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Xenotransplantation of Transgenic Oligodendrocyte-Lineage Cells into Spinal Cord-Injured Adult Rats
EXPERIMENTAL NEUROLOGY ARTICLE NO.
147, 172–182 (1997)
EN976583
Xenotransplantation of Transgenic Oligodendrocyte-Lineage Cells into Spinal Cord-Injured Adult Rats Jack Rosenbluth,*,1 Rolf Schiff,* Wei-Lan Liang,* Gaetano Menna,† and Wise Young† *Department of Physiology and Neuroscience, Rusk Institute of Rehabilitation Medicine; and †Department of Neurosurgery, N.Y.U. School of Medicine, New York, New York 10016
Spinal cord trauma is associated not only with loss of nerve cells and fibers but also with damage to oligodendrocytes and demyelination. In order to assess the potential of transplanted oligodendrocyte-lineage cells to repair the demyelination that follows spinal cord injury, we have used donor glia derived from a transgenic mouse line containing the LacZ transgene under control of the myelin basic protein promoter (16). Glia derived from fetal or neonatal transgenic mice were injected into the spinal cords of immunosuppressed adult rats at the site of an experimental traumatic lesion 1–16 days after injury. Cells expressing LacZ were identified 15–18 days later in cryosections rostral and caudal to the transplant site, most conspicuously within white matter defects. Some of these cells within the dorsal columns gave rise to D30- to 60-mm processes, consistent with myelin segments, which are oriented parallel to the fiber tract. Glial transplantation may thus be a feasible means of replacing damaged host oligodendrocytes with donor oligodendrocyte-lineage cells capable of reforming myelin and potentially restoring function lost as a result of demyelination associated with spinal cord injury. r 1997 Academic Press
INTRODUCTION
Previous studies have shown that allografts of oligodendrocyte-lineage cells will form myelin in the central nervous system (CNS) of congenitally myelin-deficient animals (17, reviewed in 4) in developing normal CNS (19, 26) and in demyelinated lesions of the CNS (3, 12, 15, 27). Mouse glial cell xenografts into rat CNS have also succeeded in immunosuppressed hosts (12, 23). With both allografts and xenografts, the best results are produced not by adult oligodendrocytes but rather by precursor cells obtained from fetal or neonatal donors (22, 27, 28).
1 To whom reprint requests should be addressed at RR 714, NYU Medical Center, 400 East 34 Street, New York, NY 10016. Fax: (212) 263 8007. E-mail: [email protected].
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Spinal cord injury has been shown to result not only in damage to neural elements but also in a significant amount of demyelination, which has been documented in an extensive series of studies (5–11). Glial transplantation here as well could potentially result in some degree of remyelination beyond what occurs spontaneously (10) and might therefore lead to additional restoration of function. Transplanted glia could also serve to form myelin around regenerated axons. Studies of embryonic rat spinal cord transplanted into surgical lesions of adult rat spinal cord have shown survival, growth, and maturation of the transplants as well as extensive myelination of the donor tissue (20). Thus the environment of the lesioned adult spinal cord does not preclude myelin formation. In impact lesions, however, the success of transplantation could be affected by the conspicuous inflammation that follows this form of trauma (7, 8), which could damage not only endogenous but also exogenous oligodendrocyte-lineage cells. The environment of the traumatized spinal cord could thus compromise the survival of transplanted oligodendrocytes or their ability to form myelin. In order to investigate the feasibility of glial transplantation after spinal cord injury, we injected mouse oligodendrocyte-lineage cells into experimental traumatic lesions of rat spinal cord caused by controlled impact (2, 18). Because of the presence of residual host-derived myelin-forming cells in the traumatized spinal cord, it would be difficult to distinguish them from donor myelin-forming cells without a marker. For this reason, we used donor cells obtained from a transgenic mouse line which carries the LacZ (bacterial galactosidase) gene under control of the MBP promoter (14, 16). Oligodendrocyte-lineage cells from these animals do not express LacZ constituitively. Thus O2A progenitors derived from neonatal donors would be LacZ2 and would become LacZ1 only after differentiating to the stage at which myelin basic protein (MBP) is expressed. The marker, therefore, does not identify all donor cells or even all oligodendrocyte-lineage donor cells, but only
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those oligodendrocytes that have matured sufficiently to transcribe the MBP gene. MATERIALS AND METHODS
Female Long–Evans hooded rats weighing ,300 g were anesthetized with pentobarbital (45 mg/kg ip), and laminectomy was performed at the T9–10 level. During surgery and postoperatively, body temperature was maintained at 37 6 1°C by means of a heating pad. Spinal cord injury was inflicted by the NYU Impactor (2, 18) using a 10-g rod dropped from a height of 12.5 mm. Muscle and skin were then closed over the laminectomy site. This trauma produces ,50–70% loss of white matter at the impact site by 6 weeks after injury. The lesion is centered in the central gray matter and extends concentrically outward to involve white matter and rostrocaudally about 5 mm. Rats injured by a 12.5-mm weight drop suffer immediate paraplegia but typically recover locomotor function within 4 weeks, including ability to support weight and stepping, but without forelimb-hindlimb coordination. The bladders are paralyzed, and the rats require twice daily bladder expression until automatic micturation recovers. In order to prevent urinary tract infection, the rats are treated with an