosis-inducing factor deficiency decreases the proliferation rate and protects the subventricular zone against ionizing radiation Yoshiaki Sato 1,2 , Kazuhiro Osato 1,3, Tomoyo Ochiishi 1,4, Akari Osato 1,3, Changlian Zhu 1,9, Machiko Sato 1,6, Janos Swanpalmer 5, Nazanine Modjtahedi 8, Guido Kroemer 8, Georg H. Kuhn 1, Klas Blomgren 1,7 1 Center for Brain Repair and Rehabilitation, University of Gothenburg, Gothenburg, Sweden 2 Maternity & Perinatal Care Center, Nagoya University Hospital, Nagoya, Japan 3 Department of Obstetrics and Gynecology, Miyazaki Medical College, University of Miyazaki, Kiyotake, Japan 4 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan 5 Department of Radiation Physics, Sahlgrenska University Hospital, Gothenburg, Sweden 6 Department of Obstetrics and Gynecology, Narita Hospital, Nagoya, Japan 7 Department of Pediat. Oncology, Queen Silvia Children’s Hospital, Gothenburg, Sweden 8 Inst. National de la Santé et de la Recherche Médicale, U848, Villejuif, France 9 Department of Pediat., Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China Cranial radiotherapy in children often leads to progressive cognitive decline. We have established a rodent model of irradiation-induced injury to the young brain. A single dose of 8 Gy was administered to the left hemisphere of postnatal day 10 (P10) mice. Harlequin (Hq) mice, carrying the hypomorphic apoptosis-inducing factor AIFHq mutation, express 60% less AIF at P10 and displayed significantly fewer dying cells in the subventricular zone (SVZ) 6 h after IR, compared with wild type (Wt) littermates. Irradiated cyclophilin A-deficient (CypA−/−) mice confirmed that CypA plays an essential role in AIF-induced apoptosis after IR. Hq mice displayed no reduction in SVZ size 7 days after IR, whereas 48% of the SVZ was lost in Wt mice. The proliferation rate was lower in the SVZ of Hq mice. Cultured neural precursor cells from the SVZ of Hq mice displayed a slower proliferation rate and were more resistant to IR. IR preferentially kills proliferating cells, and the slower proliferation rate in the SVZ of Hq mice may, at least partly, explain the protective effect of the Hq mutation. Together, these results indicate that targeting AIF may provide a fruitful strategy for protection of normal brain tissue against the detrimental side effects of IR. doi:10.1016/j.neures.2010.07.1149 P2-f08 Proteomic analysis of macrophages temporarily stimulated with albumin, oxidized or reduced galectin-1 Kazunori Yoshimura 1 , Fuyuki Kametani 2, Takashi Miyazaki 3, Hiromichi Suzuki 3, Yasushi Sakaoto 4, Mayumi Kato 1, Masami Nishina 5, Hidenori Horie 6, Toshihiko Kadoya 7 1 Dept Rehab, Nihon Inst Med Sci, Saitama, Japan 2 Tokyo Inst Psychia, Tokyo Metro Org Med Res, Tokyo, Japan 3 Commun Health Sci Ctr, Saitama Med Univ, Saitama, Japan 4 Bio Med Res Ctr, Saitama Med Univ, Saitama, Japan 5 Med Res Ctr, Saitama Med Univ, Saitama, Japan 6 Res Ctr Brain Oral Sci, Kanagawa Dent Col, Kanagawa, Japan 7 Dept Biotechnol, Facul Eng, Maebashi Inst Technol, Gunma, Japan Oxidized galectin-1 was discovered as a factor that regulates initial axonal growth in the peripheral nerve after axotomy (Horie et al., 1999; Inagaki et al., 2000). Galectin-1 is well known as a lectin which binds beta-galactoside when it is in a reduced form. Once galctin-1 has been oxidized, it exhibits marked peripheral nerve regeneration-promoting activity though it loses the lectin activity. Furthermore, we have demonstrated that the oxidized galectin-1 promotes the axonal regeneration via macrophages (Horie et al., 2004). At the present study, to explore the proteins secreted from macrophages and the intracellular proteins of macrophages stimulated with oxidized galectin-1 (Ox-Gal), we performed 2-dimensional electrophoresis (2-D) following Sypro Rubby and LC–MS/MS, and 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE) of macrophages treated with bovine serum albumin, Ox-Gal or galectin-1 mutant, in which all six cysteine residues were replaced by serine (CS-Gal), or with bovine serum albumin. As a result, 2-D showed that macrophages secreted several proteins by stimulation of Ox-Gal. Proteomic analysis including both 2D-DIGE and LC–MS/MS identified several proteins such as an arginase1 and GRP78 as the changed proteins after the treatment of Ox-Gal. Moreover, Real-Time RT-PCR analysis of the macrophages showed that different stimulants gave expression of different mRNAs. Therefore these results suggest that the signaling of Ox-Gal is different from that of reduced galectin-1. doi:10.1016/j.neures.2010.07.1150 P2-f09 Combining extensive treadmill training with a selective semaphorin3A inhibitor treatment enhances locomotor functional recovery by wiring regenerated axons in adult spinal cord-transected rats Liang Zhang 1,2,3 , Shinjiro Kaneko 3,6, Akihiko Sano 5, Miho Maeda 5, Akiyoshi Kishino 5, Masahiko Mukaino 1,4, Yoshiaki Toyama 3, Meigen Liu 1, Masaya Nakamura 3, Hideyuki Okano 2 1 Dept Rehabil Med, Univ Keio, Tokyo 2 Dept Physiol, Univ Keio, Tokyo 3 Dept Orthop, Univ Keio, Tokyo 4 Rehabil Center, Univ Keio, Tokyo 5 Dainippon Sumitomo Pharma Co., Ltd., Otsuka 6 Murayama Med Center, Tokyo It has been known that completely spinal cord-transected (SCT) adult rats exhibit extremely limited functional recovery.We previously reported the efficacy of semaphorin3A (Sema3A) inhibitor treatment in this model. One of the remaining questions is whether combining rehabilitation, such as treadmill training, with Sema3A inhibitor treatment can enhance the effect of Sema3A inhibitor. In this study we employed the combinatorial therapy of selective Sema3A inhibitor (SM-34543) and treadmill training for adult SCT rats to examine its combinatorial effect. For SM-345431 administration, we employed the newly developed drug delivery system to secure the continuous drug delivery during the period of the experiment. By anatomical and kinematic analysis we found that, being consistent with our previous study rats treated with Sema3A inhibitor showed significantly enhanced axonal regeneration, but limited functional recovery. Enhanced axonal regeneration was also observed in the combinatorial group, and interestingly, significantly enhanced locomotor functional recovery was observed in this combinatorial group (compared with Sema3A inhibitor alone group). Moreover, we found that this combinatorial intervention could recruit specific populations of spinal circuits, enhancing motor function presumably via increased sensory input and functional remodeling of locomotor pathways. Conceivably, although axonal regeneration is very important in spinal cord injury (SCI) treatment, it alone cannot result in substantial locomotor functional recovery. It is speculated that treadmill training might be able to recruit regenerated axons and make them orient to their target more accurately (wiring effect), as a result, lead to improved locomotor functional recovery after SCT. These results provide a new prospect for this combinatorial ther-