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Survival of oligodendrocytes in chronic relapsing experimental autoimmune encephalomyelitis
Journal of the Neurological Sciences, 1984, 65:137-145 Elsevier
137
Survival of Oligodendrocytes in Chronic Relapsing Experimental Autoimmune Encephalomyelitis G.R. Wayne Moore, Ute Traugott and Cedric S. Raine Departments of Neuropathology, Neurology and Neuroscience and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (U.S.A.) (Received 19 December, 1983) (Revised, received 13 March, 1984) (Accepted 26 March, 1984)
SUMMARY
Demyelinated plaques of chronic relapsing experimental autoimmune encephalomyelitis (EAE) have been examined in Strain 13 guinea pigs. Oligodendrocytes could be identified within these lesions adjacent to naked axons and astrocytic processes. Oligodendrocytes were identified both ultrastructurally and immunocytochemically. Many of these cells showed bizarre shapes and myelin within their cytoplasm. The survival of oligodendrocytes within these lesions suggests that the myelin sheath, not the oligodendrocyte, is the primary target in autoimmune demyelination. A similar sequence of events has been proposed in multiple sclerosis, for which chronic relapsing EAE serves as a laboratory model. The persistence of myelinating cells in areas of chronic demyelination and gliosis might have significant reparatory implications.
Key words: A u t o i m m u n i t y - E x p e r i m e n t a l allergic encephalomyelitis - Multiple sclerosis - Myelin - Oligodendrocytes
INTRODUCTION
It has long been debated whether the myelin sheath or the oligodendrocyte, the cell responsible for myelin elaboration, is the primary target in the central nervous Supported in part by research grants NMSS i~G-1001-D-4 from the National Multiple Sclerosis Society; and USPHS grants NS-08952; NS-11920; and NS-07098. 0022-510X/84/$03.00 ~) 1984 Elsevier Science Publishers B.V.
138 system (CNS) demyelinating disorders, of which multiple sclerosis (MS) is the prime example. Previous reports addressing this problem have arrived at differing conclusions but it is clear that many were based upon chronically established lesions wherein active demyelination had long passed, while others were written at a time when the criteria for identification of the various glial types were not well defined (Lumsden 1955). It has now been established that oligodendroglial hyperplasia (Ibrahim and Adams 1963, 1965; Brown et al. 1982) occurs in these CNS disorders, and since this is usually seen at the periphery of the lesion, it is suggested that proliferating oligodendrocytes arise from cells in adjacent white matter. However, recently unequivocal survival of oligodendrocytes within chronically demyelinated areas in active MS has been reported (Raine et al. 1981b). The present report addresses this problem in CNS lesions from Strain 13 guinea pigs with chronic relapsing experimental autoimmune encephalomyelitis (EAE), an experimental model which most closely resembles MS (Raine and Stone 1977). Oligodendrocytes could be identified within plaques both by morphologic and immunocytochemical criteria. Many of these cells assumed peculiar shapes and contained abundant intracytoplasmic debris. The findings support the notion that the myelin sheath, and not the oligodendrocyte, is the initial target during immune-mediated CNS demyelination. MATERIALS AND METHODS The induction and clinical course of chronic relapsing EAE in Strain 13 guinea pigs has been described in detail previously (Stone and Lerner 1965; Raine et ai. 1974; Snyder et al. 1975). Essentially, 0.5~o ml of an emulsion of syngeneic spinal cord and complete Freund's adjuvant (CFA) was inoculated intracutaneously in the nuchal area of juvenile male Strain 13 guinea pigs and this produced a delayed onset paraparesis which invariably remitted. After 4-16 months of chronic relapsing or progressive neurologic illness, animals were perfused under ether anaesthesia with 4 ~o paraformaldehyde followed by 5.0~o glutaraldehyde both buffered to pH 7.4 with phosphate. Representative slices were taken from the brain and spinal cord, osmicated, dehydrated and embedded in Epon for electron microscopy (EM) as described previously (Raine