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The Journal of Neuroscience, April 1, 1998, 18(7):2498-2505
Acceleration in the Rate of CNS Remyelination in
Lysolecithin-Induced Demyelination
Kevin D.
Pavelko1,
Baziel G. M.
van Engelen1, 2, and
Moses
Rodriguez1
1 Departments of Neurology and Immunology, Mayo Clinic
and Mayo Foundation, Rochester, Minnesota 55905, and
2 Institute of Neurology, University Hospital Nijmegen,
6500 HB Nijmegen, The Netherlands
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ABSTRACT |
One important therapeutic goal during CNS injury from trauma or
demyelinating diseases such as multiple sclerosis is to develop methods
to promote remyelination. We tested the hypothesis that spontaneous
remyelination in the toxic nonimmune model of lysolecithin-induced demyelination can be enhanced by manipulating the inflammatory response. In PBS-treated SJL/J mice, the number of remyelinating axons
per square millimeter of lesion area increased significantly 3 and 5 weeks after lysolecithin injection in the spinal cord. However,
methylprednisolone or a monoclonal antibody (mAb), SCH94.03, developed
for its ability to promote remyelination in the Theiler's virus murine
model of demyelination, further increased the number of remyelinating
axons per lesion area at 3 weeks by a factor of 2.6 and 1.9, respectively, but did not increase the ratio of myelin sheath thickness
to axon diameter or the number of cells incorporating tritiated
thymidine in the lesion. After 3 weeks, the number of remyelinating
axons in the methylprednisolone or mAb SCH94.03 treatment groups was
similar to the spontaneous remyelination in the 5 week PBS
control-treated group, indicating that these treatments promoted
remyelination by increasing its rate rather than its extent. To address
a mechanism for promoting remyelination, through an effect on scavenger
function, we assessed morphometrically the number of macrophages in
lesions after methylprednisolone and mAb SCH94.03 treatment.
Methylprednisolone reduced the number of macrophages, but SCH94.03 did
not, although both enhanced remyelination. This study supports the
hypothesis that even in toxic nonprimary immune demyelination,
manipulating the inflammatory response is a benefit in myelin
repair.
Key words:
lysolecithin; CNS; injury; demyelination; remyelination; immunoglobulin; corticosteroids; autoantibodies
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INTRODUCTION |
Three general categories of
experimental CNS demyelination can be distinguished: (1) toxin-induced
[cuprizone (Blakemore, 1973 ), ethidium bromide (Yajima and Suzuki,
1979), and lysolecithin (Hall, 1972)]; (2) autoimmune-induced
[experimental autoimmune encephalomyelitis (EAE) (Raine and Traugott,
1985)]; and (3) virus-induced [corona virus (Herndon, 1977) and
Theiler's murine encephalomyelitis virus (TMEV) (Rodriguez and Lennon,
1990)]. Spontaneous CNS remyelination has been observed in each of
these models of demyelination in rodents. Some CNS lesions of multiple
sclerosis (MS) in humans are remyelinated by oligodendrocytes (Prineas
and Connell, 1979; Rodriguez and Sheithauer, 1994) or Schwann cells
(Ghatak et al., 1973), although most lesions examined at autopsy show
extensive demyelination and gliosis without myelin repair. In addition, demyelination and remyelination have been observed after acute spinal
cord compression (Gledhill et al., 1973). These observations suggest
that there is the potential for complete remyelination after
demyelination in spinal cord injury or multiple sclerosis, but there
are factors that prevent the full expression.
Two hypotheses have been proposed to explain the absence of full
remyelination in the human CNS (Rodriguez and Lindsley, 1992). The
absence of significant spontaneous remyelination may be attributable to
either the presence of inhibitory elements preventing myelin repair or
the absence of cells or factors necessary for new myelin synthesis. In
the experimental model of demyelination induced by TMEV, a natural
enteric picornavirus in mice, CNS remyelination can be promoted either
by immunosuppression (Rodriguez and Lindsley, 1992) or by
immunostimulation by passive transfer of Igs such as CNS-specific
antiserum (Rodriguez et al., 1987), purified Ig (Rodriguez and Lennon,
1990), polyclonal mouse IgG (van Engelen et al., 1995), or a monoclonal
autoantibody (mAb) designated SCH94.03 (Miller et al., 1994). The
purpose of these experiments was to determine in a toxic nonimmune
model of demyelination whether corticosteroids suppress inhibitory
elements for myelin repair, which would allow for increasing the rate
of spontaneous remyelination, and whether stimulation of the humoral
immune response by administration of polyclonal or monoclonal
antibodies provides factors for new myelin synthesis.
