Cell death and birth in multiple sclerosis brain

Journal of Neurological Sciences 149 (1997) 1–11

Cell death and birth in multiple sclerosis brain Peter Dowling a

a,b,

*, Walter Husar a , b , Joseph Menonna a , b , Hyman Donnenfeld c , Stuart Cook a , b , Mohinderjit Sidhu a , b , 1

Neurology Service, Department of Veterans Affairs, New Jersey Health Care System, East Orange, NJ 07018, USA b Department of Neurosciences, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA c Department of Neurology, Saint Vincent’ s Hospital, New York, NY 10011, USA Received 24 May 1996; revised 12 August 1996; accepted 22 August 1996

Abstract The hallmark of the brain pathology in multiple sclerosis is the white matter plaque, characterized by myelin destruction and oligodendrocyte loss. To examine the role that cell death plays in the development of MS lesions, we used the in situ TUNEL technique, a method that sensitively detects DNA fragmentation associated with death at the single cell level. We found that patchy areas within acute MS lesions have massive numbers of inflammatory and glial cells undergoing cell death. The punched out areas of some long-standing chronic lesions also had labeled glial cells showing that the attack was not a single event. Immunocytochemical identification of the dying cells with glial specific marker co-labeling showed that 14–40% were the myelin-sustaining oligodendroglial cell. Confocal microscopic evaluation of fluorescein-labeled TUNEL positive cells revealed nuclei with morphologic characteristics of apoptosis, and electrophoresed MS brain DNA produced a ladder characteristic of apoptotic DNA cleavage confirming that substantial numbers of labeled cells, but not necessarily all, were dying by apoptotic mechanisms rather than cell necrosis. Companion studies using a marker for cell proliferation on MS lesions revealed that unexpectedly large populations of perivascular inflammatory cells and parenchymal glial cells had entered the cell proliferation cycle. These findings establish that two opposing glial cell responses – relentless cell death and coincident brisk cellular proliferation – are important features of MS pathology. In the end, however, glial cell loss prevails, and we suspect apoptosis may be the critical death mechanism responsible for the depletion of myelin observed in this condition.  1997 Elsevier Science B.V. Keywords: Apoptosis; Multiple sclerosis; Proliferation; EAE

1. Introduction Multiple sclerosis (MS) is a common chronic central nervous system white matter disease of unknown pathogenesis. It is widely held to be an autoimmune inflammatory condition, but the antigen has resisted identification and the mechanism behind the glial cell destruction and loss of myelin sheaths is equally mysterious (ffrenchConstant, 1994; Lassman, 1983; Prineas, 1988). It is clear *Corresponding author: Tel.: 11 201 9825359; Fax: 11 201 6781648. Present address: Department of Viral Vaccine Research, Wyeth-Ledere Vaccines and Pediatrics, 401 North Middleton Road, Pearl River, NY 10965-1299. 1

0022-510X / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0022-510X( 97 )05213-1

from MS pathology that nerve cell bodies and axons are spared and that active lesions continue to develop within different parts of the brain and spinal cord for many years. Prineas and others have shown that the disease is not unremittingly destructive as evidenced by early modest remyelination and reappearance within recently formed MS lesions of newly generated glial cells with the features of immature oligodendroglia (Prineas et al., 1993, 1989; Raine et al., 1981; Raine and Wu, 1993). Recent work also indicates that the myelin repair process is often transient (Prineas et al., 1993). The fate of the new myelin-forming oligodendrocytes is also unclear, but their presence in the mature MS plaque is uncommon (Bruck et al., 1994; Prineas, 1988; Yao et al.,

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1994). To investigate the fate of cells within the MS brain, we used the recently developed in situ TUNEL technique that can detect nuclear DNA fragmentation associated with cell death within tissue sections (Gavrielli et al., 1992; Schmitz et al., 1991; Ozawa et al., 1994; Dowling et al., 1995; Vartarian et al., 1995). This method provided the first opportunity to seek cells undergoing programmed cell death (apoptosis) within developing and mature lesions of the MS brain and to compare the extent of the dying population found in MS lesions to the number of apoptotic cells previously described in the central nervous system of the animal prototype of MS called experimental allergic encephalomyelitis (EAE) (Schmeid et al., 1993). The striking magnitude of the glial cell death encountered in some MS tissue suggested to us that glial cell loss might be balanced by an active glial regenerative process. For this reason, we re-examined MS brains using immunocytochemical techniques and found a vigorous counterbalancing proliferative response within both acute and long term MS lesions.