et al. 1974). One-micron epoxy sections were stained with toluidine blue for light microscopy (LM) and thin sections for EM were stained with lead citrate and uranyl acetate. The latter were examined with a Siemens 101 transmission electron microscope. For immunocytochemistry, 1/~m epon sections were etched with 10~o H202 for 10 min or sodium ethoxide for 3-5 min. After appropriate rinses this was followed by incubation with 0.3} 0 H20 2, 3~o normal swine serum containing Tris/saline, rabbit antigalactocerebroside serum (Raine et al. 1981a) diluted 1:50, swine anti-rabbit Ig (1 : 10) and rabbit PAP (1 : 50) (Traugott, in prep.). RESULTS The presence of perivascular cuffs" of inflammatory cells, demyelinated axons, gliosis and remyelination in chronic demyelinated plaques of chronic relapsing EAE has
139
Fig. 1. One-#mEpon section of a plaque in which numerous demyelinatedaxons are seen. The cells which have prominent heterochromatin and a dark rim of relatively well delineated cytoplasm are probably oligodendrocytes(arrows). Immunocytochemistry(see Fig. 7) and electronmicroscopy(see Figs. 2 through 6) are required to demonstrate definitivelytheir oligodendroglialnature. In this section,astrocytesare also seen whichhave paler nuclei and lightercytoplasmwith ill-definedcell boundaries.Toluidineblue; x 1000.
been described in detail previously (Raine et al. 1974; Snyder et al. 1975). Within these lesions, cells with small nuclei rich in heterochromatin, the identification of which could not be previously discerned by LM, were apparent (Fig. 1). Under the EM, such cells fulfilled the ultrastructural criteria for oligodendrocytes (Fig. 2) being clearly distinguishable from the many fibrous astrocytes having a thin rim of well delineated, relatively electron-dense cytoplasm containing many 24-nm microtubules (Fig. 3) but no intermediate filaments, rounded nuclei with clumped heterochromatin which comprised most of the nuclear volume and associated prominent perinuclear cisterns. These cells could be found deep within the gliotic lesion adjacent to demyelinated axons and/or astroglial processes. Some of these oligodendrocytes contained myelin in varying amounts from simple whorls to large collections of electron-dense membranes (Figs. 4 and 5). In the latter case, the cell appeared to encompass the myelin material which on occasion occupied such a large portion of its cytoplasm that it encroached upon the nucleus. Such distorted cells were common (Fig. 5). While many of these were difficult to discern from macrophages, some were most likely oligodendroglia as evidenced by a rounded profile and 24-nm microtubules and an absence of intermediate filaments (Fig. 6). To substantiate the ultrastructural criteria, these cells were also stained by immunocytochemical means. One-/~m sections reacted against rabbit anti-GC serum (Raine et al. 1981a)
140
Fig. 2. Electron micrograph of an oligodendrocyte in a chronically demyelinated plaque. This cell is surrounded by astroglial cell processes with numerous intermediate filaments (arrows). A portion of a demyelinated axon is seen in the upper left. The area of oligodendroglial cytoplasm indicated by the asterisk is shown at higher magnification in Fig. 3. x 18000.
showed extensive amounts of positively labelled myelin within unreactive macrophages which were abundant in perivascular spaces (Fig. 7) and within the parenchyma. In addition, between naked axons within the lesion proper, small rounded cells were seen and these demonstrated reaction product both on the cell surface and within the cytoplasm as a rim surrounding an unstained nucleus (Fig. 7). In view of the proven specificity of anti-GC serum for oligodendroglia (Raft et al. 1978; N o r t o n et al. 1983) and C N S myelin, these cells were clearly oligodendrocytes. M a n y of these, as noted by electron microscopy, contained GC-positive myelin accumulations. Axons, astrocytes, blood vessels and macrophages failed to stain with anti-GC serum, supporting the specificity of the staining.
Fig. 3. High magnification of area of oligodendrocyte indicated by asterisk in previous figure (Fig. 2). Note the microtubules (arrows) in the thin rim of cytoplasm. No intermediate filaments are seen. x 80000.
