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

https://doi.org/10.1016/j.expneurol.2004.01.021Get rights and content

Abstract

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 μl 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.

Introduction

Cell transplantation is at present considered to be the most effective way to repair spinal cord injuries. So far, several different kinds of cells have been used as transplants for spinal cord regeneration, including Schwann cells (Martin et al., 1996), embryonic spinal cord (Iwashita et al., 1994), olfactory ensheathing cells (Doucette, 1995), macrophages Lazarov-Spiegler et al., 1996, Rapalino et al., 1998, choroid plexus ependymal cells Ide et al., 2001, Kitada et al., 2001, and neural stem cells (Ogawa et al., 2002). Among these cells, embryonic neural stem cells have been most enthusiastically studied Kordower et al., 1995, McDonald et al., 1999. However, ethical problems make it impossible to use human fetal tissue as a practical and immediate source for therapeutic treatment Bjorklund and Linvall, 2000, Constantini and Young, 1994, Krause et al., 2001.

Pittenger et al. (1999) reported that mesenchymal stem cell (MSC) derived from the bone marrow differentiated into osteocytes, chondrocytes, and adipocytes. It is also reported that multipotent adult progenitor cells (MAPC) derived from the bone marrow Reyes et al., 2002, Reyes and Verfaillie, 2001, which comprise approximately 0.125% of the total marrow cells (Blanco et al., 2001), are multipotent stem cells with the capacity to differentiate, under specific experimental conditions Hofstetter et al., 2002, Prockop, 1997, Wu et al., 2003, into several different types of cells including osteoblasts, adipocytes, chondrocytes (Phinney et al., 1999), skeletal muscle fibers (Ferrari et al., 1998), cardiomyocytes (Orlic et al., 2001), hepatocytes (Petersen et al., 1999), neural cells (Sanchez-Ramos et al., 2000), and epithelial cells of the lung and intestinal tract (Krause et al., 2001). It has recently been reported that bone marrow-derived cells have also the potential to develop into neural lineages, such as neurons and astrocytes, both in vivo Eglitis and Mezey, 1997, Kopen et al., 1999, Mezey et al., 2003 and in vitro Deng et al., 2001, Schwarz et al., 1999, Woodbury et al., 2000. These are noteworthy characteristics of bone marrow-derived non-hematopoietic cells from the practical point of view of cell transplantation, since these bone marrow-derived cells can readily be used as autografts for cell transplantation.

BMSCs, which are adherent cells in the culture of bone marrow aspirates, but are not so homogeneous as MSC or MAPC, have already been used for the treatment of the injured spinal cord Chopp et al., 2000, Hofstetter et al., 2002 and brain Chen et al., 2000, Chen et al., 2002, Lu et al., 2001, Mahmood et al., 2001, Martin et al., 1996, Sanchez-Ramos et al., 2000. Our recent study indicated that transplantation of BMSCs by direct injection into the lesion might promote tissue repair in the injured spinal cord by reducing the size of the cavity at the lesion (Wu et al., 2003). There is no evidence that the engrafted BMSCs differentiate into neural cells. The effects of transplanted BMSCs on tissue repair as described above suggest that some trophic factors might be released from BMSCs to promote the tissue repair Chen et al., 2002, Mahmood et al., 2002, Wang et al., 2002, West et al., 2001, Widenfalk et al., 2001.

In previous studies, we reported a new technique to transplant neural stem cells through CSF by injecting cells into the 4th ventricle in the rat Bai et al., 2003, Wu et al., 2001, Wu et al., 2002a, Wu et al., 2002b. The injected cells were conveyed though the CSF, invaded the injured region, and integrated with the host tissue. Considering the risk of direct local injection of cells into the lesion causing additional damage to the spinal cord, the injection through CSF is regarded as a promising way with minimum damage to the host tissue.

In the present study, we studied the effect of BMSCs transplanted via the CSF on the repair of the injured rat spinal cord. Our previous experiment, in which BMSCs were transplanted directly into a spinal cord lesion, showed that the transplanted BMSCs suppressed cavity formation and promoted some behavioral recovery in the rat (Wu et al., 2003). The present study was carried out to examine whether BMSCs administered through CSF have beneficial effects on the tissue repair of the injured spinal cord and on behavioral recovery of the rat. The distribution of BMSCs on the surface of the spinal cord as well as within the lesion was examined up to 5 weeks after cell injection through the 4th ventricle. The behavioral improvement was estimated by BBB score. Histological examination was focused on the tissue repair of the spinal cord lesion in terms of the reduction in size of cavities. In addition, we showed in vitro that the CSF obtained from the rats to which BMSCs had been injected through the 4th ventricle 2 days previously had effects on adhesion and spreading of neurosphere cells derived from the spinal cord as well as from the hippocampus.