2. Materials and methods

2.1. Autopsy specimens The study was performed on fresh frozen multiple sclerosis cerebrum obtained at autopsy after an average post-mortem interval of 16 h and 6 specimens were obtained early within 2–6 h of death (n523). Some MS samples and controls were obtained from the Rocky Mountain Multiple Sclerosis Center (Englewood, CO, USA) and others were obtained from the multiple sclerosis human neurospecimen bank (West Los Angeles, CA, USA). In addition, CNS tissue from 6 children with subacute sclerosing panencephalitis (SSPE) (average postmortem interval 8 h) (Sidhu et al., 1994) and 16 non-MS brains (average post-mortem interval 13 h) were examined. The other neurological disease controls (OND) included nine non-MS CNS diseases: motor neuron disease, Huntington’s disease (2), schizophrenia (2), drug overdose (1) and seven normal control brains with no neuropathologic abnormalities who died of acute myocardial infarction, hepatic failure, trauma or malignant neoplasm. The average age of the four test groups was: MS, 55.9 years; normal controls, 56.6 years; OND controls, 77 years; and SSPE, 10.6 years. The tissue diagnosis was established from clinical records and neuropathological examination. Paraffin-embedded acute MS plaques were obtained from Dr. John Prineas (Newark, NJ, USA) and the remainder came from the two specimen banks listed above and material collected over the past 23 years at St. Vincent’s Hospital (New York, NY, USA). The average post-mortem interval for MS paraffin embedded material was 11.4 h and 13 were obtained early within 4.7 h of death. The paraffin-embedded non-MS control brains

included a highly selected group of 10 ALS patients whose autopsies were performed usually within 3–6 h of death, HIV/AIDS encephalitis (12), Alzheimer’s disease (2), post-polio (1), Parkinson’s disease (1), and an additional child’s brain with SSPE (1). Paraffin embedded glioblastoma multiforme tumor and embedded lymph nodes containing predominant germinal centers served as the positive controls for the studies using Ki-67 as a marker for cells in the proliferative cycle.

2.2. In situ detection of nuclear DNA fragmentation in human and EAE tissue To ascertain if the in situ TUNEL method efficiently detected programmed cell death (apoptosis) at the single cell level, we tested it on a well characterized fibroblast cell line transformed with the myc oncogene. Most of the cell layer was induced to lift up under conditions of serum deprivation, with the cells undergoing apoptosis within several hours (Wagner et al., 1993). Apoptotic cells are characterized by extensive DNA fragmentation and we could readily determine that greater than 80% of the floating apoptotic cell nuclei were heavily labeled by the TUNEL assay (TdT mediated dUTP-digoxigenin end labeling of DNA fragments) a method which labels DNA breaks. For experiments on frozen brain, 10 micron cryosections were cut and several different sections were mounted on the same electrostatically treated glass slide. Each slide contained sections known to be highly positive and a negative control section as well as 4–5 test sections. We used a modification of the TUNEL procedure (Gavrielli et al., 1992) in which cells containing fragmented DNA are labeled with digoxigenin-11-dUTP and subsequently detected by an immunoperoxidase localization system. All cells displaying a positive signal within the entire cryosection were scored by two observers. The in situ experiments were performed using kits purchased from Oncor (ApopTag in situ kit) with minor modifications to the manufacturer’s directions and no counterstain was usually employed. When testing 3 micron thick paraffin-embedded sections, we greatly curtailed the suggested deproteinization times with proteinase K (0 to 10 min) and reduced the terminal deoxynucleotidyl transferase (TdT) incubation time from the suggested 60 min to a range of 3 to 60 min. Fluorescent TUNEL labeling was used to analyze the nuclear morphology of apotag positive cells in several experiments and these sections were evaluated by confocal microscopy. EAE was induced in 10 female Lewis rats, 8–12 weeks of age by subcutaneous immunization of emulsified guinea pig spinal cord in complete Freunds adjuvant (100 ml). After euthanasia, the spinal cords of inoculated animals were rapidly collected and quick frozen at the time points shown in Fig. 1. The cryosections from EAE and 3 control animals were mounted on the same slide and the number

P. Dowling et al. / Journal of Neurological Sciences 149 (1997) 1 – 11

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Fig. 1. Left and Right: TUNEL positive cell time course within spinal cords of the EAE Lewis rat model. Labeled cells were rarely found in three control spinal cords (2–3 cells / entire spinal cord cross section). The figure shows there is a 100-fold increase in the population of dying cells coincident with clinical symptoms, and moderately increased numbers of dying cells were detected as late as day 23 post inoculation. The rat spinal cord cross sectional area averages 5–7.5 mm 2 and the photomicrograph on the right shows a small field from an EAE cord. The dying cell density within frozen EAE spinal cord cross-sections at their peak is comparable to the density within MS brains shown in Fig. 2. Short post-mortem interval rapidly frozen tissues were used in this initial survey rather than routine paraffin embedded tissues since we wished to minimize post-mortem interval and fixation time as a potential source of false-positive cells, original magnification: 4003.

of TdT positive cells within the complete spinal cord cross-section was quantified by two observers.