antibiotic (Keflin, 30 mg/day for 7 days after injury). On the BBB scale (1, 2), for assessing behavioral performance, the rats typically achieve a score of 10–11 at 2 weeks and 12–13 at 3 weeks out of a total scale of 0–21. Oligodendrocyte-lineage cells were obtained from MbP5 transgenic mouse embryo (E16–20) or neonatal (P1–2) brains (16). (Transgenic breeders were kindly provided by Dr. R. Lazzarini.) The tissue was minced, dissociated with trypsin, and separated on a Percoll gradient according to methods used previously, which yield mixed glial cultures free of neurons (22). MbP mouse cells contain multiple copies of a transgene consisting of the LacZ reporter under control of MBP promoter/enhancer elements. Thus, transgenic cells of the oligodendrocyte lineage express LacZ only after they have differentiated to the point of MBP expression. The transgene is not expressed by astrocytes or microglia. Glia obtained from the transgenic mice were either cultured in a CO2 incubator for 8–20 days in vitro (DIV) or, in one case, purified, stored overnight at 4°C, and then used directly. Cells to be transplanted were suspended in L15 culture medium. An aliquot was mixed with trypan blue and the concentration of live cells determined from counts in a hemocytometer. (Any cells stained with trypan blue were excluded from the count.) Cell concentration was adjusted to ,5 3 107 cells/ml, and 0.02 ml was then injected into the spinal cord of anesthetized rats (1–16 days postinjury) through the original laminectomy site, directed at the epicenter of
the trauma. Cyclosporine (10 mg/kg/ip) was administered on the day of transplant and daily thereafter. Fifteen to eighteen days later, the rats were reanesthetized and fixed by vascular perfusion with 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) containing 2 mM MgCl2 and 1.25 mM EGTA. Previous studies have shown this to be the approximate time at which myelin formation by transplanted glia has plateaued and can be demonstrated reliably (22, 23). Spinal cords were divided into segments, infiltrated with sucrose, frozen, and sectioned either transversely or horizontally at ,30–50 µm. Sections were processed in X-gal reaction solution overnight at neutral pH (16) and then mounted for examination and photography. Cells expressing bacterial b-galactosidase are stained blue by this method. The mammalian enzyme, which is active at a much lower pH, produces no detectable reaction product when incubated under these conditions. Positive controls consisted of spinal cord sections from a transgenic mouse, processed together with the experimental sections in each staining run, to show that the reaction conditions and reagents were adequate to yield reaction product. A negative control consisted of a rat spinal cord subjected to equivalent trauma and not given a transplant, but incubated in X-gal reaction solution 21 days later. Transverse cryosections were cut from four successive lengths of spinal cord, each ,2–3 mm long, extending rostrally from the lesion site and from four successive lengths extending caudally. Some segments were sectioned horizontally. After incubation in the X-gal reaction solution, sections were scanned for bluestained cells and photographed using a red filter. For each animal, the number of stained cells was counted in a randomly chosen single transverse section taken at each of the eight spinal cord levels adjacent to the lesion, and the counts were summed. RESULTS
Spinal cord sections from our negative control showed no stained cells after incubation in the X-gal reaction solution (Fig. 1A), indicating that under the conditions we used, mammalian galactosidases in host macrophages or other host cells do not generate detectable reaction product from the X-gal reaction mixture. This negative result is consistent with results we have obtained in control animals from other studies (unpublished) in which traumatized or untraumatized rat spinal cords have been exposed to X-gal reaction mixture and also fail to show reaction product due to endogenous enzyme. In contrast, sections from LacZ1 adult mice from our transgenic breeding colony, used as positive controls, showed strong staining in all white matter tracts, as well as scattered stained cells in gray
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FIG. 1. (A) Negative control. Spinal cord-injured rat, no transplant, incubated in X-gal. Arrows indicate lesions within dorsal and lateral columns. Note absence of reaction product. (B) Positive control. Transgenic mouse spinal cord; incubation in X-gal results in dense staining of white matter and of scattered cells in gray matter. (C) Same as B without X-gal incubation to show density of unstained myelin under comparable optical conditions.
FIG. 2. Sections incubated in X-gal. From successive 2- to 3-mm lengths (A–D rostral and E–G caudal to injury site). Transgenic glial transplant performed 13 days after injury. Fixed 17 days after transplant. Clusters of stained cells are visible primarily in dorsal and lateral white matter regions at all levels shown. In several cases (e.g., at arrow) a lucent area of defective myelin surrounds the cluster of stained cells. Stained cells are also scattered within gray matter (cf. Fig. 1B).
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TABLE 1 Summary of Findings Interval, trauma to transplant (days) 1 1 4 6 9 13 16 b
Donor source (mouse) P1 1 10 DIV P1 1 10 DIV P1 1 13 DIV E19/20 1 18 DIV E17/18 1 4°C, 18 h P1/2 1 20 DIV E16–18 1 8 DIV None (control)
Interval, transplant Cell to fixation (days) counta 18 18 15 16 17 17 18 b
60 29 55 41 6 .400 69 0
a Cell count 5 o(L . . . L ), where L represents the number of 1 8 x stained cells from a randomly chosen transverse section at level x. Levels 1–8 consist of the four 2- to 3-mm lengths of spinal cord just rostral to the level of the lesion plus the four just caudal to that level. b Rat fixed 21 days after trauma.
