Journal qf the neurological Sciences, 1975, 24:1-11
1
Elsevier Scientific Publishing Company, Amsterdam-Printed in Tile Netherlands
Immunoglobulin G Distribution in Multiple Sclerosis Brain An ImmunofluorescenceStudy BRUNO F. TAVOLATO Clinica delle Malattie Nervose e Mentali. University o] Padova, Padol~a (Italy)
(Received 6 June, 1974)
INTRODUCTION
The elevation of the IgG: total protein ratio in the cerebrospinal fluid (CSF) of a large majority of multiple sclerosis (MS) patients has been considered as indicating an immunological reaction taking place in the central nervous system (CNS). Subsequently a positive gradient for IgG between brain and CSF was demonstrated (Tourtellotte and Parker 1966a), in addition to elevation of the IgG: albumin ratio in the brains of the majority of MS patients (Tourtellotte and Parker 1966b). It was also observed that IgG is maximally elevated in plaques, but usually the normal white matter in MS also has an elevated lgG: albumin ratio (Tourtellotte and Parker 1967). At a later stage an attempt was made to evaluate a correlation between IgG elevation and plaque activity (Tourtellotte, Itabashi and Parker 1967). This experiment involved using two adjacent coronal sections, one for histology, the other for IgG immunochemical determination and a positive correlation was found between perivascular mononuclear cuffing and the IgG level. However we believe that this important experiment left two questions unresolved. Firstly mononuclear perivascular infiltrates cannot be considered as the sole indicator of the complex phenomena associated with the different stages of demyelinating activity. Secondly plaque dissection for IgG determination was made on unstained coronal sections and it is well known that the demyelinating activity in MS plaques varies even within a very restricted area. For instance an old sclerotic plaque may show a zone in a small area at its margin where demyelination is active; moreover isolated areas of recent demyelination are very difficult to distinguish in unstained fresh sections. Therefore we believe that some confusion of plaque types may have occurred in these experiments. A possible approach to this problem is offered by the immunofluorescence (IF) technique, to demonstrate the distribution of IgG (or other proteins) specifically at the microscopical level. This technique has already been used by Ter Meulen, EndersRuckle, Mfiller and Joppich (1969), to show IgG, IgA and IgM in brains from patients affected by subacute sclerosing panencephalitis. The IF was used in MS brains by Simpson, Tourtellotte, Kokmen, Parker and Ita-
2
B. F. TAVOLATO
bashi (1969), who were able to demonstrate IgG accumulation in plaques, apparently located in the enlarged extracellular space, as well as in the surrounding " n o r m a l " white matter. Lumsden (1970) obtained similar results in 1 case of MS and concluded, on morphological grounds, that the IgG is "myelin-bound" and consequently auto-aggressive in nature. In the same paper Lumsden showed some clear lymphoid cells with the IgG-rich cytoplasm. The same cells have also been demonstrated by Frick (1969) and Tourtellotte, Kokmen, Tavolato and Kruger (1971 ) in MS brains, collected and frozen very quickly after death ( < 3 hr). The aim of the present study was to correlate if possible IgG levels and demyelinating activity in MS plaques. The 1F method appears especially suited for this purpose, since it permits the localisation of IgG at a histological level. The demyelinating activity can be evaluated and compared in alternate serial sections. Some further observations have been mad e concerning elution of IgG fro m M S brain to demonstrate the site(s) of possible antigen-antibody reaction.
MATERIALS AND METHODS
(1) Brain tis'sue The brains from 5 MS patients and 3 controls without evidence of CNS disease were used in the present study. The autopsies were done between 3 and 24 hr after death. The brains were collected, sliced, quick-frozen and stored at 90°C. A total o f 49 MS and 6 control samples derived from 22 coronal sections and 5 samples of cord or brain stem were studied. More than one sample was generally taken from each MS coronal section and we attempted to include portions of plaque, plaque margin, normal white matter near the plaque, normal white matter distant from the plaque and grey matter.
(2) Preparation of brain tissue./or IF staining The coronal sections were warmed to - 2 0 ° C just before use and samples were cut out with a cork-bore, having an inside diameter of 1 cm. Ten-#m thick serial slices were then cut with the cryostat and carefully mounted in cold gelatinized microscope slides. The slices were then thawed, air dried for 10 min and fixed in ethanol (95 °£1)-ether (1 : 1 ) for 10 min and then in 95 o//,,ethanol for 20 min at room temperature. Sections were stained with fluorescent antisera after evaporation of the ethanol. In each experiment some sections were washed in physiological saline for 1-10 min and then fixed and stained as previously described.
(3) Fluorescent antibody staining Only direct staining was used in the present study. The sections were incubated with the fluorescent antisera for 30 min at 37°C in a moist chamber and then washed 3 times for 10 min in phosphate-buffered saline. The slides were quickly rinsed in distilled water, air dried and mounted in buffered glycerine. Several commercial and home-made fluorescent antisera were tested and one commercial* R A H IgG F1 serum and a home made R A H Alb Fl were chosen and * Antibodies Incorporated, Davies, Calif.
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
3
used throughout. The RAH Alb serum was labelled with fluorescein isothiocyanate by the method of Hijmans, Schuit and Klein (1969). Controls : two methods were used in each experiment to control the specificity of fluorescent staining: the blocking test and a fluorescent antiserum in which the specific antibodies were previously precipitated with the specific antigen.
(4) Microscopy A Zeiss Ultraphot II microscope was used for fluorescence microscopy. Phase contrast microscopy (Zernicke) was used for the immunofluorescence-stained sections to distinguish the plaques from other structures. Polarized light was used with the Oil Red O stains to demonstrate the different classes of myelin breakdown products.
(5) Histology Alternate serial sections were used for immunofluorescence and for normal microscopy. Sections for histology were fixed in 10 ~ buffered formalin and stained with hematoxylin and eosin and oil red 0. Sometimes the Klfiver-Barrera stain for myelin sheaths was also used.