2.3. Double staining immunochemistry Three micron paraffin embedded sections were attached to slides precoated with vectorbond adhesive and dewaxed. Endogenous peroxidase activity was blocked by washing with 2% hydrogen peroxide for 5 min and antigenic sites were exposed by immersion in target unmasking fluid (TUF-Signet Labs) preheated to 908C for 10 min. The ABC system was used for glial cell marker immunocytochemistry following the TUNEL procedure and visualization of the TUNEL reaction products with diaminobenzidine. Monoclonal primary antibody against undiluted Human NK marker (HNK-1) (Becton-Dickinson), astrocytic glial fibrillary acidic protein (GFAP); dilution 1:300, CD-3 pan T-cell marker; dilution 1:1200, and the proliferation marker Ki-67; 1:10 dilution (all from Dako Corporation) was applied overnight at 48C. The slides were rinsed and incubated for 30 min with biotinylated secondary antibody and the procedure repeated with peroxidase-conjugated avidin–biotin complex (Elite Peroxidase ABC kit; Vector Laboratories). Reaction product within double labeled cells was visualized with Vector SG chromagen. The RCA-1 biotinylated lectin probe; dilution 1:1500 (Vector Laboratories) was reacted for 1 h and reactions in the absence of primary antibody were used to control for nonspecific binding. Double labeled cells were quantified by two observers by light microscopy and video projection using a 24-mm eyepiece disk reticle (535 mm in 1-mm subdivisions) calibrated with a stage micrometer.

2.4. Electrophoretic detection of apoptotic DNA Low molecular weight DNA was extracted by digesting brain tissue homogenates or tissue culture cells in 10 mM Tris–HCI, pH 8.0, 0.5% SDS, 0.1 M NaCI 2 , 5 mM EDTA with Proteinase K 100 mg / ml for 2 h at 378C (Hirt, 1967). Sodium chloride was then added to a final concentration of 1 M and samples incubated overnight at 48C. The next morning, the preparations were centrifuged for 15 min and the supernatant was ethanol-precipitated after phenol and chloroform extraction. In order to detect very low levels of apoptotic DNA, we enhanced our detection system by ¨ (Rosl, ¨ using a radiolabeling method described by Rosl 1992). One microgram of DNA was labeled with 0.5 mCi of 32 P-labeled dATP in the presence of 10 mM Tris–HCI, pH 7.5, 5 mM MgCl 2 , and 5 U of Klenow polymerase. The mixture was incubated for 10 min at room temperature and the reaction terminated by adding EDTA to 10 mM. Unincorporated nucleotides were removed by Centricone30 filtration units (Amicon) and identical amounts of sample DNA were electrophoresed on 1.5% agarose gels. The gels were dried on DEAE cellulose filters and exposed for autoradiography. The control for the apoptotic DNA ladder was DNA extracted from a myc transformed Ratla fibroblast cell line that undergoes apoptotic DNA fragmentation on serum withdrawal (Wagner et al., 1993).

3. Results

3.1. Frozen tissue – cell death in EAE spinal cord We first tested the specificity and sensitivity of the in

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situ TUNEL detection system on EAE spinal cord frozen sections by determining the time course of dying cells after antigen administration in the Lewis rat model. TUNEL positive cells were rarely found in three control spinal cords (2–3 cells per entire spinal cord cross section), and our system was able to document a 100-fold increase in dying cells over control levels co-incident with clinical signs and both resolved by day 30 (see Fig. 1).

3.2. Frozen tissue – cell death in MS and controls The TUNEL procedure was then used to screen for apoptosis in tissue samples from frozen human control CNS blocks obtained at autopsy. Brain sections from three normal individuals and four others who expired from nonneurologic disease were tested for apoptotic cells. We found that brain from these sources rarely contained many apoptotic-positive cells. Fig. 2 (see nl group) shows that the maximum number of labeled cells encountered ranged from 2 to 10 per entire frozen section. This finding indicates that cell loss by apoptosis occurs but is extremely low in normal human adult brain. To determine how widespread cell death is in neurologic conditions, we tested diseased brain from nine non-MS patients with other chronic CNS disease. Fig. 2 shows the values for this group [other neurological diseases (OND)] and there were either few labeled nuclei or a modest number of cells within entire sections (average 18 cells). We then tested

Fig. 2. Log value of TUNEL positive cells in the four frozen cerebral cortex test groups. The control range shown is derived from the other neurological disease group (OND) mean value (18 cells12SD544 cells). The MS group (closed circles –d–) is significantly different from all other groups by the Kruskal-Wallis one way analysis of variance P5 0.001, and also significantly different from the OND group P20.004 (Mann–Whitney U test). The MS column also contains additional values from a subset of eight untreated MS patients (open circles –s–), mean value is 477. The untreated MS subset is also significantly different from the OND control group (P50.004). Mean values for the other groups NL, 6.7; OND, 18; MS, 199; SSPE, 92 cells. To be certain that the section area of experimental groups was comparable, the area of each cryosection was measured by image analysis and the mean values in square millimeters for each group: NL, 23.9; OND, 22.3; MS, 20.2; SSPE, 21.5 are not significantly different.