matter (Fig. 1B), compared with adjacent sections not exposed to the reaction solution (Fig. 1C). Of the six rats that received transplants of cultured glia grown from fetal or neonatal transgenic mice, five, transplanted at 1, 4, 6, and 16 days postinjury, showed moderate numbers of stained cells at multiple spinal cord levels, and the remaining one, transplanted at 13 days, showed very extensive staining (Fig. 2). The rat injected with donor cells that had not first been cultured showed few stained cells. Table 1 summarizes these findings. In all of the experimental animals, the distribution of stained cells was to some extent related to the underlying defects induced by the trauma. Cryosections of the traumatized but untransplanted control showed defects in lateral and dorsal fiber tracts rostral and caudal to the level of the injury (Fig. 1A). Typically, these defects are focal, variably shaped, low-density, sometimes cystic areas within white matter. Their irregular distribution suggests that they represent rostral or caudal extensions of the primary traumatic lesion. Comparable defects have also been identified in specimens prepared for ultrastructural analysis (unpublished). In one case such a defect within dorsal column sensory tracts, examined 6 weeks after injury, was traced more than 1 cm caudally from the lesion site. This defect was found to contain fluid and macrophages and was surrounded by a cellular rim of tissue containing conspicuous astrocyte processes, replacing the ascending myelinated fiber tracts normally present in this location. In the traumatized animals that received transplanted transgenic glia, incubation in the X-gal reaction solution revealed prominent clusters of LacZ1 cells associated with equivalent regions showing focal defects in myelin staining, primarily in the dorsal and
lateral white matter (Figs. 2–4). Such clusters were identified at multiple levels rostral and caudal to the injury site. Stained cells were found not only in clusters associated with white matter defects but also scattered through the central gray (Figs. 2C, 3, and 4), sometimes in linear patterns, especially in regions of decussating fibers (Fig. 3A). Stained cells also formed conspicuous linear streaks within transversely sectioned white matter (Figs. 2B, 2G, and 3A). Otherwise, intact white matter regions showed relatively few stained cells in comparison with the adjacent gray matter (Figs. 3A and 4A). Dense, ovoid stained cells were also clearly associated with the margins of blood vessels (Fig. 5). The morphology of the stained cells was difficult to assess critically in cryosections because of distortions introduced during tissue processing, which included detergent treatment as well as freezing. Nevertheless, in transverse sections through white matter, it was possible to identify cellular processes arising from stained cell bodies (Fig. 3B) and running perpendicular to the axis of the fiber tract. Horizontal sections through white matter were even more revealing. In this plane, it was possible to identify cells giving rise to elongated LacZ1 processes, ,30–60 µm in length, running parallel to the fiber tract (Figs. 6A and 6B). The intensity of the stain varied along the length of these processes. In some cases, these cells appeared as doublets, suggesting that a single cell underwent cell division and that both daughter cells remained at that site without further migration. Occasional oblique (Fig. 6A) and transverse (Fig. 6B) processes also could be seen arising from the same cells. In contrast to the cells bearing multiple parallel proceses within fiber tracts (Fig. 6), the small, dense, ovoid LacZ1 cells within gray matter and those associated with the margins of blood vessels (Fig. 5) gave rise to few recognizable processes. Thus, we identified two types of LacZ1 cells in these specimens. DISCUSSION
Our results show LacZ1 cells within the traumatized rat spinal cord after xenografting transgenically marked glia carrying the LacZ gene under control of the MBP promoter into the site of experimental spinal cord lesions. Despite the rigors of the preparative and staining procedures used for demonstration of X-gal reaction product, we were able to identify stained cells within fiber tracts that gave rise to multiple, relatively straight LacZ1 processes running parallel to the nerve fibers. These process-bearing cells resemble differentiated oligodendrocytes, as shown in previous in vivo studies (28), and correspond to the type II oligodendrocytes described by Rio Hortega (reviewed in ref. 25).
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FIG. 3. (A) Detail showing clusters of stained cells, each surrounded by a lucent area of defective myelin (arrows) within lateral and dorsal column white matter. Stained cells are also scattered through gray matter and form linear strings within the central gray and within the lateral white matter at lower left. Same level as Fig. 2B. (B) Detail of Fig. 2F showing clusters of stained cells within central and dorsal horn gray matter and in white matter defects in dorsal and lateral white columns. Arrow indicates one such defect and, beyond it, a stained cell bearing a process.
FIG. 4. (A) Stained cells within a lucent area in dorsal column white matter (arrow). Section from the level just caudal to Fig. 2G. (B) Detail of lateral column white matter showing defect (arrows) containing cluster of stained cells. Scattered LacZ1 cells are also present within dorsal gray horn (upper left) and central gray below.
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FIG. 5. Brain stem section from same animal as in Fig. 2 showing small dense, strongly LacZ1 cells associated with blood vessel margins.
The length of the processes is consistent with the length of myelin segments. Since the lacZ reaction product is cytoplasmic in location, these stained segments are most simply explained as cytoplasmic components of the myelin sheath, i.e., the external and internal tongues, the paranodal loops, and perhaps other Schmidt–Lanterman-like cytoplasmic channels within the sheath. Such cytoplasmic elements are more conspicuous in newly formed, incompletely compacted myelin segments. Variation in the intensity of the stain could reflect a periodic disposition of these cytoplasmic channels. Transversely or obliquely oriented processes probably represent the connecting elements that extend between the oligodendrocyte cell body and the outer tongues of the myelin sheaths. They may appear either as extended processes or as stained circular cross sections of small caliber (Figs. 6A and 6B). Such connecting processes are seen as well in transverse sections through fiber tracts, but the multiple elon-
gated processes occur only in the horizontal sections, in which the myelin segments are cut longitudinally. The second population of LacZ1 cells consists of small dense cells that correspond in morphology and location to the type I oligodendrocytes of Rio Hortega (25). In silver-stained preparations, these give rise to tenuous processes which, however, may be too fine to accommodate the LacZ reaction product and are, therefore, not well demonstrated in our preparations. In view of the inflammation that follows spinal cord trauma, we also considered the possibility that some of these small dense cells could represent macrophages which engulfed LacZ1 donor cells that did not survive and in which the bacterial enzyme remained sufficiently active to yield reaction product. The size and morphology of these cells is, however, not consistent with that of macrophages laden with cell fragments. Moreover, when sections were immunostained with HAM56, an antibody that recognizes macrophages, the small ovoid LacZ1 cells were HAM562, while a popula-
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FIG. 6. (A) Horizontal section through dorsal columns showing a pair of LacZ1 cells, ,6–9 µm in diameter, associated with slender, relatively straight longitudinally oriented LacZ1 processes (vertical arrows) measuring ,1–2 µm in diameter and ,30 µm in length, consistent with myelin segments. Two other stained processes (arrowheads) are not clearly connected to these cells. Diagonal arrows indicate obliquely and transversely cut processes. (B) Horizontal section through dorsal columns showing LacZ1 cells, one of which appears to give rise to a longitudinal process ,60 µm long and 2–3 µm wide, which ends abruptly, consistent with a myelin segment. Vertical arrows indicate putative nodes of Ranvier. Periodic densities in the left half of this segment, also visible in A, could represent cytoplasmic expansions. Diagonal arrows show obliquely and transversely cut processes.
