A cta Neuropathologica
Acta neuropathol. (Berl.) 43, 43-53 (1978)
(~ Springer-Verlag 1978
Chronic Relapsing Experimental Allergic Encephalomyelitis: CNS Plaque Development in Unsuppressed and Suppressed Animals C. S. Raine, U. Traugott, and S. H. Stone Departments of Pathology (Neuropathology) and Neuroscience, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, The Bronx, N.Y. 10461; and NIAID, Bethesda, Md. 20014, U.S.A.
Summary. Central nervous system (CNS) lesion morphology has been studied in inbred Strain 13 guinea pigs sensitized for chronic relapsing EAE in which the disease was either left to develop (unsuppressed) or was suppressed with injections containing myelin basic protein (MBP). Pathologic changes correlated well with clinical activity. In unsuppressed chronic EAE animals, active clinical disease was invariably matched by acute inflammation in the CNS. In more chronic states, the CNS displayed fibrosis and remyelination while relapses showed the CNS to contain recent changes superimposed upon old lesions. In animals in which the disease was suppressed by injections of MBP, clinical signs did not develop. However, some early subclinical changes were seen morphologically. These lesions were able to remyelinate early on and there was no progression in lesion formation. Apparently, therefore, MBP had a beneficial effect upon the course of the disease and had promoted structural repair. It thus appears that MBP therapy might be one effective approach for the prevention of chronic relapsing EAE. The findings should prove relevant to future MBP trials in multiple sclerosis. Key words: A u t o i m m u n e demyelination - Multiple sclerosis - Remyelination - Suppression - Myelin basic protein - Chronic relapsing EAE.
The clinical and pathological courses of multiple sclerosis, the type example of the h u m a n inflammatory demyelinating diseases, are believed to be better represented by the animal model, chronic experimental allergic encephalomyelitis (EAE), in inbred Strain 13 guinea pigs, than by the more usual acute forms of EAE (Raine and Stone, 1977). This chronic experimental Offprint requests to : Dr. Cedric S. Raine, Department of Pathology (Neuropathology),Albert Einstein Collegeof Medicine, Bronx, N.Y. 10461, U.S.A.
analog of MS, induced by sensitization of juvenile animals, has a delayed clinical onset and, not unusually, a relapsing and remitting course (Stone and Lerner, 1965; Raine et al., 1974; Snyder et al., 1975; Raine et al., in press). Acute EAE, the conventional experimental autoimmune analog for MS, can be produced in a variety of species (Adams, 1959; Alvord, 1970; Paterson, 1976; Raine, 1976) and can be suppressed with injections containing myelin basic p r o t e i n - MBP (Alvord et al., 1965; Levine et al., 1972; Eylar, 1972; Driscoll et al., 1974), the encephalitogenic component of CNS myelin (Kibler and Shapira, 1968; Eylar et al., 1969; Martenson et al., 1970). Recently, we have reported on a similar suppressive approach to acute EAE in adult Strain 13 guinea pigs in addition to preliminary, short-term results from attempts to suppress chronic EAE (Raine et al., 1977). The present study extends the latter and outlines in detail the variation of CNS lesion types in long-term unsuppressed animals and compares them with changes seen in matching animals in which the disease was suppressed for up to 27 months. The findings have shown that unsuppressed animals developed lesions reminiscent of those seen in both MS and acute disseminated encephalomyelitis (ADE). Animals in which the disease had been suppressed, displayed morphologic evidence of a single, subclinical episode of CNS disease and that these lesions had completely remyelinated.
Materials and Methods A total of 47 Strain 13 guinea pigs (Strain 13/N from NIH) wereused. All animals were sensitized intracutaneously for chronic EAE as juveniles (below 250 g body weight) with 0.5 ml of an emulsion containing isologous spinal cord in complete Freund's adjuvant (CFA)--Stone and Lerner, 1965. Following this single sensitization, the animals were separated into two groups. One group comprised 27 animals which were left without further treatment to develop chronic EAE over a 27 month period. The second group of 20 animals was used for suppression. For this, beginning 1 weekpost-
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C. S. Raine et al. : Chronic E.A.E. Plaques inoculation (PI), each animal was given a course of 10 intramuscular injections of bovine MBP in incomplete Freund's adjuvant (IFA) at 3 day intervals. Each animal received a total of 1.4 mg MBP as reported earlier (Raine et al., 1977). The bovine MBP was purified by previously published techniques (Eylar et al,, 1969) and kindly supplied by Dr. E. H. Eylar, Playfair Neuroscience Institute, Toronto. After suppression the animals were observed for up to 27 mo PI. Following the onset of disease in unsuppressed animals (usually 8 - 12 weeks PI), animals from both groups were sampled at various timepoints for morphologic study. Throughout the period of observation, a number of unsuppressed and suppressed animals were bled at frequent intervals by cardiac puncture for the estimation of lymphocyte populations (Traugott et al., in prep.). For morphologic study, guinea pigs were anesthetized with ether and perfused through the heart with 100 ml of cold 4 ~ paraformaldehyde buffered with PO4, followed by 3 I of 5 }/oglutaraldehyde, similarly buffered. Slices 1 mm thick were taken from the spinal cord at C7, L6, Lv and $1, and together with corresponding nerve roots, were post-fixed in chrome osmium. Following dehydration in ethyl alcohol, the tissue was embedded flat in Epon. For light microscopy, 1 pm sections were cut and stained with toluidine blue. Thin sections for electron microscopy (EM), were placed on uncoated grids, double-stained with lead and uranium salts, carbon-coated and scanned in a Siemens 101 (Raine et al., 1974). In addition, coronal slices of brain were paraffin-embedded for routine histology.
Results
Clinical Findings Unsuppressed Chronic EAE Animals A f t e r a b o u t 8 - 12 weeks PI, m o s t animals d e v e l o p e d clinical signs consisting o f weight loss, m o u t h wetting, i n c o n t i n e n c e a n d paraparesis. The latter sometimes
Fig. 1. Chronic EAE, 16 weeks P1, no relapses. A sma11subpiaI plaque is seen in the spinalcord. Note the persistant meningeal inflammation, the fibrous astrogliosis, chronic demyelination and fibrotic blood vessels. Normal CNS white matter lies below, x 300 Fig. 2. Chronic EAE, 20 weeks PI, 2 relapses. A demyelinated plaque (right) in a dorsal column at L 7is shown. A dorsal horn lies to the left. • 120
Fig. 3. Slightly higher magnification from the plaque shown in Figure 2. Note the fibrotic blood vessels, macrophages, gliosis and many transversly sectioned naked axons, x 245 Fig. 4. Chronic EAE, 22 weeks PI, 2 relapses. A zone ofremyeIination (thin myeIin sheaths around large diameter axons) is shown from the margin of a chronic leasion (normal white matter below). Note the fibrotic blood vessels and the many oligodendroglia (arrows). x 756 Fig. 5. Chronic EAE, 22 weeks PI, 2 relapses. A recent perivascular cuff with a surrounding rim of acutely demyelinated axons (arrow) is shown from an area of previously unaffected white matter, x 756 Fig. 6. Same animal as Figure 2. At the edge of this chronically demyelinated plaque (note gliosis, macrophages with myelin debris, naked axons) macrophages with myelin debris, normal myclinated fibers lie to the top. A perivascular cuff(below, right) contains several macrophages and plasma cells (arrows). x 756
45 p r o g r e s s e d to q u a d r i p a r e s i s b u t usually remitted, albeit i n c o m p l e t e l y in m o s t cases. A t irregular intervals after the first episode, animals were s a m p l e d for m o r p h o logic study. In those m a i n t a i n e d for m o r e t h a n 6 m o PI (15 animals), clinical worsenings were n o t e d in eight. Some o f these d i s p l a y e d as m a n y as four relapses over a p e r i o d o f 2 years. The p o l y p h a s i c disease was rarely fatal a l t h o u g h a few d e a t h s were e n c o u n t e r e d due to i n t e r c u r r e n t infections o r c o m p l i c a t i o n s arising d u r i n g c a r d i a c puncture. Suppressed Chronic EAE Animals O f the 20 suppressed animals examined, n o n e dev e l o p e d overt clinical signs o f E A E over the 27 m o period. D u r i n g the p e r i o d o f injections, it was n o t i c e d t h a t a few a n i m a l s d i s p l a y e d some transient limb weakness which r e m i t t e d completely.
