Syoichi Tashiro, Masaya Nakamura, Hideyuki Okano

1 Department of Rehabilitation Medicine, Keio University School of Medicine, Tokyo, Japan

2 Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan

3 Department of Physiology, Keio University School of Medicine, Tokyo, Japan

The prospects of regenerative medicine combined with rehabilitative approaches for chronic spinal cord injury animal models

Syoichi Tashiro1, Masaya Nakamura2, Hideyuki Okano3,＊

1 Department of Rehabilitation Medicine, Keio University School of Medicine, Tokyo, Japan

2 Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan

3 Department of Physiology, Keio University School of Medicine, Tokyo, Japan

How to cite this article:Tashiro S, Nakamura M, Okano H (2017)e prospects of regenerative medicine combined with rehabilitative approaches for chronic spinal cord injury animal models. Neural Regen Res 12(1):43-46.

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Regenerative medicine has opened a window for functional recovery in acute‐to‐subacute phase spinal cord injury (SCI). By contrast, there are still only a few studies have focused on the treatment of the chron‐ically injured spinal cord, in which cell‐based regenerative medicine seems less e ff ective. Since the majority of SCI patients are in the chronic phase, representing a major challenge for the clinical application of cell‐based regenerative medicine. Although combined therapies for the treatment of chronic SCI have attracted attention of researchers and its potential importance is also widely recognized, there had been very few studies involving rehabilitative treatments to date. In a recent study, we have demonstrated for the fi rst time that treadmill training combined with cell transplantation signi fi cantly promotes functional recovery even in chronic SCI, not only in additive but also in synergistic manner. Even though we have succeeded to out‐line the pro fi les of recovery secondary to the combination therapy, the mechanism underlying the e ff ects remain unsolved. In this review article, we summarize the present progress and consider the prospect of the cell‐based regenerative medicine particularly combined with rehabilitative approaches for chronic SCI animal models.

transplantation; spinal cord injury; regenerative medicine; chronic phase; rehabilitation; treadmill training

Accepted: 2017-01-14

Introduction

Rapid progress in stem cell medicine is being realized in neural regeneration in various disease and injury animal models. With regard to spinal cord injury (SCI), researchers report remarkable recovery from sequelae such as motor paresis and spasticity, sensory disturbances, and urinary dys‐function with a variety of cell sources, like neural stem/pro‐genitor cells (NS/PCs) and mesenchymal stem cells (MSCs), especially in acute‐to‐subacute‐phase animals. By contrast, although there are more than 50‐fold more SCI patients in the chronic phase than in the acute‐to‐subacute phase, only a few studies have focused on the treatment of the chronical‐ly injured spinal cord, in which cell regenerative medicine seems less effective (Shinozaki et al., 2016). In this review, we summarize our recent study on combination therapy for chronic SCI using transplantation of NS/PCs as well as treadmill training (TMT) (Tashiro et al., 2016) and consider the prospect of combining regenerative medicine with other treatment approaches.

Early, Subacute, and Chronic Phases of SCI

The drastic microenvironmental transition loosely defines the time‐phases of injured spinal cords. In the “hyper‐acute phase”, which is before 1 day post‐injury (DPI) in the rodent model, there are still little inflammatory changes induced. Although it seems not practically treated in the clinics, most of experimental studies demonstrate good therapeutic e ff ect of cell replacement therapy.e consecutive “acute phase” is characterized as the in fl ammatory reactions by the up‐regu‐lation of various cytokines, harmful neurotransmitters, free radical and nitric oxide, those are mediated by the haem‐orrhage, the destruction of blood‐brain barrier (BBB) and the infiltration of inflammatory cells. Although neuronal plasticity may be potentially resided, those harmful factorsinterfere the e ff ect of regenerative therapy around 2–4 DPI. It is followed by the “sub‐acute phase”, in which the glial scarring is not yet progressed and the neuronal plasticity is still retained, besides the in fl ammation is reduced, shaping a time window with good therapeutic response around 7–14 DPI. However, as the glial scar and the cystic cavity formed around the lesion, it gradually transits to a “chronic phase”by 28–42 DPI (Nakamura and Okano, 2013). Although the spontaneous recovery is dried up during this transitional phase called “l(fā)ate‐subacute/early‐chronic phase”, the plastic response to treatments is still preserved to some degree. After this phase, the injured spinal cord becomes literally refractory to various therapeutic approaches including cell transplantation in the de fi nite “chronic phase” later than 42 DPI (Tashiro, 2016). Although few researchers have reported di ff erent characteristics of “delayed‐chronic phase” later than 90 DPI, this is still not clarified sufficiently (Kumamaru et al., 2013).

The refractory state of the chronically injured spinal cord is characterized as a microenvironmental change includ‐ing glial scar formation around the epicentre of the lesion, which interrupts the migration of grafted cells and results in lower regeneration of neural circuits and remyelination, the secretion of axonal growth inhibitors like semaphoring 3A that restricts the regenerative potency, and a decrease in the anti‐in fl ammatory M2 macrophages, but there is no re‐markable change in the expression of cytokines and growth factors that in fl uence the survival rate and the di ff erentiation of graed cells.e phase‐dependent transition of these fac‐tors a ff ects the therapeutic time window for transplantation therapy in SCI (Nishimura et al., 2013).