tion of LacZ2 cells that had the morphology of macrophages was HAM561 (D. C. Miller et al., unpublished data). Thus we have no evidence supporting the hypothesis that these LacZ1 cells are macrophages. Finally, recent studies have shown evidence for apop-
tosis in endogenous cells of the spinal cord following trauma (13), introducing the possibility that local conditions could induce apoptosis in transplanted cells as well. Thus, some of the small LacZ1 cells lacking apparent processes could be donor glia that differenti-
GLIAL XENOGRAFTS AFTER SPINAL CORD INJURY
ated to the point of MBP expression and subsequently underwent apoptosis with accompanying loss of processes. It is of interest, however, that very similar, small LacZ1 cells occur in the adult transgenic mouse spinal cord as well (Fig. 1B) scattered through both gray and white matter and also associated with blood vessels. Since there is no reason to postulate apoptosis in the normal control, cells of this type, in both the normal and traumatized spinal cord, could simply represent the classically described type I oligodendrocytes known to be associated with neurons in gray matter and with blood vessels and which are also present in white matter (25). The distribution of LacZ1 cells found in the present study (Fig. 2) indicates that the transplanted glia move rostrocaudally over considerable distances. To some extent this movement could be passive, within fluidfilled cystic defects resulting from the trauma. Their presence within intact regions of gray matter, however, where cellular processes are tightly packed, indicates that the cells are also capable of active migration through the tissue. In view of their association with blood vessels, these cells may also move through perivascular spaces, as Vignais et al. have proposed (27). The paucity of LacZ1 cells in intact white matter suggests, in contrast, that they avoid migrating into normally myelinated regions or fail to differentiate there. The fact that LacZ1 cells tend to become concentrated at the defects in myelinated fiber tracts that develop following the trauma could indicate that the transgenic cells move more readily through such defects. Alternatively, the cells may be drawn to such sites chemotactically, or may be retained there, by cytokines, adhesion molecules, or other factors released by the damaged tissue itself or by macrophages that invade secondarily. The cells may also proliferate to a greater extent at these sites than elsewhere or may be induced to mature there to the point of LacZ expression and myelin formation. Glial transplants from the same transgenic mouse line have been shown previously to form ultrastructurally normal myelin in the myelin-deficient rat spinal cord (21), and it is therefore highly likely that the same cells are capable of myelin formation around rat spinal cord axons in the trauma model as well. The demonstration of 30- to 60-µm LacZ1 segments in horizontal sections supports the conclusion that at least some of the transplanted cells find their way to receptive axons and do in fact form myelin segments. Transplantation of cultured glial cell suspensions is thus a potentially useful approach to replacing oligodendrocytes in traumatized spinal cord. Such transplants could remyelinate and restore functions lost as a result of demyelination secondary to trauma. Success with xenografted glia suggests, in addition, that the source
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of the cells transplanted need not be restricted to donors of the same species. Further studies will be required to assess long-term survival of LacZ1 transplanted glial cells and the myelin-like segments produced by them, to determine the optimal parameters for glial transplantation in this model and to determine whether behavioral improvement results from this procedure. ACKNOWLEDGMENTS The authors are indebted to Ron Morella for expert technical assistance. A preliminary report of this study has been presented (24). This work was supported by grants from the Paralyzed Veterans of America Spinal Cord Research Foundation and the National Multiple Sclerosis Society.
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ROSENBLUTH ET AL. Gout, O., A. Gansmuller, N. Baumann, and M. Gumpel. 1988. Remyelination by transplanted oligodendrocytes of a demyelinated lesion in the spinal cord of the adult shiverer mouse. Neurosci. Lett. 87: 195–199. Gow, A., V. L. Friedrich, Jr., and R. A. Lazzarini. 1992. Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression. J. Cell Biol. 119: 605–616. Gumpel, M., N. Baumann, M. Raoul, and C. Jacque. 1983. Survival and differentiation of oligodendrocytes from neural tissue transplanted into newborn mouse brain. Neurosci. Lett. 37: 307–311. Huang, P. P., and W. Young. 1994. The effects of arterial blood gas values on lesion volumes in a graded rat spinal cord contusion model. J. Neurotrauma 11: 547–562. Lachapelle, F., E. Duhamel-Clerin, A. Gansmuller, A. BaronVan Evercooren, H. Villarroya, and M. Gumpel. 1994. Transplanted transgenically marked oligodendrocytes survive, migrate and myelinate in the normal mouse brain as they do in the shiverer mouse brain. Eur. J. Neurosci. 6: 814–824. Reier, P. J., B. S. Bregman, and J. R. Wujek. 1986. Intraspinal transplantation of embryonic spinal cord tissue in neonatal and adult rats. J. Comp. Neurol. 247: 275–296. Rosenbluth, J. 1996. Glial transplantation in the treatment of myelin loss or deficiency. Pages 124–128 in H. Bostock, P. A. Kirkwood, and A. H. Pullen, Eds., Neurobiology and Disease: Contributions from Neuroscience to Clinical Neurology Cambridge Univ. Press, New York.
