expert reviews

http://www.expertreviews.org/ in molecular medicine

Extrinsic and intrinsic factors controlling axonal regeneration

after spinal cord injury
Extrinsic and intrinsic factors
controlling axonal regeneration
after spinal cord injury
Fardad T. Afshari*, Sunil Kappagantula and James W. Fawcett
Spinal cord injury is one of the most devastating conditions that affects the central
nervous system. It can lead to permanent disability and there are around two
million people affected worldwide. After injury, accumulation of myelin debris
and formation of an inhibitory glial scar at the site of injury leads to a physical
and chemical barrier that blocks axonal growth and regeneration. The
mammalian central nervous system thus has a limited intrinsic ability to repair
itself after injury. To improve axonal outgrowth and promote functional
recovery, it is essential to identify the various intrinsic and extrinsic factors
controlling regeneration and navigation of axons within the inhibitory
environment of the central nervous system. Recent advances in spinal cord
research have opened new avenues for the exploration of potential targets for
repairing the cord and improving functional recovery after trauma. Here, we
discuss some of the important key molecules that could be harnessed for
repairing spinal cord injury.
Worldwide, over two million people are affected cord injuries requires regeneration of damaged
by spinal cord injury, which is a heterogeneous axons or stimulation of the growth of spared
condition, depending on the cause and the axons at the site of injury to allow possibility of
severity of the injury. Injury can be caused by any functional recovery. The failure of cut axons
contusion, compression and laceration, of in the spinal cord to regenerate or sprout has
which contusion as a result of fracture many causes. These can be categorised into
dislocation of the spine is the most common extrinsic inhibitory effects and the intrinsically
(Ref. 1). Trauma to the spinal cord leads to low ability of central nervous system (CNS)
primary loss of cells at the site of injury. This axons to regenerate.
initiates various inflammatory responses, which Formation of a glial scar and the presence of
together with other cytotoxic events, leads to various inhibitory molecules at the site of injury
secondary damage and formation of fluid-filled lead to the extrinsic inhibition of axon growth,
cysts, demyelination and degeneration of axons which prevents any substantial regrowth and
at the site of injury (Ref. 2). Repair of spinal sprouting of the axons following injury.

Centre for Brain Repair, University of Cambridge, Cambridge, UK.

*Corresponding author: Fardad T. Afshari, Centre for Brain Repair, University of Cambridge, Forvie
Site, Robinson Way, Cambridge, CB2 0PY, UK. Tel: +44 1223 334121; Fax: +44 1223 331174; E-mail:
ft218@cam.ac.uk

