Abstract
Purpose :
Recent data have shown that disruption of nutrient delivery and utilization by photoreceptor cells is a common pathway in regulating cell death in retinal degenerative diseases. Experimental retinal detachment (RD) in rodents is a model of acute outer retinal nutrient stress that leads to photoreceptor apoptosis. The purpose of this study was to optimize flow cytometry markers of outer retinal stress and apoptosis in a rodent model of nutrient stress.
Methods :
Methods Experimental RD was created in mice and rats via subretinal injection of 1% hyaluronic acid. 3 days post-RD, retinas were harvested and dissociated into single cells using trypsin. Dissociated cells were either incubated with Annexin-V, propidium iodide (PI), and Phiphi lux (active caspase-8) or fixed and stained with TUNEL and cleaved caspase-8. Z-VAD, a pan-caspase inhibitor, was injected sub-retinally at the time of RD and these markers were assessed 3 days later. Tert-butyl hydroperoxide (TBH) was injected intravitreally (IVT), retinas harvested and dissociated 6 and 24 hours later, and cells incubated with DCFDA, C11-Bodipy, and Calcein-AM to examine oxidative stress using flow cytometry. Cells were gated, and data were analyzed by comparing signals to either isotype controls or unstained cells using FCS Express software (De Novo Software, Ontario, CA).
Results :
Statistically significant increases in TUNEL (>17-fold; p<0.001) and active Caspase-8 (>17-fold) were observed in RD. Treatment with Z-VAD significantly reduced these markers. To confirm these results, we repeated RD in a previously reported model of photoreceptor neuroprotection, the PKM2 conditional knockout mouse. We confirmed that compared to detached retinas in WT animals, PKM2 cKO mice showed reduced TUNEL staining (>7-fold vs >1.3-fold). In WT mice, TBH-mediated oxidative stress increased DCFDA staining (>2.3 fold) and lipid peroxidation markers (>6.7-fold) as well as reduced viability (>1.7-fold).
Conclusions :
Flow cytometry is a sensitive, quantifiable, and reproducible method for measuring apoptosis and oxidative stress in a model of outer retinal nutrient stress. It has the advantage of increased efficiency compared to traditional histologic methods. These flow cytometric methods will be key to assessing novel photoreceptor neuroprotective therapies in a multitude of retinal degeneration models.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.