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Brain, Vol. 125, No. 2, 338-349, February 1, 2002
© 2002 Oxford University Press

Oligodendrocyte precursor cells in the demyelinated multiple sclerosis spinal cord

Guus Wolswijk1

1 Netherlands Institute for Brain Research, Amsterdam, The Netherlands

Correspondence to: G. Wolswijk, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ, Amsterdam ZO, The Netherlands E-mail: G.Wolswijk{at}nih.knaw.nl


   Summary
Top
 Summary
Introduction
 Material and methods
 Results
 Discussion
 References
 
Lesions appearing in the CNS of patients in the chronic phase of the inflammatory, demyelinating disease multiple sclerosis often fail to repair, resulting in neurological dysfunction. This failure of remyelination appears, in many cases, to be due not to the destruction of the local oligodendrocyte precursor population, a source for new myelin-forming cells, but to the failure of the precursor cells to proliferate and differentiate, at least in brain lesions. The spinal cord is also a prominent site for lesions in multiple sclerosis, but nothing is known about the fate of the oligodendrocyte precursor population in this area. The present study has therefore analysed spinal cord samples with demyelination from 16 subjects with longstanding multiple sclerosis for the presence of oligodendrocyte precursor cells. Immunolabellings of 10 µm thick sections with the O4/anti-galactocerebroside (GalC) antibody combination, to visualize O4-positive, GalC-negative oligodendrocyte precursor cells, revealed that such cells were prevalent in many spinal cord lesions, with densities of up to 35 cells/mm2. Six of the spinal cord lesions contained <=3 O4-positive, GalC-negative cells/mm2, but such cells were widespread in brain lesions from these multiple sclerosis cases that were available for study (8–26 cells/mm2). The density of the O4-positive, GalC-negative oligodendrocyte precursor cells in all spinal cord and brain lesions studied thus far (n = 41) decreased significantly with declining numbers of debris-laden macrophages. In addition, lesions lacking macrophages tended to be derived from the older patients and there was a negative correlation between the density of the oligodendrocyte precursor cells and clinical age of the multiple sclerosis subject at death, and disease duration. The analysis further revealed that lesions from subjects with primary progressive and secondary progressive multiple sclerosis contained, on average, similar numbers of oligodendrocyte precursor cells/mm2 and that immature oligodendrocytes were only present in significant numbers in lesions with high precursor densities. Taken together, the present data suggest that there is a gradual reduction in the size of the O4-positive, GalC- negative oligodendrocyte precursor population with increasing age of the lesion, that the generation of new oligodendrocytes becomes increasingly more impaired and that lesions are not repopulated to a significant extent by migratory oligodendrocyte precursor cells present in the adjacent unaffected tissue. Hence, strategies intended to promote endogenous remyelination in multiple sclerosis patients should focus on both enhancing the long-term survival of oligodendrocyte precursor cells and on stimulating these cells to proliferate and differentiate into remyelinating oligodendrocytes.

Keywords: demyelination; multiple sclerosis; O4; oligodendrocyte precursor cell; remyelination

Abbreviations: GalC= galactocerebroside; GFAP = glial fibrillary acidic protein; HLA = human leucocyte antigen; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; PDGF = platelet-derived growth factor; PP = primary progressive; RR = relapsing–remitting; SP = secondary progressive


   Introduction
Top
 Summary
 Introduction
Material and methods
 Results
 Discussion
 References
 
The generation of new myelin-forming oligodendrocytes in the inflammatory, demyelinating disease multiple sclerosis fails as disease progresses, resulting in the formation of confluent areas of persistent demyelination, impairment of impulse conduction along the denuded axons and neurological symptoms (Smith, 1996Go; Lassmann et al., 1997Go; Prineas and McDonald, 1997Go; Lucchinetti et al., 2000Go; Wingerchuk et al., 2001Go). This situation is very different from that observed in experimental models of demyelinating disease where the generation of new oligodendrocytes, re-ensheathment of denuded axons, restoration of impulse conduction and functional recovery frequently are complete (Ludwin, 1981Go; Jeffery and Blakemore, 1997Go; Wolswijk, 1998Goa; Franklin, 1999Go). The remyelinating cells in such models are generated by a population of immature cells capable of proliferation, migration and differentiation along the oligodendrocyte pathway (Ludwin, 1979Go; Arenella and Herndon, 1984Go; Godfraind et al., 1989Go; Rodriguez, 1991Go; Carrol and Jennings, 1994Go; Gensert and Goldman, 1997Go; Franklin et al., 1997Go; Keirstead et al., 1998Go; Redwine and Armstrong, 1998Go; Di Bello et al., 1999Go). The expansion of the oligodendrocyte precursor population and the production of new myelin-forming cells in response to demyelination is controlled by a number of growth factors, and their mRNAs are upregulated in distinct patterns during the remyelination process depending on whether they stimulate precursor proliferation and migration or promote oligodendrocytic differentiation (Wolswijk et al., 1991Go; Komoly et al., 1992Go; Tourbah et al., 1992Go; Wolswijk and Noble, 1992Go; Yao et al., 1995Go; Engel and Wolswijk, 1996Go; Shi et al., 1998Go; Hinks and Franklin, 1999Go; Messersmith et al., 2000Go). The success of repair of experimentally induced lesions is thought to be influenced by ageing, sex, the size of the lesion and the extent to which the oligodendrocyte precursor population is affected by the disease process (Gilson and Blakemore, 1993Go; Franklin et al., 1997Go; Keirstead et al., 1998Go; Shields et al., 1999Go).