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MATERIALS AND METHODS |
Mice. Forty-six 12-week-old SJL/J
(H-2s) mice weighing 20-25 gm were obtained from
The Jackson Laboratory (Bar Harbor, ME). Mice were housed in plastic
cages, and food and water were provided ad libitum. Handling
of all animals conformed to the National Institute of Health and Mayo
Clinic institutional guidelines.
Surgery. Mice were anesthetized by intraperitoneal injection
of sodium pentabarbitol (0.08 mg/gm). Dorsal laminectomies were performed on the upper thoracic region of the spinal cord. A 34 gauge
needle attached to a Hamilton syringe mounted on a stereotactic micromanipulator was used to inject 1 µl of a 1% solution of
lysolecithin (L- -lysophosphatidylcholine) (Sigma, St.
Louis, MO) in sterile PBS, pH 7.4, with Evan's blue added as a marker.
The needle was inserted into the anterior or lateral part of the spinal
cord, lysolecithin solution was injected, and then the needle was
slowly withdrawn. The wound was sutured in two layers, and mice were allowed to recover. The day of lysolecithin injection was designated day 0.
Treatments. Mice were assigned randomly to groups (four to
nine animals per group) to receive the following treatments and were
killed on days 14 (n = 6), 21 (n = 34),
and 35 (n = 6) after lysolecithin injection. All mice
were 12 weeks of age to exclude the potential bias of age on
remyelination after demyelination (Gilson and Blakemore, 1993).
Methylprednisolone. Mice were treated with pulse doses of
methylprednisolone (Depo-Medrol, 80 mg/ml; Upjohn, Kalamazoo, MI) given
by intraperitoneal injections of 1 mg (~45 mg/kg) on days 0, 3, 7, 10, 14, and 17 to determine whether steroids would enhance remyelination. This approach was used to test whether inhibition of the
inflammatory response would enhance myelin repair. This approach also
simulated treatments used in spinal cord injury (Bracken et al.,
1990).
Polyclonal IgG. On days 7, 10, 14, and 17, mice were
injected intraperitoneally twice daily with 0.5 mg of polyclonal IgG obtained from multiple mouse donors (1 mg/ml in PBS from Sigma lot
033H8860). This approach was identical to treatments used in other
murine models in which IgG had been shown to promote remyelination (van
Engelen et al., 1995). This approach also simulated the use of
intravenous Ig (IvIg), which has been shown to be beneficial in a
subset of patients with multiple sclerosis (Fazekas et al., 1997).
Anti-SCH Ig.Polyclonal Ig directed against spinal cord
homogenate (anti-SCH Ig) was shown to enhance remyelination in the TMEV
model of demyelination (Rodriguez et al. 1987, 1990). This was injected
intraperitoneally (0.5 ml of 1.1 mg/ml solution) on days 7, 10, 14, and
17.
mAb SCH94.03 IgM- . A monoclonal antibody developed in our
laboratory for its ability to promote remyelination in the TMEV model
(Miller and Rodriguez, 1995) was injected intraperitoneally (0.1 mg) on
days 7, 10, 14, and 17.
PBS. Control mice were given intraperitoneal injections of
0.5 ml of PBS on days 7, 10, 14, and 17. Three groups of mice were killed on days 14, 21, and 35 after lysolecithin injection to address
the normal temporal profile of spontaneous remyelination in the
lysolecithin model.
