Analysis of the Effect of Substrate Stiffness on the Efficacy of Fibroblast Growth Factor 2 and Bone Morphogenetic Protein 4 in Inducing Pro-Regenerative Astrocyte Phenotype: A Research Protocol

Introduction: Astrocytes are glial cells essential for neuronal development and repair and are thus a highly promising target for regenerative therapies for neurodegenerative disease. Neurodegeneration has been connected to the degradation of extracellular matrix (ECM) components. ECM's structural properties, such as stiffness, influence the phenotypic outcomes of growing cells. Notably, astrocytes are induced into an A2 (pro-regenerative) phenotype when grown on stiff substrates or exposed to specific signalling molecules, particularly fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein 4 (BMP-4). Given that the ECM can bind and sequester growth factors, it is possible that matrix stiffness may modulate the efficacy of these molecules. Methods: In this in vitro study, rat primary cortical astrocytes will be cultured on soft and stiff substrates and exposed to varying amounts of FGF-2 and BMP-4. This study aims to determine the effect of substrate stiffness on the efficacy of FGF-2 and BMP-4 in promoting pro-regenerative phenotype. Cell proliferation and glial fibrillary acidic protein (GFAP) expression are key indicators of reactive astrocytes, including pro-regenerative astrocytes. 5-bromo-2-deoxyuridine staining will be used to analyze proliferation. GFAP expression will be determined using anti-GFAP antibody conjugated with Alexa Fluor 594. Further, pro-regenerative phenotypic genes Clcf1, Tgm2, and Ptgs2 will be detected via polymerase chain reaction to differentiate from pro-inflammatory astrocytes, a separate category of reactive astrocytes. Anticipated Results: We hypothesize there will be a greater positive correlation between the concentration of FGF-2 or BMP-4 and expression of markers of pro-regenerative phenotype under stiff substrate conditions, compared to softer substrate, thus indicating dependency or synergy between FGF-2 or BMP-4 and extracellular matrix-dependent pathways. Discussion: The correlation between FGF-2 or BMP-4 concentration and the prominence of A2 astrocyte phenotype indicators will be graphed and reported along with a comparison of these correlations between soft and stiff substrate groups. The authors will attempt to conclude whether substrate stiffness significantly effects the activities of FGF-2 or BMP-4. Conclusion: We hope the results of this proposed study will inform the development of neuroregenerative therapies involving astrocytes by indicating the necessity for greater focus on either the application of signalling molecules or modification of ECM.