Abstract
Purpose :
Alterations in extracellular matrix (ECM) stiffness have been linked to glaucoma, where increased intraocular pressure (IOP) is a major risk factor. Müller glia (MG) are able to respond to mechanical cues, including changes in ECM stiffness and IOP. In this study, we analyzed how substrate stiffness affects MG survival, behavior, and ECM deposition in vitro, and how these are influenced by high pressure (HP) conditions
Methods :
Primary rat MG were cultured on polyacrylamide (PAA) gels of 10 and 100 kPa and on a glass-slide as control. MG were labelled by immunocytochemistry against vimentin for morphology, Ki67, PCNA and BrdU for cell proliferation, GFAP for gliosis, α-SMA for dedifferentiation, collagen I, collagen IV and fibronectin for ECM deposition, and against mechanorreceptor TRPV4. A custom-made pressure chamber was used for HP conditions. Positive-labelled cells were quantified and the cell area and the intensity of ECM deposition were measured
Results :
Survival was severely reduced on PAA gels (4.37±1.07%) compared to glass (considered 100%). MG grown on softer gels exhibited a round shape and reduced cell area and became reactive as shown by the expression of GFAP. However, no significant differences were observed in the expression of proliferation markers. MG grown on stiffer gels showed a higher expression of dedifferentiation marker α-SMA (25.91±3.59% of surviving cells) compared to MG grown on softer gels (15.95±0.04%). We found a higher deposition of collagen I (47.50±0.10%), IV (64.87±7.32%), and fibronectin (50.96±5.98%) on stiffer gels compared to softer ones (7.94±1.24%; 18.45±4.51% and 17.07±0.95%, respectively), relative to deposition on glass. This deposition increased in HP. We found a higher expression of TRPV4 on stiffer substrates, which increased further in HP condition. There was an overall reduction in survival in HP
Conclusions :
MG thrive on stiffer substrates and display a hightened sensitivity to them. Moreover, HP drives further MG mechanoresponse across all substrates, reducing overall cell survival. These results suggest that mechanical cues are key to MG behavior in the context of glaucoma. Therefore, PAA gels emerge as a compelling in vitro model to study the modulation of the retinal mechanical landscape for therapeutic approaches.
Supported by ELKARTEK (KK-2019/00086), Gobierno Vasco (IT1510-22), MINECO-Retos (PID2019-111139RB-I00) and PIBA (2020_1_0026).
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.