Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2

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.

Human ex vivo spinal cord slice culture as a useful model of neural development, lesion, and allogeneic neural cell therapy

“…Moreover, they may recapitulate or mimic lesion pathology, allowing the study of pathological and regenerative pathways in tissue-like complexity [13,14]. Therefore, an established human nervous system slice culture model would allow promising repair strategies including gene [15,16] as well as neural cell therapeutic approaches [17][18][19][20] to be pre-clinically tested in human conditions. Thus, as a complementary method (allowing human spinal cord study, while reducing costs and resource demands in addition to animal use), human slice culture, when available, is of value.…”

Injectable hydrogels in stroke and spinal cord injury treatment: a review on hydrogel materials, cell–matrix interactions and glial involvement

“…This could be useful in tackling the issues of glial scarring in spinal cord repair. 171 Neural system-on-a-chip is a scaffoldbased in vitro system for studies of complex physiological tissue interactions on a functional and customizable substrate. The system may be useful as a platform to study the nerve injury model since it allows direct observation of the states of injury and regeneration.…”