The limited success of myelin repair during the chronic phase of multiple sclerosis appears, in many cases, not to be the result of the concomitant destruction of both oligodendrocytes and their precursor cells. This notion has come from recent histopathological studies demonstrating that brain lesions from subjects with longstanding multiple sclerosis often contain substantial numbers of oligodendrocyte precursor cells, identified using the O4 antibody (Wolswijk, 1998Gob), antibodies to the platelet-derived growth factor (PDGF)-{alpha} receptor (Scolding et al., 1998Go; Wolswijk, 1998Gob; Maeda et al., 2001Go) and antibodies to the NG2 chondroitin sulphate proteoglycan (Chang et al., 2000Go) (reviewed in Wolswijk, 1998Goa; Dawson et al., 2000Go; Levine et al., 2001Go). Although many oligodendrocyte precursor cells apparently survive the demyelination process in chronic stage multiple sclerosis, they appear to be in a relatively quiescent state (Wolswijk, 1998Gob, 2000). This finding raises the possibility that remyelination in chronic multiple sclerosis is scanty or absent because of the failure of the local oligodendrocyte precursor population to expand and generate new myelin-forming oligodendrocytes. Since lesion repair is more successful during the early course of multiple sclerosis (Prineas et al., 1989Go; Raine and Wu, 1993Go; Prineas and McDonald, 1997Go; Lucchinetti et al., 2000Go), it suggests that the proliferation and differentiation of oligodendrocyte precursor cells become gradually more impaired with progression of the disease. Thus, the lesion environment changes from one conducive to remyelination to one hampering endogenous repair processes, because of either the absence of growth factors implicated in remyelination, the presence of inhibitory molecules or the presence of the scar tissue formed by astrocytes (Wolswijk, 1998Goa). Another intriguing possibility that has emerged from a recent study in the rat is that the therapeutic administration of glucocorticoids may impair the proliferative capacity of the oligodendrocyte precursor population (Alonso, 2000Go).

Demyelinated lesions also develop in the spinal cord of many multiple sclerosis patients and, because of their location, they can have devastating consequences in terms of disability (Smith, 1996Go; Prineas and McDonald, 1997Go). To gain further insights into the failure of lesion repair in multiple sclerosis, the present study has analysed demyelinated spinal cord samples obtained at autopsy from 16 subjects with longstanding multiple sclerosis for the presence of oligodendrocyte precursor cells, using indirect immunofluorescence techniques and confocal laser scanning microscopy.


   Material and methods
Top
 Summary
 Introduction
 Material and methods
Results
 Discussion
 References
 
Treatment of post-mortem spinal cord tissue
Spinal cord and brain tissue was obtained from The Netherlands Brain Bank (NBB; co-ordinator, R. Ravid); the NBB received permission for performing autopsies, for the use of tissue and for the access to medical records for research purposes from the Ethical Committee of the Medical Faculty of the Free University, Amsterdam, The Netherlands. Within 4 h 50 min–16 h 45 min after death (mean 8 h 25 min ± 3 h 00 min), spinal cord samples (5–10 mm in length) and brain lesions from subjects with longstanding multiple sclerosis (Table 1) were placed in a solution of 4% paraformaldehyde in PBS (phosphate-buffered saline, pH 7.4), stored for 1–7 days at 4°C, and incubated in a solution of 30% sucrose in PBS for 1–3 days at 4°C under constant rotation. The tissue was then placed into a boat prepared from aluminium foil and filled with Tissue-Tek optimum cutting temperature embedding compound (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands), frozen on dry ice and stored at –80°C (Wolswijk, 1998Gob, 2000). Separate blocks of brain and spinal cord tissue were processed for neuropathological examination by Dr W. Kamphorst, Department of Pathology, Academic Hospital of the Free University, Amsterdam, The Netherlands.


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  		Table 1 Details of multiple sclerosis subjects
 
