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

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 effective. 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 ifrst time that treadmill training combined with cell transplantation signiifcantly 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 proifles of recovery secondary to the combination therapy, the mechanism underlying the effects 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.     

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

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 chronically injured spinal cord,in which cell-based regenerative medicine seems less effective.Since the majority of SCI patients are in the chronic phase,representing a major challenge for the clinical application of cellbased 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 first time that treadmill training combined with cell transplantation significantly promotes functional recovery even in chronic SCI,not only in additive but also in synergistic manner.Even though we have succeeded to outline the profiles of recovery secondary to the combination therapy,the mechanism underlying the effects 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.     

Proliferation of parenchymal neural progenitors in response to injury in the adult rat spinal cord.

It has long been believed that the fully developed mammalian central nervous system (CNS) lacks significant regenerative capacity. Recent advances have revealed, however, that many regions of the adult CNS contain neural progenitors that have the ability to generate new neurons and glia. Although the periventricular area has been identified as a rich source of these progenitors, their precise location in each region and details of their properties in vivo still remain poorly understood. Here we provide evidence that in the adult rat spinal cord, a significant number of neural progenitors are present, not only in the periventricular area, but also in other regions of the parenchyma. These progenitors could proliferate in vitro as neurosphere-like cell aggregates in the presence of growth factors and also gave rise to neurons and glia under appropriate conditions. We further demonstrate that these parenchymal neural progenitors were capable of proliferating in vivo in response to injury. Immunohistochemical studies suggested that proliferative progenitors emerged throughout the gray and white matter in the lesioned spinal cord. Consistently, an increased number of neurosphere-forming cells could be isolated from injured tissues, and they were able to differentiate into neurons in vitro. The widespread occurrence of neural progenitors in the parenchyma expands the possibility of repairing damaged tissue by activating the latent regenerative potential of the adult spinal cord.     

Polyethylene glycol as a promising synthetic material for repair of spinal cord injury

Polyethylene glycol is a synthetic, biodegradable, and water-soluble polyether. Owing to its good biological and material properties, polyethylene glycol shows promise in spinal cord tissue engineering applications. Although studies have examined repairing spinal cord injury with polyethylene glycol, these compellingfindings have not been recently reviewed or evaluated as a whole. Thus, we herein review and summarize the findings of studies conducted both within and beyond China that have examined the repair of spinal cord injury using polyethylene glycol. The following summarizes the results of studies using polyethylene glycol alone as well as coupled with polymers or hydrogels: (1) polyethylene glycol as an adjustable bio-molecule carrier resists nerve fiber degeneration, reduces the inflammatory response, inhibits vacuole and scar formation, and protects nerve membranes in the acute stage of spinal cord injury. (2) Polyethylene glycol-coupled polymers not only promote angiogenesis but also carry drugs or bioactive molecules to the injury site. Because such polymers cross both the blood-spinal cord and blood-brain barriers, they have been widely used as drug carriers. (3) Polyethylene glycol hydrogels have been used as supporting sub-strates for the growth of stem cells after injury, inducing cell migration, proliferation, and differentiation. Simultaneously, polyethylene glycol hydrogels isolate or reduce local glial scar invasion, promote and guide axonal regeneration, cross the transplanted area, and re-establish synaptic connections with target tissue, thereby promoting spinal cord repair. On the basis of the reviewed studies, we conclude that polyethylene glycol is a promising synthetic material for use in the repair of spinal cord injury.     

