Journal of Neuroimmunology, 46 (1993) 207-216 207 Elsevier Science Publishers B.V.

JNI 02438

On classification of post-mortem multiple sclerosis plaques for neuroscientists

Virginia Sanders a, Andrew J. Conrad b and Wallace W. Tourtel lot te c

a Brain Research Institute, UCLA, Los Angeles, CA, USA, b Department of Neurology, UCLA School of Medicine, Los Angeles, CA, USA, and c Neurology and Research Services, VAMC, Los Angeles, CA, USA

(Received 9 March 1993) (Revision received 9 April 1993)

(Accepted 9 April 1993)

Key words: Classification of plaque's demyelinating activity; Post-mortem; Major histocompatibility complex class II; Oil Red O

Summary

Plaque classification is proposed based on observation of 348 plaques from 52 post-mortem multiple sclerosis (MS) cases. Four plaque types, ranging from 'earliest lesion' to 'inactive', are described according to immunological activation and degree of demyelination, seen by expression of the Major Histocompatability Complex (MHC) Class II molecule, HLA-DR, and by Oil Red O staining, respectively. 40% of the plaques were inactive. This result highlights the need for a description of plaque activity for studies of the etiopathogenesis of MS; that is, the earliest and/or the most active plaques should contain the causative agent whereas the burnt out plaques should not.

Introduction

Multiple sclerosis is a disease of unknown etiology characterized clinically by motor and/or sensory deficits and characterized pathologically by multifocal lesions of inflammation and demyelination (Matthews et al., 1985). The cluster destruction of oligodendro- cytes-myelin sheaths forms plaques, the vast majority being in the white matter. The center of the plaque is composed of astrocytes, which replace the oligodendro- cytes-myelin sheaths; as plaques age, axons also drop out. At the plaque edge, the site of the ongoing MS disease process, there can be found polyphasic inflam- mation. The disease is not static by either clinical, magnetic resonance imaging (MRI), or pathological manifestations. Exacerbations and remissions, espe- cially during the early stages of the disease, are com- mon in disease progression, as assessed by clinical symptoms and/or magnetic resonance imaging (MRI). The presence of new lesions without new clinical symp-

Correspondence to: W. Tourtellotte, Neurology Research (127A), VAMC West Los Angeles, Los Angeles, CA 90073, USA.

toms is often reported in MRI studies (Thompson et al., 1992). This lack of correlation between symptoms and pathological lesions is an additional complication in the dynamic nature of MS.

Plaque tissue has been the obvious choice of tissue to study the pathology of MS. Histological staining has provided a picture of plaque appearance as either active with hypercellularity, immune system infiltrates, and myelin debris or inactive with hypocellularity and complete loss of myelin replaced by astrocytosis. Al- though more recent immunocytochemical staining and MRI studies have suggested gradual changes in plaques (Hauser et al., 1990; Thompson et al., 1990; Harris et al., 1991), studies still rely on an 'on-off ' description of plaque tissue. In the literature, plaques have been 'typed' by either the clinical course of the patient (acute or chronic) (Bellamy et al., 1985; Lee et al., 1990; Toms et al., 1990) or by the presence or absence of inflammatory cells (active/acute vs. inactive/ chronic) (Allen and McKeown, 1979; Adams et al., 1987; Hofman et al., 1986; Brazil et al., 1988). With a few notable exceptions (Esiri, 1980; Adams et al., 1987), most studies have had small sample numbers and did not address the appearance of plaques in a random post-mortem population.

208

TABLE 1

Demography of each case

Cases are listed in order of increasing disease duration.