gray-white matter brain sections obtained from 23 MS patients. The MS brain samples were not preselected for recent disease activity and in fact, the patients on average were quite old (56 years) with long-standing MS. In spite of the overall chronicity of the frozen brain MS series, 16 MS brains (70%) had significantly increased numbers of scattered heavily labeled cells when compared to controls (P50.001) that were clearly restricted to white matter (defined as more than 44 cells / section). Many hundreds of cells labeling for DNA breaks were present in several specimens, while a few others had only modest numbers of positive cells in this random selection of MS brains. The lack of TUNEL staining in some brain specimens probably represents a sampling error reflecting the non-homogeneous nature of the MS process within the neuraxis, because we have found that some frozen samples manifest considerable TUNEL activity whereas blocks from other regions of the same brain have little activity. In other cases, we were able to sample two or three plaques from the same brain that were consistently negative and while this finding may reflect a true lack of disease activity within these brains, more study is required. We also tested frozen brain from six children with SSPE, a chronic measles infection of brain which often has prominent inflammatory white matter destruction, and five (80%) showed significant numbers of scattered labeled subcortical white matter glial cells and an occasional labeled neuron when compared to the controls. We wished to exclude the possibility that the TUNEL positive staining in MS brain was an artifact of MS treatment with cytotoxic drugs. To this end, a highly selected subset of white matter plaques isolated from eight additional MS patient brains known to be free of medications thought to modify the course of the disease (such as corticosteroids, ACTH, cyclosporin A, azathioprine) were tested by the TUNEL technique. The number of labeled cells within the white matter MS plaques from these untreated patients are depicted within the MS column in Fig. 2 as open circles. When compared to either normals or the OND control group, a significantly elevated number of dying cells were present in this MS non-treated subgroup (P50.004). This data indicates that the cell death changes we describe accurately reflect the MS process and are not the effect of MS treatments.

3.3. Paraffin embedded tissue – cell death in MS and controls The small sized frozen sections allowed us to simultaneously test known highly positive and negative brain controls, as well as several diseased brain sections on the same slide. This design permitted us to rapidly evaluate numerous samples, and required no tissue morphology destroying proteinase K step. The use of identical experimental conditions for several samples at a time permitted greater confidence in the quantitative findings than in

P. Dowling et al. / Journal of Neurological Sciences 149 (1997) 1 – 11

the paraffin studies. A major drawback of frozen tissue, however, was the inability to make reliable morphologic distinction between brain cell types and infiltrating inflammatory cells. For this reason, we turned to archival paraffin-embedded MS plaques, and also tested paraffinembedded cerebrum from additional non-MS neurologic conditions. When five paraffin-embedded acute MS plaques were tested, cell death was massive in focal areas of the plaques, with up to a thousand or more darkly staining cells undergoing apoptosis (see Fig. 3, left panel). These young hypercellular lesions were restricted to white matter or the gray–white matter junction and the majority of positive cells were infiltrating macrophages with distended cytoplasm. Glial cells also stained, but their numbers were largely obscured by the intense macrophage response. These features were present within foci of the acute plaques examined, but the extent of cell death varied greatly at different sites within any one lesion. Cells of microglial lineage, as well as reactive astrocytes, internalized nuclei within hypertrophic astrocytes, and small dark cells resembling oligodendroglia labeled strongly by the TUNEL procedure. We next tested five chronic MS plaques of long-standing with hypocellular centers and little or no inflammation. An unexpectedly large glial population undergoing cell death was found within the punched-out plaque area in three specimens (Fig. 3, right). The number of labeled cells usually dropped abruptly as one emerged from the rim of the plaque, as though some local soluble element(s) was present within the confines of the plaque, inducing cells within these preexisting lesions to undergo apoptosis. Labeled cells were not limited to acute plaques or preexisting chronic lesions undergoing reattack. We sometimes found scattered foci of positive glial cells in normalappearing white matter remote from plaques, which we suspect may be evidence for apoptotic activation prior to

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myelin breakdown. The cell types labeling in healthy white matter appeared to be mostly cells with elongated nuclei and long bipolar cytoplasmic processes resembling microglia, but small round cells with the characteristics of oligodendroglia and an occasional astrocyte also labeled. Apoptotic neurons in gray matter were virtually never encountered in MS material except for a very rare cell at the gray–white border of acute plaques. This observation contrasts with the findings in AIDS described below. Brain samples from twelve AIDS / HIV encephalitis patients (five paraffin-embedded and seven frozen specimens) were tested as an additional type of CNS control often displaying a prominent inflammatory component and extensive macrophage infiltrates. We found marked neuronal cell death in seven of 12 patients, but the TUNEL positive cells were mainly restricted to deep cortical neurons. By contrast, the subcortical white matter in the AIDS / HIV group was largely negative except for heavily labeled multinucleated giant cells and an occasional labeled blood vessel or macrophage. Thus the pattern of gray matter TUNEL positive cells in AIDS cerebrum is much different than we encountered in MS tissue and the constellation of apoptotic AIDS brain abnormalities described above has recently been reported by several groups (Adle-Biasesette et al., 1995; Gelbard et al., 1995; Petito and Roberts, 1995). Two of 15 additional paraffin-embedded blocks from other non-MS neurologic conditions also had elevated numbers of labeled cells. Cortex from a single ALS patient out of ten, showed multiple neuronal cells undergoing cell death but the only extensive white matter labeling among non-MS conditions studied, was found in a progressive multifocal leukoencephalopathy brain.