Fig. 4. Low magnification electron micrograph of chronically demyelinated plaque showing numerous demyelinated axons and gliosis, in the midst of which is an oligodendrocyte containing whorls of myelin material, x 7200.
Fig. 5. This oligodendrocyte contains a large amount of myelin material which appears to be encroaching on and distorting the outline of the nucleus. Higher magnification of the region indicated by asterisk is shown in Fig. 6. × 22 500.
Fig. 6. High magnification of area indicated by asterisk in Fig. 5. Note the microtubules (arrows) and absence of intermediate filaments indicating this cell is oligodendroglial. × 80000.
143
Fig. 7. Staining ofa l-#m Epon section of a plaque similar to the one shown in Fig. 1 with anti-GC serum by the PAP technique. The oligodendrocytes(arrows) demonstrate a dense ring of reaction product on their cell membranes and a lighter stained rim of cytoplasm surrounding an unstained nucleus. Some of these cells are associated with intensely staining myelindroplets. Extensivedeposits of reaction product are seen over myelin debris within the cytoplasm of macrophages located in perivascular spaces or in the parenchyma.The background consists of faint outlines of unstained axons and astrocyticprocesses, x 1000. DISCUSSION The present study has demonstrated the persistence (and therefore, survival) of oligodendrocytes in chronic relapsing EAE plaques. The lesions studied were usually chronic and silent as indicated by extensive astroglial scarring but many demonstrated ongoing myelin breakdown products in perivascular and parenchymal macrophages. Within these lesions, oligodendrocytes were identified by their characteristic ultrastructural appearances including a cytoplasm which contained numerous microtubules but no intermediate filaments (Peters et al. 1976). This in contrast to the astrocyte wherein bundles of intermediate filaments predominate with microtubules occurring in lower numbers (Raine and Wisniewski 1970). Moreover, oligodendrocytes were identified immunocytochemically by their staining positively with anti-GC serum. The presence of galactocerebroside in the cell membrane has been reported to be specific for the oligodendrocyte (Raft et al. 1978). In addition to the classic morphologic appearance of oligodendrocytes, some cells containing large amounts of myelin debris, which on superficial examination might have been taken for macrophages, were identified as oligodendrocytes ultrastructurally by the presence of microtubules without intermediate filaments and immunocytochemically by positive staining for galactocerebroside. It is not clear whether these cells were actively producing or phagocytosing
144 myelin. Phagocytic activity by oligodendrocytes has been claimed previously on purely morphologic evidence (no immunocytochemistry) in an experimental study on degenerating optic nerves (Cook and Wisniewski 1973). Another alternative 0xplanation is that myelin debris within the present cells may have accumulated after ar~aputation of their connection with the myelin internode and their retraction into the cell soma. However, we believe its origin was probably due to the active production of myelin within the surviving oligodendrocyte. Synthesis of redundant myelin has been noted previously by oligodendrocytes in vitro (Norton et al. 1983). The separation of the cell from its internode during demyelination might eventually lead to its death in a manner akin to oligodendroglial dying-back (Hirano 1978; Ludwin and Johnson 1981). In any event, it is clear from the present work that at least some oligodendrocytes survive the immunologic attack on myelin and in this context, it appears that the myelin sheath and not the oligodendrocyte is the primary target in autoimmune demyelination. Oligodendrocyte survival has also been reported in chronic active MS (Raine et al. 1981b), canine distemper encephalomyelitis (Raine 1976) and murine chronic EAE (Brown et al. 1982). Other similarities between chronic relapsing EAE and the human