Abstract
Purpose :
Glaucoma is characterized by progressive loss of retinal ganglion cells (RGCs) within the optic nerve head (ONH). Elevated intraocular pressure (IOP) in glaucoma can induce pathologic biomechanical strains on the ONH. In response, local astrocytes upregulate reactivity markers such as intermediate filament glial fibrillary acidic protein (GFAP) and vimentin. They remodel their F-actin cytoskeleton to provide neurotrophic support to RGCs. However, with chronically high IOP, astrocytes become gliotic and alter the surrounding extracellular matrix (ECM). Using our established 3D hydrogel system which allows for analysis of cell-to-cell and cell-ECM interaction, we investigated mouse ONH astrocyte (MONHA) response to IOP-associated pathological mechanical strains.
Methods :
Primary MONHAs (N = 3) were isolated and cell purity confirmed. MONHA-hydrogels were engineered by mixing 2.5 x 106cells/ml with photoactive ECM components (i.e., collagen type I, hyaluronic acid) and crosslinked using 0.025% Riboflavin and UV light (405-500 nm, 10.3mW/cm2) for 5 minutes. Hydrogels were cultured for two weeks prior to mechanical loading of 0%, 3%, and 10% compressive or tensile strains for 0-24 hours. Cell viability and hydrogel stiffness were measured using MTS assays and rheometry, respectively. Astrocyte morphology was systematically analyzed; intracellular F-actin, vimentin, and GFAP, and ECM levels were quantified.
Results :
Astrocytic response and cytoskeletal remodeling were detected after 12 hours of mechanical loading. Astrocyte viability and hydrogel stiffness were unchanged post 24-hour mechanical loading. Increased compressive/tensile strains were associated with diminished astrocyte process length, complexity, and F-actin levels (~0.6-fold decrease, p<0.0005) centrally versus peripherally (~2-fold increase, p<0.05). Strained MONHAs upregulated levels of GFAP and vimentin (~1.4-fold, p<0.005), and altered organization of ECM proteins (i.e., collagen IV and fibronectin).
Conclusions :
IOP-associated mechanical strains alter MONHA morphology, cytoskeletal architecture, and matrix deposition, akin to in vivo. Our system allows for detailed examination of MONHA response to mechanical strains in glaucoma. Ongoing RNA sequencing analysis will determine differentially expressed genes/pathways in response to mechanical strains.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.