Journal of the Neurological Sciences, 1979, 41 : 81-91 © Elsevier/North-Holland Biomedical Press
8l
A HISTOLOGICAL, HISTOCHEMICAL AND BIOCHEMICAL STUDY OF THE MACROSCOPICALLY NORMAL WHITE MATTER IN MULTIPLE SCLEROSIS
INGRID V. ALLEN and STEPHANIE R. McKEOWN
Department of Pathology (Multiple Sclerosis Research Laboratory), Royal Victoria Hospital and Queen's University (Department of Pathology), Belfast ( N. Ireland) (Received 27 September, 1978) (Accepted 3 October, 1978)
SUMMARY
In a combined histological, biochemical and histochemical study of the macroscopically normal white matter in multiple sclerosis 72 ~ of samples were histologically abnormal. The significance of this fact in the interpretation of previous biochemical studies and in the design of future studies is discussed. The present study showed a significant elevation of the lysosomal enzyme fl-glucosaminidase in the microscopically normal white matter in MS as compared with controls. Studies on lysosomes separated from microscopically normal or mild to moderately gliosed white matter in multiple sclerosis showed an increase in lysosomal fragility. Histochemical study of the microscopically normal white matter in multiple sclerosis revealed an increase in the number of acid phosphate-containing cells as compared with normal and neurological control material. The significance of these findings is discussed and it is suggested that irrespective of the primary or secondary nature of these abnormalities, the white matter may be rendered more susceptible to the pathogenetic process in this disease.
INTRODUCTION
There have been frequent suggestions that in multiple sclerosis (MS) there is an inherent biochemical defect in the so-called normal white matter (Cumings 1953, 1955; Einstein et al. 1970; Gerstl et al. 1970). Such a defect, while not necessarily the immediate cause of the disease, could render the myelin susceptible to the pathogen. Unfortunately, the biochemical studies which have been carried out to test this hypothesis have produced conflicting results. Norton (1977), having reviewed the biochemical evidence, suggests that while there is undoubted biochemical abnormality
82 in m a n y samples o f m a c r o s c o p i c a l l y n o r m a l white m a t t e r in MS, the variation ~n results implies t h a t there is no inherent lipid defect, b u t r a t h e r that the a b n o r m a l i t i e s are the result o f m i c r o s c o p i c lesions. Others, however, take the view that there is a p r i m a r y biochemical a b n o r m a l i t y o f myelin and relate this to genetic, d i e t a r y and e n v i r o n m e n t a l factors ( T h o m p s o n 1973; G o l d b e r g 1974). Studies on o t h e r biochemical c o n s t i t u e n t s have also p r o d u c e d conflicting results. F o r example, Riekkinen et al. (1971) r e p o r t e d t h a t MS myelin had decreased a m o u n t s o f basic protein, but this finding has n o t been s u p p o r t e d by o t h e r w o r k ( W o l f g r a m 1972; Suzuki et al. 1973). It has also been r e p o r t e d t h a t lysosomal hydrolases are increased in some samples o f m a c r o s c o p i c a l l y n o r m a l white m a t t e r in MS (Einstein et al. 1972; A r s t i l a et al. 1973: C u z n e r et al. 1976). However, Hirsch et al. (1976) found, in a small n u m b e r o f samples, n o consistent increase in lysosomal h y d r o l a s e activity in n o r m a l a p p e a r i n g white m a t t e r . U n f o r t u n a t e l y , m a n y o f the biochemical studies on m a c r o s c o p i c a l l y n o r m a l white m a t t e r in M S have, largely for technical reasons, i n a d e q u a t e histological control. M o r e o v e r , the classical p a t h o l o g i c a l descriptions of the disease have concent r a t e d on the plaque and p e r i p l a q u e and, a l t h o u g h microscopic lesions in the m a c r o s c o p i c a l l y n o r m a l white m a t t e r are well recognised ( L u m s d e n 1970), their variety and incidence are p o o r l y d o c u m e n t e d . We are u n a w a r e o f any definitive histological study o f the m a c r o s c o p i c a l l y n o r m a l white m a t t e r in M S. The present study was u n d e r t a k e n as a c o m b i n e d histological/biochemical investigation in an a t t e m p t to o b t a i n some idea o f the incidence o f histological a b n o r m a l i t y in a p p a r e n t l y n o r m a l samples o f M S white m a t t e r t a k e n for biochemistry. O u r second aim was to relate b i o c h e m i c a l a b n o r m a l i t i e s to the histological findings using, in a d d i t i o n , histochemicat techniques.
TABLE l MS CASES Case
Sex
Cause of death Age Duration Death/ (yrs) of illness necropsy (yrs) interval (hr)
D15 D17 D24 D25 D27
F F M M F
29 60 38 23 63
10 25 6 1 27
D35 D36 D37 D39 D40 D41 D48 D49 D54
M F F F F M M F F
45 71 60 48 64 59 84 57 56
22 42 16 15 18 22 50 38 31
3 6 13 4 4 3-} 4½ 3¼ 5¼ 3 4½ 7½ 5½ 2
bronchopneumonia multiple sclerosis bronchopneumonia bronchopneumonia ruptured abdominal aneurysm bronchopneumonia pulmonary embolism bronchopneumonia pulmonary oedema bronchopneumonia bronchopneumonia bronchopneumonia bronchopneumonia bronchopneumonia
Enzyme Histostudy chemical study
Lysosomal fragility study
-i +
i ~ ---
----
-! ~t ---~ -4-F + --
~ ~i ------+ +
--i +~÷ -+ -4-
83 MATERIALS AND METHODS
Case material and sampling S a m p l e s were o b t a i n e d f r o m 14 M S cases (Table 1) a n d 11 controls (Table 2). The M S cases were, with one exception, o f the chronic variety. I n only a few cases was the d e a t h - n e c r o p s y interval greater t h a n 6 hours. C o n t r o l cases were selected because o f the s h o r t d e a t h - n e c r o p s y interval a n d the absence o f neurological illness (with the e x c e p t i o n o f one n e u r o l o g i c a l control). Necropsies were p e r f o r m e d in a m o r t u a r y a d j a c e n t to the l a b o r a t o r y a n d i m m e d i a t e l y after r e m o v a l the b r a i n was b r o u g h t to the l a b o r a t o r y f o r careful dissection. C o r o n a l slices of the fresh b r a i n were e x a m i n e d a n d samples o f m a c r o s c o p i c a l l y n o r m a l white m a t t e r were taken, in the M S cases, as far d i s t a n t as possible f r o m visible plaques (this was n o t easy a n d white m a t t e r which was a p p a r e n t l y u n r e l a t e d to a p l a q u e in the c o r o n a l plane could be close to a plaque in the sagittal plane). N o r m a l white m a t t e r f r o m c o n t r o l cases was similarly sampled.
