Development of a Platform for Studying 3D Astrocyte Mechanobiology: Compression of Astrocytes in Collagen Gels

Abstract

21Glaucoma is a common optic neuropathy characterized by retinal ganglion cell death. 22Elevated intraocular pressure (IOP), a key risk factor for glaucoma, leads to significant 23 biomechanical deformation of optic nerve head (ONH) cells and tissues. ONH astrocytes 24 respond to this deformation by transforming to a reactive, proliferative phenotype, which 25 has been implicated in the progression of glaucomatous vision loss. However, little is 26 known about the mechanisms of this transformation. In this study, we developed a 3D 27 collagen gel culture system to mimic features of ONH deformation due to elevated IOP. 28Compressive loading of astrocyte-seeded collagen gels led to cell alignment 29 perpendicular to the direction of strain, and increased astrocyte activation, as assayed by 30 GFAP, vimentin, and s100β levels, as well as MMP activity. This proof-of-concept study 31shows that this system has potential for studying mechanisms of astrocyte 32 mechanobiology as related to the pathogenesis of glaucoma. Further work is needed to 33 establish the possible interplay of mechanical stimulation, matrix properties, and hypoxia 34 on the observed response of astrocytes.

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants

“…The F-to-G actin ratio (F-actin fiber length over G-actin fluorescence) significantly decreased during reactive astrocytosis ( Figure 1E). Altogether, these data are consistent with in vitro studies using 3D culture models [38], in vivo models using an acute controlled elevation of intraocular pressure (CEI) [39], and with the generally accepted notion that mechanical forces exerted on the human optic nerve head cause disruption of the actin cytoskeleton (reviewed in [40]).…”

Optic Nerve Head Astrocytes Display Axon-Dependent and -Independent Reactivity in Response to Acutely Elevated Intraocular Pressure

“…Thus, some ONH astrocyte structural responses to elevated IOP appear to be independent of viable axons, and do not depend on yet unidentified axon signaling. This is supported by three-dimensional astrocyte culture models in which deformations of the culture matrix cause re-orientation of astrocyte, despite the absence of neurons/axons in the culture model 31. In addition, cultured ONH astrocytes in the absence of neurons display significant actin-based motility within 1 day after initiation of a wound healing assay,23 and become four to six times more motile 12 hours after exposure to elevated hydrostatic pressure in vitro 22.…”

“…Thus, understanding the events leading to ONH astrocyte reactivity is critical to defining the role of astrocyte reactivity in glaucomatous axon degeneration 17,28–30. ONH astrocyte reactivity may be initiated directly via primary local effects on astrocytes (including local biomechanical forces from IOP elevation),31 or indirectly through signaling and crosstalk between astrocytes and the axonal/extracellular environment at the site of injury. While astrocyte-axon communication and crosstalk has been well documented in several models,32–34 the role of astrocyte-axon communication in initiating astrocyte reactivity remains an open question.…”

Biomechanics of the optic nerve head and peripapillary sclera in a mouse model of glaucoma

“…The strain response of the ONH to IOP change has been characterized in post-mortem human eyes [14–16] and more recently directly in patient eyes [17–19]. Cell-scale studies of ONH astrocyte responses to mechanical loading have been limited to two-dimensional cell culture [20–23] and more recently three-dimensional cell culture methods have been proposed [24]. Cell culture methods cannot capture the complex in vivo cellular connections of ONH astrocytes to their mechanical environment, and individual cells of the human ONH are currently impossible to visualize in vivo.…”