Investigating the mechanoresponse of optic nerve head astrocytes using 3D hydrogels and its implications in glaucoma

Astrocytes residing within the optic nerve head (ONH) are master regulators of tissue homeostasis. They integrate multiple cues from the vasculature, matrix mechanical stimuli, and can functionally support neighboring neurons. In glaucoma, elevated intraocular pressure causes excessive biomechanical strain on the ONH. In response, astrocytes undergo early morphological remodeling, accompanied by reactive gliosis, and ECM dysregulation, prior to irreversible retinal ganglion cell damage. To better understand the ONH astrocyte mechanoresponse to glaucomatous injury, this thesis introduces a novel 3D ECM cell-encapsulated hydrogel system that more closely resembles the biochemical and mechanical milieu of in vivo neural tissue than existing 2D culture models. In Chapter 2, we established a photocrosslinkable ECM hydrogel by mixing natural polymers, like collagen I/hyaluronic acid, with mouse ONH astrocytes (MONHAs). This model system allowed for astrocytes to develop stellate coupled network. Exposure to pro-fibrotic cytokine TGFβ2 induced reactive astrogliosis, GFAP upregulation, F-actin remodeling, and increased fibronectin/collagen IV deposition. In Chapter 3, we subjected MONHA-encapsulated hydrogels to glaucomatous biomechanical strains and showed that astrocytes exhibited strain- and time-dependent transcriptomic and morphological changes, mirroring responses seen in in vivo models of glaucoma. Specifically, strained MONHAs decreased actin coverage, increased GFAP and HIF-1α expression, and induced remodeling of ECM collagen fibrils. In Chapter 4, we further investigated ONH astrocyte mechanoresponse to biomechanical strain by focusing on the role of mechanosensitive channel Piezo1. Here, pharmacological activation of Piezo1 in encapsulated MONHAs revealed morphological changes similar to strain-induced alterations, such as reduced actin coverage, process retraction and nuclear volume changes. Moreover, these events were rescued upon inhibition of mechanosensitive channels or the RhoA/ROCK pathway. As such, our findings implicate Piezo1 as a key regulator of ONH astrocyte mechanoresponse to biomechanical strain, potentially through RhoA/ROCK signaling. Finally, in Chapter 5 we integrate our findings within the broader aspects of astrocyte mechanobiology across CNS mechanopathologies and discuss future translational studies that aim at preserving astrocyte morphological integrity and slowing glaucomatous neurodegeneration. Collectively, this thesis establishes a novel 3D ECM hydrogel system as a more physiologically relevant in vitro platform, well-suited to dissect astrocyte mechanosensation, transduction, and response to glaucoma-related biomechanical strains.

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Investigating the mechanoresponse of optic nerve head astrocytes using 3D hydrogels and its implications in glaucoma

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