Histopathology. On days 14, 21, and 35, mice were killed for
pathological analysis. After anesthesia with sodium pentobarbital, mice
were perfused with Trump's fixative (phosphate-buffered 4% formaldehyde containing 1% glutaraldehyde, pH 7.4). Spinal columns were removed and allowed to post-fix for 1-3 d until spinal cords were
removed. Six to eight 1 mm coronal blocks were cut from the site marked
by the Evan's blue marker. This assured that the entire lesion area
was examined. Serial blocks were kept in 24-well plates and washed with
0.1 M phosphate buffer. Blocks were secondarily fixed with
osmium tetroxide and dehydrated through a graded alcohol series, washed
in propylene oxide and embedded in Araldite (Polysciences, Warrington,
PA). One-micrometer sections were cut and stained with 4%
paraphenylenediamine. Selected areas were trimmed and prepared for
electron microscopy. Ultrathin sections were placed on celloidin-coated
200-mesh grids, stained with 4% uranyl acetate in 50% methanol and
counterstained with lead citrate. The grids were examined with a JEOL
1200 electron microscope.
In vivo analysis of [3H]thymidine
incorporation. To detect proliferating cells, 40 animals were
injected intraperitoneally with 100 µCi of
[3H]thymidine (Amersham, Arlington Heights, IL)
48, 36, 24, and 12 hr before killing on days 14 (n = 5), 21 (n = 29), and 35 (n = 6).
One-micrometer sections of spinal cord from mice previously injected
intraperitoneally with [3H]thymidine were dipped
in autoradiography emulsion (NTB2; Eastman Kodak, Rochester, NY),
sealed in black boxes, and exposed for 3 weeks at 4°C. Slides were
developed in Kodak D19 developer, rinsed in a distilled water stop
bath, and fixed with Kodak fixer. Only lesion areas that were at least
0.02 mm2 in total area were studied morphometrically
to exclude the possibility that very small lesions would undergo more
rapid and extensive repair. Based on this assessment only five lesion
areas from 40 mice with lysolecithin-induced lesions were excluded from
the analysis.
Analysis of light and electron microscopic spinal cord section
images. A Zeiss (Oberkochen, Germany) interactive digital analysis system attached to a Zeiss photomicroscope was used to measure the area
(square millimeters) of each lysolecithin lesion from every block. The
lesion was defined as the area that included axons that were completely
and partially demyelinated as well as completely and partially
remyelinated (Fig. 1). The lesion areas
for each of the 35 mice killed at days 14, 21, and 35 were summed to
provide the total lesion area per mouse. This system was also used to
count the number of remyelinated axons in each lesion. Abnormally thin
myelin sheaths, relative to axonal diameter, were the criterion for
oligodendrocyte remyelination. Schwann cell remyelination was
characterized by thicker myelin sheaths, a one cell per axon
relationship, and a surrounding basement membrane. A cursor was used to
mark each remyelinated axon (oligodendrocyte or Schwann cell) with a
cross, and these were summed to determine the total number of
remyelinated axons per total lesion area.
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Figure 1.
Demonstration of the methodology for determining
lesion area measurements. A, Lysolecithin lesion from a
PBS-treated control mouse 3 weeks after lysolecithin injection.
B, Area in blue shows the normal white
matter surrounding lesion that was used to discriminate lesion.
C, Area in red demonstrates the area used
in calculating the total lesion area.
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Lysolecithin lesion areas were also viewed on a JEOL 1200 electron
microscope. Areas of demyelination and oligodendrocyte-type remyelination were photographed. Photo negatives were incorporated into
digital image files. These images were analyzed by the Zeiss digital
analysis system to determine the ratio of myelin thickness to axon
diameter.
In vivo analysis of macrophage infiltration. A Zeiss
interactive image analysis system attached to a Zeiss photomicroscope was used to measure areas of phagocytic macrophage infiltrates in 41 lysolecithin lesion areas from mice killed at days 14, 21, and 35 (Fig.
2). Macrophages were readily identified
in the lesions as cells with extensive myelin debris-containing
vacuoles. Because in certain areas macrophage infiltrates were very
dense and individual macrophages could not be distinguished, cumulative
areas of macrophage infiltrate were used to calculate the number of
macrophages per area of lesion. The total area of infiltration was
divided by the mean area of one macrophage (0.00127 mm2) to yield total macrophages. The data were
expressed as number of macrophages per square millimeter of lesion
area.
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Figure 2.
