PI3K-Akt and Ras-MAPK pathways were both implicated in cell survival and neurite-outgrowth, respectively. Akt has been suggested to be involved in neuronal survival and MAPK in neurite outgrowth during development. This study investigated if Akt, a component of PI3K-Akt pathway, and MAPK/ERK, a component of Ras-MAPK pathway, were expressed in the normal and regenerating dorsal root ganglia and their respective peripheral nerves. Western blot investigations carried out on rodent nerve tissues showed that both phospho-Akt and phospho-MAPK were expressed after axotomy. Therefore, both the phosphoproteins appeared to be important not only during nerve development but also during nerve regeneration.

Functional recovery after peripheral nerve repair in humans is often disappointing. A major reason for this is the inaccuracy of re-innervation of muscles and sensory structures. We hypothesized that promoting plasticity in the spinal cord, through digestion of chondroitin sulphate proteoglycans (CSPGs) with chondroitinase ABC (ChABC), might allow the CNS to compensate for inaccurate peripheral re-innervation and improve functional recovery. The median and ulnar nerves were injured and repaired to produce three grades of inaccuracy of peripheral re-innervation by (i) crush of both nerves; (ii) correct repair of median to median and ulnar to ulnar; and (iii) crossover of the median and ulnar nerves. Mapping of the motor neuron pool of the flexor carpi radialis muscle showed precise re-innervation after nerve crush, inaccurate regeneration after correct repair, more inaccurate after crossover repair. Recovery of forelimb function, assessed by skilled paw reaching, grip strength and sensory testing varied with accuracy of re-innervation. This was not due to differences in the number of regenerated axons. Single injections of ChABC into the spinal cord led to long-term changes in the extracellular matrix, with hyaluronan and neurocan being removed and not fully replaced after 8 weeks. ChABC treatment produce increased sprouting visualized by MAP1BP staining and improved functional recovery in skilled paw reaching after correct repair and in grip strength after crossover repair. There was no hyperalgesia. Enhanced plasticity in the spinal cord, therefore, allows the CNS to compensate for inaccurate motor and sensory re-innervation of the periphery, and may be a useful adjunct therapy to peripheral nerve repair.

A number of recent studies have established that the bacterial enzyme chondroitinase ABC promotes functional recovery in the injured CNS. The issue of how it works is rarely addressed, however. The effects of the enzyme are presumed to be due to the degradation of inhibitory chondroitin sulphate GAG chains. Here we review what is known about the composition, structure and distribution of the extracellular matrix in the CNS, and how it changes in response to injury. We summarize the data pertaining to the ability of chondroitinase to promote functional recovery, both in the context of axon regeneration and the reactivation of plasticity. We also present preliminary data on the persistence of the effects of the enzyme in vivo, and its hyaluronan-degrading activity in CNS homogenates in vitro. We then consider precisely how the enzyme might influence functional recovery in the CNS. The ability of chondroitinase to degrade hyaluronan is likely to result in greater matrix disruption than the degradation of chondroitin sulphate alone.

Bone morphogenetic proteins (BMPs) and their endogenous inhibitors, including noggin, chordin and follistatin, have roles in pattern formation and fate specification of neuronal and glial cells during nervous system development. We have examined their influence on glial reactions in the injured central nervous system (CNS). We show that penetrating injuries to the brain and spinal cord resulted in the upregulation of BMP-2/4, BMP-7, and noggin, with the latter being expressed almost exclusively by reactive astrocytes at the injury site, and we show that astrocytes in vitro produce noggin. As BMPs have been shown to drive cultured NG2-positive oligodendrocyte precursors (OPCs) towards a multipotential phenotype (type II astrocytes), we investigated the effects of inhibiting noggin with a function-blocking antibody (noggin-FbAb). In vitro, BMP-driven conversion of OPCs to type 2 astrocytes was inhibited by noggin, an effect that was reversed by noggin-FbAb. Noggin-FbAb also increased the number of type 2 astrocytes generated from cultured OPCs exposed to an astrocyte feeder layer, consistent with astrocytes producing both BMPs and noggin. In knife cut injuries in vivo, noggin-FbAb treatment resulted in an increase in the number of NG2-positive cells and small GFAP-positive cells in the injury site, and the appearance of glial cells with the morphological and antigenic characteristics of type 2 astrocytes (as generated in vitro), with coexpression of both GFAP and NG2. This potential conversion of inhibitory OPCs to type 2 astrocyte-like cells in vivo suggests that endogenous BMPs, unmasked by noggin antagonism, might be exploited to manipulate cell fate following CNS trauma.