(6) Classification o/plaque activity The major goal of this study was to find a correlation, if any, between demyelinating activity and lgG level in MS plaques. Although the histological criteria for the classification of activity in plaques are well established and generally accepted (Greenfield 1960; Seitelberger 1965; Peters 1968; Lumsden 1971), we feel it necessary to define the criteria used with regard to their critical importance in this study. Recent or acti~'e plaque: demyelination is usually incomplete with swelling and fragmentation of some myelin sheaths, while others may remain normal. The overall cellularity is increased. There are many sudanophilic granules lying free in the tissue, as well as those engulfed in microglial phagocytes. Some spherical myelin catabolic products show a Brewster cross (myelin balls) between crossed polarising filters. Later some crystallized needles with an intense double refraction can be observed. Such lipids, composed mainly of cholesterol, are not stained with Oil Red O or Sudan IV. In recent plaques the marginal area may be wide. In such a transitional zone single lipid macrophages and free sudanophilic lipids surrounded by otherwise normal myelin are observed. The oligodendroglial cells disappear soon in the lesions. However, in the early phase many abnormal oligodendroglial nuclei can be seen. The astrocytic reaction is not very prominent in recent plaques. Marked astrocytic proliferation is evident only after demyelination and the microglial reaction has taken place. At this stage many swollen or binucleated astrocytes are found. In the earliest stages neuroglial sclerosis is absent, although some reactive changes in the astrocytes are seen even in recent lesions. Old plaque: demyelination is complete and sharply defined and overall cellularity is decreased. The myelin breakdown products are collected entirely in phagocytes, while free lipids are no longer visible. The lipid phagocytes are collected mostly around the walls of the blood vessels. In the final stages lipids are almost completely removed from the tissue. In very old plaques only astrocytic nuclei are visible, while the increase of glial fibrils is marked.
4
B. F. TAVOLATO
In a single preparation, an old plaque with a marginal area with active demyelination is a frequent observation. In such instances the IgG distribution will be evaluated with special regard to such important differences. Shadow plaque : this type of lesion is usually defined as an incompletely d~'m\'clina~cd area, perhaps partially remyelinated, in which histological activity may be present (as in recent plaques, but apparently at a lower level) or may have subsided. All four examples of shadow plaque encountered in our study were classified in the latter (inactive) group. In our classification of plaque activity no mention was made of perivascular infiltrates. Although frequently seen, especially in recent lesions, such a type of reaction is difficult to evaluate: it may be present (but also absent) in recent and in old plaques, in normal white matter, near or distant from plaques, and in the meninges. Furthermore some authors (Peters 1968 ; Lumsden 1971 ) suspect that they may indicate also a resorptive inflammatory reaction, and therefore a distinction is made between primary and secondary perivascular infiltrates. For such reasons it does not seem to us justified to attribute crucial significance to these in classifying plaque demyelinating activity. Finally it must be mentioned that we have never encountered a lesion similar to that described by Seitelberger (1965) as "early stage of the focus formation", which probably should be considered as the initial injury, and this is therefore not considered in our classification. (7) Types and gradation qflfluorescence Normally myelinated areas show a dark-blue autofluorescence. However, in plaques, particularly in old demyelinated lesions, the background was black. This property, coupled with phase contrast microscopy, permitted the precise identification of plaques in IF-stained slides. A second type of autofluorescence was observed as yellow globules and was usually abundant in plaques, but was also scattered in normal white matter. Yellow or pink granules were also present in the perikarya of neurons, corresponding probably to lipofuscin and Nissl substance. Crystalline granules with white autofluorescence were only rarely observed in plaques. The specific fluorescence given by the fluorescent antisera was brilliant green and easily distinguished from the different types of autofluorescence. The gradation of the immunofluorescence in different areas of the same slide or in separate preparations is notoriously difficult to assess (Nairn 1969). Therefore ~ve devised a simplified classification system, which considers the extension of immunofluorescent material rather than the brilliance. An area without any specific fluorescence is classified as zero level, an area with single whispy fluorescence as low level and finally areas with large amounts (lakes) as high level (see also Figures). Furthermore, graded neutral filters were interposed in the primary light path and the density required to extinguish the fluorescence was n o t e d , lhe latter method is particularly useful in comparing the brightness of adjacent areas of the same preparation (Collier 1968). RESULTS
Normal brains IgG and albumin were not observed in normal brains except in or immediately around the blood vessels.
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
5
M S brains Old plaques: this type of lesion was found in 9 instances. The IgG immunofluores-
Fig. I. Old plaque (bottom right) and normal white matter (top left). In the marginal area some lipid macrophages are still visible. Cryostat section, oil red 0. x 100.
Fig. 2. The same plaque as in Fig. 1, stained with RAH IgG F1. Accumulation of lgG (lakes) is visible in the enlarged extracellular space of the plaque. Single yellow autofluorescent granules, corresponding to the myelin breakdown products, may be confused in the black-and-white picture with lhe green fluorescence of the IgG. x 100.
6
B.F. TAVOLATO
Fig. 3. Normal white matter near the plaque shown in Fig. l and 2, stained with RAH lgG Fi. Abundant specific fluorescence corresponding to tgG is visible without evidence of histological damage. × 100.
cence was rated as "high level" in 7 cases (Figs. 1 and 2). In these cases the igG was very abundant and clearly situated in the enlarged extracellular space of the plaque. A high IgG level was noticeable also in the marginal areas, extending for 50-100 #m into the adjacent white matter (Fig. 3). In areas distant from plaques the IgG level decreased, but did not disappear completely. Occasionally IgG accumulation was also noticeable in the normal white matter near the plaques. Usually such accumulation was noted in connection with small blood vessels, particularly if presenting perivascular mononuclear infiltrates, but sometimes the IgG appeared to stream out from the plaque without any relation to a particular structure or lesion. In 2 old plaques the central area o f the lesion was classified as "low level", while larger amounts of IgG were visible in the area corresponding to the margin of the plaque. In no instance was an old quiescent plaque completely deprived of IgG. In 5 samples we encountered an old plaque with a marginal area where demyelination was active (old and recent plaque together). A definite IgG distribution was evident in such cases: large amounts in the older areas and lower levels or even absence in the recent lesions. In such cases a negative IgG gradient paralleled the apparent age of the lesions. The normal white matter near or adjacent to the plaques showed high or medium levels of IgG. in general higher than in the actual areas o f demyelination. In 1 sample 2 separate plaques were observed: old quiescent and recent lesion, separated by a bridge of normal white matter. The old plaque showed a high IgG concentration, the normal white matter showed IgG, but at definitely lower levels than in the old lesion. The only area of the slide which was found to be almost deprived of IgG corresponded to the recent lesion. In 4 samples a recent lesion was observed and showed that IgG was absent or reduced, while in areas of the adjacent white matter higher levels of IgG were demon-
IMMUNOGLOBULiN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
7
Fig. 4. The same plaque as in Fig. 1 and 2, stained with RAH IgG FI, but prewashed in physiological saline. Some yellow autofluorescent drops are visible, sometimes in a perinuclear position (arrows). The specific fluorescence corresponding to lgG is completely removed, x 400.