Enzyme study S a m p l e s for b i o c h e m i c a l analysis were w r a p p e d tightly in parafilm a n d s t o r e d at - - 2 0 °C in a i r t i g h t tubes for u p to 15 m o n t h s . I n some samples blocks for histological c o n t r o l were t a k e n i m m e d i a t e l y but, in the majority, blocks were t a k e n when the samples were p r e p a r e d for h o m o g e n i z a t i o n . Since it was c o n s i d e r e d o f vital i m p o r t a n ce to o b t a i n representative blocks for histological c o n t r o l the specimens for biochemical analysis were carefully dissected before m a t e r i a l was t a k e n f r o m the centre o f the samples for histology. The following enzymes were analysed on tissue h o m o g e nates p r e p a r e d in 0.1 ~ T r i t o n X-100 as described previously ( M c K e o w n a n d Allen 1978): n-acetyl-fl-D-glucosaminidase (fl-Glm), fl-galactosidase (fl-gal), fl-glucuronidase (fl-Glon), cathepsin D (cath D) a n d aryl sulphatase (ASS). P r o t e i n was d e t e r m i n e d by the m e t h o d o f L o w r y et al. (1951). TABLE 2 CONTROL CASES Case
Sex
Age Diagnosis (yrs)
C7
M
49
C 16 C 18 C24 C27 C32 C35 C36 C37 C38 C43
M F M F F M M F M F
66 61 64 65 84 62 48 28 67 58
demyelination following CO poisoning myocardial infarction myocardial infarction myocardial infarction cor pulmonale haemorrhagic diathesis myocardial infarction myocardial infarction bronchopneumonia myocardial infarction myocardial infarction
Death/ Enzyme Histonecropsy study chemical interval (hr) study
Lysosomal fragility study
3 9 5 5½ 7 6 5 5 3½ 4 3
---+ -+ + + + + --
--+ -~ -+ ~ + Jr --
+ + --------+
84
L),sosomal fragility study A series of samples from 7 MS cases and 6 controls (Tables 1 and 2) were also assessed for lysosomal fragility according to methods already described (McKeown 1979; McKeown and Allen 1978). Histochemical study Nine samples of microscopically normal white matter from 6 cases of MS, 7 samples from 2 normal controls and 2 samples from one neurological control (Tables 1 and 2 ) w e r e studied. The neurological control was a case of carbon monoxide poisoning with extensive myelin damage resulting in death after 3 months. The longest interval between death and cooling of the tissues was 9 hr. The fresh brain was sectioned as for the biochemical studies and the blocks for histochemistry were placed on small pieces of moistened cork and snap frozen in isopentane, pre-cooled with liquid nitrogen. Cryostat sections, cut at 8 #m, were air-dried and were then placed in the incubation solution for 45 min for the demonstration of acid phosphatase (APP) by the naphthol AS-B1 phosphate method (Barka 1960) using pararosanilin hydrochloride (Hopkins and Williams, basic red 9 C. 1. 42500). The incubated sections were then stained by a variety of techniques previously developed in this laboratory (Allen, Glover, McKeown and McCormick 1979) which permit the simultaneous demonstration of APP, myelin, lipids and various cells, particularly astrocytes. For the enzyme and lysosomal fragility studies all samples were assessed histologically, staining techniques being limited to ensure sufficient tissue for biochemical analysis. Frozen sections were cut from blocks fixed in buffered formalin and were stained with Spielmeyer-scharlach Red. The remaining tissue was embedded in paraffin wax and the sections cut were stained with haematoxylin and eosin (H and E).
TABLE 3 HISTOLOGY OF MACROSCOPICALLY NORMAL WHITE MATTER
Histologically normal Incidence of histological abnormalities Gliosis Demyelination Small round cell infiltration Macrophages Perivascular deposits lipofuscin iron calcium Thickened blood vessels Corpora amylacea
MS (54)
Control (52)
15 (27.8~)
33 (63.5~)
32 7 5 7
(59.3~) (13.0~) (9.3 ~) (13.0~)
16 (29.6~) 0 1 (1.9~) 3 (5.6%) 4 (7.4~)
4 (7.7 ~) 0 0 0 4 3 0 6 6
(7.7 ~) (5.8 ~) (11.5~) (11.5~)
85
Fig. 1. MS macroscopically normal white matter. A: histologically normal; B: gliosed. H and E, x 100.
Fig. 2. MS macroscopically normal white matter. Astrocyte (arrowed). PTAH, × 250.