condition, MS, have been reviewed in detail elsewhere (Raine 1983) and the survival of oligodendrocytes in well-established chronic lesions of these two disorders is yet another point of comparison. Factors important in the stimulation of these oligodendrocytes to proliferate and perhaps remyelinate adjacent axons might be of therapeutic interest in multiple sclerosis. ACKNOWLEDGEMENTS We thank Dr. Sanford Stone for continued collaboration; Everett Swanson, Miriam Pakingan, Howard Finch and Patricia Kennedy for technical assistance; and Nanci Legg for secretarial assistance. Dr. Moore is a fellow of the Multiple Sclerosis Society of Canada. REFERENCES Brown, A., D.E. McFarlin and C.S. Raine (1982) Chronologic neuropathologyof relapsing experimental allergic encephalomyelitisin the mouse, Lab. Invest., 46: 171-185. Cook, R. D. and H. M. Wisniewski(1973)The role of oligodendrogliaand astrogliain Walleriandegeneration of the optic nerve, Brain Res., 61: 191-2(r6. Hirano, A. (1978)A possiblemechanismof demyelinationin the Syrian hamster with hindlegparalysis,Lab. Invest., 38: 115-121. Ibrahim, M. Z. M. and C. W. M. Adams (1963) The relationship between enzyme activity and neuroglia in plaques of multiple sclerosis, J. Neurol. Neurosurg. Psychiat., 26:101-110. Ibrahim, M.Z.M. and C.W.M. Adams (1965) The relation between enzyme activityand neuroglia in early plaques of multiple sclerosis, J. Path. Bact., 90: 239-243. Ludwin, S. K. and E. S. Johnson (1981) Evidencefor a "dying-back"gliopathyin demyelinatingdisease,Ann. Neurol., 9: 301-305. Lumsden, C.E. (1955) In: D. McAlpine, N.D. Compston and C.E. Lumsden (Eds.), Multiple Sclerosis, Livingstone, Edinburgh, pp. 208-293. Norton, W.T., M. Farooq, K. L. Fields and C. S. Raine (1983) The long term culture of bulk-isolated bovine oligodendroglia from adult brain, Brain Res., 270:295-310. Peters, A., S. L. Palay and H. deF. Webster (1976) In: The Fine Structure of the Nervous System - - The Neurons and Supporting Cells, W.B. Saunders Co., Philadelphia, pp. 231-263.
145 Raft, M.C., R. Mirsky, K.L. Fields, R.P. Lisak, S.H. Dorfman, D.H. Silverberg, N.A. Gregson, S. Leibowitz and M. C. Kennedy (1978) Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture, Nature (Lond.), 274: 813-816. Raine, C. S. (1976) On the development of CNS lesions in natural canine distemper encephalomyelitis, J. Neurol. Sci:, 30: 13-28. Raine, C.S. (1983) Multiple sclerosis and chronic relapsing EAE - - Comparative ultrastructural neuropathology. In: J.F. Hallpike, C.W.M. Adams and W.W. Tourtellotte (Eds.), Multiple Sclerosis, Williams and Wilkins, Baltimore, MD, pp. 413-460. Raine, C. S. and S.H. Stone (1977) Animal model for multiple sclerosis - - Chronic experimental allergic encephalomyelitis in inbred guinea pigs, N.Y. State J. Med., 77: 1693-1696. Raine, C. S. and H. Wisniewski (1970) On the occurrence of microtubules within mature astrocytes, Anat. Rec., 167: 303-308. Raine, C.S., D.H. Snyder, M.P. Valsamis and S.H. Stone (1974) Chronic experimental allergic encephalomyelitis in inbred guinea pigs - - An ultrastructural study, Lab. Invest., 31: 369-380. Raine, C. S., A. B. Johnson, D.M. Marcus, A. Suzuki and M.B. Bornstein (1981a) Demyelination in vitro Absorption studies demonstrate that galactocerebroside is a major target, J. Neurol. Sci., 52: 117-131. Raine, C.S., L. Scheinberg and J.M. Waltz (1981b) Multiple sclerosis - - Oligodendrocyte survival and proliferation in an active lesion, Lab. Invest., 45: 534-546. Snyder, D. H., M. P. Valsamis, S. H. Stone and C. S. Raine (1975) Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis,J. Neuropath. Exp. Neurol., 34:209-221. Stone, S.H. and E.M. Lerner (1965) Chronic disseminated allergic encephalomyelitis in guinea pigs, Ann. N. Y. Acad. Sci., 122: 227-241. -
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137
Survival of Oligodendrocytes in Chronic Relapsing Experimental Autoimmune Encephalomyelitis G.R. Wayne Moore, Ute Traugott and Cedric S. Raine Departments of Neuropathology, Neurology and Neuroscience and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (U.S.A.) (Received 19 December, 1983) (Revised, received 13 March, 1984) (Accepted 26 March, 1984)
