The effect of immunosuppressive protocols on spontaneous CNS remyelination following toxin-induced demyelination

Journal of Neuroimmunology 119 Ž2001. 261–268 www.elsevier.comrlocaterjneuroim

The effect of immunosuppressive protocols on spontaneous CNS remyelination following toxin-induced demyelination P.M. Smith, R.J.M. Franklin ) Department of Clinical Veterinary Medicine, UniÕersity of Cambridge, Madingley Road, Cambridge CB3 0ES, UK Received 2 May 2001; received in revised form 11 July 2001; accepted 16 July 2001

Abstract Glial cell transplantation is a potential therapy for human demyelinating disease, though obtaining large numbers of human myelinating cells for transplantation remains a major stumbling block. Autologous transplantation is currently not possible, since the adult human CNS is not a good source of oligodendrocyte precursors, and long-term immunosuppression of engrafted allogeneic or xenogeneic cells is therefore likely to be necessary. Immunosuppressive drugs may need to be used in situations where more recent, active areas of demyelination are undergoing endogenous remyelination. It is therefore pertinent to establish the extent to which immunosuppressive protocols will suppress spontaneous remyelination. In order to investigate this issue, we created demyelinating lesions in the spinal cord of adult rats and compared the extent of remyelination in animals receiving different immunosuppressive treatments. In animals given only cyclosporin A, there was no difference in the extent of either Schwann cell or oligodendrocyte remyelination of ethidium bromide-induced demyelinating lesions. However, in animals given cyclophosphamide, either alone or in combination with cyclosporin, there was a significant reduction in the extent of oligodendrocyte-mediated remyelination. These results demonstrate that cyclophosphamide is deleterious to oligodendrocyte remyelination and for this reason should be used with caution in patients with demyelinating disease. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Remyelination; Demyelination; Cyclophosphamide; Cyclosporin; Transplantation; Multiple sclerosis

1. Introduction A possible future therapy for multiple sclerosis ŽMS. is the transplantation of cultured glial cells that are capable of forming new myelin. Studies using experimental animals demonstrate that transplanted glial cells are able to remyelinate demyelinated axons ŽDuncan et al., 1997; Blakemore and Franklin, 2000.. Furthermore, transplantmediated remyelination leads to a restoration of saltatory impulse conduction and an improvement in the functional impairment associated with focal demyelination ŽHonmou et al., 1996; Jeffery et al., 1999.. Currently, the most suitable cells for effecting CNS remyelination following transplantation are oligodendrocyte precursors, cells that are committed to becoming oligodendrocytes but which remain capable of extensive proliferation and migration under appropriate conditions ŽWarrington et al., 1993; Zhang et al., 1998; Smith and Blakemore, 2000.. Although ) Corresponding author. Tel.: q44-1223-337642; fax: q44-1223337610. E-mail address: [email protected] ŽR.J.M. Franklin..

oligodendrocyte precursors can be readily isolated from the developing CNS ŽAvellana-Adalid et al., 1996; Archer et al., 1997; Satoh and Kim, 1994., obtaining large numbers of cells required for transplantation from the adult human CNS is currently not possible, eliminating the possibility of autologous transplantation and raising the prospect that allogeneic or xenogeneic sources of cells will be required if transplantation is to become a clinical reality. Since immunosuppressants will be required to prevent rejection of engrafted cells, it will be important to determine what effect immunosuppressant drugs might have on any non-transplanted demyelinating lesions within the same individual and, more specifically, to ensure that active remyelination within acute demyelinating lesions is not impeded. Cyclosporin A currently forms the mainstay of immunosuppressant protocols, both in experimental and clinical situations. Cyclosporin A acts by interfering with the signalling cascade that follows T cell receptor stimulation, preventing transcription of genes necessary for T cell activation ŽSchreiber, 1991. and both suppressing cellmediated immunity and limiting T cell help for antibody-

