Invivovue clinic software

Spectral domain optical coherence tomography (SD-OCT) images were obtained using the Bioptigen Spectral Domain Ophthalmic Imaging System (SDOIS; Envisu R2200, Bioptigen, Morrisville, NC, USA). ALMS mice were anesthetized and pupils were dilated by applying 1–2 drops of topical 0.5% tropicamide (Visufarma, Rome, Italy). To prevent corneal desiccation during the procedure, topical lubricant eye drops (Recugel; Bausch & Lomb, Rochester, NY, USA) were applied bilaterally with a small brush. Mice were positioned into the animal imaging mount and rodent alignment stage (AIM-RAS; Bioptigen, Morrisville, NC, USA); the laser source was placed in front of the mouse, and images were acquired by the InVivoVue Clinic software (Bioptigen, Morrisville, NC, USA). Three images, one central, one superior, and one inferior to the optic nerve, were taken from each eye. ONL thickness was manually measured three times from each OCT scan image and averaged.

The ocular surfaces of conscious mice were examined under a slit lamp microscope (SL-D7; Topcon, Tokyo, Japan). Images were taken with a digital camera (D800; Nikon, Tokyo, Japan) using identical settings.
A separate cohort of mice was anesthetized with i.p. ketamine/xylazine (100 mg ketamine + 10 mg xylazine/kg body weight) for bilateral measurement of central corneal thickness (CCT) with spectral domain optical coherence tomography (SD-OCT; Bioptigen, Durham, SC, USA). Scans of the anterior segment were obtained with a 12 mm telecentric bore centered over the pupil, with the following parameters: 2.0 mm radial volume scans, 1000 A scans/B scan, 100 B scans/volume, 1 frame/B scan, 1 volume. CCT, epithelial thickness, and stromal thickness were measured with vertical-locked B scan calipers in the Bioptigen InVivoVue Clinic software. Mice were omitted from analysis if variation in CCT between left and right eyes exceeded 5 μm.

Mice were injected with a standard mixture of ketamine/xylazine (intraperitoneal injection of (100 mg ketamine +10 mg xylazine)/kg body weight (KetasetÒ, Fort Dodge Animal Health; AnaSedÒ, Lloyd Laboratories). Upon anesthesia, eyes were hydrated with balanced salt solution (Alcon Laboratories). To obtain retinal images, the tear film was wicked away and eyes were imaged with a Bioptigen spectral domain optical coherence tomographer (Bioptigen, Inc) using the mouse retinal bore. The volume intensity projection was centered on the optic nerve. Scan parameters were as follows: rectangular volume scans 1.4 mm in diameter, 1000 A-scans/B-scan, 100 B-scans/volume, 1 frame/B-scan, and 1 volume. After imaging, eyes were hydrated with artificial tears and mice were provided supplemental indirect warmth for anesthesia recovery. Retinal thickness was measured from retinal images within the Bioptigen InVivoVueClinic Software four times per eye using vertical angle-locked B-scan calipers from the internal limiting membrane to the retinal pigment epithelium. The six retinal thickness measurements obtained from two eyes of each animal were averaged together to produce a single retinal thickness measurement per animal. All retinal thickness measurement values are reported as an average ± SD.