Immunohistochemistry
Sections of 10 µm were cut from each tissue block using a Reichert-Jung 2800 cryostat (cutting temperature –20 to –25°C), mounted onto SuperFrost*/Plus microscope slides (Menzel-Gläser, Braunschweig, Germany) and immunolabelled, either directly or after storage at –20°C, using either indirect immunofluorescence or immunoperoxidase techniques, as described before (Wolswijk, 1998Gob, 2000). Sections were incubated for 1–7 days at 4°C in the primary antibody solutions, rinsed several times in TBS (Tris-buffered saline, pH 7.6) and then incubated for 2 h at room temperature or overnight at 4°C with the fluorochrome [FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine isothiocyanate), Cy3 or Cy5]-conjugated or biotinylated anti-rabbit or mouse IgG (H+L), or anti-mouse Ig subclass-specific antibodies (purchased from either Southern Biotechnology Associates, Inc., Birmingham, Ala., USA or Jackson ImmunoResearch, West Grove, Pa., USA). The binding of the biotinylated antibodies was visualized by incubating sections in the presence of the Vectastain ABC kit reagents A and B (Vector Laboratories, Inc., Burlingham, Calif., USA) followed by substrate {a filtered solution of 0.42 mg/ml 3-amino-9-ethylcarbazole [dissolved in dimethyl formamide (Merck, Darmstadt, Germany)] and 0.01% H2O2 (Merck) in 0.05 M sodium acetate (Sigma Chemical Company, St Louis, Miss., USA) buffer, pH 5.0} or by incubating sections in the presence of fluorochrome-coupled streptavidin (Vector). The primary antibodies used in the present study were: (i) the mouse O4 monoclonal antibody (Sommer and Schachner, 1981Go); (ii) a mouse IgG3 anti-galactocerebroside (GalC) monoclonal antibody (the Ranscht antibody; Ranscht et al., 1982Go); (iii) a rabbit antiserum to myelin basic protein (MBP; a gift from Dr H. van Noort, TNO, Leiden, The Netherlands); (iv) a mouse IgG1 anti-myelin oligodendrocyte glycoprotein (MOG) antibody [the Y10 antibody (Piddlesden et al., 1993Go), a gift from Dr S. Piddlesden]; (v) a rabbit antiserum to the NG2 chondroitin sulphate proteoglycan (a gift from Dr J. Levine, State University of New York, Stony Brook, NY, USA); (vi) an IgG2a mouse monoclonal antibody to chondroitin sulphate proteoglycan (the 9.2.27 antibody; PharMingen, San Diego, Calif., USA); (vii) a rabbit antiserum to the PDGF-{alpha} receptor [the R7 rabbit polyclonal antibody raised against a synthetic peptide from the C-terminal end, which was also used in the study of Scolding et al., 1998Go); a gift of Dr C.-H. Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden]; (viii) a mouse IgG1 anti-neurofilament monoclonal antibody [the RT97 antibody (Wood and Anderton, 1981Go); Boehringer Mannheim, Germany]; and (ix) a mouse IgG1 anti-human leucocyte antigen (HLA)-DP, DQ, DR antigen (major histocompatibility complex class II) monoclonal antibody (Dako A/S, Glostrup, Denmark). Antibodies were diluted in TBS containing 0.25% Triton X-100 (Sigma) and/or 5% heat-inactivated calf serum (Sigma). Nuclei were visualized by incubating sections in 1 mg/ml Hoechst 33258 (Sigma) (for conventional fluorescence microscopical analysis), in 1 mM TO-PRO-3 iodide (Molecular Probes, Eugene, Oreg., USA) (for confocal laser scanning microscopical analysis) or in a haematoxylin solution (for bright-field microscopical analysis). At the end of the staining procedure, a drop of glycerol containing 22 mM 1,4-diazobicyclo [2,2,2] octane (Sigma) was placed on the section (to reduce fading of the fluorochromes), followed by a glass coverslip. The excess glycerol was removed and the coverslip was then sealed using clear nail varnish. Sections were viewed on a Zeiss Axiophot microscope equipped with phase-contrast, bright-field and dark-field optics, epi-UV illumination and selective filters optimized for distinguishing between FITC and TRITC/Cy3 and Hoechst emission, or on a Zeiss 410 inverted confocal laser scanning microscope with three different lasers emitting at 488, 543 and 633 nm to excite FITC, TRITC/Cy3 and TO-PRO-3 iodide/Cy5, respectively, and with bright-field optics.

Cell counts and data analysis
The density of the populations of O4-positive, GalC-negative oligodendrocyte precursor cells, process-bearing GalC-positive oligodendrocytes and phase-bright macrophages in completely demyelinated areas of the spinal cord sections was calculated from the number of cells present in microscope fields with a size of 1/16 (0.0625) mm2 [>=30 fields/section (n = 3 sections)]. Depending on the size of the lesion area, either every adjacent microscope field or up to every fifth field was analysed. Microcal Origin 5.0 software was used to plot and analyse the data.


   Results
Top
 Summary
 Introduction
 Material and methods
 Results
Discussion
 References
 
Characteristics spinal cord tissue with demyelination
Seventeen spinal cord samples (5–10 mm in length) from 16 subjects with chronic multiple sclerosis (Table 1) were analysed for the presence of oligodendrocyte precursor cells. The collection included samples from subjects with relapsing–remitting (RR) multiple sclerosis (1 case), primary progressive (PP) multiple sclerosis (two cases) and secondary progressive (SP) multiple sclerosis (seven cases); the remaining six subjects died during the progressive phase of the disease, but it was not clear from their medical records whether they had suffered from the PP or SP form of multiple sclerosis. The 17 blocks had areas of complete demyelination ranging from <5% to >95% of a complete spinal cord cross-section, including the grey matter region (Fig. 1 and Table 2). These areas contained numerous axons, identified using antibodies to neurofilament, but it is likely that axon loss had occurred in these regions, either because of injury occurring in the lesion area itself or because of the presence of other lesions along the length of the spinal cord (secondary Wallerian degeneration) (Prineas and McDonald, 1997Go). Areas with reduced numbers of myelinated axons with or without demyelinated axons were observed in most samples and, in seven cases, >95% of the cross-section was affected by the disease process (Table 2). None of the spinal cord samples contained significant areas lacking both myelin and axons, i.e. areas larger than the size of a microscope field of 0.0625 mm2. Mature oligodendrocytes lacking myelin-forming processes were either absent or present in only small numbers in the affected regions. Remyelinated axons, i.e. axons surrounded by weakly MOG-positive, thin myelin sheaths, were rarely observed. The lesion areas were packed with glial fibrillary acidic protein (GFAP)-positive filaments (Fig. ), an intermediate filament found in astrocytes.



