Harnessing cell-encapsulated hydrogels to study astrocyte mechanoresponse in 4D

In glaucoma, the optic nerve head (ONH) is exposed to increased biomechanical strain, impacting the resident astrocytes that maintain neural homeostasis. After injury, astrocytes exhibit morphologic and metabolic shifts; however, the specific impact of glaucoma-related biomechanical strains on astrocyte behavior remains poorly understood. To address this, we utilized our previously established 3D cell-encapsulated ECM hydrogel to elucidate ONH astrocyte transcriptomic and cellular responses to varying biomechanical strain levels over time. Murine ONH astrocyte-encapsulated hydrogels were subjected to 0, 3, or 10% cyclic strain for 4h and 24h. Using confocal reflectance microscopy, we observed that hydrogel porosity was adequate for nutrient supplementation, while bulk hydrogel stiffness and cell viability remained unchanged after biomechanical strain. Mechanotranscriptional responses were robustly altered within 4h in a hydrogel region-, strain-, and time-dependent manner. RNA sequencing revealed changes in gene expression related to cell morphology, division, senescence, hypoxia, metabolism, and ECM regulation. Morphometric analyses of strained ONH astrocytes showed reduced F-actin area coverage, increased GFAP, HIF-1α, fibronectin, and collagen fibril reorganization. Our findings demonstrate that ONH astrocyte transcriptional responses are highly dependent on duration/magnitude of biomechanical strain and surrounding ECM density, corresponding with altered cell morphology, hypoxia, and ECM modification. This ONH astrocyte-encapsulated hydrogel provides a valuable platform for nuanced future manipulation of porosity, ECM composition, and cellularity to study the impact of biomechanical strain on ONH pathophysiology.