Published papers

The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, . ffLuc-cp156, which consists of a yellow variant of . Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic . ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies. © 2012 Elsevier Inc.

Study Design: Retrospective case series of chronic atlantoaxial rotatory fixation (AARF) treated by a novel closed reduction method (remodeling therapy) and review of the literature. Objective: To describe clinical features of chronic AARF and the rationale of the remodeling therapy. Summary of Background Data: Treatment strategy for chronic recurrent AARF remains controversial. Posterior C1-C2 arthrodesis has been widely used for recurrent unstable AARFs after the closed reduction, and a variety of posterior fusion techniques have been advocated. In contrast, several investigators reported a chronic AARF case treated by a simple traction or closed reduction followed by cervical immobilization. Previously, we have found that a deformity of the superior C2 facet joint (C2 facet deformity) on three dimensional computed tomography (3D CT) reconstructions is a risk factor for recurrent subluxation. In addition, the remodeling of this C2 facet deformity by careful closed manipulation followed by halo fixation (remodeling therapy) could prevent the recurrence of subluxation. Methods: Twelve children with chronic AARF who sustained torticollis for an average of 4.4 months after the onset were referred to our clinic. The mean age at the initial visit was 7.8 years. All patients underwent the remodeling therapy as reported previously. Radiographic findings and clinical courses were retrospectively reviewed. Treatment methodology, pearls, and pitfalls of the remodeling therapy were discussed with review of the literature. Results: 3D CT images before reduction revealed persistent atlantoaxial subluxation and the C2 facet deformity in the dislocated side in all cases. Follow-up 3D CT scans demonstrated the remodeling of the C2 facet deformity at an average of 2.4 months after successful reduction of subluxation. Neither recurrence of symptoms nor subluxation was observed at a mean follow-up of 42 months in any case. The cervical range of motion was 73.9° at the dislocated and 83.5° at the contra-dislocated side from the midline. Conclusion: Chronic irreducible and recurrent unstable AARF should be initially managed by remodeling therapy using the facet deformity sign as a clinical index, if the C1 and C2 have not been osseously fused. Copyright © 2012 Lippincott Williams & Wilkins.

PURPOSE:: To describe patients with cervical myelopathy due to cervical disc herniation (CDH) who showed spontaneous regression, we conducted a retrospective study. METHODS:: A review of clinical records since 1990 identified 19 patients (12 male and 7 female; mean age, 42.4 y) with mild cervical myelopathy due to CDH who exhibited spontaneous CDH regression after conservative treatment. RESULTS:: The mean Japanese Orthopaedic Association scores were 13.7±1.4 before conservative treatment, 14.8±1.2 at 3 months, and 15.9±0.7 at final follow-up. On magnetic resonance imaging (MRI), the CDH regression was observed at 7.5 months on average. Intramedullary high-signal intensity was observed in 15 patients (78.9%) before treatment and 9 patients (47.4%) at follow-up. The mean area of the spinal cord was 58.4±8.3 mm before treatment and 62.4±9.7 mm at follow-up. CONCLUSIONS:: Spontaneous regression of the CDH in 19 patients with mild cervical myelopathy was observed, with improvements in neurological status and in intramedullary high-signal intensity on MRI. © 2012 by Lippincott Williams & Wilkins.

Background: Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice.Methods: MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-γ and tumor necrosis factor-α) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-γ and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia.Results: LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-γ-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior.Conclusion: The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages. © 2012 Guerrero et al; licensee BioMed Central Ltd.

Once the safety issue has been overcome, induced pluripotent stem cells (iPSCs), which do not entail ethical or immunological concerns, may become the preferred cell source for regenerative medicine. Various types of iPSCs have been established by different methods, and each type exhibits different biological properties. Before iPSC-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. Recently, we demonstrated the directed neural differentiation of mouse iPSCs and examined their therapeutic potential in a mouse spinal cord injury (SCI) model. Mouse iPSC-derived neural stem/progenitor cells (NS/PCs), which had been pre-evaluated as non-tumorigenic by their transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-scid) mouse brain, were transplanted into the spinal cord 9 days after SCI. Mouse iPSC-derived NS/PCs differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in re-myelination and induced the axonal re-growth, promoting motor functional recovery. Nevertheless, our results constitute only the first step toward clinical application. The safety and effectiveness of human iPSC-derived NS/PCs need to be more intensively investigated in future preclinical studies, for example, using non-human primate SCI models. In particular, human iPSCs established by delivering reprogramming factors using a safer method than retrovirus system, such as an integration-free virus system, virus-free system, or transgene-free system should be evaluated.

Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call "Schwann-spheres." The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells in vitro. Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these "Schwann-spheres" would provide a more potential autologous cell source for such transplantation. © 2011 Takagi et al.

Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively. © 2011 Nakamura et al.

Abstract. Background: The olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos. Results: Examination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa. Conclusion: Our data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration. © 2011Katoh et al; licensee BioMed Central Ltd.

Transplantation of human neural stem/progenitor cells (hNSPCs) is a promising method to regenerate tissue from damage and recover function in various neurological diseases including brain ischemia. Galectin-1(Gal1) is a lectin that is expressed in damaged brain areas after ischemia. Here, we characterized the detailed Gal1 expression pattern in an animal model of brain ischemia. After brain ischemia, Gal1 was expressed in reactive astrocytes within and around the infarcted region, and its expression diminished over time. Previously, we showed that infusion of human Gal1 protein (hGal1) resulted in functional recovery after brain ischemia but failed to reduce the volume of the ischemic region. This prompted us to examine whether the combination of hNSPCs-transplantation and stable delivery of hGal1 around the ischemic region could reduce the ischemic volume and promote better functional recovery after brain ischemia. In this study, we transplanted hNSPCs that stably overexpressed hGal1 (hGal1-hNSPCs) in a model of unilateral focal brain ischemia using Mongolian gerbils. Indeed, we found that transplantation of hGal1-hNSPCs both reduced the ischemic volume and improved deficits in motor function after brain ischemia to a greater extent than the transplantation of hNSPCs alone. This study provides evidence for a potential application of hGal1 with hNSPCs-transplantation in the treatment of brain ischemia. © 2011Yamane et al; licensee BioMed Central Ltd.

To investigate potential cures for spinal cord injury (SCI), several researchers have transplanted neural stem/progenitor cells (NS/PCs) into the injured spinal cord by different procedures, including intralesional (IL), intrathecal (IT), and intravenous (IV) injection. However, there are no reports quantifying or comparing the number of cells successfully transplanted to the lesion site by each procedure in vivo. The purpose of the present study was to determine the optimal method of cell transplantation to the SCI site in terms of grafted cell survival and safety. For this purpose, we developed mouse NS/PCs that expressed a novel Venus-luciferase fusion protein that enabled us to detect a minimum of 1,000 grafted cells in vivo by biolumines-cence imaging (BLI). After inducing contusive SCI at the T10 level in mice, NS/PCs were transplanted into the injured animals three different ways: by IL, IT, or IV injection. Six weeks after the transplantation, BLI analysis showed that in the IL group, the luminescence intensity of the grafted cells had decreased to about 10% of its initial level, and appeared at the site of injury. In the IT group, the luminescence of the grafted cells, which was distributed throughout the entire subarachnoid space immediately after transplantation, was detected at the injured site 1 week later, and by 6 weeks had gradually decreased to about 0.3% of its initial level. In the IV group, no grafted cells were detected at the site of injury, but all of these mice showed luminescence in the bilateral chest, suggesting pulmonary embolism. In addition, one third of these mice died immediately after the IV injection. In terms of grafted cell survival and safety, we conclude that the IL application of NS/PCs is the most effective and feasible method for transplanting NS/PCs into the SCI site.

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. © 2011 Munehisa Shinozaki et al.

Although minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) has widely been developed in patients with lumbar diseases, surgeons risk exposure to fluoroscopic radiation. However, to date, there is no studies quantifying the effective dose during MIS-TLIF procedure, and the radiation dose distribution is still unclear. In this study, the surgeons' radiation doses at 5 places on the bodies were measured and the effective doses were assessed during 31 consecutive 1- to 3-level MIS-TLIF surgeries. The operating surgeon, assisting surgeon, and radiological technologist wore thermoluminescent dosimeter on the unshielded thyroid, chest, genitals, right middle finger, and on the chest beneath a lead apron. The doses at the lens and the effective doses were also calculated. Mean fluoroscopy times were 38.7, 53.1, and 58.5 seconds for 1, 2, or 3 fusion levels, respectively. The operating surgeon's mean exposures at the lens, thyroid, chest, genitals, finger, and the chest beneath the shield, respectively, were 0.07, 0.07, 0.09, 0.14, 0.32, and 0.05 mSv in 1-level MIS-TLIF; 0.07, 0.08, 0.09, 0.18, 0.34, and 0.05 mSv in 2-level; 0.08, 0.09, 0.14, 0.15, 0.36, and 0.06 mSv in 3-level; and 0.07, 0.08, 0.10, 0.15, 0.33, and 0.05 mSv in all cases. Mean dose at the operating surgeon's right finger was significantly higher than other measurements parts (P,0.001). The operating surgeon's effective doses (0.06, 0.06, and 0.07 mSv for 1, 2, and 3 fusion levels) were low, and didn't differ significantly from those of the assisting surgeon or radiological technologist. Revision MIS-TLIF was not associated with higher surgeons' radiation doses compared to primary MIS-TLIF. There were significantly higher surgeons' radiation doses in over-weight than in normal-weight patients. The surgeons' radiation exposure during MIS-TLIF was within the safe level by the International Commission on Radiological Protection's guidelines. The accumulated radiation exposure, especially to surgeon's hands, should be carefully monitored. © 2014 Funao et al.

