Cell Mol Neurobiol (2010) 30:101–111
DOI 10.1007/s10571-009-9435-x
Expression of Transforming Growth Factor-b Receptors
in Meningeal Fibroblasts of the Injured Mouse Brain
Yukari Komuta Æ Xichuan Teng Æ Hiroko Yanagisawa Æ
Kazunori Sango Æ Koki Kawamura Æ Hitoshi Kawano
Received: 13 May 2009 / Accepted: 17 July 2009 / Published online: 4 August 2009
Ó Springer Science+Business Media, LLC 2009
Abstract The fibrotic scar which is formed after trau-
matic damage of the central nervous system (CNS) is
considered as a major impediment for axonal regeneration.
In the process of the fibrotic scar formation, meningeal
fibroblasts invade and proliferate in the lesion site to
secrete extracellular matrix proteins, such as collagen and
laminin. Thereafter, end feet of reactive astrocytes elabo-
rate a glia limitans surrounding the fibrotic scar. Trans-
forming growth factor-b1 (TGF-b1), a potent scar-inducing
factor, which is upregulated after CNS injury, has been
implicated in the formation of the fibrotic scar and glia
limitans. In the present study, expression of receptors to
TGF-b1 was examined by in situ hybridization histo-
chemistry in transcortical knife lesions of the striatum in
the mouse brain in combination with immunofluorescent
staining for fibroblasts and astrocytes. Type I and type II
TGF-b receptor mRNAs were barely detected in the intact
brain and first found in meningeal cells near the lesion
1 day postinjury. Many cells expressing TGF-b receptors
were found around the lesion site 3 days postinjury, and
some of them were immunoreactive for fibronectin. After
5 days postinjury, many fibroblasts migrated from the
meninges to the lesion site formed the fibrotic scar, and
Y. Komuta
X. Teng
H. Yanagisawa
K. Sango

K. Kawamura
H. Kawano (&)
Department of Developmental Morphology,
Tokyo Metropolitan Institute for Neuroscience,
2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan
e-mail: kawano-ht@igakuken.or.jp
X. Teng
Department of Pathology, Eastern Liaoning
University Medical College, 118000 Dandong, China
most of them expressed TGF-b receptors. In contrast, few
of reactive astrocytes expressed the receptors throughout
the postinjury period examined. These results indicate that
meningeal fibroblasts not reactive astrocytes are a major
target of TGF-b1 that is upregulated after CNS injury.
Keywords Transforming growth factor-b
Receptor

Fibrotic scar
Astrocyte
Immunohistochemistry

In situ hybridization
Introduction
After traumatic injury of the central nervous system (CNS),
fibroblasts derived from meninges invade and proliferate in
the lesion site. They secrete extracellular matrix molecules
(ECMs) such as type IV collagen, laminin and fibronectin
(FN) to create a fibrotic scar that contains of dense layers of
basal lamina. Thereafter, end feet of reactive astrocytes
form a glia limitans and envelop the fibrotic scar tissue
(Berry et al. 1983; Mathewson and Berry 1985; Maxwell
et al. 1990). These events are considered as a result of a
stepwise cascade of post-traumatic inflammatory responses
that wall off the lesion site from the surrounding neural
tissue and protect the CNS from further infection (Shearer
and Fawcett 2001). Simultaneously, the fibrotic scar and
surrounding glia limitans can be a physical and chemical
obstacle for axonal regeneration in the damaged CNS.
Elimination of the fibrotic scar has been shown to be
required for axonal regeneration in a variety of animal
models (reviewed in Klapka and Muller 2006; Kawano
et al. 2007), such as by suppression of type IV collagen
synthesis (Stichel et al. 1999; Klapka et al. 2005), in
newborn mouse (Kawano et al. 2005), in the mouse
hypothalamic arcuate nucleus (Homma et al. 2006), by
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degradation of glycochains of chondroitin sulfate proteo-
glycans with chondroitinase ABC (Li et al. 2007), and by
transplantation of olfactory ensheathing cells (Teng et al.
2008).