86 RESULTS
Histology Of the 54 specimens of macroscopically normal white matter available from the 12 MS cases used for the lysosomal enzyme and fragility studies, 7 2 ~ were histologically abnormal (Table 3). The 8 control cases used for these studies had an incidence of 3 6 ~ histological abnormality but the nature and severity of the abnormality in the MS cases differed markedly from that of the controls. By far the most common abnormality in the MS cases was gliosis which in this study was assessed subjectively (Fig. 1). The gliosis, even with H and E staining only, could often be attributed to proliferation of astrocytes (Fig. 2) and although no attempt was made to quantitate the abnormalities the assessment was relatively easy. Unsuspected demyelination was found in 13.0 ~ of the samples from the MS cases. The incidence of perivascular small round cell infiltration was only 9 ~ and the degree of infiltration in the 5 samples showing this change was slight and usually associated with perivascular macrophage infiltration and deposition of lipofuscin (Fig. 3). The other histological abnormalities in the MS cases listed in Table 3 were only found in a few samples. Although 19 of the control samples were histologically abnormal, the degree of abnormality was slight and in most cases consisted of minor vascular changes or corpora amylacea deposition. Most of the control samples would have been passed as normal in routine histology.
Fig. 3. MS macroseopically normal white matter. Lipofusein in perivascular spaces. H and E, periodic acid-Schiff (PAS), Sudan Btaek (SB), APP; × 200.
87 TABLE 4 LYSOSOMAL ENZYMES IN MICROSCOPICALLY NORMAL WHITE MATTER FROM MS AND CONTROL CASES Results reported ± SEM. The number of estimations is given in parenthesis. Enzyme
/~-Glm fl-gal ASS (nmoles MUB~/ (nmoles MUB/ (roMp-nitromin/mg protein) min/mgprotein) catechol/h/mg protein)
cath D (mM tyrosine/ h/mg protein)
fl-Glon (mmoles MUB/ min/ mg protein)
Control
4.16 2_ 0.22 (34) 5.30 £- 0.27 b (16)
0.52 ± 0.02 (34) 0.56 -k 0.05 (16)
0.30 ± 0.01 (23) 0.33 4- 0.02 (10)
MS
0.27 d- 0.03 (33) 0.27 d_ 0.02 (15)
1.22 ± 0.11 (34) 1.24 zt- 0.24 (13)
4-Methylumbelliferone. b p < 0.0005 (Results analysed with a Student t-test were compared to the control values).
Lysosomal enzyme study B i o ch em ic a l assay was carried o u t on 38 samples; o f these 16 were histologically n o r m a l , 18 showed mild to m o d e r a t e gliosis an d 4 were demyelinated. The results f o r the 16 microscopically n o r m a l specimens are given in Tab l e 4. fl-Glm was the only e n z y m e which was significantly increased.
Lysosomalfragility study O f the 19 samples used in this study 3 were histologically n o r m a l , 12 showed mild to m o d e r a t e gliosis a n d 4 were demyelinated. Because o f the small n u m b e r o f histologically n o r m a l samples available f o r study, the gliosed samples were included in the calculation o f the results. H o w e v e r , the results for the microscopically n o r m a l
TABLE 5 N-ACETYL-fl-D-GLUCOSAMINIDASE IN WHITE MATTER FROM CONTROL AND MS BRAINS Each control value was obtained as an average of 4 determinations (in duplicate) obtained by investigation of 4 different samples from one brain. Each MS value is a duplicate determination of a sample dissected from one of 7 MS brains as macroscopically normal white matter. (Histological control of these samples led to 4 samples with frank demyelination being eliminated, see Results.) Mean values 4- SEM obtained by analysis of raw data only.
Control (6) MS (15)
/3-Glucosaminidase a
Non-sedimentable activity b ( ~ )
fl-Glucosaminidase Protein recovery recovery ( ~ ) (%)
3.65 ]_ 0.20 4.37 i 0.34
41.7 i 4.0 52.4 _L 4.0
94.7 97.4
a N-acetyl-fl-D-glucosaminidase activity as in Table 4. b For definition of non-sedimentable activity see Materials and Methods.
99.5 97.5
88
Fig. 4. Microscopically normal white matter. Normal control (N); neurological control (OND); MS. APP, x 100. samples did not appear to differ from those of the gliosed samples. Comparison of the MS cases and the controls (Table 5) showed a greater degree of lysosomal rupture as measured by the non-sedimentable activity (P < 0.05).
APP histochemistry This study was done in an attempt to demonstrate the cellular source of lysosomal enzymes in the microscopically normal white matter in MS. Nine blocks of macroscopically normal white matter from 6 MS cases were used. Of the 9 MS specimens, 1 showed slight gliosis, 7 contained a few small round cells in perivascular spaces and 3 contained a few small deposits of lipofuscin. The overall degree of histological abnormality, however, was slight and most specimens in routine histology would have been passed as normal. The 9 blocks available from the control cases were all histologically normal and a striking finding in the neurological control was the normality of the white matter except immediately surrounding the demyelinated areas. Despite the fact that both the MS and control specimens were essentially normal histologically, APP-positive cells were much more numerous in the MS cases (Fig. 4). In the MS cases combination of APP staining with H and E or Spielmeyer-scharlach Red confirmed the increase in APP-positive cells despite the normal myelin stains. Combined APP and phosphotungstic acid haematoxylin (PTAH) staining showed that most of the APP-positive cells were astrocytes (Fig. 5). Although oligodendrocytes were not specifically stained the techniques used did not demonstrate APP in interfascicular oligodendrocytes. DISCUSSION The histological assessment of the macroscopically normal white matter in MS is of importance first in the understanding of other studies (e.g, biochemical) in the
89
Fig. 5. MS microscopicallynormal white matter. Astrocytes(arrowed). APP/PTAH, × 250.
