Ch Chau

Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded guidance channels after spinal cord injury

Grafting of Schwann cell‐seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured cord. Despite success in guiding axonal growth into the graft, regeneration across the distal graft–host interface into the host spinal cord was limited. We hypothesized that chondroitin sulfate (CS) glycoforms deposited at the gliotic front of the interface constitute a molecular barrier to axonal growth into the host cord. Because CS glycoforms deposited by purified astrocytes in vitro were removable by digestion with chondroitinase ABC, we attempted to achieve likewise by infusion of the enzyme to the host side of the interface. By 1 month post‐treatment, significant numbers of regenerating axons crossed an interface that was subdued in macrophage/microglia reaction and decreased in CS‐immunopositivity. The axons extended as far into the caudal cord as 5 mm, in contrast to nil in vehicle‐infused controls. Fascicular organizations of axon−Schwann cell units within the regenerated tissue cable were better‐preserved in enzyme‐treated cords than in vehicle‐infused controls. We conclude that CS glycoforms deposited during gliosis at the distal graft–host interface could be cleared by the in vivo action of chondroitinase ABC to improve prospects of axonal regeneration into the host spinal cord.

Upregulation of chondroitin 6-sulphotransferase-1 facilitates Schwann cell migration during axonal growth

Cell migration is central to development and post-traumatic regeneration. The differential increase in 6-sulphated chondroitins during axonal growth in both crushed sciatic nerves and brain development suggests that chondroitin 6-sulphotransferase-1 (C6ST-1) is a key enzyme that mediates cell migration in the process. We have cloned the cDNA of the C6ST-1 gene (C6st1) (GenBank accession number AF178689) from crushed sciatic nerves of adult rats and produced ribonucleotide probes accordingly to track signs of 6-sulphated chondroitins at the site of injury. We found C6st1 mRNA expression in Schwann cells emigrating from explants of both sciatic nerve segments and embryonic dorsal root ganglia. Immunocytochemistry indicated pericellular 6-sulphated chondroitin products around C6ST-1-expressing frontier cells. Motility analysis of frontier cells in cultures subjected to staged treatment with chondroitinase ABC indicated that freshly produced 6-sulphated chondroitin moieties facilitated Schwann cell motility, unlike restrictions resulting from proteoglycan interaction with matrix components. Sciatic nerve crush provided further evidence of in vivo upregulation of the C6ST-1 gene in mobile Schwann cells that guided axonal regrowth 1-14 days post crush; downregulation then accompanied declining mobility of Schwann cells as they engaged in the myelination of re-growing axons. These findings are the first to identify upregulated C6st1 gene expression correlating with the motility of Schwann cells that guide growing axons through both developmental and injured environments.

Heparan sulphates upregulate regeneration of transected sciatic nerves of adult guinea-pigs

The increased content of soluble glycosaminoglycan‐containing forms in sciatic nerves during recovery from crush injury [Shum & Chau (1996) J. Neurosci. Res., 46, 465] suggests that the glycosaminoglycans modulate the environment for post‐traumatic tissue remodelling and axonal regrowth. To test this, defined amounts of soluble heparan sulphates from bovine kidney or guinea‐pig nerve were introduced into the regenerating environment via silicone conduits that bridged 8‐mm gaps of transected sciatic nerves of adult guinea‐pigs. Controls were bridged using the phosphate‐buffered saline (PBS) vehicle or a chondroition sulphate preparation from whale cartilage. After timed periods of recovery, the animals were assessed for electromyographic signals at the target gastrocnemius muscle to determine the conduction velocity across the bridged nerve. Sections of the bridge were also histologically examined for nerve fibres. Transected sciatic nerves bridged with heparan sulphates or chondroitin sulphate showed earlier stimulated myelination of axons (week 5–6) than PBS‐bridged nerves (week 9). Initial electromyographic indication of reconnection with the target was at week 9 post‐transection. In the course of 20 weeks, transected sections of the bridge indicated similar numbers of unmyelinated axons irrespective of bridge material, but distinctly higher numbers of myelinated axons in heparan sulphate‐bridged nerves than either PBS‐ or chondroitin sulphate‐bridged nerves. At the end of the same period, heparan sulphate‐bridged nerves resumed normal conduction velocities, but both PBS‐ and chondroitin sulphate‐bridged nerves remained at 50% of that of the intact contralateral nerves. These results are the first to demonstrate that supplementation of soluble heparan sulphate to the fluid regenerative neural environment can restore functional, axonal reconnection of the severed nerve with the target muscle.

Changes in glycosaminoglycans during regeneration of post-crush sciatic nerves of adult guinea pigs

The glycosaminoglycans of sciatic nerves recovering from crush‐injury were studied in adult guinea pigs and compared with those of non‐injured mature neural tissues. The glycosaminoglycans were recovered from the 1,900 g supernatant and pellet of the tissue homogenates and assayed for hexuronate contents and susceptibilities to hyaluronidase, chondroitinase ABC, and nitrous acid. In the normal brain and central nerve tracts, the glycosoaminoglycans were distributed both in the supernatant and pellet fractions; the brain showed a predominance of chondroitin sulphates but the tracts showed a predominance of heparan sulphates. Twice as much glycosaminoglycans were found in normal sciatic nerves, only in the pellet fraction and with heparan sulphate predominant. In the 2 weeks post‐crush, progressive increase in hexuronate was observed, due mainly to additional chondroitin sulphate forms in the supernatant; the pellet fraction in the same period was however similar to the untreated controls in relative abundance of glycosaminoglycan classes and hexuronate content. At 4 weeks post‐crush, although the total hexuronate returned to the control level, a significant proportion of glycosaminoglycans remained in the supernatant fraction. Evidence is thus provided for the need to modulate the glycosaminoglycan expression pattern in adult neural tissue to allow post‐traumatic tissue remodelling and axonal regrowth.

Mapping heparanase expression in the spinal cord of adult rats

This work addresses the cellular localization of heparanase and its colocalization with syndecan‐3, a transmembrane heparan sulfate proteoglycan in spinal cords of adult rats. Reverse transcriptase/polymerase chain reaction (RT‐PCR) and in situ hybridization for the heparanase transcript revealed expression in neurons and white matter glia. This was confirmed by immunohistochemistry showing cytoplasmic localization of the heparanase protein. Double immunofluorescence for heparanase and syndecan‐3 revealed colocalization of the proteins in cell bodies of neurons and oligodendrocytes, suggestive of constitutive expression in these cell types. In contrast, only subpopulations of astrocytes and NG2‐expressing glia in the white matter expressed heparanase, and these did not show expression of syndecan‐3. Cultures of astrocytes further evidenced upregulation of heparanase expression with TGF‐β1 treatment, but no accompanying upregulation of syndecan‐3 was detectable. These first findings of heparanase expression in the adult cord therefore provide the cellular basis for understanding functional interactions of heparanase and syndecan‐3 in the normal neural network or otherwise in glial reactions to spinal cord injury. J. Comp. Neurol. 494:345–357, 2006.

Heparanse-syndecan-3 interaction in neurons of the adult spinal cord

pp. 111–156 of this Free journal issue entitled: Abstracts of the 24th and the 25th Scientific Meeting of the Hong Kong