Role of the lesion scar in the response to damage and repair of the central nervous system
Hong Peng Li
H. P. Li Department of Human Anatomy, College of Basic Medical Sciences, China Medical University
, Shenyang 110001,
This study was supported with funding from the Ministry of Education,
, Sports and Culture of Japan (23500422)
Traumatic damage to the central nervous system (CNS) destroys the blood-brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.
After damage to the central nervous system (CNS) of adult
mammals, regeneration of transected axons barely occurs.
There is a growing view that severed central axons are
capable of regeneration and that the failure to regenerate is
due to the blocking effect of the scar formed at the lesion
site. This scar consists in both glial (mainly astrocytic) and
fibrotic components (Fitch and Silver 2008) and may have
its inhibitory effects both by the production of inhibitory
molecules and by the physical abrogation of aligned
pathways for regenerating axons to cross the lesion. At the
same time, the scarring process fulfils the vital functions
of restoring the bloodbrain barrier (BBB) and limiting
the damage to the site of injury. This review will cover a
number of recent studies in which manipulations of the
fibroblastic component of the scar by reagents, such as
blockers of collagen synthesis, have been found to be
beneficial for axon regeneration.
Various kinds of inhibiting factors that are upregulated
around the lesion site have been postulated to prevent the
regrowth of severed axons beyond the lesion site. These
include molecules of the chondroitin sulfate
proteoglycan (CSPG) family (for review, see Asher et al. 2001;
Morgenstern et al. 2002; Tan et al. 2005), tenascin
(McKeon et al. 1991), semaphorin 3A (Pasterkamp et
al. 1999), myelin-associated molecules (reviewed by
Zrner and Schwab 2010) and subtypes of the Eph
receptors and their ligands ephrins (reviewed by Goldshmit
et al. 2006). These molecules have axonal growth-repelling
activities in vitro and play important roles in axon guidance in
the developing CNS. Attempts to eliminate the molecules or
neutralize the inhibitory effect have been reported to enhance
axonal regeneration in the damaged brain and spinal cord
(Bradbury et al. 2002; Goldshmit et al. 2006; Kaneko et al.
2006; Moon et al. 2001).
The molecular changes in the glial and fibrotic scar
are closely related with the tissue repair process of the
CNS lesion site. Following CNS injury, bleeding occurs
and the BBB is broken down. The infiltration of blood
proteins such as thrombin (Nishino et al. 1993) and
fibrinogen (Ryu et al. 2009) triggers the inflammatory
reaction. At the same time, hematogenous cells including
leukocytes, macrophages and lymphocytes also invade from
the lesion site to the surrounding neural tissue and secrete
various cytokines and chemokines (Donnelly and Popovich
2008; Merrill and Benveniste 1996). Under the influence of
these factors, astrocytes, microglia and oligodendrocyte
progenitor cells are activated and constitute the glial
scar around the lesion site. On the other hand, from
several days after injury, fibroblasts intrude from the
damaged meninges to the lesion site, proliferate and
secrete extracellular matrix molecules (ECMs) including
type IV collagen (Type IV collagen), fibronectin and
laminin to form the fibrotic scar (Fig. 1). The
composition and arrangements of cells in these lesion scars are
postulated to play important roles for the protection of
damaged tissue, re-establishment of the BBB and isolation of
the lesion site from the surrounding neural tissue (Berry et al.
1983; Mathewson and Berry 1985; Maxwell et al. 1990a; for
review, see Shearer and Fawcett 2001). Simultaneously, the
cells of these scars express the above-mentioned axonal
growth-inhibiting molecules, which are believed to
prevent the axonal regeneration and functional recovery in
the injured CNS.