apparently normal white matter, and, secondly, in the formulation of fundamental hypotheses concerning mechanisms of myelin damage in this disease. While many pathological descriptions of MS make some reference to the white matter at a distance from plaques, we are unaware of any systematic study of this tissue combined with biochemical and histochemical assessment. Much of the controversy over biochemical results on the macroscopically normal white matter has arisen probably because of inadequate histological control. Many biochemical techniques, particularly for lipid analysis, require large amounts of tissue and, therefore, in these histological control may be difficult or impossible until microtechniques have been evolved. The present study has shown, however, that some 72 ~ of histological specimens from the macroscopically normal white matter were histologically abnormal and one may question therefore the validity of any biochemical analysis on "normal white matter" when large samples are required. The present study has also shown that reasonable histological control is possible in many biochemical studies and that the histological interpretation of samples, deep-frozen for biochemical analysis, is relatively easy. Several points emerge from the histological assessment of the biochemical samples. The most frequent histological abnormality was diffuse gliosis. Astrocytic proliferation in the white matter away from plaques has frequently been described both with the light microscope (Lumsden 1970) and with the electron microscope (Andrews 1972; Prineas and Raine 1976). However, no attempts have been made to assess the extent of this gliosis, the assumption usually being made that it is a secondary phenomenon in reaction to the demyelinating process. Distortion of
90 biochemical results in apparently normal white matter can also be attributed, in a proportion of cases, to the presence of demyelination which, despite the care taken in sampling, was found on microscopic assessment in 13 ~ of the samples. Similarly, biochemical estimations may be influenced by the lipofuscin deposition found in 30 % of samples and the presence of perivascular round cell infiltration which was found in 9 ~ of samples. These infiltrates contained few cells and were often associated with macrophage infiltration and lipofuscin deposition. Detailed analysis of the biochemical results shows the importance of histological control. Of the enzymes studied, only /3-Glm was significantly elevated in the microscopically normal white matter. However, when the results were calculated using all samples of the macroscopically normal white matter, they showed a significant elevation, not only of/3-Glm, but also of/3-Gal and/%Glon; cathepsin D was also elevated in several samples although the overall result was not significant. Unfortunately, only 3 of the 15 samples used for the investigation of lysosomal fragility were histologically normal. The results, supported by further studies on the remaining intact lysosomes (McKeown and Allen 1979), suggest that there is an inherent instability of cerebral lysosomes in MS. To confirm that this is not due to the gliosis found in the majority of the samples analysed it is hoped to obtain more results from samples free of histological abnormalities. It is possible that these results could be caused by differences in the terminal illnesses between the control patients, the majority of whom died as a result of myocardial infarction, and the MS patients in whom the terminal event was in most cases bronchopneumonia. However, in both these gloups the terminal illnesses were relatively short and it is possible that the controls were subjected to a greater degree of cerebral damage due to repeated episodes of ischaemia, than the MS cases where no attempts were made at resuscitation. The charts of all patients were scrutinized for any other common factor between the two groups but no consistent differences, apart from MS in the test group, were found. In the histochemical study in microscopically normal white matter in MS it would obviously have been preferable to have assessed /%Glm activity since in the biochemical study this was the only enzyme which was significantly increased. Unfortunately, up to the present it has not proved technically possible to combine this reaction with the othe~ staining techniques and it was therefore decided to visualise APP using the relatively insoluble dye pararosanilin. Sections in which APP was demonstrated could then be stained for myelin, lipid, glial fibres, etc. Our finding of an increase in APP positive cells in microscopically normal white matter in MS compared to controls has not, to our knowledge, been reported previously. Moreover, the demonstration that the majority of these cells are astrocytic raises the question of the significance of an elevation of at least some of the lysosomal enzymes in this cell (for discussion of this point see Allen et al. 1979). Irrespective of the primary or secondary nature of this phenomenon, it clearly has important implications. An increase in lysosomal enzymes in astrocytes in the microscopically normal white matter in MS, especially if these lysosomes are more fragile, may render this tissue more susceptible to the pathogen, whatever that may be.
91 REFERENCES Allen, I. V., G. Glover, S. R. McKeown and D. McCormick (1979) The cellular origin of lysosomal enzymes in the plaque in multiple sclerosis, Part 2 (An histochemical study with combined demonstration of myelin and acid phosphatase), Neuropath. appl. Neurobiol., In press. Andrews, J. M. (1972) The ultrastructural neuropathology of multiple sclerosis. In: F. Wolfgram, G. W. Ellison, J. G. Stevens and J. M. Andrews (Eds.), Multiple Sclerosis - - Immunology, Virology and Ultrastructure, Academic Press, New York and London, pp. 23-52. Arstila, A. U., P. Riekkinen, U. K. Rinne and L. Laitinen (1973) Studies on the pathogenesis of multiple sclerosis - - Participation of lysosomes on demyelination in the central nervous system white matter outside plaques, Europ. Neurol., 9: 1-20. Barka, T. (1960) A simple azo-dye method for histochemical demonstration of acid phopshatase, Nature (Lond.), 187: 248-249. Cumings, J. M. (1953) The cerebral lipids in disseminated sclerosis and in amaurotic family idiocy, Brain, 76: 551-563. Cumings, J. M. (1955) Lipid chemistry of the brain in demyelinating diseases, Brain, 78: 554-563. Cuzner, M. L., R. O. Barnard, B. J. L. MacGregor, N. J. Borshell and A. N. Davison (1976) Myelin composition in acute and chronic multiple sclerosis in relation to cerebral lysosomal activity, J. neurol. Sci., 29: 323-334. Einstein, E. R., K. B. Dalai and J. Csejtey (1970) Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques, J. neurol. Sci., 11: 109-121. Einstein, E. R., J. Csejtey, K. B. Dalai, C. W. M. Adams, O. B. Bayliss and J. F. Hallpike (1972) Proteolytic activity and basic protein loss in and around multiple sclerosis plaques - - Combined biochemical and histochemical observations, J. Neurochem., 19: 653-662. Gerstl, B., L. F. Eng, M. Tavaststjerna, J. K. Smith and S. L. Kruse (1970) Lipids and proteins in multiple sclerosis white matter, J. Neurochem., 17: 677-689. Goldberg, P. (1974) Multiple sclerosis - - Vitamin D and calcium as environmental determinants of prevalence, Part 1 (Sunlight, dietary factors and epidemiology), Int. J. Environm. Stud., 6: 19. Hirsch, H. E., P. Duquette and M. E. Parks (1976) The quantitative histochemistry of multiple sclerosis plaques - - Acid proteinase and other acid hydrolases, J. Neurochem., 26: 505-512. