Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

Chondroitin sulfates affect the formation of the segmental motor nerves in zebrafish embryos

Chondroitin sulfates have been implicated in the promotion and in the inhibition of axon growth. In the zebrafish embryo, chondroitin sulfates are present at the interface of the somites and the notochord where spinal motor axons extend ventrally to establish the midsegmental ventral motor nerves. Injection of chondroitinase ABC prior to motor axon outgrowth effectively removed all chondroitin sulfate immunoreactivity and induced abnormal axonal outgrowth in many (39%) of the ventral motor nerves. The most common abnormality was the formation of side branches, approximately half of which extended posteriorly, the others anteriorly. The effect was specific to the removal of chondroitin sulfates, since injections of vehicle solution or of heparinase III did not affect the ventral motor nerves. Electron microscopic examination demonstrated that the injections caused no damage to spinal cord, somite, and notochord. This suggests that chondroitin sulfates normally constrain the outgrowth of the ventral motor nerves. Consistent with this hypothesis, injections of soluble chondroitin sulfates, either as a mixture or individually, led to truncated or missing ventral motor nerves. Truncations were most frequent after injection of chondroitin sulfate-B (up to 23%) while chondroitin sulfate-A had a lesser, and chondroitin sulfate-C no apparent, effect.     

Axon routing at the optic chiasm after enzymatic removal of chondroitin sulfate in mouse embryos

The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on guidance of retinal ganglion cell axons at the optic chiasm were investigated in a brain slice preparation of mouse embryos of embryonic day 13 to 15. Slices were grown for 5 hours and growth of dye-labeled axons was traced through the chiasm. After continuous enzymatic digestion of the chondroitin sulfate proteoglycans with chondroitinase ABC, which removes the glycosaminoglycan chains, navigation of retinal axons was disrupted. At embryonic day 13, before the uncrossed projection forms in normal development, many axons deviated from their normal course, crossing the midline at aberrant positions and invading the ventral diencephalon. In slices from embryonic day 14 embryos, axons that would normally form the uncrossed projection at this stage failed to turn into the ipsilateral optic tract. In embryonic day 15 slices, enzyme treatment caused a reduction of the uncrossed projection that develops at this stage. Growth cones in enzyme-treated slices showed a significant increase in the size both before and after they crossed the midline. This indicates that responses of retinal axons to guidance signals at the chiasm have changed after removal of the chondroitin sulfate epitope. We concluded that the chondroitin sulfate moieties of the proteoglycans are involved in patterning the early phase of axonal growth across the midline and at a later stage controlling the axon divergence at the chiasm.     

A heparin-binding growth factor, midkine, binds to a chondroitin sulfate proteoglycan, PG-M/versican.

Midkine is a heparin-binding growth factor with survival-promoting and migration-enhancing activities. In order to understand the regulation of midkine signaling, we isolated midkine-binding proteoglycans from day 13 mouse embryos, when midkine is intensely expressed. Deglycosylation followed by SDS/PAGE revealed various protein bands; one of these was identified as PG-M/versican by in gel trypsin digestion and sequencing the resulting peptides. PG-M/versican isolated from day 13 mouse embryos bound midkine with a Kd of 1.0 nM. Pleiotrophin/heparin-binding growth-associated molecule, which has a structure related to midkine, was also bound similarly. Digestion with chondroitinase ABC, AC-I or B abolished the binding to midkine. Heparin as well as chondroitin sulfate D and E inhibited the binding. After chondroitinase ABC digestion, the midkine-binding PG-M/versican released 4-sulfated, 6-sulfated, 2, 6-disulfated and 4,6-disulfated unsaturated disaccharides. These results suggest that midkine binds to a polysulfated domain in the chondroitin sulfate chain with a region of dermatan sulfate structure. This proteoglycan may modulate the midkine activity, as binding to midkine can enhance midkine action by concentrating it to the cell periphery or inhibit the action by competing with the binding to a signaling receptor.     

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac

Summary In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

The involvement of axonin-1/SC2 in mediating notochord-derived chemorepulsive activities for dorsal root ganglion neurites