3.4. Identity of dying inflammatory and glial cells in MS plaques To determine the precise identity of the TdT-positive

Fig. 3. Left: Acute MS white matter plaque from 20 year old male with nine-month history of neurologic illness labeled by TUNEL procedure for fragmented DNA. Numerous scattered cells throughout the white matter are densely labeled (dark brown). The vessel on left is surrounded by a dense mononuclear cell infiltrate and the hypercellularity of the white matter lesion contrasts with the chronic MS plaque on the right, original magnification: 2003. Right: TUNEL stained chronic hypocellular MS plaque showing several intensely labeled glial cells (dark stain, arrows) scattered amongst numerous non-reactive cells, original magnification: 4003.

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Table 1 Identity of TdT labeled cells dying in MS plaques (% of TdT-positive cells) Probe

Specificity

Acute plaques (%) 1

RCA-1 Lectin GFAP HNK-1 CD-3

Macrophage / microglia Astrocyte Oligodendroglia Pan T-cell

a

68 18 14 4

Chronic plaques (%)

b

2

3

4

5

6

7

65 21 15

61 22 16 0

30 33 27 2

18 40 39 0

21 3 40 0

21 25 31 0

17 6 25

1a

b

c

Cell type of TUNEL positive cells using cell specific markers on acute and chronic MS plaques assessed by light microscopy as described in the methods. Inflammatory cells (mainly macrophages and a few T cells) make up the bulk of all dying cells in the three acute evolving lesions. In contrast, the chronic plaques from five patients had far less dying inflammatory cells and there was a pronounced shift towards a higher proportion of dying glial cells. a Patient number. b Additional acute plaque from patient 1. c No tissue available.

cells previously identified by light microscopy, we carried out double labeling with cell specific markers for macrophages, glial and T cells. This data is summarized in Table 1 which details the markers tested and shows that lectin

stained dying macrophage / microglial lineage cells clearly formed the bulk of the TdT-positive cells in the three acute lesions studied, (see Fig. 4A) in contrast macrophages were far less frequent in chronic plaques where resident dying

Fig. 4. Acute MS lesion. (A) Co-labeled by TUNEL procedure (brown stain) and RCA-1 lectin macrophage / microglial marker double labeled cells (blue stain-arrows). A large TUNEL stained dying astrocyte is visible in the upper left. (B) Two large apoptag-positive dying parenchymal cells which resemble reactive astrocytes (arrows, brown stain). The cells contain numerous ‘‘internalized’’ nuclei which are apoptag positive and their cytoplasm stains diffusely positive for DNA fragments since this cell type appears to actively phagocytose dying cells. (C) Co-localization experiment on the same lesion in which the astrocyte marker glial fibrillary acidic protein (GFAP) (dark blue stain) was used to establish the astrocytic nature of the labeled apoptotic cells (brown stain). (D) Co-labeled by TUNEL procedure (brown stain) and HNK-1 oligodendroglia-specific marker (blue stain) showing double labeled oligodendroglia (arrows). HNK-1 positive oligodendroglia made up 15% of the TUNEL positive cells in the acute plaque whereas 25–40% of the dying cells in chronic plaques were of oligodendroglial lineage, original magnification: 4003.

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astrocytes and oligodendroglial cells predominated. Fig. 4B shows two TUNEL labeled brain parenchymal cells in an acute lesion which morphologically appear to be reactive hypertrophic astrocytes. Fig. 4C shows a colocalization experiment on this lesion in which GFAP was used as an astrocyte marker. Twenty percent of all cells dying in acute lesions were astrocytes and Table 1 shows the number of dying astrocytes was much more variable in chronic lesions. The oligodendrocyte is the glial cell responsible for myelin support and remyelination and when the TUNEL processed MS lesions were co-labeled for dying cells of oligodendrocyte lineage with HNK-1, intensely reactive double labeled HNK-1 cells were prominent within the white matter lesion (see Fig. 4D). Oligodendrocytes were a major component of the dying glial cell population in both acute and chronic lesions and comprised 14 to 40% of all cells labeled. These values may underestimate the number of dying oligodendroglia present in MS lesions since not all developmental stages of oligodendroglia are recognized by antisera against the HNK-1 epitope. Labeling with a CD-3 lymphocyte T cell marker revealed striking numbers of reactive lymphoid cells within the massive perivascular infiltrates of acute lesions and there were also substantial numbers of infiltrating T cells scattered throughout the parenchyma of the acutely evolving plaque. Double label quantification, however, revealed that remarkably few dying T cells were present in the perivascular cuffs, whereas nearly 4% of the infiltrating CD3 1 T cells within remote brain parenchyma were TdT positive in one acute plaque (see Table 1). Another acute plaque also had large numbers of infiltrating T cells throughout the brain parenchyma, but none were dying as judged by TdT staining.