1
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Reactive gliosis is an injury-induced change in the
behaviour of cells at the site of injury, primarily by
astrocytes and meningeal cells, which ultimately
culminates in the formation of glial scar tissue at
the site of injury (Ref. 3). Reactive gliosis is
associated with proliferation and hypertrophy
of astrocytes and oligodendrocyte precursors,
with invasion of fibroblastic meningeal cells
following the disruption of the blood – brain
barrier (BBB) (Refs 3, 4). Reactive gliosis and
glial scar formation are both important in the
early stages of injury. They limit further
damage by suppressing inflammation and
preserve function by establishing a boundary
between damaged tissue and salvageable tissue
(Refs 5, 6, 7). Depletion of reactive astrocytosis
after injury prevents re-establishment of the
BBB and is associated with increased leukocyte
infiltration, severe demyelination and increased
cell death (Ref. 7). Experiments have shown
that interaction of invading meningeal cells
with reactive astrocytes is important in the
formation of a new glia limitans and resealing
of the BBB (Refs 7, 8, 9). However, although
glial scar formation is very important for tissue
preservation, it acts as a physical (Ref. 10) and
chemical barrier to axon regeneration (Refs 11,
12). Within the glial scar, there is upregulation
of various inhibitory factors capable of blocking
axonal growth (Ref. 4). There is no single
trigger to the formation of glial scars. Instead,
there are many cytokines and chemokines
involved, including TGF-b (TGFB) (Ref. 13),
interferon-g (IFNG) (Ref. 14), interleukin (IL)-1
(Ref. 15) and IL-6 (IL6) (Refs 5, 6), which are
released at the site of injury as a result of
inflammation and immune cell infiltration. This
then leads to astrocytic hypertrophy,
proliferation and upregulation of inhibitory
factors, such as chondroitin sulphate
proteoglycans (Ref. 3). The other major sources
of inhibitory molecules for axon regeneration in
the damaged spinal cord are myelin and myelin
debris, which also have an important role in
preventing axonal regeneration and growth
(Refs 16, 17).
For axons to regenerate successfully, they must
be able to initiate the process of regrowth followed
by a dynamic process of continuation to reach the
necessary target for synapse formation. However,
axons vary greatly in their intrinsic regenerative
ability, with most CNS axons being poor
regenerators. Soon after injury, an axon has to
Accession information: doi:10.1017/S1462399409001288; Vol. 11; e37; December 2009
expert reviews
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
undergo a series of cell-autonomous changes at
the injury site. The first step is the sealing of the
plasma membrane at the cut end, followed by
disintegration of the cytoskeletal elements,
retraction of axon from the injury site,
formation of a growth cone, regrowth to the
target area, cessation of growth and
synaptogenesis. The ability of axons to
complete this process varies. It is dependent
on the neuronal type (peripheral neurons
regenerate better than central neurons), age
(embryonic and neonatal neurons regenerate
better than adult neurons) and distance of site
of injury from the cell body (axonal injuries
closer to the cell body regenerate better). In
addition, there are also changes in the pattern
of gene expression, which modulate the
responses to axonal injury.
To achieve efficient axonal regeneration and
functional recovery, a combined approach will
be essential, in which the external environment
of the axon is made more permissive by (1)
promoting the factors supporting regeneration
and (2) blocking the external elements
inhibiting regeneration. In addition, the
intrinsic ability of axonal outgrowth must be
manipulated to enhance the vigour of the
regenerative response.
Extrinsic factors
Modification of the extrinsic environment for
promoting axonal regeneration has progressed
significantly in the past two decades. Some of
these interventions work by removing the
inhibitory elements within the CNS and glial
scar, and others by promoting and creating a
more permissive environment to support
regeneration and sprouting of the axons.
Myelin inhibitors
Myelin is a major inhibitory component of the
CNS and many studies have examined the
mechanism of inhibition of axons by myelin
inhibitors. One myelin-associated molecule,
Nogo-A (RTN4) was the first inhibitor to be
isolated (Refs 18, 19). Application of the Nogo-
A-blocking monoclonal antibody IN-1 in vitro
(Ref. 18) and in vivo inhibits the effect of the
Nogo-A isoform and leads to substantial axonal
sprouting in the injured adult CNS (Ref. 20).
Other monoclonal antibodies, mostly targeting
the extracellular N-terminus of the molecule,
also block the inhibitory effects of Nogo-A on
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axons, and promote axon regeneration in the
CNS. Anti-Nogo antibodies have also been
shown to promote the sprouting of
corticospinal axons in marmosets (Ref. 21) and
macaque monkey (Ref. 22) after spinal cord
injury. A humanised version of the anti-N-
terminus Nogo monoclonal antibody is
currently undergoing clinical trials in acute
spinal cord injury (Ref. 23).
Inhibition of Nogo has been attributed to two
separate domains on the molecule. One is a 66-
residue loop (Nogo-66) located between two
putative transmembrane portions of Nogo
(Ref. 24) and the other is at the N-terminus
(Ref. 25). Nogo-66 interacts with a glycosyl
phosphatidylinosital (GPI)-linked receptor – the
Nogo receptor (NgR or RTN4R) – that is
expressed in various neurons (Ref. 26).
Introduction of NgR into cells that normally do
not respond to this myelin inhibitor confers
sensitivity to Nogo. Both p75 (NGFR) (Ref. 27)
and Lingo-1 (LINGO1) (Ref. 28) have been
identified as coreceptors for NgR. A truncated
peptide comprising residues 1-40 of Nogo
(NEP1-40) has been shown to act as a
competitive antagonist for the binding of Nogo-
66 to NgR. Application of NEP1-40 in spinal
cord injury models in rats has led to increased
corticospinal sprouting with significant
functional recovery (Ref. 29).
It is important to remember that Nogo is,
however, only one of many inhibitors present
within the CNS. Knockout mice produced to
study the effect of Nogo after spinal cord injury
have provided variable and conflicting results
on regeneration of corticospinal tracts (Refs 30,
31). There are three Nogo isoforms, and some
authors have proposed that the lack of
regeneration in studies targeting only one or two
of these can be explained by a compensatory
increase in the remaining isoforms, and some
studies have been confounded by axon-labelling
artefacts (Ref. 32). A recent report reassessed the
effect of Nogo by removing most of the
previous confounding factors, using a reliable
tracing dye and deleting all Nogo isoforms, in
addition to reanalysing the mouse line
previously reported to promote regeneration
(Ref. 33). The authors conclude that deletion of
all Nogo isoforms and other confounding
factors did not lead to any corticospinal tract
regeneration. Therefore, the removal or
neutralisation of this molecule alone is not
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expert reviews
in molecular medicine
Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
sufficient to significantly promote axon
regeneration (Ref. 33), and might not provide
any measurable therapeutic effect.
Other myelin-associated inhibitory molecules
include myelin-associated glycoprotein (MAG)
and oligodendrocyte myelin glycoprotein
(OMG). MAG is a transmembrane protein
belonging to the immunoglobulin superfamily
and is present in both the CNS and peripheral
nervous system (PNS) (Ref. 34). MAG inhibits
axon growth from adult dorsal root ganglion
(DRG) and cerebellar granule cells (Refs 35, 36),