SUMMARY
Demyelinated plaques of chronic relapsing experimental autoimmune encephalomyelitis (EAE) have been examined in Strain 13 guinea pigs. Oligodendrocytes could be identified within these lesions adjacent to naked axons and astrocytic processes. Oligodendrocytes were identified both ultrastructurally and immunocytochemically. Many of these cells showed bizarre shapes and myelin within their cytoplasm. The survival of oligodendrocytes within these lesions suggests that the myelin sheath, not the oligodendrocyte, is the primary target in autoimmune demyelination. A similar sequence of events has been proposed in multiple sclerosis, for which chronic relapsing EAE serves as a laboratory model. The persistence of myelinating cells in areas of chronic demyelination and gliosis might have significant reparatory implications.
Key words: A u t o i m m u n i t y - E x p e r i m e n t a l allergic encephalomyelitis - Multiple sclerosis - Myelin - Oligodendrocytes
INTRODUCTION
It has long been debated whether the myelin sheath or the oligodendrocyte, the cell responsible for myelin elaboration, is the primary target in the central nervous Supported in part by research grants NMSS i~G-1001-D-4 from the National Multiple Sclerosis Society; and USPHS grants NS-08952; NS-11920; and NS-07098. 0022-510X/84/$03.00 ~) 1984 Elsevier Science Publishers B.V.
138 system (CNS) demyelinating disorders, of which multiple sclerosis (MS) is the prime example. Previous reports addressing this problem have arrived at differing conclusions but it is clear that many were based upon chronically established lesions wherein active demyelination had long passed, while others were written at a time when the criteria for identification of the various glial types were not well defined (Lumsden 1955). It has now been established that oligodendroglial hyperplasia (Ibrahim and Adams 1963, 1965; Brown et al. 1982) occurs in these CNS disorders, and since this is usually seen at the periphery of the lesion, it is suggested that proliferating oligodendrocytes arise from cells in adjacent white matter. However, recently unequivocal survival of oligodendrocytes within chronically demyelinated areas in active MS has been reported (Raine et al. 1981b). The present report addresses this problem in CNS lesions from Strain 13 guinea pigs with chronic relapsing experimental autoimmune encephalomyelitis (EAE), an experimental model which most closely resembles MS (Raine and Stone 1977). Oligodendrocytes could be identified within plaques both by morphologic and immunocytochemical criteria. Many of these cells assumed peculiar shapes and contained abundant intracytoplasmic debris. The findings support the notion that the myelin sheath, and not the oligodendrocyte, is the initial target during immune-mediated CNS demyelination. MATERIALS AND METHODS The induction and clinical course of chronic relapsing EAE in Strain 13 guinea pigs has been described in detail previously (Stone and Lerner 1965; Raine et ai. 1974; Snyder et al. 1975). Essentially, 0.5~o ml of an emulsion of syngeneic spinal cord and complete Freund's adjuvant (CFA) was inoculated intracutaneously in the nuchal area of juvenile male Strain 13 guinea pigs and this produced a delayed onset paraparesis which invariably remitted. After 4-16 months of chronic relapsing or progressive neurologic illness, animals were perfused under ether anaesthesia with 4 ~o paraformaldehyde followed by 5.0~o glutaraldehyde both buffered to pH 7.4 with phosphate. Representative slices were taken from the brain and spinal cord, osmicated, dehydrated and embedded in Epon for electron microscopy (EM) as described previously (Raine et al. 1974). One-micron epoxy sections were stained with toluidine blue for light microscopy (LM) and thin sections for EM were stained with lead citrate and uranyl acetate. The latter were examined with a Siemens 101 transmission electron microscope. For immunocytochemistry, 1/~m epon sections were etched with 10~o H202 for 10 min or sodium ethoxide for 3-5 min. After appropriate rinses this was followed by incubation with 0.3} 0 H20 2, 3~o normal swine serum containing Tris/saline, rabbit antigalactocerebroside serum (Raine et al. 1981a) diluted 1:50, swine anti-rabbit Ig (1 : 10) and rabbit PAP (1 : 50) (Traugott, in prep.). RESULTS The presence of perivascular cuffs" of inflammatory cells, demyelinated axons, gliosis and remyelination in chronic demyelinated plaques of chronic relapsing EAE has