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 0 1 . 0 0 3 9 6 - 4

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producing B cells. However, evidence from studies of organ rejection suggests that suppression of the T cell response alone is insufficient to prevent graft rejection in many circumstances, so that cyclosporin will need to be used in combination with other drugs for immunosuppression to be effective ŽLim et al., 1991; van den Bogaarde et al., 1991; Davis et al., 1996; Kobayashi et al., 1997; Miyatake et al., 1998.. One combination that may prove effective in MS patients transplanted with allogeneic or xenogeneic glial cells is that of cyclosporin and cyclophosphamide. Animals with cardiac xenografts demonstrate markedly extended graft survival when treated with both drugs over treatment with cyclosporin alone ŽHasan et al., 1992., indicating that cyclophosphamide overcomes non-T cell dependent rejection responses. Since both cyclosporin and cyclophosphamide have been used in the primary treatment of MS to suppress the immune-mediated destruction of myelin ŽCompston, 1998., it is possible that such a treatment protocol may both prevent graft rejection and reduce the severity or incidence of new demyelinating lesions. In order to investigate the effect of cyclosporin and cyclophosphamide on endogenous remyelination in the CNS, we have examined toxin-induced demyelinating lesions in the spinal cord of adult rats and compared the extent of remyelination in animals receiving different combinations of these drugs.

2. Materials and methods 2.1. Creation of focal demyelinating lesions Focal demyelinating lesions were created in the spinal cord of 50 adult female Sprague–Dawley rats Ž180–200 g.. The animals were anaesthetised using halothane and oxygen and a dorsal laminectomy carried out at the level of the thoracolumbar junction. One microliter of a solution of 0.1% ethidium bromide in saline was then injected into the dorsal funiculus using a glass micropipette-tipped Hamilton syringe held in a micromanipulator, and tissues were surgically closed in routine fashion. All animals were given the analgesic carprofen ŽRimadyl; Pfizer, Sandwich, Kent, UK; 10 mgrkg; s.c.. and treatment with cyclosporin andror cyclophosphamide was initiated 2 days following surgery. Cyclosporin ŽSandimmun; Sandoz, Camberley, Surrey, UK. was administered subcutaneously at a dose of 15 mgrkg, daily throughout the course of the experiment. Cyclophosphamide ŽEndoxana; Pharmacia and Upjohn, Milton Keynes, Beds, UK. was administered by intraperitoneal injection at a dose rate of 20 mgrkg on days 2, 5, 8 and 11 following surgery. This protocol has proven to be an effective immunosuppressive regime when used in conjunction with cyclosporin, while avoiding the development of the side-effects that accompany daily treatment ŽHasan et al., 1992; PMS, unpublished observations..

2.2. Tissue processing and histological analysis The rats were killed by perfusion fixation either 21, 28 or 42 days following ethidium bromide injection. The animals were deeply anaesthetised with pentobarbitone ŽSagatal; Rhone Merieux, Harlow, Essex, UK; i.v.. and perfused via the aorta with 4% glutaraldehyde in phosphate buffer. The thoracic and lumbar spinal cord was removed and the isolated cord immersed for a minimum of 1 h in 4% glutaraldehyde. The section of spinal cord containing the demyelinating lesion was cut into coronal sections and washed in phosphate buffer prior to immersion in 2% osmium tetroxide at 4 8C overnight. The tissue blocks were then dehydrated through sequential ethanol washed and embedded in TAAB resin. One-micron sections were cut from hardened resin blocks and stained using toluidine blue and examined using light microscopy. Schwann cell and oligodendrocyte remyelinated axons could be readily distinguished at the level of the light microscope using previously documented criteria ŽCrang and Blakemore, 1992., enabling the proportion of dorsal column axons remyelinated by each cell type to be estimated by a blinded observer. The extremities of the lesion were not scored, so as to avoid any bias arising through the preponderance of oligodendrocyte remyelination in these regions. In addition, the small diameter axons of the corticospinal tract were not included in the analysis, since they are invariably remyelinated by oligodendrocytes but were not demyelinated in all animals. The mean percentage of oligodendrocyte and Schwann cell remyelinated axons was then calculated for each animal and the different groups of animals were compared using standard statistical analyses. In the first experiment, a Kruskal–Wallis test was used to identify significant variation between the different groups, followed by a pairwise comparison of individual groups with control groups ŽMann–Whitney.. In the other experiments, the Mann–Whitney test was used to identify significant differences between the control group and treatment group. A value of p F 0.05 was taken as statistically significant.

