Spinal cord injury is one of the severest traumatic life-changing events suffered by the human body, frequently incurring devastating physical loss of voluntary control and somatosensation. Due to poor outcomes, research has identified many of the pathophysiological processes that occur following SCI. It is dominated (in part) by the formation of a complex and inhibitory scar at the site of injury. Central to its formation is the development of a complex neuro-immune interaction between sentinel immune cells, microglia and injury-responsive astroglial cells (astrocytes) close to the lesion site. Both cell types contribute to the development and maturation of dense cell layers that regrowing axons find impossible to traverse without trophic support.
In conjunction with industry partners, the tissue engineering research group (TERG) have driven considerable successes in the area of advanced biomaterials for peripheral nerve repair. Recently we have begun a project in partnership with the Irish Rugby Football Union Charitable Trust and the Science Foundation Ireland funded Advanced Materials and BioEngineering Research (AMBER) Centre to design and build a next-generation smart bioscaffold system to treat spinal cord injury. While comprising a multifunctional axon guidance scaffold (AGS) containing extracellular matrix to trophically support axons it will also carry microencapsulated nanoparticle delivery systems containing diverse molecular cargoes (trophic factors, DNA, siRNA) for local targeting of injury responsive cells. Using this combinatorial approach, we aim to provide the optimal engineered conditions required for axons to not only cross the lesion site but also to extend into the distal cord and reform functional connections.
Injured axons are surrounded and damaged by injury responsive ‘reactive’ astrocytes and microglia, including a scar forming ‘A1’-type astrocyte and M1-type microglia subtypes that can arise from injuryinduced astroglial-microglial interaction. We have identified these cells as prime targets for therapeutic intervention. Using standard 2-dimensional culturing techniques, in combination with the use of microfluidic isolation methods and a 3D biomimetic spinal cord scaffold system (SCSS) this project will investigate and parse the different signalling pathways that contribute to astrocyte-microglial activation following injury in vitro. Using different physical- and chemical- induced injury methods the spatiotemporal genotypic and phenotypic changes in reactive cells to produce the A1 and M1 phenotypes will be identified. Using pharmacological manipulation, the different astrocyte-microglia and microglial-astrocyte signalling pathways will be investigated. Several poorly understood signalling pathways have been implicated such as the NFκB pathway, inflammatory cytokines and the P2Y1 purinergic signalling. These pathways will be targeted using selective agonists or antagonists via bath application in 2D cultures or through bolus delivery via a fibrin matrix to the SCSS. In addition, by using immunohistochemical analysis to a panel of markers in conjunction with RNA sequencing under specifically controlled injury conditions the spatiotemporal changes in phenotype in both cell types will be mapped to transcriptomic changes in order to further identify novel pathways.
This novel approach to study isolated and naïve astrocytes and microglia together in 2D and 3D architectures will provide an unsurpassed ability to parse the effects of physical damage and the neuroinflammatory pathways that contribute to glial scar formation. The deliverables from the project will identify key targets and/or pathways and will inform the design of therapeutic DNA- and RNA-containing nanoparticle cargoes for inclusion into the AGS system.
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