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

PURPOSE: We aimed to establish a rabbit model with retinal atrophy induced by an iatrogenic retinal pigment epithelium (RPE) removal, for future testing of the efficacy and safety of cell therapy strategies. METHODS: A localized detachment of the retina from the RPE/choroid layer was created in 18 pigmented rabbits. The RPE was removed by scraping with a custom-made extendable loop instrument. The resulting RPE wound was observed over a time course of 12 weeks with optical coherence tomography and angiography. After 4 days (group 1) and 12 weeks (group 2), histology was done and staining with hematoxylin and eosin, as well as immunofluorescence performed to further investigate the effects of debridement on the RPE and the overlying retina. RESULTS: Already after 4 days, we observed a closure of the RPE wound by proliferating RPE and microglia/macrophage cells forming a multilayered clump. This pattern continued over the observation time course of 12 weeks, whereby the inner and outer nuclear layer of the retina became atrophic. No neovascularization was observed in the angiograms or histology. The observed changes were limited to the site of the former RPE wound. CONCLUSIONS: Localized surgical RPE removal induced an adjacent progressive retinal atrophy. Altering the natural course of this model may serve as a basis to test RPE cell therapeutics.

Imaging of Therapeutic Effects of Anti-Vascular Endothelial Growth Factor Inhibitors by Optical Coherence Tomography Angiography in a Rat Model.

Purpose: The aim of the study was to investigate optical coherence tomography angiography (OCTA) as a high-resolution in vivo imaging modality for monitoring therapeutic response to different vascular endothelial growth factor inhibitors in the rat model of laser-induced choroidal neovascularization (CNV). Further, OCTA findings were compared with fluorescein angiography (FA) and fluorescence microscopy. Methods: Laser treatment at day (D)0 was followed by intravitreal injection of aflibercept, AF564, and NaCl in dark agouti rats. Imaging with OCTA and FA was performed at D2, D7, D14, and D21. OCTA was compared to FA as well as confocal imaged flat mounts and analysis included quantification of CNV area, pixel intensity, vessel density, and number of vessel junctions. Results: Within laser lesions, neovascularization were visible especially in deeper retinal layers on OCTA, but not on FA images. Using OCTA, mean CNV area (D21) at the level of the outer nuclear layer (ONL) was 0.017 mm² following aflibercept administration, 0.016 mm² following AF564 and 0.026 mm² following NaCl injection (P = 0.04 and P = 0.03). Similar differences between treatment groups were determined by FA and histology, although the overall CNV area was always larger on FA due to dye leakage (P ≤ 0.0001, all layers). Conclusions: Compared to FA, OCTA imaging allows for a more precise and quantitative analysis of new blood vessel formation and therapeutic response to vascular endothelial growth factor (VEGF)-inhibitors, whereas it does not permit assessment of leakage. Translational Relevance: These findings suggest that OCTA may be particularly useful for the investigation of new treatment targets in the animal model.

Optical coherence tomography angiography (OCT-A) in an animal model of laser-induced choroidal neovascularization.

Aim of the study was to compare optical coherence tomography angiography (OCT-A) and conventional fluorescein angiography (FA) for quantitative analysis of the retinal and choroidal vasculature in the animal model of laser-induced choroidal neovascularization (CNV). Therefore, Dark Agouti rats underwent argon laser photocoagulation to induce CNV at D0. In vivo imaging using combined confocal scanner laser ophthalmoscopy (cSLO)-based FA and OCT-A (Heidelberg Engineering GmbH, Heidelberg, Germany) was performed before and immediately after laser treatment as well as at day 2, 7, 14 and 21. OCT-A en-face images were compared to cSLO images obtained by conventional FA topographic uptake recorded using a series of different pre-defined focus settings. For a quantitative comparison of CNV imaging by OCT-A and FA, CNV area, vessel density, number of vessel junctions, total vessel length and number of vessel end points were analyzed. Subsequent ex vivo analyses of the CNV included immunofluorescence staining of vessels in retinal and RPE/choroidal/scleral flatmount preparations. We found, that OCT-A allowed for high-resolution non-invasive imaging of the superficial, intermediate and deep retinal capillary plexus as well as the choroidal blood vessels in rats. Compared with OCT-A, visualization of CNV progression by invasive FA was less accurate, in particular the deep vascular plexus was visualized in more detail by OCT-A. The area of neovascularization was mainly detected in the deep retinal vascular plexus, outer nuclear layer (ONL), ellipsoid zone (EZ) and the choroid. Within the laser lesions, signs of CNV formation occurred at day 7 with progression in size and number of small vessels until day 21. Due to leakage and staining effects, CNV areas appeared significantly larger in FA compared to OCT-A images (p ≤ 0.0001 for all tested layers). Vessel density, number of vessel junctions, total vessel length and number of vessel end points were significantly higher in intermediate vascular plexus (IVP) and deep vascular plexus (DVP) in OCT-A compared to FA images. Overall, CNV area in flatmounts was similar to OCT-A results and much smaller compared to the area of dye leakage by FA. This study demonstrates that in vivo OCT-A imaging in small animals is feasible and allows for precise analysis of the formation of new blood vessel formation in the animal model of laser-induced CNV. Given its superior axial resolution, sensitivity and non-invasiveness compared to conventional FA imaging, OCT-A opens the door for a more detailed evaluation of CNV development in such a model and, thus, enables the analysis of the response to novel therapeutic interventions in longitudinal in vivo studies.

A Step by Step Protocol for Subretinal Surgery in Rabbits.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision. Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology. The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.