3. Results 3.1. Cyclophosphamide is deleterious to oligodendrocyte remyelination In order to investigate the effect of cyclosporin and cyclophosphamide on spontaneous CNS remyelination, we created focal demyelinating lesions in the dorsal funiculus of the spinal cord in 20 adult rats and divided animals into four groups. Group A received cyclophosphamide alone, group B received both cyclophosphamide and cyclosporin, group C received cyclosporin alone and group D remained untreated. Cyclosporin was administered at a dose of 15

P.M. Smith, R.J.M. Franklinr Journal of Neuroimmunology 119 (2001) 261–268

mgrkg subcutaneously, a dose that we have previously found to be effective in preventing rejection of allogeneic cells. Cyclophosphamide was injected in accordance with

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the protocol described by Hasan et al. Ž1992., at a dose rate of 20 mgrkg every third day for four treatments. This regimen significantly improves the survival of organ and

Fig. 1. Variation in the extent of oligodendrocyte remyelination at 3 weeks following ethidium bromide-induced demyelination of the spinal cord. Cross-section of the spinal cord at the level of the thoracolumbar junction taken from a control animal ŽA and C. and an animal treated with cyclophosphamide ŽB and D.. A large demyelinating lesion can be clearly identified in both sections ŽA and B., occupying the majority of the dorsal funiculus. Many of the axons in each section are pale-stained due to the absence of myelin, though a darker-stained region of Schwann cell-remyelinated axons can be identified in the centre of each lesion Žhighlighted by the dotted line.. Dark-stained macrophages filled with myelin debris are scattered throughout both lesions. C and D show higher power views of the same sections taken from the periphery of the lesion, adjacent to the grey matter of the dorsal horns Ž GM .. In C, Schwann cell-remyelinated axons are present on the right of the section and can be easily distinguished from the large numbers of oligodendrocyte-remyelinated axons seen in between these and the grey matter Ž OL.. A similar region from the cyclophosphamide-treated animal ŽD. reveals that, despite the similar extent of Schwann cell remyelination, there is no oligodendrocyte remyelination in this section and axons around the periphery of the lesion remain demyelinated Ž DM .. Scale bars: A and B, 100 mm; C and D, 50 mm.

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Table 1 Mean percentage of axons remyelinated by oligodendrocytes and Schwann cells 3 weeks following demyelination Ž%. Oligo RM Significancea % SrC RM

Group

Treatment

A Ž ns 4. B Ž ns 5. C Ž ns 5. D Ž ns 5.

CyP 4.4"0.89 CyP and CyA 4.0"0.80 CyA 12"2.0 mock 16"4.2 injection

ps 0.0159 ps 0.0079 ps 0.6905

24"9.3 27"5.0 42"4.8 48"4.1

a

Significance of a pairwise comparison between the percentage of oligodendrocyte remyelination in treatment and control groups. Variation in the extent of Schwann cell remyelination between the different groups was not significant.

cellular xenografts ŽHasan et al., 1992; PMS, unpublished observations., while minimising the side-effects that accompany prolonged treatment with anti-mitotic agents. In all animals, the demyelinating lesion extended up to 4 mm cranially and caudally from the site of ethidium bromide injection, occupying the majority of the dorsal funiculus in the central blocks of the lesion and tapering towards the tips. Both demyelinated and remyelinated axons could be clearly identified in all lesions, with debris-laden macrophages interspersed throughout. In the centre of the lesion, Schwann cell remyelination predominated, whereas oligodendrocyte remyelination was restricted to the more peripheral regions, in the ventral and lateral parts of the dorsal funiculus and at the cranial and caudal limits of the lesion ŽFig. 1.. In lesions in which the small diameter axons of the corticospinal tract had been demyelinated, they were invariably remyelinated by oligodendrocytes and not by Schwann cells. In addition to remyelinated axons, a significant proportion of the axons remained demyelinated. In some areas, demyelinated axons were closely apposed to one another in clusters, and in other regions, they were less densely packed and were separated by extracellular space. Lesions were scored by estimating the proportion of demyelinated, oligodendrocyte-remyelinated and Schwann cell-remyelinated axons in each lesion. So as to eliminate any bias arising through the predominance of oligodendrocyte remyelination in the tips of the lesion, counting was only carried out on central

Fig. 2. Extent of oligodendrocyte ŽA. and Schwann cell remyelination ŽB. 3 weeks following demyelination using ethidium bromide. Following demyelination, animals were treated with cyclophosphamide alone Žgroup A., cyclophosphamide and cyclosporin Žgroup B., cyclosporin alone Žgroup C. or mock injections Žgroup D.. Animals in groups A and B demonstrated a statistically significant reduction Ž p- 0.05. in the extent of oligodendrocyte remyelination. ŽC. A comparison of the extent of oligodendrocyte and Schwann cell remyelination in animals treated with a short course of cyclophosphamide at 6 weeks following ethidium bromide-induced demyelination revealed no significant difference from controls. Each point represents the mean remyelination score from an individual animal. Abbreviations: CyP, cyclophosphamide treated group; SrC RM, Schwann cell remyelination; Oligo RM, oligodendrocyte remyelination.

