Streampix software

Fundus examination and optical coherence tomography (OCT) were performed on adult injected and uninjected animals. Animals were anesthetized with a mixture of ketamine (1 mg/mL) and xylazine (0.4 mg/mL) and pupils were dilated with 1% tropicamide. Fundus images were taken using the Micron III Retinal Imaging Camera and Stream Pix software (Phoenix Research Laboratories, Pleasanton, CA, USA). Following fundus imaging, OCT was performed using the Bioptigen OCT scanner and software. Mice were restrained in a mounting tube and the fundus camera in the optical head of the apparatus and alignment was guided by monitoring and optimizing the real time OCT image of the retina. Four rotational cross section scans (dorsal–ventral and nasal–caudal) with 100 series/scan were taken for each retina. Data was analyzed using Bioptigen OCT software (N = 10/strain/experimental group).

Fundus color video and FA during the IR surgery were taken using the Micron III Retinal Imaging Microscope (Phoenix Research Laboratories, Pleasanton, CA, USA). For FA, 0.1 ml of 2% fluorescein sodium (Akorn, Lake Forest, IL, USA) was injected intraperitoneally. Images were taken by attaching the cornea covered with 2.5% Goniovisc (HUB Pharmaceuticals, Rancho Cucamonga, CA, USA) to the Micron camera lens using StreamPix software (Phoenix Research Laboratories, Pleasanton, CA, USA).

Mice were anesthetized with an i.p. injection of avertin (Sigma) diluted in saline. Pupils were dilated with one drop of a solution of 0.5% (w/v) tropicamide and 5% (w/v) phenylephrine (MEEI pharmacy). Goniovisc (2.5%, HUB pharmaceuticals) was used as a corneal medium during imaging. A Micron III Retinal Imaging Microscope and StreamPix software (Phoenix Research Labs) were used to image the fundus. Scoring was as reported previously [7 (link)],[26 (link)]. Briefly, retinal infiltrates, optic disc changes, vascularity, and structural damage were scored on a scale of 0–4. Infiltrates: 1 = 1–4 small lesions or 1 linear lesion, 2 = 5–10 small lesions or 2–3 linear lesions, 3 = >10 small lesions or >3 linear lesions, 4 = confluent linear lesions. Optic disc: 1 = minimal inflammation, 2 = mild inflammation, 3 = moderate inflammation, 4 = severe inflammation. Retinal vessels: 1 = engorged vessels, no cuffing, 2 = engorged vessels, 1–4 mild cuffs, 3 = >4 mild cuffs or 1–3 moderate cuffs, 4 = >3 moderate cuffs, >1 severe cuff. Structural damage: 1 = lesions or atrophy on <1/4 of retina, 2 = lesions or atrophy on 1/4-3/4 of retina, 3 = pan retinal atrophy with multiple small scars or <3 linear scars, 4 = pan retinal atrophy with >3 linear or confluent scars. Clinical score was calculated by averaging the score for each of the four criteria.

Full-field electroretinograms (ERGs) were recorded as described previously [26 (link)]. Briefly, mice were dark-adapted overnight and were anesthetized using 85 mg/kg ketamine and 14 mg/kg xylazine (Henry Schein Animal Health, Dublin, OH, USA). Eyes were dilated with 1% cyclopentolate and covered in Gonak (Akorn Pharmaceuticals, Lake Forest, IL, USA). Platinum wire loops were placed in contact with the cornea through a layer of Gonak. Using the UTAS system (LKC, Gaithersburg, MD, USA) full-field scotopic ERG responses were recorded from each eye in response to a single 157-cd s/m² flash. Fundus imaging was performed using the Micron IV system (Phoenix Research Laboratories, Pleasanton, CA, USA). Animals were anaesthetized/dilated as for ERG but were not dark-adapted. Brightfield images were captured first, and then HA-NS distribution was analyzed by imaging with the green filters (451.5–486.5 nm excitation and 488 nm emission, for fluorescein) or red filters (553 nm excitation and 627 nm emission). All images were captured using StreamPix^® software (Phoenix Research Laboratories, Pleasanton, CA, USA). For OCT, animals were anesthetized and eyes dilated as for fundus imaging. OCT images were captured using an Image Guided OCT2 (Phoenix Research Laboratories). Images were captured at 100 fpm using the Reveal OCT software.

Fundus images were taken using the Micron III Retinal Imaging Microscope (Phoenix Research Laboratories, Pleasanton, CA). Mice were anesthetized with Avertin (Sigma, St. Louis, MO) and placed on the 37°C heating pad to keep body temperature. The pupils were dilated with 5% phenylephrine and 0.5% tropicamide. Images were taken by attaching cornea covered with 2.5% Goniovisc (HUB Pharmaceuticals, Rancho Cucamonga, CA) to the Micron camera lens using the StreamPix software (Phoenix Research Laboratories, Pleasanton, CA).