Among several studies focusing specifically on chronic SCI in rodents, only a few, in which transplantation was carried out at the relatively early stage of approximately 21 to 30 DPI, report signi fi cant recovery. By contrast, the rest of the studies, in which the intervention was administered aer 42 DPI, all conclude ine ff ectiveness of the intervention, as far as we know. Based on these previous studies, the time window seems to close by the end of the subacute phase to the early‐chronic phase, and then, during the de fi nite chron‐ic phase in the rodent model, the SCI becomes refractory to transplantation as a single therapy.is is therefore a major challenge for the clinical application of cell transplantation for most SCI patients in the chronic phase.

Combinationerapies toreat Chronically Injured Spinal Cord

On these grounds, combination therapies that include cell transplantation and administration of neurotrophic factors and medication have been investigated for chronic SCI. In particular, medicine‐based treatments have attracted at‐tention, as represented by the use of chondroitinase ABC (C‐ABC) infusion to degrade chondroitin sulfate proteo‐glycans, which are major constituents of the glial scar, and a semaphorin3A inhibitor to block a major axonal growth inhibitor, for which a silicon sheet preparation was recent‐ly developed for use as artificial dura mater (Zhang et al., 2014). Because the factors that interfere with recovery are quite complex, various preparations of medications can have remarkable superiority. So far, studies have revealed that a combination of C‐ABC and neurotrophic factors with NS/ PCs and C‐ABC with TMT can both induce signi fi cant loco‐motor recovery in rats with chronic SCI (Karimi‐Abdolrezaee et al., 2010; Shinozaki et al., 2016). Although it has not yet been investigated for chronic SCI, semaphorin3A inhibitors may also be promising. By contrast, there are few studies on combination therapies that involve rehabilitative intervention, and there were none on combination therapies involving cell transplantation in the chronic phase until our recent study.

Combinationerapy ofransplantation and Rehabilitation

Although some rehabilitative interventions are inappropriate due to the di ffi culty in employing them for motor‐impaired SCI animals, bipedal treadmill training (TMT) is a method that can be easily employed with bodyweight support, par‐ticularly in rats with SCI, in order to e ff ectively rehabilitate their paretic limbs (Tashiro et al., 2014). TMT has been used for rats with SCI to facilitate their functional recovery aer transplantation of a variety of cell sources, including NS/ PCs, MSCs and a combination of Schwann cells with olfac‐tory ensheathing cells. However, surprisingly, only a few ar‐ticles treating the acute‐to‐subacute SCI were available, nev‐ertheless, their potential importance as combination therapy candidates is widely recognised.

The reason why there are only a few numbers of studies is partially explained by the fact that the experimental animal species suitable for cell transplantation and rehabilitation are different. Generally, research on cell transplantation is per‐formed using mice because of their smaller size and amenabil‐ity to genetic manipulation, as well as the availability of biolu‐minescencein vivoimaging (BLI) for transplanted cells. On the other hand, rehabilitative intervention, like TMT, is far more established for rat models because of their calmer tempera‐ment, greater endurance and larger body size, which allows foreasier handling.us, the lack of an optimized animal model well‐suited for both training and cell transplantation may be a remarkable issue to be resolved in this area. We addressed this problem by employing a novel method introduced by Fong et al., namely partial body weight‐supported bipedal tread‐mill‐gait training for mice with SCI (Fong et al., 2005).

Figure 1 Behavioural manifestations in each group.

Challenges to a Combination of Rehabilitation and Cellerapy for Chronic SCI

Aiming for the clinical application of regenerative medicine for chronic SCI patients, we investigated the effect of com‐bination therapy with the transplantation of mouse NS/PCs and rehabilitation using 80 severely thoracic cord‐contused C57BL/6J mice in the chronic phase. A four‐armed design with groups composed of combination therapy (Tp‐RH), transplan‐tation single therapy (Tp), TMT‐rehabilitation single therapy (RH) and a control group (Control) was applied. TMT was used for two purposes during two di ff erent periods: 1 week of conditioning training was conducted for all animals from 42 DPI, and 8 weeks of intervention training was conducted for the RH and Tp‐RH groups aer the NS/PC vehicle injection. NS/PCs were transplanted at 49 DPI as neurospheres three passages after they were harvested. The cell source was the striata of E14 transgenic mice established from C57BL/6J that ubiquitously express ff Luc‐cp156, which is a fusion protein of a yellow variant of Aequorea GFP and fi re fl y luciferase, which enables the assessment of transplanted cell viability using BLI. In particular, no intervention was performed during the acute to “early‐chronic” phase in this study.

Figure 2 A scheme summarizing the mechanisms of locomotor recovery brought about by each intervention.