blocks, where the cross-sectional area of the lesion was maximal. In addition, small diameter corticospinal axons were excluded from the study, since they were not demyelinated in all animals. There were pronounced differences in the extent of remyelination in tissue blocks from different animals, rang-

P.M. Smith, R.J.M. Franklinr Journal of Neuroimmunology 119 (2001) 261–268 Table 2 Mean percentage of axons remyelinated by oligodendrocytes and Schwann cells 6 weeks following demyelination Treatment

Ž%. Oligo RM

Significancea

Ž%. SrC RM

CyP Ž ns 5. Mock injection Ž ns 5.

29.2"2.3 30.8"2.1

ps 0.5476

27.2"5.4 47.7"5.1

a

Significance of a pairwise comparison between the percentage of oligodendrocyte remyelination in treatment and control groups. Variation in the extent of Schwann cell remyelination between the different groups was not significant.

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no greater than at 3 weeks in either the control group or in the group treated with cyclophosphamide. However, the proportion of oligodendrocyte-remyelinated axons was significantly greater at 6 weeks in both the control and cyclophosphamide-treated groups of animals, indicating that oligodendrocyte remyelination had progressed in both untreated and treated animals over this period ŽTables 1 and 2; p s 0.0317, 0.0159, respectively.. There was no difference in the proportion of either Schwann cell- or oligodendrocyte-remyelinated axons between the cyclophosphamide-treated animals and the control animals at 6 weeks, indicating that the impairment of oligodendrocyte remyelination was not permanent ŽTable 2..

ing from areas showing almost complete remyelination of the demyelinated axons to those in which there were almost no remyelinated axons. The percentages of demyelinated and remyelinated axons are shown in Table 1. A Kruskal–Wallis analysis of the proportions of Schwann cell and oligodendrocyte-remyelinated axons revealed that there was significant variation in the extent of oligodendrocyte remyelination in the different groups of animals. Although the extent of Schwann cell remyelination varied between the different groups, this was not statistically significant. A subsequent pairwise comparison of each treatment group with the control group identified that animals in groups A Žcyclophosphamide only. and B Žcyclophosphamide and cyclosporin. showed significantly less oligodendrocyte remyelination than control animals, indicating a detrimental effect on oligodendrocyte remyelination ŽTable 1, Fig. 2.. Animals in group C Žcyclosporin only. were not significantly different from control animals, indicating that treatment with cyclosporin alone had no effect on remyelination. Furthermore, group B animals were no worse than group A animals, indicating that the use of cyclosporin alongside cyclophosphamide was no more deleterious to oligodendrocyte remyelination than using cyclophosphamide alone ŽTable 1.. There was no obvious difference in the size of the lesions in the different experimental groups, which was best estimated by examining the area of intact white matter in the dorsal funiculus around the lesion. 3.2. Cyclophosphamide impairment of remyelination is reÕersible The reduced extent of oligodendrocyte remyelination in animals treated with a short course of cyclophosphamide could be either from a permanent or a reversible effect on endogenous remyelinating capability. In order to distinguish these two possibilities, we examined the extent of remyelination 6 weeks following ethidium bromide injection in two groups of animals, one given a course of four cyclophosphamide injections on days 2, 5, 8 and 11 following demyelination and the other given mock intraperitoneal injections according to the same schedule. The extent of Schwann cell remyelination in these animals was

Fig. 3. In order to examine the influence of stress on endogenous CNS remyelination, we created demyelinating lesions in the spinal cord and examined the extent of remyelination 3 weeks ŽA. and 4 weeks ŽB. later. There was no significant difference in the extent of either Schwann cell or oligodendrocyte remyelination at either time point. Abbreviations: SrC RM, Schwann cell remyelination; Oligo RM, oligodendrocyte remyelination.

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Table 3 Mean percentage of axons remyelinated by oligodendrocytes and Schwann cells in mock-injected animals Survival Time

Treatment

3 weeks

mock injection Ž n s 5. control Ž n s 4. mock injection Ž n s 5. control Ž n s 5.