Fig. 1 Schematic drawings represent the process of the lesion scar
formation in the mouse brain. One day after traumatic CNS injury, the
BBB is disrupted and macrophages infiltrate the BBB-free area. a
Upregulation of GFAP immunoreactivity in reactive astrocytes is
already observed. b Three days after the injury, reactive astrocytes
significantly increase around the lesion site but they are absent from
the lesion center where the BBB is destroyed. Fibroblasts intrude from
the damaged meninges to the lesion site. c By 1 week after injury,
fibroblasts actively proliferate and secrete ECMs to form the fibrotic
scar. Reactive astrocytes re-occupy the surrounding area of the lesion
site and the BBB-free area around the lesion site is eliminated. d At
2 weeks after, processes of reactive astrocytes seal the lesion site to
form a glia limitans
Glial scar and tissue repair
The formation of a glial scar is generally referred to reactive
gliosis. After CNS injury, astrocytes, microglia and glial
progenitor cells around the lesion site are activated and
increase in number. They express and release various
bioactive substances, which play important roles in tissue repair
processes including inflammation, BBB repair and neural
protection (Rolls et al. 2009). Above all, there is a major
rearrangement of the anatomical structure. Immediately after
CNS injury, resident astrocytes become hypertrophic and
extend thick processes together with increased glial fibrillary
acidic protein (GFAP) immunoreactivity. Upregulation of
GFAP immunoreactivity in astrocytes is observed as early as
1 day after injury (Fig. 1a). Recent imaging shows that
astrocytic processes react within hours of the injury (Sibson et al.
2008). A recent study suggests that glial progenitors around
the lesion site also generate reactive astrocytes (Yoshioka et al.
2012). At 35 days after the injury, there is a significant
increase in the numbers of reactive astrocytes around the
lesion site but they are absent from the lesion center where
the BBB is broken down (Fig. 1b). Concomitant with the
accumulation of reactive astrocytes surrounding the lesion site
by 1 week after injury (Fig. 1c), the BBB-disrupted area
becomes confined as a result of reactive astrocytes enclosing
the lesion site to form a glia limitans (Fig. 1d; Mathewson and
Berry 1985; Yoshioka et al. 2010).
The astrocytic sealing of the lesion site contributes to
homeostatic functions including maintenance of extracellular
ion and fluid balance, clearance of extracellular glutamate,
water transport, production of pro- or anti-inflammatory
cytokines and chemokines, production of growth factors and free
radical scavenging (Rolls et al. 2009; Sofroniew 2009).
Recent studies using gene manipulation to suppress reactive
gliosis showed that in the traumatic CNS injury reactive
astrocytes played roles essential in prevention of neuronal
death, repair of the destructed BBB and restriction of
postinjury inflammation (Bush et al. 1999; Faulkner et al. 2004;
Herrmann et al. 2008; Okada et al. 2006; Pekny et al. 1999).
The vasculature of the CNS constructs a highly specialized
biological interface: the BBB, which helps to maintain
homeostasis within the CNS. To accomplish this, brain
capillaries possess extensive tight junctions between endothelial
cells and the astrocytic processes directly invest the
endothelia. The ability of endothelial cells to form a BBB is not
intrinsic to these cells but instead is induced by astrocytes.
Grafts of astrocytes induce BBB-like properties in peripheral
endothelia in vivo (Janzer and Raff 1987) and fetal astrocytes
induce various properties of the BBB in cultured endothelial
cells (Hayashi et al. 1997). In mice deficient for both GFAP
and vimentin intermediate filaments in astrocytes, glial
formation was impaired and bleeding occurred frequently
after brain and spinal cord injury (Pekny et al. 1999). A lack
of these intermediate filaments in perivascular astrocytes
decreases the mechanical strength of blood vessels, suggesting
that astrocytes normally support the structure of the blood
vessels in the CNS. The genetic ablation of proliferating
reactive astrocytes from the injured CNS also causes the
failure of BBB repair (Bush et al. 1999; Faulkner et al. 2004).
These studies also demonstrate that the glial scar plays a
crucial beneficial role in the restriction of leukocyte
spreading after CNS trauma. Increased invasion of leukocytes was
observed in mice with the genetic ablation of dividing
reactive astrocytes from the injured CNS (Bush et al.
1999; Faulkner et al. 2004). In mice with the genetic
depletion of Stat3, a mediator of cytokine action, astrocyte
migration toward the lesion site is disrupted and leukocytes
abnormally spread (Herrmann et al. 2008; Okada et al. 2006).
