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The Journal of Neuroscience, October 1, 2002, 22(19):8574-8585
Absence of Fibroblast Growth Factor 2 Promotes Oligodendroglial
Repopulation of Demyelinated White Matter
Regina C.
Armstrong1, 2,
Tuan Q.
Le1,
Emma E.
Frost1,
Rosemary C.
Borke1, 2, and
Adam C.
Vana1, 2
1 Department of Anatomy, Physiology, and Genetics and
2 Neuroscience Program at the Uniformed Services University
of the Health Sciences, Bethesda, Maryland 20814-4799
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ABSTRACT |
This study takes advantage of fibroblast growth factor 2 (FGF2)
knock-out mice to determine the contribution of FGF2 to the regeneration of oligodendrocytes in the adult CNS. The role of FGF2 during spontaneous remyelination was examined using two
complementary mouse models of experimental demyelination. The murine
hepatitis virus strain A59 (MHV-A59) model produces focal areas of
spinal cord demyelination with inflammation. The cuprizone
neurotoxicant model causes extensive corpus callosum demyelination
without a lymphocytic cell response. In both models, FGF2 expression is upregulated in areas of demyelination in wild-type mice. Surprisingly, in both models, oligodendrocyte repopulation of demyelinated white matter was significantly increased in FGF2  / mice
compared with wild-type mice and even surpassed the oligodendrocyte
density of nonlesioned mice. This dramatic result indicated that the
absence of FGF2 promoted oligodendrocyte regeneration, possibly by
enhancing oligodendrocyte progenitor proliferation and/or
differentiation. FGF2 / and +/+ mice had similar
oligodendrocyte progenitor densities in normal adult CNS, as well as
similar progenitor proliferation and accumulation during demyelination.
To directly analyze progenitor differentiation, glial cultures from
spinal cords of wild-type mice undergoing remyelination after MHV-A59
demyelination were treated for 3 d with either exogenous FGF2 or
an FGF2 neutralizing antibody. Elevating FGF2 favored progenitor
proliferation, whereas attenuating endogenous FGF2 activity promoted
the differentiation of progenitors into oligodendrocytes. These
in vitro results are consistent with enhanced progenitor
differentiation in FGF2 / mice. These studies
demonstrate that the FGF2 genotype regulates oligodendrocyte regeneration and that FGF2 appears to inhibit oligodendrocyte lineage differentiation during remyelination.
Key words:
FGF2; oligodendrocyte; glia; remyelination; cuprizone; demyelinating disease
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INTRODUCTION |
Oligodendrocytes form and maintain
the insulating myelin sheaths that facilitate conduction of nerve
impulses and protect axons. Myelin damage, as in multiple sclerosis
(MS) and other demyelinating diseases, renders affected axons useless
for communicating neural signals. Remyelination brings about limited
myelin repair of MS lesions over time (Prineas et al., 1993 ; Raine and
Wu, 1993). Interventions for demyelinating diseases are sought to
arrest disease progression and to promote remyelination, which may both restore nerve conduction and prevent permanent axonal damage. Growth
factors are candidates for intervention therapies as potential regulators of the generation, differentiation, and survival of new
oligodendrocytes that are required for remyelination.
In vitro studies of neonatal oligodendrocyte lineage cells
suggest a bipartite function for FGF2 in regulating oligodendrocyte lineage development. FGF2 promotes migration and proliferation of cells
during the early stages of the oligodendrocyte lineage (McKinnon et
al., 1990; Decker et al., 2000; Jiang et al., 2001). In contrast, at
more mature stages of the lineage, FGF2 inhibits terminal
differentiation and impairs myelination (Bansal and Pfeiffer, 1997;
Goddard et al., 2001). These differential effects of FGF2 at different
stages of the oligodendrocyte lineage may occur through differential
expression of FGF receptor (FGFR) isoforms (Bansal and Pfeiffer, 1997).
Indeed, oligodendrocyte progenitors and mature oligodendrocytes express
multiple FGFR isoforms in situ (Miyake and Itoh, 1996; Jiang
et al., 1999; Messersmith et al., 2000). However, these multiple
potential effects of FGF2 have not been demonstrated in vivo
during normal CNS development. Furthermore, it is not known whether the
effective roles of FGF2 during development correspond with functions of
FGF2 in the adult CNS and which potential actions of FGF2 are important
during remyelination.
Multiple lines of evidence indicate that FGF2 is likely to regulate
oligodendrocyte lineage responses in the adult CNS, particularly during
remyelination. Anti-FGFR antibodies and radiolabeled FGF2 ligand bind
to progenitors isolated from optic nerves of adult rats (Wolswijk and
Noble, 1992). Importantly, FGF2 is a potent mitogen for these adult
progenitors, especially in combination with platelet-derived growth
factor (PDGF; Wolswijk and Noble, 1992). Our previous analysis of
spinal cord remyelination after infection with murine hepatitis virus
(MHV-A59) demonstrated dramatic increases in the expression of FGF2
ligand and relevant FGFRs in lesioned white matter (Messersmith et al.,
2000). Therefore, we sought to determine the role of FGF2 in
remyelination using this viral model of demyelination. A neurotoxicant
model of demyelination was similarly analyzed to deduce general effects
of FGF2 on the oligodendrocyte lineage, rather than model-specific
effects. We show that wild-type and FGF2 null mice exhibited
similarity with respect to oligodendrocyte progenitor density in normal
CNS as well as proliferation and accumulation in response to
demyelination. An important finding is that mice lacking FGF2 exhibited
more vigorous regeneration of oligodendrocytes during the spontaneous remyelination phase in both models of experimental demyelination. Retroviral lineage analysis of glial cultures from remyelinating spinal
cord indicated that FGF2 inhibited differentiation of adult progenitors
into mature oligodendrocytes. Taken together, these experiments
demonstrate a significant inhibitory effect of FGF2 for progenitor
differentiation during remyelination.
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MATERIALS AND METHODS |
Animals. Mice were bred and maintained in a
pathogen-free barrier facility, and all procedures were performed in
accordance with guidelines of the National Institutes of Health, the
Uniformed Services University of the Health Sciences Institutional
Animal Care and Use Committee, and the Society for Neuroscience.
FGF2 knock-out mice and wild-type mice of the same 129 Sv-Ev:Black
Swiss genetic background were obtained from breeding heterozygous pairs
(generously provided by Dr. Doetschman, University of Cincinnati). This
FGF2 knock-out was generated by a targeted deletion replacing a 0.5 kb
portion of the FGF2 gene including 121 bp of the promoter and the entire first exon with an Hprt mini-gene (Zhou et
al., 1998). Mice were genotyped using PCR analysis of tail DNA to
identify wild-type FGF2 and the targeted allele, as
described in Zhou et al. (1998). The knock-out (/) mice do not have
detectable wild-type or truncated messages containing exon 2 and 3 sequences (Zhou et al., 1998). The absence of FGF2 transcripts in our
FGF2 mice with homozygous null (/) genotype was
confirmed using kinetic RT-PCR, as detailed in Messersmith et al.
(2000), with homozygous wild-type (+/+) mice and an FGF2 cDNA plasmid
as positive controls (Y. X. Zhou and R. C. Armstrong,
unpublished observation).
Because both the MHV-A59 and cuprizone models have been characterized
previously in the C57BL/6 strain, C57BL/6 mice were purchased from
Charles River (Wilmington, MA) for use as a comparison of genetic
background with the FGF2 +/+ and / mice.
