Genteal

Optical coherence tomography (OCT) (Envisu OCT, Bioptigen, Inc., Morrisville, NC, USA) was performed on mice kept anesthetized with isoflurane/O₂. Pupils of the anesthetized mice were dilated with 1% tropicamide ophthalmic drops prior to image acquisition. Lubricant eye gel (GenTeal; Novartis Pharmaceuticals, East Hanover, NJ, USA) was used throughout the procedure to maintain corneal moisture and clarity [26 (link)]. Morphometric analysis of retinal thickness was assessed using ImageJ software (version 1.49, National Institutes of Health, Bethesda, MD, USA).

To assess the presence of angiogenesis, imaging of eyes was performed after 1 and 6 weeks using OCT imaging device with a 25-diopter lens fitted on a 30-degree angle lens. The pupils of the anesthetized mice were dilated with 1% tropicamide eye drops before the images were acquired. A lubricant eye gel (GenTeal; Novartis Pharmaceuticals, East Hanover, NJ, USA) was used throughout the procedure to maintain moisture and clarity in the cornea. Three repeated-volume intensity projections were acquired for each eye. The images consisted of 50–100 averaged B-scans.

Cross-sectional images of live mouse retinas were taken 1 to the optic nerve using a Spectralis HRA + OCT device (Heidelberg Engineering, Heidelberg, Germany) as previously described^{46 (link)}. Eye gel (GenTeal; Novartis, NSW, AUS) was administered to both eyes for recovery.
Using OCT cross-sectional retinal images or retinal cryosections, and ImageJ software (National Institutes of Health, Bethesda, MD, USA), outer nuclear layer (ONL) thickness was either calculated as the ratio of the thickness of the ONL to the whole retinal thickness (outer limiting membrane to the inner limiting membrane) for OCT images, or ONL thickness (μM) for retinal cryosections. The length (μM) of photoreceptor inner and outer segments (IS and OS) were also measured. ONL, IS and OS thickness was measured five times at 1-mm intervals across the retina (superior for IS and OS) and averaged. In addition, the thickness of the ONL was determined by counting the number of rows of nuclei (photoreceptor cell bodies) in the area of retinal lesion development (1 mm superior to the optic nerve head), to quantify photoreceptor survival. The process of ONL photoreceptor cell row quantification was performed five times per retina, on two retinal sections at comparable locations per mouse.

For in vivo confocal microscopy, mice were anesthetized via an intraperitoneal injection of ketamine (120 mg/kg; Phoenix Scientific, St. Joseph, MO, USA) and xylazine (20 mg/kg; Phoenix Scientific). Eyes were treated with a drop of Proparacaine 0.5% Ophthalmic Solution, a topical anesthetic. A drop of Genteal (Novartis, St. Louis, MO, USA) lubrican puralube eye gel was placed on the tip of the objective lens to maintain immersion contact between the objective lens and the eye. The contralateral eye was moisturized with the same gel and subsequently imaged. During the procedure, anesthetized mice were placed on a heated platform to maintain physiological temperature. Images were acquired using an HRT II/RCM (Heidelberg Engineering GmbH, Heidelberg, Germany) in vivo confocal microscope with a diode laser with a wavelength of 670 nm and a 60× objective immersion lens. An area of 400 × 400 μm with a transverse optical resolution of approximately 1 mm/pixel was imaged. For all eyes, 30 images of each layer of the cornea (total 150 images), including the superficial and basal epithelium, anterior and posterior stroma, and endothelium, were recorded.

Cross-sectional and fundus images of live mouse retinas were taken using a MICRON® IV device (Phoenix-Micron, Inc., OR, United States). Cross-sectional images were taken at 1 mm increments from the optic nerve. Eye gel (GenTeal; Novartis, NSW, Australia) was administered to both eyes for recovery. Using OCT cross-sectional retinal images, and ImageJ V2.0 software (National Institutes of Health, Bethesda, MD, United States), the thickness of the outer nuclear layer (ONL), was calculated as the ratio to the whole retinal thickness (outer limiting membrane to the inner limiting membrane).

Six-week-old hWtEPOR, hMtEPOR and litter mate control mWtEPOR mice were anesthetized with intraperitoneal injections of 100 mg/kg ketamine (Bioniche Teoranta, Ireland) and 10 mg/kg xylazine (Akorn, IL) following mydriasis with 0.5% tropicamide ophthalmic solution (Bausch & Lomb, NY). Mice were raised onto a platform in front of the Phoenix Micron IV Imaging System (Phoenix research Labs, Pleasanton, CA), and a coupling agent, GenTeal (Novartis, NJ), was applied to the cornea. Laser photocoagulation was performed using the Micron laser module (450 mW intensity, 100 ms duration; Phoenix Research Labs, Pleasanton, CA). Three laser spots per eye were applied approximately 2 disc diameters from the optic nerve, avoiding major vessels. Disruption of Bruch’s membrane was confirmed by the appearance of a cavitation bubble. Eyes were collected three and seven days post-laser and used for either flatmounts or RT-PCR.