Fig. 5. Area of recent dcmyelination. Many microglial phagocytes and lipid granules lying free in the tissue are visible. Cryostat section, Oil Red O, × 250.
8
B. F. I'AVOLATO
strated (Figs. 4 and 5). In a further 4 samples a shadow plaque (quiescent type) was observed showing high levels of IgG. The samples without plaques had no tgG or low levels. However such IgG was localised not only around the blood vessels as in normal brains, but was in many instances diffused in the tissue, or accumulated in areas without evidence of a lesion. Some IgG was often observed in the gray matter but apparently at levels lower than in the adjacent white matter. Albumin : abundant specific fluorescence corresponding to albumin was found only in 2 old plaques and trace amounts were found in a further 5. Albumin was absent in new and shadow plaques. The normal white matter near or distant from plaques was deprived of albumin except in the perivascular area. In each experiment some slides were washed in physiological saline and then fixed and stained with the immunofluorescent antisera and these were completely deprived of both lgG and albumin (Fig. 6). The time of washing was not critical between I min and
Fig. 6. The same plaque as in Fig. 5, stained with R A H IgG FL Trace a m o u n t s of lgG are visible. Most of the fluorescent material is composed of yellow autofluorescent granules corresponding to myelin breakdown products. × 100.
20 min and stirring was unnecessary. In exceptional cases IgG persisted alter washing in the cytoplasm of perivascular, mononuclear cells (immunocytes). Such cells were found only in brains where autopsy and tissue freezing was done within 3 hr of death. thus probably preserving the integrity of the cellular membranes. No albumincontaining cells were ever observed. I) ISCUSSION
This study was based on the inspection of immunofluorescent material and on classi-
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
9
fication of plaque activity and the discussion will consider mainly the extreme variations, to obtain a further margin of error. However, even considering only the presence or absence of IgG some conclusions may be drawn. Firstly, it is confirmed that IgG is present in quiescent plaques and can also be observed in the white matter of MS brains without evidence of histological damage. Similar results have been reported with an immunochemical method at the macroscopical level by Tourtellotte and Parker (1967). Furthermore we must consider the fact that recent actively demyelinating plaques were observed without evidence of IgG. Finally it is evident that IgG may be easily and completely eluted from MS brains with physiological saline; therefore it is difficult to conceive the IgG as being immunologically combined with components of the nervous tissue (i.e. basic protein). The known sensitivity of the immunofluorescent method makes it improbable that IgG is myelin-bound in a conventional immunological manner even if it is widely distributed around the myelin sheaths. However, the possibility of an antigen-antibody reaction with special features, weak combination or adhesion on macrophages of cytophilic antibodies, for instance, cannot be ruled out completely in this experiment. The significance of IgG-containing cells is discussed fully elsewhere (Tourtellotte et al. 1971). Obviously these observations do not exclude other possible immunopathological mechanisms in the pathogenesis ofdemyelination in MS brains and these are reviewed in detail by Johnson and Weiner (1972). In our case a cell-mediated immune system has been examined, similar to that which seems to play an important role in the pathogenesis of experimental allergic encephalomyelitis. A possible approach to this problem is offered by the leucocyte migration inhibition test, using lymphocytes from peripheral blood of MS patients and basic protein (BP) as the antigen, The fairly extensive studies now available (Caspary and Field 1972; Bartfeld, Atonyatan and Donnenfeld 1972; Rocklin 1972; Myers 1972) seem to indicate that BP-sensitive lymphocytes are present in MS, but also in other diseases characterized by destruction of nervous tissue. Furthermore~ from a histological point of view, we must remember that demyelinating activity in MS does not correlate strictly with the presence of lymphocytes; conversely it is possible to observe perivascular cuffings in otherwise normal white matter. Moreover, while in EAE the passive transfer of serum from already immunized animals was shown to protect against or to delay the onset of the disease (Harwin and Paterson 1962), the ventricular infusion of serum from animals with acute EAE can evoke a disease similar to EAE (Jankovic, Dra~koci and Janjic 1965). It may also be relevant to consider the fluorescent antibody studies of Rauch and Raffel (1964) which showed that antibodies prepared against BP were able to react in vitro with myelinated fibres of the CNS. In such experiments the antigen-antibody reaction was stable after repeated washing in phosphate buffered saline. From our data, the IgG in MS brains and spinal cords, does not seem to be lesion-, nor myelin-bound, at least as described in the experiments of Rauch and Raffel (1964). On the other hand, ifIgG in MS is not myelin-bound, we must also admit that we could not demonstrate IgG fixed to any other structure. This finding may be explained
10
B.F. TAVOLATO
by considering that specific antibodies of the IgG class are unable to cross normal cell membranes to react with an intracellular antigen (virus for instance!. In summary it seems more reasonable to suppose that IgG in MS brains is behaving as a normal antibody protecting the tissue from further damage if present in sufficient amount, but being unable to eradicate an intracellular latent infection. The finding concerning the presence of albumin in some old plaques is suggestive of possible damage to the blood-brain barrier. Similar observations have been reported by Broman (1946) and Gonssette and Andr6-Balisaux (1965) using different methods. From our data the damage of the blood--brain barrier in MS brain seems to be slight and present only in some lesions. This point is important considering the immunospecificity of the IgG in MS brains. In fact it is possible that a portion of such IgG is merely a result of blood-brain barrier damage. ° However, the presence of IgG-producing cells in MS brains and the available data for CSF IgG in MS (Tourtellotte 1970) are indicative that at least a parl of the brain IgG is produced in Ioco, by specific clones of immunocytes.