3.5. Agarose gel confirmation and confocal microscopy of apoptosis in chronic MS brain The in situ TUNEL procedure distinguishes between dead and living cells and provides exquisite single cell localization. It cannot, however, reliably distinguish between cell death by apoptosis and cells undergoing necrosis. In conventional necrosis, the DNA breaks down into random length fragments producing a long smear on electrophoresis. The pattern of fragmentation in apoptosis is non-random, with apoptotic DNA commonly breaking into fragments which persist as distinct multiples of 180– 200 base pairs (Wyllie, 1980). This fragmentation typically produces a laddering pattern if DNA extracted from apoptotic cells is evaluated by gel electrophoresis. This method gauges DNA fragmentation within an entire tissue sample and is thus much less sensitive than in situ testing. A substantial percentage of cells within the tissue must be undergoing apoptosis before a ladder will be apparent. With this in mind, we extracted DNA from the frozen MS cerebrum with the greatest number of positive cells, and

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from blocks of three non-MS neurologic disorders relatively free of apoptotic cells as judged by the TUNEL procedure. The positive control for this assay was fibroblast control cell DNA that was induced to undergo rapid apoptosis by serum deprivation. Fig. 5 left panel is an autoradiograph from an agarose gel of electrophoresed DNA from the highly positive MS brain. In this figure, the DNA shows a strong laddering pattern similar to that found in the positive control. Conversely little low molecular weight signal was found in non-MS brain DNA. This finding was subsequently extended by testing six additional chronic MS brain extracts of which three showed positive DNA ladders. These findings confirm that many labeled cells in the MS brain die by apoptosis, but cannot exclude the possibility that some cell populations are dying by non-apoptotic mechanisms. We also repeated the in situ TUNEL procedure using FITC-conjugated anti-digoxigenin detection on acute plaques and several chronic lesions and the slides were evaluated by laser scanning confocal microscopy. Fig. 5 (right) shows that the FITC labeled cells displayed several of the nuclear characteristics of cells undergoing apoptotic death.

3.6. Proliferation in MS brain involves glial and perivascular T cells The present study was initially designed to investigate cell death in MS brain and while dying, infiltrating, inflammatory cells might be expected, based on their prior identification in EAE lesions, the number of glial cells dying in chronic MS lesions was unexpectedly massive. It seemed unlikely that the large number of oligodendroglia and astrocytic cells undergoing destruction could be sustained long term unless newly generated glial cells were augmenting those dying in the plaque area. In order to determine if simultaneous cell proliferation occurs in multiple sclerosis lesions with large dying cell populations, tissue sections from available TdT cell-rich plaques used in the glial cell identification studies were reassayed for proliferation by the cell proliferation marker Ki-67. Ki-67 is a nuclear protein expressed during the late Gap 1, S, Gap 2 and M phase of the cell cycle (Brown and Gatter, 1990). We found that the Ki-67 antibody accurately and specifically detected proliferation within the germinal centers of lymph node control and also reacted strongly with the malignant cells from the glioma positive control. When tested on acute MS lesions, we found many hundreds of intensely labeled Ki-67 cells within the confines of the white matter lesions as shown in Fig. 6. Fig. 6 also shows the maximum focal density of positive cells within MS white matter was 310 / mm 2 . Variable smaller numbers of Ki-67 positive glial cells were encountered in the demyelinated zone of twelve chronic plaques tested. Sections from 21 OND controls including patients with SSPE, HIV, PML, vascular disease, acute hemorrhagic

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Fig. 5. Left: Autoradiograph showing DNA analysis by 1.5% agarose gel electrophoresis. Lane 1: Rat fibroblast DNA after serum starvation (positive control); Lane 2: SSPE brain DNA; Lane 3: schizophrenic brain DNA; and Lane 4: motor neuron disease brain DNA from TUNEL negative blocks. Lane 5 is DNA from TUNEL positive chronic MS brain. A strong low molecular weight DNA smear with an apoptotic ladder is present in the MS DNA but not in controls (which show DNA migration limited to a small area in the higher molecular weight gel zone–see lanes 2, 3, 4). Right: Acute MS white matter plaque from 20 year old male with nine-month history of illness FITC labeled by TUNEL procedure for fragmented DNA. Numerous cells scattered throughout the white matter are intensely labeled. The nuclear staining pattern of several positive cells show marked chromatin margination (arrows) and while other cells show nuclear condensation, all changes are characteristic of apoptotic cell death. The large cell in center (arrowheads) is a hypertrophic astrocyte which has engulfed several apoptag positive corpses. This phenomenon is also shown in Fig. 4B, original magnification: 6303.

leucoencephalitis and ALS showed little or no proliferation in white matter. The Ki-67 positive cells morphologically appeared to represent glial and inflammatory cell types in the approximate proportions defined in the cell death study with the exception of the T cell population and the four cell types were quantified by double labeling with cell