through interaction with the NgR and
gangliosides (Refs 37, 38, 39). OMG is a GPI-
linked protein that is abundant in the CNS. It
causes growth-cone collapse in many neuronal
populations (Refs 40, 41). Remarkably,
both of these molecules have also been
shown to act through the NgR receptor.
A polyclonal antibody to NgR, a soluble
version of NgR and a dominant-negative NgR
mutant are all capable of blocking inhibition of
neurite outgrowth by MAG (Ref. 39). A
separate study identified NgR as an OMG
receptor and showed that expression of NgR
in DRG neurons confers sensitivity to OMG
(Ref. 42).
Engagement of NgR with myelin ligands has
been associated with activation of RhoA and
downstream pathways, which is known to
inhibit neurite extension (Refs 43, 44).
Identification of the same receptor complex for
three major myelin inhibitors, Nogo, MAG and
OMG, therefore opens up a new therapeutic
avenue that is capable of intercepting several
myelin-associated inhibitors at once. However,
the mechanism of action of the N-terminus of
Nogo is not clearly understood, although recent
studies suggest a role in the disruption of
integrin function (Ref. 25).
Chondroitin sulphate proteoglycans
Proteoglycans are a major inhibitory component
of the glial scar (Ref. 45). Astrocytes, the main
cell type of the glial scar, are capable of
producing four classes of proteoglycans,
including heparan sulphate proteoglycan,
dermatan sulphate proteoglycan, keratan
sulphate protoglycan and chondroitin sulphate
proteoglycan (CSPG) (Ref. 46). CSPGs consist of
a core protein to which one or more
unbranched glycosaminoglycan (GAG) chains
are attached (Ref. 46). The chondroitin sulphate
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GAG chains are sulphated polysaccharides made
of repeating disaccharides of glucuronic acid and
N-acetylgalactosamine (Refs 3, 47). Various
studies have shown that mature reactive
astrocytes upregulate several proteoglycans that
contribute to the inhibition of axonal outgrowth
(Refs 48, 49). One of the major tools for
studying proteoglycans is the bacterial enzyme
chondroitinase ABC derived from Proteus
vulgaris. This enzyme digests the CSPG GAG
chains into disaccharides (Ref. 47). In vivo
application of this enzyme has been shown to
be effective in reversing the inhibitory nature
of CSPGs. Application of chondroitinase ABC
to the brain after nigrostriatal tract lesioning
promotes regeneration of dopaminergic
neurons back to their targets (Ref. 50).
Intrathecal application of the enzyme after
spinal cord injury in rats has also been shown
to enhance regeneration and sprouting of the
axons, and functional recovery in several
models of injury, including contusion injury
(Refs 51, 52). Transgenic expression of
chondroitinase under the control of the GFAP
promoter permitted regeneration of sensory
axons after dorsal rhizotomy (Ref. 53). These
studies with chondroitinase ABC demonstrated
that one of the major proteoglycan inhibitory
components is the sulphated GAG chains on
CSPG.
Further work supporting the role of GAG
chains in the inhibition of axonal growth came
from studies that interfered with enzymes
involved in GAG synthesis, such as
polymerases and sulphotransferases (Refs 54,
55). Treatment of inhibitory astrocytes with
siRNA targeting chondroitin-polymerising
factor (CHPF) reduced their inhibitory nature,
facilitating axon growth from cerebellar granule
neurons (Ref. 56).
Together, these and other experiments show
that GAG chains have an important role in
inhibiting the regeneration of axons. The
mechanism by which CSPGs mediate the
inhibitory effect is not clearly understood, and
to date, no specific receptors for CSPGs have
been identified. However, accumulating data
suggest that CSPGs mediate their inhibitory
effect via disruption of integrin function on
axons (Refs 49, 57, 58), and therefore prevent
axon growth cones from responding to the
growth-promoting extracellular cues in the
microenvironment.
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
Decorin
Although CSPGs are established as one of the
main inhibitory components of glial scar, not all
CSPGs are considered inhibitory. One of the
exceptions to this is decorin. Decorin (DCN) is
a small leucine-rich dermatan- or chondroitin-
sulphate proteoglycan found naturally in
many tissues. Decorin is known to inhibit the
activity of three isoforms of TGF-b, which
are involved in astrocytosis and promoting the
reactive astrocyte response at the site of injury
(Ref. 59).
Continuous infusion of decorin in acute stab
injuries of spinal cord in rats leads to a
significant reduction in the astrocytic response,
macrophage accumulation and deposition of
the CSPGs, such as neurocan (NCAN), NG2
(CSPG4), phosphacan (PTPRZ1) and brevican
(BCAN) in scar tissue (Ref. 60). The mechanism
is probably related to the ability of decorin to
block the effects of TGF-b and EGFR
(epidermal growth factor receptor) activation,
which are involved in reactive astrocytosis
(Ref. 61). The modification of the environment
of the injured cord leads to an increase in
neurite outgrowth from microtransplanted
sensory neurons at the site of injury (Ref. 60).
Decorin also appears to have direct effects on
neurite outgrowth and branching in axons
(Ref. 62). How decorin promotes neurite
outgrowth is yet to be elucidated.
Semaphorins
Semaphorins are a large family of guidance
molecules that are either secreted or membrane
bound. Vertebrates express secreted class III,
transmembrane (Class IV– VI) and GPI-linked
(Class VII) semaphorins. Semaphorins have
important roles in development, including
midline guidance (Ref. 63), through their ability
to repel axons by interacting with their
receptors – neuropilins and plexins (Ref. 64).
The best studied semaphorin in the context of
spinal cord injury is semaphorin-3A (SEMA3A),
which is produced by meningeal fibroblasts of
the glial scar (Refs 65, 66). Treatment of spinal
cord injury with a specific pharmacological
inhibitor of SEMA3A, xanthofulvin or SM-
216189 (Ref. 67), leads to significant
regeneration of axons, Schwann cell infiltration
and functional recovery (Ref. 68). Semaphorins
are therefore a possible target with the potential
to improve outcome after spinal cord injury.
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Ephs and ephrins
Eph receptor tyrosine kinases and their ligands
have many roles in development and axonal
pathfinding (Ref. 69). The Eph family has 16
receptor members in vertebrates (EPHA1 –
EPHA10 and EPHB1 – EPHB6) and they interact
with nine separate ephrin ligands (Ref. 70).
Both Eph receptors and ephrins are subdivided
into class A and B, depending on their binding
affinity: EPHA receptors bind mainly to class A
ephrins and EPHB receptors interact with class
B ephrins. The exceptions to this rule are
EPHA4 and EPHB2, which can be activated by
both classes of ephrins (Ref. 71). Class A
ephrins attach to the membrane via a GPI link,
whereas class B are transmembrane molecules
with a highly conserved C-terminal tail
(Refs 70, 72). Although Ephs are generally
assumed to be the receptors and the ephrins
their ligands, it has also been shown that
ephrins themselves are capable of acting as
receptors after engagement with the
corresponding Eph receptor, leading to reverse
signalling (Ref. 73). Ephs and ephrins have
been shown to be involved in the repulsion of
growth cones in different neuronal types in
vitro (Refs 74, 75). EphrinB3 (EFNB3)-knockout
mice show abnormal axonal guidance at the
midline of the spinal cord, suggesting a role for
EPHA4 in mediating the inhibition of
corticospinal axons from passing the EFNB3-
positive midline during development (Ref. 76).
Several studies have indicated the importance
of Ephs and ephrins in the prevention of axon
regeneration after spinal cord injury. Several
Eph receptors are upregulated after injury,