Although the mechanism underlying fibrotic scar for-
mation remains to be elucidated, one of the important
factors that are involved in the formation is transforming
growth factor-b1 (TGF-b1), a fibrogenic factor that
enhances proliferation and ECM production of fibroblasts
(Ignotz and Massague 1986; Moses et al. 1987). TGF-b1
expression increases around the lesion site during the
period of fibrotic scar formation, increasing from 2 days,
reaching a peak at 4 days, and declining, but with still
enhanced levels at 2 weeks after CNS injury (Semple-
Rowland et al. 1995; Streit et al. 1998; Lagord et al. 2002;
Nakamura et al. 2003). Experimentally, the formation of
fibrotic scar and surrounding glia limitans in injured rat
brains is promoted by exogenously administered TGF-b1
and prevented by neutralization with TGF-b1 antibody
(Logan et al. 1994).
The biological action of TGF-b1 is mediated through
binding to both type I and type II TGF-b receptors (TRI
and TRII). TRII binds to its proper ligand, but TRI requires
the presence of bound TRII to interact with TGF-b1
(Wrana et al. 1992). Therefore, TRI and TRII are colo-
calized in many cases on same cells. In normal mouse
brains, the expression of TRI and TRII is at very low level,
while they are upregulated after traumatic injury of the
CNS (McTigue et al. 2000; Fee et al. 2004) and in multiple
sclerosis lesion (de Groot et al. 1999). After CNS lesioning,
TRI and TRII are found in neurons and astrocytes (Vivien
et al. 1998; de Groot et al. 1999), endothelial cells of the
blood vessels (de Groot et al. 1999; Fee et al. 2004) and
macrophages (de Groot et al. 1999; McTigue et al. 2000).
The localization of TGF-b receptors in the meningeal
fibroblasts, however, has not been reported so far. In the
present study, expression of TGF-b receptors was exam-
ined using in situ hybridization histochemistry with special
attention to meningeal fibroblasts and reactive astrocytes
that constitute the fibrotic scar and glia limitans after CNS
injury.
Methods
Surgical lesion of the striatum
The animals were obtained from Clea Japan, Inc (Tokyo,
Japan). The unilateral brain injury was made as previously
reported (Kawano et al. 2005). Briefly, adult male mice of
ICR strain (30–35 g) were anesthetized with intraperitoneal
injection of sodium pentobarbital (40 mg/kg body weight)
and transferred to a stereotaxic frame (Narishige, Tokyo,
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Cell Mol Neurobiol (2010) 30:101–111
Japan) with the incisor bar set 3 mm below the interaural
line. A midline skin incision was made on the pre-shaved
scalp, the periosteum cleared from the cranium, and a small
oval-shaped hole was made with a dental drill where the
knife was inserted. A special knife with a width of 2.0 mm
made of a razor blade was attached to the vertical bar of the
stereotaxic frame, and the tip of the knife was inserted into
the right side of the brain at 0.5 mm lateral to the midline,
at 1.5 mm posterior to the bregma and at a depth of 6.0 mm
from the surface of the brain. The knife was allowed to
remain in place for a further 1 min and withdrawn. The
experimental protocols mentioned were approved by the
Animal Care and Use Committee of the Tokyo Metropol-
itan Institute for Neuroscience.
Tissue preparation
The numbers of mice analyzed were 3 at 1 day, 3 days and
5 days, 4 at 7 days, 5 at 14 days and 3 at 28 days after
injury. Intact 3 mice were used as a control. Mice were
deeply anesthetized with isoflurane, and the brains were
immediately dissected out, snap-frozen in cold isopentane
on dry ice and stored at -80°C until sectioning. Horizontal
sections were serially cut at 16 lm thickness and collected
on MAS-coated glass slides (Matsunami Co, Tokyo, Japan)
and kept at -80°C until use.
Immunohistochemistry
Fresh-frozen sections were dried well and fixed with 4%
paraformaldehyde in 0.1 M phosphate-buffered saline
(PBS) on ice for 30 min. After washing twice in PBS,
endogenous peroxidase activity was eliminated with 3%
hydrogen peroxide in 0.1% Triton X-100 for 15 min at
room temperature. Sections were washed twice in PBS and
incubated with primary antibodies against glial fibrillary
acidic protein (GFAP, DAKO Corporation, Carpenteria,
CA, 1:25) or FN (Sigma, Saint Louis, MO, 1:5,000) in
0.5% skim milk at 4°C overnight. Following two washes in
PBS, sections were sequentially incubated in biotinylated
anti-rabbit IgG (1:100; Vector Laboratories, Burlingame,
CA) for 1 h at 37°C. These signals were amplified
with Vectastain Elite ABC kit (Vector) and visualized in
0.01% diaminobenzidine tetrahydrochloride (DAB)/0.01%
hydrogen peroxide at 37°C.
