A rabbit model for outer retinal atrophy caused by surgical RPE removal

**Background:** Age-related macular degeneration (AMD), particularly the dry form (geographic atrophy), lacks effective treatments. The retinal pigment epithelium (RPE) is crucial for photoreceptor health, and its dysfunction is a key factor in AMD. Replacing dysfunctional RPE with healthy cells is a promising therapeutic strategy, but requires a method to remove the native RPE in the target area. This study aimed to establish a rabbit model of outer retinal atrophy induced by surgical RPE removal, using an improved instrument, to enable future testing of RPE cell therapies.

**Methods:** 18 pigmented Dutch Belted rabbits (2-2.5 kg) were used. In 15 rabbits, a localized bleb retinal detachment (bRD) was created by subretinal injection of 20-30 μL balanced salt solution via a 25/38 G cannula. A 1.5 mm retinotomy was made, and the RPE was scraped using a custom prolene loop instrument. In 3 control rabbits, the bRD was created without RPE scraping. Animals were divided into two groups: 8 rabbits were euthanized at 4 days (short-term), and 10 at 12 weeks (long-term). Post-operative follow-up included scanning laser ophthalmoscopy/spectral-domain optical coherence tomography (SLO/SD-OCT), fluorescein angiography (FA), and indocyanine green angiography (ICGA) at 4 days, 1, 2, 4, and 12 weeks. Histological analysis (hematoxylin and eosin, immunofluorescence) was performed on enucleated eyes. Markers included Ki67 (proliferation), laminin and collagen IV (Bruch's membrane), pan cytokeratin (RPE), ZO-1 (tight junctions), and isolectin B4 (endothelial cells, microglia, macrophages).

**Key Results:** The improved loop instrument allowed RPE removal without subretinal viscoelastic, creating a 5-9 mm² wound. At 4 days, OCT showed RPE irregularity and loss of the ellipsoid zone over the wound. FA/ICGA showed hyperfluorescence without leakage, indicating no choroidal neovascularization (CNV). Histology revealed photoreceptor degeneration and outer nuclear layer (ONL) thinning limited to the scraping site. The RPE wound was closed by proliferating RPE cells (panCK-positive) and microglia/macrophages (IB4-positive), forming a multilayered clump. Ki67 staining showed proliferation at the wound, but no colocalization with panCK. Collagen IV deposition was observed beneath the ONL. At 12 weeks, OCT showed progressive retinal atrophy and RPE hypertrophy. FA/ICGA showed subtle hyperfluorescence with pigment blockage. Histology confirmed atrophy extending to inner retinal layers, with a contiguous Bruch's membrane and no CNV. Microglia/macrophages were no longer detectable (IB4-negative), and Ki67 signals were absent. The RPE remained multilayered and hypertrophic, with weak, non-polarized ZO-1 expression. In controls (bRD without scraping), only minor RPE hypertrophy around the retinotomy and few microglia/macrophages were observed, with no ONL atrophy.

**Clinical Implications:** This study presents a reproducible, cost-effective large animal model of outer retinal atrophy induced by localized RPE removal, without causing CNV. The model mimics key features of geographic atrophy, including progressive photoreceptor loss and RPE dysfunction. It provides a platform for testing the efficacy and safety of stem cell-derived RPE replacement therapies. The improved surgical technique minimizes iatrogenic damage, making it suitable for preclinical studies. Limitations include the use of healthy rabbit RPE (not diseased) and the merangiotic rabbit retina, which may lead to faster atrophy than in humans. Future studies should investigate the survival and functionality of transplanted RPE cells in this model.