Abstract
Purpose :
Retinal ganglion cell (RGC) degeneration is a defining feature of glaucoma. While research on RGC survival mechanism is pertinent for development of neuroprotective therapies, the lack of an effective method to purify and culture human RGCs hinders translational progress. In this study, we established an immunomagnetic bead-based method to isolate RGCs from human donor retina that can be cultured up to 3 months and facilitates live cell imaging to monitor mitochondrial membrane potential (MMP) after laser-induced axon injury.
Methods :
Human donor retinas were collected from the Hospital Authority Eye Bank with written informed consent. All study protocols followed tenets of the Declaration of Helsinki and were approved by the Joint CUHK-NTEC Clinical Research Ethics Committee and REC/IRB. There were no known ocular or brain disorders. Primary antibodies CD90 Microbeads and biotinylated CX3CR1 clone 2A9-1 (Miltenyi Biotec) were used for RGC purification and glial depletion, respectively.
Results :
16 retinas from 8 donors (age range: 9-90) were collected for this study. Immunostaining of RBPMS and TUJ1 (RGC-specific markers) or GFAP showed that most cells were RGCs(97.8%) with low glial contaminants(2.3%). RGC axon and total neurite lengths from 9- to 76-year-old donors decreased with age(p<0.001) (Figure 1). Change in MMP after laser-induced axon injury was detected by TMRM imaging up to 12 hours. 79% survived after 60 minutes post-axon injury, while 21% showed signs of blebbing or apoptosis <60 minutes post-injury(n=62 RGCs) (Figure 2). For RGCs that survived beyond 60 minutes, TMRM signals dropped 14±0.02% at 2 minutes after injury, and 25±5% at 12 hours post-injury. Declining TMRM signals were mitigated after treatment with CSA(inhibitor of the mitochondrial permeability transition pore), delaying mitochondrial dysfunction and promoting survival.
Conclusions :
The method described here –modified from a previously described immunomagnetic bead method to target human RGCs –is reproducible and supports long-term culture viability that facilitates live-cell microscopy for intracellular tracking experiments.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.