HSB a Age/gender Cause of death

735 39/M Pneumonia 470 47/M Septicemia 944 33/F Septicemia 644 37/F Cardiac arrest 573 50/F Overdose 678 40/M Cardiac arrest 536 31/M Cardiac arrest

1 864 37/F Cerebral depression 1035 66/M Pneumonia

511 40/M Respiratory failure 1 238 36/F G.I. bleeding 1 862 69/M Septicemia 1 011 44/M Pneumonia 1 337 58/F MS 1291 44/F Cardiac arrest

996 41 / F Fibrosarcoma 1 682 50/F Septicemia

888 50/M Pneumonia 1 145 42/M Anoxic

encepholopathy 1 206 61/F Pneumonia 1 354 73/M Cardiac arrest

344 40/M Pneumonia 845 45/M Drowning 985 66/F Undetermined

1588 46/F Resp. distress syndrome

1 735 79/M Infected decubitis ulcers

1061 50/M MS 1 324 67/M Pneumonia

934 50/F Pneumonia 1 449 59/F Cardiac arrest 1 342 64/M Pneumonia 1 174 65/M Septicemia 1334 58/M Cardiac arrest

640 51/M Pneumonia 1 446 51/F Pneumonia 1 270 67/F Septicemia 1396 66/F Cardiac arrest 1489 72/F Septicemia 1 668 59/F Septicemia

228 80/M Pneumonia 1 657 53/F Pneumonia 1934 60/F Met. colon cancer 1023 71/F Septicemia 1 721 68/F Respiratory arrest

682 71/F Stroke 909 70/F Undetermined

1088 64/F Renal failure 1 151 74/F Met. breast cancer

579 61/M Pneumonia 1 897 68/F Met. breast cancer 1 292 62/M Pneumonia 1 779 69/F Met. lung cancer

Duration (years)

Clinical Neurological disability preagonal state of death (modified Kurtzke scale)

Autolysis time (h)

4 5 6 8 9

10 11 11 12 13 13 14 16 16 19 20 20 21 22

22 22 23 23 23 23

23

24 24 25 25 26 27 27 28 29 30 30 30 32 33 34 34 36 37 40 40 40 40 41 44 45 47

7.5 8 8.5 9 7 7.5 6.5 9 8 8 9 7.5 8 8 8 8 9 8.5 8

9 7 7.5 6.5 6 8.5

9 8 9 8.5 8 7.5 9 9 9 8 8.5 8 8.5 9 8.5 7.5 7.5 9 8.5 6.5 8.5 7.5 7.5 8 7.5 8.5

a Human Specimen Bank Number.

6 3.5

22.5 11 15 5.5

25 12 10.5 12 19.5 9 4.5

14 7

14 13 8

31.5

9 9.5 6

28 18 23.75

12 6

11.5 18 9

12 8

23 4.5

23.5 8 6.5 9.5 5

41.5 10 18 8.5

20 31 11.5 13 12 7

14.5 6.5

Number of Blocks

2 3

21 7

13 20 14 2 4

20 4 1 2 2

17 12 6

19 3

15 4 4

16 10 5

4 6 2

14 9 7

10 22

1 5 3 9

19 3 5 4 7 2 5 2 1 2

16 1 7

16

Cell-mediated immune function is dependent on antigen presentation to CD4 + and CD8 ÷ T cells by the Class I and Class II major histocompatability complex (MHC) molecules, respectively. The CNS was long considered to be immunologically isolated; however, recent evidence has suggested that there are low levels of constitutive expression of the MHC class II molecule, HLA-DR, on perivascular cells (Partridge et al., 1989) and that expression may be induced on microglial cells and macrophages by activating factors such as gamma- interferon. With the advent and improvements in im- munocytochemical staining, more specific characteriza- tion of plaques has been done. Both Class I and Class II MHC molecules have been identified in plaque tissue on endothelial cells, infiltrating lymphocytes, and astroglia (Woodroofe et al., 1986; Hayashi et al., 1988), and endothelial cells, macrophages, microglia, and as- troglia (Woodroofe et al., 1986; Traugott, 1987; Lee et al., 1990), respectively. The associated T cell subset molecules, CD8 and CD4, have also been identified in the lesions and surrounding normal appearing white matter (Woodroofe et al., 1986; McCallum et al., 1987; Sobel, 1989; Estes et al., 1990; Gambi et al., 1991). The immunocytochemical data suggest that the mechanism in plaque formation is T cell-mediated, that it is depen- dent on a very specific but as yet not well characterized interrelationship between antigen-presenting cells, T cells, cytokines, and, of course the ultimate target, myelin, and that, at any given time, the amount present of any of the above components varies a great deal.