Murine hepatitis virus experimental demyelination. As
previously described (Redwine and Armstrong, 1998), stocks of murine hepatitis virus strain A59 (MHV-A59) were prepared, and each mouse was
injected intracranially with 1000 plaque-forming units (pfu) in a 10 µl volume. MHV-A59 infection results in widespread focal demyelinating lesions throughout the spinal cord white matter over a
1-3 week period followed by extensive spontaneous remyelination within
8 weeks after injection (Armstrong et al., 1990). Throughout the
disease progression, a clinical score was assigned to quantify the
extent of paralysis and/or paresis as follows: 0 for no symptoms, 1-5
for paresis/paralysis in one to five appendages, and 6 for morbidity
(Redwine and Armstrong, 1998).
Unfortunately, after MHV-A59 infection, mice of the FGF2
line had extremely high mortality, which was not abrogated by either lowering the infectious dose (1, 100, and 1000 pfu tested; data not
shown) or increasing the age of the mice at the time of MHV-A59 injection (4 and 6 weeks tested; data not shown). The high incidence of
mortality was dependent on the 129 Sv-Ev:Black Swiss genetic background
and not the FGF2 genotype based on the following outcomes: FGF2 +/+, 16 of 16 became symptomatic and all died (100%);
FGF2 +/, 40 of 42 became symptomatic and 36 died (86%);
FGF2 /, 58 of 60 became symptomatic and 54 died (90%).
Deaths occurred ~7-10 d after MHV-A59 injection. In C57BL/6 mice the
typical mortality rate has been ~11% (Armstrong et al., 1990), and
this relatively low mortality rate was confirmed in the present
experiments with only 3 deaths (15%) among 20 symptomatic mice of 20 C57BL/6 mice injected in parallel with mice from the FGF2 line.
Cuprizone experimental demyelination. Male 8-week-old mice
were placed on a diet of 0.3% (w/w) cuprizone [oxalic
bis(cyclohexylidenehydrazide); Aldrich, St. Louis, MI] mixed into
milled chow (Harlan Teklad Certified LM-485 code 7012CM), which was
available ad libitum. Mice were maintained on the cuprizone
diet until killing, as noted in Results, or until being returned
to normal chow pellets after 6 weeks. Cuprizone ingestion results in a
reproducible pattern of corpus callosum demyelination over this 6 week
period followed by extensive spontaneous remyelination within the
subsequent 2-3 weeks on normal chow (Matsushima and Morell, 2001).
Mice of the FGF2 line were tested with 0.2% and 0.3%
cuprizone to determine the optimal dose in this genetic background. The
0.3% dosage produced more extensive corpus callosum demyelination in
mice of the FGF2 line so that the pattern of resulting
demyelination matched with 0.2% cuprizone in C57BL/6 mice (data not
shown), as previously characterized (Hiremath et al., 1998). Thus,
subsequent studies in the FGF2 mice used 0.3% cuprizone.
The cuprizone-treated mice did not exhibit any overt behavioral
symptoms and the treatment did not cause mortality at this 0.3%
dosage. Control "nontreated" mice were maintained on normal chow
pellets (Harlan Teklad Certified LM-485 code 7012C).
Tissue preparation and histopathological analysis. Mice were
perfused with 4% paraformaldehyde, then brains and spinal cords were
dissected before overnight postfixation in 4% paraformaldehyde (Redwine and Armstrong, 1998). Segments of spinal cord and brain were
either embedded in paraffin for histological analysis and detection of
apoptosis, or cryoprotected and embedded in OCT compound for
immunostaining and in situ hybridization. Demyelination was evaluated in 7 µm paraffin sections of spinal cord and corpus callosum using Luxol fast blue with periodic acid-Schiff reaction (Morell et al., 1998; Hiremath et al., 1998).
In situ hybridization. In situ hybridization and preparation
of digoxigenin-labeled riboprobes were performed as previously detailed
(Redwine and Armstrong, 1998; Messersmith et al., 2000). Digoxigenin-labeled antisense riboprobes were used to detect mRNA transcripts for FGF2 (gift from Dr. Gail Martin, University of California at San Francisco; Hebert et al., 1990; Messersmith et al.,
2000), proteolipid protein (PLP; gift from Dr. Lynn Hudson; National
Institutes of Health; Hudson et al., 1987; Redwine and Armstrong,
1998), and PDGF  receptor (PDGFR; gift from Dr. Bill Richardson,
University College London; Lee et al., 1990; Mudhar et al., 1993;
Redwine and Armstrong, 1998). An in vitro transcription kit
(Ambion, Austin, TX) was used to incorporate digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN). The digoxigenin-labeled riboprobes were hybridized to 15 µm cryosections of brain or spinal cord tissues. Digoxigenin was detected with an alkaline
phosphatase-conjugated sheep anti-digoxigenin antibody (Boehringer
Mannheim), followed by reaction with
nitroblue-tetrazolium-chloride-5-bromo-4-chlor-indolyl-phosphate substrate (Dako, Carpinteria, CA).
BrdU incorporation and detection. In situ hybridization
combined with bromodeoxyuridine incorporation was performed as detailed previously (Redwine and Armstrong, 1998). Mice were injected
intraperitoneally with 200 mg/kg bromodeoxyuridine (BrdU; Sigma) at 4 and 2 hr before killing. After in situ hybridization
detection, sections were treated to permeabilize the tissue and
denature the DNA. Sections were incubated overnight with a monoclonal
anti-BrdU conjugated to horseradish peroxidase (diluted 1:15;
Boehringer Mannheim). Peroxidase activity was detected by incubation
with 3,3'-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA)
Immunohistochemistry. To identify oligodendrocyte
progenitors in situ, 15 µm cryosections were immunostained
for NG2 and PDGFR (Redwine and Armstrong, 1998; Messersmith et al.,
2000). Primary antibodies used were rabbit polyclonal anti-NG2 antibody
(1:500; gift from Dr. William Stallcup, La Jolla, CA) and rat
monoclonal anti-PDGFR antibody (APA5 at 1:200; PharMingen, San
Diego, CA). Donkey anti-rabbit IgG Cy3 conjugate (Jackson
ImmunoResearch, West Grove, PA) was used to detect NG2, whereas the
PDGFR was detected with biotinylated donkey anti-rat secondary
antibody (Jackson ImmunoResearch) with fluorescein tyramide
amplification (New England Nuclear, Boston, MA). Sections were stained
with DAPI (Sigma) before mounting.
Mature oligodendrocytes were identified with CC1, which
immunostains oligodendrocyte cell bodies without labeling myelin
(Fuss et al., 2000). The CC1 antibody (Oncogene Research Products,
Cambridge, MA) was detected with donkey anti-mouse IgG FITC (Jackson
ImmunoResearch). The CC1 immunostaining conditions were previously
tested to ensure that CC1 did not label astrocytes or NG2-labeled cells
(Messersmith et al., 2000).