including EPHA3, EPHA4, EPHA6, EPHA8 and
EPHB3 (Refs 77, 78). In addition, some ephrins
are upregulated at the site of injury by
astrocytes (Ref. 8). After injury, there is
accumulation of EPHA4 in the stumps of
injured corticospinal neurons, with the
complementary upregulation of EFNB2 in
astrocytes (Ref. 79). In addition, injury-induced
upregulation of EPHA4 has been shown to be
linked to reactive gliosis after injury. EPHA4-
knockout mice show significant reductions in
astrocytosis and glial scar formation after spinal
cord injury. Astrocytes from EphA4 – / – mice do
not respond to interferon-g or leukaemia
inhibitory factor (LIF) (Ref. 80), which are both
involved in inducing reactive astrocytosis after
injury (Ref. 81). Two groups have demonstrated
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expert reviews
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
that EPHA4-mediated inhibition has an
important role in preventing regeneration after
spinal cord injury. EphA4 – / – mice show robust
regeneration and functional recovery after
spinal cord injury (Ref. 81). In addition,
application of an EPHA4-blocking peptide after
corticospinal cord injury in adult rats preserved
corticospinal tract axons and sprouting after
injury, with significant functional recovery in
paw control, a function that is specific to the
corticospinal tract pathway (Ref. 82).
Various mechanisms of action have been
described in different cell types; however, a
common theme emerging from various studies
is the role of ephrins and Ephs in modulating
integrins. Ephs and ephrins have been shown
to mediate various integrin-mediated functions,
such as adhesion, migration and axonal
elongation (Refs 83, 84). In addition, repulsive
ephrins and Ephs have been shown to mediate
their effect by activating RhoA and downstream
inhibitory pathways involved in inhibition of
neurite outgrowth (Refs 83, 85). Ephrins and
Ephs are therefore one of the most promising
targets for intervention in spinal cord injury.
More specific tools will allow a better
understanding and targeting of this class of
molecules.
Polysialic acid
Polysialic acid (PSA) is a cell-surface glycan and a
linear polymer of N-acetylneuraminic acid in
a2-8 linkage. It is added to glycoproteins,
particularly to neural cell adhesion molecule
(NCAM), as a post-translational modification in
the Golgi complex by two polysialytransferase
enzymes (Ref. 86). PSA, when present on the
cell surface, has the general effect of preventing
cell adhesive interactions because of its large
hydrated volume, which serves to modulate cell
interactions and the distance between cells
(Ref. 86). PSA – NCAM expression is higher in
embryonic brain than in adult brain, but in the
adult CNS, it continues to be present in areas of
high plasticity and neurogenesis (Ref. 87).
During development, PSA – NCAM is involved
in processes such as cell migration, axonal
guidance and targeting (Ref. 88). In the adult
brain, PSA is expressed in specific regions
such as the hippocampus (Ref. 89) and
hypothalamus (Ref. 90), areas that are
specifically associated with structural plasticity.
Its presence is also necessary for the migration
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of progenitor cells from the subventricular zone to
the olfactory bulb (Ref. 91). After injury, the most
persistent axonal growth occurs in close
apposition to a small subpopulation of astrocytes
that express PSA–NCAM after injury (Ref. 92).
These results all led to the idea that forced
expression of PSA on NCAM might improve
axon regeneration and sprouting, by shielding
the axons from negative cues and allowing
positive cues to take over. Various studies have
examined the effect of forced expression of
polysialyltransferase to increase PSA – NCAM.
This strategy has led to some regrowth and
sprouting of corticospinal tract axons (Ref. 93)
in a dorsal column crush lesion model. It
induces a 20-fold increase in axons growing
into the lesion cavity when combined with a
peripheral conditioning lesion, which primes
the neurons (Ref. 94). Increased PSA-NCAM on
Schwann cells greatly increases their migration
into a spinal cord injury by providing a
substrate for axonal regeneration (Refs 94, 95).
As a result of the difficulty in manipulating
PSA – NCAM itself, mimetic peptides have
been produced that have been used to
improve recovery in mouse spinal cord
injury (Ref. 96).
Neurotrophic factors
Many neurotrophic factors are released in the
normal spinal cord, and many of these are
up- or downregulated after injury. The
neurotrophins nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NTF3) and glial-derived
neurotrophic factor (GDNF) all promote the
regrowth of populations of axons. Efficient
regenerative ability of the PNS is partly
dependent on the ability of Schwann cells to
secrete growth factors, supporting axon
elongation after nerve injury. CNS axons
also respond to exogenously introduced
neurotrophic factors. Different studies have
shown the effectiveness of genetically
engineered cells that secrete growth factors,
or matrices containing trophic factors, in
promoting the regeneration and sprouting of
the injured axons (Ref. 97).
Some of the trophic factors that are capable of
promoting axon growth after injury include
NGF (Ref. 98), BDNF (Ref. 99), NTF3 (Ref. 100),
NTF4, NTF5 (Ref. 101) and GDNF (Ref. 102),
with different factors slightly favouring the
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
outgrowth of different populations of neuronal
axons. An example of a specific-acting
neurotrophic factor is artemin (ARTN). Artemin
is a member of the GDNF family of
neurotrophic factors that has been shown to
favour neurite extension in DRG neurons and it
prevents the loss of C-fibres following injury
(Ref. 103). Virus-mediated transfection of
neurons to express artemin has been shown to
promote neurite extension by DRG cells, both
in vitro and following dorsal hemisection
lesions in vivo (Ref. 104). The effects of artemin
are mediated through several pathways
involving transcriptional changes and
upregulation of genes necessary for neurite
extension (Ref. 105), and activation of Src-
family kinase activity (Ref. 106).
The use of trophic factors is therefore likely to be
one of the essential components of any
combinatorial therapeutic strategy that is used
to promote axon regeneration at the site of
injury. Overall, to improve regeneration of the
axon, the surrounding microenvironment must
be rendered more permissive, either by
promoting positive cues or by reducing the
negative cues (Fig. 1).
Macrophages and inflammation
Immune cells have a pivotal role in injury and
repair. Although inflammation is traditionally
considered to be harmful to the surrounding
tissues (Ref. 107), increasing evidence
demonstrates the beneficial role of inflammation
following injury. Crucial evidence came from
studies investigating the role of activated
macrophages in spinal cord injury repair
(Ref. 101), with some results demonstrating the
ability of macrophages to promote regeneration
by secreting growth factors and removing debris
from the site of injury (Refs 108, 109).
In an elegant series of experiments, it was
shown that macrophages infiltrating through an
opened BBB have a crucial role in promoting
recovery by expressing IL-10 (IL10) and acting
as local anti-inflammatory agents at the site of
injury, leading to functional recovery. This task
cannot be performed by activated resident
microglia (Ref. 110). CSPGs, which are
discussed earlier in this review, have also been
shown to interact with inflammatory cells, such
as macrophages and microglia, in the acute
phase of injury. Interactions with CSPG are
mediated via the CD44 receptor and modulate
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Extrinsic and intrinsic factors controlling axonal regeneration

