Abstract
The failure of the spinal cord to regenerate can be attributed both to a lack of trophic support for regenerating axons and to upregulation of inhibitory factors such as chondroitin sulphate proteoglycans including NG2 following injury. Lentiviral vector-mediated gene therapy is a possible strategy for treating spinal cord injury (SCI). This study investigated the effect of lentiviral vectors expressing Neurotrophin-3 (NT-3) and short-hairpin RNA against NG2 (NG2 sh) to enhance neurite outgrowth in in vitro and ex vivo transection injury models. Conditioned medium from cells transduced with NT-3 or shNG2 lentiviruses caused a significant increase in neurite length of primary dorsal root ganglia neurons compared to the control group in vitro. In an ex vivo organotypic slice culture (OSC) transduction with Lenti-NT-3 promoted axonal growth. Transducing OSCs with a combination of Lenti-NT-3/NG2 sh lead to a further increase in axonal growth but only in injured slices and only within the region adjacent to the site of injury. These findings suggest that the combination of lentiviral NT-3 and NG2 sh reduced NG2 levels and provided a more favourable microenvironment for neuronal regeneration after SCI. This study also shows that OSCs may be a useful platform for studying glial scarring and potential SCI treatments.
Highlights
The inability of the spinal cord to regenerate after damage makes spinal cord injury (SCI) a devastating condition
Lentiviral vectors encoding short hairpin RNA (shRNA) targeting the NG2 transcript of Mus musculus but not Rattus norvegicus were commercially available at the initiation of this study
Ex vivo models of SCI allow researchers to mimic in vivo injury and to optimise novel interventions before moving into preclinical studies supporting the three Rs of animal usage
Summary
The inability of the spinal cord to regenerate after damage makes spinal cord injury (SCI) a devastating condition. SCI represents a clear unmet medical need where new treatments are desperately required, and these need to be evaluated in preclinical studies prior to clinical trials [2]. Animal models of SCI have been developed to better understand the physiology of damage and repair [3]. These animal models are important for evaluating the efficacy and safety of new therapies to promote regeneration and repair of damaged tissue. The animal-based models of SCI require substantial post-operative care and are time consuming and expensive [4]. There is an international effort to replace, reduce and refine animal-based studies (the three Rs)
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