Background: Rats 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. Results: Treatment 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). Conclusions: This 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. © 2014 Zhang et al.; licensee BioMed Central Ltd.

Background: Cervical compressive myelopathy (CCM) is caused by chronic spinal cord compression due to spondylosis, a degenerative disc disease, and ossification of the ligaments. Tip-toe walking Yoshimura (twy) mice are reported to be an ideal animal model for CCM-related neuronal dysfunction, because they develop spontaneous spinal cord compression without any artificial manipulation. Previous histological studies showed that neurons are lost due to apoptosis in CCM, but the mechanism underlying this neurodegeneration was not fully elucidated. The purpose of this study was to investigate the pathophysiology of CCM by evaluating the global gene expression of the compressed spinal cord and comparing the transcriptome analysis with the physical and histological findings in twy mice. Methods: Twenty-week-old twy mice were divided into two groups according to the magnetic resonance imaging (MRI) findings: a severe compression (S) group and a mild compression (M) group. The transcriptome was analyzed by microarray and RT-PCR. The cellular pathophysiology was examined by immunohistological analysis and immuno-electron microscopy. Motor function was assessed by Rotarod treadmill latency and stride-length tests. Results: Severe cervical calcification caused spinal canal stenosis and low functional capacity in twy mice. The microarray analysis revealed 215 genes that showed significantly different expression levels between the S and the M groups. Pathway analysis revealed that genes expressed at higher levels in the S group were enriched for terms related to the regulation of inflammation in the compressed spinal cord. M1 macrophage-dominant inflammation was present in the S group, and cysteine-rich protein 61 (Cyr61), an inducer of M1 macrophages, was markedly upregulated in these spinal cords. Furthermore, C1q, which initiates the classical complement cascade, was more upregulated in the S group than in the M group. The confocal and electron microscopy observations indicated that classically activated microglia/macrophages had migrated to the compressed spinal cord and eliminated synaptic terminals. Conclusions: We revealed the detailed pathophysiology of the inflammatory response in an animal model of chronic spinal cord compression. Our findings suggest that complement-mediated synapse elimination is a central mechanism underlying the neurodegeneration in CCM. © 2014 Takano et al.; licensee BioMed Central Ltd.

Secondary degeneration after spinal cord injury (SCI) is caused by increased vascular permeability, infiltration of inflammatory cells, and subsequent focal edema. Therapeutic interventions using neurotrophic factors have focused on the prevention of such reactions to reduce cell death and promote tissue regeneration. Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor. However, the effect of VEGF on SCI remains controversial. VEGF signaling is primarily regulated through two primary receptors, VEGF receptor 1 (VEGF-R1) and VEGF receptor 2 (VEGF-R2). The purpose of this study was to examine the effects of intraperitoneal administration of VEGF-R1- and VEGF-R2-neutralizing antibodies on a mouse model of SCI. VEGF-R1 blockade, but not VEGF-R2 blockade, decreased the permeability and infiltration of inflammatory cells, and VEGF-R2 blockade caused a significant increase in neuronal apoptosis in the acute phase of SCI. VEGF-R2 blockade decreased the residual tissue area and the number of neural fibers in the chronic phase of SCI. VEGF-R2 blockade worsened the functional recovery and prolonged the latency of motor evoked potentials. These data suggest that endogenous VEGF-R2 plays a crucial role in neuronal protection after SCI. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.