Fig. 6. Graph of proliferating Ki-67 reactive cells in MS, other neurological disease control brains (OND) and glioblastoma multiforme tissue (positive control). Note that several MS lesions contained levels of proliferating cells comparable to the proliferation within the malignant glioblastoma. The OND group includes nine subjects with marked CNS inflammatory disease including SSPE, HIV, PML, and acute hemorrhagic leukoencephalitis. The mean value for Ki-67 positive cells in the controls is 17, and the mean value for the MS group is 133; the difference between groups is highly significant by two-tailed t testing (P,0.0001).

specific markers as shown in Fig. 8. For T cells, there were large numbers heavily labeled with Ki-67 within many of the thick perivascular cuffs [see Fig. 7 (left panel)], and this is in marked contrast with the finding of only a rare dying T cell in this location. We found that up to 50% of the T cell infiltrate in some areas of perivascular cuffs stained for Ki-67, and the labeled mass of perivascular cells assumed a staining appearance equal in intensity to the germinal centers contained in lymph node positive control tissue. T cell proliferation of this magnitude is not encountered in the peripheral blood or CSF of MS patients, and was an unexpected finding, implying the presence of a highly antigenic stimulus within the perivascular microenvironment of the early evolving MS plaque. Double labeling for macrophage / microglia, oligodendroglia, astrocytes, and T cells, was performed on three acute plaques and selected chronic MS lesions. Fig. 8 shows double labeled proliferating macrophage lineage cells were by far the most common cells in the parenchyma of acute evolving MS lesions (43 to 52%). In chronic plaques, however, labeled macrophage numbers were much lower and more variable (1–20%). Double labeled macrophages were also prominent along with proliferating T cells within the perivascular mononuclear cell infiltrates. Fig. 8 also shows that oligodendroglia specific HNK-1 positive staining was present in 17–37% of proliferating cells in both acute and chronic lesions, whereas proliferative astrocytes were present in variable

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Fig. 7. Left: Acute MS lesion. Ki-67 proliferation marker stained section of a vessel within an evolving plaque showing extensive labeling of mononuclear cells in the perivascular infiltrate (brown stain). Right: Chronic hypocellular MS plaque. The Ki-67 antibody strongly reacts with scattered cells throughout the hypocellular lesion indicating numerous cells have entered the proliferative cycle, original magnification: 4003.

numbers. Proliferating CD3-positive T lymphocytes were found only in acute lesions and the percent of perivascular cells proliferating ranged from 12 to 25%. The finding of simultaneous proliferative activity within white matter MS lesions during the same period that cell death is marked, extend the morphological and immunocytochemical studies of Prineas and co-workers who have long proposed that the early evolution of some MS plaques is characterized by oligodendrocyte loss subsequently followed by a phase of oligodendrocyte proliferation (Prineas, 1988; Prineas et al., 1989). Neither the intense T cell proliferative response nor the macrophage proliferation in perivascular cuffs of hyperacute MS lesions has been reported previously. However, evidence consistent with new oligodendroglia production in an MS brain as judged

Fig. 8. Graph of proliferating Ki-67 cells in acute and chronic lesions by cell type. The macrophage / microglia is the most common proliferating cell in acute MS lesions (range, 43–52%; median, 49%). By contrast, labeled macrophages in chronic lesions are much lower and more variable (range, 1–20%; median, 5%). Labeled astrocytes were present in widely variable amounts in lesions of all ages (acute: range, 1–34%; median, 3%; chronic: range, 1–33%; median, 17%). Oligodendroglia specific HNK-1 positive proliferating cells are present in both acute and chronic plaques in substantial numbers (acute: median, 22.5%; chronic: median, 27%). Proliferating CD3 positive T lymphocytes were found only in acute lesions and only in the perivascular cuffs (range, 12–25%; median, 18.5%).

by staining with antibody to proliferating cell nuclear antigen (PCNA) has been reported (Morris et al., 1994).

4. Discussion In spite of several decades of investigation, the pathogenesis of the white matter lesion in MS has eluded definition. The key finding in this study establishes a significant correlation between increased numbers of dUTP stained inflammatory / glial cells undergoing apoptosis and white matter pathology in the MS brain. This mode of cell death probably escaped attention because cells undergoing genetically programmed death and their subsequent phagocytosis are thought to occur rapidly and the subtle late morphologic changes of apoptosis in diseased tissue can be difficult to recognize by conventional histopathology (Walker et al., 1988). The recent introduction of in situ biochemical markers of cell death permitted us to identify apoptotic cells in tissues where cell death was largely undetectable by prior methods and double staining with cell specific markers allowed us to distinguish between a population of blood-derived inflammatory immune cells and a large population of dying resident glial cells. We obtained our quantitative observations by testing a large rigorously controlled series of quick frozen MS and non-MS unfixed brains where group post-mortem intervals were comparable and the individual case post-mortem interval was often shorter than 6 h. Recent evidence derived from animal studies indicates that post-mortem intervals as long as 72 h do not substantially increase the number of neural cells staining positive by the TUNEL technique (Petito and Roberts, 1995). Specificity of the TUNEL test for CNS tissues was further validated on spinal cord cryosections from EAE animals where the quantitative findings were remarkably similar to those found subsequently in the human tissue studies. In EAE, we identified only rare background dying cells in spinal