4 weeks

a

Significancea

Ž%. SrC RM

7.6 " 1.6

p s 0.413

36.6 " 5.1

12.7 " 3.8 12.2 " 0.8

p s 0.841

46.2 " 7.4 33.2 " 5.0

Ž%. Oligo RM

14.2 " 2.5

33.7 " 2.1

Significance of a pairwise comparison between the percentage of oligodendrocyte remyelination in the control and mock-injected groups of animals.

3.3. Daily mock injections do not inhibit oligodendrocyte remyelination It has previously been shown that treatment with exogenously administered glucocorticoids can alter the rate of remyelination following lysolecithin-induced demyelination ŽTriarhou and Herndon, 1986; Pavelko et al., 1998., raising the possibility that the stress of daily injections— mock or otherwise—might raise endogenous glucocorticoid levels and therefore effect remyelination. In order to address this hypothesis, we again created demyelinating lesions in the spinal cord of 10 adult rats and assigned animals to groups given either daily mock injections for 3 weeks, or no injection at all. The proportion of axons remyelinated by Schwann cells and oligodendrocytes was scored as detailed previously and the results were analysed using the Mann–Whitney test. This demonstrated that there was no significant difference in either the proportion of axons remyelinated by Schwann cells or by oligodendrocytes, suggesting that the daily injections had no effect on the efficiency of remyelination ŽFig. 3, Table 3.. However, since the effect of stress on remyelination is likely to be small, and because oligodendrocyte remyelination was relatively poor at 3 weeks, it is possible that resolving such a subtle effect was beyond the power of this experiment. In an attempt to accentuate any possible effect of stress, we therefore repeated this experiment, allowing the animals to survive for 4 weeks, when oligodendrocyte remyelination is more extensive ŽFig. 3.. In addition, we ensured that control animals receiving no injection were housed in a separate room from the mock-injected animals, so as to eliminate the possibility that group distress caused by the injection procedure might contribute to individual stress. This study once again demonstrated that there was no significant difference in the extent of either Schwann cell or oligodendrocyte remyelination ŽTable 3..

4. Discussion In this study, we investigated the effects on endogenous CNS remyelination of treatment with the immunosuppressive drugs cyclosporin and cyclophosphamide, and found

that cyclophosphamide caused a suppression of remyelination that was reversible once the treatment was stopped. However, neither cyclosporin nor stress altered remyelination. Our results, although broadly in support of a morphological study by Bondan et al. Ž2000., are in contrast with an earlier study, which demonstrated that cyclophosphamide was able to substantially increase the area of remyelination in the spinal cord following virus-induced demyelinating disease ŽRodriguez and Lindsley, 1992.. This effect of cyclophosphamide was considered to arise through the suppression of T cell-mediated interference with oligodendrocyte remyelination. A critical difference between Theiler’s virus infection and ethidium bromide induced demyelination is that the latter causes demyelination through direct toxicity to oligodendrocytes ŽYajima and Suzuki, 1979. and lymphocytes are not a prominent feature of gliotoxin-induced demyelinating lesions ŽFernandes et al., 1998; Ousman and David, 2000.. The absence of an inhibitory role for T cells in the ethidium bromide model, is reinforced by the failure of cyclosporin—a potent T cell suppressant—to have any significant effect on remyelination in our experiments ŽTable 1.. These results suggest that cyclophosphamide may have some benefit in permitting remyelination to occur in the presence of a sustained demyelinating T cell presence but that once an environment is permissive to remyelination, remyelination is rendered less efficient than would otherwise be the case. Cyclophosphamide is a cytotoxic drug that is able to alkylate DNA and therefore prevent duplication of the genome in dividing cells. Since proliferation of oligodendrocyte progenitors is a prerequisite for CNS remyelination ŽBlakemore and Patterson, 1978; Prayoonwiwat and Rodriguez, 1993., cyclophosphamide may have restricted oligodendrocyte progenitor division during the treatment period and therefore delayed the generation of adequate numbers of remyelinating cells. Furthermore, evidence from in vitro studies indicates that oligodendrocyte lineage cells are particularly poor at repairing damage to their mitochondrial DNA arising through exposure to alkylating agents ŽLedoux et al., 1998.. This shortcoming makes them prone to undergo apoptosis following alkylation and could make dividing oligodendrocyte progenitors