It is proposed that a better measurement of activity within the brain is the degree of myelin breakdown in conjunction with the degree of immunological activity of microglial cells as seen by the expression of the Class II major histocompatability complex molecule, HLA-DR. While it is not known yet what antigen(s) is being presented, it has been demonstrated that macrophages filled with myelin debris within the plaque express the HLA-DR molecule and that microglia at the plaque edge and within the 'normal' appearing white matter and gray matter express it also (Boyle and McGeer, 1990). The following is the proposed classifi- cation: Type I, 'earliest lesion' is defined as an area of hypercellularity, positive for HLA-DR with no ORO staining. Activated microglia are responding to initial inflammatory events in the lesion and can form a microglial nodule. There is no evidence of breakdown of myelin, no digested myelin debris and therefore no conversion to neutral lipids. These nodules are found in the normal appearing white matter, frequently adja- cent to plaques. Type II, 'active' is defined as an area of foamy macrophages which are positive for HLA-DR and ORO. Activated macrophages towards the center of the plaque have converted debris to neutral lipid therefore they are ORO ÷. This type is often accompa- nied by a shelf of HLA-DR ÷ microglia. Type III,

209

'modestly active' is defined as a 'shelf' of plump HLA- DR ÷ cells at the edge loaded with ORO stained lipid. A region of hypocellularity is observed at the center of the plaque. Activated microglia/macrophages have completed phagocytosing myelin debris centrally. How- ever, activity remains at the plaque border. Type IV, 'least active' or 'inactive' is defined as scattered HLA- DR + cells within the plaque or only at the plaque edge with little or no ORO staining. Activated microglia/ macrophages are present but no neutral lipid or myelin debris remains.

This classification system is not only more descrip- tive than the currently used on-off classification but it is more related to the MS disease process, allowing for more sensitive correlations in a patient between plaque type and duration of disease as well as the utilization of sensitive and specific techniques, such as in situ hybridization (ISH) or polymerase chain reaction (PCR) to search for the etiopathogenesis of MS.

Materials and Methods

All tissue was obtained from the National Neurolog- ical Research Specimen Bank (NNSRB), V.A. Medical Center, Los Angeles, CA. The standard procedure for cryopreservation of the brains is placing 4-7 mm thick coronal sections in moisture-resistant, heat-sealable plastic bags after photomacrographs have been taken and then 'quick-freezing' them between 400-g alu- minum plates pre-chilled in liquid nitrogen (Tourtel- lotte and Berman, 1987). This method eliminates ice artifact and allows for long-term storage at -70°C of excellently preserved tissue that can then be cut into specific 'blocks' to be fixed or cryosectioned unfixed. Gross examination of frozen coronal sections com- pared with photomacrographs of the brain slices al- lowed for precise identification of plaque areas. Focal plaques, periventricular plaques, gray matter plaques (if present), and normal appearing white matter were dissected from random areas of the brain from random cases. Coronal sections were picked from the brains to insure proper sampling of different brain areas. For example, blocks were dissected from coronal sections 2, 5, 10, 15, and 20 in order to obtain frontal lobe white matter, temporal lobe white matter, subcortical struc- tures, occipital lobe white matter, etc.

A total of 435 blocks were dissected from 52 multi- ple sclerosis cases. Of these, 348 blocks contained plaque regions. Age of patients ranged from 33 to 80 years, mean age 56. Duration of disease ranged from 5 to 47 years, mean duration 25 years. Average autolysis time was 13.4 h (Table 1). Preagonal state of death was determined according to the Self-Rated Overall Func- tion Scale, a modification of Kurtzke's Disability Scale. Numbers range for least severe (0) to most severe (9).

210

t"-I

i

212

0 . . . . . . . . . . . . . . . . . . . .

