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Cell Tissue Res (1998) 294:19 Springer-Verlag 1998

Christine C. Stichel Hans Werner Mller
The CNS lesion scar: new vistas on an old regeneration barrier
Received: 19 June 1998 / Accepted: 23 July 1998
Studies in the authors' laboratory were supported by the Deutsche
Forschungsgemeinschaft (SFB 194/B5 and Graduiertenkolleg) and
a Heisenberg Stipendium to C.C.S.
C.C. Stichel (
) H.W. Mller
Molecular Neurobiology Laboratory, Department of Neurology,
Heinrich-Heine University, Moorenstrasse 5,
D-40225 Dsseldorf, Germany
Tel.: +49 0211 81 17822; Fax: +49 0211 81 18485;
Abstract Scar formation represents a reaction of nervous
tissue to any form of physical injury. Research over the
past decade has demonstrated that the scar composed of
glial cells and several extracellular matrix molecules con-
stitutes an obstacle to axon regeneration in the CNS. This
review briefly summarizes the current knowledge on (a)
the structural and functional features of the lesion scar
and (b) the development of therapeutic interventions to
override this regeneration barrier.
Key words Axonal regeneration CNS injury
Growth inhibition Gliosis Extracellular matrix
Basal membrane Therapeutic approaches
Mechanical injury to the adult mammalian CNS always
results in the formation of a scar. This cardinal histopath-
ological reaction at the site of lesion was first noticed by
Ramn y Cajal (1928), who also postulated a causal role
of this response in axonal regeneration failure: Neither is
it rare to see conductors that turn back on encountering
the scar, near which they accumulate, forming complicat-
ed plexuses, as though the edge of the nerve presented an
obstacle that the cones find it very difficult to cross.
Glial cell transplantation studies and the recent concept
of an extrinsic determination of axonal regeneration have
renewed the interest in lesion scar influences in axonal re-
growth. Over the past decade considerable advances have
been made in identifying the scar components and in de-
fining those factors that are responsible for axonal growth
arrest. Most importantly, recent studies have led to signif-
icant progress in the development of new concepts to
overcome the regeneration-inhibiting scar. They demon-
strate that the reestablishment of injured fiber tracts
across the lesion site and through the original pathway
is a goal that can be achieved. These new regeneration-
stimulating strategies hold promise as therapeutic ap-
proaches for traumatic injuries and neurodegenerative dis-
eases of brain and spinal cord.
Cellular and molecular composition of lesion scars
Injury to the CNS leads, like in all other organs of the
body, to the formation of a scar located at the site of le-
sion. This rapid and complex process includes both meso-
dermal cells, such as fibroblasts, endothelial and hema-
togenous cells, and glial cells, such as astrocytes and mi-
croglia/macrophages (Table 1). After lesion many glial
cells start to proliferate and change their morphological
and functional properties. Together with exudated serum
factors they induce the second major type of histopatho-
logical alteration within the scar area: a profound change
in the composition of the local extracellular matrix
(ECM) (Table 1).
Although different glial cells participate in CNS scar
formation, the reactive astrocytes play the predominant
role (Lindsay 1986). The vigorous response of astrocytes
Table 1 Lesion scar components
Cellular Extracellular
Reactive astrocytes Collagen IV
Microglia/macrophages Laminin
Endothelial cells Fibronectin
Fibroblasts Chondroitin sulfate proteoglycans
Heparan sulfate proteoglycans (perlecan)
Keratan sulfate proteoglycans
Tenascin C
Basal membrane
as reflected by morphological changes and by an increase
in the synthesis of glial fibrillary acidic protein (GFAP) is
a stereotyped reaction to any type of CNS insult (Fig. 1F).
It is also well conserved across a variety of species and
extends even in areas distant from the lesion interface
(Frank and Wolburg 1996; Maxwell et al. 1990; McLoon
1986; Puckett et al. 1997; Sivron et al. 1990; Stichel and
Mller 1994a). The reactive astrocytes form a dense web
of interdigitated processes, called (astro)gliosis, which
fills the space vacated by the dead or dying cells. These
structural changes are accompanied by diverse physiolog-
ical alterations. While the upregulation of GFAP is a
global response of astrocytes (Fig. 1F), various other mol-
ecules have been identified which are up- or downregulat-
ed in all or only in subsets of reactive astrocytes (Fig. 1J
L). Interestingly, many of these molecules are already ex-
pressed during normal development and are downregulat-
ed when development is complete (Brodkey et al. 1993;
Aubert et al. 1995). There are excellent reviews with a
comprehensive listing of astrocytic molecules already
available (Eddleston and Mucke 1993; Ridet et al.