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  		Fig. 1 Details of multiple sclerosis spinal cord samples with demyelination. (A) Low power view of a cross-section through a multiple sclerosis spinal cord (Case 5) immunolabelled with antibodies to myelin basic protein (MBP; green, FITC) and neurofilament (red, TRITC), an intermediate filament found in axons. Note that the central portion of this spinal cord section, including the grey matter, contains virtually only demyelinated axons, (see also Table 2). Very few debris-laden macrophages were present in this spinal cord sample (Table 3), suggesting that the demyelinating activity had occurred some time ago. Diameter of the spinal cord, 7 mm. (B) Low power view of a section cut from the spinal cord sample from multiple sclerosis case 14 that was immunolabelled with antibodies to HLA-DP, DQ, DR antigens (green, FITC), which label activated cells of the microglial/macrophage lineage, and antibodies to MBP (red, Cy3). The activated microglia/macrophages were found mostly at the borders of the lesional areas. Note that the grey matter is partially affected by the demyelination process (Table 2). Diameter of the spinal cord, 9 mm. (C) Detail of an area of a multiple sclerosis spinal cord (Case 3) with very few myelinated axons. Axons were visualized with antibodies to neurofilament (red, TRITC), while the myelin rings were visualized with antibodies to MBP (green, FITC). Image size, 200 µm x 200 µm. (D) HLA-DP, DQ, DR-positive microglia/macrophages (arrow; green, FITC) were sometimes observed within layers of MBP-positive myelin segments (red, Cy3); nuclei were visualized with the nuclear dye TO-PRO-3 iodide (blue). Immunoreactivity for HLA- DP, DQ, DR is also visible in another myelin ring (arrowhead). Multiple sclerosis case 9. Image size, 35 µm x 35 µm. (E) Most demyelinated areas of the spinal cord samples were packed with GFAP-positive filaments (green, FITC), an intermediate filament found in astrocytes. The small numbers of myelin sheaths in the lesion area shown were labelled with antibodies to GalC (red, TRITC). Multiple sclerosis case 3. Image size, 200 µm x 200 µm.

 

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  		Table 2 Details of spinal cord samples with demyelination
 
Numerous macrophages filled with myelin degradation products were present in 11 of the 17 blocks and they occupied up to 50% of the spinal cord cross-sections, with densities of up to 900–950 cells/mm2 section (10 µm thick) (Table 2). They were distributed either throughout the lesion area, concentrated at the lesion edges (see Fig. ) or present in small clusters in areas with still many myelinated axons. Immunolabellings involving antibodies to HLA-DP, DQ, DR [major histocompatibility complex class II] antigens showed that most samples contained activated cells of the microglia/macrophage lineage (Fig. ). Confocal laser scanning microscopic analysis revealed many examples in which the activated microglia/macrophages appeared to have engulfed individual myelin rings, or contained either MBP-positive myelin fragments or diffuse MBP immunoreactivity. Moreover, HLA-DP, DQ, DR-positive cells were sometimes observed within layers of the myelin sheaths (Fig. ). The myelin-free areas of the sections contained lower numbers of phase-bright macrophages (Table 3) and these cells tended to lack immunoreactivity for MBP.


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  		Table 3 Densities of oligodendrocyte precursor cells, immature oligodendrocytes and phase-bright macrophages in demyelinated spinal cord and brain lesions
 
Oligodendrocyte precursor cells in demyelinated spinal cord lesions
The multiple sclerosis spinal cord sections were immunolabelled with three different antibody markers for oligodendrocyte precursor cells, i.e. the O4 monoclonal antibody (Sommer and Schachner, 1981Go) [in combination with antibodies to GalC to distinguish between O4-positive, GalC-negative oligodendrocyte precursor cells and O4-positive, GalC-positive oligodendrocytes (Wolswijk, 1998Gob)] and antibodies to the PDGF-{alpha} receptor and NG2 chondroitin sulphate proteoglycan. Consistent and reliable labelling in all spinal cord samples (and spinal nerves) of oligodendrocyte lineage cells and/or of myelin was observed only with the O4 antibody (Fig. 2), with no obvious deleterious effects of autolysis time of the tissue and length of fixation (see also Back et al., 2001Go). With two different antibodies to NG2 [a rabbit antiserum and the 9.2.27 monoclonal antibody used by Chang et al. (2000Go)], only consistent labelling was found of blood vessels and of cells in the spinal nerves (Fig. ), which are probably non-myelinating Schwann cells (Schneider et al., 2001Go). Only very occasionally, a cell with an oligodendrocyte precursor-like morphology expressed NG2 in the spinal cord sections. NG2-labelling in the spinal cord sections was, however, only seen following long incubations with the primary antibody and using an amplification step (Chang et al., 2000Go). Using this improved immunolabelling protocol, NG2-positive, oligodendrocyte precursor-like cells were now observed in brain lesions derived from multiple sclerosis cases 13 and 14 (Fig. ), in contrast to that reported previously (Wolswijk, 1998Gob). These results suggest that if oligodendrocyte precursor cells in the multiple sclerosis spinal cord express NG2, they do so at much lower levels than oligodendrocyte precursor cells in brain lesions and non-myelinating Schwann cells in spinal nerves. No convincing labelling of oligodendrocyte precursor-like cells in the spinal cord sections was obtained with the R7 polyclonal antibody to the C-terminus of the human PDGF-{alpha} receptor (Claesson-Welsh et al., 1989Go), which was also used in the study of Scolding and co-workers (Scolding et al., 1998Go); using the same immunolabelling procedure, these antibodies did label reproducibly process-bearing, oligodendrocyte precursor-like cells in sections of marmoset and rhesus monkey brain tissue (G. Wolswijk, B. ’t Hart and H. Brok, unpublished observations).