Demonstration of the methodology for macrophage
enumeration. A, Region of macrophage infiltration that
does not allow for distinction of individual cells. B,
Region of macrophages from A discriminated to calculate
total macrophage area. C, Region where individual
macrophages can be distinguished. D, Region
discriminated from C used to calculate individual size
of macrophages. Note examples of axons remyelinated by Schwann cells
(arrows).
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Statistical analysis. Analyses were performed in a blinded
manner on coded slides. Pathological abnormalities were scored without
knowledge of treatment groups. Differences between treatment groups
were analyzed using Kruskal-Wallis one-way ANOVA, and pairwise multiple comparisons were done using Dunn's method
(p < 0.05). Data that were normally distributed
were analyzed by one-way ANOVA, and pairwise multiple comparisons were
done by Dunnett's method (p < 0.05).
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RESULTS |
Demyelination and remyelination in PBS control-treated
SJL/J mice
Light and electron microscopy in the PBS-treated animals revealed
focal areas of demyelination, invading macrophages laden with myelin
debris, and thinly myelinated axons at 14, 21, and 35 days after
lysolecithin injection (Fig.
3A). No differences in lesion
areas were found between the PBS-treated groups; the mean area ± SEM of lesion for these groups was 0.0709 ± 0.015 mm2
(n = 17). Few if any lymphocytes were identified in
lesions, as would be expected from toxin-induced demyelination. In
previous pilot experiments we found that spontaneous oligodendrocyte
remyelination, as detected by abnormally thin myelin sheaths in
relation to axon diameter, started ~1 week after lysolecithin
injection, as has been reported in the mouse (Jeffery and Blakemore,
1995) and rat (Blakemore, 1976) previously. Most animals also had some
evidence of PNS-type remyelination, characterized by the one-to-one
relationship between CNS axons and Schwann cells, and a basement
membrane surrounding newly myelinated axons. Five weeks after
lysolecithin injection the total number of remyelinated axons per
square millimeter of lesion area increased significantly compared with
the 2 and 3 week groups (Table 1). The
number of remyelinated axons per square millimeter increased 2.6-fold
from 2 to 3 weeks and 6.2-fold from 2 to 5 weeks (Table 1). Myelin
sheath thickness in axons remyelinated by oligodendrocytes also
increased spontaneously from 2 to 5 weeks (Table 1, Fig.
4) but never reached the thickness
observed in normal axons.
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Figure 3.
Spinal cord sections from SJL/J mice injected with
lysolecithin and treated with A, PBS control;
B, methylprednisolone; C, mAb 94.03; or
D, polyclonal Ig directed against spinal cord homogenate (SCH/Ig). Treatment was begun on day 0 (methylprednisolone) or day 7 (PBS control, mAb 94.03 or SCH/Ig) and continued until day 21 at time
of kill. Note focal area of demyelination with dense macrophage
infiltration with minimal remyelination in the mouse treated with PBS
control (A). In contrast, there is
oligodendrocyte remyelination, characterized by abnormally thin myelin
sheath relative to axon diameter, and Schwann cell remyelination in
mice treated with methylprednisolone (B), mAb
SCH94.03 (C) or SCH/Ig (D). The number of remyelinated axons from
sections such as these are shown in Tables 1 and 2 (× 750).
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Figure 4.
Relative myelin sheath thickness as a function of
axon diameter in axons remyelinated by oligodendrocytes at 2, 3 or 5 weeks in PBS-treated SJL/J mice injected with lysolecithin.
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Demyelination and remyelination in Ig- or
methylprednisolone-treated animals
At 3 weeks after lysolecithin injection, the total lysolecithin
lesion area was 0.098 ± 0.01 mm2 (mean ± SEM), based on the analysis of the 23 mice shown in Table 2. There was no statistical difference in
the lesion areas when comparing different treatment groups. In
contrast, there was a continuous increase in the number of
remyelinating axons per square millimeter of lesion area from the
lowest in the PBS control group, increasing in the polyclonal IgG,
anti-SCH Ig, and mAb SCH94.03 to the highest in the methylprednisone
group (Table 2).
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Table 2.