To outline the profiles of recovery secondary to the com‐bination therapy, we carried out cross‐sectional analyses on various aspects of the changes induced in each group. Al‐though no signi fi cant changes were observed in transplanted cell viability, residual spinal volume or spared fibres within the lesion epicentre in our investigations, the following new insights were gained. NS/PC transplantation both promotes the recovery in spinal conductivity and upregulates excitatory central pattern generator (CPG) activity, and TMT facilitates the suppressive regulation related to coordinated rhythmic motor control and the amelioration of spasticity, respectively. Although no signi fi cant locomotor recovery was detected in either of the Tp and RH single therapy groups, in comparison with the control group, the above‐mentioned results indicate that even each single therapy induced potentially beneficial changes in chronically injured spinal cord (Figure 1). Fur‐thermore, neuronal differentiation and the phenomenon of appropriate inhibition recovery and/or synaptic regeneration, which we have termed “CPG maturation”, were specifically promoted by the combination therapy. Overall, significant locomotor recovery was observed in the mice of the Tp‐RH group, compared with that in the control mice, due to the not only additive but also synergistic e ff ect of transplantation and rehabilitation. We have summarized the mechanisms in a scheme in the previous study (Figure 2). On the other hand, no significant difference was functionally detected between the Tp‐TMT and TMT groups in our study; thus, some other treatment is still required for the clinical application of cell transplantation in chronic SCI (Tashiro et al., 2016).

What is the Key to the Bene fi cial Changes of Rehabilitation-ransplantation Combinationerapy?

Although we have outlined the changes brought about by the interventions, the mechanisms behind them remain to be determined. Our current hypothesis is that neurotrophic factors and rehabilitation‐specific effects are contributing factors. Summarizing the previous reports, while combined infusion or genetic expression of neurotrophic factors facil‐itate the recovery induced by transplantation or TMT single therapy, both NS/PC transplantation and TMT also have thepotential to independently supply neurotrophic factors to the injured spinal cord (Houle and Cote, 2013; Kumamaru et al., 2013).erefore, such trophic support can enhance the intrinsic ability of these interventions to interact with one another and thereby induce locomotor recovery.

Next, the functional recovery secondary to TMT can be explained by following rehabilitation‐speci fi c e ff ects: 1) ac‐tivity‐dependent neuronal plasticity and modification and 2) the treatment of disuse and learned non‐use. Voluntary and/or instrumental training can promote both task‐speci fi c and use‐dependent neuronal plasticity and modification, involving the formation of new neuronal circuits, the rein‐forcement of locomotor networks in a more selective and stable manner, the strengthening of synaptic connectivity with long‐term structural change, the limiting of maladap‐tive plasticity, spinal fi xation through the peripheral sensory input and the reorganisation of the cortical network. Fur‐thermore, we hypothesize that chronic SCI animals are in a state of disuse and learned non‐use, triggering dysfunction and less recovery, which could further suppress the impaired hindlimb activity, thereby latently masking the beneficial effects of transplantation. Interestingly, in our study, loco‐motor function gradually improved in the transplantation single therapy group, in which animals were exposed to 1 week of conditioning training, which disagrees with previous reports on transplantation therapy for chronic SCI. Because even 1 week of TMT intervention stimulates the endogenous expression of BDNF, which further induces spinal learning (Gomez‐Pinilla et al., 2007), conditioning training as we in‐corporated it may be e ff ective in improving the behavioural states of SCI animals to some degree (Tashiro et al., 2016).

Perspectives on Regenerative Medicine in Combination with Variouserapeutic Modalities

Although there are almost no reports on the mechanisms underlying the changes induced by the combination therapy of TMT and cell transplantation in detail, such studies would be a major step in exploring more e ff ective approaches and preventing adverse effects. Therefore, further investigation into the neurotrophic factors, electrophysiological changes and neural plasticity is needed, as well as an examination of adverse e ff ects related to pain. In particular, pro fi ling the changes in the expression of neurotrophic factors will pro‐vide us with the most desirable composition of such factors to be used in a combination therapy.

In conclusion, we have demonstrated that rehabilitative treatments can be a third therapeutic option to facilitate locomotor recovery after NS/PC transplantation, even in chronic SCI. TMT promotes functional recovery not only additively but also synergistically with cell transplantation through neuronal differentiation and CPG maturation.erefore, a comprehensive regenerative approach, involving medication and rehabilitation as well as the cell replacement therapy, will have a remarkable e ff ect on the treatment of the chronically injured spinal cord, which is refractory to each of these interventions alone.

Acknowledgments:We appreciate the help of all the members of the spinal cord research team at the Department of Orthopaedic Surgery, Physiology, and Rehabilitation Medicine at Keio University’s School of Medicine.

Author contributions:ST, MN and HO wrote the manuscript; MN and HO edited the manuscript.

Con fl icts of interest:HO is a scienti fi c consultant for SanBio Co., Ltd (Japan), Eisai Co., Ltd (Japan), and Daiichi Sankyo Co., Ltd (Japan).

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Hideyuki Okano, M.D., Ph.D., hidokano@a2.keio.jp.

10.4103/1673-5374.198972

＊< class="emphasis_italic">Correspondence to: Hideyuki Okano, M.D., Ph.D., hidokano@a2.keio.jp.

orcid: 0000-0001-7482-5935 (Hideyuki Okano) 0000-0002-1430-0552 (Syoichi Tashiro)