Morphologic Findings U n s u p p r e s s e d c h r o n i c E A E guinea pigs d i s p l a y e d a s p e c t r u m o f C N S lesions which c o m p l e m e n t e d in m a n y r a g a r d s the diverse clinical pictures. Those s a m p l e d 3 6 m o n t h s PI, soon after the onset o f signs, h a d a variety o f C N S changes indicative o f l o n g - s t a n d i n g and ongoing disease. C h r o n i c C N S lesions were usually subpial in l o c a t i o n (Fig. I) b u t also occurred deep within the white m a t t e r (Fig. 2). Such old lesions were n o t comp a t i b l e with the recent clinical onset a n d p r o b a b l y arose d u r i n g the latent p e r i o d o f the disease (before the d e l a y e d onset o f signs) when subclinical acute inf l a m m a t o r y changes have been described ( R a i n e et al., 1974). C h r o n i c lesions a p p e a r e d as large areas o f d e m y e l i n a t e d axons s e p a r a t e d by glial scar tissue, d e b r i s - l a d e n m a c r o p h a g e s a n d fibrotic b l o o d vessels (Fig. 3). Sometimes, n a r r o w zones o f r e m y e l i n a t i o n with a b u n d a n t o l i g o d e n d r o c y t e s were present a r o u n d the peripheries o f the plaques (Fig. 4). Active lesions, which better reflected the clinical state o f the animals, consisted o f regions o f white m a t t e r with extensive perivascular infiltrations, a r o u n d which o n g o i n g dem y e l i n a t i o n was a p p a r e n t (Fig. 5). A c u t e inflamm a t o r y changes were also evident at the m a r g i n s o f some chronic lesions (Fig. 6). In such areas, p l a s m a cells were c o m m o n constituents. U n s u p p r e s s e d a n i m a l s which h a d experienced chronic disease with e x a c e r b a t i o n s ( a b o u t 3 0 - 40 ~ o f those studied), r e v e a l e d C N S changes c o n s i s t e n t with relapsing disease. In such cases, chronically demyelina t e d lesions were the p r e d o m i n a n t type s u p e r i m p o s e d u p o n which were m a r k e d , acute p e r i v a s c u l a r cuffing a n d d e m y e l i n a t i o n (Figs. 7 a n d 8). F i b e r s recently dem y e l i n a t e d (some p r o b a b l y for a second time) were t i g h t l y - p a c k e d , while c h r o n i c a l l y d e m y e l i n a t e d a x o n s were s e p a r a t e d by glial scar tissue (Figs. 9 a n d 10).
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Acta neuropathot. (Berl.) 43 (1978)
Fig. 7. Chronic EAE, 24 weeks, 2 relapses. In this L6 lateral column, note the chronically demyelinated plaque with superimposed acute inflammation around many of the blood vessels (arrow). Meningeal inflammation is seen to the left. x 75
Fig. 8. Another section from the lesion shown in Figure 7. Acute perivascular inflammation (note the densely staining plasma cellsarrows) and recent demyelination-manifested by tightly-packed axons, are seen. x 190
Oligodendrocytes were rare or absent. Lesion dimensions were greatest in chronic relapsing animals and in some cases, lesions were grossly visible (Fig. 11). In addition to plaques of the mixed type, the spinal cord in relapsing animals also demonstrated lesions containing only recent, inflammatory activity. Long-standing disease, characterized by large burnt-out plaques, was common in a few animals maintained for 6 months or more and in which single or multiple clinical episodes had been documented with marked residual deficits (sometimes para- or quadriparesis). In one animal which had experienced 3 episodes of para- or quadriparesis and had demonstrated seizure activity, symmetrical lesions were seen in the lateral columns of the cervical cord at C6 and C7. In these grossly visible lesions (Fig. 12), marked gliotic and fibrotic changes, chronic demyelination and remyelination were present (Fig. 13). Oligodendrocytes were numerous in these areas. A number of nonspecific changes were also seen in these longstanding CNS lesions. These included Schwann cell invasion and PNS myelination (Figs. 14, and 15), glial bridges between subpial astrocytes and the leptomeninges-phenomena recently analyzedin chronic relapsing EAE (Raine et al., in press), fenestrated blood vessels (Snyder et al., 1975a)-Fig.16, and myelinated oligodendroglial cell somata (Fig. 17). Axonal degeneration was rare although in the latter type of lesion, the massive parenchymalchanges had probably evolvedat the expense of many axons. Except for an occasionalperivascular cuffwhichhad no apparent deleterious effect upon the tissue, grey matter was never involved. Spinal nerve roots did not consistently show changesbut when present, there was equal involvement of both the anterior and posterior roots. In contrast to the above, the 20 suppressed animals never displayed large CNS lesions. Such a negative
finding would have been suggested by the clinical observations. However, when sampled after 2 or more months PI, morphologic evidence of CNS disease was invariably apparent. These lesions were related to subclinical inflammatory changes occurring 2 4 weeks PI. The early genesis of these changes in s u p p r e s s e d - E A E guinea pigs was discussed in our preliminary report (Raine et al,, 1977). By 2 months PI, subpial remyelination was seen against a background of chronic meningeal inflammation (Figs. / 8 - 2 0 ) . This remyelination represented the repair of previously demyelinated axons, The degree of remyelination was uniform, suggesting a single wave of disease. This remyelination was accompanied by large numbers of oligodendrocytes. At 27 mo PI, remyelination of subpial fibers in long-term suppressed animals was more advanced but still distinguishable from unaffected fibers (Figs. 21, and 22). Some residual meningeal and vascular fibrosis and fibrous astrogliosis were present but not prominent. Schwann cell invasion and fenestration of parenchymal blood vessels were not features of these areas of remyelination. Evidence of ongoing disease (inflammation and active demyelination) was not seen.
