Kazuya Kataoka

Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation

The effects of bone marrow stromal cells (BMSCs) on the repair of injured spinal cord and on the behavioral improvement were studied in the rat. The spinal cord was injured by contusion using a weight-drop at the level of T8-9, and the BMSCs from the bone marrow of the same strain were infused into the cerebrospinal fluid (CSF) through the 4th ventricle. BMSCs were conveyed through the CSF to the spinal cord, where most BMSCs attached to the spinal surface although a few invaded the lesion. The BBB score was higher, and the cavity volume was smaller in the rats with transplantation than in the control rats. Transplanted cells gradually decreased in number and disappeared from the spinal cord 3 weeks after injection. The medium supplemented by CSF (250 microl in 3 ml medium) harvested from the rats in which BMSCs had been injected 2 days previously promoted the neurosphere cells to adhere to the culture dish and to spread into the periphery. These results suggest that BMSCs can exert effects by producing some trophic factors into the CSF or by contacting with host spinal tissues on the reduction of cavities and on the improvement of behavioral function in the rat. Considering that BMSCs can be used for autologous transplantation, and that the CSF infusion of transplants imposes a minimal burden on patients, the results of the present study are important and promising for the clinical use of BMSCs in spinal cord injury treatment.

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord‐derived neurosphere cells by either cell mixing or making monolayered‐feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered‐feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.

Alginate Enhances Elongation of Early Regenerating Axons in Spinal Cord of Young Rats

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.

Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord

Hippocampus-derived neurospheres were prepared from transgenic rat fetuses expressing green fluorescent protein (GFP), and transplanted into an alginate-filled lesion of young rat spinal cord. One, two and four weeks after transplantation, a large number of grafted cells survived, many of which expressed immunoreactivity for glial fibrillary acidic protein, and a few expressed immunoreactivity for beta-tubulin III. The grafted cells closely attached to the host tissue including astrocytes at the border of the lesion. It was notable that numerous GFP-positive cells had migrated within host spinal cord tissue up to 2 mm away from the implanted site 4 weeks postoperation. These results demonstrate that rat fetal hippocampus-derived neurosphere cells could survive, differentiate, extensively migrate, and integrate well into the host spinal cord tissue.

Alginate, a bioresorbable material derived from brown seaweed, enhances elongation of amputated axons of spinal cord in infant rats

Freeze-dried alginate sponge crosslinked with covalent bonds was developed in our laboratory and has been demonstrated to enhance peripheral nerve regeneration. In this study, we examined spinal cord repair using alginate sponge in infant rats. On postnatal day 8-12, the spinal cord was transversely resected at Th7-Th8 to produce a 2-mm gap. The gap was filled with alginate sponge in the alginate group. For the control group, the gap was left empty. In the alginate group, the recovery of evoked electromyogram and sensory-evoked potentials 6 weeks after surgery indicated that elongation of axons could establish electrophysiologically functional projections through the gap. A histological study revealed that myelinated and unmyelinated axons, surrounded by a perineurial-like structure, had elongated across the gap. An immunohistochemical examination revealed that elongation of astrocytic processes and/or migration of astrocytes into the alginate sponge was induced, whereas astrocyte gliosis was reduced at the interface between the implanted alginate and the host spinal cord, compared with the control group. However, a horseradish peroxidase tracing study revealed ascending and descending fibers had also elongated into the gap and reentered the other stump of the transected spinal cord beyond the gap. These results suggest that alginate might provide a permissive microenvironment for elongation of spinal cord axons.

New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat

Here we report a novel method of supplying cultured neurosphere cells to the injured spinal cord, by injection of cells into the cerebrospinal fluid (CSF) through the fourth ventricle or cisterna magna. Hippocampus-derived neurosphere cells, isolated from a transgenic rat fetus expressing green fluorescent protein, were transplanted into the CSF of a rat with spinal cord injury. It was found that injected cells were extensively transported by CSF within the subarachnoidal space, and survived as clusters on the pial surface of the spinal cord. The most notable finding was that a large number of injected cells migrated into the lesion site and integrated into the injured spinal cord tissues.