ACKNOWLEDGEMENTS
I am indebted to many colleagues and friends who in different ways helped my work. I would like to thank particularly Dr. W. W. Tourtellotte who made this research possible by supplying the materials. SUMMARY
The distribution of IgG in MS brains (plaques and normal zones) was studied with immunofluorescence. The histological activity was evaluated on alternate serial sections. The following results were achieved : high levels of IgG were usually connected with old plaques; IgG could also be observed in normal white matter, especially near old plaques. In recent plaques IgG was found to be absent or at very low levels, while higher levels of IgG were noticeable in the surrounding, apparently normal, areas. Finally it was observed that IgG can be completely removed from the tissue with a brief washing in physiological saline before fixation. The results are discussed and it is suggested that lgG in MS brains is acting more as a protective than as an autoimmune antibody. REFERENCES BARTFELD, H., T. A. ATONYATANAND H. DONNENFELD(1972) In vitro cellular immunity to central nervous system antigenes in multiple sclerosis. In: F. WOLFGRAM, G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Sclerosis. Immunology, Virology and Ultrastructure, Academic Press, New York and London, pp. 333-364. BROMAN~ T. 0946) Blood brain barrier damage in multiple sclerosis. Supravital test-observations, Acta neurol, scand, 40 (Suppl. 10): 2l-24. CASPARY, E. A. AND E. J. FIELD (1972) Sensitized lymphocytes in blood: a study of human neurological disease and experimental allergic encephalomyelitis. In: U. LEII3OWlTZ (Ed.), Pro,qress in Multiple
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
11
Sclerosis. Research and Treatment, Academic Press, New York and London, pp. 154-163. COLLIER, L. H. (1968) The use of graded density filters made by photography in fluorescence microscopy, J. Path. Bact., 96:238 242. FRICK, E. (1969) Zur Pathogenese entziindlicher Nervenerkrankungen. Fortsehr. Med., 87:1191 1194. GONSETTE, R. AND G. ANDRE-BALISAUX(1965) La perm6abilit6 des vaisseaux c~r6braux, Partie 4 (l~tude des 16sions de la barri6re h6mato-enc6phalique dans la scl6rose en plaques, Acta neurol, psychiat, belg., 65:19 34. GREENFIELD, J. G. (1960) Neuropatholoyy, Arnold, London, pp. 441-474. HARWIN, S. M. AND P. Y. PATERSON(1962) Anti-brain antibodies of the 19 S gamma-globulin type in rats with allergic encephalomyelitis, Nature (Lond.), 194: 391-392. HIJMANS, W., H. R. E. SCHUITAND F. KLEIN (1969) An immunofluorescence procedure for the detection of intracellular immunoglobulins, Clin. exp. lmmunol., 4: 457-472. JANKOVIC, B. D., M. DRASKOCIAND M. JANJIC [1965) Passive transfer of "'allergic" encephalomyelitis with antibrain serum injected into the lateral ventricle of the brain, Nature (Lond.) 207: 428-429. JOHNSON. R. T. AND L. P. WEINER (1972) The role of viral infections in demyelinating diseases. In: F. WOLI~GRAM,G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Sclerosis. Immunoh~gy. Virology and UItrastructure, Academic Press, New York and London, pp. 245 264. LUMSDEN,C. E. (1970) The neuropathology of multiple sclerosis. In: P. J. VINKEN AND G. W. BRUYN (Eds.), Handbook of Clinical Neurology, Vol. 9 (Multiple Sclerosis and other Demyelinatin9 Diseases), NorthHolland, Amsterdam, pp. 217 309. LUMSDEN, C. E. (1971) The immunogenesis of the multiple sclerosis plaque, Brain Res., 28: 365-390. M VERS,L. W. (1972) Leukocyte migration inhibition in multiple sclerosis. In: F. WOLEGRAM,G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Selerosis. Immunology, Virology and UItrastrueture, Academic Press, New York and London, pp. 383-402. NAIRN, R. C. (1969)Fluorescent Protein Tracing, Livingstone, Edinburgh and London. PETERS, G. (1968) Multiple sclerosis. In: J. MINKLER (Ed.), Pathology o/' the Ner~rous System, Vol. 1, McGraw-Hill, New York, N.Y., pp. 821 842. RAUCH, H. C. AND S. RAFFEL(1964) Immunofluorescent localization ofencephalitogenic protein in myelin, J. lmmunol., 92: 452-455. ROCKLIN. R. R. (1972) What is the role of lymphocyte in multiple sclerosis? In: F. WOLFGRAM,G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Sclerosis. Immunology. Virology and UItrastructure, Academic Press, New York and London, pp. 365-382. SEITELBERGER, F. (1965) Histochemistry of demyelinating diseases proper, including allergic encephalomyelitis and Pelizaeus-Merzbacher's disease. In: Modern Scient('fic Aspects of Neurology, Arnold. London, pp. 14~187. SIMPSON. J. F., W. W. TOURTELEOTTE,E. KOKMEN,J. A. PARKERAND H. H. ITABASHI (1969) Fluorescent protein tracing in multiple sclerosis brain tissue, Arch. Neurol. (Chic.), 20: 373-377. TER MEULEN, V.. G. ENDERS-RUCKLE,D. MULLER AND G. JOPPICH (1969) lmmunohistological, microscopical and neurochemical studies on encephalitides, Part 3, Acta neuropath. (Berl.), 12: 244-259. TOURTELLOTTE, W. W. (1970) Cerebrospinal fluid in multiple sclerosis. In: P. J. VINKEN AND G. W. BRUYN (Eds.), Handbook qf Clinical Neurology, Vol. 9 (Multiple Sclerosis and other Demyelinatin,q Diseases). North-Holland, Amsterdam. pp. 324-382. TOURTEELOTTE, W. W. AND J. A. PARKER(1966a) Multiple sclerosis: correlation between immunoglobulinG in cerebrospinal fluid and brain, Science, 154: 1044-1046. TOURTELLOTTE. W. W. AND J. A. PARKER (1966b) Immunoglobulin in multiple sclerosis white matter, J. Neuropath. exp. Neurol., 25:167 169. TDURTEELOTTE, W. W. AND PARKER (1967) Multiple sclerosis: brain immunoglobulin-G and albumin, Nature (Lond.), 214: 683-686. TOURTELLOTTE, W. W , H. H. ITABASHIAND J. A. PARKER (1967) Multifocal areas of synthesis of immunoglobulin-G in multiple sclerosis brain tissue and the sink action of the cerebrospinal fluid. Trans. Amer. neurol. Ass.. 92:288 290. TDURTELLOTTE, W. W.. E. KOKMEN, B. TAVOLATO AND P. S. KRUGER (1971) Fluorescent cell tracing in multiple sclerosis brain tissue, Trans. Amer. neurol. Ass., 96: 318-320.