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exquisitely sensitive to the toxic effects of cyclophosphamide and therefore markedly reduce the rate of remyelination. Another possibility is that the effect of cyclophosphamide on remyelination is due to an indirect action on oligodendrocyte lineage cells. Remyelination has been postulated as a two-stage process ŽFranklin and Hinks, 1999., beginning with the recruitment of nearby oligodendrocyte progenitors, which proliferate and migrate into the area of demyelination ŽFranklin et al., 1997; Gensert and Goldman, 1997. before differentiating into mature oligodendrocytes. With age, the rate of remyelination slows and this is associated with a delay in the upregulation of PDGF-A and a delay in the onset of TGF-b1 and IGF-1 expression, suggesting that these growth factors play an integral role in the coordination of remyelination ŽHinks and Franklin, 2000.. The temporal and spatial correlation of these altered growth factor profiles in elderly animals with expression of the macrophage scavenger-B receptor suggests that macrophages are a major source of these growth factors and may play a pivotal role in remyelination. This therefore raises the possibility that cyclophosphamide might interfere with remyelination by diminishing the macrophage response following demyelination. However, the effect of the macrophage response in models of demyelinating disease remains a moot point. Following lysolecithin-induced demyelination, an increase in the rate of remyelination in animals treated with methylprednisolone has been associated with a decrease in the presence of debris-filled macrophages, suggesting that they might impede remyelination ŽPavelko et al., 1998.. In contrast, others have drawn attention to the close association between a robust macrophage response and efficient remyelination and inferred a causal link between the two ŽLudwin, 1980; Graca and Blakemore, 1986; Morell et al., 2000.. Moreover, removing the monocyte contribution to macrophages within toxin-induced demyelinating lesions leads to a decrease in oligodendrocyte remyelination ŽKotter et al., in press.. This study also showed that the Schwann cell component of remyelination was unaffected by monocyte depletion, indicating that the absence of an effect on Schwann cell remyelination in the cyclophosphamide-treated animals in the current study is compatible with an effect on macrophages. 4.1. Clinical implications The reduction in the rate of oligodendrocyte remyelination during treatment with cyclophosphamide indicates that this drug is not an appropriate component of immunosuppressive regimes designed to prevent the rejection of transplanted glial cells. Cyclosporin forms the mainstay of current immunosuppressive regimes, in both experimental and clinical situations. Since it can be used in conjunction with a number of other drugs to prevent graft rejection, this deleterious effect of cyclophosphamide should not

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significantly hinder the development of glial cell transplantation as a therapy for clinical demyelinating disease. However, cyclophosphamide is also used in the primary treatment of multiple sclerosis, to restrict immune-mediated damage to CNS myelin. Clinical trials carried out over the last 20 years suggest that intermittent ‘pulse’ therapy with cyclophosphamide can generally be well-tolerated ŽLikosky et al., 1991; Martin et al., 1997. but that the benefits of treatment are limited ŽHauser et al., 1983; Killian et al., 1988. or absent ŽLikosky et al., 1991. and that it may even have a detrimental effect ŽThe Canadian Cooperative Multiple Sclerosis Study Group, 1991.. Our observations indicate that a side-effect of this treatment might be to delay remyelination of acutely demyelinating lesions, which could prolong the period over which demyelinated axons are susceptible to damage by inflammatory mediators and contribute to long-term disability.

Acknowledgements We should like to thank Clare Ready for excellent technical assistance. This work was supported by the British Trust for the Myelin Project ŽPMS. and the Wellcome Trust.