139
Fig. 1. One-#mEpon section of a plaque in which numerous demyelinatedaxons are seen. The cells which have prominent heterochromatin and a dark rim of relatively well delineated cytoplasm are probably oligodendrocytes(arrows). Immunocytochemistry(see Fig. 7) and electronmicroscopy(see Figs. 2 through 6) are required to demonstrate definitivelytheir oligodendroglialnature. In this section,astrocytesare also seen whichhave paler nuclei and lightercytoplasmwith ill-definedcell boundaries.Toluidineblue; x 1000.
been described in detail previously (Raine et al. 1974; Snyder et al. 1975). Within these lesions, cells with small nuclei rich in heterochromatin, the identification of which could not be previously discerned by LM, were apparent (Fig. 1). Under the EM, such cells fulfilled the ultrastructural criteria for oligodendrocytes (Fig. 2) being clearly distinguishable from the many fibrous astrocytes having a thin rim of well delineated, relatively electron-dense cytoplasm containing many 24-nm microtubules (Fig. 3) but no intermediate filaments, rounded nuclei with clumped heterochromatin which comprised most of the nuclear volume and associated prominent perinuclear cisterns. These cells could be found deep within the gliotic lesion adjacent to demyelinated axons and/or astroglial processes. Some of these oligodendrocytes contained myelin in varying amounts from simple whorls to large collections of electron-dense membranes (Figs. 4 and 5). In the latter case, the cell appeared to encompass the myelin material which on occasion occupied such a large portion of its cytoplasm that it encroached upon the nucleus. Such distorted cells were common (Fig. 5). While many of these were difficult to discern from macrophages, some were most likely oligodendroglia as evidenced by a rounded profile and 24-nm microtubules and an absence of intermediate filaments (Fig. 6). To substantiate the ultrastructural criteria, these cells were also stained by immunocytochemical means. One-/~m sections reacted against rabbit anti-GC serum (Raine et al. 1981a)
140
Fig. 2. Electron micrograph of an oligodendrocyte in a chronically demyelinated plaque. This cell is surrounded by astroglial cell processes with numerous intermediate filaments (arrows). A portion of a demyelinated axon is seen in the upper left. The area of oligodendroglial cytoplasm indicated by the asterisk is shown at higher magnification in Fig. 3. x 18000.
showed extensive amounts of positively labelled myelin within unreactive macrophages which were abundant in perivascular spaces (Fig. 7) and within the parenchyma. In addition, between naked axons within the lesion proper, small rounded cells were seen and these demonstrated reaction product both on the cell surface and within the cytoplasm as a rim surrounding an unstained nucleus (Fig. 7). In view of the proven specificity of anti-GC serum for oligodendroglia (Raft et al. 1978; N o r t o n et al. 1983) and C N S myelin, these cells were clearly oligodendrocytes. M a n y of these, as noted by electron microscopy, contained GC-positive myelin accumulations. Axons, astrocytes, blood vessels and macrophages failed to stain with anti-GC serum, supporting the specificity of the staining.
Fig. 3. High magnification of area of oligodendrocyte indicated by asterisk in previous figure (Fig. 2). Note the microtubules (arrows) in the thin rim of cytoplasm. No intermediate filaments are seen. x 80000.