Myelin was immunostained with the Rip monoclonal antibody (a
gift from Dr. Beth Friedman, Regeneron, Tarrytown, NY). Cryostat sections (15 µm) were washed with PBS, incubated at 4°C overnight with undiluted Rip hybridoma supernatant containing 2% Triton-X 100. Rip antibody binding was detected with donkey anti-mouse IgG FITC
(Jackson ImmunoResearch). Myelin was also immunostained with monoclonal
antibody 8-18C5, which recognizes myelin oligodendrocyte glycoprotein
(MOG; hybridoma cells provided by Dr. Minetta Gardinier, University of
Iowa, Iowa City, IA; Linnington et al., 1984). Paraffin sections (7 µm) were dewaxed, washed with PBS, and incubated at 4°C overnight
with hybridoma supernatant diluted 1:5. MOG immunoreactivity was
detected with the ABC elite kit for mouse (Vector Laboratories) with
DAB substrate to reveal peroxidase enzymatic activity.
Apoptosis. Paraffin sections (7 µm; coronal) of brains
were prepared (see above) and processed with a modified terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling (TUNEL) assay (ApopTag Plus peroxidase in situ
apoptosis detection kit; Intergen, Purchase, NY). The 3'-OH DNA ends,
generated by DNA fragmentation typically observed with apoptotic cells,
were labeled with digoxigenin-dUTP using terminal deoxynucleotidyl
transferase. The digoxigenin tag was then detected with an
anti-digoxigenin antibody conjugated with peroxidase to yield a dark
brown reaction product with DAB substrate. The sections were lightly
counterstained with methyl green to detect nuclei.
Preparation and retroviral labeling of glial cell cultures from
remyelinating spinal cord. Female C57BL/6 mice (Charles River, Wilmington, MA) were infected with 1000 pfu of MHV-A59 at 4 weeks of
age (see above). Mice were killed during the onset of remyelination (4 weeks after infection; Redwine and Armstrong, 1998), and glial cell
cultures were prepared from the spinal cords, as previously detailed
(Armstrong et al., 1990). Spinal cords were combined from six mice for
preparation of glial cell cultures. Cells were grown in DMEM
supplemented with 10% FBS (Invitrogen, Gaithersburg, MD) for
2 d, and then the medium was replaced with Sato defined medium (Bottenstein and Sato, 1979) either with no treatment (control), or with exogenous FGF2 (10 ng/ml; human recombinant FGF2; R & D
Systems, Minneapolis, MN), or with FGF2 neutralizing antibody (FGF2
nAb; AF-233-NA; used at 1 µg/ml which is the recommended ND50; R & D Systems). This FGF2 nAb has been
previously used successfully to block FGF2 activity in vivo
(Nilsson et al., 2001). We have characterized the in vitro
specificity of this FGF2 nAb as inhibiting the proliferation of
neonatal oligodendrocyte lineage cells in response to FGF2 but with no
inhibition of the PDGF-AA response (E. E. Frost and R. C. Armstrong, unpublished observation). After a further 24 hr, the
 -galactosidase at gag (BAG) replication-deficient retrovirus
(80 pfu; generously provided by Dr. Steve Levison, Penn State
University, Hershey, PA) (Price et al., 1987; Levison et al., 1999) was
added to the cultures, which were returned to the incubator for 48 hr.
The BAG retrovirus becomes incorporated into the chromosome after
mitosis, and then -galactosidase (-gal) is expressed as a
heritable marker in clonally derived cells.
Three-color immunocytochemistry was used to simultaneously identify
oligodendrocyte progenitor cells with anti-NG2 (see above) and
differentiated oligodendrocytes with O1 monoclonal antibody (Bansal and
Pfeiffer, 1992) within clones expressing -gal. O1 supernatant (1:10
supernatant from hybridoma cultures containing 10% FBS in DMEM) was
added to the medium for the final 60 min. The dishes were then fixed in
2% paraformaldehyde, and O1 binding was detected with a goat
anti-mouse IgM secondary antibody conjugated with tetramethylrhodamine
isothiocyanate (TRITC). The cultures were then incubated for 24 hr at
4°C with anti-NG2 polyclonal antibody (1:500) and -gal using the
40-1A monoclonal antibody (1:2 dilution of supernatant from hybridoma
cultures containing 15% FBS in DMEM). The 40-1A hybridoma cells were
obtained from the Developmental Studies Hybridoma Bank, developed under
the auspices of the National Institute of Child Health and Human
Development and maintained by the University of Iowa, Department of
Biological Sciences (Iowa City, IA). Monoclonal 40-1A was detected
with donkey anti-mouse IgG FITC, whereas NG2 was detected with donkey
anti-rabbit IgG 7-amino-4-methylcoumarin-3-acetic acid (AMCA) secondary
antibody. All secondary antibodies were purchased from Jackson
ImmunoResearch. All labeled clones were counted within each dish.
Imaging, quantitation, and statistical analysis. Images of
immunostaining and in situ hybridization results were
captured with a Spot 2 digital camera on an Olympus IX-70 microscope.
Fluorescence channels were imaged singly or in combination using narrow
bandpass filter sets for Cy3/TRITC, FITC, and AMCA or a triple
bandpass filter (Chroma Technologies, Brattleboro, VT). Images were
prepared as panels using Abobe Photoshop. Immunofluorescence signal
intensity within a given area was quantitated from digital images using Metamorph Software (Universal Imaging Corporation, West Chester, PA) to
select the region of interest and calculate the average pixel intensity
of the region.
For cell density quantitation related to the cuprizone model, cells in
the corpus callosum expressing PLP mRNA were quantitated using unbiased
stereological morphometric analysis (Messersmith et al., 2000). The
cell density (cells per cubic millimeter) was estimated using
the Stereologer System (Systems Planning and Analysis, Inc.,
Alexandria, VA). Analysis was restricted to the corpus callosum region,
from the midline, and extending laterally to below the cingulum in
15-µm-thick coronal sections. The unbiased stereological method could
not be used appropriately for conditions with relatively few cells of
interest in any particular category. Therefore, quantitation of
PDGFR/BrdU single- and double-labeled categories in the corpus callosum required counting all labeled cells and measuring the area
sampled. Using the Stereologer System, the thickness is sampled as part
of the definition of each "dissector" volume, so that density
measurements reflect cells per cubic millimeter. However, without the
Stereologer System, section thickness could not be sampled in the
mounted specimen, and so the density measurements are stated as cells
per square millimeter.
For cell density quantitation related to the MHV-A59 model, entire
15-µm-thick transverse sections of spinal cord were sampled. In
contrast to corpus callosum, unbiased stereological methods were not
appropriate in the spinal cord because it is not an anatomically homogeneous structure (i.e., comprised of gray matter and white matter,
as well as diverse fiber tracts within white matter). For each
experiment, all labeled cells within each section were counted, and the
area was measured to determine cells per square millimeter.
Each category analyzed included three or more tissue sections per mouse
and three or more mice per condition. Specific numbers of animals per
sample are noted in text and figures. Unpaired Student's t
tests were used to identify significant differences between genotypes
and/or treatments.