Probe preparation
The oligo RNA sense and antisense probes were designed
against rat TRI (576-2064 of NM_012775) and TRII (446-
2084 of NM_031132). After reverse transcription-PCR was
performed, products were purified using Qiagen gel puri-
fication kit (Qiagen, Tokyo, Japan) and ligated in pGEM-T
Cell Mol Neurobiol (2010) 30:101–111
Fig. 1 Spatiotemporal localization of FN (left column) and GFAP
(right column) immunoreactivity in horizontal sections of intact brain
(a, b) and in brains at 1 (d), 3 (c), 5 (e, f) and 14 days (g, h) after injury.
a In the intact brain, FN immunoreactivity is localized in the meninges
(M) and blood vessels (BV). In the lesion site, FN-immunoreactive
cells are first found at 3 days (c, arrows), accumulated at 5 days
(e, arrow) and formed the fibrotic scar (FS) at 14 days (g) after brain
easy vector (Promega Co, Madison, WI). After sequences
were confirmed, the product was used for in vitro tran-
scription with T7 or SP6 RNA polymerase. Dig-labeling
103
injury. b Scattered GFAP-immunoreactive cells are distributed in intact
brain. d Cells with intense GFAP immunoreactivity already appear
around the lesion site at 1 day. f At 5 days, reactive astrocytes are
localized in the region distant from the lesion site. (h) By 14 days after
injury, reactive astrocytes form the glia limitans (arrows) to surround
the FS. CC cystic cavity, DG dentate gyrus, HF hippocampal fissure, S
subiculum. Scale bar = 400 lm
RNA probes were generated according to recommended
protocol for Dig RNA labeling mix (Roche Diagnostics,
Mannheim, Germany).
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In situ hybridization histochemistry
and immunofluorescent staining
In situ hybridization histochemistry was performed accord-
ing to the recommended protocol by Roche. Only the first
day of the protocol was modified for fresh-frozen sections as
follows. Before the experiment, sections on glass slides were
well dried and fixed in ice-cold 4% paraformaldehyde in
PBS for 30 min. After rinsing in 0.1 M PBS, sections were
equilibrated in 5 9 SSC for 15 min at room temperature and
hybridized with 2 ng/ll Dig-labeled probes in hybridization
solution containing 50% formamide, 5 9 SSC and 1% SDS
C over night at 57°. After sufficient signals were detected,
the sections were washed in 0.1 M PBS twice, incubated in
LAB solution (Polyscience, Warrington, PA, USA) for
15 min at room temperature to activate antigens and washed
in 0.1 M PBS. Immunofluorescent staining following in situ
hybridization was carried out with above-mentioned anti-
bodies to GFAP (1:25) and FN (1:100). The sections were
incubated with diluted primary antibodies overnight at 4°C,
washed twice and sequentially incubated with biotinylated
anti-rabbit IgG (1:100; Vector) and streptavidin-Alexa
FluorÒ 488 conjugate (1:100; Molecular Probes, Inc., OR,
USA) for 1 h each at 37°C.
Image analysis
Micrographs of sections doubly stained with in situ
hybridization and immunofluorescence were obtained
using an Axiocam microscope (Zeiss) equipped with color
CCD camera, and the contrast and brightness were adjusted
on Adobe Photoshop Elements (v. 6.0). Some photographs
of in situ hybridization were reversed to dark field images,
and the color of the positive reaction was converted to
magenta. Then, the images of in situ hybridization and
immunofluorescence of the same sections were merged to
demonstrate magenta–green combination for the benefit of
people with red–green color blindness.
Results
Fibrotic scar formation and astrocytic response after
brain injury
The spatiotemporal localization of FN and GFAP immu-
noreactivity after brain injury accords well with that repor-
ted previously (Berry et al. 1983; Mathewson and Berry
1985; Maxwell et al. 1990). In the intact brain, FN immu-
noreactivity was found in the meninges of the brain surface
and the wall of blood vessels (Fig. 1a). No FN immunore-
activity was enhanced around the lesion site 1 day postin-
jury, and a small number of FN-immunoreactive cells were
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Cell Mol Neurobiol (2010) 30:101–111
first detected in the lesion site at 3 days after injury (Fig. 1c).
