Ex Vivo slice culture model of rat spinal cord injury as a platform for testing proposed treatment strategies

Ab Patar, Mohd Nor Azim
There are several detrimental events that occur in the aftermath of a spinal cord injury (SCI); these include glial scar formation, accumulation of axon growth inhibitors and immune mediators. These events present challenges to SCI repair and therapy development. Ex vivo organotypic slice cultures provide a reliable model platform for the study of cell dynamics and therapeutic intervention following SCI. Four research chapters (chapters 3-6) are presented here. In research chapter 3 of this thesis, three SCI models (stab, contusion and transection injury) were developed to study glial scar formation with a focus on reactive astrocytes and NG2 proteoglycans. Stereological analysis was carried out on scar zone (SZ) and injury zone (IZ) in each model of SCI. The transection injury model was chosen as the best model to study axonal growth after SCI. Ex vivo spinal culture experiments consumed many Millicell® inserts. In chapter 4, we optimised a cheap reproducible method of preparing homemade inserts. Chondroitinase ABC (ChABC) successfully promotes neurite outgrowth and functional recovery after SCI in in vivo rat studies. In chapter 5, the effect of ChABC on the cellular environment was examined after SCI in the SZ, near scar zone (NSZ) and far scar zone (FSZ). We show that ChABC enzymatic treatment causes significant change in the injured microenvironment and may promote axonal regeneration. Lentiviral vectors expressing neurotrophin-3 (NT3) and short hairpin NG2 (shNG2) knockdown were tested in vitro and in ex vivo transected spinal cord slice cultures in chapter 6. NT3 promotes axonal sprouting following SCI. NG2 is upregulated after SCI and inhibits neurite outgrowth. We show that NG2 sh1 causes NG2 knockdown and promotes neurite outgrowth in vitro. Combination treatment of Lenti NT3/NG2 sh1 promotes axonal sprouting at day 7 post transection injury. This study shows how ex vivo spinal cord slices can be used as a platform for studying glial scarring and potential SCI treatments.
NUI Galway
Publisher DOI
Attribution-NonCommercial-NoDerivs 3.0 Ireland