Effects of corticosteroids and Ig treatment on
remyelination 3 weeks after lysolecithin-induced demyelination in the
spinal cord
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Polyclonal IgG and anti-SCH Ig did not statistically improve
remyelination, whereas mAb SCH94.03 and methylprednisolone
treatment statistically increased the number of remyelinated axons per
square millimeter of lesion area at the 3 week time point by a factor of 1.9 and 2.6, respectively (Table 2, Fig. 3). After 3 weeks the total
number of remyelinated axons per square millimeter in the mAb SCH94.03
or methylprednisolone treatment groups (Table 2) was comparable with
the spontaneous remyelination observed in the 5 week PBS control group
(Table 1). The various treatments did not increase the ratio of myelin
sheath thickness to axon diameter in fibers undergoing
oligodendrocyte-type remyelination (Table 2).
In vivo analysis of
[3H]thymidine incorporation
Although the number of cells incorporating
[3H]thymidine decreased from 2 to 5 weeks after
lysolecithin injection, no statistical differences were observed in the
PBS control groups (Fig. 5). Furthermore,
the number of cells incorporating [3H]thymidine in
the various treatments did not differ statistically from the 3 week PBS
control group (Fig. 5). The greatest number of proliferating cells was
observed in mice receiving poly-IgG, but this was not statistically
significant (Fig. 5). Although corticosteroids are potent
antiproliferative agents for macrophages and other inflammatory cells,
cells incorporating [3H]thymidine (presumed glial
cells) were observed in mice receiving this treatment. Some
[3H]thymidine-positive cells were identified as
inflammatory (i.e., surrounding blood vessels or infiltrating the
spinal cord parenchyma from the meninges). A few proliferating Schwann
cells were observed near the root entry zone associated with PNS-type
remyelination. Many of the [3H]thymidine-positive
noninflammatory cells were presumptive glia near areas of
oligodendrocyte remyelination. As described previously (Rodriguez and
Lindsley, 1992), these cells had large nuclei without a definite
nucleolus, a non-electron-dense cytoplasm, and no distinct glial
fibrils and were usually at a distance from dense inflammatory infiltrates.
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Figure 5.
Number of proliferating cells per
mm2 of lesion in PBS-treated groups and treatment
groups at 3 weeks after lysolecithin injection (data expressed as
proliferating cells per square millimeter of lesion + SEM).
n, Number of mice studied.
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In vivo analysis of macrophage infiltration
One potential mechanism for enhancing remyelination is by
effecting the recruitment or function of scavenger macrophages in the
lesion. Removal of myelin debris from the lesion could then allow
progenitor glia to proliferate and differentiate to initiate myelination. We first assessed the number of macrophages in
lysolecithin lesions induced at 2, 3, and 5 weeks before killing. The
number of macrophages/mm2 of lesion for PBS-treated
mice injected with lysolecithin was 731 ± 79 (n = 6) at 2 weeks (mean ± SEM), 1044 ± 185 at 3 weeks, and
643 ± 69 (n = 6) at 5 weeks. Although the number
of macrophages appeared to peak at 3 weeks, there was no statistical
difference in the data. To test the hypothesis that a change in the
macrophage infiltrate would correlate with enhanced remyelination, we
analyzed the number of macrophages per square millimeter in treatment
groups at the 3 week time point. There was a statistically significant difference between the PBS-treated group and the methylprednisolone group by Dunnett's multiple comparison procedure (Fig.
6). Of interest, however, no decrease in
the number of macrophages per lesion area was observed in the
94.03-treated group, which statistically enhanced remyelination, or in
the anti-SCH Ig and poly-IgG groups, which did not significantly
enhance remyelination. This result indicates that the mechanism of
enhanced remyelination between methylprednisolone and 94.03 may be
fundamentally different.
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Figure 6.
Number of macrophages per lesion area in treatment
groups (data expressed as macrophages per square millimeter of lesion + SEM). n, Number of mice studied. Data marked by * denote
statistical significance by Dunnett's multiple comparison procedure
with the PBS-treated group serving as the control.