Electrophysiological and horseradish peroxidase-tracing studies of nerve regeneration through alginate-filled gap in adult rat spinal cord

The spinal cord segments at T(9-10) were totally excised and the resulting gap was filled by implantation of alginate sponge in adult rats. A horseradish peroxidase-tracing study at 21 weeks after operation showed that numerous ascending and many but less numerous descending regenerating fibres traversed the alginate-filled gap, and that after re-entering the distal stump of the transected spinal cord, they extended randomly over a long distance away from the gap. Intracellular electrophysiological recording at the same postoperative time showed that both ascending and descending regenerating axons formed functional synapses with host neurons located beyond the gap. These findings suggest that alginate could be a promising material for the support of regenerating axons in the spinal cord.

Dissemination and proliferation of neural stem cells on the spinal cord by injection into the fourth ventricle of the rat: a method for cell transplantation.

We examined the distribution of hippocampus-derived neural stem cells on the spinal cord surface for up to 3 weeks following injection through the fourth ventricle. The injected cells were disseminated as tiny spots on the pia mater of the spinal cord and proliferated into large cell-clusters. On both the dorsal and ventral side, cell clusters increased in number rapidly up to 5 days after injection and thereafter decreased gradually due to the coalition of neighbouring clusters. Concomitantly, individual cell clusters continuously increased in size, occupying almost 50% of the spinal cord surface. Cell attachment was usually found around blood vessels, along which cells invaded into the spinal cord. In the injured site, cells migrated into the lesion and were integrated into the spinal cord tissue, some of which had differentiated into astrocytes 1-2 weeks after injection. BrdU-uptake experiments demonstrated that the transplanted cells proliferated within the host cerebrospinal fluid. These results indicate that application of neural stem cells through the ventricle is an effective method to disseminate cells all over the spinal cord and that they can migrate and be integrated into the injured spinal cord.

Induction of activating transcription factor 3 after different sciatic nerve injuries in adult rats

Staining by activating transcription factor 3 (ATF3), a neuronal marker of nerve injury, was examined by immunocytochemistry in neurons and Schwann cells after crush or transection (regeneration inhibited) of rat sciatic nerve. ATF3 immunoreactivity peaked in neurons after three days and then gradually subsided to normal within 12 weeks after the crush. The response lasted somewhat longer and declined over time in spinal cord neurons but not in those of dorsal root ganglia (DRG) after transection, indicating a differential regulation of sensory and motor neurons. ATF3 expression was more pronounced in Schwann cells, and remained longer after transection, implying that to some extent regenerating axons produce signals that reduce ATF3 expression in Schwann cells. However, even after transection without repair (no contact with regenerating axons), ATF3 expression in Schwann cells in the distal segment decreased over time suggesting that regenerating axons are not entirely responsible for the down-regulation. These findings have clinical implications on when it is worthwhile to reconstruct nerve injuries.

Peripheral nerve regeneration by transplantation of BMSC-derived Schwann cells as chitosan gel sponge scaffolds

It is said that bone marrow stromal cells (BMSCs) are able to differentiate into different kinds of cells, including Schwann cells, under relevant conditions (Dezawa et al., Eur J Neurosci 2001;14:1771-1776). In the previous paper, we demonstrated that chitosan gel sponge is one of the effective scaffolds for regenerating axons of the rat sciatic nerve (Ishikawa et al., J Biomed Mater Res A 2007;83:33-40). In the present study, we examined whether BMSC-derived Schwann cells with chitosan gel sponges were one of the effective scaffolds for peripheral nerve regeneration in rats. BMSC-derived cells with Schwann cell characteristics were labeled by green fluorescent protein using a retrovirus. An 8-mm gap was made by removing a nerve segment from the rat peripheral nerve, and chitosan gel sponges containing BMSC-derived Schwann cells were grafted, sandwiching the proximal and distal stumps of the transected nerve. Rats were sacrificed at 7, 14, and 28 days, and 2 and 4 months after transplantation. Immunohistochemistry demonstrated that regenerating axons were found near transplanted Schwann cells 7 days after surgery and extended into the host distal nerve segment at 14 days after surgery. Electron microscopy showed that transplanted Schwann cells formed myelin sheaths on regenerating axons 1 month after transplantation. The mean diameter of myelinated fibers was increased from 2.58 mum at 2 months to 2.84 mum at 4 months postsurgery. This study indicates that chitosan gel sponges containing BMSC-derived Schwann cells have strong potentiality as a graft that can be used for peripheral nerve regeneration.