Limiting the infiltration of inflammatory leukocytes and
restoring the BBB are considered to reduce the post-traumatic
secondary injury after spinal cord injury (Donnelly and
The earlier concept (e.g., Banker 1980) that astrocytes
provide nutritive, neurotrophic and other supportive functions
for neurons has been greatly strengthened by the recent
demonstration that the energy metabolism of neuronal
mitochondria is dependent on lactate energy supplied by
the adjoining astrocytes (Herrero-Mendez et al. 2009;
Tsacopoulos and Magistretti 1996).
Glial scar as an impediment for axonal regeneration
Closing off the lesion site and re-establishment of a glia-pial
barrier produce a re-duplicated tangle of astrocytic processes,
that completely abrogate any pathways which regenerating
axons might have used to cross the lesion (Raisman and Li
2007). The scar further contributes to the failure of axonal
regeneration through the axonal growth-inhibiting property of
ECMs produced by reactive astrocytes (reviewed by Asher et
al. 2001; Hke and Silver 1996). Among them, CSPGs
produced by glial scar have been considered as major
impediments for axonal regeneration (for review, see Carulli et al.
2005 ; Morgenstern et al. 2002; Silver and Miller 2004; Yiu
and He 2006). Phosphacan, neurocan, brevican and NG2 have
been reported to have an axonal growth-inhibiting property
(Dou and Levine. 1994; Friedlander et al. 1994; Milev et al.
1994; Yamada et al. 1997) and increase markedly after CNS
injury (Tang et al. 2003). The inhibitory property of CSPGs
may reside in the chondroitin sulfate (CS) side chains, since
the administration of chondroitinase ABC (ChABC), a
CS-degrading enzyme, into the lesion site, effectively
promotes regeneration of severed axons in the nigrostriatal
ascending (Moon et al. 2001) and spinal descending
(Bradbury et al. 2002) pathways. In addition, administration
of a DNA enzyme whose target is the mRNA of a critical
enzyme, xylotransferase-1, which initiates glycosylation of
the protein backbone of CSPGs, also promotes axonal
regeneration in the injured rat spinal cord (Grimpe and Silver 2004).
Recently, a transmembrane tyrosine phosphatase, PTP, was
reported to act as a receptor for CS and mediate the axonal
growth-inhibiting signal of CSPGs (Shen et al. 2009).
Others have questioned to what extent the glial scar and
CSPGs are inhibitory to axon regeneration. NG2
proteoglycan is a major CSPG upregulated after CNS injury and
considered as a potent inhibitor of axonal growth in the glial
scar (reviewed in Tan et al. 2005). Regenerating axons have
been reported to pass through the glial scar (Camand et al.
2004), an area abundant in NG2 proteoglycan (Jones et al.
2003). More recently, after the spinal cord lesion, growth of
serotonergic axons was shown to be suppressed in the scar
tissue in mice lacking NG2 (de Castro et al. 2005). Finally,
NG2 cells, generally referred as oligodendrocyte precursor
cells, which are abundant in the glial scar, do not inhibit but
promote axonal growth even in the presence of elevated
level of NG2 (Yang et al. 2006).
Experiments aimed at genetic suppression of the glial
scar have been introduced to evaluate axonal regeneration
after CNS injury. The double genetic deletion of GFAP and
vimentin, cytoskeletal proteins of astrocytes, has been
reported to promote axonal sprouting and functional
recovery after spinal cord injury (Menet et al. 2003). In contrast, a
line of evidence indicates that genetic ablation of reactive
astrocytes prevents the glial scar formation in damaged CNS
but fails to promote axonal regeneration (Herrmann et al.
2008; Okada et al. 2006). Although randomly oriented
nerve fibers were increased along the wound margin in mice
deleted with astrocytes, they did not extend for long
distances (Bush et al. 1999). Failure of the axonal regeneration
in mice with glial scar deletion may be attributed to the
enlarged inflammation in these animals as mentioned in the
previous chapter. Although glial scar may be an obstacle to
axonal regeneration in damaged CNS, suppression of the
glial scar formation cannot be useful for the treatment of
traumatic injury in the CNS.