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  		Fig. 2 Details of oligodendrocyte precursor cells in demyelinated spinal cord lesions. (A) Two O4-positive (green, FITC), GalC-negative (blue, Cy5) oligodendrocyte precursor cells in an area with large numbers of neurofilament-positive (red, TRITC), demyelinated axons. The arrowhead points to a GalC-positive process that has enwrapped a denuded axon; the oligodendrocyte to which this process belonged was in another section. Multiple sclerosis case 9. Image size, 135 µm x 135 µm. (B) Oligodendrocyte precursor cell that had bound the O4 antibody (red, TRITC), but not antibodies to GalC (blue, cy5), in an area with macrophages expressing HLA-DP, DQ, DR antigens (green, FITC). Multiple sclerosis case 14. Image size, 45 µm x 45 µm. (C) O4-positive, GalC-negative oligodendrocyte precursor cells appear to lack the astrocyte-specific intermediate filament GFAP, as shown previously for O4-positive, GalC-negative cells in brain lesions (Wolswijk, 1998Gob). The expression of GFAP in most demyelinated areas of the spinal cords studied was much more pronounced than in the image shown here (see Fig. 1E) (O4 = red, TRITC; GalC = blue, Cy5; GFAP = green, FITC). Multiple sclerosis case 5. Image size, 50 µm x 50 µm. (D) Some processes of the GalC-positive cell (red, TRITC) shown were connected to MBP-positive (green, FITC) myelin sheaths (arrow), while others had encircled individual neurofilament-positive (blue, Cy5) axons, but had not formed myelin (arrowheads). The cell body of this, presumably immature, oligodendrocyte contained some MBP immunoreactivity. Multiple sclerosis case 2. Image size, 105 µm x 105 µm. (E) NG2-positive (green, FITC) oligodendrocyte precursor cell in a brain lesion from multiple sclerosis case 13. The cell shown was present in a demyelinated area containing many HLA-DP, DQ, DR-positive macrophages (red, TRITC); nuclei were visualized with the nuclear dye TO-PRO-3 iodide (blue). Although NG2-positive cells were observed in a brain lesion from multiple sclerosis case 13, no NG2-positive cells were observed in the spinal cord sample from this subject. However, the antibodies to NG2 did label cells in spinal nerve tissue from all multiple sclerosis subjects (see Fig. 2F). Image size, 90 µm x 90 µm. (F) NG2-positive cell (red, Cy3) in a spinal nerve attached to the spinal cord sample obtained from multiple sclerosis case 8; nuclei in the section were visualized with TO-PRO-3 iodide (blue). A recent study has suggested that NG2-positive cells in adult rat peripheral nerves are non-myelinating Schwann cells (Schneider et al., 2001Go). Image size, 80 µm x 80 µm.

 
O4-positive, GalC-negative cells were observed throughout the demyelinated areas of most spinal cord samples, including the affected grey matter areas (Table 3). They had an oval-shaped cell body containing an oval or irregular-shaped nucleus and little cytoplasm (Fig. ). The small number of processes (1–4) that emanated from their cell body were fine, and sometimes long (Fig. ); an occasional O4-positive, GalC-negative cell had a more elongated morphology. These cells were in close proximity to numerous demyelinated axons and were embedded in an often dense network of GFAP-containing astrocytic processes (Figs. and ).

The highest numbers of O4-positive, GalC-negative cells were observed in the demyelinated areas of the spinal cord sections from multiple sclerosis cases 2 and 3 (33–35 cells/mm2 section; 10 µm thick sections) (Table 3 and Fig. 3), with up to seven cells/microscope field (size: 1/16 mm2). Although both samples harboured comparable numbers of O4-positive GalC-negative cells, the spinal cord lesion from multiple sclerosis case 2 contained many phase-bright macrophages (245.0 ± 37.3 cells/mm2), while the spinal cord lesion from multiple sclerosis case 3 virtually lacked macrophages (Table 3 and Fig. ), suggesting it was a relatively old lesion; it has been suggested that it can take >6 months for debris-laden macrophages to disappear from demyelinated areas (Brück et al., 1995Go). Eight of the spinal cord samples contained between 10 and 20 O4-positive, GalC-negative cells/mm2, while oligodendrocyte precursor cells were rare (<=3.0 cells/mm2) in the demyelinated samples from multiple sclerosis cases 4, 6, 8, 11, 12 and 13 (Table 3). Oligodendrocyte precursor cells were detected in demyelinated spinal cord tissue derived from both subjects with PP multiple sclerosis (9.1 ± 2.3 and 10.6 ± 1.3 cells/mm2) and from six out of seven subjects with SP multiple sclerosis (up to 34.8 ± 1.8 cells/mm2) (Table 3).



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  		Fig. 3 There is a positive correlation between the density of the oligodendrocyte precursor population and the macrophage population in spinal cord and brain lesions from subjects with chronic multiple sclerosis. The data suggest that the number of O4-positive, GalC-negative oligodendrocyte precursor cells/mm2 section was higher in lesions containing numerous macrophages than in lesions with only small numbers of macrophages [data from 17 spinal cord (squares) and 24 brain lesions (triangles)]; 15 brain lesions were analysed in a previous study (Wolswijk, 1998Gob; see Table 1 for details of these multiple sclerosis cases). A clear exception was the spinal cord sample with demyelination obtained from Case 3, which had one of the highest precursor densities (35 ± 2 cells/mm2), but virtually lacked macrophages (<3 cells/mm2). Since a higher proportion of the lesions obtained from the older multiple sclerosis subjects, who also had a longer disease duration, lacked macrophages, there was a negative correlation between the density of the O4-positive, GalC-negative oligodendrocyte precursor population and the age of the multiple sclerosis subject at death, and length of the disease process. MS = multiple sclerosis.

