Effect on RAS and MMP Pathways After Q-switched Frequency-doubled Nd:YLF with RTF Laser Treatment in Hard Drusen Mice Model

Abstract

Purpose : To evaluate the impact of Q-switched frequency-doubled Nd:YLF Laser with Real-Time Feedback (RTF) technology (R:GEN) treatment on retinal and RPE wound healing process in hard drusen mice model using RAS metabolomics and gene expression.

Methods : Aged Cx3cr1-/- mice were randomly assigned non-treatment group versus R:GEN treated that were evaluated from 1 hour to 90 days post treatment. Retinal imaging included fundus photos, OCT and FA. At each timepoint, RAS metabolomics were performed on blood and ocular fluids. H&E and ZO-1 staining to determine the histological impact of laser. Additionally, gene expression for MMPs and RAS components were performed on both retina and RPE specimens.

Results : Fundus imaging showed visible lesions at 1 and 4hrs, whereby day 7 the lesions were completely healed. FA showed hyperfluorescent leaking patterns in early time points (1hr, 4hrs and day 3) only. Neither hypo nor hyperfluorescent patterns were observed beyond day 7. OCT scans showed cellular disruption along the RPE layer with minimal disruption along the ONL. By day 7, ONL and RPE recovery with no visible changes detected. H&E showed disruption of the RPE up to day 7, while recovery of the RPE was observed on day 7 with minimal disruption to the RPE. ZO-1 demonstrated RPE defects up to day 3, revealing RPE polymegathism to cover the lesions by day 7. RT-PCR showed upregulation of the MasR and TGF-B1 at 1hr, 3 days and 90 days. RAS-metabolomics showed vitreous Ang(1-7) corresponded with MasR regulation. These findings preceded MMP changes in the RPE (MMP3, MMP9 and TIMP2). Downregulation on Ang(1-9), AngII, and Ang(2-8) in comparison with non-treated eyes (p <0.05). Spearman correlation showed a negative correlation of ocular and plasma fold changes of Ang(1-10) and Ang (1-5) (r = -0.7857 (p = 0.0480) and r = -0.8214 (p = 0.0341) respectively.

Conclusions : Our study demonstrates that R:GEN laser was well-tolerated in the Cx3cr1-/- mice that promoted upregulation of RAS components that preceded MMP upregulation, which may earlier biomarker of wound healing. These findings suggest R:GEN treatment in an animal model of early AMD can promote minimally fibrotic RPE healing. The negative correlation between plasma and ocular levels of Ang(1-10) highlights the potential for a blood-based biomarker capable to elucidate wound healing activity.

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