Fibrotic scar and tissue repair
After CNS trauma, fibroblasts invade the lesion site,
proliferate and secrete ECMs, such as Type IV collagen, fibronectin
and laminin. The invading fibroblasts cooperate with the
astrocytes to lay down a continuous basal lamina on the
outer-facing astrocytic surface, thus re-establishing the
gliapial barrier referred to as the glia limitans (Mathewson and
Berry 1985; Maxwell et al. 1990a; Shearer and Fawcett 2001).
Morphological evidence suggests that the fibrotic scar appears
to seal off the lesion site and encloses leukocytes infiltrated
after brain injury (Berry et al. 1983; Maxwell et al. 1990a).
However, suppression of fibrotic scar formation with an
administration of iron chelator 2,2-dipyridyl (DPY), an inhibitor
of Type IV collagen synthesis (Ikeda et al. 1992), or with
suppression of the function of transforming growth factor-
(TGF-), significantly reduces the recruitment of
inflammatory leukocytes (Logan et al. 1999b; Yoshioka et al. 2010;
2011). Furthermore, the astrocytic reconstitution of the BBB
still occurs in the absence of the fibrotic scar (Yoshioka et al.
2010, 2011). Therefore, it is unlikely that the fibrotic scar
plays a crucial role in the repair of the damaged CNS tissue.
Fibrotic scar as an impediment for axonal regeneration
There are reports that transected axons stop at the border of
the fibrotic scar (Fig. 2a; Camand et al. 2004; Stichel and
Mller 1994) and fibroblasts have been shown to express
various axonal growth-inhibitory molecules including NG2
proteoglycan (Tang et al. 2003), phosphacan (Tang et al.
2003), tenascin-C (Tang et al. 2003), semaphorin 3A
(Pasterkamp et al. 1999) and EphB2 (Bundesen et al.
2003). However, since the CNS tissue is rapidly walled off by
astrocytes, the ability of the fibrotic scar to present either a
physical or molecular obstacle to the regeneration of severed
axons depends upon the extent to which the axons come into
contact with it.
DPY treatment (Fig. 2b)
Elimination of the fibrotic scar has been shown to allow
axonal regeneration in a variety of animal models (reviewed
in Kawano et al. 2007; Klapka and Mller 2006; Fig. 2).
The idea of inhibiting the formation of the fibrotic scar was
first introduced by the group of Mller (Stichel and Mller
1998; Stichel et al. 1999a, b). Inhibition of Type IV collagen
synthesis by administration of DPY into the lesion site
prevents the fibrotic scar formation and has been associated
with regeneration of postcommissural fornix axons in the
injured rat brain (Stichel and Mller 1998; Stichel et al.
1999a, b). This treatment also promoted the regeneration
of mouse nigrostriatal dopaminergic axons (Kawano et al.
2005). These results suggest that Type IV collagen is
required for the fibrotic response to adult brain injury. Local
injection of antibodies against Type IV collagen also
suppressed the fibrotic scar formation after the transection of
the postcommissural fornix in adult rats (Stichel et al.
1999a). DPY treatment was also applied to the spinal cord
injury but was unable to suppress the larger amounts of
Type IV collagen that were deposited in this site
(Hermanns et al. 2001). Thereafter, treatment with both
DPY and cyclic AMP, which inhibits fibroblast proliferation,
Fig. 2 Elimination of the
fibrotic scar in the mouse and
rat brain has been shown to
promote axonal regeneration in
a variety of animal models. a In
injured brain, axons stop at the
border of the fibrotic scar and
do not regenerate. b In neonatal
and DPY-treated animals, axons
regenerate despite of the
presence of glial scar and
chondroitin sulfate proteoglycan
(CSPG) (Stichel et al. 1999a;
Kawano et al. 2005). c In the
hypothalamic arcuate nucleus
(ARC) and by chondroitinase
ABC (ChABC) treatment,
upregulation of chondroitin
sulfate is prevented and axons
regenerate (Homma et al. 2006;
Li et al. 2007). d In olfactory
(OEC)transplanted rats, fibrotic scar
is not formed and axons
regenerate (Teng et al. 2008)
transiently suppressed the fibrotic scar formation in the injured
spinal cord and has been reported to promote regeneration of
corticospinal tract axons and recovery of motor function
(Klapka et al. 2005).