Figs. 1-10. (Publisher's magnification: 0.66 X .) Fig. 1. Microglial nodules (arrows) in normal appearing white matter, HLA-DR (40 x ). Fig. 2. Different morphological forms of immunologically activated microglia: resting microglia (arrowhead); activated microglia (closed arrow) and foamy macrophage (open arrow), HLA-DR (400 × ). Fig. 3. Type 1 plaque. Distinct area of increased HLA-DR expression (top). 'Landmark' blood vessel noted with an asterisk, HLA-DR (40 x ). Fig. 4. Type 1 plaque. Sequential section to Fig. 3. Complete absence of lipid-filled macrophages in plaque area. 'Landmark' blood vessel noted with an asterisk, ORO (40 × ). Fig. 5. Type 2 plaque. Large focal plaque filled with immunologically activated macrophages. Activated microglia form distinct rim of this plaque. 'Landmark' blood vessels noted with an asterisk, HLA-DR (40 x ). Fig. 6. Type 2 plaque. Sequential section of Fig. 5. Large focal plaque comprised of lipid-filled macrophages. 'Landmark' blood vessels noted with an asterisk, ORO (40 X ). Fig. 7. Type 3 plaque. Strong border of immunologically activated macrophages and microglia (arrows). Hypocellular center of plaque is at top of the figure, HLA-DR (40 × ). Fig. 8. Type 3 plaque. Strong border of lipid-filled macrophages (arrows). Hypocellular center of plaque is at the top of the figure, ORO (40 x ). Fig. 9. Type 4 plaque. Large plaque (bottom right) with only a few immunologically active macrophages remaining at the edge (arrow). 'Landmark' blood vessels noted with an asterisk, HLA-DR (40 × ). Fig. 10. Type 4 plaque. Sequential section of Fig. 9. Large plaque with only a few lipid-filled macrophges remaining at edge (arrow). 'Landmark'

blood vessels noted with an asterisk, ORO (40 × ).

A rat ing of 6 implies assistance on one side is requi red to walk approximately one city block. A rat ing of 9 implies restr ict ion to bed with no use of arms (Kurtzke, 1983)

This study arose out of a service offered by the NNSRB, providing classified p laque tissue and controls to MS researchers. Because this is a retrospective study, the n u m b e r of blocks dissected from each case varies. Availabil i ty of tissue also affected the n u m b e r of blocks per case. The n u m b e r of cases wi thin each dura t ion subset vary because of the na tu re of the sample (pos t -mor tem tissue). A n average of 8.4 blocks

was dissected for each case and an average of 6.7 p laques was examined per case (Table 2).

Blocks were e i ther fixed in 4% paraformaldehyde (PFA), cryoprotected in 15% sucrose, and s ta ined for H L A - D R as free-f loat ing sections (Boyle and McGeer , 1990) or m o u n t e d unfixed on Fisher ® b rand Super-frost Plus ® slides and immersion-f ixed in 95% ethanol . Af- ter mount ing , histological s ta ining (LFB, O R O , H & E) was essentially ident ical (Bancroft and Cook, 1987). Whi le fixation in 4% P F A resul ted in be t te r preserva- t ion of an t igen and thus be t te r cell morphology, cryosect ioning of unfixed tissue and later immers ion

TABLE 2

Number of plaques dissected per case within each duration subgroup

Duration (years) Plaques/cases Average number of plaques/case

~< 5 5/2 2.5 6-10 29/3 9.7

10-19 66/10 6.6 20-29 156/20 7.8 30-39 62/9 6.9

>_- 40 30/8 3.8

fixation was the method of choice because non-fixation allowed the use of the same block for further studies.

Immunocytochemical staining was performed using the ABC (avidin-biotin complex) method as per in- structions in the Vector Elite ® kit. After fixation, tissue sections were briefly rinsed in PBS, treated with 0.5% H202 to block endogenous peroxidase activity, rinsed in PBS, and treated for 30 min with 2% normal horse serum. Primary antibody, HLA-DR (DAKO Corp., Carpenteria. M704) was diluted 1:20 for the free-floating sections, or 1:100 for the mounted sec- tions. Sections were treated with primary antibody for either 24 h (4°C.) for free-floating or 1 h (room temper- ature) for mounted sections. Sections were rinsed briefly with PBS and then treated with a 1 : 200 dilution of biotinylated horse anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, B-1000) for 1 h followed by PBS rinse and ABC (Vector Laboratories, Burlingame, CA, PK-6100) for 1 h. Finally, visualiza- tion of the reaction was achieved by treating with 0.01% DAB, 0.6% nickel, 0.05% imidazole, and 0.0003% H202 in 0.05 M Tris.