By 5 days after injury, FN immunoreactivity deposited in
the lesion site was the first sign of the formation of a fibrotic
scar (Fig. 1e). After 14 days, FN-immunoreactive fibrils
were well developed and clearly delineated the fibrotic scar
(Fig. 1g).
In the intact brain, dispersed astrocytes were weakly
GFAP immunoreactive (Fig. 1b). One day after injury,
reactive astrocytes with intense GFAP immunoreactivity
were already detected around the lesion site (Fig. 1d). At
5 days postinjury, the zone of reactive astrocytes encom-
passed the entire lesioned side of the brain. Reactive
astrocytes were localized in the region distant from the
lesion site and did not extend their processes to coalesce
around the lesion site (Fig. 1f). By 14 days after injury, foot
processes of reactive astrocytes attached to one another to
form a lining zone around the fibrotic scar, the glia limitans
(Fig. 1h).
Expression of TRI and TRII mRNAs after brain lesion
The results of the present in situ hybridization histochem-
istry were consistent among mice of the same postinjury
periods. TRI and TRII mRNAs were barely detected in the
brain of intact mice (Fig. 2a, b). Only faint positive signals
were found in the meninges and blood vessels in the brain
surface and choroid plexus. One day after the brain injury,
TRI and TRII messages were not observed in the lesion site
(Fig. 2c, d), while in all 3 mice examined they were
localized in the meninges, choroid plexus and blood vessels
close to the site of knife insertion. At 3 days postinjury,
TRI and TRII expression was first detected in and around
the lesion site (Fig. 2e, f). In one case, messages extended
from the lesion site to the entire striatum. Subsequently,
TRI and TRII mRNAs came to be expressed in the lesion
site and became dense in the lesion center at 14 days after
injury (Fig. 2g, h). The localizations of TRI and TRII
mRNAs were similar, but staining intensity of TRII mRNA
was stronger than that of TRI in all mouse brains exam-
ined. The positive reactions observed were considered as
specific, since no reactions were found in sections
hybridized with sense probes (Fig. 2i, j).
Localization of TRI and TRII mRNAs in meningeal
fibroblasts
As mentioned earlier, TRI and TRII mRNAs were found in
the meninges, blood vessels and choroid plexus in the area
around the lesion at 1 day after the injury. When FN
immunoreactivity was demonstrated with TGF-b receptor
mRNAs in the same sections, receptors were expressed in
FN-immunoreactive cells in the meninges (Fig. 3a–c), cells
Cell Mol Neurobiol (2010) 30:101–111 105

Fig. 2 Localization of TRI

(a, c, e, g) and TRII (b, d, f, h)
mRNAs in horizontal sections
of intact brain (a, b) and in
brains at 1 day (c, d), 3 days
(e, f) and 14 days (g, h) after
injury. No positive signals are
found in intact brain (a, b) and
around the lesion site (asterisks)
at 1 day after injury (c, d).
Intense signals are localized in
the lesion site from 3 days after
(e, f, asterisks) and in the
fibrotic scar (FS) at 14 days
after (g, h). No reactions are
found around the lesion site
(asterisks) with sense probes of
TRI (i) and TRII (j) in
sequential sections with c and d.
Scale bar = 400 lm in a–f, i, j;
200 lm in g and h

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around the blood vessels (Fig. 3d–f) and choroid plexus
(Fig. 3g–i). At 3 days after injury, when the first receptor
signals were detected in the lesion site, the receptor
mRNAs were localized in cell nucleus and cytoplasm
(Fig. 3j), and FN immunoreactivity was found in cyto-
plasm and outside the cells (Fig. 3k). In doubly stained
sections, the majority of fibroblasts were proved to express
TRI and TRII (Fig. 3l).