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DISCUSSION |
This study demonstrated that spontaneous remyelination in a
toxic-traumatic model of demyelination is a normal physiological response that can be promoted either by treatment with corticosteroids or by immunostimulation using mAb SCH94.03. These findings are comparable with experiments published previously in an immune-mediated virus model of demyelination, in which immunosuppression (Rodriguez and
Lindsley, 1992) or mAb SCH94.03 (Miller et al., 1994) was able to
promote CNS-type remyelination. Although there may be differences in
the role of the inflammatory response in the genesis of
toxic-traumatic versus virus-induced demyelination, the present study showed that a beneficial therapeutic effect by agents that potentially influence inflammation is not restricted to immune-mediated demyelination.
Theoretically a beneficial therapeutic effect could be caused either by
decreasing demyelination or by increasing remyelination. Corticosteroid
treatment could have been beneficial by inhibiting directly
lysolecithin-induced demyelination, as reported by Triarhou and Herndon
(1986). Lysolecithin injection causes primary myelin breakdown by
increasing phospholipase A2 activity contained in activated
macrophages (Trotter and Smith, 1986), which degrades membrane
phosphatidylcholine into lysolecithin (Weltzien, 1979). Because
corticosteroids inhibit phospholipase A2 (Crompton et al.,
1988), they could counteract directly the demyelinating effect of
lysolecithin on phospholipase A2. However, we found that
corticosteroid administered within 0.5-1 hr after lysolecithin
injection did not significantly decrease the lesion area. This is in
contrast with the report of Triarhou and Herndon (1986) on
dexamethasone acetate administration in rats, but the latter study did
not specify the interval between lysolecithin injection and
corticosteroid administration. In patients with acute spinal cord
injury, treatment with methylprednisolone (as a bolus of 30 mg/kg
followed by 5.4 mg · kg 1 · hr1 for 23 hr) improves neurological recovery when the medication is given within
the first 8 hr (Bracken et al., 1990), possibly by preventing the
breakdown of membranes (Braughler and Hall, 1983). In our lysolecithin
experiments, methylprednisolone was administered for 17 d instead
of 23 hr as in the human trial (Bracken et al., 1990). Our results
raise the possibility that the therapeutic effect of methylprednisolone
in toxic or traumatic spinal cord injury may in part be the result of
enhanced remyelination.
Two potential mechanisms exist by which treatment may promote
spontaneous remyelination (Miller et al., 1994). First, some pathogenic
component preventing full remyelination may be inhibited. If the
outcome of the demyelinating process is based on a balance between
tissue destruction and repair, inhibiting those components that prevent
healing would allow a physiological repair response to predominate.
Thus, corticosteroids may have promoted spontaneous CNS remyelination
by their anti-inflammatory effects; i.e., perturbation of leukocyte
trafficking, lytic action on lymphocytes, and inhibition of
T-lymphocyte activation (Cupps and Fauci, 1982), or by interfering with
locally secreted cytokines and lymphokines of interest. In these
experiments the number of macrophages infiltrating the lesion at 3 weeks was significantly reduced in the methylprednisolone-treated group, which is consistent with the first hypothesis. Of interest, methylprednisolone has also been shown to inhibit inflammatory processes in experimental spinal cord lesions in the rat (Bartholdi and
Schwab, 1995). Corticosteroids also directly effect lymphokine production, especially interleukin 1 (Lew et al., 1988), and
interleukin-2 (IL-2) (Arya et al., 1984; Boumpas et al., 1991). IL-2
has been shown to stimulate (Benveniste and Merrill, 1986) or inhibit
(Knobler et al., 1988) differentiation of oligodendrocytes depending on the microenvironment. Therefore, corticosteroids may have promoted remyelination in vivo by inhibiting a pathogenic component
from the microenvironment, thereby allowing the normal physiological response to predominate. Of interest, SCH94.03 has also been shown in vivo to have local immunosuppressive properties because
treatment with the antibody successfully prevents further relapses of
experimental autoimmune encephalomyelitis (Miller et al., 1997) and
suppresses CNS inflammation after Theiler's virus-induced
demyelination (Miller et al., 1996). This effect appears to be
primarily a T-cell rather than macrophage phenomenon, which may explain
the dissociation of mechanism of action between methylprednisolone and
SCH94.03, as supported by the results (Fig. 6).