A high proportion of NG2 in the adult rat spinal cord is saline-soluble and migrates slightly faster than intact NG2 on SDS–PAGE, suggesting that it represents the shed ectodomain of NG2. In the injured cerebral cortex, much of the overall increase in NG2 is due to the saline-soluble (shed), rather than the detergent-soluble (intact), form. Hydroxamic acid metalloproteinase inhibitors, but not TIMPs, were able to prevent NG2 shedding in oligodendrocyte precursor cells (OPCs) in vitro. The generation of another truncated form of NG2 was, however, sensitive to TIMP-2 and TIMP-3. Two observations suggest that NG2 is involved in PDGF signaling in OPCs: the rate of NG2 shedding increased with cell density and NG2 expression was increased in the absence of PDGF. Ectodomain shedding converts NG2 into a diffusible entity able to interact with the growth cone, and we suggest that this release is likely to enhance its axon growth-inhibitory activity.

Chondroitin sulphate proteoglycans (CSPGs) are up-regulated in the CNS after injury and inhibit axon regeneration mainly through their glycosaminoglycan (CS-GAG) chains. We have analysed the mRNA levels of the CS-GAG synthesizing enzymes and measured the CS-GAG disaccharide composition by chromatography and immunocytochemistry. Chondroitin 6-sulfotransferase 1 (C6ST1) is up-regulated in most glial types around cortical injuries, and its sulphated product CS-C is also selectively up-regulated. Treatment with TGFalpha and TGFbeta, which are released after brain injury, promotes the expression of C6ST1 and the synthesis of 6-sulphated CS-GAGs in primary astrocytes. Oligodendrocytes, oligodendrocyte precursors and meningeal cells are all inhibitory to axon regeneration, and all express high levels of CS-GAG, including high levels of 6-sulphated GAG. In axon growth-inhibitory Neu7 astrocytes C6ST1 and 6-sulphated GAGs are expressed at high levels, whereas in permissive A7 astrocytes they are not detectable. These results suggest that the up-regulation of CSPG after CNS injury is associated with a specific sulphation pattern on CS-GAGs, mediating the inhibitory properties of proteoglycans on axonal regeneration.

Chondroitin sulphate proteoglycans (CSPGs) are up-regulated in the central nervous system after injury, specifically around the lesion site where the glial scar forms. This structure contains astrocytes, oligodendrocyte precursor cells, microglia and meningeal cells, and forms an inhibitory substrate for axon re-growth. CSPGs have been shown to be closely involved in this neuronal growth inhibition, specifically through their sugar chains. These chains are composed of repeats of the same disaccharide unit carrying sulphate groups in different positions. The sulphation pattern directly influences the CSPG binding properties and function; the specific sulphation pattern required for the inhibitory activity of these molecules on axon growth is unknown at present. The expression of the chondroitin sulphotransferases, which sulphate the disaccharide residues of CSPGs and thus are responsible for the structural diversity of the chondroitin sulphate sugar chains, is regulated differently in central nervous system during development and after injury, suggesting the implication of a specific sulphation pattern in the inhibitory activity of CSPGs.

We have investigated expression of the axon growth-inhibitory proteoglycan NG2 in peripheral nerve. In the adult, NG2 was present on endoneurial and perineurial fibroblasts, but not on Schwann cells. At birth, peripheral nerve NG2 was heavily glycanated, but was much less so in the adult. In vitro, sciatic nerve fibroblasts also produced heavily glycanated NG2. After peripheral nerve injury in rats and humans, an accumulation of NG2-positive cells was observed at the injury site. In the rat, there was an increase in NG2 glycanation for at least 2 weeks following injury. In mixed cultures of Schwann cells and peripheral nerve fibroblasts, the axons preferred to grow on the Schwann cells and seldom crossed onto the fibroblasts. Three-dimensional cultures of sciatic nerve fibroblasts were inhibitory to the growth of dorsal root ganglion axons. Inhibition of proteoglycan synthesis made the cells more permissive. NG2 may play a part in blocking axon regeneration through scar tissue in injured human peripheral nerve.