after spinal cord injury
Myelin

Intact axon Macrophage/

microglia

Reactive
astrocyte
Oligodendrocyte

Meningeal
fibroblast

Cystic
cavity Myelin
debris

↑ TGF-β
↑ IFN-γ
↑ IL-1
↑ IL-6 Demyelinated
axon
Dystrophic
axon
Degenerating
axon
Glial scar formation after injury
Expert Reviews in Molecular Medicine © Cambridge University Press 2009

Figure 1. Glial scar formation after injury. Injury to the central nervous system is accompanied by the
formation of a glial scar and upregulation of inhibitory molecules, such as TGF-b, IFN-g, IL-1 and IL-6,
posing a physical and chemical barrier. Inflammatory cells and immune cells surround the central cavity, in
addition to being present throughout the site of injury. Meningeal fibroblasts are mainly present at the core of
the lesion, disrupting the blood–brain barrier. Oligodendrocytes are dispersed throughout the scar.
Astrocytes, the main cellular component in the mature glial scar, undergo proliferation and hypertrophy in
response to injury. Damaged axons at the site of injury undergo Wallerian degeneration and demyelination.
Cut axons attempting to regenerate face the hostile environment of the scar, which contains inhibitory
factors, such as chondroitin sulphate proteoglycans and myelin debris and are unable to mount an efficient
regenerative response or penetrate the dense glial scar, and form dystrophic axons.