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951) Protein measurement with the Folin phenol reagent, J. biol. Chem., 193: 265-275. Lumsden, C. E. (1970) The neuropathology of multiple sclerosis. In: P. J. Vinken and G. W. Bruyn (Eds.), Handbook o f Clinical Neurology, Vol. 9 (Multiple Sclerosis and Other Demyelinating Diseases), North-Holland Publishing Co., Amsterdam, pp. 217 309. McKeown, S. R. (1979) Postmortem autolytic response in rat brain lysosomes, J. Neurochem., 32: 391-396. McKeown, S. R. and 1. V. Allen (1978) The cellular origin of lysosomal enzymes in the plaque in multiple sclerosis, Part 1 (A combined histological and biochemical study), Neuropcth. appl. Neurobiol., 4: 471-482. McKeown, S. R. and I. V. Allen (1979) The fragility of cerebral enzymes in multiple sclerosis, Submitted for publication. Norton, W. T. (1977) Chemical pathology of diseases involving myelin. In: P. Morell (Ed.), Myelin, Plenum Press, New York and London, pp. 383-413. Prineas, J. W. and C. S. Raine (1976) Electron microscopy and immunoperoxidase studies of early multiple sclerosis lesions. Neurology (Minneap.), 26, No. 6 (2): 29-32. Riekkinen, P. J., J. Palo, A. U. Arstila, H. J. Savolainen, U. K. Rinne, E. K. Kivalo and H. Frey (1971) Protein composition of multiple sclerosis myelin, Arch. NeuroL (Chic.), 24: 545-549. Suzuki, K., Y. Eto and J. O. Gonatas (1973) Myelin in multiple sclerosis - - Composition of myelin from normal-appearing white matter, Neurology (Minneap.j, 28: 293-297. Thompson, R. H. S. (1973) Fatty acid metabolism in multiple sclerosis, Biochem. Soc. Syrup., 35: 103-111. Wolfgram, F. (1972) Chemical theories of the demyelination in multiple sclerosis. In: F. Wolfgram, G. W. Ellison, J. G. Stevens and J. M. Andrews (Eds.), Multiple Sclerosis - - Immunology, Virology and Ultrastructure, Academic Press, New York and London, pp. 173-181.
8l
A HISTOLOGICAL, HISTOCHEMICAL AND BIOCHEMICAL STUDY OF THE MACROSCOPICALLY NORMAL WHITE MATTER IN MULTIPLE SCLEROSIS
INGRID V. ALLEN and STEPHANIE R. McKEOWN
Department of Pathology (Multiple Sclerosis Research Laboratory), Royal Victoria Hospital and Queen's University (Department of Pathology), Belfast ( N. Ireland) (Received 27 September, 1978) (Accepted 3 October, 1978)
SUMMARY
In a combined histological, biochemical and histochemical study of the macroscopically normal white matter in multiple sclerosis 72 ~ of samples were histologically abnormal. The significance of this fact in the interpretation of previous biochemical studies and in the design of future studies is discussed. The present study showed a significant elevation of the lysosomal enzyme fl-glucosaminidase in the microscopically normal white matter in MS as compared with controls. Studies on lysosomes separated from microscopically normal or mild to moderately gliosed white matter in multiple sclerosis showed an increase in lysosomal fragility. Histochemical study of the microscopically normal white matter in multiple sclerosis revealed an increase in the number of acid phosphate-containing cells as compared with normal and neurological control material. The significance of these findings is discussed and it is suggested that irrespective of the primary or secondary nature of these abnormalities, the white matter may be rendered more susceptible to the pathogenetic process in this disease.
INTRODUCTION
There have been frequent suggestions that in multiple sclerosis (MS) there is an inherent biochemical defect in the so-called normal white matter (Cumings 1953, 1955; Einstein et al. 1970; Gerstl et al. 1970). Such a defect, while not necessarily the immediate cause of the disease, could render the myelin susceptible to the pathogen. Unfortunately, the biochemical studies which have been carried out to test this hypothesis have produced conflicting results. Norton (1977), having reviewed the biochemical evidence, suggests that while there is undoubted biochemical abnormality
82 in m a n y samples o f m a c r o s c o p i c a l l y n o r m a l white m a t t e r in MS, the variation ~n results implies t h a t there is no inherent lipid defect, b u t r a t h e r that the a b n o r m a l i t i e s are the result o f m i c r o s c o p i c lesions. Others, however, take the view that there is a p r i m a r y biochemical a b n o r m a l i t y o f myelin and relate this to genetic, d i e t a r y and e n v i r o n m e n t a l factors ( T h o m p s o n 1973; G o l d b e r g 1974). Studies on o t h e r biochemical c o n s t i t u e n t s have also p r o d u c e d conflicting results. F o r example, Riekkinen et al. (1971) r e p o r t e d t h a t MS myelin had decreased a m o u n t s o f basic protein, but this finding has n o t been s u p p o r t e d by o t h e r w o r k ( W o l f g r a m 1972; Suzuki et al. 1973). It has also been r e p o r t e d t h a t lysosomal hydrolases are increased in some samples o f m a c r o s c o p i c a l l y n o r m a l white m a t t e r in MS (Einstein et al. 1972; A r s t i l a et al. 1973: C u z n e r et al. 1976). However, Hirsch et al. (1976) found, in a small n u m b e r o f samples, n o consistent increase in lysosomal h y d r o l a s e activity in n o r m a l a p p e a r i n g white m a t t e r . U n f o r t u n a t e l y , m a n y o f the biochemical studies on m a c r o s c o p i c a l l y n o r m a l white m a t t e r in M S have, largely for technical reasons, i n a d e q u a t e histological control. M o r e o v e r , the classical p a t h o l o g i c a l descriptions of the disease have concent r a t e d on the plaque and p e r i p l a q u e and, a l t h o u g h microscopic lesions in the m a c r o s c o p i c a l l y n o r m a l white m a t t e r are well recognised ( L u m s d e n 1970), their variety and incidence are p o o r l y d o c u m e n t e d . We are u n a w a r e o f any definitive histological study o f the m a c r o s c o p i c a l l y n o r m a l white m a t t e r in M S. The present study was u n d e r t a k e n as a c o m b i n e d histological/biochemical investigation in an a t t e m p t to o b t a i n some idea o f the incidence o f histological a b n o r m a l i t y in a p p a r e n t l y n o r m a l samples o f M S white m a t t e r t a k e n for biochemistry. O u r second aim was to relate b i o c h e m i c a l a b n o r m a l i t i e s to the histological findings using, in a d d i t i o n , histochemicat techniques.