Many chondroitin sulfate proteoglycans (CSPGs) have been shown to influence CNS axon growth in vitro and in vivo. These interactions can be mediated through the core protein or through the chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains. We have shown previously that degrading CS GAG side chains using chondroitinase ABC enhances dopaminergic nigrostriatal axon regeneration in vivo. We test the hypothesis that interfering with complete CSPGs also limit axon growth in vivo. Neurocan, versican, aggrecan, and brevican CSPGs may be anchored within extracellular matrix through binding to hyaluronan glycosaminoglycan. We examine whether degradation of hyaluronan using hyaluronidase might release these inhibitory CSPGs from the extracellular matrix and thereby enhance regeneration of cut nigrostriatal axons. Anesthetized adult rats were given knife cut lesions of the right hemisphere nigrostriatal tract and cannulae were secured transcranially thereby allowing repeated perilesional infusion of saline or saline containing hyaluronidase once daily for 10 days post-axotomy. Eleven days post-transection brains from animals under terminal anesthesia were recovered for histological evaluation. Effective delivery of substance was inferred from the observed reduction in perilesional immunoreactivity for neurocan and versican after treatment with hyaluronidase (relative to saline). Immunolabeling using antibodies against tyrosine hydroxylase was used to examine the response of cut dopaminergic nigral neurons. After transection and treatment with saline, dopaminergic nigral neurons sprouted in a region lacking astrocytes, neurocan and versican. Axons did not regenerate into the lesion surround that contained astrocytes and abundant neurocan and versican. After transection and treatment with hyaluronidase, there was a significant increase in the number of cut dopaminergic nigral axons growing up to 800 microm anterior to the site of transection. However, cut dopaminergic nigral axons still did not regenerate into the lesion surround that contained reduced (albeit residual) neurocan and versican immunoreactivity. Thus, partial degradation of hyaluronan and chondroitin sulfate and depletion of hyaluronan-binding CSPGs enhances local sprouting of cut CNS axons, but long-distance regeneration fails in regions containing residual hyaluronan-binding CSPGs. Hyaluronan, chondroitin sulfate and hyaluronan-binding CSPGs therefore likely contribute toward the failure of spontaneous axon regeneration in the injured adult mammalian brain and spinal cord.

Invading meningeal cells form a barrier to axon regeneration after damage to the spinal cord and other parts of the CNS, axons stopping at the interface between meningeal cells and astrocytes. Axon behavior was examined using an in vitro model of astrocyte/meningeal cell interfaces, created by plating aggregates of astrocytes and meningeal cells onto coverslips. At these interfaces growth of dorsal root ganglion axons attempting to grow from astrocytes to meningeal cells was blocked, but axons grew rapidly from meningeal cells onto astrocytes. Meningeal cells were examined for expression of axon growth inhibitory molecules, and found to express NG2, versican, and semaphorins 3A and 3C. Astrocytes express growth promoting molecules, including N-Cadherin, laminin, fibronectin, and tenascin-C. We treated cultures in various ways to attempt to promote axon growth across the inhibitory boundaries. Blockade of NG2 with antibody and blockade of neuropilin 2 but not neuropilin 1 both promoted axon growth from astrocytes to meningeal cells. Blockade of permissive molecules on astrocytes with N-Cadherin blocking peptide or anti beta-1 integrin had no effect. Manipulation of axonal signalling pathways also increased axon growth from astrocytes to meningeal cells. Increasing cAMP levels and inactivation of rho were both effective when the cultures were fixed in paraformaldehyde, demonstrating that their effect is on axons and not via effects on the glial cells.

As the preceding discussion has demonstrated, experimental data now indicate that the expression of a number of different CSPGs is increased following CNS injury. The hyalectans neurocan, versican and [figure: see text] brevican, plus NG2 and phosphacan are upregulated following injury and all have been shown to exhibit inhibitory effects on neurite outgrowth in vitro. It is likely therefore that the increased expression of these molecules contributes to the non-permissive nature of the glial scar. The relative contributions of individual molecules remain, however, to be determined. It is important to remember also that not only does the glial scar contain many different inhibitory molecules, but that these are the products of a number of different cells, including not just astrocytes, but also oligodendrocyte progenitor and meningeal cells. It is arguable that the latter two cell types make a greater contribution than astrocytes to the inhibitory environment of the injured CNS. Recently, attempts have been made to alter the CSPG component of the glial scar in the hope that this will facilitate improved axonal regeneration. Three studies (Bradbury et al., 2002; Yick et al., 2000; Moon et al., 2001) have reported an improved regenerative response following treatment of the injured CNS with chondroitinase ABC. CSPGs represent a significant source of inhibition within the injured CNS; these studies indicate that successful CNS regeneration may be brought about by interventions which target these molecules and/or the cells which produce them.