 
Statistical analysis indicated that there was a significant negative correlation between the density of O4-positive, GalC-negative precursor cells in the demyelinated spinal cord areas, and the age of the multiple sclerosis subject at death (P = 0.0002) (Fig. ) and clinical disease duration (P = 0.006); as expected, the length of the disease process increased significantly with age of the multiple sclerosis subject (P = 0.004). Moreover, there was a positive correlation between the density of the macrophage population, which gives an indication of the relative age of the lesion (Ozawa et al., 1994Go; Brück et al., 1995Go), and the density of the oligodendrocyte precursor cell population (P = 0.0132), suggesting that lesions with few if any macrophages, i.e. relatively old lesions, contained fewer O4-positive, GalC-negative cells than lesions with numerous macrophages, i.e. relatively fresh lesions. A clear exception was the spinal cord sample from multiple sclerosis case 3, which harboured many O4-positive, GalC-negative cells, but lacked macrophages. The analysis further showed that spinal cord lesions with only small numbers of macrophages (<10 cells/mm2) were most often derived from subjects over the age of 65 (Fig. ).

Immature GalC-positive oligodendrocytes were rare in the demyelinated areas of most chronic multiple sclerosis spinal cord samples (<0.5 cell/mm2 section). The three exceptions were those derived from multiple sclerosis cases 2 (4.2 ± 2.5 cells/mm2), 3 (3.5 ± 1.8 cells/mm2) and 9 (0.9 ± 0.6 cells/mm2); these lesions also harboured the highest numbers of O4-positive, GalC-negative cells/mm2 section (Table 3). These presumably newly generated oligodendrocytes tended to be present in clusters [with up to four cells/microscope field (1/16 mm2)] and these clusters also contained O4-positive, GalC-negative cells (Fig. ). The processes of some of the GalC-positive cells had encircled individual denuded axons in the lesion area and/or were connected to MBP-positive myelin sheaths (Fig. ).

Oligodendrocyte precursor cells in brain lesions
Appropriately fixed brain lesions were available from three of the six chronic multiple sclerosis cases with no or only small numbers of O4-positive, GalC-negative cells in their spinal cord lesions (multiple sclerosis cases 8, 11 and 13). Immunolabellings demonstrated that these brain lesions retained numerous O4-positive, GalC-negative cells (8.3 ± 1.3, 11.7 ± 2.0 and 25.6 ± 4.4 cells/mm2) (Table 3). These cells were also abundant in demyelinated brain lesions analysed from some of the other multiple sclerosis cases (n = 7; range 10.5 ± 2.0 to 38.4 ± 3.8 cells/mm2; Table 3). GalC-positive cells with an immature morphology were present in only very small numbers in the brain lesions (Table 3), as shown previously (Wolswijk, 2000Go). The highest density was observed in a brain lesion from multiple sclerosis case 14 (4.3 ± 1.1 cells/mm2), which also contained the highest density of O4-positive, GalC-negative cells (38.4 ± 3.8 cells/mm2) and phase-bright macrophages (188.1 ± 9.3 cells/mm2) (Table 3).

Factors influencing the density of the oligodendrocyte precursor population in multiple sclerosis lesions
The combined results from the spinal cord and brain lesions (41 lesions in total) analysed in the present study and previously [Wolswijk, 1998Gob; see Table 1 for details of the multiple sclerosis subjects whose brain lesions (n = 15) were studied previously] further supported the indications that the density of the O4-positive, GalC-negative cells in the lesion areas gradually decreased with increasing age of the multiple sclerosis subject (P < 0.0001) (Fig. ), length of the disease process (P = 0.006) and with declining numbers of macrophages (P < 0.0001.) Moreover, the density of the macrophage populations in the lesions also decreased with age of the multiple sclerosis subject at death (P = 0.040) (see Fig. ). Furthermore, the density of the O4-positive, GalC-negative cell population (and macrophage population) in lesions (n = 5) derived from subjects with the PP form of multiple sclerosis (n = 4) was not significantly different from that in lesions (n = 16) derived from subjects with the SP form of multiple sclerosis (n = 12) [12.0 ± 7.6 versus 16.5 ± 8.8 O4-positive, GalC- negative cells/mm2 (and 28.8 ± 62.3 versus 47.9 ± 69.6 macrophages/mm2)].


   Discussion
Top
 Summary
 Introduction
 Material and methods
 Results
 Discussion
References
 
The present study establishes for the first time that O4-positive, GalC-negative oligodendrocyte precursors are abundant in demyelinated spinal cord lesions from most subjects with longstanding multiple sclerosis (n = 16), including those with the PP and SP form, with densities of up to 35 cells/mm2. The present findings thus extend those of previous histopathological studies demonstrating the presence of a sizeable population of oligodendrocyte precursor cells in demyelinated brain lesions from subjects with chronic multiple sclerosis, identified using either the O4/anti-GalC antibody combination (Wolswijk, 1998Gob; 14 multiple sclerosis cases; range of densities 2–34 cells/mm2), antibodies to the NG2 chondroitin sulphate proteoglycan [Chang et al., 2000Go; five multiple sclerosis cases; densities were only reported for three inactive lesions from a single chronic multiple sclerosis case (40–80 NG2-positive cells/mm2 in 30 µm thick sections, which corresponds to a density of 13–27 cells/mm2 cells in 10 µm thick sections)] or antibodies to the PDGF-{alpha} receptor (Scolding et al., 1998Go; five multiple sclerosis cases; range of densities 1–3 PDGF-{alpha} receptor-positive cells/100 nuclei). A recent study has reported, however, that brain lesions often contain much higher numbers of PDGF-{alpha} receptor-positive cells and that many of these cells are not oligodendrocyte precursor cells, but either oligodendrocytes or astrocytes (Maeda et al., 2001Go).

