On classification of post-mortem multiple sclerosis plaques for neuroscientists

On classification of post-mortem multiple sclerosis plaques for neuroscientists

Journal of Neuroimmunology, 46 (1993) 207-216 Elsevier Science Publishers B.V. 207 JNI 02438 On classification of post-mortem multiple sclerosis pl...

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

207

JNI 02438

On classification of post-mortem multiple sclerosis plaques for neuroscientists Virginia Sanders

a, A n d r e w J. C o n r a d b and Wallace W. Tourtellotte 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 a n d / o r 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 a n d / o r sensory deficits and characterized pathologically by multifocal lesions of inflammation and demyelination (Matthews et al., 1985). The cluster destruction of oligodendrocytes-myelin sheaths forms plaques, the vast majority being in the white matter. The center of the plaque is composed of astrocytes, which replace the oligodendrocytes-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 inflammation. The disease is not static by either clinical, magnetic resonance imaging (MRI), or pathological manifestations. Exacerbations and remissions, especially during the early stages of the disease, are common in disease progression, as assessed by clinical symptoms a n d / o r 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. Although 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

735 470 944 644 573 678 536 1 864 1035 511 1 238 1 862 1 011 1 337 1291 996 1 682 888 1 145

39/M 47/M 33/F 37/F 50/F 40/M 31/M 37/F 66/M 40/M 36/F 69/M 44/M 58/F 44/F 41 / F 50/F 50/M 42/M

1 206 1 354 344 845 985 1588

61/F 73/M 40/M 45/M 66/F 46/F

1 735

79/M

1061 1 324 934 1 449 1 342 1 174 1334 640 1 446 1 270 1396 1489 1 668 228 1 657 1934 1023 1 721 682 909 1088 1 151 579 1 897 1 292 1 779

50/M 67/M 50/F 59/F 64/M 65/M 58/M 51/M 51/F 67/F 66/F 72/F 59/F 80/M 53/F 60/F 71/F 68/F 71/F 70/F 64/F 74/F 61/M 68/F 62/M 69/F

Cause of death

Pneumonia Septicemia Septicemia Cardiac arrest Overdose Cardiac arrest Cardiac arrest Cerebral depression Pneumonia Respiratory failure G.I. bleeding Septicemia Pneumonia MS Cardiac arrest Fibrosarcoma Septicemia Pneumonia Anoxic encepholopathy Pneumonia Cardiac arrest Pneumonia Drowning Undetermined Resp. distress syndrome Infected decubitis ulcers MS Pneumonia Pneumonia Cardiac arrest Pneumonia Septicemia Cardiac arrest Pneumonia Pneumonia Septicemia Cardiac arrest Septicemia Septicemia Pneumonia Pneumonia Met. colon cancer Septicemia Respiratory arrest Stroke Undetermined Renal failure Met. breast cancer Pneumonia Met. breast cancer Pneumonia Met. lung cancer

a Human Specimen Bank Number.

Duration (years)

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

Clinical Neurological disability preagonal state of death (modified Kurtzke scale) 7.5 8

8.5 9 7 7.5 6.5 9 8 8

9 7.5

Autolysis time

Number of Blocks

(h) 6 3.5 22.5 11 15 5.5 25 12 10.5 12 19.5 9 4.5 14 7 14 13

2 3 21 7 13 20 14 2 4 20 4 1

8 8 9 8.5 8

31.5

2 2 17 12 6 19 3

9 7 7.5 6.5 6 8.5

9 9.5 6 28 18 23.75

15 4 4 16 10 5

9

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

4 6 2 14 9 7 10 22

8 8

8

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

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

1

5 3 9 19 3 5 4 7 2 5 2 1

2 16 1

7 16

209 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 gammainterferon. With the advent and improvements in immunocytochemical staining, more specific characterization 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 astroglia (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 dependent 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 classification: 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 adjacent 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 accompanied by a shelf of HLA-DR ÷ microglia. Type III,

'modestly active' is defined as a 'shelf' of plump HLADR ÷ 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. However, activity remains at the plaque border. Type IV, 'least active' or 'inactive' is defined as scattered HLADR + 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 descriptive 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 Neurological 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 aluminum plates pre-chilled in liquid nitrogen (Tourtellotte 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 compared with photomacrographs of the brain slices allowed 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 structures, occipital lobe white matter, etc. A total of 435 blocks were dissected from 52 multiple 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 Function 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 immunologicallyactive 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 rating of 6 implies assistance o n o n e side is r e q u i r e d to walk a p p r o x i m a t e l y o n e city block. A rating of 9 implies restriction to b e d with n o use of arms (Kurtzke, 1983) This study arose out of a service offered by the N N S R B , providing classified p l a q u e tissue a n d controls to MS researchers. B e c a u s e this is a retrospective study, the n u m b e r of blocks dissected from each case varies. Availability of tissue also affected the n u m b e r of blocks p e r case. T h e n u m b e r of cases w i t h i n each d u r a t i o n subset vary b e c a u s e of the n a t u r e of the s a m p l e ( p o s t - m o r t e m tissue). A n average of 8.4 blocks