Acrylic hydrogel implants after spinal cord lesion in the adult rat

Abstract

Acrylic hydrogels, like the polymer of 2-hydroxyethyl methacrylate, are biocompatible, mechanically stable, porous materials that can be coated with collagen or laminin acting as bioadhesive substrates. Poly- 2-hydroxyethyl methacrylate sponges have been proposed for restoring the anatomical continuity of damaged neural structures. In the present work, the ability of poly-2-hydroxyethylmethacrylate sponges to provide the injured spinal cord neurons with a conductive substrate for their regenerating axons was investigatedin 32 adultWistar rats. Collagen impregnated poly-2-hydroxyethylmethacrylatesponges were implanted into suction cavities of the dorsal funiculus of the spinal cord. Two to four months after implantation, the spinal cord was removed and processed for histology, and S100 and GFAP immunohistochemistry. To study axonal regeneration into the sponge, the spinal cord or the sensorimotor cortex were injected with 0.05–0.1 ml of an 8% solution of lectin-conjugated horseradish peroxidase or 10% dextran tetramethylrhodamine. The fibroglial reaction, accumulation of mononuclear cells, and angiogenesis at the interface between the spinal cord and the sponge were minimal. Cystic cavitation in the spinal cord was virtually absent. Anterograde labeled axons were seen to penetrate and to elongate the full length of the sponge. These results demonstrate that poly-2-hydroxyethylmethacrylate sponges represent a safe supportive material for regenerating spinal cord axons.     

Acrylic hydrogel implants after spinal cord lesion in the adult rat.

Acrylic hydrogels, like the polymer of 2-hydroxyethyl methacrylate, are biocompatible, mechanically stable, porous materials that can be coated with collagen or laminin acting as bioadhesive substrates. Poly-2-hydroxyethyl methacrylate sponges have been proposed for restoring the anatomical continuity of damaged neural structures. In the present work, the ability of poly-2-hydroxyethyl methacrylate sponges to provide the injured spinal cord neurons with a conductive substrate for their regenerating axons was investigated in 32 adult Wistar rats. Collagen impregnated poly-2-hydroxyethyl methacrylate sponges were implanted into suction cavities of the dorsal funiculus of the spinal cord. Two to four months after implantation, the spinal cord was removed and processed for histology, and S100 and GFAP immunohistochemistry. To study axonal regeneration into the sponge, the spinal cord or the sensorimotor cortex were injected with 0.05-0.1 microl of an 8% solution of lectin-conjugated horseradish peroxidase or 10% dextran tetramethylrhodamine. The fibroglial reaction, accumulation of mononuclear cells, and angiogenesis at the interface between the spinal cord and the sponge were minimal. Cystic cavitation in the spinal cord was virtually absent. Anterograde labeled axons were seen to penetrate and to elongate the full length of the sponge. These results demonstrate that poly-2-hydroxyethyl methacrylate sponges represent a safe supportive material for regenerating spinal cord axons.     

Spinal cord grafts: An intraocular approach to enigmas of nerve growth regulation

The possible usefulness of intraocular transplantation in studies of spinal cord growth and regeneration has been evaluated. Defined segments of fetal rat spinal cord were grafted to the anterior chamber of the eye of adult rats. Such grafts become vascularized from the host iris, grow and develop neuron types, myelinated fiber bundles, astroglial populations (as shown by GFA-immunoreactivity), and electrical activity reminiscent of such features in normal spinal cord tissue. The intraocular technique permits studies of intrinsic circuitries as well as conditions for formation of afferent and efferent connections with the host iris and with other central or peripheral tissues which can be grafted into contact with the spinal cord grafts. One example of an intrinsic system preserved in the grafts is a rich network of nerve fibers with enkephalin-like immunoreactivity. When combined with cerebral cortex, the enkephalin-positive neurons of the spinal cord graft are able to form only very limited projections to the cortex graft. Special emphasis was given the possible formation of adrenergic afferents to spinal cord grafts. No appreciable ingrowth of peripheral sympathetic nerves occurred. Locus coeruleus grafts have many organotypical electrophysiological characteristics and were able to innervate adjacent spinal cord grafts provided that the sensory innervation of the host iris was removed. Experiments such as these suggest that "negative neurotropic" factors may be present in spinal cord and possibly relate to the unique relationship between spinal ganglia and spinal cord.     

Advances in regenerative therapies for spinal cord injury: a biomaterials approach

Spinal cord injury results in the permanent loss of function, causing enormous personal, social and economic problems. Even though neural regeneration has been proven to be a natural mechanism, central nervous system repair mechanisms are ineffective due to the imbalance of the inhibitory and excitatory factors implicated in neuroregeneration. Therefore, there is growing research interest on discovering a novel therapeutic strategy for effective spinal cord injury repair. To this direction, cell-based delivery strategies, biomolecule delivery strategies as well as scaffold-based therapeutic strategies have been developed with a tendency to seek for the answer to a combinatorial approach of all the above. Here we review the recent advances on regenerative/neural engineering therapies for spinal cord injury, aiming at providing an insight to the most promising repair strategies, in order to facilitate future research conduction.     

Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts

Background

Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process.

Results

We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response.

Conclusions

These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for meningeal and glial cells in facilitating axon regeneration. Given that these cell types interact to form inhibitory barriers in mammals, identifying the mechanisms underlying their permissive behaviors in the newt will provide new insights for improving spinal cord regeneration in mammals.     

Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord

Olfactory ensheathing glia (OEG) are a specialized type of glia that guide primary olfactory axons from the neuroepithelium in the nasal cavity to the brain. The primary olfactory system is able to regenerate after a lesion and OEG contribute to this process by providing a growth-supportive environment for newly formed axons. In the spinal cord, axons are not able to restore connections after an injury. The effects of OEG transplants on the regeneration of the injured spinal cord have been studied for over a decade. To date, of all the studies using only OEG as a transplant, 41 showed positive effects, while 13 studies showed limited or no effects. There are several contradictory reports on the migratory and axon growth-supporting properties of transplanted OEG. Hence, the regenerative potential of OEG has become the subject of intense discussion. In this review, we first provide an overview of the molecular and cellular characteristics of OEG in their natural environment, the primary olfactory nervous system. Second, their potential to stimulate regeneration in the injured spinal cord is discussed. OEG influence scar formation by their ability to interact with astrocytes, they are able to remyelinate axons and promote angiogenesis. The ability of OEG to interact with scar tissue cells is an important difference with Schwann cells and may be a unique characteristic of OEG. Because of these effects after transplantation and because of their role in primary olfactory system regeneration, the OEG can be considered as a source of neuroregeneration-promoting molecules. To identify these molecules, more insight into the molecular biology of OEG is required. We believe that genome-wide gene expression studies of OEG in their native environment, in culture and after transplantation will ultimately reveal unique combinations of molecules involved in the regeneration-promoting potential of OEG.     

Current status of cell-mediated regenerative therapies for human spinal cord injury

During the past decade, significant advances have been made in refinements for regenerative therapies following human spinal cord injury （SCI）. Positive results have been achieved with different types of cells in various clinical studies of SCI. In this review, we summarize recently-completed clinical trials using cell- mediated regenerative therapies for human SCI, together with ongoing trials using neural stem cells. Specifically, clinical studies published in Chinese journals are included. These studies show that current transplantation therapies are relatively safe, and have provided varying degrees of neurological recovery. However, many obstacles exist, hindering the introduction of a specific clinical therapy, including complications and their causes, selection of the target population, and optimization of transplantation material. Despite these and other challenges, with the collaboration of research groups and strong support from various organizations, cell-mediated regenerative therapies will open new perspectives for SCI treatment.     

Role and prospects of regenerative biomaterials in the repair of spinal cord injury

Axonal junction defects and an inhibitory environment after spinal cord injury seriously hinder the regeneration of damaged tissues and neuronal functions. At the site of spinal cord injury, regenerative biomaterials can fill cavities, deliver curative drugs, and provide adsorption sites for transplanted or host cells. Some regenerative biomaterials can also inhibit apoptosis, inflammation and glial scar formation, or further promote neurogenesis, axonal growth and angiogenesis. This review summarized a variety of biomaterial scaffolds made of natural, synthetic, and combined materials applied to spinal cord injury repair. Although these biomaterial scaffolds have shown a certain therapeutic effect in spinal cord injury repair, there are still many problems to be resolved, such as product standards and material safety and effectiveness.     

