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Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection.

ABSTRACT: Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.

SUBMITTER: Cheng CH

PROVIDER: S-EPMC4579094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSpinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord.MethodsENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair.ResultsENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours.ConclusionsRegenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.

Project description:BackgroundThere is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury.Methodology/principal findingsTo investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of β-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the β-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth.Conclusions / significanceThe novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson's disease.

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Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection.

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