Typing was done on an Olympus Vanox-S micro- scope at 40 and 100 x magnification. All four stains were examined to determine cellularity by Hematoxylin and Eosin (H&E), demyelination/remyelination by Luxol fast blue (LFB), degree of myelin debris by Oil Red O (ORO), and degree of immunological activity (HLA-DR Ab).

Results

Standard histological stains were done to identify the location, degree of cellularity, and degree of di- gested myelin debris of each plaque region. Often, areas of partial remyelination and /o r areas filled with myelin debris were identified in grossly 'normal' ap- pearing white matter. The standard myelin stain, Luxol fast blue, was used to identify areas of demyelination or partial remyelination, appearing as myelin pallor, and even was able to (show) individual fibers in the areas of myelin pallor. Hematoxyin and Eosin allowed for the distinction between focal plaques and small

213

areas of gray matter. It also gave a fair picture of degree of cellularity and /o r astrocytosis within the plaque region. Oil Red O stained normal myelin pink and myelin debris converted to neutral lipids bright red. The converted myelin debris could be seen inside large foamy macrophages as well as in the form of extracellular 'globules'. The lipid filled macrophages were found infiltrating the normal appearing white matter. Phagocytosing macrophages could be found immediately adjacent to the normal appearing white matter or separated by a shelf of macrophages presum- ably filled with myelin debris that had not been con- verted to neutral lipid yet. Occasionally both circum- stances occurred in the same plaque

Microglia and macrophages expressing the MHC Class II molecule, HLA-DR, were identified by stan- dard anti-HLA-DR immunocytochemistry. Small focal areas of HLA-DR ÷ microglia, resembling microglial nodules, were seen (Fig. 1). Within the normal parenchyma, microglia with well-described 'resting' or ramified morphology were sometimes stained. More often, 'activated' or ameboid microglia and darkly staining, foamy macrophages were identified (Fig. 2).

Areas of increased HLA-DR staining in relation to nearby normal appearing white matter were described as the earliest, 'pre-plaques' and designated Type 1. These areas were LFB positive (indicating normal myelin), ORO negative, and may or may not have shown hypercellularity (Figs. 3, 4). More easily identi- fied because of the strong red ORO staining, were the 'active' Type 2s. These were hypercellular areas of HLA-DR + and ORO + macrophages. Microglia posi- tive for HLA-DR were seen at the plaque edge and to a lesser concentration in the surrounding normal ap- pearing white matter (Figs. 5, 6). These plaques were often small focal lesions, surrounding blood vessels. They could also be seen as larger areas confluent with either Type 3 or even Type 4 plaques (see below). Type 2s were sometimes identified in LFB positive (normal myelin), suggesting areas of infiltrating macrophages or partial remyelination. Type 3 plaques appeared as a hypocellular region devoid of HLA-DR ÷ and ORO + macrophages but retaining a distinct HLA-DR ÷ and ORO ÷ edge. Like the Type 2s, these plaques often were surrounded by a lower concentration of HLA- DR ÷ microglia (Figs. 7, 8). Hypocellular regions of demyelination with few, if any, HLA-DR ÷, ORO ÷ macrophages were characterized as inactive Type 4 plaques (Figs. 9, 10). They were seen as small focal plaques and, more often, larger, irregularly shaped plaques. The focal Type 4s were surrounded by an edge of HLA-DR ÷, O R O - microglia (not shown). Some of the larger Type 4s were surrounded by a high concentration of HLA-DR ÷ microglia; however, most showed no adjacent regions of microglia with MHC expression.

