Micron 4 system

Mice were dark adaptated over night and pupils dilated using Cyclogyl 1% (Alcon Pharmaceuticals, Fribourg, Switzerland) and Neosynephrine 5% (Ursapharm Schweiz GmbH, Roggwil, Switzerland) 20 min before recording. Mice were anesthetized by a subcutaneous injection of ketamine (85 mg/kg, Parke-Davis, Berlin, Germany) and Xylazine (4 mg/kg, Bayer AG, Leverkusen, Germany). A drop of mydriacticum dispersa (OmniVision AG, Neuhausen, Switzerland) was applied to each cornea to induce mydriasis and to keep the tissue moist. Recordings were done using flashes of 13 different light intensities ranging from −50 db (0.000025 cd*s/m²) to 15 db (79 cd*s/m²) for scotopic and flashes of 8 different light intensities ranging from −10 db (25 cd*s/m²) to 25 db (790 cd*s/m²) for photopic ERG as described.^{69 (link)} Ten recordings were averaged per light intensity.
Fundus imaging and OCT were done essentially as described.^{70 (link)} In brief, pupils of mice were dilated and mice anesthetized as describe as above. Eyes were kept moist with 2% methocel (OmniVision AG) and fundus images and OCT scans recorded using the Micron IV system (Phoenix Research Labs, Pleasanton, CA, USA).

OCT was performed with the Micron IV system (Phoenix Research Labs, Pleasanton, CA, USA). The mice were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 5 mg/kg Xylazine. The pupils were dilated with 1% tropicamide eye drops. All drugs used with animals were obtained from the University of Illinois at Chicago Pharmacy. For each eye, four OCT sections were imaged (superior, inferior, temporal, nasal) 350 μm from the optic nerve head. The corneal surface was protected using polyethylene glycol and propylene glycol eye gel during and after the imaging process. The thickness of the following layers was measured and averaged in both eyes with the InSight2D software (Phoenix Research Labs, Pleasanton, CA, USA): ganglion cell complex (GCC), including the nerve fiber layer–ganglion layer, inner plexiform layer), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), inner segment/outer segment (IS/OS) and total retina. For each OCT image, three points were selected to represent the thickness of the retina. These points were chosen to not include blood vessels, which overestimate the total retinal thickness. The data obtained from both eyes of the same animal were averaged.

Pupils of the mice were dilated using drops of 1% Cyclogyl (Alcon Pharmaceuticals, Fribourg, Switzerland) and 5% Neosynephrine (Ursapharm Schweiz GmbH, Roggwil, Switzerland) 20 min before anesthesia with a subcutaneous injection of 51 μl ketamine (50 mg/kg, Parke-Davis) and 6 μl Rompun (2%, Bayer AG, Leverkusen, Germany) per 30 g body weight. Eyes were kept moist throughout the experiment with the topical application of 2% Methocel (OmniVision AG, Neuhausen, Switzerland). OCT and fundus imaging were carried out with the Micron IV system (Phoenix Research Labs, Pleasanton, CA, USA). Mice were kept on a heating pad throughout the procedure.

After oral administration was completed, mice were treated with tribromoethanol (1329-86-8, Hefei TNJ Chemical Industry Co., Ltd.) for anesthesia and treated with 0.5% tropicamide for mydriasis. Electroretinography was performed using the Micron IV system (Phoenix Research Labs, USA).

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.