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the release of insulin-like growth factor-1 (IGF-1)
from the activated microglia and macrophages
(Ref. 111). Therefore, removal of CSPGs
immediately following injury has adverse
effects, because of disruption of immune-
mediated recovery, whereas delayed removal
of CSPGs preserves the beneficial effects of
inflammation at the site of injury (Ref. 111).
Inflammation can thus be harnessed to promote
recovery. This demonstrates that careful
attention should be given to the temporal and
dynamic nature of an injury and injury-
mediated mechanisms during treatment.
Intrinsic factors
There is little merit in rendering the external
environment of the axon more permissive for
regeneration if the cut axons themselves make
no attempt to regenerate. Unfortunately, many
damaged axons in the spinal cord appear to
make no regenerative response after they have
been cut, and some regenerate only feebly. In
this part of the review, we shall describe some
of the most important intrinsic factors that
influence the ability of axon to regenerate.
Peripheral versus central regenerative
ability
Axons from the PNS generally have a superior
ability to regenerate compared with those of the
CNS. This could be a result of the evolution of
a complex nervous system in higher animals,
such as mammals. PNS axons are more prone
to injury from daily life events such as
contusions, cuts and other injuries; whereas
those of the CNS are protected by a bony skull
and vertebral column as well as cerebrospinal
fluid, which together absorb most of the
external shocks. The reduced regenerative
ability could alternatively have evolved as a
protective mechanism to prevent excess
sprouting and the possibility of miswiring
within the complex CNS circuits, which would
have detrimental consequences to the animal.
However, there are some distinct factors, both
extrinsic and intrinsic, which differentiate the
PNS and CNS in their regenerative ability.
Understanding these factors has important
implications for finding new therapeutic
approaches to injuries and disorders involving
axonal degeneration.
Superior PNS regeneration is due partly to the
permissive environment created by Schwann cells
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Extrinsic and intrinsic factors controlling axonal regeneration
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and partly to the absence of the inhibitory
molecules, which are always present within the
CNS. There are also molecular differences in the
response of PNS and CNS axons to axotomy.
For example, cut PNS axons activate
regeneration-associated genes such as JUN,
GAP43 (growth-associated protein-43) and
genes involved in the JAK – STAT3 pathway
(Refs 112, 113, 114), whereas injury to CNS
axons (such as those of Purkinje cells)
does not result in activation of these genes
(Ref. 115). There are two main phases of
axon regeneration that we address. The first is
the rapid production of a new axon growth
cone at the site of axotomy. The second is the
continued growth of the axon through the
damaged CNS.
Calcium
Calcium has a very important role in axon growth
guidance and its local elevation in axoplasm is
necessary for the rapid formation of a growth
cone at the end of cut axons (Refs 116, 117).
After axotomy, there is a brief influx of calcium
into the axon until the membrane reseals. The
calcium has two main effects on the process of
growth cone regeneration. First, it is essential
for membrane sealing after axonal transection
(Refs 118, 119). After this, calcium plays an
important role in the rearrangement of
cytoskeletal proteins required for the formation
of the growth cone. This cytoskeletal
reorganisation results in the formation of a
locus for microtubules to polymerise and
extend into a new growth cone, as well as
creating a compartment in which membrane
vesicles remain trapped and are readily
available for the advancing growth cone
(Ref. 120). The importance of influx of calcium
on the assembly and formation of growth cones
from the extracellular medium has also been
demonstrated in mammalian neurons in vitro
(Refs 117, 121). Calcium probably has several
roles. It is involved in the activation of kinases
such as protein kinase A (PKA) and
extracellular signal-related protein kinases
(ERKs), which in turn can induce cytoskeletal
reorganisation and axonal protein synthesis
(Ref. 117). Influx of calcium also activates
proteolytic machinery involving calpains.
Calpain has been shown to be involved in
degradation of the submembrane protein
spectrin, promoting decoupling of cytoskeletal
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elements from plasma membrane and
rearrangement of microtubules and
neurofilaments, which is necessary for axonal
growth cone formation (Refs 120, 122).
cAMP and conditioning lesions
Cyclic AMP (cAMP) is a key intracellular
signalling molecule, so it is not surprising that
it influences axonal regeneration. Raising the
cAMP level in neurons allows them to
overcome the inhibitory effects of myelin and
MAG (Ref. 123). In vivo, a stronger regenerative
response can be obtained by elevated levels of
cAMP in retinal ganglion cells (Refs 117, 124),
and in the damaged spinal cord (Refs 125, 126).
The effect of cAMP on regeneration is mediated
through various mechanisms. Elevated cAMP
levels promote the regenerative ability of
central axons by increasing growth factor
receptor translocation from the cytoplasm to the
cell membrane (Ref. 127). cAMP also induces
activation of PKA and the subsequent
activation of CREB transcription factor. This
pathway has been shown to be responsible
for mediating the neuritogenic effects of
neurotrophic factors (Ref. 128). One of the
products of CREB-mediated gene upregulation
is arginase I (ARG1). It stimulates synthesis of
polyamines, which are capable of overcoming
the effects of inhibitors such as myelin, by
hydrolysing arginine to urea and ornithine
(Ref. 129).
One setting where cAMP has a role in
enhancing regeneration is in conditioning
lesions. Conditioning is a process by which the
regenerative ability of CNS axons can be
enhanced after injury by priming the neuron
with an initial injury. In other words, the first
injury to the nerve leads to the priming of the
neuron, which then leads to better axonal
growth in the subsequent injury. This effect has
been well studied in DRG axons, which have a
peripheral and a central branch. Studies have
shown that lesions to the peripheral branch of
DRG axons, condition the neuron into a
growth-promoting mode. A subsequent injury
to the central axon of the DRG is followed by
enhanced regenerative ability compared with
the nonconditioned state. The timing of this
conditioning lesion is important, with the best
results obtained when peripheral axotomy is
performed 1 week before the injury to the
central funiculus; no beneficial effect is seen
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
when central axons are injured before the
peripheral branch (Ref. 130). The growth-
promoting action of conditioning lesions has
been mostly attributed to changes within the
cell body of the DRG neurons, involving an
increase in cAMP concentration in the perikarya
in the first days after injury (Ref. 125). Further
details of enhanced regeneration mediated by
conditioning lesions have emerged that involve
changes such as upregulation of arginase I
(Ref. 131) and IL-6 (Ref. 132). Conditioning
lesions in the peripheral branch of DRGs also
upregulate other regeneration-associated genes