214

TABLE 3

A. Percentage of plaque occurrence in total population

Total Type

1 2 3 4

15% 30% 14% 41% (51/348) (103/348) (46/348) (137/348)

B. Duration of MS vs. plaque activity

Duration Type

(years) 1 2 3 4

~< 5 80% 0% 0% 20% (4/5) (1/5)

6-9 17% 66% 17% 0% (5/29) (19/29) (5/29)

10-19 11% 44% 15% 30% (7/66) (29/66) (10/66) (20/66)

20-29 19% 31% 17% 33% (30/156) (48/156) (26/156) (52/156)

30-39 2% 12% 2% 85% (1/52) (6/52) (1/52) (44/52)

>~ 40 10% 3% 10% 77% (4/30) (1/30) (4/30) (21/30)

435 blocks were dissected from 52 patients. Of these, 348 contained plaques. 15% of the total number of plaques were Type ls; 30% were Type 2s, and 14% were Type 3s. The majority (41%) were the inactive, Type 4 plaques (Table 3A). Breaking the data down further into six groups of increasing disease duration, there was little difference between duration subgroups for Type 1 and Type 3 plaque occurrence. The percent- age of Type 2 plaques decreased as duration increased while the percentage of Type 4s increased as duration increased (Table 3B). Linear regression analysis re- vealed significant correlation between percentage of plaque occurrence and disease duration for Types 2 and 4 ( r = - 0 . 9 9 and r = +0.93, respectively)., whereas slopes for Types 1 and 3 showed only a small negative correlation ( r = -0 .7 and r = -0.6, respec- tively) (Fig. 11).

I '- Z

o,,, r e l.IJ a .

o-Q ,0:,9 2o-~ 30-39 D U R A T I O N O F D I S E A S E ( Y R S )

Fig. 11. Plot of percentage of plaque occurrence for each duration subgroup.

100"

80

60

40

20

Discussion

The current view of the multiple sclerosis disease progression is one of an ever changing ebb and flow of immune activation, inflammation, and demyelination. However, the current method of characterizing MS plaques does not reflect this view because it fails to take into account the interdependent components in- volved in cellular activation and plaque formation. Accordingly, we have proposed, based on observations made from 348 plaques, a classification that reflects the dynamic nature of the MS disease process. The steps necessary to obtain the information needed for this descriptive classification are technically simple, involving standard myelin and cellular histological stains, H&E, LFB, and ORO, and one immunocyto- chemical stain for the surface marker, HLA-DR.

The high expression of HLA-DR in MS brains, particularly in plaque regions, suggests that there are abnormally high levels of activating factors, such as gamma-interferon, within these areas and that antigen is being presented to T cells (Traugott, et at., 1983). With this in mind, we interpret our Type 1 lesion as the earliest lesion or 'pre-plaque'. It is seen as a distinct area of high concentration of HLA-DR expres- sion in relation to the surrounding parenchyma. Some resemble microglial nodules, others are less focal but still distinct. Increased microglial HLA-DR expression not specific to immunological abnormalities has been noted throughout the white matter parenchyma of older human and rat brains (Perlmutter et al., 1992; Perry et al., 1993). Brain trauma, abcess, or primary axonal degeneration can also result in non-immune microglial activation (Finsen et al., 1993; Flaris et al., 1993; McGeer et al., 1993). Because, however, there is no evidence of trauma or degeneration, it is believed that activated microglia and/or macrophages are presum- ably responding to initial immune-mediated events of the lesion.

This classification system allows for a conceptualiza- tion of temporal plaque progression. The focal activa- tion of microglial cells could be essential for the subse- quent transformation into macrophages and infiltration of macrophages leading to demyelination, as seen in Type 2 plaques. The Type 3 plaque may be interpreted as continuous outward progression from .Type 2 or reactivation of the edge of a Type 4 plaque.

The least active or inactive plaque, Type 4, is the equivalent of the chronic or burnt-out plaque. Type 1 plaques were observed near Type 4 plaques, suggesting a reactivation of the area that may, in fact, give rise to further demyelination and ultimately converting a Type 4 to a Type 3. This appearance supports the concept of the dynamic MS disease process. The majority of Type 4s had no surrounding activated microglia; this may represent an effective defense response of the body to

the putative causative agent. We propose the disease process has stopped altogether in these areas.