In the research for the treatment of spinal cord injury (SCI), the evaluation of motor function in model rats must be as objective, noninvasive, and ethical as possible. The maximum speed and acceleration of a mouse measured using a SCANET system were previously reported to vary significantly according to severity of SCI. In the present study, the motor performance of SCI model rats was examined with SCANET and assessed for Basso-Beattie-Bresnahan (BBB) score to determine the usefulness of the SCANET system in evaluating functional recovery after SCI. Maximum speed and acceleration within the measurement period correlated significantly with BBB scores. Furthermore, among several phased kinematic factors used in BBB scores, the capability of "plantar stepping" was associated with a drastic increase in maximum speed and acceleration after SCI. Therefore, evaluation of maximum speed and acceleration using a SCANET system is a useful method for rat models of SCI and can complement open field scoring scales. © 2013 by The Japan Neurosurgical Society.

This paper describes human induced pluripotent stem (iPS) cell-derived neural stem cell (hiPSC-NSC) bundles covered with growth factor-encapsulated amphiphilic chitosan. The hiPSC-NSC bundle is formed by 4-12 hiPSC-NSC microfibers that are made by core-shell hydrogel microfibers encapsulating hiPSC-NSCs. The hiPSC-NSC microfibers can be cultured for over two weeks and induced to differentiate into neurons predominantly. We modified the coating material to make bundle structures that have growth factor enrichment capability (Figure 1). The coating material, amphiphilic chitosan, can release growth factors slowly for a long time, allowing hiPSC-NSCs to survive, grow and differentiate into neural lineages in the bundle structure. We believe that our hiPSC-NSC bundle would be an extremely effective tool for neural transplantation in neurodegenerative diseases such as stroke, spinal cord injury and peripheral nerve injury, because hiPSC-NSCs in the bundle structure have long-term survival ability in vivo.

Extracellular factors that inhibit axon growth and intrinsic factors that promote it affect neural regeneration. Therapies targeting any single gene have not yet simultaneously optimized both types of factors. Chondroitin sulphate (CS), a glycosaminoglycan, is the most abundant extracellular inhibitor of axon growth. Here we show that mice carrying a gene knockout for CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, recover more completely from spinal cord injury than wild-type mice and even chondroitinase ABC-treated mice. Notably, synthesis of heparan sulphate (HS), a glycosaminoglycan promoting axonal growth, is also upregulated in TI knockout mice because HS-synthesis enzymes are induced in the mutant neurons. Moreover, chondroitinase ABC treatment never induces HS upregulation. Taken together, our results indicate that regulation of a single gene, T1, mediates excellent recovery from spinal cord injury by optimizing counteracting effectors of axon regeneration - an extracellular inhibitor of CS and intrinsic promoters, namely, HS-synthesis enzymes. © 2013 Macmillan Publishers Limited. All rights reserved.

Background: The transplantation of neural stem/progenitor cells (NS/PCs) at the sub-acute phase of spinal cord injury, but not at the chronic phase, can promote functional recovery. However, the reasons for this difference and whether it involves the survival and/or fate of grafted cells under these two conditions remain unclear. To address this question, NS/PC transplantation was performed after contusive spinal cord injury in adult mice at the sub-acute and chronic phases. Results: Quantitative analyses using bio-imaging, which can noninvasively detect surviving grafted cells in living animals, revealed no significant difference in the survival rate of grafted cells between the sub-acute and chronic transplantation groups. Additionally, immunohistology revealed no significant difference in the differentiation phenotypes of grafted cells between the two groups. Microarray analysis revealed no significant differences in the expression of genes encoding inflammatory cytokines or growth factors, which affect the survival and/or fate of grafted cells, in the injured spinal cord between the sub-acute and chronic phases. By contrast, the distribution of chronically grafted NS/PCs was restricted compared to NS/PCs grafted at the sub-acute phase because a more prominent glial scar located around the lesion epicenter enclosed the grafted cells. Furthermore, microarray and histological analysis revealed that the infiltration of macrophages, especially M2 macrophages, which have anti-inflammatory role, was significantly higher at the sub-acute phase than the chronic phase. Ultimately, NS/PCs that were transplanted in the sub-acute phase, but not the chronic phase, promoted functional recovery compared with the vehicle control group. Conclusions: The extent of glial scar formation and the characteristics of inflammation is the most remarkable difference in the injured spinal cord microenvironment between the sub-acute and chronic phases. To achieve functional recovery by NS/PC transplantation in cases at the chronic phase, modification of the microenvironment of the injured spinal cord focusing on glial scar formation and inflammatory phenotype should be considered. © 2013 Nishimura et al.; licensee BioMed Central Ltd.