was dissected for each case a n d a n average of 6.7 p l a q u e s was e x a m i n e d p e r case ( T a b l e 2). Blocks were e i t h e r fixed in 4 % p a r a f o r m a l d e h y d e (PFA), cryoprotected in 15% sucrose, a n d s t a i n e d for H L A - D R as free-floating sections (Boyle a n d M c G e e r , 1990) or m o u n t e d u n f i x e d o n F i s h e r ® b r a n d Super-frost Plus ® slides a n d i m m e r s i o n - f i x e d in 95% ethanol. After m o u n t i n g , histological s t a i n i n g (LFB, O R O , H & E) was essentially identical (Bancroft a n d Cook, 1987). W h i l e fixation in 4 % P F A r e s u l t e d in b e t t e r preservation of a n t i g e n a n d thus b e t t e r cell morphology, cryosectioning of unfixed tissue a n d later i m m e r s i o n

213 TABLE 2 Number of plaques dissected per case within each duration subgroup Duration (years)

Plaques/cases

~<5 6-10 10-19 20-29 30-39 >_-40

5/2 29/3 66/10 156/20 62/9 30/8

Averagenumber of plaques/case 2.5 9.7 6.6 7.8 6.9 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 instructions 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 sections. Sections were treated with primary antibody for either 24 h (4°C.) for free-floating or 1 h (room temperature) 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, visualization 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 microscope 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 digested myelin debris of each plaque region. Often, areas of partial remyelination a n d / o r areas filled with myelin debris were identified in grossly 'normal' appearing 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

areas of gray matter. It also gave a fair picture of degree of cellularity a n d / 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 presumably filled with myelin debris that had not been converted to neutral lipid yet. Occasionally both circumstances occurred in the same plaque Microglia and macrophages expressing the MHC Class II molecule, HLA-DR, were identified by standard 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 identified because of the strong red ORO staining, were the 'active' Type 2s. These were hypercellular areas of HLA-DR + and ORO + macrophages. Microglia positive for HLA-DR were seen at the plaque edge and to a lesser concentration in the surrounding normal appearing 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 HLADR ÷ 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

Discussion

A. Percentage of plaque occurrence in total population Total

Type 1

2

3

4

15%

30% (51/348)

14% (103/348)

41% (46/348)

(137/348)

2

3

4

0%

0%

66% (19/29) 44% (29/66) 31% (48/156) 12% (6/52) 3% (1/30)

17% (5/29) 15% (10/66) 17% (26/156) 2% (1/52) 10% (4/30)

20% (1/5) 0%

B. Duration of MS vs. plaque activity Duration (years) ~<5 6-9 10-19 20-29 30-39 >~40

Type 1 80% (4/5) 17% (5/29) 11% (7/66) 19% (30/156) 2% (1/52) 10% (4/30)

30% (20/66) 33% (52/156) 85% (44/52) 77% (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 percentage of Type 2 plaques decreased as duration increased while the percentage of Type 4s increased as duration increased (Table 3B). Linear regression analysis revealed 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, respectively) (Fig. 11). 100"

80 I'Z

o,,, re l.IJ a.

60

40

20

o-Q

,0:,9 DURATION

2o-~ 30-39 OF DISEASE (YRS)

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

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 involved 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 immunocytochemical 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 expression 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 a n d / o r macrophages are presumably responding to initial immune-mediated events of the lesion. This classification system allows for a conceptualization of temporal plaque progression. The focal activation of microglial cells could be essential for the subsequent 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

215 the putative causative agent. We propose the disease process has stopped altogether in these areas. Since we were working with a post-mortem population, 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 persistent. 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 process. 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 communication). 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 classification 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

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 control 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 correlations between percentage of plaque types and disease duration. These correlations demonstrate that as MS progresses, more lesions become inactive. It is therefore 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 technical aid in preparation and staining of the tissue. Specimens were obtained from the Multiple Sclerosis Human Neurospecimen Bank sponsored by N I N D S / N I M H , Comprehensive Epilepsy Program (NINDS), Hereditary Disease Foundation, Dystonia Medical Research Foundation and Tourette's Syndrome Association, the Veterans Health Services and Research Administration, Department of Veteran's Affairs, Merit Review Funding and the National Multiple Sclerosis Society, Award No. RG 829-L-35.

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