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RESULTS |
MHV-A59 demyelination in FGF2 null mice
Similar to multiple sclerosis lesions, MHV-A59 lesions involve
oligodendrocyte cell loss and a complex immunological response (Houtman
and Fleming, 1996; Redwine and Armstrong, 1998). Therefore, FGF2 that
is locally upregulated in MHV-A59 lesions could potentially act
directly on the oligodendroglial regeneration process or could have a
role in the immune response to MHV-A59. Oligodendrocyte populations
were assessed by counting the density of PLP mRNA-positive (+) cells in
transverse sections of spinal cord from noninfected mice and from mice
that had recovered from transient demyelination at 8 weeks after
MHV-A59 infection (Fig. 1). Control
noninfected FGF2 / mice had a similar density of PLP
mRNA+ cells compared with wild-type mice of the C57BL/6 background,
which is the strain used previously to characterize this MHV-A59 model
and upregulation of FGF2 in lesions (Jordan et al., 1989;
Messersmith et al., 2000). After remyelination and after transient
MHV-A59-induced demyelination, C57BL/6 mice recovered normal densities
of white matter PLP mRNA+ oligodendrocytes compared with the
age-matched adult noninfected C57BL/6 mice. In contrast,
FGF2 / mice had a significantly higher density of white
matter PLP mRNA+ oligodendrocytes after recovery from MHV-A59, compared
with age-matched noninfected FGF2 / mice (p < 0.0001). In addition, comparing across
responses to MHV-A59, the FGF2 / mice had a
significantly greater recovery of white matter PLP mRNA+ cells than did
the C57BL/6 mice (p = 0.0031). Thus, the absence
of FGF2 enhanced the oligodendroglial repopulation of white matter
lesions during this spontaneous remyelination.
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Figure 1.
Oligodendrocyte repopulation after MHV-A59
demyelination. C57BL/6 mice (C57) were used as the calibrator strain in
which the MHV-A59 model is well characterized. FGF2 null
(/) mice were compared with and without MHV-A59 infection, relative
to the C57BL/6 strain. All MHV-A59 infected mice had a clinical score
of at least 2, indicating limb paralysis and/or paresis associated with
spinal cord demyelination. All mice were killed 8 weeks after MHV-A59
infection, when remyelination is well underway. Control mice were
age-matched noninjected mice. In situ hybridization for
PLP mRNA was used to identify oligodendrocytes, as shown in
representative ventrolateral quadrants of spinal cord sections for a
MHV-A59 infected C57BL/6 mouse (A) and a MHV-A59
infected FGF2 null mouse (B). For comparison, the
images are aligned at the midline (A, central canal,
top right; B, central canal, top
left). Oligodendrocytes were counted in entire transverse 15 µm sections of lumbar spinal cord (C). The
number of oligodendrocytes was similar in control mice of both
genotypes. However, FGF2 null mice recovering from
MHV-A59 had a significantly (p < 0.0031)
higher density of white matter oligodendrocytes compared with C57BL/6
mice after MHV-A59. Number of mice sampled for each genotype and
condition is shown in parentheses in the symbol legend.
Values shown are mean ± SD. Scale bar, 100 µm.
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Generation of new oligodendrocytes after transient demyelination is
believed to involve a proliferative response of oligodendrocyte progenitor cells that persist in the normal adult CNS (Redwine and
Armstrong, 1998; Reynolds et al., 2001). To examine the resting population of progenitors in adult FGF2 / mice,
oligodendrocyte progenitors were identified (Fig.
2) using double immunostaining to
simultaneously detect two characteristic markers, PDGFR and NG2
chondroitin sulfate proteoglycan (Nishiyama et al., 1999). Progenitor
distribution did not appear to be altered in FGF2 / mice, as compared with either FGF2 +/+ mice or C57BL/6 mice.
Quantitative analysis of transverse spinal cord sections showed a
similar density of PDGFR+/NG2+ oligodendrocyte progenitors present
between C57BL/6 mice (8 weeks of age; 53.18 ± 6.62 cells/mm2; n = 4) and in
FGF2 / mice (54.72 ± 8.03 cells/mm2; n = 5) and
between FGF2 genotypes (12 weeks of age; +/+, 64.38 ± 4.06 cells/mm2, n = 4;
/, 55.20 ± 11.25 cells/mm2,
n = 6). This similarity indicates that FGF2 is not
required for the generation and survival of the normal density of
oligodendrocyte progenitors in the nonlesioned CNS. In addition, this
normal density of both oligodendrocytes and progenitors in the CNS of
FGF2 / mice before infection delimits the effect of FGF2
absence to within the period of MHV-A59 disease progression.
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Figure 2.
Oligodendrocyte progenitors in normal adult spinal
cord. Oligodendrocyte progenitors were identified in transverse
sections of lumbar spinal cord using immunostaining for NG2
(red) and PDGFR (green) in
combination with a DAPI nuclear stain (blue).
Colocalization of immunoreactivity for NG2 and for PDGFR appears
yellow because of the integration of red
and green signal in those pixels. FGF2
+/+ mice (A), FGF2 / mice
(B), and C57BL/6 (C57; C)
wild-type mice had grossly similar populations of oligodendrocyte
progenitors (examples at arrows) with highly variable
branched processes, as shown in representative areas of transverse
sections of ventral spinal cord. Scale bar, 50 µm.
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Further analysis of the cellular responses during remyelination in
FGF2 null mice after MHV-A59 infection was not attempted because of an unusually high incidence of mortality 7-10 d after MHV-A59 injection in the FGF2 genetic background (see
Materials and Methods). Relatively few mice of the 129 Sv-Ev:Black
Swiss background survived until the remyelination phase for analysis, regardless of the FGF2 genotype.
Cuprizone demyelination of FGF2 null mice
Cuprizone is a well established means to reproducibly demyelinate
the corpus callosum of mice over a 6 week period of daily ingestion
(Matsushima and Morell, 2001). Cuprizone toxicity results in
oligodendrocyte and myelin loss, with clearing of debris by macrophages, but does not involve lymphocytic cell invasion or breakdown of the blood-brain barrier. Therefore, cuprizone
demyelination was selected as an optimal model to compare and contrast
with the MHV-A59 model in evaluating the role of FGF2 in remyelination.
The FGF2 mice were fed 0.3% cuprizone in milled chow.
Lesion progression was evaluated using Luxol fast blue with
periodic-acid Schiff (PAS) reaction in which myelin stained dark blue,
and demyelinated tracts reacted with only the PAS, resulting in a pink
color (Fig. 3A,C,E).
Oligodendrocytes were identified using immunostaining for CC1, a
specific marker of oligodendrocyte cell bodies (Fuss et al., 2000). In
nontreated adult (8-week-old) FGF2 / mice, the corpus
callosum was well myelinated (Fig. 3A) with characteristic rows of interfascicular oligodendrocytes (Fig. 3B) and no
apparent differences compared with FGF2 +/+ mice or C57BL/6
mice (data not shown). After 3 weeks of cuprizone ingestion,
FGF2 / mice exhibited patches of myelin loss in the
corpus callosum (Fig. 3C). In addition, oligodendrocytes
were rarely detected by CC1 immunostaining (Fig. 3D), which
is consistent with the stage of substantial oligodendrocyte loss and
maximal reduction of myelin gene transcript abundance (Ludwin, 1978;
Morell et al., 1998; Mason et al., 2000). After 6 weeks of cuprizone
ingestion, the corpus callosum was extensively demyelinated in
FGF2 / mice (Fig. 3E), corresponding with the
stage of maximal myelin loss (Morell et al., 1998). In addition,
initial recovery of the oligodendrocyte population was evident at the 6 week cuprizone stage (Fig. 3F) (Mason et al., 2000).
This analysis demonstrates that the cuprizone effects on
oligodendrocyte and myelin loss were not altered in the FGF2
line or as a consequence of FGF2 absence in the null mice.
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Figure 3.
Myelin and oligodendrocyte loss in cuprizone
demyelination of FGF2 / mice. Mice were fed normal chow
continuously (A, B) or 0.3% cuprizone for either 3 weeks (C, D) or 6 weeks (E, F).