Discussion The present neuropathologic account highlights the spectrum of lesion variability in Strain 13 guinea pigs afflicted with chronic, relapsing EAE and compares the picture with changes occurring in similar animals in which the disease had been successfully suppressed for
C. S. Raine et al. : Chronic E.A.E. Plaques
Fig, 9, A group of recently demyelinated axons with beginning gliosis. Note the tight packing of the fibers, x 9 000 Fig. 10, An area of chronic demyelination depicts fibrous gliosis between the separate naked axons, x 9 000
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Acta neuropathol. (Berl.) 43 (1978)
Fig. 11. Chronic EAE, 16 weeks, 2 relapses. Note the disseminated nature of the lesions in this spinal cord at L7- Chronic plaques are seen in the dorsal columns, anterior columns and the right lateral column, while a recent, acute plaque is seen in the left lateral column, x 20
Fig. 13. Detail from Figure 12. Note the perivascular and parenchymal collagen deposition, the demyelinated and remyelinated fibers and the many, densely staining oligodendroglial cells (arrows). • 756
Fig. 12. Chronic EAE, quadriparetic, several seizures over 10 months. A large, burnt out plaque is seen in the right lateral column of this spinal cord at C 7. x 20
Fig. 14. Detail from Figure 12. An area of spinal cord similar to Figure 14 also shows Schwann cell invasion and PNS myelination (arrows). x 756
m o r e t h a n 2 years. T h i s s u p p r e s s i o n was a c h i e v e d w i t h a single s h o r t c o u r s e o f i n t r a m u s c u l a r i n j e c t i o n s c o n taining myelin basic protein (MBP). Of particular i n t e r e s t has b e e n the s t r i k i n g m o r p h o l o g i c s i m i l a r i t y b e t w e e n lesion p a t t e r n s seen in this c h r o n i c r e l a p s i n g m o d e l a n d a p p e a r a n c e s e n c o u n t e r e d in M S (Prineas, 1975; R a i n e , 1977), as well as the s t r o n g c o r r e l a t i o n b e t w e e n l y m p h o c y t e f l u c t u a t i o n s a n d clinical a n d m o r p h o l o g i c a c t i v i t y ( R a i n e et al., in p r e s s ; T r a u g o t t et al., in prep.).
Fig. 15. EM appearance of the lesion shown in Figure 12. Extensive collagen deposition (C) is present. Also note the PNS myelination (arrows), CNS remyelination (left) and several "free floating" oligodendrocytes (*). x 3 400 Fig. 16. A fenestrated blood vessel (lumen above) is shown from a chronic EAE lesion. Fenestrae are indicated by arrows, x 28 000 Fig. 17. An oligodendrocyte is encompassed by 2 layers of myelin (arrows) and surrounded by several fibers at early stages of remyelination, x 13000
C. S. Raine et al. : Chronic E.A.E. Plaques
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Acta neuropathol. (Berl.) 43 (1978)
C. S. Raine et al. : Chronic E.A.E. Plaques Active clinical disease was invariably matched by acute inflammation and demyelination while longstanding stable states were paralleled by chronic fibrotic changes and repair. Similar clinico-pathologic associations have also been speculated in MS but have been difficult to document. Although the lesion topography in our chronic EAE model (subpial margins) frequently approximated more closely that of ADE, e.g., post-rabies immunization encephalomyelitis, the size of the lesions, their location deep within the CNS parenchyma, their periventricular location (as seen in paraffin sections) and their structural similarities to chronic MS render the model one highly relevant to investigations on the pathogenesis of MS. Other points in favor of this model as an analog for MS have recently been discussed (Raine, 1976; Raine and Stone, 1977). In addition to its application to MS research, chronic EAE has proven useful in the analysis of a variety of pathologic events associated with chronic demyelinative activity and CNS repair, viz., remyelination fibrosis, gliosis, Schwann cell invasion and fenestrated blood vessels. The latter phenomenon, one probably resulting from repeated damage to the vascular endothelium (Snyder et al., 1975 a), was a constant feature of old sclerotic lesions. The reported failure to confirm the presence of fenestrated endothelium in our model (Kristensson, 1977) was probably due to poor sampling or to more recent lesions being studied. The present investigation had given much insight to the capacity of CNS axons to remyelinate under these conditions. Remyelination in general was a phenomenon first observed 1 - 2 months after the initial loss of myelin, was rapid in lesions which underwent only one demyelinative insult and was invariably accompanied by an abundance of active-looking oligodendrocytes. Herndon et al., (1977) showed that the increased number ofoligodendrocytes during remyelin-
Fig. 18. Suppressedchronic EAE, 8 weeksPI. A subpiai lesionshows early remyelinationof all fibers and fibrotic changes in the leptomeninges, x 480 Fig. 19. Higher magnificationfrom Figure 18. Note the thin myelin around the remyelinatedfibers and the densely staining, rounded oligodendrocytes(arrows). x 756 Fig. 20. EM appearance of Figure 19. The leptomeninges (not shown) lie above. The many subpial fibers display early remyelinatiou. x 4 300 Fig. 21. Suppressedchronic EAE, 25 months PI. AdvancedremyeIination (arrows) can be distinguished in the subpial layers • 756 Fig. 22. Suppressed chronic EAE, 27 months PI. An area of dorsal column at L7 shows advanced remyelination (arrows), fibrotic changes around a blood vesseland severaldenselystaining oligodendrocytes (lower, left center), x 756
51 ation were the result of local mitosis of surviving cells. The presence of rims of oligodendrocytes around the margins of chronic EAE lesions is also suggestive of their having been derived from local mitotic activity. Only in old, silent lesions was remyelination encountered deep within plaques. More usually, it was restricted to marginal strips. The occurrence ofmyelinated celt somata has been discussed on a number of occasions (see Raine and Bornstein, 1974) where it was pointed out that one reason for such redundant myelination may be an overabundance of oligodendrocytes and a relative paucity of axons. This may also be the case in old demyelinated lesions. In terms of the rapidity and degree of CNS remyelination, a previous study by Prineas et al. (1969) showed that in comparison to the PNS, the CNS remyelinated slowly and incompletely. The present observations are in agreement with this conclusion and also show that remyelination is more apparent after a single episode of disease. The extensive remyelination in CNS lesions observed in long-term suppressed guinea pigs showed that as a result of the arrest of the disease process, remyelination and oligodendroglial cell proliferation were able to progress apparently unimpeded. Thus, the ability to favor remyelination in this way may be of therapeutic significance to MS research although it must be realized that in the present experiments, suppression commenced prior to the first onset of signs. MS can only be diagnosed with certainty after several exacerbations and therefore, comparable degrees of repair could not be anticipated in the human CNS. Nevertheless, if MBP therapy were beneficial to MS, then more recent CNS lesions might remyelinate and lesion progression might be abated. However, previous MBP trials in MS have not yielded positive results (see Gonsette et al., 1977). Scattered remyelinated fibers have been described on several occasions in MS (Suzuki et al., 1969; Prineas, 1977), but probably do not occur in numbers sufficient to be detectable clinically. The common occurrence of Schwann cells and PNS myelination in older EAE lesions once more underscores this as a common sequela to damage to CNS vasculature and glia limitans (Blakemore and Paterson, 1975; Raine et al., in press). Schwann cell invasion of the CNS has also been reported in MS (Ghatak et al., 1973; Prineas, 1977). The pathogenetic mechanism in chronic relapsing EAE probably has an immunologic basis. Previous studies (Traugott and Raine, I977; Traugott et at., 1978) and recent findings (Raine et al., in press; Traugott et al., in prep.), have shown that during the latent periods of acute and chronic EAE in Strain 13 guinea pigs, the percentages of circulating early (highaffinity rosetting) T cells, the lymphocytes responsible