1997) that need not be rereviewed in this paper. However,
one important group of astrocytic proteins should be men-
tioned in a paper dealing with axonal regeneration. These
are the glycoproteins laminin (Fig. 1G), fibronectin, ten-
ascin C (Fig. 1J) and the large family of proteoglycans
(Fig. 1K). In vitro studies, showing their various and
sometimes conflicting growth-modulating effects as well
as their neurotrophic activities, imply that they play an
important role in the regeneration process (for reviews
see Faissner 1997; Fawcett 1997; Hke and Silver 1996;
Mller et al. 1996).
During recent years evidence has accumulated that the
astrocytic response is heterogeneous and differs depend-
ing on the nature of the injury, the proximity to the lesion
and the microenvironment at the lesion site (David and
Ness 1993; Fernaud-Espinosa et al. 1993; Hatten et al.
1991). Accordingly, gliosis can be classified as anisomor-
phic or isomorphic, based on whether or not the eliciting
stimulus is an open injury or not. In the case of anisomor-
phic injury, astrocytes proliferate and express a different
set of molecules than their isotropic counterparts
(Fernaud-Espinosa et al. 1993; Ridet et al. 1997;
Bovolenta et al. 1992). Moreover, their functional proper-
ties may also depend on the distance to the lesion site.
Thus, reactive astrocytes in the immediate vicinity of a le-
sion generally display more excessive changes in gene ex-
pression patterns, e.g., increased expression of ECM mol-
ecules. This astrocytic heterogeneity might reflect differ-
ences in the factors or signals that induce the reactive
changes and depend on the extent of damage or degener-
A similar heterogeneous response has been reported
for the second prominent cell type of the scar, the micro-
glial cells. They are rapidly activated in a wide area
around the lesion site and undergo a profound change in
cell shape and phenotype (Fig. 1D) (Perry and Gordon
1988; Streit et al. 1988). Microglial cells and their phago-
cytic form, the macrophages (Fig. 1E), are found to se-
crete a number of cytokines, bioactive lipids, coagulation
factors, reactive oxygen intermediates and also neurotro-
phic factors (Auger and Ross 1992; Elkabes et al. 1996;
Chamak et al. 1994). The expression of these molecules
depends on the proximity to the lesion site, but is always
more prominent in the immediate surroundings of the le-
sion center. Within the latter these cells intermingle with
several blood vessels. Indeed, stimulated by various an-
giogenic factors (Klagsbrun and D'Amore 1991), endo-
thelial cells start to proliferate, to migrate and to synthe-
size matrix proteins (Landis 1994). After injury the mean
number of capillary profiles in the lesioned area is twice
that of uninjured regions and the new blood vessels exhib-
it an increased total luminal area and supernumerary lay-
ers of basal lamina (Jaeger and Blight 1997; Blight 1991;
Kalaria and Pax 1995).
All of these cells produce a variety of cytokines,
growth factors, cell adhesion and matrix molecules after
injury. Most of these molecules are soluble and are liber-
ated into the extracellular space (ECS) (Fig. 1GM)
(Nicholson and Sykova 1998). Formerly referred to as
ground substance, the matrix of this ECS, the so-called
extracellular matrix (ECM), represents a sort of reservoir,
in which the secreted factors organize into a superstruc-
ture of macromolecules (Rutka et al. 1988; Faissner
1997; Carbonetto 1984) and imbed the neurons and glial
cells. However, the ECM is a dynamic structure and
changes in composition, density and architecture after in-
jury. One of the most dominant histopathological ECM
features formed is the basal membrane (BM) (Timpl
and Dziadek 1986; Choi 1997; Yurchenco and O'Rear
1994; Paulsson 1992). Within the lesion site extensive de-
posits of BM are laid down (Fig. 1H) (Feringa et al. 1980;
Berry et al. 1983; Bernstein et al. 1985; Maxwell et al.
