Abstract
Spinal cord injury (SCI) results in a cascade of secondary events including inflammation, extracellular matrix (ECM) remodelling, and scarring, which all contribute to the chronic non-resolving tissue pathology and lack of repair. There are inherent links and complex interactions between these secondary pathologies. Glial scar formation is important for limiting inflammation to the immediate injury epicentre. However, scar-associated cells dramatically change their production of ECM components, and this ECM remodelling creates bioactive fragments that act as chemoattractants for immune cells. ECM components, such as chondroitinase sulfate proteoglycans (CSPGs), and molecules released by damaged/dying cells can activate inflammatory responses through immune receptors including toll-like receptor 4 (TLR4). Once activated, immune cells can then release molecules that further modulate the ECM composition, and can further propagate inflammation. Thus, immune-matrix interactions are complex and bi-directional. Targeting immune-matrix interactions may help to identify key bioactive mediators and targets for improving tissue pathology and recovery after SCI.We have used two approaches to modulate immune-matrix interactions after T10 spinal contusion in adult rats: pharmacological blockade of the immune toll-like receptor 4 (TLR4), and digestion of inhibitory CSPGs. For TLR4 inhibition studies, rats were treated with the TLR4 inhibitor, TAK-242, acutely after injury. This dosing regimen significantly reduced pro-inflammatory IL-6 and iNOS gene expression at 24 hours postinjury, and the percentage of iNOS+ macrophages at 7 days post-injury. For CSPG digestion studies, animals received intraspinal injections of a chondroitinase ABC lentiviral vector (LV-ChABC), which enabled digestion of CSPGs in spinal injured tissue. We assessed the effects of our immune-blocking and matrix modifying approaches on a number of parameters. Functional recovery was assessed using Basso, Beattie and Bresnahan (BBB) score and horizontal ladder tasks. Histological assessments included tissue sparing and neuronal survival. To identify potential bioactive mediators of inflammatory pathology, we performed proteomics analysis of spinal cord tissue, collected 7 days and 8 weeks post-injury. Samples were biochemically processed to collect cellular and extracellular tissue subfractions for analysis with high-throughput proteomics. Proteomics datasets were analysed using MaxQuant, Perseus and Proteus R package to identify changes after TLR4 inhibition or digestion of CSPGs. Potential targets identified through this analysis were subsequently validated by Western blotting and included osteopontin, Hapln1 and STAT1. Preliminary glycomics was perfomed on naive and injured tissue, and early results suggest there may be changes in fucosylation patterns after SCI.
Using a combination of behavioural, histological, and proteomics data we aimed to determine the effects of TLR4 and CSPG inhibition on modulating functional outcome, extent of injury pathology, and identify key components involved in immune and ECM pathologies. Proteomics analysis on SCI tissue treated with these two interventions has identified several targets of interest which can be studied further in future mechanistic studies. Although glycomics analysis presented in this thesis was only preliminary, these studies provide a basis for further research into this underexplored area. Finally, as proteomics was performed on naive, early, and late stage SCI tissue, in both rat and mouse, these datasets provide a valuable resource for future researchers to investigate their proteins of interest.
| Date of Award | 1 Aug 2021 |
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| Original language | English |
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| Supervisor | Elizabeth Bradbury (Supervisor) & Tanya Shaw (Supervisor) |