Previous studies have suggested that the developing notochord secretes diffusible axon guidance molecules that repel dorsal root ganglion (DRG) neurites (R. Keynes et al., 1997, Neuron 18, 889-897; K. Nakamoto and T. Shiga, 1998, Dev. Biol. 202, 304-314). Neither notochord-derived chemorepellents nor their receptors on DRG neurites are, however, known. Here we investigated whether cell adhesion molecules (CAMs) of the immunoglobulin/fibronectin type III subfamily present on DRG neurites, including axonin-1/SC2, N-CAM, Ng-CAM, and Nr-CAM, are required for mediating the notochord-derived chemorepulsion. Using collagen gel cocultures of DRGs and notochord explants, we found that an antibody against axonin-1/SC2 diminished the effects of the chemorepulsive activity from the notochord, whereas antibodies against N-CAM, Ng-CAM, and Nr-CAM had no effect. We further showed that the removal of glycosylphosphatidylinositol-anchored cell surface molecules, including axonin-1/SC2, from DRG neurites diminished the effects of the notochord-derived chemorepulsive activity to an extent similar to that of treatment with the anti-axonin-1/SC2 antibody. These results suggest that axonin-1/SC2 expressed on DRG neurites may be involved in mediating the notochord-derived chemorepulsive activity.     

Embryonic lens repels retinal ganglion cell axons.

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc.     

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.     

Differential non-target-derived repulsive signals play a critical role in shaping initial axonal growth of dorsal root ganglion neurons

Initial trajectories of dorsal root ganglion (DRG) axons are shaped by chemorepulsive signals from surrounding tissues. Although we have previously shown that axonin-1/SC2 expression on DRG axons is required to mediate a notochord-derived chemorepulsive signal, Dev. Biol. 224, 112-121), other molecules involved in the non-target-derived repulsive signals are largely unknown. Using coculture assays composed of tissues derived from the chick embryo or mutant mice treated with function-blocking antibodies and phosphatidylinositol-specific phospholipase C, we report here that the chemorepellent semaphorin 3A (Sema3A) and its receptor neuropilin-1 are required for mediating the dermamyotome- and notochord-derived, but not the ventral spinal cord-derived, chemorepulsive signal for DRG axons. The dermamyotome-derived chemorepulsion is exclusively dependent on Sema3A/neuropilin-1, whereas other molecules are also involved in the notochord-derived chemorepulsion. Chemorepulsion from the ventral spinal cord does not depend on Sema3A/neuropilin-1 but requires axonin-1/SC2 to repel DRG axons. Thus, differential chemorepulsive signals help shape the initial trajectories of DRG axons and are critical for the proper wiring of the nervous system.     

Contactin 1 knockdown in the hindbrain induces abnormal development of the trigeminal sensory nerve in <Emphasis Type="Italic">Xenopus</Emphasis> embryos

In Xenopus tailbud embryos, the mandibular branch of trigeminal sensory nerve has a transient pathway innervating the cement gland. This pathway is settled by pioneer neurons in the trigeminal ganglion and along which extend later-growing axons from the trigeminal ganglion and the hindbrain. Axons in this branch express a neuronal recognition molecule, Contactin 1, from the initial stage of its outgrowth in early tailbud embryos and form a tightly joined, strongly Contactin 1-positive fascicle in the later stages. When the expression vector encoding the enhanced green fluorescent protein was electrotransfected into the brain neurons of early tailbud embryos, the fluorescence was detected in the hindbrain and the trigeminal nerve at late tailbud stages. Cotransfection of antisense vector caused knockdown of Contactin 1 concurrent with defasciculation and misguidance of the sensory axons in the trigeminal mandibular branch. The results suggest that Contactin 1 is required for the growing axon of hindbrain sensory neurons to recognize and follow the pathway settled by the pioneer neurons.     

Depolymerization of chondroitin C Sulfate by immobilized Proteus vulgaris cells

Chondroitinase ABC catalyzing the depolymerization of chondroitin sulfate was induced by incubating the Proteus vulgaris cells in a medium containing chondroitin C sulfate as an inducer. Incubation of P. vulgaris cells for 12 h in the presence of 0.3% inducer was optimal to obtain the cells with highly active chondroitinase ABC. Such cells were immobilized in k-carrageenan gel lattice, and some properties of chondroitinase ABC in immobilized cells were studied in comparison with those of the enzyme without immobilization (free enzyme). The stabilities of the enzyme toward heat and storage were remarkably improved by immobilizing the cells in k-carrageenan gel lattice. Optimal pH and temperature for activity of the enzyme were slightly shifted to the alkaline region and higher temperature by immobilization and were 9.0 and 35 degrees C, respectively.     

Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.     

Isolation and characterization of type IX collagen-proteoglycan from the Swarm rat chondrosarcoma.