TABLE l MS CASES Case
Sex
Cause of death Age Duration Death/ (yrs) of illness necropsy (yrs) interval (hr)
D15 D17 D24 D25 D27
F F M M F
29 60 38 23 63
10 25 6 1 27
D35 D36 D37 D39 D40 D41 D48 D49 D54
M F F F F M M F F
45 71 60 48 64 59 84 57 56
22 42 16 15 18 22 50 38 31
3 6 13 4 4 3-} 4½ 3¼ 5¼ 3 4½ 7½ 5½ 2
bronchopneumonia multiple sclerosis bronchopneumonia bronchopneumonia ruptured abdominal aneurysm bronchopneumonia pulmonary embolism bronchopneumonia pulmonary oedema bronchopneumonia bronchopneumonia bronchopneumonia bronchopneumonia bronchopneumonia
Enzyme Histostudy chemical study
Lysosomal fragility study
-i +
i ~ ---
----
-! ~t ---~ -4-F + --
~ ~i ------+ +
--i +~÷ -+ -4-
83 MATERIALS AND METHODS
Case material and sampling S a m p l e s were o b t a i n e d f r o m 14 M S cases (Table 1) a n d 11 controls (Table 2). The M S cases were, with one exception, o f the chronic variety. I n only a few cases was the d e a t h - n e c r o p s y interval greater t h a n 6 hours. C o n t r o l cases were selected because o f the s h o r t d e a t h - n e c r o p s y interval a n d the absence o f neurological illness (with the e x c e p t i o n o f one n e u r o l o g i c a l control). Necropsies were p e r f o r m e d in a m o r t u a r y a d j a c e n t to the l a b o r a t o r y a n d i m m e d i a t e l y after r e m o v a l the b r a i n was b r o u g h t to the l a b o r a t o r y f o r careful dissection. C o r o n a l slices of the fresh b r a i n were e x a m i n e d a n d samples o f m a c r o s c o p i c a l l y n o r m a l white m a t t e r were taken, in the M S cases, as far d i s t a n t as possible f r o m visible plaques (this was n o t easy a n d white m a t t e r which was a p p a r e n t l y u n r e l a t e d to a p l a q u e in the c o r o n a l plane could be close to a plaque in the sagittal plane). N o r m a l white m a t t e r f r o m c o n t r o l cases was similarly sampled.
Enzyme study S a m p l e s for b i o c h e m i c a l analysis were w r a p p e d tightly in parafilm a n d s t o r e d at - - 2 0 °C in a i r t i g h t tubes for u p to 15 m o n t h s . I n some samples blocks for histological c o n t r o l were t a k e n i m m e d i a t e l y but, in the majority, blocks were t a k e n when the samples were p r e p a r e d for h o m o g e n i z a t i o n . Since it was c o n s i d e r e d o f vital i m p o r t a n ce to o b t a i n representative blocks for histological c o n t r o l the specimens for biochemical analysis were carefully dissected before m a t e r i a l was t a k e n f r o m the centre o f the samples for histology. The following enzymes were analysed on tissue h o m o g e nates p r e p a r e d in 0.1 ~ T r i t o n X-100 as described previously ( M c K e o w n a n d Allen 1978): n-acetyl-fl-D-glucosaminidase (fl-Glm), fl-galactosidase (fl-gal), fl-glucuronidase (fl-Glon), cathepsin D (cath D) a n d aryl sulphatase (ASS). P r o t e i n was d e t e r m i n e d by the m e t h o d o f L o w r y et al. (1951). TABLE 2 CONTROL CASES Case
Sex
Age Diagnosis (yrs)
C7
M
49
C 16 C 18 C24 C27 C32 C35 C36 C37 C38 C43
M F M F F M M F M F
66 61 64 65 84 62 48 28 67 58
demyelination following CO poisoning myocardial infarction myocardial infarction myocardial infarction cor pulmonale haemorrhagic diathesis myocardial infarction myocardial infarction bronchopneumonia myocardial infarction myocardial infarction
Death/ Enzyme Histonecropsy study chemical interval (hr) study
Lysosomal fragility study
3 9 5 5½ 7 6 5 5 3½ 4 3
---+ -+ + + + + --
--+ -~ -+ ~ + Jr --
+ + --------+
84
L),sosomal fragility study A series of samples from 7 MS cases and 6 controls (Tables 1 and 2) were also assessed for lysosomal fragility according to methods already described (McKeown 1979; McKeown and Allen 1978). Histochemical study Nine samples of microscopically normal white matter from 6 cases of MS, 7 samples from 2 normal controls and 2 samples from one neurological control (Tables 1 and 2 ) w e r e studied. The neurological control was a case of carbon monoxide poisoning with extensive myelin damage resulting in death after 3 months. The longest interval between death and cooling of the tissues was 9 hr. The fresh brain was sectioned as for the biochemical studies and the blocks for histochemistry were placed on small pieces of moistened cork and snap frozen in isopentane, pre-cooled with liquid nitrogen. Cryostat sections, cut at 8 #m, were air-dried and were then placed in the incubation solution for 45 min for the demonstration of acid phosphatase (APP) by the naphthol AS-B1 phosphate method (Barka 1960) using pararosanilin hydrochloride (Hopkins and Williams, basic red 9 C. 1. 42500). The incubated sections were then stained by a variety of techniques previously developed in this laboratory (Allen, Glover, McKeown and McCormick 1979) which permit the simultaneous demonstration of APP, myelin, lipids and various cells, particularly astrocytes. For the enzyme and lysosomal fragility studies all samples were assessed histologically, staining techniques being limited to ensure sufficient tissue for biochemical analysis. Frozen sections were cut from blocks fixed in buffered formalin and were stained with Spielmeyer-scharlach Red. The remaining tissue was embedded in paraffin wax and the sections cut were stained with haematoxylin and eosin (H and E).