1984), the density of BM around blood vessels increases
(Kalaria and Pax 1995) and even astrocytic processes be-
come partially covered by thin BM sheets (Jaeger and
Blight 1997). The BM shows a three-layered structure
with the basal lamina as one prominent layer. The mole-
cular architecture of the BM is created by glycoprotein
and proteoglycan protomers, with two independent net-
works one formed by collagen IV (Coll IV) (Fig. 1H)
and the other by laminin (LN) (Fig. 1G). While it is
known from studies in the kidney and tumor tissue that
within this Coll IV/LN scaffold 50 or more constituents
may be anchored, the list of well-characterized BM com-
ponents, especially in the CNS, is rather short. Besides
Coll IV and LN, the major BM components, entactin/nid-
ogen, fibronectin, BM-40/osteonectin/SPARC (secreted
protein acidic and rich in cysteine), fibulin-1 and the hep-
aran sulfate proteoglycan perlecan were identified and
also the presence of chondroitin sulfate proteoglycans in
Fig. 1AM Summary of cellular and molecular changes in the
proximal postcommissural fornix and the transection site at 9
14 days post-lesion. At this time point the spontaneously sprouting
fibers reached the lesion site (A). Scale bars 50 m (reprinted with
permission from Stichel-Gunkel 1997)
brain BM has been shown (Couchman et al. 1984; Stichel
et al. 1998).
The proteins LN and Coll IV are major mediators of
contacts between BM and cells. Many but not necessarily
all signals provided by these two proteins are transduced
by the integrin class of cellular receptors (Hall et al. 1990;
Kramer and Marks 1989; Joosten 1996). Thus, it is known
that astrocytes attach to the BM and both together form
the so-called glia limitans, which surrounds the blood ves-
sels and covers the CNS.
Finally, the lesion scar might be characterized as a
gross cellular and molecular rearrangement of tissue mor-
phology with a series of common motifs (i.e., the overex-
pression of GFAP or the formation of a BM) to which
several other specific factors are added, depending on
the type of injury and the brain region damaged. Today,
we are far from having a complete description of all scar
components. However, more and more pieces are becom-
ing available and give rise to hope that this scar puzzle
will be completed soon.
Lesion scar functions
Having explored the dynamics and cellular and molecular
features of the lesion scar, the next issue to be addressed
is that of the functional consequences of such a tremen-
dous reorganization of tissue. What is the purpose of this
profound modulation, and does the CNS benefit from
scarring? The answer is, it certainly does. The lesion scar
reestablishes the integrity of the CNS by sealing it off
from the external environment. The latter effect allows re-
establishment of the diffusional barrier between the CNS
and its environment and prevents the CNS from infection
and further spread of damage. The BM exhibits contrac-
tile properties and therefore another job of scarring a
wound is to bring the wound margins toward one another
in order to shrink the lesion cavity and to reconstruct the
damaged parts. The lesion scar is then heavily invaded by
epithelial cells that reorganize the blood vessels of the
scar tissue. The resulting increased revascularization pro-
vides the nutritional, trophic and metabolic support essen-
tial for wound healing.
Scarring and regeneration failure
The above-mentioned beneficial effects of the scarring
process contrast with its clear inhibitory effects on the re-
generation of interrupted projections. In a long series of
experiments stretching back many years the lesion scar
has been identified as one critical element that impedes
axonal regeneration in the adult mammalian CNS (Ramn
Fig. 2A, B The influence of Schwann cell suspension implants on
axonal regeneration in the transected postcommissural fornix. A In
animals with transection only axons sprout up to the lesion site (ar-
row), where they stop. B In animals with an additional Schwann cell
implant the axons cross the lesion/implantation site (arrow) and en-
ter their former pathway, the distal fornix stump (d distal, p proxi-
mal). Scale bars 100 m
y Cajal 1928; Reier et al. 1983; Guth et al. 1983). After
lesion CNS axons start to sprout over short distances,
but almost all of them stop abruptly at the lesion scar bor-
der and fail to traverse it (Figs. 1A, 3A,B) (Stichel and
Mller 1994b; Blaugrund et al. 1993; Richardson et al.
1982; Schnell and Schwab 1993). Only single axons ei-
ther emit short sprouts into the scar, where they end local-
ly (Frisn et al. 1993; Li and Raisman 1995), or they are
deflected and bypass the lesion scar via bridges of con-
nective tissue, blood vessels or surviving tissue (Clemente
1955; Guth et al. 1983; Krger et al. 1986).
Reasons offered to explain the inhibitory nature of the
scar include (a) the accumulation of growth-inhibitory
molecules (molecular barrier) and (b) the formation of a
dense, impenetrable matrix within this area (physical bar-
Due to their dominant role in scar formation and their
diverse physiological changes after lesion, reactive astro-
cytes are suggested to play a key role in the formation of a
regeneration barrier. They (a) express several potential
growth-inhibitory molecules, such as tenascin C or
proteoglycans (Ajemian et al. 1994; Grierson et al.
