Atropine sulfate ophthalmic solution

The light-aversion assay was performed as previously described with modifications.⁵⁷ (link)^,58 (link) A two-chamber box with open, light and closed, dark sections was used to measure time spent in the light compartment. An overhead LED lighting system, with adjustable illumination from 0 to 1000 lux calibrated with a light meter (HHLM-2; Omega Engineering, Norwalk, CT, USA), a standard LED spectrum, and diffusers provided uniform illumination in the open, lit side of the chamber. Behavior was monitored using an infrared light source and video camera with white light filter, and automated tracking and analysis were performed with a video tracker (Med Associates, St. Albans, VT, USA) and an activity monitor (Med Associates), respectively. Mice were acclimated to a dimly lit room (less that 10 lux) for at least 15 minutes and dark adapted prior to testing for at least 10 minutes. Light aversion was tested at 0 and 1000 lux: the 0-lux test was used as baseline to calculate aversion indices. A 1% Atropine Sulfate Ophthalmic Solution (Akorn, Lake Forest, IL, USA) was used as a dilating agent where indicated.

For electoretinography (ERG), mice were dark-adapted overnight. Prior to injection of anesthesia, the eyes were dilated twice (10–15 min apart) with one drop each of 2.5% phenylephrine hydrochloride ophthalmic solution (Paragon BioTek, Inc., Portland, OR) and 1% atropine sulfate ophthalmic solution (Akorn, Inc., Lake Forest, IL). The mice were anesthetized with a mixture of ketamine (95 mg/kg) and xylazine (5–10 mg/kg) by intraperitoneal injection. Prior to placing electrodes to record ERG, one drop of sterile lubricant eye drops (CVS Pharmacy, Inc., Woonsocket, RI) was applied to each of the eyes to prevent the eye from drying during procedures. To examine retinal responses, scotopic a-, b- and c-wave amplitudes from each eye were measured using Espion full-field ERG system (Diagnosys LLC, Lowell, MA 01851) at a flash intensity of 20 cds/m²) [37 (link)]; the results from treated and untreated eyes were compared.

iPSC-RPE cells were injected into the subretinal space of Mertk^–/– mice (129 genetic background) and BALB/cJ albino mice. Cultures of iPSC-RPE cells were washed thoroughly with PBS before enzymatic dissociation with TrypLE™. BSS PLUS™ (0065080050; Alcon Laboratories) was added to create a suspension at a concentration of 50,000 cells/μl. Mice (postnatal day (P) 10–16) were anesthetized by isoflurane inhalation. Their pupils were dilated with a drop of 1% (w/v) Atropine Sulfate ophthalmic solution (17478-215-02; Akorn Pharmaceuticals), and the corneas were kept moist with Hypromellose ophthalmic demulcent 2.5% solution (51394-315-15; Wilson Ophthalmic Corp.). A 1-μl suspension of iPSC-RPE cells was injected into the subretinal space of each eye, under a Zeiss Stemi 2000 microscope, as described previously [26 (link)]. Ophthalmic ointment (Neomycin & Polymyxin B sulfates and Dexamethasone, 61314-631-36; Falcon Pharmaceuticals) was applied to each eye immediately following injection. Cyclosporine (200 mg/l, 0078-0109-61; Novartis) was added to the drinking water of the dam from 1 day prior to the injection until the pups were weaned at P28. Mice were kept on a 12-h dark/12-h light cycle. For experiments concerning phagosomes, they were killed between 15 and 30 min after lights on.