References Archer, D.R., Cuddon, P.A., Lipsitz, D., Duncan, I.D., 1997. Myelination of the canine central nervous system by glial cell transplantation: a model for repair of human myelin disease. Nat. Med. 3, 54–59. Avellana-Adalid, V., Nait-Oumesmar, B., Lachapelle, F., Baron-Van Evercooren, A., 1996. Expansion of rat oligodendrocyte progenitors into proliferative AoligospheresB that retain differentiation potential. J. Neurosci. Res. 45, 558–570. Blakemore, W.F., Franklin, R.J.M., 2000. Transplantation options for therapeutic central nervous system remyelination. Cell Transplant. 9, 289–294. Blakemore, W.F., Patterson, R.C., 1978. Suppression of remyelination in the CNS by X-irradiation. Acta Neuropathol. 42, 105–113. Bondan, E.F., Lallo, I.L., Sinhorini, I.L., Pereira, L.A.V., Graca, D.L., 2000. The effect of cyclophosphamide on brainstem remyelination following local ethidium bromide injection in Wistar rats. J. Submicrosc. Cytol. Pathol. 32, 603–612. Compston, D.A.S., 1998. Treatment and management of multiple sclerosis. In: Compston, D.A.S., Ebers, G.C., Lassman, H., McDonald, I., Matthews, B., Wekerle, H. ŽEds.., Multiple Sclerosis. 3rd edn. Churchill Livingstone, London, pp. 437–583. Crang, A.J., Blakemore, W.F., 1992. Transplantation of glial cells into areas of demyelination in the adult rat spinal cord. In: Dunnet, S.B., Bjorklund, A. ŽEds.., Neural Transplantation: a Practical Approach. Oxford Univ. Press, Oxford, pp. 105–123. Davis, E.A., Pruitt, S.K., Greene, P.S., Ibrahim, S., Lam, T.T., Levin, J.L., Baldwin, W.M., Sanfilippo, F., 1996. Inhibition of complement, evoked antibody and cellular response prevents rejection of pig to primate cardiac xenografts. Transplantation 62, 1018–1023. Duncan, I.D., Grever, W.E., Zhang, S.C., 1997. Repair of myelin disease: strategies and progress in animal models. Mol. Med. Today 3, 554– 561. Fernandes, C.G., Graca, D.L., Pereira, L.A., 1998. Inflammatory response

268

P.M. Smith, R.J.M. Franklinr Journal of Neuroimmunology 119 (2001) 261–268

of the spinal cord to multiple episodes of blood–brain barrier disruption and toxic demyelination in Wistar rats. Braz. J. Med. Biol. Res. 31, 933–936. Franklin, R.J.M., Hinks, G.L., 1999. Understanding CNS remyelination: clues from developmental and regeneration biology. J. Neurosci. Res. 58, 207–213. Franklin, R.J.M., Gilson, J., Blakemore, W.F., 1997. Local recruitment of remyelinating cells in the repair of demyelination in the central nervous system. J. Neurosci. Res. 50, 337–344. Gensert, J.M., Goldman, J.E., 1997. Endogenous progenitors remyelinate demyelinated axons in the adult CNS. Neuron 19, 197–203. Graca, D.L., Blakemore, W.F., 1986. Delayed remyelination in rat spinal cord following ethidium bromide injection. Neuropathol. Appl. Neurobiol. 12, 593–605. Hasan, R.I.R., van den Bogaarde, J., Forty, J., Wright, L., Wallwork, J., White, D.J.G., 1992. Prolonged survival of hamster to rat heart xenografts with cyclophosphamide therapy. Transplant. Proc. 24, 517–518. Hauser, S.L., Dawson, D.M., Lehrich, J.R., Beal, M.F., Kevy, S.V., Propper, R.D., Mills, J.A., Weiner, H.L., 1983. Intensive immunosuppression in progressive multiple sclerosis. A randomized, three-arm study of high-dose intravenous cyclophosphamide, plasma exchange, and ACTH. N. Engl. J. Med. 308, 173–180. Hinks, G.L., Franklin, R.J.M., 2000. Delayed changes in growth factor gene expression during slow remyelination in the CNS of aged rats. Mol. Cell. Neurosci. 16, 542–556. Honmou, O., Felts, P.A., Waxman, S.G., Kocsis, J.D., 1996. Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cells. J. Neurosci. 16, 3199–3208. Jeffery, N.D., Crang, A.J., O’Leary, M.T., Hodge, S.J., Blakemore, W.F., 1999. Behavioural consequences of oligodendrocyte progenitor cell transplantation into experimental demyelinating lesions in the rat spinal cord. Eur. J. Neurosci. 11, 1508–1514. Killian, J.M., Bressler, R.B., Armstrong, R.M., Huston, D.P., 1988. Controlled pilot trial of monthly intravenous cyclophosphamide in multiple sclerosis. Arch. Neurol. 45, 27–30. Kobayashi, T., Taniguchi, S., Neethling, F.A., Tose, A.G., Hancock, W.W., Ye, Y., Miekrasz, M., Kosanke, S., Wright, L.J., White, D.J.G., Cooper, D.K.C., 1997. Delayed xenograft rejection of pig to baboon cardiac transplants after cobra venom factor therapy. Transplantation 64, 1255–1261. Kotter, M.R., Setzu, A., Sim, F.J., van Rooijen, N., Franklin, R.J.M., 2001. Macrophage depletion impairs oligodendrocyte remyelination following lysolecithin-induced demyelination. Glia 35, 204–212. Ledoux, S.P., Shen, C.-C., Grishko, V.I., Fields, P.A., Gard, A.L., Wilson, G.L., 1998. Glial cell specific differences in response to alkylation damage. Glia 24, 304–312. Likosky, W.H., Fireman, B., Elmore, R., Eno, G., Gale, K., Goode, G.B., Ikeda, K., Laster, J., Mosher, C., Rozance, J., 1991. Intense immunosuppression in chronic progressive multiple sclerosis: the Kaiser study. J. Neurol., Neurosurg. Psychiatry 54, 1055–1060. Lim, S.M.L., Li, S.Q., Wee, A., Chong, S.M., Hu, C., Rauff, A., White, D.J.G., 1991. Both concordant and discordant heart xenografts are