Neonatal animals (Fig. 2b)
At earlier stages of development, there are a number of
situations in which axons have been reported to regenerate
successfully, with recovery of function after injury (for
review, see Xu and Martin 1991; Nicholls and Saunders
1996). When nigrostriatal dopaminergic axons are
unilaterally transected in mice aged postnatal day 7, dopamine
axons regenerate across the lesion site, while they stop and
do not extend across the lesion site in mice transected at
postnatal day 14 or older (Kawano et al. 2005). Reactive
astrocytes bearing CSPGs were increased around the lesion
in mice transected at all ages. However, a fibrotic scar
containing Type IV collagen deposits was not formed in
mice lesioned at postnatal day 7. The fibrotic response is
operational in adults but does not occur in wounds of the rat
cerebral cortex before 810 days after birth (Berry et al.
1983; Maxwell et al. 1990b). Thus, the period of failure of
axonal regeneration is correlated with the postnatal
development of the Type IV collagen deposition in the lesion site,
suggesting that the formation of the fibrotic scar could be a
contributory cause of the age-related failure of axonal
regeneration in the ascending dopaminergic system.
ChABC treatment (Fig. 2c)
After injury to the adult CNS, the increase in CSPG around
the lesion site is generally believed to constitute a major
impediment for axonal regeneration (for review, see Carulli
et al. 2005; Hke and Silver 1996; Morgenstern et al. 2002;
Silver and Miller 2004). It has been reported that degradation
of CSPG by injection of ChABC into the lesion site enhances
regeneration of nigrostriatal ascending (Moon et al. 2001) and
spinal descending (Bradbury et al. 2002) systems.
Subsequently, this treatment was shown to suppress the fibrotic
scar formation and promote axonal regeneration of the
ascending dopaminergic pathway (Li et al. 2007).
NPY neurons in the hypothalamic arcuate nucleus (Fig. 2c)
Some neurons in the adult mammalian CNS seem to have
relatively high capacity to regenerate after transection. An
example is the system of neuropeptide Y (NPY) containing
neurons in the hypothalamic arcuate nucleus. Since arcuate
NPY neurons exert a potent orexigenic function, many
experiments have been performed to examine the effect of
their destruction by electrolytic or chemical lesions and
surgical deafferentation of the projection. Alonso and Privat
(1993a) surgically cut NPY axons from the arcuate nucleus
and reported axonal regeneration beyond the lesion site.
They further found that the astrocytic response in this region
differs from other brain regions and suggested that axonal
regeneration of arcuate NPY neurons is attributed to the
particular organization of the glial scar in this region
(Alonso and Privat 1993b).
Administration of gold thioglucose, a neurotoxic glucose
analog, to mice increased their body weight and produced a
hypothalamic lesion that extended from the ventromedial
part of the hypothalamus (Marshall et al. 1955). This
treatment transected axons from arcuate NPY neurons
but 2 weeks later, they regenerated and extended across
the lesion site (Homma et al. 2006). The lesion site was
identified by accumulation of reactive astrocytes but a
fibrotic scar was not formed, suggesting that the absence of a
fibrotic scar may be a permissive factor in the regeneration of
the axons of arcuate NPY neurons.
Over the past 10 years, transplantation of OECs into the
lesion site of the spinal cord has been shown to promote
axonal regeneration and functional recovery (Franklin et al.
1996; Li et al. 1997). In the olfactory bulb, OECs have been
described as opening a pathway through the astrocytic
covering of the CNS (Raisman 1985). The pathway hypothesis
of axon regeneration proposes that nerve fibers will
regenerate if they are able to access an aligned pathway of glial
cell surfaces (Raisman and Li 2007). OECs transplanted into
the lesion site are postulated to provide a pathway for the
regeneration of transected axons by opening up the
arrangement of the astrocytic processes at the scar
interfaces. Possible beneficial effects of OEC transplantation
may also include protection of neuronal degeneration,
secretion of growth factors, tissue sparing, angiogenesis
and remyelination (reviewed in Barnett and Riddell
2004; Radtke et al. 2009). OECs transplanted into injured
rat spinal cord were reported to reduce the formation of glial
scar (Garcia-Alias et al. 2004; Ramer et al. 2004), while others
observed strong astrocytic reaction around the transplanted
tissue (Ramn-Cueto et al. 2000; Teng et al. 2008). Although
CSPG expression around the lesion was also reported to be
reduced by OEC transplantation, neurocan immunoreactivity
was unchanged (Garcia-Alias et al. 2004). In other
studies, high levels of CSPG expression after lesion
were not affected by cell transplantation (Jones et al.
