Dataset Information

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury.

ABSTRACT:

Aims

Usually, spinal cord injury (SCI) develops into a glial scar containing extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABC (ChABC), from Proteus vulgaris degrading the glycosaminoglycan (GAG) side chains of CSPGs, offers the opportunity to improve the final outcome of SCI. However, ChABC usage is limited by its thermal instability, requiring protein structure modifications, consecutive injections at the lesion site, or implantation of infusion pumps.

Methods

Aiming at more feasible strategy to preserve ChABC catalytic activity, we assessed various stabilizing agents in different solutions and demonstrated, via a spectrophotometric protocol, that the 2.5 mol/L Sucrose solution best stabilized ChABC as far as 14 days in vitro.

Results

ChABC activity was improved in both stabilizing and diluted solutions at +37°C, that is, mimicking their usage in vivo. We also verified the safety of the proposed aqueous sucrose solution in terms of viability/cytotoxicity of mouse neural stem cells (NSCs) in both proliferating and differentiating conditions in vitro. Furthermore, we showed that a single intraspinal treatment with ChABC and sucrose reduced reactive gliosis at the injury site in chronic contusive SCI in rats and slightly enhanced their locomotor recovery.

Conclusion

Usage of aqueous sucrose solutions may be a feasible strategy, in combination with rehabilitation, to ameliorate ChABC-based treatments to promote the regeneration of central nervous system injuries.

SUBMITTER: Raspa A

PROVIDER: S-EPMC6436590 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury.

Raspa Andrea A Bolla Edoardo E Cuscona Claudia C Gelain Fabrizio F

CNS neuroscience & therapeutics 20180531 1

<h4>Aims</h4>Usually, spinal cord injury (SCI) develops into a glial scar containing extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABC (ChABC), from Proteus vulgaris degrading the glycosaminoglycan (GAG) side chains of CSPGs, offers the opportunity to improve the final outcome of SCI. However, ChABC usage is limited by its thermal instability, requiring protein structure modifications, consecutive injections at the lesion site, or implantation ...[more]

PMID: 29855151

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Project description:Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1?+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.

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.

Dataset Information

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury.

Aims

Methods

Results

Conclusion

Publications

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury.

Similar Datasets

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