Abstract
Purpose :
Endothelin-1(ET-1) and its receptors contribute to the etiology of glaucoma. Previously, we reported that ET-1-treatment induced reactivation of astrocytes (ASTs) and apoptosis of retinal ganglion cells (RGCs). Recently, studies indicate that two newly-identified AST subtypes have neurotoxic and neuroprotective effects on RGCs. However, ET-mediated subtype changes of ASTs and their effect on RGC survival are largely unknown. This study aimed to study the roles of ET-1 in the interaction between RGCs and AST subtypes and investigate the distribution of astrocytes at optic nerve head and retina with glaucomatous damage.
Methods :
Primary rat RGCs were isolated from rat pup retinas and ASTs from their optic nerves. ASTs were seeded directly into RGC culture or seeded into a filter insert and placed over cultured RGCs. Both co-cultures were treated with 100nM endothelin-1 for 24-72 hours and RGC survival was determined by LIVE/DEAD assay and synapse proteins were detected using immunocytochemistry. ET-1-mediated intracellular calcium was monitored in RGCs, ASTs and co-culture of RGCs and ASTs using Fura-2 AM calcium imaging. Expression of astrocytic markers in cultured ASTs was determined by immunocytochemistry and some were confirmed by immunoblot analysis. Optic nerve and retina sections obtained from rats following ET-1 intravitreal injection were analyzed by immunochemistry for astrocytic marker detection.
Results :
ET-1 upregulated GFAP, Ki67, c-Jun, and JNK in primary rat ASTs. ET-1-induced elevation of [Ca2+]i was significantly attenuated in co-culture of RGCs and ASTs, and correspondingly less cell death was also observed in both co-culture systems. More synapse formation was detected in RGC-AST co-cultures. Increased duration of co-culture produced more synapses. ET-1 treatment or c-Jun overexpression in ASTs changes the gene and protein expression profile of several astrocytic markers. ET-1 intravitreal injection in rats also upregulated GFAP, Serping1 and CD14. Similar results were observed in human optic nerve head astrocytes treated with ET-1 and in optic nerve and retina sections from glaucoma patients.
Conclusions :
Co-culture of ASTs with RGCs stimulated more synapse formation in RGCs and decrease RGC death. ET-1 treatment induces not only the reactivation of ASTs but also the switch of astrocyte subtypes, which could lead to dysfunction of axon transport in the optic nerve and affect RGC survival.
This is a 2020 ARVO Annual Meeting abstract.