Viability and differentiation of neural precursors on hyaluronic acid hydrogel scaffold

The traditional notion that injured neurons are unable to regenerate in the adult mammalian brain and spinal cord has long been a concern. This view has led to methodology designed to overcome this problem, most recently by advancements in tissue engineering. Here, neural precursor cells (NPCs) and the Nogo receptor antibody (NgR‐Ab) or poly‐L‐lysine (PLL) were tested in concert with hyaluronic acid hydrogel scaffolds (HA). In particular, we wished to optimize viability and differentiation of NPCs within HA hydrogel scaffolds. Our results show that HA hydrogels can be modified physically or chemically to improve NPCs attachment on the scaffolding doped with NgR‐Ab or PLL. Both the HA hydrogels and their modifications support the viability of NPCs. NPCs were also able to differentiate into neurons and glial cells on HA hydrogels, although this was affected by the different modifications. Immunofluorescence showed that fewer β‐III‐tubulin antibody and antineurofilament antibody‐positive cells were found on HA‐PLL hydrogel compared with HA or HA NgR‐Ab hydrogels. This indicates that the PLL‐modified HA hydrogels may inhibit differentiation of NPCs, whereas modification by NgR‐Ab had no such effect. Finally, the NgR‐Ab‐modified HA scaffold can be used as not only a NPC delivery system but also a bioactive factor transportation system for CNS repair. © 2009 Wiley‐Liss, Inc.     

Ultrastructure of spinal cord grafts with and without cografts of locus coeruleus in oculo

The ultrastructure of spinal cord and spinal cord-locus coeruleus double grafts transplanted to the anterior chamber of the eye of adult rats was studied. The present results show that single spinal cord grafts express several morphologically organotypical characteristics of normal spinal cord at the ultrastructural level. Thus, the parenchyma was divided into a cell-rich layer corresponding to the normal gray matter and an axonal layer with a large amount of myelinated fibers and immature oligodendrocytes. In the cellular layer, a variety of cell types of different sizes were observed. The neurons had different patterns of cytoplasmic organelles, including Nissl bodies, Golgi apparatus, mitochondria, and polysomes. The Nissl substance was variable and some neurons appeared to be immature. Although the spinal cord grafts are in a state of relative gliosis, surrounded by a glial barrier, cografted fetal locus coeruleus catecholamine neurons are able to innervate the spinal cord grafts and form anatomically relevant synapses with the spinal cord neuronal elements as revealed by TH-immunoelectron microscopy. In conclusion, several organotypical features of normal spinal cord are found. Examples also were found, however, of a disturbed and delayed development that have to be considered when evaluating the functional potential of grafted cells.     

Selective expression of CSPG receptors PTPσ and LAR in poorly regenerating reticulospinal neurons of lamprey

Disability following spinal cord injury is due to failure of axon regeneration, which has been ascribed to environmental factors in the central nervous system and a developmental loss of intrinsic growth capacity in neurons. Recently, the receptor‐like protein tyrosine phosphatases, protein tyrosine phosphatase σ (PTPσ) and leukocyte common antigen‐related phosphatase (LAR), have been identified as specific receptors for the regeneration‐inhibiting matrix molecules chondroitin sulfate proteoglycans (CSPGs). After spinal cord transection in lampreys, axons of the large, identified reticulospinal neurons have heterogeneous regenerative abilities. The bad‐regenerating neurons also undergo a delayed form of axotomy‐induced apoptosis. In the present study, a lamprey genomic database was used to identify homologs of CSPGs, clone PTPσ and LAR, and examine their mRNA expression. CSPG immunoreactivity was increased significantly near the lesion at 2 weeks post transection, and decreased thereafter. Both receptors were expressed selectively in the bad‐regenerating neurons and had overlapping cellular distributions. PTPσ was upregulated with age (LAR was not evaluated). By 2 weeks post transection, neurons expressing PTPσ also showed caspase activation, suggesting apoptosis. The probability of axon regeneration for individual identified neurons was negatively correlated with the expression level of PTPσ in both control and spinal cord–transected lampreys. In an animal 7 weeks post transection, regenerated axons were labeled retrogradely from beyond the transection. PTPσ expression and caspase labeling was seen only in neurons whose axon had not regenerated. These results are consistent with a possible role for PTPσ (and LAR) in both retrograde neuronal death and the poor intrinsic regenerative ability of bad‐regenerating neurons. J. Comp. Neurol. 522:2209–2229, 2014. © 2013 Wiley Periodicals, Inc.     

Therapeutic potential of induced pluripotent stem cells for spinal cord injury

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.     