Fig. 3 Double staining of TRII mRNA (magenta) and FN immuno-
reactivity (green) in single horizontal sections of brains at 1 day (a–i)
and 3 days (j–k) after injury. TRII (left column), FN (middle column)
and merged images of TRII and FN (right column). TRII mRNA is
detected in the meninges close to the lesion site (a) which is
immunoreactive for FN (b, c). Arachnoid (Ar) and pia mater (PM) are
clearly distinguished in b. (d–f) Around the blood vessels (BV) close
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Cell Mol Neurobiol (2010) 30:101–111
At 5 days after injury, cells expressing TRI and TRII
mRNAs were localized in and around the lesion center
where they corresponded well with FN immunoreactivity
(Fig. 4a–c). At 14 days postinjury, cells expressing TRI
and TRII were concentrated in the fibrotic scar, and vir-
tually all of receptor-expressing cells in the scar were
immunoreactive for FN (Fig. 4d–f). In sections of a mouse
brain in which the lesion accidentally extended to the
to the meninges near the lesion site, TRII mRNA is localized in
FN-immunoreactive cells (arrows). (g–i) In the choroids plexus, TRII
mRNA is detected in FN-immunoreactive cells (arrows). (j–k) At
3 days postinjury, many cells contain TRII mRNA around the lesion
site, and some of them are overlapped with FN-immunoreactive cells
(arrows). Asterisks indicate extracellular deposition of FN. Scale
bar = 100 lm
Cell Mol Neurobiol (2010) 30:101–111
basolateral surface of the forebrain, cells expressing TRI
and TRII mRNAs were observed to localize contiguously
from the pial surface to the fibrotic scar (Fig. 4g). In the
same section, they overlapped FN-immunoreactive cells
(Fig. 4h, i), clearly indicating that meningeal fibroblasts
expressing TGF-b receptors invaded to the lesion site to
form the fibrotic scar.
Localization of TRI and TRII mRNAs in reactive
astrocytes
After brain injury, reactive astrocytes already increased
around the lesion site at 1 day postinjury. At 3 and 7 days
after injury, a number of reactive astrocytes were situated
close to cells expressing TRI and TRII mRNA, but few
GFAP-immunoreactive cells colocalized with TRI and TRII
Fig. 4 Double staining of TRII mRNA (magenta) and FN immuno-
reactivity (green) in single horizontal sections of lesioned brain at 5
(a–c) and 14 days (d–i) after injury. TRII (left column), FN (middle
column) and merged images of TRII and FN (right column). (a–c)
Cells containing TRII mRNA signals are localized in and around the
lesion site, and cells in the lesion center (asterisks) contain both TRII
and FN immunoreactivity. At 14 days after, TRII mRNA-positive
cells are accumulated in the lesion core, the fibrotic scar (FS) (d), in
107
mRNAs (Fig. 5a–f). After 14 days postinjury, processes of
reactive astrocytes surrounded the lesion site (fibrotic scar)
and formed the glia limitans (Fig. 5g), whereas receptor
mRNAs were localized mainly in the fibrotic scar (Fig. 5h).
In these sections, TRI and TRII mRNAs were absent from
the reactive astrocytes (Fig. 5i). Consequently, reactive
astrocytes proved to be devoid of TRI and TRII in all mouse
brains examined.
Discussion
Expression of TGF-b receptors after CNS injury
After penetrating injuries of the CNS, various kinds of
inflammatory cytokines are increasingly expressed with
which FN-immunoreactive cells are aggregated (e). Majority of TRII-
positive cells are overlapped with FN-positive cells (f). (g–i) In one
mice, surgical transection accidentally reaches the basolateral brain
surface (arrows). TRII-positive cells are contiguously localized from
the pial surface to the fibrotic scar (asterisks). FN-immunoreactive
cells exhibit similar distribution (h), overlapping with TRII-contain-
ing cells (i). Scale bar = 100 lm
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Fig. 5 Double staining of TRII mRNA (magenta) and GFAP
immunoreactivity (green) in single sections of lesioned brain at
3 days (a–c), 7 days (d–f) and 14 days (g–i) after injury. TRII (left
column), GFAP (middle column) and merged images of TRII and
GFAP (right column). TRII mRNA (a, d) and reactive astrocytes (b,
characteristic spatiotemporal patterns. Expression of pro-
inflammatory cytokines including interleukin 1a, 1b and 6,
tumor necrosis factor a and leukemia inhibitory factor are
acute and only transient after CNS injury, steeply increase
by 6 h, reach a peak at 12 h and decline at 24 h after injury
(Bartholdi and Schwab 1997; Streit et al. 1998; Nakamura
et al. 2003). In contrast, expression of TGF-b1, an anti-
inflammatory cytokine, is delayed and continuous after
CNS injury, increases from 2 days, reaches a peak at
4 days, and declines, but is still enhanced at 14 days after
injury (Logan et al. 1992; Semple-Rowland et al. 1995;
Streit et al. 1998; Lagord et al. 2002; Nakamura et al.