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Rosenbluth, J., M. Hasegawa, N. Shirasaki, C. L. Rosen, and Z. Liu. 1990. Myelin formation after transplantation of normal fetal spinal cord fragments or cultures into myelin-deficient rat spinal cord. J. Neurocytol. 19: 718–730. Rosenbluth, J., Z. Liu, D. Guo, and R. Schiff. 1991. Myelin formation by mouse glia in myelin-deficient rats treated with cyclosporine. J. Neurocytol. 22: 967–977. Rosenbluth, J., R. Schiff, W.-L. Liang, G. Menna, and W. Young. 1995. Survival, migration and differentiation of transgenic oligodendrocyte-lineage glia transplanted into spinal cordinjured adult rats. J. Neurotrauma 12: 972. Szuchet, S. 1995. The morphology and ultrastructure of oligodendrocytes and their functional implications. Pages 23–43 in H. Kettenmann and B. R. Ransom, Eds., Neuroglia. Oxford Univ. Press, New York. Tontsch, U., D. R. Archer, and I. D. Duncan. 1994. Transplantation of an oligodendrocyte cell line leading to extensive myelination. Proc. Natl. Acad. Sci. USA 91: 11616–11620. Vignais, L., B. Nait Oumesmar, F. Mellouk, O. Gout, G. Labourdette, A. Baron-Van Evercooren, and M. Gumpel. 1993. Migration of oligodendrocytes. Transplantation of oligodendrocyte precursors in the adult demyelinated spinal cord: Migration and remyelination. Int. J. Dev. Neurosci. 11: 603–612. Warrington, A. E., E. Barbarese, and S. E. Pfeiffer. 1993. Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts. J. Neurosci. Res. 34: 1–13.
147, 172–182 (1997)
EN976583
Xenotransplantation of Transgenic Oligodendrocyte-Lineage Cells into Spinal Cord-Injured Adult Rats Jack Rosenbluth,*,1 Rolf Schiff,* Wei-Lan Liang,* Gaetano Menna,† and Wise Young† *Department of Physiology and Neuroscience, Rusk Institute of Rehabilitation Medicine; and †Department of Neurosurgery, N.Y.U. School of Medicine, New York, New York 10016
Spinal cord trauma is associated not only with loss of nerve cells and fibers but also with damage to oligodendrocytes and demyelination. In order to assess the potential of transplanted oligodendrocyte-lineage cells to repair the demyelination that follows spinal cord injury, we have used donor glia derived from a transgenic mouse line containing the LacZ transgene under control of the myelin basic protein promoter (16). Glia derived from fetal or neonatal transgenic mice were injected into the spinal cords of immunosuppressed adult rats at the site of an experimental traumatic lesion 1–16 days after injury. Cells expressing LacZ were identified 15–18 days later in cryosections rostral and caudal to the transplant site, most conspicuously within white matter defects. Some of these cells within the dorsal columns gave rise to D30- to 60-mm processes, consistent with myelin segments, which are oriented parallel to the fiber tract. Glial transplantation may thus be a feasible means of replacing damaged host oligodendrocytes with donor oligodendrocyte-lineage cells capable of reforming myelin and potentially restoring function lost as a result of demyelination associated with spinal cord injury. r 1997 Academic Press
INTRODUCTION
Previous studies have shown that allografts of oligodendrocyte-lineage cells will form myelin in the central nervous system (CNS) of congenitally myelin-deficient animals (17, reviewed in 4) in developing normal CNS (19, 26) and in demyelinated lesions of the CNS (3, 12, 15, 27). Mouse glial cell xenografts into rat CNS have also succeeded in immunosuppressed hosts (12, 23). With both allografts and xenografts, the best results are produced not by adult oligodendrocytes but rather by precursor cells obtained from fetal or neonatal donors (22, 27, 28).
1 To whom reprint requests should be addressed at RR 714, NYU Medical Center, 400 East 34 Street, New York, NY 10016. Fax: (212) 263 8007. E-mail: [email protected].