1
Elsevier Scientific Publishing Company, Amsterdam-Printed in Tile Netherlands
Immunoglobulin G Distribution in Multiple Sclerosis Brain An ImmunofluorescenceStudy BRUNO F. TAVOLATO Clinica delle Malattie Nervose e Mentali. University o] Padova, Padol~a (Italy)
(Received 6 June, 1974)
INTRODUCTION
The elevation of the IgG: total protein ratio in the cerebrospinal fluid (CSF) of a large majority of multiple sclerosis (MS) patients has been considered as indicating an immunological reaction taking place in the central nervous system (CNS). Subsequently a positive gradient for IgG between brain and CSF was demonstrated (Tourtellotte and Parker 1966a), in addition to elevation of the IgG: albumin ratio in the brains of the majority of MS patients (Tourtellotte and Parker 1966b). It was also observed that IgG is maximally elevated in plaques, but usually the normal white matter in MS also has an elevated lgG: albumin ratio (Tourtellotte and Parker 1967). At a later stage an attempt was made to evaluate a correlation between IgG elevation and plaque activity (Tourtellotte, Itabashi and Parker 1967). This experiment involved using two adjacent coronal sections, one for histology, the other for IgG immunochemical determination and a positive correlation was found between perivascular mononuclear cuffing and the IgG level. However we believe that this important experiment left two questions unresolved. Firstly mononuclear perivascular infiltrates cannot be considered as the sole indicator of the complex phenomena associated with the different stages of demyelinating activity. Secondly plaque dissection for IgG determination was made on unstained coronal sections and it is well known that the demyelinating activity in MS plaques varies even within a very restricted area. For instance an old sclerotic plaque may show a zone in a small area at its margin where demyelination is active; moreover isolated areas of recent demyelination are very difficult to distinguish in unstained fresh sections. Therefore we believe that some confusion of plaque types may have occurred in these experiments. A possible approach to this problem is offered by the immunofluorescence (IF) technique, to demonstrate the distribution of IgG (or other proteins) specifically at the microscopical level. This technique has already been used by Ter Meulen, EndersRuckle, Mfiller and Joppich (1969), to show IgG, IgA and IgM in brains from patients affected by subacute sclerosing panencephalitis. The IF was used in MS brains by Simpson, Tourtellotte, Kokmen, Parker and Ita-
2
B. F. TAVOLATO
bashi (1969), who were able to demonstrate IgG accumulation in plaques, apparently located in the enlarged extracellular space, as well as in the surrounding " n o r m a l " white matter. Lumsden (1970) obtained similar results in 1 case of MS and concluded, on morphological grounds, that the IgG is "myelin-bound" and consequently auto-aggressive in nature. In the same paper Lumsden showed some clear lymphoid cells with the IgG-rich cytoplasm. The same cells have also been demonstrated by Frick (1969) and Tourtellotte, Kokmen, Tavolato and Kruger (1971 ) in MS brains, collected and frozen very quickly after death ( < 3 hr). The aim of the present study was to correlate if possible IgG levels and demyelinating activity in MS plaques. The 1F method appears especially suited for this purpose, since it permits the localisation of IgG at a histological level. The demyelinating activity can be evaluated and compared in alternate serial sections. Some further observations have been mad e concerning elution of IgG fro m M S brain to demonstrate the site(s) of possible antigen-antibody reaction.
MATERIALS AND METHODS
(1) Brain tis'sue The brains from 5 MS patients and 3 controls without evidence of CNS disease were used in the present study. The autopsies were done between 3 and 24 hr after death. The brains were collected, sliced, quick-frozen and stored at 90°C. A total o f 49 MS and 6 control samples derived from 22 coronal sections and 5 samples of cord or brain stem were studied. More than one sample was generally taken from each MS coronal section and we attempted to include portions of plaque, plaque margin, normal white matter near the plaque, normal white matter distant from the plaque and grey matter.
(2) Preparation of brain tissue./or IF staining The coronal sections were warmed to - 2 0 ° C just before use and samples were cut out with a cork-bore, having an inside diameter of 1 cm. Ten-#m thick serial slices were then cut with the cryostat and carefully mounted in cold gelatinized microscope slides. The slices were then thawed, air dried for 10 min and fixed in ethanol (95 °£1)-ether (1 : 1 ) for 10 min and then in 95 o//,,ethanol for 20 min at room temperature. Sections were stained with fluorescent antisera after evaporation of the ethanol. In each experiment some sections were washed in physiological saline for 1-10 min and then fixed and stained as previously described.
(3) Fluorescent antibody staining Only direct staining was used in the present study. The sections were incubated with the fluorescent antisera for 30 min at 37°C in a moist chamber and then washed 3 times for 10 min in phosphate-buffered saline. The slides were quickly rinsed in distilled water, air dried and mounted in buffered glycerine. Several commercial and home-made fluorescent antisera were tested and one commercial* R A H IgG F1 serum and a home made R A H Alb Fl were chosen and * Antibodies Incorporated, Davies, Calif.
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
3
used throughout. The RAH Alb serum was labelled with fluorescein isothiocyanate by the method of Hijmans, Schuit and Klein (1969). Controls : two methods were used in each experiment to control the specificity of fluorescent staining: the blocking test and a fluorescent antiserum in which the specific antibodies were previously precipitated with the specific antigen.
(4) Microscopy A Zeiss Ultraphot II microscope was used for fluorescence microscopy. Phase contrast microscopy (Zernicke) was used for the immunofluorescence-stained sections to distinguish the plaques from other structures. Polarized light was used with the Oil Red O stains to demonstrate the different classes of myelin breakdown products.
(5) Histology Alternate serial sections were used for immunofluorescence and for normal microscopy. Sections for histology were fixed in 10 ~ buffered formalin and stained with hematoxylin and eosin and oil red 0. Sometimes the Klfiver-Barrera stain for myelin sheaths was also used.