52 for cell-mediated immune reactions, increase and then decrease dramatically with the onset o f signs. The decrease o f this T cell population from the circulation has been shown to be due to T cell migration to the C N S (Traugott et al., 1978). Recent w o r k on chronic relapsing E A E has shown that subsequent to exacerbations, early T cells also decrease significantly and then rise to slightly elevated levels during remissions (Traugott et al., in prep.). Exactly which subpopulations o f T cells are responsible for these fluctuations are not k n o w n but it has been tentatively suggested in suppressed acute E A E that the elevations are due to MBP-generated suppressor T cells (Swierkosz and Swanborg, 1977; Raine et al., in press). O n g o i n g experiments are investigating this possibility. Regarding the permanence o f the protection afforded by the MBP-suppression, we have recently shown that long-term (2 years PI) suppressed animals are resistant to a second rechallenge with C N S tissue in C E A while long-term unsuppressed animals experience an acut~ episode o f E A E 2 weeks after similar rechallenge (Raine et al., in press; T r a u g o t t et al., in prep.). This protection in suppressed animals might be an indication that the activity o f certain m e m o r y T cells has been restricted while in unsuppressed animals, m e m o r y cells persist and are able to m o u n t a response. The fact that MBP, a single c o m p o n e n t o f C N S myelin, is capable o f suppressing EAE, a disease induced in the present study by whole brain tissue in C F A , is remarkable and would suggest that M B P is the specific etiologic antigen in this condition. However, it should be pointed out that acute E A E can also be suppressed by n o n - C N S antigens, e.g., complete F r e u n d ' s adjuvant (Alvord et al., 1965). This raises the interesting point that since M B P has been the favored antigen for suppressive trials in MS, perhaps the use o f other n o n - C N S antigens might be equally feasible. Since not all MS patients display cell-mediated immunity to M B P (Paterson, 1973), perhaps M B P therapy would be ineffective in nonresponders, if M B P responsiveness is at all related to MS pathogenesis or relevant to its therapy. Therefore, there is the remote possibility that suppressive trials using non-neural c o m p o u n d s m a y be equally helpful in MS. While cell-mediated immunity to C N S antigens, in particular MBP, in MS remains a controversial issue (Paterson, 1973; Raine, 1977), the majority o f the current evidence tends to support an immunogenic basis for plaque development. Should the immunologic mechanism in chronic relapsing E A E represent a true simulation o f that occurring in MS, and in view o f the close pathologic and clinical similarities between the two conditions, then the present results on the suppression o f this chronic relapsing condition using M B P therapy should be encouraging for future M B P trials on
Acta neuropathol. (Berl.) 43 (1978) MS. It should be stressed, however, that the vehicle (IFA) used here m a y not be ideal for h u m a n trials and that other dep6t-forming reagents (e.g., liposomes) m a y be considered. T a k e n in concert, this demonstration of long-term clinical and m o r p h o l o g i c suppression o f a chronic relapsing immune-mediated demyelinating condition represents the first o f its kind and m a y prove to be a milestone in this research area. Acknowledgements. The authors thank Drs. Robert D. Terry, John W. Prineas, Murray B. Bornstein, and David H. Snyder for discussion and constructive criticism; Everett Swanson, Howard Finch, Marianne van Hooren, Frances Cross, and Miriam Pakingan for technical assistance; Mary Palumbo and Violet Hantz for secretarial assistance, and Dr. Edwin H. Eylar for the bovine myelin basic protein. Supported in part by National Multiple Sclerosis Society grant RG 1001-A-1; U.S.P.H.S. grants NS 08952 and NS 11920; and a grant from the Kroc Foundation.
References Adams, R. D.: A comparison of the morphology of the human demyelinative diseases and experimental "allergic" encephalomyelitis. In: "Allergic" Encephalomyelitis; (edited by M. W. Kies and E. C. Alvord), pp. 183-209. Springfield: Thomas 1959 Alvord, E. C. : Acute disseminated encephalomyelitis and "allergic" neuro-encephalopathies. In: Handbook of Clinical Neurology, Vol. 9 Multiple Sclerosis and Other Demyelinating Diseases; edited by P. J. Vinken and G. W. Bruyn, pp. 500-571. Amsterdam: North Holland 1970 Alvord, E. C., Shaw, C.-M., Hruby, S., Kies, M. W. : Encephalitogeninduced inhibition of experimental allergic encephalomyelitis: Prevention, suppression and therapy. Ann. N. Y. Acad. Sci. 122, 333- 345 (1965) Driscoll, B. F., Kies, M. W., Alvord, E. C. : Successful treatment of experimental allergic encephalomyelitis (EAE) in guinea pigs with homologous myelin basic protein. J. Immunol. 112, 392397 (1974) Eylar, E. H. : Experimental allergic encephalomyelitis and multiple sclerosis. In: Multiple Sclerosis: Immunology, Virology and Ultrastructure; edited by F. Wolfgram, G. W. Ellison, J. G. Stevens and J. M. Andrews, pp. 449 - 486. New York: Academic Press 1972 Eylar, E. H., Salk, J., Beveridge, G. C., Brown, L. V. : Experimental allergic encephalomyelitis. An encephalitogenic basic protein from bovine myelin. Arch. Biochem. Biophys. 132, 34-48 (1969) Ghatak, H., Hirano, A., Doron, Y., Zimmerman, H. M. : Remyelination in MS with peripheral type myelin. Arch. Neurol. 29, 262 267 (1973) Gonsette, R. E., Delmotte, P., Demonty, L. : Failure of basic protein therapy for multiple sclerosis. J. Neurol. 216, 27-31 (1977) Herndon, R. M., Price, D. L., Weiner, L. P.: Regeneration of oligodendroglia during recovery from demyelinating disease. Science 195, 693-694 (1977) Kibler, R. F., Shapira, R. : Isolation and properties of an encephalitogenic protein from bovine, rabbit and human central nervous system tissue. J. Biol. Chem. 243, 281-286 (1968) Kristensson, K., Wisniewski, H. : Chronic relapsing experimental allergic encephalomyelitis: Studies in vascular permeability changes. Acta neuropath. (Berl.) 39, 189-194 (1977) Levine, S., Sowinski, R., Kies, M. W. : Treatment of experimental allergic encephalomyelitis with encephalitogenic basic proteins. Proc. Soc. Exp. Biol. Med. 139, 506-510 (1972) Martenson, R. E., Deibler, G. E., Kies, M. W. : Myelin basic proteins of the rat central nervous system. Purification, encephalitogenic