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Spinal cord injury has been shown to result not only in damage to neural elements but also in a significant amount of demyelination, which has been documented in an extensive series of studies (5–11). Glial transplantation here as well could potentially result in some degree of remyelination beyond what occurs spontaneously (10) and might therefore lead to additional restoration of function. Transplanted glia could also serve to form myelin around regenerated axons. Studies of embryonic rat spinal cord transplanted into surgical lesions of adult rat spinal cord have shown survival, growth, and maturation of the transplants as well as extensive myelination of the donor tissue (20). Thus the environment of the lesioned adult spinal cord does not preclude myelin formation. In impact lesions, however, the success of transplantation could be affected by the conspicuous inflammation that follows this form of trauma (7, 8), which could damage not only endogenous but also exogenous oligodendrocyte-lineage cells. The environment of the traumatized spinal cord could thus compromise the survival of transplanted oligodendrocytes or their ability to form myelin. In order to investigate the feasibility of glial transplantation after spinal cord injury, we injected mouse oligodendrocyte-lineage cells into experimental traumatic lesions of rat spinal cord caused by controlled impact (2, 18). Because of the presence of residual host-derived myelin-forming cells in the traumatized spinal cord, it would be difficult to distinguish them from donor myelin-forming cells without a marker. For this reason, we used donor cells obtained from a transgenic mouse line which carries the LacZ (bacterial galactosidase) gene under control of the MBP promoter (14, 16). Oligodendrocyte-lineage cells from these animals do not express LacZ constituitively. Thus O2A progenitors derived from neonatal donors would be LacZ2 and would become LacZ1 only after differentiating to the stage at which myelin basic protein (MBP) is expressed. The marker, therefore, does not identify all donor cells or even all oligodendrocyte-lineage donor cells, but only
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those oligodendrocytes that have matured sufficiently to transcribe the MBP gene. MATERIALS AND METHODS
Female Long–Evans hooded rats weighing ,300 g were anesthetized with pentobarbital (45 mg/kg ip), and laminectomy was performed at the T9–10 level. During surgery and postoperatively, body temperature was maintained at 37 6 1°C by means of a heating pad. Spinal cord injury was inflicted by the NYU Impactor (2, 18) using a 10-g rod dropped from a height of 12.5 mm. Muscle and skin were then closed over the laminectomy site. This trauma produces ,50–70% loss of white matter at the impact site by 6 weeks after injury. The lesion is centered in the central gray matter and extends concentrically outward to involve white matter and rostrocaudally about 5 mm. Rats injured by a 12.5-mm weight drop suffer immediate paraplegia but typically recover locomotor function within 4 weeks, including ability to support weight and stepping, but without forelimb-hindlimb coordination. The bladders are paralyzed, and the rats require twice daily bladder expression until automatic micturation recovers. In order to prevent urinary tract infection, the rats are treated with an antibiotic (Keflin, 30 mg/day for 7 days after injury). On the BBB scale (1, 2), for assessing behavioral performance, the rats typically achieve a score of 10–11 at 2 weeks and 12–13 at 3 weeks out of a total scale of 0–21. Oligodendrocyte-lineage cells were obtained from MbP5 transgenic mouse embryo (E16–20) or neonatal (P1–2) brains (16). (Transgenic breeders were kindly provided by Dr. R. Lazzarini.) The tissue was minced, dissociated with trypsin, and separated on a Percoll gradient according to methods used previously, which yield mixed glial cultures free of neurons (22). MbP mouse cells contain multiple copies of a transgene consisting of the LacZ reporter under control of MBP promoter/enhancer elements. Thus, transgenic cells of the oligodendrocyte lineage express LacZ only after they have differentiated to the point of MBP expression. The transgene is not expressed by astrocytes or microglia. Glia obtained from the transgenic mice were either cultured in a CO2 incubator for 8–20 days in vitro (DIV) or, in one case, purified, stored overnight at 4°C, and then used directly. Cells to be transplanted were suspended in L15 culture medium. An aliquot was mixed with trypan blue and the concentration of live cells determined from counts in a hemocytometer. (Any cells stained with trypan blue were excluded from the count.) Cell concentration was adjusted to ,5 3 107 cells/ml, and 0.02 ml was then injected into the spinal cord of anesthetized rats (1–16 days postinjury) through the original laminectomy site, directed at the epicenter of
the trauma. Cyclosporine (10 mg/kg/ip) was administered on the day of transplant and daily thereafter. Fifteen to eighteen days later, the rats were reanesthetized and fixed by vascular perfusion with 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) containing 2 mM MgCl2 and 1.25 mM EGTA. Previous studies have shown this to be the approximate time at which myelin formation by transplanted glia has plateaued and can be demonstrated reliably (22, 23). Spinal cords were divided into segments, infiltrated with sucrose, frozen, and sectioned either transversely or horizontally at ,30–50 µm. Sections were processed in X-gal reaction solution overnight at neutral pH (16) and then mounted for examination and photography. Cells expressing bacterial b-galactosidase are stained blue by this method. The mammalian enzyme, which is active at a much lower pH, produces no detectable reaction product when incubated under these conditions. Positive controls consisted of spinal cord sections from a transgenic mouse, processed together with the experimental sections in each staining run, to show that the reaction conditions and reagents were adequate to yield reaction product. A negative control consisted of a rat spinal cord subjected to equivalent trauma and not given a transplant, but incubated in X-gal reaction solution 21 days later. Transverse cryosections were cut from four successive lengths of spinal cord, each ,2–3 mm long, extending rostrally from the lesion site and from four successive lengths extending caudally. Some segments were sectioned horizontally. After incubation in the X-gal reaction solution, sections were scanned for bluestained cells and photographed using a red filter. For each animal, the number of stained cells was counted in a randomly chosen single transverse section taken at each of the eight spinal cord levels adjacent to the lesion, and the counts were summed. RESULTS
Spinal cord sections from our negative control showed no stained cells after incubation in the X-gal reaction solution (Fig. 1A), indicating that under the conditions we used, mammalian galactosidases in host macrophages or other host cells do not generate detectable reaction product from the X-gal reaction mixture. This negative result is consistent with results we have obtained in control animals from other studies (unpublished) in which traumatized or untraumatized rat spinal cords have been exposed to X-gal reaction mixture and also fail to show reaction product due to endogenous enzyme. In contrast, sections from LacZ1 adult mice from our transgenic breeding colony, used as positive controls, showed strong staining in all white matter tracts, as well as scattered stained cells in gray
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FIG. 1. (A) Negative control. Spinal cord-injured rat, no transplant, incubated in X-gal. Arrows indicate lesions within dorsal and lateral columns. Note absence of reaction product. (B) Positive control. Transgenic mouse spinal cord; incubation in X-gal results in dense staining of white matter and of scattered cells in gray matter. (C) Same as B without X-gal incubation to show density of unstained myelin under comparable optical conditions.
FIG. 2. Sections incubated in X-gal. From successive 2- to 3-mm lengths (A–D rostral and E–G caudal to injury site). Transgenic glial transplant performed 13 days after injury. Fixed 17 days after transplant. Clusters of stained cells are visible primarily in dorsal and lateral white matter regions at all levels shown. In several cases (e.g., at arrow) a lucent area of defective myelin surrounds the cluster of stained cells. Stained cells are also scattered within gray matter (cf. Fig. 1B).