2003). Other members of TGF-b family, TGF-b2 and TGF-
b3, are constitutively expressed in the normal CNS (Uns-
icker and Strelau 2000; Lagord et al. 2002) and not sig-
nificantly increased after CNS injury (Lagord et al. 2002;
Nakamura et al. 2003), although restricted upregulation of
TGF-b2 close to the lesion site has been reported (Lagord
et al. 2002). Prompt upregulation of pro-inflammatory
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Cell Mol Neurobiol (2010) 30:101–111
e) increase around the lesion, but cells hardly contain both signals (c,
f). (g–i) Cells containing TRII mRNA are localized in the lesion
center, the fibrotic scar (FS) (g) and reactive astrocytes surround the
FS (h). The localization of these cells is clearly distinguishable (i).
Scale bar = 200 lm in (a–f); 100 lm in (g–i)
cytokines is caused by macrophages that invade the lesion
site immediately after injury, while expression of TGF-b1
has been reported to be localized on meningeal cells,
endothelial cells, astrocytes, macrophages, microglia and
neurons around the lesion after injury (Logan et al. 1994;
Lagord et al. 2002).
Major biological actions of TGF-b1 are mediated
through binding to their heterodimeric receptors, TRI and
TRII serine/threonine kinase receptors (Massague 1992).
TGF-b1 acts on target cells through both TRI and TRII
(Wrana et al. 1992; Vivien et al. 1995). Therefore, TRI and
TRII are co-localized on the same cells in many cases. In
the present study, the localization and expression pattern of
TRI and TRII mRNAs were similar each other (Fig. 2). In
the present results, expression of these receptors is at very
low levels in the normal CNS and restricted to the
meninges, blood vessels and choroid plexus 1 day after
brain injury. Cells bearing TRI and TRII mRNAs accu-
mulate in and around the lesion site from 3 days postinjury.
Cell Mol Neurobiol (2010) 30:101–111
Thereafter, these cells are concentrated in the lesion center
and seen until 4 weeks postinjury. This time course in the
expression of TRI and TRII mRNAs is consistent with that
reported previously after brain injury (McTigue et al. 2000;
Fee et al. 2004) and skin excisional wounds (Gold et al.
1997), suggesting that TGF-b1 signaling is involved in the
events occurring chronically in the lesion site.
Cell types expressing TGF-b receptors after brain
injury
The cellular localization of TRI and TRII mRNAs after
traumatic CNS injury has been previously reported. These
studies have demonstrated that TRI and TRII are localized
in macrophages (McTigue et al. 2000) and endothelial cells
of blood vessels (Fee et al. 2004). Other immunohisto-
chemical studies reported TRI and TRII in neurons and
astrocytes after focal cerebral ischemia (Vivien et al. 1998)
and in endothelial cells, astrocytes, microglia and neurons
in multiple sclerosis lesions (de Groot et al. 1999), but the
signals are not necessarily increased after these lesions.
The present study has demonstrated that TRI and TRII
mRNAs are expressed in meningeal fibroblasts after CNS
injury by using combination with immunofluorescent
staining. Fibroblasts bearing receptor messages were first
detected in the meninges and around blood vessels on 1 day
after injury (Fig. 3a–d). At 3 days after injury, small
numbers of fibroblasts are localized in the lesion site, and
they contain receptor mRNAs (Fig. 3j–l). Fibroblasts in the
lesion center increase in number with postinjury period, and
the fibrotic scar develops from 5 days after injury. The
majority of fibroblasts in the scar express receptor mRNAs
(Fig. 4). Furthermore, TRI and TRII are also expressed in
the migratory pathway of fibroblasts from meninges
(Fig. 4g–i). Therefore, TRI and TRII are expressed in
fibroblasts throughout the process of fibrotic scar formation.