1990; Bartsch et al. 1992; Stichel et al. 1995a; Canning
et al. 1996; Geisert et al. 1992), and (b) are able to form
a dense network with a strikingly high number of gap
junctions (Alonso and Privat 1993b). The fact that axons
succeed in entering and elongating within dense astrogli-
otic tissue (Alonso and Privat 1993b; Lips et al. 1995;
Stichel et al. 1994b; Alonso and Privat 1993a; Frisn et
al. 1993; Li and Raisman 1995) or a proteoglycan-rich
perilesional area (Lips et al. 1995; Stichel et al. 1995a)
encourages the view that reactive astrocytes and their
products are not a priori impermeable barriers for axons.
The BM, which is always deposited in the center of a
scar, might be another candidate responsible for abortive
axonal regeneration. Due to its dense architecture and in-
teraction with several growth-modulating molecules
(Timpl and Dziadek 1986), the BM might either create
a mechanical obstacle, a molecular barrier or both for re-
growing axons.
Recent experiments in our laboratory have renewed in-
terest in the lesion-induced BM formation and have cast
new light on its role in regeneration failure. In our lesion
model, the mechanically transected postcommissural for-
nix, sprouting fornix axons cross astrogliotic tissue rich in
Fig. 3 Sections reacted for neurofilament immunohistochemistry
show that in untreated animals (A, B) sprouting axons stop abruptly
at the lesion site (arrow), while in animals with dipyridyl injection
(C, D) axons succeed in crossing the lesion site (arrow) and enter
the distal fornix stump (d distal, p proximal). Scale bars 100 m
(A, C), 50 m (B, D)
both chondroitin/keratan sulfate proteoglycans and tenas-
cin C (Stichel et al. 1995; Stichel and Mller 1994b; Lips
et al. 1995), but abruptly stop growing at the BM in the
lesion center. The BM formed within the 1st week postle-
sion and penetrated the full depth of the lesion. It ap-
peared as a parallel array of BM sheets aligned perpendic-
ular to the course of the tract and intermingled with sev-
eral blood vessels. Confocal microscopy of double-la-
beled sections revealed that axons either arrest close to
BM deposits or make hook-like turns and stop some dis-
tance from the BM (Fig. 3A,B). The temporal coincidence
of BM maturation and the arrival of regrowing axons as
well as the close apposition of growth-arrested axons at
the BM sheets suggest that the BM is the inhibitory con-
stituent of the impermeable lesion scar.
Strategies to overcome the lesion scar
In the past a substantial number of different experimental
approaches have been made to drive regrowing axons,
e.g., by the use of neurotrophins (Bregman et al. 1997;
Houle et al. 1996; Tuszynski et al. 1996), the transplanta-
tion of nerve grafts (Vidal-Sanz et al. 1987; Cheng et al.
1996), Schwann cells (Harvey et al. 1995; Paino et al.
1994; Stichel et al. 1996; Xu et al. 1995), olfactory en-
sheathing cells (Ramon-Cuto et al. 1998; Ramon-Cuto
and Nieto-Sampedro 1994; Li et al. 1997) and micro-
glia/macrophages (Franzen et al. 1998; Rabchevsky and
Streit 1997; Lazarov-Spiegler et al. 1996) or the neutral-
ization of myelin-associated growth inhibitors (Schnell
et al. 1994; Schnell and Schwab 1990). Most of these ap-
proaches succeeded in stimulating axon growth but failed
to elongate the lesioned axons within their appropriate
pathway. However, a topographically correct regeneration
is a prerequisite for the reestablishment of original topo-
graphic reinnervation, and the more precise the reinnerva-
tion, the greater the functional improvements will be.
Thus, it emerges that a crucial and outstanding problem
of regeneration-promoting strategies is the accuracy rath-
er than the vigor of regeneration.
The lesion scar seems to be a major barrier that dis-
turbs the required continuous pathway and prevents a to-
pographic reconstruction of a lesioned pathway. Several
decades ago Windle and colleagues (Windle et al. 1951)
made the pioneering studies of injecting a pyrogenic bac-
terial polysaccharide, pyromen, into the lesioned spinal
cords of cats. They reported both reduction of astrogliosis
and enhanced neuritic regeneration. Despite the fact that
the regeneration was not permanent, they provided the
first evidence that the scar constitutes a barrier to regrow-
ing axons. Subsequent studies have applied a variety of
other manipulations, to interfere with the physiology of
the reactive astrocyte. These include surgical removal of
the scar, administration of hormones (Puchala and Windle
1977) and more recent strategies such as X-irradiation
(Kalderon and Fuks 1996) and the application of gluco-
corticoids (Li and David 1996) or antibodies to transform-
ing growth factor b (TGFb) (Logan et al. 1994) (Table 2).