Proteoglycans may modulate axon growth in the intact and injured adult mammalian CNS. Here we investigate the distribution and time course of deposition of a range of proteoglycans between 4 and 14 days following unilateral axotomy of the nigrostriatal tract in anaesthetised adult rats. Immunolabelling using a variety of antibodies was used to examine the response of heparan sulphate proteoglycans, chondroitin sulphate proteoglycans and keratan sulphate proteoglycans. We observed that many proteoglycans became abundant between 1 and 2 weeks post-axotomy. Heparan sulphate proteoglycans were predominantly found within the lesion core (populated by blood vessels, amoeboid macrophages and meningeal fibroblasts) whereas chondroitin sulphate proteoglycans and keratan sulphate proteoglycans were predominantly found in the lesion surround (populated by reactive astrocytes, activated microglia and adult precursor cells). Immunolabelling indicated that cut dopaminergic nigral axons sprouted prolifically within the lesion core but rarely grew into the lesion surround. We conclude that sprouting of cut dopaminergic nigral axons may be supported by heparan sulphate proteoglycans but restricted by chondroitin sulphate proteoglycans and keratan sulphate proteoglycans.

Chondroitin sulfate proteoglycan (CS-PG) expression is increased in response to CNS injury and limits the capacity for axonal regeneration. Previously we have shown that neurocan is one of the CS-PGs that is upregulated (Asher et al., 2000). Here we show that another member of the aggrecan family, versican, is also upregulated in response to CNS injury. Labeling of frozen sections 7 d after a unilateral knife lesion to the cerebral cortex revealed a clear increase in versican immunoreactivity around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed considerably more versican in the injured tissue extract. In vitrostudies revealed versican to be a product of oligodendrocyte lineage cells (OLCs). Labeling was seen between the late A2B5-positive stage and the O1-positive pre-oligodendrocyte stage. Neither immature, bipolar A2B5-positive cells, nor differentiated, myelin-forming oligodendrocytes were labeled. The amount of versican in conditioned medium increased as these cells differentiated. Versican and tenascin-R colocalized in OLCs, and coimmunoprecipitation indicated that the two exist as a complex in oligodendrocyte-conditioned medium. Treatment of pre-oligodendrocytes with hyaluronidase led to the release of versican, indicating that its retention at the cell surface is dependent on hyaluronate (HA). In rat brain, approximately half of the versican is bound to hyaluronate. We also provide evidence of a role for CS-PGs in the axon growth-inhibitory properties of oligodendrocytes. Because large numbers of OLCs are recruited to CNS lesions, these results suggest that OLC-derived versican contributes to the inhospitable environment of the injured CNS.

Following CNS injury in the adult mammal, axon regeneration fails in scar regions containing a number of different chondroitin sulfate-bearing proteoglycans (CSPGs) 1 . Degradation of chondroitin sulfate using chondroitinase ABC reduces growth inhibition associated with many CSPGs 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . Here we demonstrate that it is possible to enhance CNS axon regeneration in the adult rat nigrostriatal tract following chondroitinase ABC degradation of chondroitin sulfate.

Injury to the CNS results in the formation of the glial scar, a primarily astrocytic structure that represents an obstacle to regrowing axons. Chondroitin sulfate proteoglycans (CSPG) are greatly upregulated in the glial scar, and a large body of evidence suggests that these molecules are inhibitory to axon regeneration. We show that the CSPG neurocan, which is expressed in the CNS, exerts a repulsive effect on growing cerebellar axons. Expression of neurocan was examined in the normal and damaged CNS. Frozen sections labeled with anti-neurocan monoclonal antibodies 7 d after a unilateral knife lesion to the cerebral cortex revealed an upregulation of neurocan around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed substantially more neurocan in the injured CNS. Western blot analysis revealed neurocan and the processed forms neurocan-C and neurocan-130 to be present in the conditioned medium of highly purified rat astrocytes. The amount detected was increased by transforming growth factor beta and to a greater extent by epidermal growth factor and was decreased by platelet-derived growth factor and, to a lesser extent, by interferon gamma. O-2A lineage cells were also capable of synthesizing and processing neurocan. Immunocytochemistry revealed neurocan to be deposited on the substrate around and under astrocytes but not on the cells. Astrocytes therefore lack the means to retain neurocan at the cell surface. These findings raise the possibility that neurocan interferes with axonal regeneration after CNS injury.

Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenitors (O2A cells) can all produce molecules that inhibit axon regeneration. We have shown previously that inhibition of axon growth by astrocytes involves proteoglycans. To identify inhibitory mechanisms, we created astrocyte cell lines that are permissive or nonpermissive and showed that nonpermissive cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We have now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PGs in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was tested using a function-blocking antibody. This allowed increased growth of dorsal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increased the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 cells; CS-56 antibody had a similar effect. Inhibitory fractions of conditioned medium contained NG2 coupled to CS glycosaminoglycan chains, whereas noninhibitory fractions contained NG2 without CS chains. Enzyme preparations that facilitated axon growth in Neu7 cultures were shown to either degrade the NG2 core protein or remove CS chains. Versican is present as patches on Neu7 monolayers, but DRG axons do not avoid these patches. Therefore, NG2 appears to be the major axon-inhibitory factor made by Neu7 astrocytes. In the CNS, NG2 is expressed by O2A cells, which react rapidly after injury to produce a dense NG2-rich network, and by some reactive astrocytes. Our results suggest that NG2 may be a major obstacle to axon regeneration.

Damage to the central nervous system (CNS) results in a glial reaction, leading eventually to the formation of a glial scar. In this environment, axon regeneration fails, and remyelination may also be unsuccessful. The glial reaction to injury recruits microglia, oligodendrocyte precursors, meningeal cells, astrocytes and stem cells. Damaged CNS also contains oligodendrocytes and myelin debris. Most of these cell types produce molecules that have been shown to be inhibitory to axon regeneration. Oligodendrocytes produce NI250, myelin-associated glycoprotein (MAG), and tenascin-R, oligodendrocyte precursors produce NG2 DSD-1/phosphacan and versican, astrocytes produce tenascin, brevican, and neurocan, and can be stimulated to produce NG2, meningeal cells produce NG2 and other proteoglycans, and activated microglia produce free radicals, nitric oxide, and arachidonic acid derivatives. Many of these molecules must participate in rendering the damaged CNS inhibitory for axon regeneration. Demyelinated plaques in multiple sclerosis consists mostly of scar-type astrocytes and naked axons. The extent to which the astrocytosis is responsible for blocking remyelination is not established, but astrocytes inhibit the migration of both oligodendrocyte precursors and Schwann cells which must restrict their access to demyelinated axons.

Monoclonal antibodies (mAbs) against the major constituents of cartilage extracellular matrix, aggrecan and link protein, were screened by indirect immunofluorescence on frozen sections of bovine spinal cord. Antibodies against aggrecan and link protein gave rise to very similar perineuronal labeling in spinal cord gray matter. Aggrecan and link protein reactivities were seen in other regions of the central nervous system (CNS), although their distributions were not always coincident. Pretreatment of the tissue section with Streptomyces hyaluronidase, which is hyaluronate‐specific, led to the loss of both reactivities. On Western blots, anti‐aggrecan mAbs reacted with a large chondroitin sulfate proteoglycan. The chondroitinase‐treated CNS proteoglycan co‐migrated with the chondroitinase‐ and keratanase‐treated cartilage proteoglycan. In CNS tissue homogenates, the addition of Streptomyces hyaluronidase brought about the release of the proteoglycan from the tissue. Anti‐link protein mAbs were reactive with two species in the bovine CNS, the mobilities of which were very similar to those of the cartilage link proteins. The release of these species from the tissue required hyaluronidase. A rabbit antiserum against aggrecan was used to identify a similar proteoglycan in the rat CNS. In spinal cord‐derived cell cultures, the labeled material was associated with astrocytes. An aggrecan cDNA hybridized to a 9.5 kb mRNA in the rat CNS. We conclude that the perineuronal matrix consists, in part, of a hyaluronate‐bound aggrecan‐like proteoglycan and link proteins, and that the former is produced by astrocytes. © 1995 Wiley‐Liss, Inc.

Glial hyaluronate-binding protein (GHAP) is a 60 kDa glycoprotein with an amino acid sequence identical to that of the hyaluronate-binding region of versican, a large fibroblast aggregating proteoglycan found in the brain. Both GHAP and versican were identified by immunoblot in bovine brain extracts prepared only minutes after death. Human recombinant collagenase, stromelysin, mouse gelatinase and gelatinases isolated from human brain by affinity chromatography digest versican and give rise to a polypeptide with electrophoretic mobility identical to GHAP. Immunoblot analysis, peptide mapping and C-terminal amino acid sequencing indicate that the polypeptide generated by digestion with human brain gelatinases is identical to GHAP. We suggest that GHAP is a naturally occurring versican degradation product.