We have also examined the presence of TRI and TRII
on reactive astrocytes using GFAP immunoreactivity, since
the glial scar is profoundly involved in the inflammatory
response, protection of damaged neurons, tissue repair
process and regeneration of transected axons after CNS
injury (reviewed in Gimenez y Ribotta et al. 2001; Silver
and Miller 2004). In our results, although reactive astro-
cytes and receptor-bearing cells are situated close to each
other after injury, most of them did not overlap at any
postinjury period examined (Fig. 5). TGF-b1 is known to
suppress the proliferation and enhance GFAP expression
and ECM production in cultured astrocytes (Lindholm
et al. 1992; Baghdassarian et al. 1993). However, the
reactive gliosis occurring around the wound is apparently
unaffected by the manipulation of TGF-b1 function (Logan
et al. 1994; Moon and Fawcett 2001) or TGF-b2 function
(Logan et al. 1999) in the lesion site. Therefore, it is
109
unlikely at least in the traumatic brain that TGF-bs directly
regulate the function of astrocytes, although the possibility
that TGF-bs affect astrocytes via other TGF-b receptors
including type III receptor (betaglycan) still remains.
In the present study, many TRI- and TRII-bearing cells
were detected at 3 days after injury in and around the
lesion site (Fig. 2e, f) in which only a few FN-positive cells
were detected (Fig. 3j–l). These FN-negative cells are
likely to be microglia/macrophages, which are known to
increase in the lesion site at this period (Popovich et al.
1997). The general distribution of TGF-b receptors and
microglia/macrophage was reported to overlap on day 7
after spinal cord injury (McTigue et al. 2000). TGF-b1 is
known to suppress the proliferation and function of
microglia/macrophage, while pro-inflammatory cytokines
stimulate them (reviewed by Merrill and Benveniste 1996).
Therefore, in the acute phase of inflammation in the CNS
pro-inflammatory cytokines induce reactive microgliosis
and invasion of macrophages, whereas in the chronic phase
of inflammation, TGF-b1 may suppress the function of
these cells. However, both increment and attenuation of
TGF-b1 levels in the lesion site increased ED1 immuno-
reactivity, a marker of microglia/macrophage (Logan et al.
1994), suggesting that the effect of TGF-b1 on these cells
is clearly complicated in the damaged brain.
Functional significance of TGF-b receptors
upregulation in meningeal fibroblasts after brain injury
TGF-b1 is a multipotent molecule that decreases astrocytic
proliferation, promotes neuron survival and inhibits cyto-
kine release from microglia and macrophages after CNS
lesioning (reviewed by Flanders et al. 1998). The best
known function of TGF-b1 is, however, promotion of
proliferation and ECM production in fibroblasts and vari-
ous mesenchymal cells (Ignotz and Massague 1986; Moses
et al. 1987). Upregulation of TGF-b1 and its receptors
commonly occurs in various organs after traumatic injuries.
For example, after excision, incision (Gold et al. 1997) and
burn wounds (Schmid et al. 1998) of skin, expression of
TRI and TRII chronically increased in fibroblasts. In the
liver, lung and kidney, various kinds of damages including
trauma, diabetes and viral infection are known to enhance
local expression of TGF-b1 that results in abnormal cell
proliferation and deposition of ECMs and leads to fibrosis
(reviewed by Hinz et al. 2007).
The present finding that TRI and TRII are expressed by
the fibroblasts of the fibrotic scar from the initial stage of the
formation suggests that TGF-b1 increased after CNS injury
directly activates meningeal fibroblasts. Fibrotic scar for-
mation is promoted by exogenous TGF-b1 and prevented by
anti-TGF-b1 antibody in injured rat brains (Logan et al.
1994). In addition, decorin, a small chondroitin sulfate
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proteoglycan that inhibits TGF-b function, also reduces the
size of the fibrotic scar (Logan et al. 1999). More recently,
blocking of TGF-b function by cAMP in combination with
collagen synthesis inhibitor can transiently reduce the
fibrotic scar formation and promote axonal regeneration in
the injured spinal cord (Klapka et al. 2005). Taken together,
it is concluded that after CNS injury TGF-bs promote the
proliferation and ECM production of meningeal fibroblasts
to form the fibrotic scar.
Acknowledgments The authors would like to thank Dr. Geoffrey
Raisman, UCL Institute of Neurology, Queen Square, London for
reviewing the manuscript and valuable advice. This work was sup-
ported by the Ministry of Education, Science, Sports and Culture of
Japan (20500318).
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