(6) Classification o/plaque activity The major goal of this study was to find a correlation, if any, between demyelinating activity and lgG level in MS plaques. Although the histological criteria for the classification of activity in plaques are well established and generally accepted (Greenfield 1960; Seitelberger 1965; Peters 1968; Lumsden 1971), we feel it necessary to define the criteria used with regard to their critical importance in this study. Recent or acti~'e plaque: demyelination is usually incomplete with swelling and fragmentation of some myelin sheaths, while others may remain normal. The overall cellularity is increased. There are many sudanophilic granules lying free in the tissue, as well as those engulfed in microglial phagocytes. Some spherical myelin catabolic products show a Brewster cross (myelin balls) between crossed polarising filters. Later some crystallized needles with an intense double refraction can be observed. Such lipids, composed mainly of cholesterol, are not stained with Oil Red O or Sudan IV. In recent plaques the marginal area may be wide. In such a transitional zone single lipid macrophages and free sudanophilic lipids surrounded by otherwise normal myelin are observed. The oligodendroglial cells disappear soon in the lesions. However, in the early phase many abnormal oligodendroglial nuclei can be seen. The astrocytic reaction is not very prominent in recent plaques. Marked astrocytic proliferation is evident only after demyelination and the microglial reaction has taken place. At this stage many swollen or binucleated astrocytes are found. In the earliest stages neuroglial sclerosis is absent, although some reactive changes in the astrocytes are seen even in recent lesions. Old plaque: demyelination is complete and sharply defined and overall cellularity is decreased. The myelin breakdown products are collected entirely in phagocytes, while free lipids are no longer visible. The lipid phagocytes are collected mostly around the walls of the blood vessels. In the final stages lipids are almost completely removed from the tissue. In very old plaques only astrocytic nuclei are visible, while the increase of glial fibrils is marked.
4
B. F. TAVOLATO
In a single preparation, an old plaque with a marginal area with active demyelination is a frequent observation. In such instances the IgG distribution will be evaluated with special regard to such important differences. Shadow plaque : this type of lesion is usually defined as an incompletely d~'m\'clina~cd area, perhaps partially remyelinated, in which histological activity may be present (as in recent plaques, but apparently at a lower level) or may have subsided. All four examples of shadow plaque encountered in our study were classified in the latter (inactive) group. In our classification of plaque activity no mention was made of perivascular infiltrates. Although frequently seen, especially in recent lesions, such a type of reaction is difficult to evaluate: it may be present (but also absent) in recent and in old plaques, in normal white matter, near or distant from plaques, and in the meninges. Furthermore some authors (Peters 1968 ; Lumsden 1971 ) suspect that they may indicate also a resorptive inflammatory reaction, and therefore a distinction is made between primary and secondary perivascular infiltrates. For such reasons it does not seem to us justified to attribute crucial significance to these in classifying plaque demyelinating activity. Finally it must be mentioned that we have never encountered a lesion similar to that described by Seitelberger (1965) as "early stage of the focus formation", which probably should be considered as the initial injury, and this is therefore not considered in our classification. (7) Types and gradation qflfluorescence Normally myelinated areas show a dark-blue autofluorescence. However, in plaques, particularly in old demyelinated lesions, the background was black. This property, coupled with phase contrast microscopy, permitted the precise identification of plaques in IF-stained slides. A second type of autofluorescence was observed as yellow globules and was usually abundant in plaques, but was also scattered in normal white matter. Yellow or pink granules were also present in the perikarya of neurons, corresponding probably to lipofuscin and Nissl substance. Crystalline granules with white autofluorescence were only rarely observed in plaques. The specific fluorescence given by the fluorescent antisera was brilliant green and easily distinguished from the different types of autofluorescence. The gradation of the immunofluorescence in different areas of the same slide or in separate preparations is notoriously difficult to assess (Nairn 1969). Therefore ~ve devised a simplified classification system, which considers the extension of immunofluorescent material rather than the brilliance. An area without any specific fluorescence is classified as zero level, an area with single whispy fluorescence as low level and finally areas with large amounts (lakes) as high level (see also Figures). Furthermore, graded neutral filters were interposed in the primary light path and the density required to extinguish the fluorescence was n o t e d , lhe latter method is particularly useful in comparing the brightness of adjacent areas of the same preparation (Collier 1968). RESULTS
Normal brains IgG and albumin were not observed in normal brains except in or immediately around the blood vessels.
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
5
M S brains Old plaques: this type of lesion was found in 9 instances. The IgG immunofluores-
Fig. I. Old plaque (bottom right) and normal white matter (top left). In the marginal area some lipid macrophages are still visible. Cryostat section, oil red 0. x 100.
Fig. 2. The same plaque as in Fig. 1, stained with RAH IgG F1. Accumulation of lgG (lakes) is visible in the enlarged extracellular space of the plaque. Single yellow autofluorescent granules, corresponding to the myelin breakdown products, may be confused in the black-and-white picture with lhe green fluorescence of the IgG. x 100.
6
B.F. TAVOLATO
Fig. 3. Normal white matter near the plaque shown in Fig. l and 2, stained with RAH lgG Fi. Abundant specific fluorescence corresponding to tgG is visible without evidence of histological damage. × 100.
cence was rated as "high level" in 7 cases (Figs. 1 and 2). In these cases the igG was very abundant and clearly situated in the enlarged extracellular space of the plaque. A high IgG level was noticeable also in the marginal areas, extending for 50-100 #m into the adjacent white matter (Fig. 3). In areas distant from plaques the IgG level decreased, but did not disappear completely. Occasionally IgG accumulation was also noticeable in the normal white matter near the plaques. Usually such accumulation was noted in connection with small blood vessels, particularly if presenting perivascular mononuclear infiltrates, but sometimes the IgG appeared to stream out from the plaque without any relation to a particular structure or lesion. In 2 old plaques the central area o f the lesion was classified as "low level", while larger amounts of IgG were visible in the area corresponding to the margin of the plaque. In no instance was an old quiescent plaque completely deprived of IgG. In 5 samples we encountered an old plaque with a marginal area where demyelination was active (old and recent plaque together). A definite IgG distribution was evident in such cases: large amounts in the older areas and lower levels or even absence in the recent lesions. In such cases a negative IgG gradient paralleled the apparent age of the lesions. The normal white matter near or adjacent to the plaques showed high or medium levels of IgG. in general higher than in the actual areas o f demyelination. In 1 sample 2 separate plaques were observed: old quiescent and recent lesion, separated by a bridge of normal white matter. The old plaque showed a high IgG concentration, the normal white matter showed IgG, but at definitely lower levels than in the old lesion. The only area of the slide which was found to be almost deprived of IgG corresponded to the recent lesion. In 4 samples a recent lesion was observed and showed that IgG was absent or reduced, while in areas of the adjacent white matter higher levels of IgG were demon-
IMMUNOGLOBULiN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
7
Fig. 4. The same plaque as in Fig. 1 and 2, stained with RAH IgG FI, but prewashed in physiological saline. Some yellow autofluorescent drops are visible, sometimes in a perinuclear position (arrows). The specific fluorescence corresponding to lgG is completely removed, x 400.
