Quantification of ROS in vascular cells using DCF-DA probe and Flow-Cytometry assay

VAGLIENTI MV; SUBIRADA, PAULA V.; PAZ MC; BARCELONA PF; BONACCI GR; SANCHEZ MC

Congreso; XIII Congreso de investigaciones en Visión y Oftalmología; 2021

AIVO

Purpose: Inflammation and oxidative stressare involved in neovascular retinopathies (NR).Nitro fatty acids are important electrophilic signalingmediators with anti-inflammatory andantioxidant properties (Keap1/Nrf2 pathway).Here, our aim is to evaluate the effect of nitrooleicacid (NO2-OA) in oxygen-induced retinopathymouse model (OIR).Materials and methods: OIR mice wereintraocular (i.o.) injected at P12 with 5 μM ofNO2-OA or vehicle and intraperitoneal (i.p) atP14, P17, P20, P23 with 15 mg/Kg of NO2-OAor vehicle. At P17 or P26 mice were sacrificed.Age-match mice in RA were used as controls.Some eyes were fixed to obtain whole mountfor microscopy and other retinas were used forWestern blot or RT-PCR assays. The electricalactivity of the retina in response to a light stimuluswas measured by scotopic electroretinography(ERG). Amplitudes and latencies of a- andb-waves from scotopic ERG were recorded atP17 y P26. Finally, the NO2-OA effect on neovascularizationwas evaluated by tube formationassay. GraphPad prism was used for statisticalanalysis.Results: NO2-OA induced vascular regrowth,reduced neovascularization and produced significantchanges in GS and GFAP at P17 OIR. AtP26 NO2-OA prevented the decrease in b-waveamplitude as well as the diminished expressionof total Caspase-3 protein. Finally, a significantdecrease in the total segment length and numberof tubular structures was observed afterNO2-OA treatment.Conclusions: These findings suggest thatNO2-OA could be beneficial or cytoprotectivefor retinal cells in OIR model.