C. S. Raine et al. : Chronic E.A.E. Plaques properties, and amino acid compositions. Biochim. Biophys. Acta. 200, 353-362 (1970) Paterson, P. Y. : Multiple Sclerosis: An immunologic reassessment. J. Chron. Dis. 26, 119-126 (1973) Paterson, P. Y. : Experimental autoimmune (allergic) encephalomyelitis. Induction, pathogenesis and suppression. In: Textbook of immunopathology, Second edition; edited by P. A. Miescher and J. J. Mueller-Eberhard, pp. 179 - 213. New York: Grune and Stratton 1976 Prineas, J. : Paramyxovirus-Iike particles in acute lesions in multiple sclerosis. In: The Aetiology and Pathogenesis of the Demyelinating Diseases; edited by H. Shiraki, T. Yonezawa and Y. Kuroiwa, pp. 311-320. Tokyo: Japan Science Press 1976 Prineas, J., Raine, C. S., Wisniewski, H. : An ultrastructural study of experimental demyelination and remyelination. III. Chronic experimental allergic encephalomyelitis in the central nervous system. Lab. Invest. 21, 472-483 (1969) Raine, C. S. : Experimental allergic encephalomyelitis and related conditions. In: Progress in Neuropathology, Vol. 3; edited by H. M. Zimmerman, pp. 225-251, New York: Grune and Stratton 1976 Raine, C. S. : The etiology and pathogenesis of multiple sclerosisrecent developments. Pathobiol. Annual 7, 347-384 (1977) Raine, C. S., Bornstein, M. B.: Unusual profiles in organotypic cuItures of central nervous tissue. J. Neurocytology 3, 3 t3 - 325 (1974) Raine, C. S., Snyder, D. H., Stone, S. H., Bornstein, M. B.: Suppression of acute and chronic experimental allergic encephalomyelitis in Strain 13 guinea pigs. J. Neurol. Sci. 31,355-367 (1977) Raine, C. S., Snyder, D. H., Valsamis, M. P., Stone, S. H. : Chronic experimental allergic encephalomyelitis in inbred guinea pigsan ultrastructural study. Lab. Invest. 31,369-380 (1974) Raine, C, S., Stone, S. H. : Animal model for multiple sclerosis. Chronic experimental allergic encephalomyelitis in inbred guinea pig. N.Y. State J. Med. 77, 1693-1696 (1977)
53 Raine, C. S., Traugott, U., Stone, S. H. : Glial bridges and Schwann cell invasion of the CNS during chronic demyelination. J. Neurocytol. (in press) Raine, C. S., Traugott, U., Stone, S. H. : Chronic relapsing allergic encephalomyelitis: Suppression and relevance to multiple sclerosis. Science (in press) Snyder, D. H., Hirano, A., Raine, C. S.: Fenestrated CNS blood vessels in chronic experimental allergic encephalomyelitis. Brain Res. 100, 645-649 (1975a) Snyder, D. H., Valsamis, M. P., Stone, S. H., Raine, C. S.: Progressive and reparatory events in chronic experimental allergie encephalomyelitis. J. Neuropath. Exp. Neurol. 34, 209 221 (1975b) Stone, S. H., Lerner, E. M. : Chronic disseminated allergic encephalomyelitis in guinea pigs. Ann. N. Y. Acad. Sci. 122, 227-24l (1965) Suzuki, K., Andrews, J. M., Waltz, J. M., Terry, R. D. : Ultrastructural studies of multiple sclerosis. Lab. Invest. 20, 444-454 (1969) Swierkosz, J. E., Swanborg, R. H. : Immunoregulation of experimental allergic encephalomyelitis: Conditions for induction of suppressor cells and analysis of mechanism. J. Immunol. 119, 1501-1506 (1977) Traugott, U., Rain< C. S. : Experimental allergic encephalomyelitis in inbred guinea pigs. Correlation of decrease in early T cells with clinical signs in suppressed and unsuppressed animals, Celt. Immunol. 34, 146-155 (1977) Traugott, U., Stone, S. H., Raine, C. S.: Experimental allergic encephalomyelitis: T cell migration to the central nervous system. J. Neurol. Sci. 36, 55-61 (1978) Traugott, U., Stone, S. H., Raine, C. S.: Suppression of chronic experimental allergic encephalomyelitis: Correlation of circulating lymphocyte fluctuations with relapsing disease. J. Exp. Med. (submitted)
Received April 12, 1978/Accepted April 18, 1978
Acta neuropathol. (Berl.) 43, 43-53 (1978)
(~ Springer-Verlag 1978
Chronic Relapsing Experimental Allergic Encephalomyelitis: CNS Plaque Development in Unsuppressed and Suppressed Animals C. S. Raine, U. Traugott, and S. H. Stone Departments of Pathology (Neuropathology) and Neuroscience, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, The Bronx, N.Y. 10461; and NIAID, Bethesda, Md. 20014, U.S.A.
Summary. Central nervous system (CNS) lesion morphology has been studied in inbred Strain 13 guinea pigs sensitized for chronic relapsing EAE in which the disease was either left to develop (unsuppressed) or was suppressed with injections containing myelin basic protein (MBP). Pathologic changes correlated well with clinical activity. In unsuppressed chronic EAE animals, active clinical disease was invariably matched by acute inflammation in the CNS. In more chronic states, the CNS displayed fibrosis and remyelination while relapses showed the CNS to contain recent changes superimposed upon old lesions. In animals in which the disease was suppressed by injections of MBP, clinical signs did not develop. However, some early subclinical changes were seen morphologically. These lesions were able to remyelinate early on and there was no progression in lesion formation. Apparently, therefore, MBP had a beneficial effect upon the course of the disease and had promoted structural repair. It thus appears that MBP therapy might be one effective approach for the prevention of chronic relapsing EAE. The findings should prove relevant to future MBP trials in multiple sclerosis. Key words: A u t o i m m u n e demyelination - Multiple sclerosis - Remyelination - Suppression - Myelin basic protein - Chronic relapsing EAE.
The clinical and pathological courses of multiple sclerosis, the type example of the h u m a n inflammatory demyelinating diseases, are believed to be better represented by the animal model, chronic experimental allergic encephalomyelitis (EAE), in inbred Strain 13 guinea pigs, than by the more usual acute forms of EAE (Raine and Stone, 1977). This chronic experimental Offprint requests to : Dr. Cedric S. Raine, Department of Pathology (Neuropathology),Albert Einstein Collegeof Medicine, Bronx, N.Y. 10461, U.S.A.
analog of MS, induced by sensitization of juvenile animals, has a delayed clinical onset and, not unusually, a relapsing and remitting course (Stone and Lerner, 1965; Raine et al., 1974; Snyder et al., 1975; Raine et al., in press). Acute EAE, the conventional experimental autoimmune analog for MS, can be produced in a variety of species (Adams, 1959; Alvord, 1970; Paterson, 1976; Raine, 1976) and can be suppressed with injections containing myelin basic p r o t e i n - MBP (Alvord et al., 1965; Levine et al., 1972; Eylar, 1972; Driscoll et al., 1974), the encephalitogenic component of CNS myelin (Kibler and Shapira, 1968; Eylar et al., 1969; Martenson et al., 1970). Recently, we have reported on a similar suppressive approach to acute EAE in adult Strain 13 guinea pigs in addition to preliminary, short-term results from attempts to suppress chronic EAE (Raine et al., 1977). The present study extends the latter and outlines in detail the variation of CNS lesion types in long-term unsuppressed animals and compares them with changes seen in matching animals in which the disease was suppressed for up to 27 months. The findings have shown that unsuppressed animals developed lesions reminiscent of those seen in both MS and acute disseminated encephalomyelitis (ADE). Animals in which the disease had been suppressed, displayed morphologic evidence of a single, subclinical episode of CNS disease and that these lesions had completely remyelinated.