2003; Ramer et al. 2004; Teng et al. 2008).
In rats transected with a nigrostriatal dopaminergic
pathway, few dopaminergic axons extended across the lesion
at 2 weeks after the transection. When OECs were
transplanted into the lesion site, many dopaminergic
axons regenerate to extend over the lesion. In these
animals, the deposition of Type IV collagen and the
fibrotic scar formation were not detectable in the lesion site
(Teng et al. 2008).
Mechanism of fibrotic scar formation
Meningeal cells make an important contribution to the
formation of the fibrotic scar (reviewed by Shearer and Fawcett
2001), although a recent study has reported that a specific
subtype of vascular pericytes also gives rise to scar-forming
stromal cells (Gritz et al. 2011). In rat spinal cord lesions,
where the dura mater was left intact, fibroblastic infiltration
to the lesion site was much reduced (Fernandez and Pallini
1985) and axons regenerated beyond the lesion (Seitz et al.
2002). Duraplasty with cadaveric rat dura mater allograft
over the injury site of the rat spinal cord reduced fibrotic
scarring at the lesion site (Iannotti et al. 2006).
In the intact brain, meningeal fibroblasts are stable or
quiescent, i.e. they express low levels of ECMs and do not
actively proliferate. When the brain is damaged, meningeal
cells are stimulated; they gain mobility to migrate to the
lesion site where they actively proliferate and produce the
ECMs. Although the exact mechanism underlying the
stimulation of meningeal fibroblasts after traumatic injury is not
yet known, it is highly likely that the bleeding in the CNS is
involved in the activation of meningeal fibroblasts. When
the transection of spinal cord was performed by a sharp
knife so that bleeding was minimized, a fibrotic scar was
not formed (Iseda et al. 2003).
After breakdown of the BBB, infiltrating leukocytes and
CNS-resident microglia secrete various cytokines and growth
factors that are involved in the inflammatory response
(reviewed in Donnelly and Popovich 2008; Lenzlinger et al.
2001; Merrill and Benveniste 1996) and they are increasingly
expressed with characteristic spatiotemporal patterns.
Expression of pro-inflammatory cytokines including interleukin 1,
1 and 6, tumor necrosis factor 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 by 24 h after
injury (Bartholdi and Schwab 1997; Nakamura et al. 2003;
Streit et al. 1998). In contrast, expression of TGF-1, an
antiinflammatory cytokine, is delayed and continuous after CNS
injury. TGF-1 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 (Lagord et al.
2002; Nakamura et al. 2003; Semple-Rowland et al. 1995;
Streit et al. 1998). Considering that TGF-1 is a potent
fibrogenic factor that enhances its proliferation and ECM
production of fibroblasts (Ignotz and Massague 1986; Moses
et al. 1987), it seems apparent that TGF-1 is involved in the
activation of meningeal fibroblasts after CNS injury.
The biological action of TGF-1 is mediated through
binding to both type I and type II TGF- receptors (TRI and
TRII). TRII binds to its specific ligand but TRI requires the
presence of bound TRII to interact with TGF-s (Wrana et
al. 1992). As a result, TRI and TRII are co-localized in
many cases on same cells. In adult normal mouse brains,
the expression of TRI and TRII is at a very low level, while
it is upregulated after traumatic CNS injury (Fee et al. 2004;
McTigue et al. 2000) and in multiple sclerosis lesion (de
Groot et al. 1999). After CNS lesioning, TRI and TRII are
expressed on neurons and astrocytes (de Groot et al. 1999),
endothelial cells of the blood vessels (de Groot et al. 1999;
Fee et al. 2004), macrophages (de Groot et al. 1999;
McTigue et al. 2000) and fibroblasts (Komuta et al. 2010).