TABLE 3 HISTOLOGY OF MACROSCOPICALLY NORMAL WHITE MATTER
Histologically normal Incidence of histological abnormalities Gliosis Demyelination Small round cell infiltration Macrophages Perivascular deposits lipofuscin iron calcium Thickened blood vessels Corpora amylacea
MS (54)
Control (52)
15 (27.8~)
33 (63.5~)
32 7 5 7
(59.3~) (13.0~) (9.3 ~) (13.0~)
16 (29.6~) 0 1 (1.9~) 3 (5.6%) 4 (7.4~)
4 (7.7 ~) 0 0 0 4 3 0 6 6
(7.7 ~) (5.8 ~) (11.5~) (11.5~)
85
Fig. 1. MS macroscopically normal white matter. A: histologically normal; B: gliosed. H and E, x 100.
Fig. 2. MS macroscopically normal white matter. Astrocyte (arrowed). PTAH, × 250.
86 RESULTS
Histology Of the 54 specimens of macroscopically normal white matter available from the 12 MS cases used for the lysosomal enzyme and fragility studies, 7 2 ~ were histologically abnormal (Table 3). The 8 control cases used for these studies had an incidence of 3 6 ~ histological abnormality but the nature and severity of the abnormality in the MS cases differed markedly from that of the controls. By far the most common abnormality in the MS cases was gliosis which in this study was assessed subjectively (Fig. 1). The gliosis, even with H and E staining only, could often be attributed to proliferation of astrocytes (Fig. 2) and although no attempt was made to quantitate the abnormalities the assessment was relatively easy. Unsuspected demyelination was found in 13.0 ~ of the samples from the MS cases. The incidence of perivascular small round cell infiltration was only 9 ~ and the degree of infiltration in the 5 samples showing this change was slight and usually associated with perivascular macrophage infiltration and deposition of lipofuscin (Fig. 3). The other histological abnormalities in the MS cases listed in Table 3 were only found in a few samples. Although 19 of the control samples were histologically abnormal, the degree of abnormality was slight and in most cases consisted of minor vascular changes or corpora amylacea deposition. Most of the control samples would have been passed as normal in routine histology.
Fig. 3. MS macroseopically normal white matter. Lipofusein in perivascular spaces. H and E, periodic acid-Schiff (PAS), Sudan Btaek (SB), APP; × 200.
87 TABLE 4 LYSOSOMAL ENZYMES IN MICROSCOPICALLY NORMAL WHITE MATTER FROM MS AND CONTROL CASES Results reported ± SEM. The number of estimations is given in parenthesis. Enzyme
/~-Glm fl-gal ASS (nmoles MUB~/ (nmoles MUB/ (roMp-nitromin/mg protein) min/mgprotein) catechol/h/mg protein)
cath D (mM tyrosine/ h/mg protein)
fl-Glon (mmoles MUB/ min/ mg protein)
Control
4.16 2_ 0.22 (34) 5.30 £- 0.27 b (16)
0.52 ± 0.02 (34) 0.56 -k 0.05 (16)
0.30 ± 0.01 (23) 0.33 4- 0.02 (10)
MS
0.27 d- 0.03 (33) 0.27 d_ 0.02 (15)
1.22 ± 0.11 (34) 1.24 zt- 0.24 (13)
4-Methylumbelliferone. b p < 0.0005 (Results analysed with a Student t-test were compared to the control values).
Lysosomal enzyme study B i o ch em ic a l assay was carried o u t on 38 samples; o f these 16 were histologically n o r m a l , 18 showed mild to m o d e r a t e gliosis an d 4 were demyelinated. The results f o r the 16 microscopically n o r m a l specimens are given in Tab l e 4. fl-Glm was the only e n z y m e which was significantly increased.
Lysosomalfragility study O f the 19 samples used in this study 3 were histologically n o r m a l , 12 showed mild to m o d e r a t e gliosis a n d 4 were demyelinated. Because o f the small n u m b e r o f histologically n o r m a l samples available f o r study, the gliosed samples were included in the calculation o f the results. H o w e v e r , the results for the microscopically n o r m a l
TABLE 5 N-ACETYL-fl-D-GLUCOSAMINIDASE IN WHITE MATTER FROM CONTROL AND MS BRAINS Each control value was obtained as an average of 4 determinations (in duplicate) obtained by investigation of 4 different samples from one brain. Each MS value is a duplicate determination of a sample dissected from one of 7 MS brains as macroscopically normal white matter. (Histological control of these samples led to 4 samples with frank demyelination being eliminated, see Results.) Mean values 4- SEM obtained by analysis of raw data only.
Control (6) MS (15)
/3-Glucosaminidase a
Non-sedimentable activity b ( ~ )
fl-Glucosaminidase Protein recovery recovery ( ~ ) (%)
3.65 ]_ 0.20 4.37 i 0.34
41.7 i 4.0 52.4 _L 4.0
94.7 97.4
a N-acetyl-fl-D-glucosaminidase activity as in Table 4. b For definition of non-sedimentable activity see Materials and Methods.