Analysis of the density of O4-positive, GalC-negative oligodendrocyte precursor cells in demyelinated spinal cord (n = 17) and brain lesions (n = 24) from 26 subjects with chronic multiple sclerosis suggests that their density decreased significantly with increasing age of the multiple sclerosis subject, duration of clinical symptoms and increasing age of the lesion, as judged by the presence and number of macrophages filled with myelin degradation products. The factor most likely to influence the density of the oligodendrocyte precursor population in the lesions is probably the age of the lesion, because, as would be expected, lesions with only small numbers of macrophages were derived most commonly from the older subjects with a long duration of clinical symptoms. If this is indeed the case, it suggests that O4-positive, GalC-negative precursor cells slowly disappear from demyelinated areas with lesion progression, possibly due to diminishing amounts of appropriate survival factors. Moreover, the presence of only small numbers of oligodendrocyte precursor cells in relatively old lesions suggests that migration of precursor cells from unaffected spinal cord regions into lesion areas is limited, in contrast to what is observed in some models of CNS demyelination (Franklin et al., 1997Go; Keirstead et al., 1998Go). Repeated damage may also play an important role in the depletion of the oligodendrocyte precursor pool, as suggested by experimental studies (Keirstead et al., 1998Go). The finding that significant numbers of immature oligodendrocytes were only present in lesions with high precursor densities suggests that the ability of precursor cells to differentiate becomes increasingly more impaired with lesion evolution. Although some studies have provided evidence that lesions in the CNS of patients with PP multiple sclerosis differ in some aspects from those present in the CNS of patients with the SP form (e.g. Revesz et al., 1994Go; Lycklama à Nijeholt et al., 2001Go), no significant difference in oligodendrocyte precursor densities was found between lesions from these patient groups.

Demyelinated lesions with no or only few O4-positive, GalC-negative oligodendrocyte precursor cells (<=3.0 cells/mm2) were more common in the spinal cord than in the brain of subjects with longstanding multiple sclerosis analysed thus far [six out of 17 spinal cord lesions (35%) versus one out of 24 brain lesions (4%) studied; Wolswijk, 1998Gob; present study]. This difference appeared not to be patient-specific, as immunolabellings of brain lesions from three of the six multiple sclerosis subjects with low precursor densities that were available for study did contain numerous O4-positive, GalC-negative cells (8–26 cells/mm2). Since a higher proportion of the brain lesions contained >=10 phase-bright macrophages/mm2 than the spinal cord lesions [54% (13 out of 24) versus 29% (five out of 17)], it suggests that the spinal cord lesions analysed were on average older than the brain lesions studied, and, because of this, frequently harboured only small numbers of O4-positive, GalC-negative precursor cells.

As reported previously (Wolswijk, 1998Gob), the O4/anti-GalC antibody combination was not useful for the detection of O4-positive, GalC-negative oligodendrocyte precursor cells in areas with large numbers of O4-positive, GalC-positive oligodendrocytes and myelin sheaths, and it was thus not possible to assess the density of the precursor population in control and unaffected multiple sclerosis spinal cord tissue. It was thus also not possible to determine whether the density of the oligodendrocyte precursor population decreases significantly with age. However, studies in the rat have indicated that up to 8% of cells in the adult rat CNS are oligodendrocyte precursor cells (Dawson et al., 2000Go; Levine et al., 2001Go). If this is also true for the human CNS, it suggests that up to 37 ± 4 cells/mm2 in the adult human spinal cord are oligodendrocyte precursor cells [spinal cord white matter from three subjects without neurological disease (54 ± 21 years of age) contained 463 ± 50 nuclei/mm2 (10 µm thick sections)]. This estimate corresponds very well with that reported for the intact adult rat spinal cord (McTigue et al., 2001Go). Furthermore, Chang et al. (2000Go) found that between 140 and 150 cells/mm2 were NG2-positive in the white matter surrounding three inactive brain lesions derived from a single chronic multiple sclerosis case (30 µm thick sections; this corresponds to a density of 47–50 cells/mm2 in a 10 µm section). These figures are very similar to the highest density for O4-positive, GalC-negative oligodendrocyte precursor cells found in both brain (38 cells/mm2) and spinal cord lesions (35 cells/mm2), suggesting that death of precursor cells during the actual myelin destruction phase in many multiple sclerosis cases may be limited. Instead, the data suggest that the size of the oligodendrocyte precursor population gradually decreases with advancing age of the lesion. However, there are clearly some exceptions. For example, one of the two spinal cord lesions with the highest density of precursor cells completely lacked macrophages (Table 3), while two distinct regions of a brain lesion studied previously (Wolswijk, 1998Gob) harboured comparable numbers of oligodendrocyte precursor cells, but one area was devoid of macrophages, while the other contained numerous macrophages laden with myelin degradation products (Wolswijk, 1998Gob). These findings thus suggest that the number of oligodendrocyte precursor cells in some lesions may remain high for prolonged periods of time.