Fig. 4. Low magnification electron micrograph of chronically demyelinated plaque showing numerous demyelinated axons and gliosis, in the midst of which is an oligodendrocyte containing whorls of myelin material, x 7200.
Fig. 5. This oligodendrocyte contains a large amount of myelin material which appears to be encroaching on and distorting the outline of the nucleus. Higher magnification of the region indicated by asterisk is shown in Fig. 6. × 22 500.
Fig. 6. High magnification of area indicated by asterisk in Fig. 5. Note the microtubules (arrows) and absence of intermediate filaments indicating this cell is oligodendroglial. × 80000.
143
Fig. 7. Staining ofa l-#m Epon section of a plaque similar to the one shown in Fig. 1 with anti-GC serum by the PAP technique. The oligodendrocytes(arrows) demonstrate a dense ring of reaction product on their cell membranes and a lighter stained rim of cytoplasm surrounding an unstained nucleus. Some of these cells are associated with intensely staining myelindroplets. Extensivedeposits of reaction product are seen over myelin debris within the cytoplasm of macrophages located in perivascular spaces or in the parenchyma.The background consists of faint outlines of unstained axons and astrocyticprocesses, x 1000. DISCUSSION The present study has demonstrated the persistence (and therefore, survival) of oligodendrocytes in chronic relapsing EAE plaques. The lesions studied were usually chronic and silent as indicated by extensive astroglial scarring but many demonstrated ongoing myelin breakdown products in perivascular and parenchymal macrophages. Within these lesions, oligodendrocytes were identified by their characteristic ultrastructural appearances including a cytoplasm which contained numerous microtubules but no intermediate filaments (Peters et al. 1976). This in contrast to the astrocyte wherein bundles of intermediate filaments predominate with microtubules occurring in lower numbers (Raine and Wisniewski 1970). Moreover, oligodendrocytes were identified immunocytochemically by their staining positively with anti-GC serum. The presence of galactocerebroside in the cell membrane has been reported to be specific for the oligodendrocyte (Raft et al. 1978). In addition to the classic morphologic appearance of oligodendrocytes, some cells containing large amounts of myelin debris, which on superficial examination might have been taken for macrophages, were identified as oligodendrocytes ultrastructurally by the presence of microtubules without intermediate filaments and immunocytochemically by positive staining for galactocerebroside. It is not clear whether these cells were actively producing or phagocytosing
144 myelin. Phagocytic activity by oligodendrocytes has been claimed previously on purely morphologic evidence (no immunocytochemistry) in an experimental study on degenerating optic nerves (Cook and Wisniewski 1973). Another alternative 0xplanation is that myelin debris within the present cells may have accumulated after ar~aputation of their connection with the myelin internode and their retraction into the cell soma. However, we believe its origin was probably due to the active production of myelin within the surviving oligodendrocyte. Synthesis of redundant myelin has been noted previously by oligodendrocytes in vitro (Norton et al. 1983). The separation of the cell from its internode during demyelination might eventually lead to its death in a manner akin to oligodendroglial dying-back (Hirano 1978; Ludwin and Johnson 1981). In any event, it is clear from the present work that at least some oligodendrocytes survive the immunologic attack on myelin and in this context, it appears that the myelin sheath and not the oligodendrocyte is the primary target in autoimmune demyelination. Oligodendrocyte survival has also been reported in chronic active MS (Raine et al. 1981b), canine distemper encephalomyelitis (Raine 1976) and murine