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TABLE 1 Summary of Findings Interval, trauma to transplant (days) 1 1 4 6 9 13 16 b
Donor source (mouse) P1 1 10 DIV P1 1 10 DIV P1 1 13 DIV E19/20 1 18 DIV E17/18 1 4°C, 18 h P1/2 1 20 DIV E16–18 1 8 DIV None (control)
Interval, transplant Cell to fixation (days) counta 18 18 15 16 17 17 18 b
60 29 55 41 6 .400 69 0
a Cell count 5 o(L . . . L ), where L represents the number of 1 8 x stained cells from a randomly chosen transverse section at level x. Levels 1–8 consist of the four 2- to 3-mm lengths of spinal cord just rostral to the level of the lesion plus the four just caudal to that level. b Rat fixed 21 days after trauma.
matter (Fig. 1B), compared with adjacent sections not exposed to the reaction solution (Fig. 1C). Of the six rats that received transplants of cultured glia grown from fetal or neonatal transgenic mice, five, transplanted at 1, 4, 6, and 16 days postinjury, showed moderate numbers of stained cells at multiple spinal cord levels, and the remaining one, transplanted at 13 days, showed very extensive staining (Fig. 2). The rat injected with donor cells that had not first been cultured showed few stained cells. Table 1 summarizes these findings. In all of the experimental animals, the distribution of stained cells was to some extent related to the underlying defects induced by the trauma. Cryosections of the traumatized but untransplanted control showed defects in lateral and dorsal fiber tracts rostral and caudal to the level of the injury (Fig. 1A). Typically, these defects are focal, variably shaped, low-density, sometimes cystic areas within white matter. Their irregular distribution suggests that they represent rostral or caudal extensions of the primary traumatic lesion. Comparable defects have also been identified in specimens prepared for ultrastructural analysis (unpublished). In one case such a defect within dorsal column sensory tracts, examined 6 weeks after injury, was traced more than 1 cm caudally from the lesion site. This defect was found to contain fluid and macrophages and was surrounded by a cellular rim of tissue containing conspicuous astrocyte processes, replacing the ascending myelinated fiber tracts normally present in this location. In the traumatized animals that received transplanted transgenic glia, incubation in the X-gal reaction solution revealed prominent clusters of LacZ1 cells associated with equivalent regions showing focal defects in myelin staining, primarily in the dorsal and
lateral white matter (Figs. 2–4). Such clusters were identified at multiple levels rostral and caudal to the injury site. Stained cells were found not only in clusters associated with white matter defects but also scattered through the central gray (Figs. 2C, 3, and 4), sometimes in linear patterns, especially in regions of decussating fibers (Fig. 3A). Stained cells also formed conspicuous linear streaks within transversely sectioned white matter (Figs. 2B, 2G, and 3A). Otherwise, intact white matter regions showed relatively few stained cells in comparison with the adjacent gray matter (Figs. 3A and 4A). Dense, ovoid stained cells were also clearly associated with the margins of blood vessels (Fig. 5). The morphology of the stained cells was difficult to assess critically in cryosections because of distortions introduced during tissue processing, which included detergent treatment as well as freezing. Nevertheless, in transverse sections through white matter, it was possible to identify cellular processes arising from stained cell bodies (Fig. 3B) and running perpendicular to the axis of the fiber tract. Horizontal sections through white matter were even more revealing. In this plane, it was possible to identify cells giving rise to elongated LacZ1 processes, ,30–60 µm in length, running parallel to the fiber tract (Figs. 6A and 6B). The intensity of the stain varied along the length of these processes. In some cases, these cells appeared as doublets, suggesting that a single cell underwent cell division and that both daughter cells remained at that site without further migration. Occasional oblique (Fig. 6A) and transverse (Fig. 6B) processes also could be seen arising from the same cells. In contrast to the cells bearing multiple parallel proceses within fiber tracts (Fig. 6), the small, dense, ovoid LacZ1 cells within gray matter and those associated with the margins of blood vessels (Fig. 5) gave rise to few recognizable processes. Thus, we identified two types of LacZ1 cells in these specimens. DISCUSSION
Our results show LacZ1 cells within the traumatized rat spinal cord after xenografting transgenically marked glia carrying the LacZ gene under control of the MBP promoter into the site of experimental spinal cord lesions. Despite the rigors of the preparative and staining procedures used for demonstration of X-gal reaction product, we were able to identify stained cells within fiber tracts that gave rise to multiple, relatively straight LacZ1 processes running parallel to the nerve fibers. These process-bearing cells resemble differentiated oligodendrocytes, as shown in previous in vivo studies (28), and correspond to the type II oligodendrocytes described by Rio Hortega (reviewed in ref. 25).
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FIG. 3. (A) Detail showing clusters of stained cells, each surrounded by a lucent area of defective myelin (arrows) within lateral and dorsal column white matter. Stained cells are also scattered through gray matter and form linear strings within the central gray and within the lateral white matter at lower left. Same level as Fig. 2B. (B) Detail of Fig. 2F showing clusters of stained cells within central and dorsal horn gray matter and in white matter defects in dorsal and lateral white columns. Arrow indicates one such defect and, beyond it, a stained cell bearing a process.
FIG. 4. (A) Stained cells within a lucent area in dorsal column white matter (arrow). Section from the level just caudal to Fig. 2G. (B) Detail of lateral column white matter showing defect (arrows) containing cluster of stained cells. Scattered LacZ1 cells are also present within dorsal gray horn (upper left) and central gray below.
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FIG. 5. Brain stem section from same animal as in Fig. 2 showing small dense, strongly LacZ1 cells associated with blood vessel margins.