Complete destruction or severe depletion of the oligodendrocyte precursor population may occur in some cases of multiple sclerosis. This indication has come from the study of Chang et al. (2000Go) who found that two actively demyelinating brain lesions derived from two multiple sclerosis subjects with short clinical duration (<1 year) completely lacked NG2-positive cells. Oligodendrocyte precursor cells may die as a result of non-specific mechanisms or of a specific immunological response to a molecule expressed on the surface of oligodendrocyte precursor cells or to a surface molecule that these cells share with oligodendrocytes and myelin [e.g. the antigen(s) recognized by the O4 antibody]. In this respect, it is interesting to note that Niehaus and colleagues found that patients with active RR multiple sclerosis synthesize antibodies recognizing a protein expressed on the surface of rat oligodendrocyte precursor cells (Niehaus et al., 2000Go), a molecule which appears to be homologous to NG2 (Diers-Fenger et al., 2001Go). Thus, it is possible that there are distinct multiple sclerosis subtypes with respect to oligodendrocyte precursor survival and destruction, as appears to be the case for patterns of oligodendrocyte pathology (Lucchinetti et al., 2000Go). To gain further insights into this issue, it will be necessary to analyse in detail actively demyelinating lesions from both acute and chronic multiple sclerosis cases, but this material unfortunately is rare.

Oligodendrocyte precursor cells in multiple sclerosis lesions and control human CNS tissue have been identified using different markers. NG2-positive cells in the control adult human (and rodent) CNS display a highly complex morphology that is distinct from that of ramified microglia, astrocytes and myelinating oligodendrocytes (Levine et al., 1993Go; Nishiyama et al., 1996Go; Reynolds and Hardy, 1997Go; Oumesmar et al., 1997Go; Chang et al., 2000Go; Dawson et al., 2000Go). The results obtained with antibodies to the PDGF-{alpha} receptor are more confusing. Chang et al. (2000Go) reported that PDGF-{alpha} receptor-expressing cells in the human CNS express a morphology that is very similar to that of NG2-positive cells, as do PDGF-{alpha} receptor-positive cells in the marmoset and rhesus monkey CNS (G. Wolswijk, B. ’t Hart and H. Brok, unpublished observations). In contrast, Scolding et al. (1998Go) found that such cells were either round or bipolar, while Maeda et al. (2001Go) found that antibodies to the PDGF-{alpha} receptor labelled the cell body of mature, 2',3'-CNPase (cyclic nucleotide phosphohydrolase)-expressing oligodendrocytes in control human brain white matter. O4-positive, GalC-negative cells in multiple sclerosis lesions are process bearing and resemble morphologically those expressing NG2 [compare images provided in Wolswijk (1998Gob); Chang et al. (2000Go) and in the present study; see also Dawson et al. (2000Go)], although some NG2-positive cells in some lesions appear to express a more elongated morphology (Chang et al., 2000Go). PDGF-{alpha} receptor-positive cells in multiple sclerosis lesions have been reported to express a rounded or bipolar morphology, with none expressing the oligodendrocyte markers GalC or Rip and astrocyte marker GFAP (Scolding et al., 1998Go), while another study presented data indicating that cells expressing the PDGF-{alpha} receptor in lesion areas frequently express CNPase or GFAP (Maeda et al., 2001Go), suggesting that they are either oligodendrocytes or astrocytes, respectively. Clearly, further studies are needed to clarify the identity of the various antigenically identified populations and to determine whether there are antigenically and morphologically distinct subsets of oligodendrocyte precursor cells. That some overlap may exist is suggested by the observations that NG2-positive cells in the cerebral cortex of the adult rat bind the O4 antibody (Reynolds and Hardy, 1997Go), that NG2-positive cells in the developing rat CNS express the PDGF-{alpha} receptor (Nishiyama et al., 1996Go) and that oligodendrocyte precursor cells freshly isolated from adult rat optic nerves and spinal cords bind the O4 antibody and divide in vitro in response to PDGF (Wolswijk et al., 1991Go; Wolswijk and Noble, 1992Go; Engel and Wolswijk, 1996Go; Shi et al., 1998Go), suggesting that the O4-positive cells have receptors for PDGF. Indeed, in situ hybridization experiments have shown that cultured O4-positive cells from the adult human CNS contain transcripts for the PDGF-{alpha} receptor (Gogate et al., 1994Go).


   Acknowledgements
 
Human brain tissue was obtained from the Netherlands Brain Bank in Amsterdam (Co-ordinator, R. Ravid). I wish to thank the team of the Netherlands Brain Bank (L. Bergers, C. Beugel, D. van Beurden, A. de Boer, J. Bot, H. Daniëls, L. Dubelaar, B. Fisser, M. Fodor, A. Goessen, S. Guldenaar, A. Holtrop, J. Jonges, M. Kahlmann, W. Kamphorst, E. Koopman, M. Kooreman, M. Langeveld, S. van Liempt, J. Meinardi, E. de Nijs, S. Pindak, R. Ravid, R. Riemersma, R. Roelofs, A. Salehi, D. Swaab, U. Unmehopa, P. van der Valk, M. Vermaak, R. Vos, H. Vrenken, R. de Vries, H. Winters and J. Wouda) for collecting the multiple sclerosis and control tissue, for analysing the patients’ medical records and for advice. I also wish to thank W. Kamphorst for helpful discussions, C.-H. Heldin, J. Levine, H. van Noort and S. Piddlesden for their gift of antibodies, T. Put for his assistance with the preparation of Figs and 2, and W. Kamphorst and R. Balesar for their comments on the manuscript. Financial support for this study came from the Netherlands Foundation ‘Friends of MS Research’.


   References
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 Summary
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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Received June 19, 2001. Revised September 24, 2001. Accepted October 4, 2001.


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