99.5 97.5
88
Fig. 4. Microscopically normal white matter. Normal control (N); neurological control (OND); MS. APP, x 100. samples did not appear to differ from those of the gliosed samples. Comparison of the MS cases and the controls (Table 5) showed a greater degree of lysosomal rupture as measured by the non-sedimentable activity (P < 0.05).
APP histochemistry This study was done in an attempt to demonstrate the cellular source of lysosomal enzymes in the microscopically normal white matter in MS. Nine blocks of macroscopically normal white matter from 6 MS cases were used. Of the 9 MS specimens, 1 showed slight gliosis, 7 contained a few small round cells in perivascular spaces and 3 contained a few small deposits of lipofuscin. The overall degree of histological abnormality, however, was slight and most specimens in routine histology would have been passed as normal. The 9 blocks available from the control cases were all histologically normal and a striking finding in the neurological control was the normality of the white matter except immediately surrounding the demyelinated areas. Despite the fact that both the MS and control specimens were essentially normal histologically, APP-positive cells were much more numerous in the MS cases (Fig. 4). In the MS cases combination of APP staining with H and E or Spielmeyer-scharlach Red confirmed the increase in APP-positive cells despite the normal myelin stains. Combined APP and phosphotungstic acid haematoxylin (PTAH) staining showed that most of the APP-positive cells were astrocytes (Fig. 5). Although oligodendrocytes were not specifically stained the techniques used did not demonstrate APP in interfascicular oligodendrocytes. DISCUSSION The histological assessment of the macroscopically normal white matter in MS is of importance first in the understanding of other studies (e.g, biochemical) in the
89
Fig. 5. MS microscopicallynormal white matter. Astrocytes(arrowed). APP/PTAH, × 250.
apparently normal white matter, and, secondly, in the formulation of fundamental hypotheses concerning mechanisms of myelin damage in this disease. While many pathological descriptions of MS make some reference to the white matter at a distance from plaques, we are unaware of any systematic study of this tissue combined with biochemical and histochemical assessment. Much of the controversy over biochemical results on the macroscopically normal white matter has arisen probably because of inadequate histological control. Many biochemical techniques, particularly for lipid analysis, require large amounts of tissue and, therefore, in these histological control may be difficult or impossible until microtechniques have been evolved. The present study has shown, however, that some 72 ~ of histological specimens from the macroscopically normal white matter were histologically abnormal and one may question therefore the validity of any biochemical analysis on "normal white matter" when large samples are required. The present study has also shown that reasonable histological control is possible in many biochemical studies and that the histological interpretation of samples, deep-frozen for biochemical analysis, is relatively easy. Several points emerge from the histological assessment of the biochemical samples. The most frequent histological abnormality was diffuse gliosis. Astrocytic proliferation in the white matter away from plaques has frequently been described both with the light microscope (Lumsden 1970) and with the electron microscope (Andrews 1972; Prineas and Raine 1976). However, no attempts have been made to assess the extent of this gliosis, the assumption usually being made that it is a secondary phenomenon in reaction to the demyelinating process. Distortion of
90 biochemical results in apparently normal white matter can also be attributed, in a proportion of cases, to the presence of demyelination which, despite the care taken in sampling, was found on microscopic assessment in 13 ~ of the samples. Similarly, biochemical estimations may be influenced by the lipofuscin deposition found in 30 % of samples and the presence of perivascular round cell infiltration which was found in 9 ~ of samples. These infiltrates contained few cells and were often associated with macrophage infiltration and lipofuscin deposition. Detailed analysis of the biochemical results shows the importance of histological control. Of the enzymes studied, only /3-Glm was significantly elevated in the microscopically normal white matter. However, when the results were calculated using all samples of the macroscopically normal white matter, they showed a significant elevation, not only of/3-Glm, but also of/3-Gal and/%Glon; cathepsin D was also elevated in several samples although the overall result was not significant. Unfortunately, only 3 of the 15 samples used for the investigation of lysosomal fragility were histologically normal. The results, supported by further studies on the remaining intact lysosomes (McKeown and Allen 1979), suggest that there is an inherent instability of cerebral lysosomes in MS. To confirm that this is not due to the gliosis found in the majority of the samples analysed it is hoped to obtain more results from samples free of histological abnormalities. It is possible that these results could be caused by differences in the terminal illnesses between the control patients, the majority of whom died as a result of myocardial infarction, and the MS patients in whom the terminal event was in most cases bronchopneumonia. However, in both these gloups the terminal illnesses were relatively short and it is possible that the controls were subjected to a greater degree of cerebral damage due to repeated episodes of ischaemia, than the MS cases where no attempts were made at resuscitation. The charts of all patients were scrutinized for any other common factor between the two groups but no consistent differences, apart from MS in the test group, were found. In the histochemical study in microscopically normal white matter in MS it would obviously have been preferable to have assessed /%Glm activity since in the biochemical study this was the only enzyme which was significantly increased. Unfortunately, up to the present it has not proved technically possible to combine this reaction with the othe~ staining techniques and it was therefore decided to visualise APP using the relatively insoluble dye pararosanilin. Sections in which APP was demonstrated could then be stained for myelin, lipid, glial fibres, etc. Our finding of an increase in APP positive cells in microscopically normal white matter in MS compared to controls has not, to our knowledge, been reported previously. Moreover, the demonstration that the majority of these cells are astrocytic raises the question of the significance of an elevation of at least some of the lysosomal enzymes in this cell (for discussion of this point see Allen et al. 1979). Irrespective of the primary or secondary nature of this phenomenon, it clearly has important implications. An increase in lysosomal enzymes in astrocytes in the microscopically normal white matter in MS, especially if these lysosomes are more fragile, may render this tissue more susceptible to the pathogen, whatever that may be.
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