Type IX collagen was partially purified from the Swarm rat chondrosarcoma by a series of a conventional salting-out procedures. The preparation was further separated by anion exchange chromatography into an unbound and a bound fraction in an A230 ratio of about 5:1. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the bound fraction appeared as a broad band, whose molecular mass ranged from 250 to 270 kDa. Digestion with chondroitinase ABC reduced the apparent molecular mass of the bound fraction to about 250 kDa, a value comparable to the molecular mass of the unbound fraction. Tryptic peptide maps of the protein moieties of unbound and bound forms showed that their molecular structures were basically identical. A monoclonal antibody specific for LMW, one of the pepsin-resistant fragments of the rat sarcoma type IX, reacted with both the unbound and bound fractions. Together the results indicate that the unbound and bound fractions represent a type IX collagen devoid of the chondroitin sulfate chain and its proteoglycan form with covalently bound chondroitin sulfate, respectively. The extent of glycosaminoglycan attachment to type IX collagen molecules in rat chondrosarcoma (about 16%) is quite different from the extents described in chick embryo cartilage (about 80%), chick vitreous humour (100%) and bovine cartilage (less than 5%). Further studies on the neoplastic tissue will offer additional information regarding the biological basis and biological consequences of the glycosaminoglycan attachment to type IX collagen molecules.     

A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.     

Variations in the composition of arterial wall isomeric chondroitin sulfate proteoglycans among different animal species

1. Isomeric chondroitin sulfate proteoglycans were extracted from human, bovine, swine and rabbit aortas by 4 M guanidine-HCl and were fractionated and purified by CsCl isopycnic centrifugation, Sepharose CL-4B gel filtration, DEAE-Sepharose ion-exchange chromatography and octyl-Sepharose hydrophobic interaction chromatography. 2. The molecular size and the composition of isomeric chondroitin sulfate proteoglycans varied among species. Variations were also noted in the composition and molecular weight of constituent glycosaminoglycan chains. 3. Observations made on chondroitinase ABC and chondroitinase AC digests of proteoglycans indicate that dermatan sulfate is linked to the core proteins through chondroitin sulfates.     

Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

A Chondroitin/Dermatan Sulfate Form of CD44 Is a Receptor for Collagen XIV (Undulin)

Collagen XIV, a fibril-associated collagen with interrupted triple helices, is expressed in differentiated soft connective tissues and in cartilage. However, a cellular receptor for this protein has not been identified. Here we show that human placental collagen XIV, isolated by a mild and simple two-step method, serves as adhesive protein for a variety of mesenchymal and some epithelial cells. Cell adhesion could be inhibited by preincubation of the collagen XIV substrate with heparin or with the chondroitin/dermatan sulfate proteoglycan decorin and by pretreatment of cells with chondroitinase ABC or heparinase III, suggesting a cell membrane proteoglycan as receptor. Affinity chromatography of¹²⁵I-labeled fibroblast cell surface proteins on collagen XIV–Sepharose yielded a chondroitin/dermatan sulfate proteoglycan with a molecular mass of 97–105 kDa after chondroitinase ABC digestion and of 60–70 kDa after further treatment withN-glycosidase F. The eluates contained also some high-molecular-weight material that was susceptible to digestion with heparinase but no detectable integrins. Immunoprecipitation with a specific monoclonal antibody identified the prominent chondroitin/dermatan sulfate proteoglycan as a member of the CD44 family. The interaction between collagen XIV and cells appears to be finely tuned, since matrix-associated glycosaminoglycans, and particularly proteoglycans like decorin, could compete with cells for the binding site(s) on collagen XIV under physiological conditions.     

Arylsulfatase B Improves Locomotor Function after Mouse Spinal Cord Injury

Bacterial chondroitinase ABC (ChaseABC) has been used to remove the inhibitory chondroitin sulfate chains from chondroitin sulfate proteoglycans to improve regeneration after rodent spinal cord injury. We hypothesized that the mammalian enzyme arylsulfatase B (ARSB) would also enhance recovery after mouse spinal cord injury. Application of the mammalian enzyme would be an attractive alternative to ChaseABC because of its more robust chemical stability and reduced immunogenicity. A one-time injection of human ARSB into injured mouse spinal cord eliminated immunoreactivity for chondroitin sulfates within five days, and up to 9 weeks after injury. After a moderate spinal cord injury, we observed improvements of locomotor recovery assessed by the Basso Mouse Scale (BMS) in ARSB treated mice, compared to the buffer-treated control group, at 6 weeks after injection. After a severe spinal cord injury, mice injected with equivalent units of ARSB or ChaseABC improved similarly and both groups achieved significantly more locomotor recovery than the buffer-treated control mice. Serotonin and tyrosine hydroxylase immunoreactive axons were more extensively present in mouse spinal cords treated with ARSB and ChaseABC, and the immunoreactive axons penetrated further beyond the injury site in ARSB or ChaseABC treated mice than in control mice. These results indicate that mammalian ARSB improves functional recovery after CNS injury. The structural/molecular mechanisms underlying the observed functional improvement remain to be elucidated.