Second, treatment may have promoted remyelination by actively
stimulating myelination in vivo. Corticosteroids, either
exogenously administered or endogenously synthesized by glial cells
(Jung-Testas et al., 1989), have been shown to promote gene
transcription (Gronemeyer, 1992), in particular on rat glial cultures
(Kumar et al., 1989). Glucocorticoids are also co-mitogens for Schwann
cells (Neuberger et al., 1994). The mAb SCH94.03 may also have had a
direct stimulatory effect on oligodendrocytes (Asakura et al., 1996a).
mAb SCH94.03 labels a surface antigen on a subpopulation of mature
murine oligodendrocytes raising the possibility that it promotes
remyelination by binding directly to a surface receptor (Asakura et
al., 1996). The mAb SCH94.03 also localizes to the lesions after
in vivo administration and binds to glia, myelin,
macrophages, and some axons (Hunter et al., 1997). Other antibodies
that directly stimulate oligodendrocyte differentiation include the O4
mAb (Bansal et al., 1988) and an mAb against a reovirus receptor
expressed by oligodendrocytes (Cohen et al., 1991).
Therefore, methylprednisone and mAb SCH94.03 may have promoted
remyelination by stimulating directly remyelination by
oligodendrocytes.
At the inception of these experiments we predicted that those
therapeutic strategies that would have enhanced remyelination also
would have resulted in increased [H3]thymidine
uptake in the lesion. This was a logical conclusion based on elegant
previous studies demonstrating that proliferation of progenitor glial
cells is necessary before remyelination (Ludwin, 1979; Armstrong et
al., 1990; Prayoonwiwat and Rodriguez, 1993; Carrol and Jennings,
1994). However, no statistical differences were observed in the number
of proliferating cells in the lesions at 3 weeks after the various
strategies to promote remyelination. We cannot exclude the possibility
that a change in the number of proliferating cells between the various
groups would have been seen if examined before 3 weeks. Although we did
not do double-labeling experiments to identify the phenotype of these
cells, previous experiments in other models of demyelination indicate
that the majority of the proliferating cells are glial (Armstrong et
al., 1990; Prayoonwiwat and Rodriguez, 1993). The results of our
experiments indicate that the stimuli for inducing proliferation, a
likely prerequisite for remyelination, are independent of the treatment paradigms we used. The result suggests that once these cells have proliferated, the approach used here allows for more rapid and efficient myelin synthesis.
Treatment with mAb SCH94.03 increased statistically the number of
remyelinated axons per lysolecithin lesion area at 3 weeks, whereas
polyclonal Ig and anti-SCH Ig did not enhance remyelination using the
same dose and duration of treatment as in our TMEV studies (Rodriguez
and Lennon, 1990; van Engelen et al., 1995). These results are
intriguing and clearly indicate that with increasing specificity of the
Ig preparation (from polyclonal Ig, polyclonal anti-SCH Ig, to mAb
SCH94.03) remyelination also increased (Table 2). This raises the
possibility that remyelination-promoting antibodies may be present in
normal serum but at a low concentration. The mAb SCH94.03 has the
phenotype of naturally occurring or physiological autoantibodies, which
are found in the serum of normal humans and mice, are polyreactive with
a wide range of antigens (Asakura et al., 1996b), and are encoded by
germ line Ig light- and heavy-chain genes without definitive somatic
mutations (Miller et al., 1995). The enhancement of remyelination in
the lysolecithin model implies that certain natural autoantibodies may
participate in a beneficial physiological response to CNS injury, even
in nonimmune demyelination.
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FOOTNOTES |
Received Nov. 6, 1997; revised Jan. 13, 1998; accepted Jan. 20, 1998.
This work was supported by the Dutch Society for Support of Research on
Multiple Sclerosis (Grant 92-115m to B.v.E.) and by National Institutes
of Health Grant NS24180. We also appreciate the generous financial
support of the Myelin Project.
K.D.P. and B.G.M.v.E. contributed equally to this manuscript.
Correspondence should be addressed to Dr. Moses Rodriguez, Department
of Neurology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905.
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Abstract
Introduction