The length of the processes is consistent with the length of myelin segments. Since the lacZ reaction product is cytoplasmic in location, these stained segments are most simply explained as cytoplasmic components of the myelin sheath, i.e., the external and internal tongues, the paranodal loops, and perhaps other Schmidt–Lanterman-like cytoplasmic channels within the sheath. Such cytoplasmic elements are more conspicuous in newly formed, incompletely compacted myelin segments. Variation in the intensity of the stain could reflect a periodic disposition of these cytoplasmic channels. Transversely or obliquely oriented processes probably represent the connecting elements that extend between the oligodendrocyte cell body and the outer tongues of the myelin sheaths. They may appear either as extended processes or as stained circular cross sections of small caliber (Figs. 6A and 6B). Such connecting processes are seen as well in transverse sections through fiber tracts, but the multiple elon-
gated processes occur only in the horizontal sections, in which the myelin segments are cut longitudinally. The second population of LacZ1 cells consists of small dense cells that correspond in morphology and location to the type I oligodendrocytes of Rio Hortega (25). In silver-stained preparations, these give rise to tenuous processes which, however, may be too fine to accommodate the LacZ reaction product and are, therefore, not well demonstrated in our preparations. In view of the inflammation that follows spinal cord trauma, we also considered the possibility that some of these small dense cells could represent macrophages which engulfed LacZ1 donor cells that did not survive and in which the bacterial enzyme remained sufficiently active to yield reaction product. The size and morphology of these cells is, however, not consistent with that of macrophages laden with cell fragments. Moreover, when sections were immunostained with HAM56, an antibody that recognizes macrophages, the small ovoid LacZ1 cells were HAM562, while a popula-
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FIG. 6. (A) Horizontal section through dorsal columns showing a pair of LacZ1 cells, ,6–9 µm in diameter, associated with slender, relatively straight longitudinally oriented LacZ1 processes (vertical arrows) measuring ,1–2 µm in diameter and ,30 µm in length, consistent with myelin segments. Two other stained processes (arrowheads) are not clearly connected to these cells. Diagonal arrows indicate obliquely and transversely cut processes. (B) Horizontal section through dorsal columns showing LacZ1 cells, one of which appears to give rise to a longitudinal process ,60 µm long and 2–3 µm wide, which ends abruptly, consistent with a myelin segment. Vertical arrows indicate putative nodes of Ranvier. Periodic densities in the left half of this segment, also visible in A, could represent cytoplasmic expansions. Diagonal arrows show obliquely and transversely cut processes.
tion of LacZ2 cells that had the morphology of macrophages was HAM561 (D. C. Miller et al., unpublished data). Thus we have no evidence supporting the hypothesis that these LacZ1 cells are macrophages. Finally, recent studies have shown evidence for apop-
tosis in endogenous cells of the spinal cord following trauma (13), introducing the possibility that local conditions could induce apoptosis in transplanted cells as well. Thus, some of the small LacZ1 cells lacking apparent processes could be donor glia that differenti-
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ated to the point of MBP expression and subsequently underwent apoptosis with accompanying loss of processes. It is of interest, however, that very similar, small LacZ1 cells occur in the adult transgenic mouse spinal cord as well (Fig. 1B) scattered through both gray and white matter and also associated with blood vessels. Since there is no reason to postulate apoptosis in the normal control, cells of this type, in both the normal and traumatized spinal cord, could simply represent the classically described type I oligodendrocytes known to be associated with neurons in gray matter and with blood vessels and which are also present in white matter (25). The distribution of LacZ1 cells found in the present study (Fig. 2) indicates that the transplanted glia move rostrocaudally over considerable distances. To some extent this movement could be passive, within fluidfilled cystic defects resulting from the trauma. Their presence within intact regions of gray matter, however, where cellular processes are tightly packed, indicates that the cells are also capable of active migration through the tissue. In view of their association with blood vessels, these cells may also move through perivascular spaces, as Vignais et al. have proposed (27). The paucity of LacZ1 cells in intact white matter suggests, in contrast, that they avoid migrating into normally myelinated regions or fail to differentiate there. The fact that LacZ1 cells tend to become concentrated at the defects in myelinated fiber tracts that develop following the trauma could indicate that the transgenic cells move more readily through such defects. Alternatively, the cells may be drawn to such sites chemotactically, or may be retained there, by cytokines, adhesion molecules, or other factors released by the damaged tissue itself or by macrophages that invade secondarily. The cells may also proliferate to a greater extent at these sites than elsewhere or may be induced to mature there to the point of LacZ expression and myelin formation. Glial transplants from the same transgenic mouse line have been shown previously to form ultrastructurally normal myelin in the myelin-deficient rat spinal cord (21), and it is therefore highly likely that the same cells are capable of myelin formation around rat spinal cord axons in the trauma model as well. The demonstration of 30- to 60-µm LacZ1 segments in horizontal sections supports the conclusion that at least some of the transplanted cells find their way to receptive axons and do in fact form myelin segments. Transplantation of cultured glial cell suspensions is thus a potentially useful approach to replacing oligodendrocytes in traumatized spinal cord. Such transplants could remyelinate and restore functions lost as a result of demyelination secondary to trauma. Success with xenografted glia suggests, in addition, that the source
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of the cells transplanted need not be restricted to donors of the same species. Further studies will be required to assess long-term survival of LacZ1 transplanted glial cells and the myelin-like segments produced by them, to determine the optimal parameters for glial transplantation in this model and to determine whether behavioral improvement results from this procedure. ACKNOWLEDGMENTS The authors are indebted to Ron Morella for expert technical assistance. A preliminary report of this study has been presented (24). This work was supported by grants from the Paralyzed Veterans of America Spinal Cord Research Foundation and the National Multiple Sclerosis Society.
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