Section snippets

Isolation of BMSCs

Bone marrow was isolated in sterile conditions from 8-week-old male transgenic Sprague–Dawley (SD) rats expressing green fluorescent protein (GFP) (Ito et al., 2001, Okabe et al., 1997, Genome Information Research Center Osaka University, and also from 8-week-old male wild-type Wistar rats. Almost all the tissues of the transgenic rat are green under an excitation light (Mezey et al., 2000). Isolation of BMSCs from bone marrow was performed according to the method described by Azizi et al.

MSCs attachment on the surface and invasion into the lesion of the spinal cord

Four days after injection, GFP-positive cells were found as cell clusters in brown-colored fine strands on the surface of spinal cord in the DAB-stained spinal cord. Such fine strands of cell clusters were mostly found at the cervical and thoracic segments, and at the lesion of the spinal cord. There was a tendency for clusters on the ventral surface to be somewhat larger than those on the dorsal surface. These strands of cell clusters gradually increased in number up to 7 days after injection

Discussion

The present study has demonstrated that injection of BMSCs from the 4th ventricle can improve locomotion with reduced cavity formation in the injured rat spinal cord. The injected BMSCs attached on the surface of spinal cord, and at the same time, some of them invaded into the cord lesion. However, BMSCs on the surface as well as within the lesion disappeared by 3 weeks after injection. It is probable that BMSCs produce into the CSF some substances effective for the repair of the lesion,

Acknowledgements

We thank Professor M. Okabe, Genome Information Research Center, Osaka University, for donating GFP-transgenic rats. This study was supported by Grants-in-Aid for Scientific Research (13357014, 14207073, and 14657458 for Y.S. and 14657004 and 15300114 for C.I.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

References (59)

  • L. Wang et al.

    Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture

    Exp. Hematol

    (2002)
  • S.F. Wu et al.

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

    Neurosci. Lett

    (2001)
  • S.F. Wu et al.

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

    Neurosci. Lett

    (2002)
  • Z. Zhang et al.

    Experimental analysis of progressive necrosis after spinal cord trauma in the rat: etiological role of the inflammatory response

    Exp. Neurol

    (1997)
  • S.A. Azizi et al.

    Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—Similarities to astrocyte grafts

    Proc. Natl. Acad. Sci. U. S. A

    (1998)
  • D.M. Basso et al.

    A sensitive and reliable locomotor rating scale for open field-testing in rats

    J. Neurotrauma

    (1995)
  • A. Bjorklund et al.

    Cell replacement therapies for central nervous system disorders

    Nat. Neurosci

    (2000)
  • P. Blanco et al.

    Bone marrow stromal cells: nature, biology, and potential applications

    Stem Cells

    (2001)
  • X. Chen et al.

    Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production

    J. Neurosci. Res

    (2002)
  • M. Chopp et al.

    Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation

    NeuroReport

    (2000)
  • S. Constantini et al.

    The effects of methylprednisolone and the ganglioside GM1 on acute spinal cord injury in rats

    J. Neurosurg

    (1994)
  • M. Dezawa et al.

    Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells

    Eur. J. Neurosci

    (2001)
  • R. Doucette

    Olfactory ensheathing cells: potential for glial cell transplantation into area of CNS injury

    Histol. Histopathol

    (1995)
  • M.A. Eglitis et al.

    Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice

    Proc. Natl. Acad. Sci. U. S. A

    (1997)
  • G. Ferrari et al.

    Muscle regeneration by bone marrow-derived myogenic progenitors

    Science

    (1998)
  • M.T. Fitch et al.

    Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma

    J. Neurosci

    (1999)
  • C.P. Hofstetter et al.

    Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery

    Proc. Natl. Acad. Sci. U. S. A

    (2002)
  • T. Ito et al.

    Application of bone marrow-derived stem cells in experimental nephrology

    Exp. Nephrol

    (2001)
  • Y. Iwashita et al.

    Restoration of function by replacement of spinal cord segments in the rat

    Nature

    (1994)
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