Materials and Methods A total of 47 Strain 13 guinea pigs (Strain 13/N from NIH) wereused. All animals were sensitized intracutaneously for chronic EAE as juveniles (below 250 g body weight) with 0.5 ml of an emulsion containing isologous spinal cord in complete Freund's adjuvant (CFA)--Stone and Lerner, 1965. Following this single sensitization, the animals were separated into two groups. One group comprised 27 animals which were left without further treatment to develop chronic EAE over a 27 month period. The second group of 20 animals was used for suppression. For this, beginning 1 weekpost-
0001-6322/78/0043/0043/8 2.20
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C. S. Raine et al. : Chronic E.A.E. Plaques inoculation (PI), each animal was given a course of 10 intramuscular injections of bovine MBP in incomplete Freund's adjuvant (IFA) at 3 day intervals. Each animal received a total of 1.4 mg MBP as reported earlier (Raine et al., 1977). The bovine MBP was purified by previously published techniques (Eylar et al,, 1969) and kindly supplied by Dr. E. H. Eylar, Playfair Neuroscience Institute, Toronto. After suppression the animals were observed for up to 27 mo PI. Following the onset of disease in unsuppressed animals (usually 8 - 12 weeks PI), animals from both groups were sampled at various timepoints for morphologic study. Throughout the period of observation, a number of unsuppressed and suppressed animals were bled at frequent intervals by cardiac puncture for the estimation of lymphocyte populations (Traugott et al., in prep.). For morphologic study, guinea pigs were anesthetized with ether and perfused through the heart with 100 ml of cold 4 ~ paraformaldehyde buffered with PO4, followed by 3 I of 5 }/oglutaraldehyde, similarly buffered. Slices 1 mm thick were taken from the spinal cord at C7, L6, Lv and $1, and together with corresponding nerve roots, were post-fixed in chrome osmium. Following dehydration in ethyl alcohol, the tissue was embedded flat in Epon. For light microscopy, 1 pm sections were cut and stained with toluidine blue. Thin sections for electron microscopy (EM), were placed on uncoated grids, double-stained with lead and uranium salts, carbon-coated and scanned in a Siemens 101 (Raine et al., 1974). In addition, coronal slices of brain were paraffin-embedded for routine histology.
Results
Clinical Findings Unsuppressed Chronic EAE Animals A f t e r a b o u t 8 - 12 weeks PI, m o s t animals d e v e l o p e d clinical signs consisting o f weight loss, m o u t h wetting, i n c o n t i n e n c e a n d paraparesis. The latter sometimes
Fig. 1. Chronic EAE, 16 weeks P1, no relapses. A sma11subpiaI plaque is seen in the spinalcord. Note the persistant meningeal inflammation, the fibrous astrogliosis, chronic demyelination and fibrotic blood vessels. Normal CNS white matter lies below, x 300 Fig. 2. Chronic EAE, 20 weeks PI, 2 relapses. A demyelinated plaque (right) in a dorsal column at L 7is shown. A dorsal horn lies to the left. • 120
Fig. 3. Slightly higher magnification from the plaque shown in Figure 2. Note the fibrotic blood vessels, macrophages, gliosis and many transversly sectioned naked axons, x 245 Fig. 4. Chronic EAE, 22 weeks PI, 2 relapses. A zone ofremyeIination (thin myeIin sheaths around large diameter axons) is shown from the margin of a chronic leasion (normal white matter below). Note the fibrotic blood vessels and the many oligodendroglia (arrows). x 756 Fig. 5. Chronic EAE, 22 weeks PI, 2 relapses. A recent perivascular cuff with a surrounding rim of acutely demyelinated axons (arrow) is shown from an area of previously unaffected white matter, x 756 Fig. 6. Same animal as Figure 2. At the edge of this chronically demyelinated plaque (note gliosis, macrophages with myelin debris, naked axons) macrophages with myelin debris, normal myclinated fibers lie to the top. A perivascular cuff(below, right) contains several macrophages and plasma cells (arrows). x 756
45 p r o g r e s s e d to q u a d r i p a r e s i s b u t usually remitted, albeit i n c o m p l e t e l y in m o s t cases. A t irregular intervals after the first episode, animals were s a m p l e d for m o r p h o logic study. In those m a i n t a i n e d for m o r e t h a n 6 m o PI (15 animals), clinical worsenings were n o t e d in eight. Some o f these d i s p l a y e d as m a n y as four relapses over a p e r i o d o f 2 years. The p o l y p h a s i c disease was rarely fatal a l t h o u g h a few d e a t h s were e n c o u n t e r e d due to i n t e r c u r r e n t infections o r c o m p l i c a t i o n s arising d u r i n g c a r d i a c puncture. Suppressed Chronic EAE Animals O f the 20 suppressed animals examined, n o n e dev e l o p e d overt clinical signs o f E A E over the 27 m o period. D u r i n g the p e r i o d o f injections, it was n o t i c e d t h a t a few a n i m a l s d i s p l a y e d some transient limb weakness which r e m i t t e d completely.
Morphologic Findings U n s u p p r e s s e d c h r o n i c E A E guinea pigs d i s p l a y e d a s p e c t r u m o f C N S lesions which c o m p l e m e n t e d in m a n y r a g a r d s the diverse clinical pictures. Those s a m p l e d 3 6 m o n t h s PI, soon after the onset o f signs, h a d a variety o f C N S changes indicative o f l o n g - s t a n d i n g and ongoing disease. C h r o n i c C N S lesions were usually subpial in l o c a t i o n (Fig. I) b u t also occurred deep within the white m a t t e r (Fig. 2). Such old lesions were n o t comp a t i b l e with the recent clinical onset a n d p r o b a b l y arose d u r i n g the latent p e r i o d o f the disease (before the d e l a y e d onset o f signs) when subclinical acute inf l a m m a t o r y changes have been described ( R a i n e et al., 1974). C h r o n i c lesions a p p e a r e d as large areas o f d e m y e l i n a t e d axons s e p a r a t e d by glial scar tissue, d e b r i s - l a d e n m a c r o p h a g e s a n d fibrotic b l o o d vessels (Fig. 3). Sometimes, n a r r o w zones o f r e m y e l i n a t i o n with a b u n d a n t o l i g o d e n d r o c y t e s were present a r o u n d the peripheries o f the plaques (Fig. 4). Active lesions, which better reflected the clinical state o f the animals, consisted o f regions o f white m a t t e r with extensive perivascular infiltrations, a r o u n d which o n g o i n g dem y e l i n a t i o n was a p p a r e n t (Fig. 5). A c u t e inflamm a t o r y changes were also evident at the m a r g i n s o f some chronic lesions (Fig. 6). In such areas, p l a s m a cells were c o m m o n constituents. U n s u p p r e s s e d a n i m a l s which h a d experienced chronic disease with e x a c e r b a t i o n s ( a b o u t 3 0 - 40 ~ o f those studied), r e v e a l e d C N S changes c o n s i s t e n t with relapsing disease. In such cases, chronically demyelina t e d lesions were the p r e d o m i n a n t type s u p e r i m p o s e d u p o n which were m a r k e d , acute p e r i v a s c u l a r cuffing a n d d e m y e l i n a t i o n (Figs. 7 a n d 8). F i b e r s recently dem y e l i n a t e d (some p r o b a b l y for a second time) were t i g h t l y - p a c k e d , while c h r o n i c a l l y d e m y e l i n a t e d a x o n s were s e p a r a t e d by glial scar tissue (Figs. 9 a n d 10).
