A Time Window for Rescuing Dying Retinal Ganglion Cells

Abstract

Purpose : Cell death of retinal ganglion cells (RGCs) causes vision loss in patients with glaucoma. Programmed cell death, like apoptosis, is generally considered to be irreversible. However, recent studies reported that dying cells could reverse the cell death program even at late stage of apoptosis. This may have therapeutic implications in restoring cells that are difficult to regenerate, like neurons. In this study, we investigated neuronal cell death with the aim of rescuing dying RGCs in glaucoma.

Methods : We studied the reversibility of cell death program in primary rat RGCs by exposing cells to cell death stimulus ethanol, followed by washing away ethanol and further culturing in fresh medium. Live cell imaging with fluorescent probes (Annexin V-FITC, Mito-tracker, TMRM, Fluo-8AM) were used to visualize the progression of the cell death program in individual RGCs with high-resolution live cell spinning disk microscopy. Electron microscopy (EM) was used to observe the ultrastructure of mitochondria. Immunostaining was used to detect cytochrome c translocation.

Results : Mitochondrial injury, including fragmentation and membrane potential loss, occurred early in the cell death process. Interestingly, fragmented mitochondria could recover their reticular morphology and regain normal membrane potential when ethanol was removed at this stage (p<0.001). This cellular recovery was not dependent on providing exogenous neurotrophic factor CNTF and BDNF in culture medium. EM showed that the length of mitochondrial profiles decreased from 1.94 ± 1.0 μm to 0.69 ± 0.44 μm after ethanol treatment (p<0.001), and recovered to 1.93 ± 1.0 μm within 20 h after removing ethanol (p<0.001), confirming the reversibility of mitochondrial fragmentation. In addition, this reversible mitochondrial injury was accompanied by elevation of intracellular Ca²⁺ (p<0.01). However, at this stage, mitochondrial cytochrome c has not been released yet. Exposure of phosphatidylserine (PS) at cell surface occurred later in the cell death process. This stage of PS exposure, which may be mediated by caspase activation, was not reversible.

Conclusions : Our study indicates that there is a time window for rescuing injured or dying RGCs. Targeting mitochondria may hold promise as therapeutic strategy in glaucoma to reduce its vision loss.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.