Fibroblasts bearing receptor mRNAs are first detected in
the meninges and around blood vessels at a 1 day after
injury. Three days after the injury onward, fibroblasts with
receptor messages increase in the lesion site and the
majority of fibroblasts in the fibrotic scar express receptor
mRNAs. Furthermore, TRI and TRII are also expressed in
fibroblasts along the migratory pathway from meninges to
the lesion site (Komuta et al. 2010), indicating that the
meningeal fibroblasts that form the fibrotic scar are a major
target of TGF-1 upregulated after CNS injury.
The manipulation of TGF- signaling in the injured CNS
modulates formation of the fibrotic scar in the lesion site.
The administration of TGF-1 to injured CNS increases the
deposition of ECMs in the lesion site (Hamada et al. 1996;
Logan et al. 1994), while antibodies to TGF-1 and
TGF2 and the endogenous TGF- inhibitor decorin, a small
leucine-rich CSPG, conversely reduce the size of fibrotic
scar (Logan et al. 1994, 1999a, b), which suggests the
involvement of TGF-s in the formation of fibrotic scar.
Receptor activation by TGF-s leads to phospholylation
of Smad2 and Smad3 by the TRI (reviewed in Derynck and
Zhang 2003; Heldin et al. 1997). Phospholylated Smads
interact with a diverse array of transcription factors to bring
about TGF--regulated transcription (Feng and Derynck
2005; ten Dijke and Hill 2004). When LY-364947, a small
molecule inhibitor of TRI, is continuously infused in the
lesion site of mouse brain, the fibrotic scar formation is
completely suppressed (Yoshioka et al. 2011). In Smad3
null mice, expression of fibronectin and laminin was also
reduced (Wang et al. 2007). These results indicate that
inhibition of TGF- signaling is likely to suppress the
formation of the fibrotic scar.
The effect of the inhibition of TGF- signaling on axonal
regeneration is controversial. The majority of authors did
not find the regeneration of transected axons by the
inhibition of TGF- signaling despite the reduction of the scar
tissue (Logan et al. 1994, 1999a, b; Moon and Fawcett
2001; King et al. 2004). In contrast, Davies et al. (2004)
demonstrated that decorin suppressed the deposition of
CSPGs in the lesion site and promoted axon growth from
transplanted sensory neurons, although axonal regeneration
of intrinsic neurons was not described. In mice with
unilateral transection of the nigrostriatal dopaminergic
pathway, dopaminergic axons scarcely extended beyond
the fibrotic scar, while they regenerated over the fibrotic
scar-free lesion site in mice treated with the inhibitor of
TRI, LY-364947 (Fig. 3ac) (Yoshioka et al. 2011).
Microtubule dynamics regulate key processes during
scarring, including cell proliferation, migration and secretion of
ECMs (Liu et al. 2005; Westermann and Weber 2003).
Moderate microtubule stabilization with Taxol reduces the
formation of fibrotic scar and allows axonal regeneration
and functional recovery after spinal cord injury (Hellal et al.
2011). Taxol treatment hinders Smad2 trafficking in TGF-
signaling, reduces the TGF-1-stimulated production of
fibronectin in cultured meningeal cells and impairs
TGF-1stimulated migration, thus reducing fibrotic scarring after
spinal cord injury.
In vitro model of the CNS lesion site
Attempts to reproduce the regeneration-inhibitory property
of the CNS lesion site in vitro have been repeatedly
performed using coculture of cerebral astrocytes and meningeal
fibroblasts (Abnet et al. 1991; Hirsch and Bahr 1999;
Struckhoff 1995). When cocultured, the two kinds of cells
separately form flat colonies and rarely overlap each other
(Fig. 3dg). Such an interface may be considered as
providing an in vitro model of the glia limitans (Abnet et al. 1991),
which is the lining of astrocytic processes surrounding the
fibrotic scar in the CNS lesion site (Berry et al. 1983). In a
similar co-culture system, meningeal fibroblasts express
moderate amounts of the axonal growth-inhibitory
molecules NG2 proteoglycan, versican and class 3 semaphorins,
while astrocytes express the axonal growth-promoting
molecules N-cadherin and laminin (Shearer et al. 2003). More
recently, Wanner et al. (2008) demonstrated that an addition
of meningeal fibroblasts to cultured astrocytes enhanced
expression of GFAP, the CSPGs phosphacan and neurocan and
tenascin-C in astrocytes. Modeling traumatic injury by
mechanically stretching the co-culture did not further activate
astrocytes. In these co-cultures, major characteristics of the
fibrotic scar, i.e., proliferation of fibroblasts, dense
accumulation of ECMs and high expression of axonal
growthinhibitory molecules are not observed (Shearer and Fawcett
2001; Shearer et al. 2003; Wanner et al. 2008).