Fig. 5. Area of recent dcmyelination. Many microglial phagocytes and lipid granules lying free in the tissue are visible. Cryostat section, Oil Red O, × 250.
8
B. F. I'AVOLATO
strated (Figs. 4 and 5). In a further 4 samples a shadow plaque (quiescent type) was observed showing high levels of IgG. The samples without plaques had no tgG or low levels. However such IgG was localised not only around the blood vessels as in normal brains, but was in many instances diffused in the tissue, or accumulated in areas without evidence of a lesion. Some IgG was often observed in the gray matter but apparently at levels lower than in the adjacent white matter. Albumin : abundant specific fluorescence corresponding to albumin was found only in 2 old plaques and trace amounts were found in a further 5. Albumin was absent in new and shadow plaques. The normal white matter near or distant from plaques was deprived of albumin except in the perivascular area. In each experiment some slides were washed in physiological saline and then fixed and stained with the immunofluorescent antisera and these were completely deprived of both lgG and albumin (Fig. 6). The time of washing was not critical between I min and
Fig. 6. The same plaque as in Fig. 5, stained with R A H IgG FL Trace a m o u n t s of lgG are visible. Most of the fluorescent material is composed of yellow autofluorescent granules corresponding to myelin breakdown products. × 100.
20 min and stirring was unnecessary. In exceptional cases IgG persisted alter washing in the cytoplasm of perivascular, mononuclear cells (immunocytes). Such cells were found only in brains where autopsy and tissue freezing was done within 3 hr of death. thus probably preserving the integrity of the cellular membranes. No albumincontaining cells were ever observed. I) ISCUSSION
This study was based on the inspection of immunofluorescent material and on classi-
IMMUNOGLOBULIN G DISTRIBUTION IN MULTIPLE SCLEROSIS BRAIN
9
fication of plaque activity and the discussion will consider mainly the extreme variations, to obtain a further margin of error. However, even considering only the presence or absence of IgG some conclusions may be drawn. Firstly, it is confirmed that IgG is present in quiescent plaques and can also be observed in the white matter of MS brains without evidence of histological damage. Similar results have been reported with an immunochemical method at the macroscopical level by Tourtellotte and Parker (1967). Furthermore we must consider the fact that recent actively demyelinating plaques were observed without evidence of IgG. Finally it is evident that IgG may be easily and completely eluted from MS brains with physiological saline; therefore it is difficult to conceive the IgG as being immunologically combined with components of the nervous tissue (i.e. basic protein). The known sensitivity of the immunofluorescent method makes it improbable that IgG is myelin-bound in a conventional immunological manner even if it is widely distributed around the myelin sheaths. However, the possibility of an antigen-antibody reaction with special features, weak combination or adhesion on macrophages of cytophilic antibodies, for instance, cannot be ruled out completely in this experiment. The significance of IgG-containing cells is discussed fully elsewhere (Tourtellotte et al. 1971). Obviously these observations do not exclude other possible immunopathological mechanisms in the pathogenesis ofdemyelination in MS brains and these are reviewed in detail by Johnson and Weiner (1972). In our case a cell-mediated immune system has been examined, similar to that which seems to play an important role in the pathogenesis of experimental allergic encephalomyelitis. A possible approach to this problem is offered by the leucocyte migration inhibition test, using lymphocytes from peripheral blood of MS patients and basic protein (BP) as the antigen, The fairly extensive studies now available (Caspary and Field 1972; Bartfeld, Atonyatan and Donnenfeld 1972; Rocklin 1972; Myers 1972) seem to indicate that BP-sensitive lymphocytes are present in MS, but also in other diseases characterized by destruction of nervous tissue. Furthermore~ from a histological point of view, we must remember that demyelinating activity in MS does not correlate strictly with the presence of lymphocytes; conversely it is possible to observe perivascular cuffings in otherwise normal white matter. Moreover, while in EAE the passive transfer of serum from already immunized animals was shown to protect against or to delay the onset of the disease (Harwin and Paterson 1962), the ventricular infusion of serum from animals with acute EAE can evoke a disease similar to EAE (Jankovic, Dra~koci and Janjic 1965). It may also be relevant to consider the fluorescent antibody studies of Rauch and Raffel (1964) which showed that antibodies prepared against BP were able to react in vitro with myelinated fibres of the CNS. In such experiments the antigen-antibody reaction was stable after repeated washing in phosphate buffered saline. From our data, the IgG in MS brains and spinal cords, does not seem to be lesion-, nor myelin-bound, at least as described in the experiments of Rauch and Raffel (1964). On the other hand, ifIgG in MS is not myelin-bound, we must also admit that we could not demonstrate IgG fixed to any other structure. This finding may be explained
10
B.F. TAVOLATO
by considering that specific antibodies of the IgG class are unable to cross normal cell membranes to react with an intracellular antigen (virus for instance!. In summary it seems more reasonable to suppose that IgG in MS brains is behaving as a normal antibody protecting the tissue from further damage if present in sufficient amount, but being unable to eradicate an intracellular latent infection. The finding concerning the presence of albumin in some old plaques is suggestive of possible damage to the blood-brain barrier. Similar observations have been reported by Broman (1946) and Gonssette and Andr6-Balisaux (1965) using different methods. From our data the damage of the blood--brain barrier in MS brain seems to be slight and present only in some lesions. This point is important considering the immunospecificity of the IgG in MS brains. In fact it is possible that a portion of such IgG is merely a result of blood-brain barrier damage. ° However, the presence of IgG-producing cells in MS brains and the available data for CSF IgG in MS (Tourtellotte 1970) are indicative that at least a parl of the brain IgG is produced in Ioco, by specific clones of immunocytes.