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Acta neuropathot. (Berl.) 43 (1978)
Fig. 7. Chronic EAE, 24 weeks, 2 relapses. In this L6 lateral column, note the chronically demyelinated plaque with superimposed acute inflammation around many of the blood vessels (arrow). Meningeal inflammation is seen to the left. x 75
Fig. 8. Another section from the lesion shown in Figure 7. Acute perivascular inflammation (note the densely staining plasma cellsarrows) and recent demyelination-manifested by tightly-packed axons, are seen. x 190
Oligodendrocytes were rare or absent. Lesion dimensions were greatest in chronic relapsing animals and in some cases, lesions were grossly visible (Fig. 11). In addition to plaques of the mixed type, the spinal cord in relapsing animals also demonstrated lesions containing only recent, inflammatory activity. Long-standing disease, characterized by large burnt-out plaques, was common in a few animals maintained for 6 months or more and in which single or multiple clinical episodes had been documented with marked residual deficits (sometimes para- or quadriparesis). In one animal which had experienced 3 episodes of para- or quadriparesis and had demonstrated seizure activity, symmetrical lesions were seen in the lateral columns of the cervical cord at C6 and C7. In these grossly visible lesions (Fig. 12), marked gliotic and fibrotic changes, chronic demyelination and remyelination were present (Fig. 13). Oligodendrocytes were numerous in these areas. A number of nonspecific changes were also seen in these longstanding CNS lesions. These included Schwann cell invasion and PNS myelination (Figs. 14, and 15), glial bridges between subpial astrocytes and the leptomeninges-phenomena recently analyzedin chronic relapsing EAE (Raine et al., in press), fenestrated blood vessels (Snyder et al., 1975a)-Fig.16, and myelinated oligodendroglial cell somata (Fig. 17). Axonal degeneration was rare although in the latter type of lesion, the massive parenchymalchanges had probably evolvedat the expense of many axons. Except for an occasionalperivascular cuffwhichhad no apparent deleterious effect upon the tissue, grey matter was never involved. Spinal nerve roots did not consistently show changesbut when present, there was equal involvement of both the anterior and posterior roots. In contrast to the above, the 20 suppressed animals never displayed large CNS lesions. Such a negative
finding would have been suggested by the clinical observations. However, when sampled after 2 or more months PI, morphologic evidence of CNS disease was invariably apparent. These lesions were related to subclinical inflammatory changes occurring 2 4 weeks PI. The early genesis of these changes in s u p p r e s s e d - E A E guinea pigs was discussed in our preliminary report (Raine et al,, 1977). By 2 months PI, subpial remyelination was seen against a background of chronic meningeal inflammation (Figs. / 8 - 2 0 ) . This remyelination represented the repair of previously demyelinated axons, The degree of remyelination was uniform, suggesting a single wave of disease. This remyelination was accompanied by large numbers of oligodendrocytes. At 27 mo PI, remyelination of subpial fibers in long-term suppressed animals was more advanced but still distinguishable from unaffected fibers (Figs. 21, and 22). Some residual meningeal and vascular fibrosis and fibrous astrogliosis were present but not prominent. Schwann cell invasion and fenestration of parenchymal blood vessels were not features of these areas of remyelination. Evidence of ongoing disease (inflammation and active demyelination) was not seen.
Discussion The present neuropathologic account highlights the spectrum of lesion variability in Strain 13 guinea pigs afflicted with chronic, relapsing EAE and compares the picture with changes occurring in similar animals in which the disease had been successfully suppressed for
C. S. Raine et al. : Chronic E.A.E. Plaques
Fig, 9, A group of recently demyelinated axons with beginning gliosis. Note the tight packing of the fibers, x 9 000 Fig. 10, An area of chronic demyelination depicts fibrous gliosis between the separate naked axons, x 9 000
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Acta neuropathol. (Berl.) 43 (1978)
Fig. 11. Chronic EAE, 16 weeks, 2 relapses. Note the disseminated nature of the lesions in this spinal cord at L7- Chronic plaques are seen in the dorsal columns, anterior columns and the right lateral column, while a recent, acute plaque is seen in the left lateral column, x 20
Fig. 13. Detail from Figure 12. Note the perivascular and parenchymal collagen deposition, the demyelinated and remyelinated fibers and the many, densely staining oligodendroglial cells (arrows). • 756
Fig. 12. Chronic EAE, quadriparetic, several seizures over 10 months. A large, burnt out plaque is seen in the right lateral column of this spinal cord at C 7. x 20
Fig. 14. Detail from Figure 12. An area of spinal cord similar to Figure 14 also shows Schwann cell invasion and PNS myelination (arrows). x 756
m o r e t h a n 2 years. T h i s s u p p r e s s i o n was a c h i e v e d w i t h a single s h o r t c o u r s e o f i n t r a m u s c u l a r i n j e c t i o n s c o n taining myelin basic protein (MBP). Of particular i n t e r e s t has b e e n the s t r i k i n g m o r p h o l o g i c s i m i l a r i t y b e t w e e n lesion p a t t e r n s seen in this c h r o n i c r e l a p s i n g m o d e l a n d a p p e a r a n c e s e n c o u n t e r e d in M S (Prineas, 1975; R a i n e , 1977), as well as the s t r o n g c o r r e l a t i o n b e t w e e n l y m p h o c y t e f l u c t u a t i o n s a n d clinical a n d m o r p h o l o g i c a c t i v i t y ( R a i n e et al., in p r e s s ; T r a u g o t t et al., in prep.).