Coronal brain sections were then examined by Luxol fast blue with
periodic-acid Schiff reaction to stain myelin blue (A, C,
E) and by immunostaining with CC1 antibody to identify
oligodendrocyte cell bodies (green; B, D,
F). The corpus callosum between the midline and cingulum
bilaterally demyelinates, progressing between 3 and 6 weeks of
cuprizone ingestion (A, C, E). Oligodendrocytes were
abundant and distributed in characteristic rows in the normal corpus
callosum (B). However, the frequency of
oligodendrocytes was dramatically decreased in number in the corpus
callosum after 3 weeks of cuprizone (D) yet began
to increase after 6 weeks on cuprizone (F). Scale
bars: A, C, E, 500 µm; B, D, F, 25 µm.
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Cuprizone demyelination induces FGF2 expression in the
corpus callosum
To determine whether FGF2 expression is increased in
cuprizone-mediated demyelination as was observed in MHV-A59 lesions
(Messersmith et al., 2000), the relative abundance of FGF2 mRNA
transcripts was examined by in situ hybridization (Fig.
4). In nontreated mice, the corpus
callosum and other white matter tracts contained relatively few cells
with detectable FGF2 mRNA signal, in contrast to various adjacent
neuronal populations that exhibited strong signal in the same tissue
sections. After 5-6 weeks of 0.3% cuprizone treatment, FGF2 mRNA
hybridization signal was dramatically increased in the regions of the
corpus callosum that corresponded with areas of demyelination. Similar
results were obtained with C57BL/6 mice (n = 2, nontreated; n = 2, cuprizone) and FGF2 +/+
mice (n = 5, cuprizone). This relatively high FGF2
expression localized to lesions is consistent with a potential role for
FGF2 in oligodendrocyte lineage responses to demyelination.
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Figure 4.
Increased FGF2 mRNA expression in
cuprizone demyelinated corpus callosum. In situ
hybridization was used to detect FGF2 mRNA expression in coronal
sections of C57BL/6 mice. The nontreated mice (A)
had few FGF2 mRNA labeled cells in the corpus callosum relative to the
abundant FGF2 mRNA expression in various adjacent neuronal populations.
In contrast, mice treated with cuprizone for 6 weeks
(B) had strong FGF2 mRNA expression in many cells
within the lesion area of the corpus callosum (above lateral ventricle,
LV). For comparison, the images are aligned at
the midline (A, right side; B, left
side). Scale bar, 250 µm.
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FGF2 null mice show enhanced oligodendrocyte repopulation of
demyelinated lesions
Oligodendrocyte populations were quantitated in coronal sections
of corpus callosum from nontreated mice and from specific stages in the
progression of cuprizone mediated demyelination and remyelination (Fig.
5). Unbiased stereological techniques were used to determine the density of oligodendrocytes, identified by
PLP mRNA expression. This quantitation corresponded with cell density
and did not appear to be attributable to a PLP transcriptional effect,
because CC1 immunostaining (Fig. 3) demonstrated qualitative changes in
the oligodendrocyte density that paralleled the results for PLP mRNA+
cells. In nontreated FGF2 / mice, the density of
oligodendrocytes remained stable between 8 weeks (age to match the
start of cuprizone feeding) and 17 weeks of age (age to match the
oldest experimental mice). Extensive oligodendrocyte loss was evident
after 3 weeks of cuprizone ingestion in both FGF2 +/+ and
/ mice (no significant difference between genotypes; p = 0.1686). After 6 weeks on cuprizone, myelin had
degraded and been cleared (Fig. 3), which coincided with the initial
regeneration of the oligodendrocyte population
(p = 0.0313 between genotypes). After 6 weeks on
cuprizone followed by 3 weeks of normal chow, the oligodendrocyte
density showed extensive repopulation of the corpus callosum. At this 3 week recovery point, the oligodendrocyte density in the FGF2
+/+ mice had returned to 30% below the level in the nontreated
17-week-old mice. In contrast, the oligodendrocyte density in the
FGF2 / mice significantly surpassed the level in the
nontreated 17-week-old mice by 31% (p = 0.0115;
6 weeks cuprizone, 3 weeks off). Even more striking is the direct
comparison of genotypes that showed the recovery of oligodendrocytes
was 87% greater in FGF2 / mice relative to
FGF2 +/+ mice (p = 0.0053; 6 weeks
cuprizone, 3 weeks off). Clearly, although the extent and timing of
oligodendrocyte loss was similar in both genotypes, the extent of
oligodendrocyte repopulation was promoted by the absence of FGF2.
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Figure 5.
Oligodendrocyte population changes during
cuprizone demyelination and remyelination. In situ
hybridization for PLP mRNA was used to identify oligodendrocytes, as
shown in representative images of the corpus callosum from
FGF2 +/+ mice (A) and
FGF2 / mice (B). In both
A and B, mice were treated with cuprizone
for 6 weeks and then taken off cuprizone for a 3 week recovery period.
For comparison, both images show the corpus callosum from the midline
laterally to under the cingulum and are aligned at the midline
(A, right side; B, left side). The PLP
mRNA expression appears markedly increased in the FGF2
/ (B) compared with the FGF2
+/+ section (A). Unbiased stereological
techniques were used to determine the density of oligodendrocytes,
identified by PLP mRNA expression, in the corpus callosum from the
midline laterally to under the cingulum (C).
White bars denote nontreated FGF2 /
mice that are age-matched to the start of cuprizone (8 weeks) and the
end of the recovery after cuprizone (17 weeks). For cuprizone-treated
mice, the gray bars denote FGF2 +/+ mice,
and black bars denote FGF2 / mice.
Dramatic oligodendrocyte loss was evident after 3 weeks of cuprizone
(3 wk cup). Cuprizone treatment for 6 weeks (6 wk
cup) coincided with the initial regeneration of the
oligodendrocyte population. After 6 weeks on cuprizone followed by 3 weeks of normal chow (6 wk cup, 3 wk off), the
oligodendrocyte density showed extensive repopulation of the corpus
callosum in cuprizone-treated mice. Compared with the
FGF2 +/+ mice, the FGF2 / mice had
dramatically enhanced recovery of oligodendrocytes
(p = 0.0053; 6 wk cup, 3 wk
off). The number of mice sampled for each value is as
follows: nontreated FGF2 / at 8 weeks,
n = 3; 3 week cup, n = 4 for
both genotypes; 6 week cup, n = 3 FGF2 +/+, n = 4 FGF2
/; 6 week cup with 3 weeks off, n = 3 for both
genotypes; nontreated FGF2 / at 17 weeks,
n = 6. Values shown are mean ± SD. Scale bar,
100 µm.
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Previous in vitro studies have reported that mature
oligodendrocytes were induced to undergo cell death by apoptosis when exposed to FGF2 (Muir and Compston, 1996). To determine whether this
potential negative effect of FGF2 might be an underlying mechanism
limiting oligodendrocyte repopulation of lesions, a modified TUNEL
assay was used for in situ detection of apoptotic cells.