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cord controls and found striking 100-fold increases in apoptotic cells during the phase when neurologic signs are present. The dying cell peak densities within MS CNS tissue and EAE spinal cord are comparable and also similar to peak cell death values quantified in other experimental systems, e.g. hepatic tumors (Kong and Ringer, 1995). We were also able to determine that some of the cell death detected in MS was apoptotic because electrophoresed DNA extracted from MS brain demonstrated a clear-cut low molecular weight internucleosomal banding pattern characteristic of apoptotic cell death. This finding was confirmed at the single cell level by evaluating fluorescent dUTP TUNEL stained cell nuclei by confocal microscopy. The stained nuclei from both early and chronic lesions showed marked nuclear chromatin margination, blebbing, and nuclear condensation – all characteristics of apoptotic cell death. The findings above taken in concert indicate that many of the cells dying in MS tissue are dying by apoptotic mechanisms. The composition of the dying cell types in MS plaques is complex and varies with age of the lesion. It is clear that infiltrating inflammatory cells (mainly macrophages and small numbers of T cells) contribute the bulk of the dying cells in the early evolving lesion and dying macrophagemicroglia still comprise 20% of all dying cells in the mature chronic lesion. Of particular note, there is also a significant population of resident glial cells dying in MS lesions and this is true of plaques of all ages. We consistently detected a large population of oligodendroglia undergoing cell death in lesions of all ages and there were also variable numbers of dying astrocytes present. The observation that 14–40% of all cells dying in lesions are of oligodendroglial lineage is of critical importance regardless of whether the death mode is apoptotic or necrotic, because this cell type is responsible for myelin sheath maintenance and myelin destruction is characteristic of MS lesions. We found dying macrophage / microglia were the most common cells dying in acute MS tissue and this appears to contrast with the literature on apoptosis in EAE, where the bulk of the dying cells within the brain are inflammatory cells of T cell origin (Nguyen et al., 1994; Pender et al., 1991; Schmeid et al., 1993). In EAE animals the greatest number of dying inflammatory T cells occurs coincident with clinical recovery and the apoptotic deletion of inflammatory cells in EAE is thought to contribute to the termination of the autoimmune response (Tabi et al., 1994). Our finding of very limited T cell death in MS lesions may reflect the difficulty in making a short duration animal model mirror the same time course of cell events that one encounters in MS, a disorder where lesions evolve over weeks to years. Within chronic MS plaques on the other hand, T lymphocytes were absent or so low (0–16 cells / section) that no valid quantification was possible, indicating that a dying T cell within chronic lesions is probably a rare event. The stimuli leading to cell death in MS brain are unknown, but there are numerous candidates which have

been shown to induce apoptosis both in vitro and in animal model systems (Locksin and Zakeri, 1991; Orrenius, 1995). Likely triggers in MS brain include cytokines such as tumor necrosis factor alpha and beta (TNF-a, TNF-b), substances known to induce apoptosis of oligodendroglia in vitro and in addition TNF-a is known to be upregulated in active MS lesions (Selmaj and Raine, 1988; Selmaj et al., 1991). We have recently described involvement of the Apo1 / Fas–Fas ligand death system in MS lesions (Dowling et al., 1996). Another possible trigger of apoptosis in MS brain is induction by the NO radical. NO synthetase is present in the lesions of both active MS and EAE and there is recent evidence that the NO molecule can induce apoptotic cell death at least in T cells (Bo¨ et al., 1994; Fehsel et al., 1995). There are yet other additional late stage cell death pathway triggers within the cytoplasm of target cells which belong to the cysteine protease family of ICE genes (interleukin 1b-converting enzyme). The ICE family has homology with the Ced3 death gene of C. elegans and they have been implicated as late stage ‘‘executioner proteins’’ in apoptosis (Kumar, 1995; Yuan, 1995). The second novel finding in our study was the remarkable amount of cell proliferation coexisting in the same white matter areas of the MS brain where glial cells are dying. It seems likely that the injured brain is attempting to maintain a dynamic equilibrium between dying glial cells and glial repopulation much like the phenomenon recently described in the lymphoreticular system of HIV infected individuals (Coffin, 1995; Ho et al., 1995; Wei et al., 1995). In HIV, continuous death of CD-4 T cells results in widespread unremitting proliferation of lymphocytes. Ultimately, the replacement mechanism fails in HIV as lymphocyte proliferative capacity gradually falters. By analogy the remarkable paucity of glial cells in chronic white matter MS lesions may represent the final outcome of a similar dynamic battle where glial cell depletion is the result of repeated cell attacks and failed regeneration. A search for factors which trigger the cell death program and the factors which augment glial cell production in MS brains may lead to novel therapies for a disease, currently without effective treatment.

Acknowledgments This work was supported by Grants VA 0013 of the Research Service of the Department of Veterans Affairs; and the Zyma Foundation. We thank Noounanong Cheewatrakoolpong for expert technical assistance; Nettie Colitti and Barbara Goldschmidt for editorial assistance.

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