Fig. 15. EM appearance of the lesion shown in Figure 12. Extensive collagen deposition (C) is present. Also note the PNS myelination (arrows), CNS remyelination (left) and several "free floating" oligodendrocytes (*). x 3 400 Fig. 16. A fenestrated blood vessel (lumen above) is shown from a chronic EAE lesion. Fenestrae are indicated by arrows, x 28 000 Fig. 17. An oligodendrocyte is encompassed by 2 layers of myelin (arrows) and surrounded by several fibers at early stages of remyelination, x 13000
C. S. Raine et al. : Chronic E.A.E. Plaques
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Acta neuropathol. (Berl.) 43 (1978)
C. S. Raine et al. : Chronic E.A.E. Plaques Active clinical disease was invariably matched by acute inflammation and demyelination while longstanding stable states were paralleled by chronic fibrotic changes and repair. Similar clinico-pathologic associations have also been speculated in MS but have been difficult to document. Although the lesion topography in our chronic EAE model (subpial margins) frequently approximated more closely that of ADE, e.g., post-rabies immunization encephalomyelitis, the size of the lesions, their location deep within the CNS parenchyma, their periventricular location (as seen in paraffin sections) and their structural similarities to chronic MS render the model one highly relevant to investigations on the pathogenesis of MS. Other points in favor of this model as an analog for MS have recently been discussed (Raine, 1976; Raine and Stone, 1977). In addition to its application to MS research, chronic EAE has proven useful in the analysis of a variety of pathologic events associated with chronic demyelinative activity and CNS repair, viz., remyelination fibrosis, gliosis, Schwann cell invasion and fenestrated blood vessels. The latter phenomenon, one probably resulting from repeated damage to the vascular endothelium (Snyder et al., 1975 a), was a constant feature of old sclerotic lesions. The reported failure to confirm the presence of fenestrated endothelium in our model (Kristensson, 1977) was probably due to poor sampling or to more recent lesions being studied. The present investigation had given much insight to the capacity of CNS axons to remyelinate under these conditions. Remyelination in general was a phenomenon first observed 1 - 2 months after the initial loss of myelin, was rapid in lesions which underwent only one demyelinative insult and was invariably accompanied by an abundance of active-looking oligodendrocytes. Herndon et al., (1977) showed that the increased number ofoligodendrocytes during remyelin-
Fig. 18. Suppressedchronic EAE, 8 weeksPI. A subpiai lesionshows early remyelinationof all fibers and fibrotic changes in the leptomeninges, x 480 Fig. 19. Higher magnificationfrom Figure 18. Note the thin myelin around the remyelinatedfibers and the densely staining, rounded oligodendrocytes(arrows). x 756 Fig. 20. EM appearance of Figure 19. The leptomeninges (not shown) lie above. The many subpial fibers display early remyelinatiou. x 4 300 Fig. 21. Suppressedchronic EAE, 25 months PI. AdvancedremyeIination (arrows) can be distinguished in the subpial layers • 756 Fig. 22. Suppressed chronic EAE, 27 months PI. An area of dorsal column at L7 shows advanced remyelination (arrows), fibrotic changes around a blood vesseland severaldenselystaining oligodendrocytes (lower, left center), x 756
51 ation were the result of local mitosis of surviving cells. The presence of rims of oligodendrocytes around the margins of chronic EAE lesions is also suggestive of their having been derived from local mitotic activity. Only in old, silent lesions was remyelination encountered deep within plaques. More usually, it was restricted to marginal strips. The occurrence ofmyelinated celt somata has been discussed on a number of occasions (see Raine and Bornstein, 1974) where it was pointed out that one reason for such redundant myelination may be an overabundance of oligodendrocytes and a relative paucity of axons. This may also be the case in old demyelinated lesions. In terms of the rapidity and degree of CNS remyelination, a previous study by Prineas et al. (1969) showed that in comparison to the PNS, the CNS remyelinated slowly and incompletely. The present observations are in agreement with this conclusion and also show that remyelination is more apparent after a single episode of disease. The extensive remyelination in CNS lesions observed in long-term suppressed guinea pigs showed that as a result of the arrest of the disease process, remyelination and oligodendroglial cell proliferation were able to progress apparently unimpeded. Thus, the ability to favor remyelination in this way may be of therapeutic significance to MS research although it must be realized that in the present experiments, suppression commenced prior to the first onset of signs. MS can only be diagnosed with certainty after several exacerbations and therefore, comparable degrees of repair could not be anticipated in the human CNS. Nevertheless, if MBP therapy were beneficial to MS, then more recent CNS lesions might remyelinate and lesion progression might be abated. However, previous MBP trials in MS have not yielded positive results (see Gonsette et al., 1977). Scattered remyelinated fibers have been described on several occasions in MS (Suzuki et al., 1969; Prineas, 1977), but probably do not occur in numbers sufficient to be detectable clinically. The common occurrence of Schwann cells and PNS myelination in older EAE lesions once more underscores this as a common sequela to damage to CNS vasculature and glia limitans (Blakemore and Paterson, 1975; Raine et al., in press). Schwann cell invasion of the CNS has also been reported in MS (Ghatak et al., 1973; Prineas, 1977). The pathogenetic mechanism in chronic relapsing EAE probably has an immunologic basis. Previous studies (Traugott and Raine, I977; Traugott et at., 1978) and recent findings (Raine et al., in press; Traugott et al., in prep.), have shown that during the latent periods of acute and chronic EAE in Strain 13 guinea pigs, the percentages of circulating early (highaffinity rosetting) T cells, the lymphocytes responsible
52 for cell-mediated immune reactions, increase and then decrease dramatically with the onset o f signs. The decrease o f this T cell population from the circulation has been shown to be due to T cell migration to the C N S (Traugott et al., 1978). Recent w o r k on chronic relapsing E A E has shown that subsequent to exacerbations, early T cells also decrease significantly and then rise to slightly elevated levels during remissions (Traugott et al., in prep.). Exactly which subpopulations o f T cells are responsible for these fluctuations are not k n o w n but it has been tentatively suggested in suppressed acute E A E that the elevations are due to MBP-generated suppressor T cells (Swierkosz and Swanborg, 1977; Raine et al., in press). O n g o i n g experiments are investigating this possibility. Regarding the permanence o f the protection afforded by the MBP-suppression, we have recently shown that long-term (2 years PI) suppressed animals are resistant to a second rechallenge with C N S tissue in C E A while long-term unsuppressed animals experience an acut~ episode o f E A E 2 weeks after similar rechallenge (Raine et al., in press; T r a u g o t t et al., in prep.). This protection in suppressed animals might be an indication that the activity o f certain m e m o r y T cells has been restricted while in unsuppressed animals, m e m o r y cells persist and are able to m o u n t a response. The fact that MBP, a single c o m p o n e n t o f C N S myelin, is capable o f suppressing EAE, a disease induced in the present study by whole brain tissue in C F A , is remarkable and would suggest that M B P is the specific etiologic antigen in this condition. However, it should be pointed out that acute E A E can also be suppressed by n o n - C N S antigens, e.g., complete F r e u n d ' s adjuvant (Alvord et al., 1965). This raises the interesting point that since M B P has been the favored antigen for suppressive trials in MS, perhaps the use o f other n o n - C N S antigens might be equally feasible. Since not all MS patients display cell-mediated immunity to M B P (Paterson, 1973), perhaps M B P therapy would be ineffective in nonresponders, if M B P responsiveness is at all related to MS pathogenesis or relevant to its therapy. Therefore, there is the remote possibility that suppressive trials using non-neural c o m p o u n d s m a y be equally helpful in MS. While cell-mediated immunity to C N S antigens, in particular MBP, in MS remains a controversial issue (Paterson, 1973; Raine, 1977), the majority o f the current evidence tends to support an immunogenic basis for plaque development. Should the immunologic mechanism in chronic relapsing E A E represent a true simulation o f that occurring in MS, and in view o f the close pathologic and clinical similarities between the two conditions, then the present results on the suppression o f this chronic relapsing condition using M B P therapy should be encouraging for future M B P trials on
Acta neuropathol. (Berl.) 43 (1978) MS. It should be stressed, however, that the vehicle (IFA) used here m a y not be ideal for h u m a n trials and that other dep6t-forming reagents (e.g., liposomes) m a y be considered. T a k e n in concert, this demonstration of long-term clinical and m o r p h o l o g i c suppression o f a chronic relapsing immune-mediated demyelinating condition represents the first o f its kind and m a y prove to be a milestone in this research area. Acknowledgements. The authors thank Drs. Robert D. Terry, John W. Prineas, Murray B. Bornstein, and David H. Snyder for discussion and constructive criticism; Everett Swanson, Howard Finch, Marianne van Hooren, Frances Cross, and Miriam Pakingan for technical assistance; Mary Palumbo and Violet Hantz for secretarial assistance, and Dr. Edwin H. Eylar for the bovine myelin basic protein. Supported in part by National Multiple Sclerosis Society grant RG 1001-A-1; U.S.P.H.S. grants NS 08952 and NS 11920; and a grant from the Kroc Foundation.
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Received April 12, 1978/Accepted April 18, 1978