Only single of these approaches improved the regenera-
tion outcome, but they failed to allow regeneration within
the normal pathway, which is a prerequisite for topo-
graphic and coordinate synaptic reinnervation.
A second series of experiments was performed to alter
the texture or density of the BM. In these early efforts en-
zymes such as collagenase, trypsin or elastase were inject-
ed into a damaged region (Freeman 1952; Puchala and
Windle 1977; Guth et al. 1980) to degrade the BM (Ta-
ble 2). Unfortunately, the unavoidable, additional attack
of the BM of blood vessels led to severe changes in vas-
cular supply and limited the regeneration success. Anoth-
er more indirect way to modify the nature of the BM is the
implantation of glial cells, such as astrocytes or Schwann
cells (Emmett et al. 1988; Smith and Miller 1991;
Wunderlich et al. 1994; Stichel et al. 1996; Raisman et
al. 1993; Harvey et al. 1995; Martin et al. 1991; Brook
et al. 1994) (Table 2). In our own work we have used
the minimal invasive microtransplantation approach to in-
troduce a Schwann cell suspension into the transected
postcommissural fornix (Stichel et al. 1996). We were
able to show that the injected Schwann cells become inte-
grated into the neuropil and promote structural recovery
of the projection. The implanted cells seem to interfere
with the scarring process by (a) establishing a guiding
framework, (b) disaggregating the dense cellular network
of the scar and/or (c) physically preventing the infiltration
of scar-promoting cells. In line with the latter effect,
Campbell and coworkers (Galuske et al. 1996) tended to
protect the lesion site from encroachment by collagenous
scar tissue by wrapping the severed ends of the spinal
cord into porous material. The porous material became
calcified and thereby impermeable to regrowing axons.
Recently, we developed a new experimental strategy to
reduce the lesion-induced deposition of the BM. A local
injection of either antibodies against Coll IV or 2, 2di-
pyridyl (DPY), an inhibitor of collagen triple helix forma-
tion and synthesis, interfered with the posttranslational
processing of the major BM component, Coll IV. This
treatment allowed massive axonal regeneration across
the former impermeable lesion scar (Fig. 1C,D). Regrow-
ing fornix axons followed their former pathway, reinner-
vated their normal target, were remyelinated and recov-
Table 2 Experimental strategies to overcome the lesion scar
Excision of scar
Implantation: Porous material
Glial cells
Infusion/injection: Pyrogenic bacterial polysaccharide
Trypsin, elastase, collagenase
Antibodies against transforming growth
factor b
Antibodies against collagen IV
Prolylhydroxylase inhibitor
ered normal conduction properties (Stichel et al. 1997,
1998a). The application of collagen-reducing agents did
not affect the quantity of vascular BM in the surrounding
neuropil (Stichel et al. 1998a) nor did the BM reduction
influence the distribution pattern and density of reactive
astrocytes or microglial cells within the scar and the peri-
lesion area (Stichel et al. 1998b).
However, up to now the nature of the inhibitory mech-
anisms mediated by the BM is still a matter of specula-
tion. Since BM might possess many holes and fenestra-
tions with a diameter of 0.55 m (Komuro 1985; Warfel
and Hull 1988), it is unlikely that the BM represents a
physical barrier for growing axons. Another potential
source of BM-mediated growth inhibition could be repul-
sive molecules associated with the membrane matrix. In
support of this assumption several reports show that
growth-inhibitory proteoglycans, such as chondroitin sul-
fate and heparan sulfate proteoglycans (Ard and Bunge
1988; Snow et al. 1990; Snow et al. 1991), are major con-
stituents of the BM (Timpl and Dziadek 1986; Stichel et
al. 1998b; Couchman et al. 1984). On the other hand, it is
apparent that the myelin-associated proteins NI35/250
and MAG are not involved in the formation of such an in-
hospitable territory. Evidence for this conclusion is (a) ab-
sence of these molecules from the scar tissue (Fig. 1C)
(Stichel et al. 1995b), (b) failure of neutralizing approach-
es to stimulate axonal regeneration across the scar tissue
(Schnell and Schwab 1990, 1993) and (c) the fact that my-
elin or myelin debris beyond the lesion scar can be highly
permissive for growing axons (Davies et al. 1997; Stichel
et al. 1995b).
Taken together, recent data provide strong evidence
that the lesion-induced BM itself is the primary determi-
nant of scar impermeability and hence a major constraint
to axonal regeneration. Exactly what aspect of this matrix
comprises the regeneration barrier is not yet defined. In
the future it will be of crucial importance to characterize
the molecular composition of the BM in CNS scar and to
define the role of its components in the process of axonal
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