rejected by athymic Žnude. rats with the same tempo as in T cell competent animals. Transplant. Proc. 23, 581–582. Ludwin, S.K., 1980. Chronic demyelination inhibits remyelination in the central nervous system. Lab. Invest. 43, 382–387. Martin, F., Lauwerys, B., Lefebvre, C., Devogelaer, J.P., Houssiau, F.A., 1997. Side-effects of intravenous cyclophosphamide pulse therapy. Lupus 6, 254–257. Miyatake, T., Sato, K., Takigami, K., Koyamada, N., Hancock, W.W., Bazin, H., Latinne, D., Bach, F.H., Soares, M.P., 1998. Complement fixing elicited antibodies are a major component in the pathogenesis of xenograft rejection. J. Immunol. 160, 4114–4123. Morell, P., Barrett, C.V., Mason, J.L., Toews, A.D., Hostettler, J.D., Knapp, G.W., Matsushima, G.K., 2000. Gene expression in brain during cuprizone-induced demyelination and remyelination. Mol. Cell. Neurosci. 12, 220–227. Ousman, S.S., David, S., 2000. Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord. Glia 30, 92–104. Pavelko, K.D., van Engelen, B.G., Rodriguez, M., 1998. Acceleration in the rate of CNS remyelination in lysolecithin-induced demyelination. J. Neurosci. 18, 2498–2505. Prayoonwiwat, N., Rodriguez, M., 1993. The potential for oligodendrocyte proliferation during demyelinating disease. J. Neuropathol. Exp. Neurol. 52, 55–63. Rodriguez, M., Lindsley, M.D., 1992. Immunosuppression promotes CNS remyelination in chronic virus-induced demyelinating disease. Neurology 42, 348–357. Satoh, J., Kim, S.U., 1994. Proliferation and differentiation of fetal human oligodendrocytes in culture. J. Neurosci. Res. 39, 260–272. Schreiber, S.L., 1991. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251, 283–287. Smith, P.M., Blakemore, W.F., 2000. Porcine neural progenitors require commitment to the oligodendrocyte lineage prior to transplantation in order to achieve significant remyelination of demyelinated lesions in the adult CNS. Eur. J. Neurosci. 12, 2414–2424. The Canadian Cooperative Multiple Sclerosis Study Group, 1991. The Canadian cooperative trial of cyclophosphamide and plasma exchange in progressive multiple sclerosis. Lancet 337, 441–446. Triarhou, L.C., Herndon, R.M., 1986. The effect of dexamethasone on L-alpha-lysophosphatidyl choline Žlysolecithin.-induced demyelination of the rat spinal cord. Arch. Neurol. 43, 121–125. van den Bogaarde, J., Aspinall, R., Wang, M.-Q., Cary, N., Lim, S., Wright, L., White, D., 1991. Induction of long-term suvival of hamster heart xenografts in rats. Transplantation 52, 15–20. Warrington, A.E., Barbarese, E., Pfeiffer, S.E., 1993. Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts. J. Neurosci. Res. 34, 1–13. Yajima, K., Suzuki, K., 1979. Ultrastructural changes of oligodendroglia and myelin sheaths induced by ethidium bromide. Neuropathol. Appl. Neurobiol. 5, 49–62. Zhang, S.-C., Lipsitz, D., Duncan, I.D., 1998. Self-renewing canine oligodendroglial progenitor expanded as oligospheres. J. Neurosci. Res. 54, 181–190.