Because during the demyelination phase cuprizone induces oligodendrocyte apoptosis (Mason et al., 2000) and the FGF2 genotype did not alter the extent of oligodendrocyte loss (Fig. 5, 3 weeks), TUNEL analysis was focused on the period of recovery of mature oligodendrocytes after removal of cuprizone. In tissue sections from
mice that had been treated with cuprizone for 6 weeks followed by 3 weeks off cuprizone to generate new oligodendrocytes, apoptotic cells
were clearly labeled but extremely rare, with only one or two cells per
section within the corpus callosum of either FGF2 / or
+/+ mice (data not shown). Therefore, during this phase of
oligodendrocyte regeneration, apoptosis does not appear to markedly
limit the repopulation response.
Absence of FGF2 does not alter progenitor proliferation in response
to demyelination
A simple explanation of how the FGF2 / mice
generate more oligodendrocytes might be that the proliferation of
oligodendrocyte progenitors in response to demyelination may be further
enhanced in FGF2 / mice compared with FGF2
+/+ mice. Because FGF2 is expected to be a mitogen for oligodendrocyte
progenitors, this effect would not be predicted but must be considered
based on an increase in oligodendrocytes. To test this possibility,
oligodendrocyte progenitors were identified by in situ
hybridization for PDGFR, and proliferation was estimated using
immunodetection of BrdU incorporated during a 4 hr terminal period
(Fig. 6). In sections from mice treated
with cuprizone for 5 weeks, the demyelinated areas of the corpus
callosum exhibited dramatic increases in the density of cells labeled
with PDGFR and/or with BrdU. However, the accumulation of PDGFR
mRNA+ cells in lesions was similar in FGF2 +/+ and /
mice. In addition, the populations that incorporated BrdU, regardless
of cell-type marker expression, were not significantly different. More
specifically, there was no significant difference in the proliferative
progenitor population, identified as double-labeled for PDGFR and
BrdU. This finding was confirmed by immunostaining simultaneously for
two progenitor cell-type markers, NG2 and PDGFR, while
counterstaining with DAPI (Fig. 6A). The frequency of
NG2+PDGFR+ double immunolabeled cells that exhibited mitotic figures
was highest in the corpus callosum of mice that had been treated with cuprizone for 6 weeks, as compared with 3 weeks on cuprizone or 6 weeks
on cuprizone followed by 3 weeks on normal chow. However, the frequency
of these cells was quite low (< 3 mitotic NG2+PDGFR+ cells/mm2), and there was no statistical
difference between FGF2 +/+ and / genotypes. Thus,
different techniques of identifying progenitor cells and two methods of
estimating proliferation demonstrate that the oligodendrocyte
progenitor proliferation induced by demyelination was similar between
FGF2 / and +/+ mice.
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Figure 6.
Oligodendrocyte progenitor proliferation and
accumulation during cuprizone demyelination. Oligodendrocyte
progenitors undergoing active proliferation were identified by two
methods. As shown in the example in A, oligodendrocyte
progenitors were immunostained for NG2 (red) and
PDGFR (green), and active cell division was
evident as mitotic figures with DAPI stain (blue).
Colocalization of immunoreactivity for NG2 and for PDGFR appears
yellow because of the integration of red
and green signal in those pixels. Oligodendrocyte
progenitors were also identified by in situ
hybridization for PDGFR, and proliferation was estimated based on
incorporation of BrdU during a 4 hr pulse before killing (B,
C). At high magnification, BrdU incorporation was detected as
brown nuclear signal, and PDGFR mRNA was detected as blue-black
cytoplasmic signal (C; border area between lesion, left,
and non-lesioned corpus callosum to the right). The frequency of
progenitors, many with BrdU labeling, was visibly different in
demyelinated areas of the corpus callosum (lesion, area
under double arrowhead) relative to adjacent normal
appearing white matter (NAWM, area under double
arrowhead). Cells labeled (+) for PDGFR mRNA and/or BrdU
were counted in the corpus callosum lesions and NAWM of
FGF2 +/+ and / mice killed after 5 weeks on
cuprizone (D). Oligodendrocyte progenitors
clearly accumulated in the lesion areas compared with NAWM (PDGFR+,
BrdU), and proliferating progenitors were more frequent in lesion
areas (PDGFR+, BrdU+). Cells other than progenitors, not identified
by cell type-specific markers, also exhibited more proliferation in
lesions (PDGFR, BrdU+). There were no significant differences
between the FGF2 +/+ mice and FGF2 /
mice for any of these cell populations (n = 6 mice
of each genotype). Values shown are mean ± SD. Scale bars:
A, 10 µm; B, 500 µm;
C, 50 µm.
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Attenuation of FGF2 promotes differentiation of
oligodendrocyte progenitors
Experiments were next designed to determine whether altered
differentiation of oligodendrocyte progenitors might contribute to the
improved oligodendroglial repopulation of demyelinated lesions in the
absence of FGF2. Glial cultures of spinal cords from MHV-A59 lesioned
C57BL/6 wild-type mice were used to perform lineage analysis of
differentiation while manipulating the level of available FGF2 ligand
(Fig. 7). We previously demonstrated that
glial cultures derived from MHV-A59 lesioned C57BL/6 mice killed at an
early stage of remyelination, 4 weeks after MHV-A59 injection,
maintained reactive astrocytes and microglia as well as the enhanced
proliferation of oligodendrocyte progenitors in response to
demyelination (Armstrong et al., 1990). These glial cultures were
infected with BAG replication-deficient retrovirus, which is integrated
into the chromosome after the infected cell undergoes mitosis
(Hajihosseini et al., 1993). BAG-infected cells expressed -gal that
served as a heritable marker of oligodendrocyte lineage cells that were
clonally derived from a progenitor cell. Differentiation of progenitors
was detected in the clones using NG2 immunoreactivity to identify the
progenitor phenotype and O1 immunostaining to distinguish mature
oligodendrocytes (Fig. 7).
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Figure 7.
Retroviral cell lineage
analysis of oligodendrocyte progenitor differentiation. Mixed glial
cultures were prepared from spinal cords of MHV-A59 infected C57BL/6
mice at the onset of remyelination (4 weeks after infection). The
cultured cells were infected with BAG replication-incompetent
retrovirus so that -gal expression served as a heritable marker of
oligodendrocyte lineage cells that were clonally derived from a
progenitor cell. Cultures were triple immunostained to simultaneously
detect -gal (A-C; green), the oligodendrocyte
progenitor marker NG2 (D-F; blue), and O1 as a marker
of differentiated oligodendrocytes (G-I; red). The
panels show examples of three separate clones illustrating
each of the phenotypes identified within the oligodendrocyte lineage.
Oligodendrocyte progenitors (pair of cells at arrows in
A, D, G; grown in defined
medium) were retrovirally infected based on -gal immunostaining and
expressed NG2 but not O1 antigens. Differentiated
oligodendrocytes (pair of cells at arrows in C,
F, G; grown in defined medium with FGF2 neutralizing antibody)
were retrovirally infected based on -gal immunostaining and
expressed O1 antigens but not NG2. A transitional stage of
differentiation was also observed (arrow in B, E,
H; grown in defined medium with FGF2 neutralizing antibody)
which expressed NG2 while also being recognized by O1. Scale bars:
A, D, G, 25 µm; B, C, E, F, H,
I, 50 µm.
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FGF2 levels within cultures were elevated by adding exogenous FGF2 to
the defined culture medium, followed by BAG infection 24 hr later and
fixation after an additional 48 hr. This elevated FGF2 condition
resulted in a 35% increase in the number of clones per culture
(p = 0.0017). Based on counts of the total
number of cells immunolabeled with NG2 and/or O1 in the clones, more of
the cells remained NG2+ progenitors (61.5 ± 1.9% in FGF2,
53.6 ± 2.7% in control medium; p = 0.0116). This
result indicated that elevated FGF2 enhanced the proportion of infected
cells remaining as proliferative progenitors.