Since we were working with a post-mortem popula- tion, it may not be surprising that the majority of the plaques were Type 4. However, unexpectedly, 31% of the total plaques were Type 2s. In examining the plaque type burden within each duration subgroup, it was clear that the overwhelming majority of Type 2s within the 9 year or less group accounted for the large percentage within the total population, that is, patients of short disease duration who died from complications resulting from a rapidly progressive case of MS or who died from factors unrelated to their MS had a higher percentage of active plaques. It is very interesting that the percentages of plaque occurrence for Type 1 and 3 did not change over the duration of the disease. It is proposed that the earlier manifestation of the MS disease process, that is, the Type 1 plaque, is persis- tent. This suggests that, whatever causes, MS never goes away. On the other hand, a defense could exist to contain the etiology since percentage of active plaques (Type 2) decreases with time and percentage of Type 4s, the oldest lesion, increases. Why is Type 3 steady across the duration of disease? Perhaps Type 3 is the result not only of rapid progression from 2 to 4 but also a regression to a more active lesion from the frequently observed active microglia at the Type 4 plaque edge. It is reasonable to propose that this process exists since gadolinium-positive lesions are frequently seen at old plaque edges (Thompson et al., 1992).

The strength of our classification lies in its simplicity and descriptive power. However, it does not include all the reported components within the MS disease pro- cess. For example, lymphocyte infiltration has been proposed as a key component in the lesion (Hauser et al., 1986). It would be of interest to re-evaluate our classification in relation to T cell subtype. Another component in plaque regions important in assessing disease progression is non-converted myelin debris in macrophages that is still myelin basic protein (MBP)- positive. MBP within the lysosomes of macrophages would aid in dating our plaques. While neutral lipids may remain in macrophages for up to 6 months, MBP is broken down within days (J. Prineas, personal com- munication). Presence of MBP within macrophages would indicate very recent myelin breakdown. Future studies could include immunocytochemically staining for MBP. It is important to note that we have not been successful in detecting MBP in macrophages, even though foamy macrophages were seen at the plaque edge without ORO.

The causative agent in MS is unknown. This classifi- cation favors infection by a persistent or latent pathogen. If MS were due to autoimmunity, we would expect plaques to be uniform in activity. On the other hand, the plaque activity variability in adjacent plaques

215

described in this report supports a persistent pathogen. In some plaques, Types 3 and 4, the core immune system may have neutralized, eradicated, or suppressed the pathogen. The causative agent is most likely to be in areas of higher immunological activity as opposed to the burnt-out plaque with no activity. Typing of each plaque from a patient allows for another type of con- trol by comparing different plaque types within a single patient. Additional controls include comparison of MS patient to MS patient, normal controls or patients with other neurological diseases.

This report presents two important issues in the study of the multiple sclerosis disease process. First, previous classifications of plaque tissue as either active or inactive is too simplistic in light of the current knowledge of the immunological components making up the lesions. Our method of categorization based on immunological activity and degree of demyelination better distinguishes the possible plaque types. Second, within our post-mortem population, there are correla- tions between percentage of plaque types and disease duration. These correlations demonstrate that as MS progresses, more lesions become inactive. It is there- fore important that any investigator studying the etiopathogenesis of MS focus on early lesions and contrast them with the older lesions.

Acknowledgements

The authors wish to thank Stephen Felisen for tech- nical aid in preparation and staining of the tissue. Specimens were obtained from the Multiple Sclero- sis Human Neurospecimen Bank sponsored by NINDS/NIMH, Comprehensive Epilepsy Program (NINDS), Hereditary Disease Foundation, Dystonia Medical Research Foundation and Tourette's Syn- drome Association, the Veterans Health Services and Research Administration, Department of Veteran's Affairs, Merit Review Funding and the National Multi- ple Sclerosis Society, Award No. RG 829-L-35.

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