Addition of TGF-1 to the coculture of cerebral astrocytes
and meningeal fibroblasts resulted in enhanced proliferation
Fig. 3 Role of the TGF- on the formation of the fibrotic scar, which
inhibits axonal regeneration. a Schematic drawing of the transection of
mouse brain (Kawano et al. 2005). Ascending dopaminergic axons that
arise from the substantia nigra and ventral tegmental area project to the
telencephalic structures are cut at the proximal part of the striatum
(green line) with a knife of 2 mm width. b The fibrotic scar containing
dense Type IV collagen (Col IV) deposits (red) is formed in the lesion
site 2 weeks after injury and transected tyrosine hydroxylase
(TH)immunoreactive dopamine (DA) axons (green) stop at the fibrotic scar.
c Continuous injection of the inhibitor of TGF-, LY-364947 into the
lesion site completely suppresses the fibrotic scar formation and
promotes axonal regeneration (Yoshioka et al. 2011). dk In vitro model
of the lesion scar (Kimura-Kuroda et al. 2010). dg Meningeal
fibroblasts (magenta) and cerebral astrocytes (green) form separate colonies
in coculture. Cerebellar neurons grow better on astoricytes than on
fibroblasts. hk When TGF-1 is added to the coculture, cells
aggregate to form a fibrotic scar-like cluster, which repels neurites of
cerebellar neurons (blue). Scale bars (b, c) 200 m, (dk) 100 m
of fibroblasts and the formation of cell clusters, which
consisted in fibroblasts inside and surrounded by astrocytes
(Kimura-Kuroda et al. 2010). The cell cluster in culture
densely accumulated the ECMs and axonal growth-inhibitory
molecules similar to the fibrotic scar. The expression of Type IV
collagen, NG2, CS, phosphacan, semaphorin 3A, EphB2 and
tenascin-C in fibroblasts and neurocan, phosphacan,
ephrinB2 and tenascin-C in astrocytes, was greatly enhanced in the
cluster induced by TGF-1. In this coculture, the neurite
outgrowth of cerebellar neurons was promoted on astrocytes,
inhibited on fibroblasts and remarkably suppressed on the
cluster (Fig. 3hk). In this aspect, this culture system mimics
a CNS lesion site and may provide a model to analyze the
inhibitory property in the lesion site of CNS (Kimura-Kuroda
et al. 2010).
In peripheral tissues, TGF-1 is known to affect various
kinds of mesodermal cells to induce physiological and
pathological fibrosis (for review, see Cutroneo 2007; Wynn
2008; Kisseleva and Brenner 2008). A variety of cultured
mesenchymal cells from kidney, heart, lung, liver, spleen
and skin form aggregates and actively produce ECMs when
stimulated by TGF-1 (Xu et al. 2007).
Virtually all lesions of the CNS open the BBB, whether
from outside, through the pia, or from blood vessels within
the CNS tissue. The rapid post-injury response by the
astrocytes serves to seal this breach in the BBB. Subsequently,
there is a fibroblastic reaction around the astrocytes.
Astrocytes and fibroblasts interact to form an organized tissue
(the scar). By sealing off the damage and restoring the
BBB, the astrocytic reaction is protective. Both astrocytes
and fibroblasts express abundant axon-repelling molecules.
Suppression of TGF- signaling has been shown to be an
effective tool for preventing formation of the fibrotic scar and
has been reported to promote axonal regeneration without
detrimental effects on the sealing process of damaged CNS.
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