(Ref. 133), including GAP43 (Ref. 134) and
activating transcription factor-3 (ATF3)
(Ref. 135). GAP43 protein is expressed mostly
during development, and is downregulated in
the adult (Ref. 113); however, it can be
upregulated following injury, especially in the
PNS, leading to a successful regeneration
response. CNS axons show a reduced ability
to upregulate GAP43 in response to injury
(Ref. 113). Exogenous expression of ATF3 in
DRG axons promotes neurite outgrowth on
permissive substrates (Ref. 135), but it remains
unclear whether ATF3 overexpression is
sufficient to overcome the inhibitory effects of
myelin.
Pathways associated with increased
regeneration
In addition to cAMP and calcium, various other
signalling pathways involved in regeneration
have been identified. ERK (Refs 136, 137) and
Akt (Refs 136, 138) are two kinases that are
activated in response to various neurotrophic
factors. Akt also mediates the formation of
colateral branches in response to local exposure
to neurotrophins (Refs 138, 139). Both ERK and
Akt also promote growth cone motility and
regeneration after axonal injury in adult DRG
axons (Ref. 117).
Following the acute phase of axon injury and
rearrangement of cytoskeletal elements, a
chronic phase is activated. This phase involves
upregulation of various genes, which are
commonly referred to as regeneration-
associated genes (Ref. 133), such as GAP43 and
ATF3, as mentioned earlier. However, another
set of genes known to be upregulated after
injury include those involved in the JAK –
STAT3 pathway, through which the
neuritogenic effect of cytokines such as LIF
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and IL-6 is mediated. IL-6 administration has
been shown to be sufficient (though not
necessary) for axons to overcome the
inhibitory effects of MAG and myelin, both in
vitro and in vivo (Ref. 132). Inhibition of
the JAK – STAT3 pathway blocks the IL-6-
mediated effect on axon regeneration
(Ref. 132). However, it should also be noted that
IL-6 is a potentially harmful proinflammatory
cytokine, which is upregulated after CNS
injury, contributing to increased cell death
(Ref. 140).
Pathways associated with inhibition of
axon regeneration
In addition to pathways involved in promoting
axon regeneration, some pathways have been
shown to be responsible for the inhibition of
axon growth and signalling downstream of
inhibitors such as CSPGs and myelin. One such
signalling pathway is via the Ras-related small
GTPase Rho. Rho functions as a molecular
switch of various cellular processes by shuttling
between the inactive, GDP-bound form and the
active, GTP-bound form (Ref. 141). Studies have
shown that Rac and Cdc42 Ras-related GTPases
generally promote neurite outgrowth, whereas
Rho inhibits neurite elongation (Ref. 85).
Commonly used agents to inhibit the Rho
pathway are the ribosylating enzyme C3 and
the Rho kinase inhibitor Y27632 (Ref. 35).
Various studies have demonstrated that
inhibiting the Rho pathway allows axonal and
neurite outgrowth from primary cortical
neurons (Ref. 142), retinal ganglion cells
(Ref. 143) and DRG neurons (Ref. 144) on
inhibitory substrates including myelin, CSPG
and aggrecan, and in doing so, have
demonstrated the role of the Rho pathway in
mediating the inhibitory effects of these
molecules.
In addition to in vitro studies, various in vivo
studies have shown the effectiveness of Rho
and ROCK (Rho-associated protein kinase)
inhibitors in promoting regeneration and
neurite outgrowth. Application of C3 or Y27632
in vivo to mice with a dorsal crush of the spinal
cord affects axonal growth and promotes
motor recovery, with an increase in forelimb –
hindlimb coordination (Ref. 142). Similarly,
application of a high dose of Y27632 to cervical
dorsal column transections in rats leads to
an increase in axonal growth distal to the lesion
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
and functional recovery compared with
animals receiving a low dose or no treatment
(Ref. 145). Owing to the important role of the
Rho – ROCK pathway in mediating the
inhibitory effect of CSPGSs (Ref. 143) and other
inhibitory molecules such as myelin (Ref. 146)
and ephrins (Ref. 147), Rho inhibitors have been
an important target in the development of
pharmacological treatments for spinal cord
injury in humans (Ref. 23).
Axonal protein synthesis and degradation
Early events in regeneration are membrane sealing,
retraction of the axon from the injury site and
formation of a new growth cone. Although
retraction involves degradation of cytoskeletal
elements such as microtubules, growth cone
formation involves de novo synthesis of a
variety of proteins, including microtubules,
neurofilaments and actin. The two separate but
inter-related processes of protein degradation
and synthesis are important for these steps to
occur. One of the earliest indications that axons
are able to synthesise proteins in situ after injury
came from the evidence that the earliest
regenerative sprouting at the axon tips occurs
within a day of axonal injury, suggesting that
growth cone formation is independent of the cell
body reaction (Ref. 148). Accordingly, various
studies have showed that inhibition of protein
synthesis in peripheral nerves attenuates their
ability to regenerate (Refs 149, 150). Further
evidence has now emerged that one of the key
differences between peripheral and central axons
is the level of protein synthesis and degradation
machinery in the axons. Comparison between
DRG and retinal ganglion cell axons has
shown strict correlation between the ability of
axons to generate a new growth cone and the
presence of proteins involved in protein
synthesis, such as the ribosomal protein P0 and
phosphorylated translation factor eIF-4E (EIF4E)
(Refs 150, 151, 152).
In addition to providing proteins for
sustaining axon regeneration, local protein
synthesis has also been shown to have a
role in retrograde signalling, as seen for
importin-b (KPNB1), which is necessary for
upregulating gene expression after injury
(Ref. 153). As well as protein synthesis
machinery, protein degradation machinery is
also crucial for the axon regeneration process
(Ref. 150).
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Membrane-associated factors for
regeneration
In addition to the axoplasmic factors, there are
many molecular events on the membrane of the
axon that modulate the ability of axons to
regenerate. Of recent interest is the role of lipid-
rich domains consisting of cholesterol and
saturated sphingolipids, commonly known as
‘rafts’. These rafts are small, highly dynamic,
liquid-ordered phases of membrane that ‘float’
in a liquid-disordered phase of the membrane.
Through a series of elegant experiments, it has
been shown that these lipid-rich domains are
important in growth cone guidance (Ref. 154).
There is also evidence for a role of the different
lipid components of ‘rafts’ in aspects of axon
growth. Disruption of cholesterol-rich domains
specifically attenuates BDNF-induced attraction,
whereas disruption of ganglioside-rich rafts
inhibits the ability of axons to elongate in the
presence of BDNF (Ref. 154). This suggests a
dichotomous role for membrane lipids, with
cholesterol-rich regions affecting growth cone
guidance and ganglioside-rich regions affecting
axonal elongation. Little is known about the
role of cholesterol in axon regeneration.
However, gangliosides have been the focus of
great interest in the past few years.
Gangliosides are saturated glycosphingolipids
that may be conjugated to one or more sialic
acid residues. Axons are enriched with only a