FGF2 levels within cultures were decreased using an FGF2 nAb to
attenuate endogenous FGF2 activity produced by reactive astrocytes and
microglia in these cultures of remyelinating spinal cords. The FGF2 nAb
did not alter the number of oligodendrocyte lineage clones per dish.
Interestingly, the FGF2 nAb significantly decreased the proportion of
oligodendrocyte lineage clones, i.e., cells expressing NG2 and/or O1,
comprised of NG2+ cells (44.1 ± 2.4% in FGF2 nAb, 53.6 ± 2.7% in control medium; p = 0.027). Therefore, when
endogenous levels of FGF2 were removed from the medium, the oligodendrocyte progenitors were more likely to differentiate to the O1
stage (55.9 ± 2.4% in FGF2 nAb, 46.4 ± 2.8% in control medium; p = 0.027). These in vitro findings
with FGF2 nAb match well with our in vivo results in
FGF2 / mice. Taken together, these findings indicate
that although FGF2 levels increase in response to demyelination,
attenuation of this FGF2 elevation allows oligodendrocyte progenitors
to differentiate more readily. The fact that our in vitro
analysis with cultures from wild-type C57BL/6 mice treated with FGF2
nAb for only 3 d produced results consistent with those found in
FGF2 / mice argues against a potential compensatory
effect in the knock-out mice and in favor of a direct role of FGF2 absence.
Myelin immunostaining during remyelination
This effect of FGF2 on differentiation extended to a corresponding
effect on myelin production as determined by immunostaining for two
myelin-specific proteins in mice. Rip immunostaining was selected as an
early marker of remyelination, because Rip immunostains pre-ensheathing
oligodendrocytes through myelinating stages and associated myelin
(Friedman et al., 1989; Butt et al., 1997). Quantitation of Rip
immunofluorescence in the corpus callosum of nontreated FGF2
/ mice with normal myelination resulted in an average pixel
intensity of 13,610 ± 1430 (n = 3 mice). Similar quantitation of Rip in the corpus callosum during remyelination was
performed using mice that had been demyelinated by a 6 week cuprizone
treatment followed by 3 weeks of recovery on normal chow. At this time
point during remyelination, the FGF2 / mice exhibited an
~50% greater recovery of Rip immunostaining of myelin (FGF2 / mice, 9337 ± 2561 average pixel intensity,
n = 3; FGF2 +/+ mice, 6254 ± 4158 average pixel intensity, n = 4). With this analysis,
within group variability was too great to allow significance to be
reached for the differences observed.
A similar trend was observed using MOG immunostaining as a qualitative
measure of myelin formation (Fig. 8). MOG
is the latest appearing myelin-specific protein, with expression
restricted to myelinating oligodendrocytes and thus associated with
myelin deposition (Solly et al., 1996). MOG immunostaining of mice that had been demyelinated by a 6 week cuprizone treatment followed by 3 weeks of recovery on normal chow showed variability during remyelination within each genotype group, as was found with the Rip
immunostaining. However, the extent of MOG immunostaining along axons
appeared to be more extensive within lesioned areas of the
FGF2 / mice as compared to the FGF2 +/+ mice
(n = 3, for each genotype). This observation is also
consistent with each of the findings presented that indicate the
absence of FGF2 promotes the differentiation phase of the remyelination
process.
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Figure 8.
Myelin immunostaining
during remyelination. In coronal brain sections from mice that had been
treated with cuprizone for 6 weeks followed by 3 weeks off cuprizone,
myelin in the corpus callosum was detected with immunostaining for MOG
(brown DAB reaction product). Variable amounts of
MOG-immunostained myelin were present in the corpus callosum of
FGF2 +/+ mice (A, C) and FGF2 / mice
(B, D) as remyelination progressed during the 3 weeks
after cuprizone treatment was discontinued. In each panel,
of three mice examined for each genotype, the one with the most
extensive MOG immunostaining is shown for that genotype.
C and D are higher- magnification images
from the corpus callosum under the cingulum within A and
B, respectively. At this higher magnification, the
FGF2 +/+ mice (C) appear to have
fewer myelin sheaths so that the immunostaining is discontinuous
along axons in contrast to the FGF2 / mice
(D) in which a similar area appears more
extensively myelinated. Scale bars: A, B, 500 µm;
C, D, 50 µm.
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DISCUSSION |
FGF2 expression is upregulated in every model of experimental CNS
demyelination examined to date, including MHV-A59 virus, cuprizone,
lysolecithin, and experimental allergic encephalomyelitis (EAE)
(present results; Liu et al., 1998; Hinks and Franklin, 1999;
Messersmith et al., 2000). Because FGF2 upregulation is a generalized
response to demyelination, it is important to discern the function of
FGF2 in the remyelination process. Our current results demonstrate that
reducing FGF2 expression enhanced oligodendroglial repopulation of
demyelinated lesions. This result in FGF2 knock-out mice was
evident with both the viral MHV-A59 model of spinal cord demyelination,
which involves a marked lymphocytic response, as well as with the
cuprizone neurotoxicant model of corpus callosum demyelination, where
lymphocyte infiltration is absent. In the cuprizone model, the extent
of oligodendrocyte loss during demyelination was similar in
FGF2/ mice compared with FGF2 +/+ mice.
Therefore, the effect of FGF2 absence corresponds with a difference in
the remyelination phase of the oligodendrocyte lineage response.
Based on our in vivo and in vitro analyses,
decreased FGF2 activity appears to promote repopulation of lesions by
promoting oligodendrocyte lineage cell differentiation, without
significantly influencing oligodendrocyte progenitor proliferation or
survival. Before remyelination, BrdU incorporation by oligodendrocyte
progenitors and accumulation of progenitors was similar in both
FGF2 genotypes. This analysis used combinations of detection
of PDGFR and/or NG2 expression, which identify oligodendrocyte
progenitors in normal and lesioned adult CNS (Nishiyama et al., 1999;
Levine et al., 2001). Because both PDGFR and NG2 markers were
considered, our findings reflect the oligodendrocyte progenitor
population response, although NG2 can be expressed by macrophages in
pathological CNS tissue (Bu et al., 2001; Jones et al., 2002). Our
interpretation of the role of FGF2 in lesion repopulation is further
supported by lineage analysis during exposure to an FGF2 neutralizing
antibody, which provided direct evidence that FGF2 activity can
modulate the differentiation of oligodendrocyte lineage cells cultured from remyelinating tissue.
FGF2 knock-out mice of this line and several similarly
designed FGF2 knock-out mice have been analyzed in previous
studies with no gross or microscopic abnormalities reported in any of the organs and tissues examined, including CNS tissues (Dono et al.,
1998; Ortega et al., 1998; Zhou et al., 1998). However, more detailed
analyses revealed that specific neuronal and glial populations in
cerebral cortex had decreased cell densities in FGF2/
mice compared with wild-type mice (Dono et al., 1998; Ortega et al., 1998; Raballo et al., 2000; Korada et al., 2002). Mature
FGF2/ mice exhibited a 30-40% decrease of overall
glial cell density in the cortex with an ~18% decrease in
subcortical white matter using general markers of glial cells,
including astrocytes (Vaccarino et al., 1999). The current study
specifically quantified oligodendrocyte progenitor and mature
oligodendrocyte populations and demonstrated normal numbers of both
cell types in the adult CNS in FGF2 / mice. This finding
is consistent with the prediction that oligodendrocyte number is
ultimately determined by axonal interactions (Barres and Raff, 1999).
