Abstract
Purpose :
Glaucoma is a neurodegenerative disease in which various triggers induce cascades of secondary events, which ultimately lead to retinal ganglion cell (RGC) death. Once started, the cell death program is generally considered to be irreversible. However, recent studies reveal that recovery of dying cells is possible, even after reaching critical events in the cell death program. This phenomenon is termed anastasis. Harnessing mechanisms of anastasis may represent a previously unrecognized therapeutic strategy to rescue dying differentiated cells that are difficult to replace. We study neuronal cell death with the aim of rescuing dying RGCs in glaucoma.
Methods :
Primary rat RGCs and differentiated PC12 cells were treated with ethanol to induce cell death. Live cell imaging with fluorescent probes (Mito-Tracker, TMRM, DCFDA, Fluo-8AM) were used to visualize the progression of the cell death program in individual RGCs and differentiated PC12 cells with high-resolution live-cell spinning disk confocal microscopy. Electron microscopy (EM) was used to observe the ultrastructure of mitochondria. Immunostaining was used to detect cytochrome c translocation.
Results :
Exposure to 5% ethanol for 24 h induced 80.7% (p<0.001) and 69.1% (p<0.001) cell death in RGCs and PC12 cells, respectively. In both cell lines, live cell imaging showed significant mitochondrial fragmentation and membrane potential loss after ethanol treatment for 3 h. The average length of mitochondria decreased from 7.2 ± 6.2 μm to 1.3 ± 0.3 μm in RGCs (p<0.0001), and 6.1 ± 4.5 μm to 1.1 ± 0.12 μm in PC12 cells (p<0.0001). Moreover, removal of ethanol and further culturing in fresh cell culture medium for 20 h restored normal mitochondrial structure (RGC, 6.1 ± 4.5 μm; PC12, 4.7 ± 3.7 μm) and membrane potential. EM results confirmed the ethanol induced mitochondrial fragmentation and its reversibility. In addition, during the mitochondrial fragmentation cells showed higher levels of reactive oxygen species (p<0.001) and intracellular Ca2+ (p<0.001), which levels returned to normal after removal of ethanol stimulus. However, immunostaining showed that no cytochrome c release from mitochondria had occurred at this stage of reversible mitochondrial injury.
Conclusions :
The results indicated that targeting and harnessing mitochondria may hold promise as therapeutic strategy to rescue dying RGCs in glaucoma and reduce its vision loss.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.