few of the various types of ganglioside that the
body can produce; these include GM1, GD1a
and GT1b (Ref. 155). During normal
development, ganglioside biosynthesis is
essential for axonogenesis (Ref. 156). Different
gangliosides have been shown to have different
effects on axon growth process. Both GD1a and
GT1b act as functional receptors of MAG
(Ref. 157), which has been shown to inhibit
regeneration of axons in CNS. Sialidase, which
digests these gangliosides to GM1, promotes
regeneration in vivo in mammalian models of
brachial plexus avulsion injuries (Ref. 158). By
contrast, antibodies against GM1 gangliosides
inhibit neurite outgrowth (Refs 159, 160),
supporting a role of this ganglioside in
promoting axon regeneration. Most of the
downstream effects of GM1 ganglioside have
been shown to be via the potentiation of the
effects of neurotrophins on the Trk family of
neurotrophin receptors. Owing to its acute
neuroprotective and long-term regenerative
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effects in several experimental models
(Refs 161, 162, 163), GM1 ganglioside has been
used in a multicentre acute spinal cord injury
study (Ref. 164). Although the first phase was
considered to be highly successful, it failed in
the subsequent stages. However, it may be
envisaged that providing GM1 ganglioside
exogenously would have different effects on
the membrane when compared with the
endogenous production. This is supported by
findings that the metabolic pathways of
endogenous and exogenous gangliosides in
cerebellar granule cells differ (Ref. 165). Thus,
interest is now shifting to the ganglioside
metabolic pathways and ways to manipulate
the cells to convert various other ‘neuronal’
gangliosides such as GD1a and GT1b
gangliosides into GM1 gangliosides using
specific enzymes, such as neuraminidase-3
(NEU3). However, it remains to be seen
whether this strategy can overcome the failures
of initial studies in spinal cord injury repair
where exogenous GM1 gangliosides were used
as therapeutic approach.
Another family of membrane-associated
proteins involved in regeneration are the
integrins. These are transmembrane receptors
that are functionally active when present as
heterodimers (consisting of an a- and a b-
subunit). In neurons, it has been shown that
the integrin activation state and change in
conformation of the integrin structure greatly
influences neurite outgrowth in culture.
Activated integrins promote high-affinity
binding of integrins to their ligands, which
promotes regeneration of axons, even under
inhibitory and nonpermissive conditions
(Refs 166, 167). There is also much indirect
evidence for involvement of integrins in
regeneration following axonal injury. For
example, a number of integrin subunits,
including a4, a5, a6, a7 and b1 are upregulated
in sensory neurons after injury (Refs 168, 169, 170).
Furthermore, upregulation of integrins after
injury to the PNS is associated with successful
regeneration (Refs 171, 172). The increase in the
vigour of axon growth after preconditioning
lesions, which promote regeneration of DRG
axons, has been shown to be at least partly
attributable to the upregulation of a5 (Ref. 170)
and a7 integrins (Ref. 171). Moreover, neurons
from integrin-a7-deficient transgenic animals
show impaired axon regeneration (Ref. 171).
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More recently, it has been shown that lentivirus-
mediated overexpression of integrins not
normally expressed by neurons allows better
interaction with the available ligands and
promotes regeneration and functional
recovery (Ref. 173). Overall, these data support
a role for integrins as key intrinsic players
in regeneration and axonal outgrowth
following injury.
Clinical implications
Advances in the identification of factors
controlling axon regeneration has opened new
avenues for the development of therapeutic
strategies after spinal cord injury. Various
clinical trials for the treatment of acute spinal
cord injury are currently in progress or recently
completed. The largest trial is to test safety and
efficacy of the myelin inhibitor neutralising
antibody anti-Nogo. Clinical trials of anti-Nogo
antibody following acute spinal cord injury
have completed Phase I and are anticipated to
start Phase II soon (Ref. 23). In addition to
changing the extracellular environment, other
clinical trials aimed at the manipulation of the
intrinsic ability of the axons to regenerate are
currently evaluating the effect of the Rho kinase
inhibitor Cethrinw in acute spinal cord injury
lesions (Ref. 23). With the identification of
more-specific targets for improving
regeneration, more therapeutic compounds are
likely to enter clinical trials in the future,
increasing the prospect for treatments for
patients suffering from spinal cord injury.
A common theme that has emerged in various
interventions aimed at improving axonal
regeneration and nerve repair has been the
superior outcome of using combinatory
treatments rather than single-agent treatments
(Ref. 97). The importance of combinatory
studies and investigating the possible
combination strategies is twofold. First, because
of the complex and multifactorial nature of
spinal cord injury, combinatory treatments are
more likely to be able to tackle and overcome
the various regenerative obstacles present
within the CNS environment, leading to a
better regenerative outcome. Second, not all
combinatory elements are suitable for use
together. Therefore, further studies on the use
of combinatory treatments are necessary to
determine strategies that can be used together
to improve the prospects for regeneration in
Accession information: doi:10.1017/S1462399409001288; Vol. 11; e37; December 2009
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Extrinsic and intrinsic factors controlling axonal regeneration
after spinal cord injury
spinal cord injury. Despite this need, the field of
spinal cord injury repair has advanced
exponentially within the past two decades,
revealing increasingly more detail about the
mechanisms of CNS regeneration.
Acknowledgements and funding
We are grateful to Dr Jessica Kwok for her input
in this review. We would also like to thank the
peer reviewers for their time and constructive
suggestions.
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Further reading, resources and contacts

Reviews
Silver, J. and Miller, J.H. (2004) Regeneration beyond the glial scar. Nature Reviews Neuroscience 5, 146-156
Rolls, A., Shechter, R. and Schwartz, M. (2009) The bright side of the glial scar in CNS repair. Nature Reviews
Neuroscience 10, 235-241
Cafferty, W.B., McGee, A.W. and Strittmatter, S.M. (2008) Axonal growth therapeutics: regeneration or sprouting
or plasticity? Trends in Neurosciences 31, 215-220
These three recent reviews provide detailed information on axon regeneration at the glial scar

Website
For the latest updates on research on spinal cord injury and clinical trials see the website of the NIH National
Institute of Neurological Disorders:
http://www.ninds.nih.gov/disorders

Features associated with this article

Figure
Figure 1. Glial scar formation after injury.

Citation detail for this article

Fardad T. Afshari, Sunil Kappagantula and James W. Fawcett (2009) Extrinsic and intrinsic factors
controlling axonal regeneration after spinal cord injury. Expert Rev. Mol. Med. Vol. 11, e37, December
2009, doi:10.1017/S1462399409001288

19
Accession information: doi:10.1017/S1462399409001288; Vol. 11; e37; December 2009
& Cambridge University Press 2009