ACKNOWLEDGEMENTS
I am indebted to many colleagues and friends who in different ways helped my work. I would like to thank particularly Dr. W. W. Tourtellotte who made this research possible by supplying the materials. SUMMARY
The distribution of IgG in MS brains (plaques and normal zones) was studied with immunofluorescence. The histological activity was evaluated on alternate serial sections. The following results were achieved : high levels of IgG were usually connected with old plaques; IgG could also be observed in normal white matter, especially near old plaques. In recent plaques IgG was found to be absent or at very low levels, while higher levels of IgG were noticeable in the surrounding, apparently normal, areas. Finally it was observed that IgG can be completely removed from the tissue with a brief washing in physiological saline before fixation. The results are discussed and it is suggested that lgG in MS brains is acting more as a protective than as an autoimmune antibody. REFERENCES BARTFELD, H., T. A. ATONYATANAND H. DONNENFELD(1972) In vitro cellular immunity to central nervous system antigenes in multiple sclerosis. In: F. WOLFGRAM, G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Sclerosis. Immunology, Virology and Ultrastructure, Academic Press, New York and London, pp. 333-364. BROMAN~ T. 0946) Blood brain barrier damage in multiple sclerosis. Supravital test-observations, Acta neurol, scand, 40 (Suppl. 10): 2l-24. CASPARY, E. A. AND E. J. FIELD (1972) Sensitized lymphocytes in blood: a study of human neurological disease and experimental allergic encephalomyelitis. In: U. LEII3OWlTZ (Ed.), Pro,qress in Multiple
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Sclerosis. Research and Treatment, Academic Press, New York and London, pp. 154-163. COLLIER, L. H. (1968) The use of graded density filters made by photography in fluorescence microscopy, J. Path. Bact., 96:238 242. FRICK, E. (1969) Zur Pathogenese entziindlicher Nervenerkrankungen. Fortsehr. Med., 87:1191 1194. GONSETTE, R. AND G. ANDRE-BALISAUX(1965) La perm6abilit6 des vaisseaux c~r6braux, Partie 4 (l~tude des 16sions de la barri6re h6mato-enc6phalique dans la scl6rose en plaques, Acta neurol, psychiat, belg., 65:19 34. GREENFIELD, J. G. (1960) Neuropatholoyy, Arnold, London, pp. 441-474. HARWIN, S. M. AND P. Y. PATERSON(1962) Anti-brain antibodies of the 19 S gamma-globulin type in rats with allergic encephalomyelitis, Nature (Lond.), 194: 391-392. HIJMANS, W., H. R. E. SCHUITAND F. KLEIN (1969) An immunofluorescence procedure for the detection of intracellular immunoglobulins, Clin. exp. lmmunol., 4: 457-472. JANKOVIC, B. D., M. DRASKOCIAND M. JANJIC [1965) Passive transfer of "'allergic" encephalomyelitis with antibrain serum injected into the lateral ventricle of the brain, Nature (Lond.) 207: 428-429. JOHNSON. R. T. AND L. P. WEINER (1972) The role of viral infections in demyelinating diseases. In: F. WOLI~GRAM,G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Sclerosis. Immunoh~gy. Virology and UItrastructure, Academic Press, New York and London, pp. 245 264. LUMSDEN,C. E. (1970) The neuropathology of multiple sclerosis. In: P. J. VINKEN AND G. W. BRUYN (Eds.), Handbook of Clinical Neurology, Vol. 9 (Multiple Sclerosis and other Demyelinatin9 Diseases), NorthHolland, Amsterdam, pp. 217 309. LUMSDEN, C. E. (1971) The immunogenesis of the multiple sclerosis plaque, Brain Res., 28: 365-390. M VERS,L. W. (1972) Leukocyte migration inhibition in multiple sclerosis. In: F. WOLEGRAM,G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Selerosis. Immunology, Virology and UItrastrueture, Academic Press, New York and London, pp. 383-402. NAIRN, R. C. (1969)Fluorescent Protein Tracing, Livingstone, Edinburgh and London. PETERS, G. (1968) Multiple sclerosis. In: J. MINKLER (Ed.), Pathology o/' the Ner~rous System, Vol. 1, McGraw-Hill, New York, N.Y., pp. 821 842. RAUCH, H. C. AND S. RAFFEL(1964) Immunofluorescent localization ofencephalitogenic protein in myelin, J. lmmunol., 92: 452-455. ROCKLIN. R. R. (1972) What is the role of lymphocyte in multiple sclerosis? In: F. WOLFGRAM,G. W. ELLISON, J. G. STEVENSAND J. M. ANDREWS (Eds.), Multiple Sclerosis. Immunology. Virology and UItrastructure, Academic Press, New York and London, pp. 365-382. SEITELBERGER, F. (1965) Histochemistry of demyelinating diseases proper, including allergic encephalomyelitis and Pelizaeus-Merzbacher's disease. In: Modern Scient('fic Aspects of Neurology, Arnold. London, pp. 14~187. SIMPSON. J. F., W. W. TOURTELEOTTE,E. KOKMEN,J. A. PARKERAND H. H. ITABASHI (1969) Fluorescent protein tracing in multiple sclerosis brain tissue, Arch. Neurol. (Chic.), 20: 373-377. TER MEULEN, V.. G. ENDERS-RUCKLE,D. MULLER AND G. JOPPICH (1969) lmmunohistological, microscopical and neurochemical studies on encephalitides, Part 3, Acta neuropath. (Berl.), 12: 244-259. TOURTELLOTTE, W. W. (1970) Cerebrospinal fluid in multiple sclerosis. In: P. J. VINKEN AND G. W. BRUYN (Eds.), Handbook qf Clinical Neurology, Vol. 9 (Multiple Sclerosis and other Demyelinatin,q Diseases). North-Holland, Amsterdam. pp. 324-382. TOURTEELOTTE, W. W. AND J. A. PARKER(1966a) Multiple sclerosis: correlation between immunoglobulinG in cerebrospinal fluid and brain, Science, 154: 1044-1046. TOURTELLOTTE. W. W. AND J. A. PARKER (1966b) Immunoglobulin in multiple sclerosis white matter, J. Neuropath. exp. Neurol., 25:167 169. TDURTEELOTTE, W. W. AND PARKER (1967) Multiple sclerosis: brain immunoglobulin-G and albumin, Nature (Lond.), 214: 683-686. TOURTELLOTTE, W. W , H. H. ITABASHIAND J. A. PARKER (1967) Multifocal areas of synthesis of immunoglobulin-G in multiple sclerosis brain tissue and the sink action of the cerebrospinal fluid. Trans. Amer. neurol. Ass.. 92:288 290. TDURTELLOTTE, W. W.. E. KOKMEN, B. TAVOLATO AND P. S. KRUGER (1971) Fluorescent cell tracing in multiple sclerosis brain tissue, Trans. Amer. neurol. Ass., 96: 318-320.