chronic EAE (Brown et al. 1982). Other similarities between chronic relapsing EAE and the human condition, MS, have been reviewed in detail elsewhere (Raine 1983) and the survival of oligodendrocytes in well-established chronic lesions of these two disorders is yet another point of comparison. Factors important in the stimulation of these oligodendrocytes to proliferate and perhaps remyelinate adjacent axons might be of therapeutic interest in multiple sclerosis. ACKNOWLEDGEMENTS We thank Dr. Sanford Stone for continued collaboration; Everett Swanson, Miriam Pakingan, Howard Finch and Patricia Kennedy for technical assistance; and Nanci Legg for secretarial assistance. Dr. Moore is a fellow of the Multiple Sclerosis Society of Canada. REFERENCES Brown, A., D.E. McFarlin and C.S. Raine (1982) Chronologic neuropathologyof relapsing experimental allergic encephalomyelitisin the mouse, Lab. Invest., 46: 171-185. Cook, R. D. and H. M. Wisniewski(1973)The role of oligodendrogliaand astrogliain Walleriandegeneration of the optic nerve, Brain Res., 61: 191-2(r6. Hirano, A. (1978)A possiblemechanismof demyelinationin the Syrian hamster with hindlegparalysis,Lab. Invest., 38: 115-121. Ibrahim, M. Z. M. and C. W. M. Adams (1963) The relationship between enzyme activity and neuroglia in plaques of multiple sclerosis, J. Neurol. Neurosurg. Psychiat., 26:101-110. Ibrahim, M.Z.M. and C.W.M. Adams (1965) The relation between enzyme activityand neuroglia in early plaques of multiple sclerosis, J. Path. Bact., 90: 239-243. Ludwin, S. K. and E. S. Johnson (1981) Evidencefor a "dying-back"gliopathyin demyelinatingdisease,Ann. Neurol., 9: 301-305. Lumsden, C.E. (1955) In: D. McAlpine, N.D. Compston and C.E. Lumsden (Eds.), Multiple Sclerosis, Livingstone, Edinburgh, pp. 208-293. Norton, W.T., M. Farooq, K. L. Fields and C. S. Raine (1983) The long term culture of bulk-isolated bovine oligodendroglia from adult brain, Brain Res., 270:295-310. Peters, A., S. L. Palay and H. deF. Webster (1976) In: The Fine Structure of the Nervous System - - The Neurons and Supporting Cells, W.B. Saunders Co., Philadelphia, pp. 231-263.
145 Raft, M.C., R. Mirsky, K.L. Fields, R.P. Lisak, S.H. Dorfman, D.H. Silverberg, N.A. Gregson, S. Leibowitz and M. C. Kennedy (1978) Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture, Nature (Lond.), 274: 813-816. Raine, C. S. (1976) On the development of CNS lesions in natural canine distemper encephalomyelitis, J. Neurol. Sci:, 30: 13-28. Raine, C.S. (1983) Multiple sclerosis and chronic relapsing EAE - - Comparative ultrastructural neuropathology. In: J.F. Hallpike, C.W.M. Adams and W.W. Tourtellotte (Eds.), Multiple Sclerosis, Williams and Wilkins, Baltimore, MD, pp. 413-460. Raine, C. S. and S.H. Stone (1977) Animal model for multiple sclerosis - - Chronic experimental allergic encephalomyelitis in inbred guinea pigs, N.Y. State J. Med., 77: 1693-1696. Raine, C. S. and H. Wisniewski (1970) On the occurrence of microtubules within mature astrocytes, Anat. Rec., 167: 303-308. Raine, C.S., D.H. Snyder, M.P. Valsamis and S.H. Stone (1974) Chronic experimental allergic encephalomyelitis in inbred guinea pigs - - An ultrastructural study, Lab. Invest., 31: 369-380. Raine, C. S., A. B. Johnson, D.M. Marcus, A. Suzuki and M.B. Bornstein (1981a) Demyelination in vitro Absorption studies demonstrate that galactocerebroside is a major target, J. Neurol. Sci., 52: 117-131. Raine, C.S., L. Scheinberg and J.M. Waltz (1981b) Multiple sclerosis - - Oligodendrocyte survival and proliferation in an active lesion, Lab. Invest., 45: 534-546. Snyder, D. H., M. P. Valsamis, S. H. Stone and C. S. Raine (1975) Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis,J. Neuropath. Exp. Neurol., 34:209-221. Stone, S.H. and E.M. Lerner (1965) Chronic disseminated allergic encephalomyelitis in guinea pigs, Ann. N. Y. Acad. Sci., 122: 227-241. -
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