The current analysis of remyelination in a constitutive knock-out mouse
model is subject to potential compensation effects caused by the
lifelong absence of FGF2. However, compensation is not a likely
explanation for the current findings because a consistent result was
observed in cultures of wild-type mice when FGF2 activity was
attenuated by application of a neutralizing antibody for only 3 d.
FGF2 may have different overriding roles as a result of the complex
interactions of growth factors, cytokines/chemokines, and cellular
receptors that vary during the progressive stages of demyelination and
remyelination. In an EAE model, intrathecal injection of a herpes
simplex virus-derived vector was used to increase the CSF
concentration of FGF2 (Ruffini et al., 2001). In this immune-mediated
disease model, FGF2 treatment initiated within 1 week of clinical onset
of EAE ameliorated the ongoing demyelination and increased the number
of oligodendrocyte progenitors and oligodendrocytes. Interestingly, a
second FGF2 treatment, later in the EAE progression, partially
abrogated the beneficial effect of the initial injection. Our in
vitro studies of glial cultures derived from MHV-A59 lesioned
spinal cord tissue demonstrated that elevated levels of FGF2 enhance
the proliferation of oligodendrocyte progenitors (present results).
Preliminary results with BrdU incorporation in cultures isolated from
spinal cords at various stages throughout the MHV-A59 disease
progression confirm this FGF2 mitogenic effect, which is more dramatic
when examined early in the disease progression and/or in combination
with PDGF treatment (Frost and Armstrong, unpublished
observation). This mitogenic role of FGF2 seems to contrast with
the enhanced oligodendroglial repopulation of demyelinated lesions
observed in our analysis of FGF2 knock-out mice. However, in
the FGF2 / mice a very strong progenitor proliferative
response still occurs during demyelination (present results) and so
must be supported by other factors, such as PDGF or tumor necrosis factor (Redwine and Armstrong, 1998; Allamargot et al., 2001; Arnett et al., 2001). The contribution of other molecules as mitogens is also indicated by our in vitro analysis; the FGF2 nAb did
not alter the number of retrovirally labeled clones per culture
(present results).
Importantly, in the course of remyelination, FGF2 appears to inhibit
differentiation of oligodendrocyte lineage cells (present results).
This role of FGF2 is consistent with findings from normal CNS
development. In vitro analysis of FGF2 effects on
differentiation at progressive stages of maturation within the
oligodendrocyte lineage indicates that FGF2 reversibly blocks terminal
differentiation of late stage progenitors into mature oligodendrocytes
(Bansal and Pfeiffer, 1997). In a myelinating culture system,
continuous activation of FGF2 signaling pathways caused a delay in
expression of myelin-specific proteins and impeded myelination (Baron
et al., 2000). In vivo administration of FGF2 resulted in an
increase of promyelinating oligodendrocytes and localized impairment of myelination in the developing anterior medullary velum (Goddard et al.,
1999). Similarly, transgenic mice expressing a dominant-negative form
of FGFR1 driven by a myelin gene promoter exhibited increased myelin
sheath thickness consistent with the suggestion that FGF2 may act as a
negative regulator of myelination (Harari et al., 1997). FGF2 in the
lesion environment may be acting in the context of other factors that
can promote oligodendrocyte differentiation, such as insulin-like
growth factor I (McMorris and Dubois-Dalcq, 1988; Mason et al., 2000)
and/or neuregulin (Park et al., 2001; Viehover et al., 2001).
Therefore, as remyelination progresses in the absence of FGF2, the
balance of regulators of oligodendrocyte lineage cell differentiation
may be altered so that progenitor cells might more efficiently
differentiate and remyelinate axons within lesions. This effect might
be useful for optimizing the extent of remyelination in lesions because
cuprizone demyelinated C57BL/6 mice remyelinate only 60-70% of axons
after 4-6 weeks of recovery as compared with 94% of axons being
myelinated in nontreated control mice (Mason et al., 2001).
Our present findings, and those of others discussed above, indicate
that FGF2 may be a critically important regulator of oligodendrocyte lineage responses to demyelinating disease. The available data can be
put together to design a hypothetical scenario of FGF2 effects in this
context. Endogenous FGF2 is upregulated at the transcriptional level in
reactive astrocytes and microglia within and near areas of demyelinated
white matter. This increase of localized FGF2 in white matter may
contribute to glial cell activation and may possibly support axon and
neuron survival in the lesion environment. Early in the disease
progression, the extent to which FGF2 induces oligodendrocyte
progenitor proliferation may be dependent on the concentration of FGF2
relative to other growth factor mitogens. However, during remyelination
after transient demyelination, FGF2 plays a significant role by
inhibiting oligodendrocyte lineage cell differentiation. Modulation of
expression of multiple FGF receptor isoforms in response to
demyelination may contribute to the differential effects of FGF2 on
proliferation and differentiation of oligodendrocyte lineage cells.
A suboptimal balance of interacting growth factors that regulate
differentiation and myelination may contribute to the limited remyelination of MS plaques (Prineas et al., 1993; Raine and Wu, 1993).
Chronic MS lesions can contain substantial populations of immature
oligodendrocytes and premyelinating oligodendrocytes that do not
effectively remyelinate throughout the extent of the lesions (Chang et
al., 2002; Wolswijk, 2002). If FGF2 expression is upregulated in MS
lesions, as it is in the animal models of experimental demyelination,
then FGF2 may be inhibiting the maturation and myelination of these
oligodendrocyte lineage cells. This inhibition may be especially
detrimental in MS lesions that may also have reduced influence of
differentiation promoting growth factors, such as the decreased
neuregulin expression reported in MS lesions (Viehover et al., 2001).
Now that approaches to modulating the immunopathogenesis of MS are
proving useful, promoting remyelination has a greater potential to
provide clinical benefit in terms of protecting axons and facilitating
saltatory conduction. Further studies are imperative to determine the
extent to which regenerative responses can be optimized from the
existing oligodendrocyte lineage cell populations that are now being
identified in MS lesions (Scolding et al., 1998; Lucchinetti et al.,
1999; Chang et al., 2000; Maeda et al., 2001; Solanky et al.,
2001; Chang et al., 2002; Wolswijk, 2002).
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FOOTNOTES |
Received June 4, 2002; revised July 22, 2002; accepted July 26, 2002.
This work was supported by National Institutes of Health Grant NS39293
(R.C.A.) We thank Dr. Thomas Doetschman for providing breeding pairs of
the FGF2 knock-out mice, Drs. William Stallcup, Minetta Gardinier, and
Beth Friedman for antibodies, Dr. Pierre Morell for advice on the
cuprizone model, and Drs. Lynn Hudson and William Richardson for
plasmids. We appreciate the comments of Joshua Murtie and Drs. Steve
Levison, Donna Messersmith, and Yong-Xing Zhou.
Correspondence should be addressed to Dr. Regina C. Armstrong,
Department of Anatomy, Physiology, and Genetics, Uniformed Services
University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD
20814-4799. E-mail: rarmstrong{at}usuhs.mil.
| |
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