Targeting proteoglycan synthesis enzymes for spinal cord injury repair: Insights into ex vivo injury models, lentiviral vector-short hairpin RNA efficacy and hydrogel delivery systems

The obstacles to regeneration after spinal cord injury (SCI) are complex and persistent. Glial scar formation and proteoglycan (PG) dysregulation are key obstacles. Modifying PG levels after SCI can improve regeneration. However, due to extensive and persistent PG dysregulation, an intervention with widespread and long-lasting effects is required. Reducing enzymes involved in PG synthesis can beneficially alter the production of entire PG subgroups to have maximum benefit. This thesis focused on three PG synthesis enzymes; Xylosyltransferase I and II (XT1/XT2) which initiate the production of all PGs, and Chondroitin Sulphate N-Acetylgalactosaminyltransferase-I (Csgal1) that initiates the synthesis of chondroitin sulphate proteoglycans (CSPGs). This project aimed to evaluate XT1, XT2, and Csgal1 as targets to enhance regeneration after SCI. The first research chapter examined these enzymes in the rat spinal cord and investigated the best age and sex from which organotypic ex vivo SCI models should be derived for glial scar and PG synthesis enzymes investigations. These enzymes were evaluated in mixed glial culture (MGC) models to gain insights into their regulation under different types of inflammation. Additionally, lentiviral vectors carrying short hairpin RNAs (LV-shRNAs) were assessed to knock down these enzymes in cell lines and two hydrogels were evaluated as carriers for LV delivery. This thesis found that the mRNA levels of XT1, XT2, and Csgal1 increased in the spinal cords of rats as they aged, with adult rats (>6 months) having significantly more of these enzymes' mRNA than rat pups, from which ex vivo cultures are commonly derived. However, adult derived ex vivo cultures were not viable. Ex vivo SCI models derived from postnatal day 11 rats were viable and showed preliminary evidence of prominent glial scar production after transection injury, suggesting they may be favourable for studying PG synthesis enzymes and glial scar modifying therapies. Only XT1 mRNA expression increased following lipopolysaccharide-induced inflammation of MGCs, but it was not increased following a physical scratch injury to MGC. CSPG levels increased after all inflammatory stimuli, regardless of changes in PG enzyme mRNA. This indicates PG synthesis enzymes can be regulated differently depending on the type of inflammation, highlighting the complexity of CSPG regulation. LV-shRNA vectors targeting XT1, XT2, and Csgal1, successfully reduced their respective mRNA in at least one cell line. The XT2 targeting LV showed the most promise, consistently lowering XT2 mRNA, significantly reducing XT2 protein compared to control non targeting LV-shRNA and altering sugars related to CSPG and heparan sulphate PGs. Preliminary tests indicate this LV improved neurite outgrowth, but on average neurites were not significantly longer than controls. Further evaluation of all LV-shRNAs is needed to assess if lack in significant improvements in neurite outgrowth is due to inefficiency in LV-shRNAs or XT2 as a target. This thesis evaluated LV delivery using tyramine-modified hyaluronic acid (HATA) and oligopoly-ethylene glycol fumarate (OPF/OPF+) hydrogels, both of which indicated beneficial properties for LV delivery and SCI treatment. The formulation of HATA gels tested was ineffective, potentially reducing LV stability. OPF and OPF+ hydrogels released 26% and 14% of the total LV loaded into them respectively, mostly within the first 24 hours, which is not an efficient total release or desirable release pattern. The inefficient release from both HATA and OPF based hydrogels were hypothesised to result from unexpected LV-biomaterial interactions. Future research should focus on optimizing both LV and hydrogel formulations to improve interactions and LV release. This work provides insights into ex vivo SCI models, the expression of PG synthesis enzymes in the rat spinal cord and glial inflammation models. It demonstrates challenges with LV-shRNA-mediated enzyme reduction and hydrogel-based LV delivery. The thesis supports further research into PG synthesis enzymes in SCI pathology, alternative approaches to reduce PG for SCI regeneration, and optimisation of hydrogel systems for LV delivery in SCI treatment.

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

cbn

Collections

University of Galway Theses (PhD Theses)