Root repair review: basic science background and clinical outcome

Spinal nerve root injuries have a profound effect on the different parts (PNS and CNS) of the root itself as well as the pertinent spinal cord segment. A root avulsion from the spinal cord is a longitudinal spinal cord injury. There is degeneration of sensory and motor axons, loss of synapses, deterioration of local segmental connections, nerve cell death and reactions among non neuronal cells with scar formation, i.e. a cascade of events similar to those known to occur in any injury to the spinal cord. For function to be restored, nerve cells must survive and there must be regrowth of new nerve fibres along a trajectory consisting of CNS growth-inhibitory tissue in the spinal cord as well as PNS growth-promoting tissue in nerves. Problems in PNS regeneration such as non directional growths and unspecific reinnervation of target organs lead to unpredictable sensorimotor activity and conspires against a useful recovery of function. From the results of basic science experiments, a surgical strategy to treat root avulsion with spinal cord injury has been developed. In humans this technique is currently the most promising treatment of any spinal cord injury, with return of useful function together with pain alleviation in cases where all nerves to the extremity have been avulsed from the spinal cord. At present the shortcomings of this technique are proportionate to the delay before surgery, which leads to death of nerve cells and incomplete and unpredictable recovery. In order to improve this situation and achieve further recovery of useful function including sensory perceptions and to fully alleviate pain it is necessary to pursue research and development of both basic and clinical science.     

Evidence for the Existence of a Functional Polysynaptic Pathway From Trigeminal Afferents to Lumbar Motoneurons in the Neonatal Rat

Stimulation of trigeminal afferents has been reported to have powerful effects on the spinal cord in adult animals of several species. In the present study, the pathway transmitting these influences was investigated in the neonatal rat. Experiments were performed on in vitro brainstem/spinal cord preparations. Stimulation of the trigeminal nerve evoked bilateral polysynaptic discharges in lumbar ventral roots. Intracellular recordings from lumbar motoneurons showed mainly excitatory responses, although a few inhibitory responses were also observed. Experiments with perfusion of different parts of the preparation with general or selective synaptic blockers revealed a synaptic relay under GABAergic control in the brainstem, and at least one synapse in the cervical and in the thoracic spinal cord. The involvement of lumbar interneurons was established by perfusing the lumbar enlargement with saline containing either a high concentration of divalent ions or mephenesin in order to reduce transmission along polysynaptic pathways. The contribution of excitatory amino acid transmission was evaluated and was found to evoke mixed receptor responses. The course of the pathway was traced by using different lesions to the brainstem and spinal cord. The pathway was found to be ipsilateral in the brainstem and to become bilateral in the spinal cord. The results of the present study demonstrate that polysynaptic sensorimotor pathways are present at birth. The results are discussed in relation to the pontomedullary locomotor strip, which has been thought to share many features with the trigeminal system.     

Grafts of embryonic tissue into spinal cord: a possible strategy for treating neuromuscular disorders.

The article describes various approaches used to bring about repair of damaged spinal cord by using embryonic grafts of neuronal tissue. One approach is to stimulate the host's neuronal elements to grow and regenerate. Indeed embryonic grafts have been found to reduce the effects of spinal cord injury, and promote regrowth of axons across a lesion site at least to a limited extent. Attempts have also been made to restore the loss of supraspinal influences with grafts from embryonic brain, and transplants of aminergic neurones have been shown to compensate for the loss of aminergic supraspinal inputs. Finally, it is possible to replace loss of highly specialised cells such as motoneurones by grafts of embryonic spinal cord. Grafted embryonic motoneurones are able to survive within adult host cord although both their chances of survival and maturation seem improved by prior depletion of the host motoneurones. They are able to innervate a skeletal muscle via its peripheral nerve if this is co-implanted at the site of grafting but no axon growth has yet been detected into the host ventral root. However, grafted embryonic neurones are able to migrate away from the graft to sites once occupied by missing motoneurones in the host anterior horn. Within the context of the treatment of neuromuscular disease, the research described suggests possible stratagems for the treatment of disorders such as amyotrophic lateral sclerosis, spinal muscular atrophies or poliomyelitis either by employing grafts that could release neuroactive substances which might prevent existing cells from dying, or even by replacing missing motoneurones with transplanted embryonic motoneurones.