Tropicamide ophthalmic solution

Mice were dark-adapted for 12 h, and then the pupils were dilated with 1% Tropicamide Ophthalmic Solution (Bausch & Lomb, Rochester, NY, USA). Mice were exposed to blue light (5000 lx) for 1 h. The blue light was obtained by filtering white fluorescent light by a filter which transmits light between 380 and 570 nm (Midnight Blue 5940, Solar Graphics, Clearwater, FL, USA). The mice were free to move in the light chamber during the light exposure. After the light exposure, the mice were returned to the low-illuminance animal room where they were housed.

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.

To induce 4 weeks of ocular hypertension in mice, we performed microbead surgeries as previously described [16 (link)]. Briefly, animals were anesthetized with isoflurane (Minrad Inc., Bethlehem, PA), and pupils were dilated with 1% tropicamide ophthalmic solution (Bausch & Lomb, Tampa, FL). Using a pulled-glass micropipette and microinjection device, one eye was injected with 1.5 µL sterile 15-µm polystyrene beads in saline (1×10⁶ microbeads/ml; Invitrogen; Eugene, OR). The contralateral eye was injected with an equivalent volume of sterile physiologic saline (Fisher Scientific, Fair Lawn, NJ). After injection, antibiotic drops (0.5% moxifloxacin HCl; Alcon, Fort Worth, TX) were placed on each eye. IOP measurements were obtained with a tonometer (TonoLab, Icare; Raleigh, NC) on unanaesthetized animals, as previously described [4 ,17 ]. IOP was determined as the mean of 10 individual measurements. Baseline IOP readings were recorded for 3 consecutive days prior to microbead or saline injections. Following microbead and saline injections, IOP readings were recorded 3 times per week for 4 weeks, when the experiment was terminated.

Rats were used for induction of diabetes as described previously [21 (link)]. Briefly, male Brown Norway (BN) rats (8 weeks old; Charles River Laboratories International, Inc., Wilmington, MA, USA) received a single intraperitoneal injection of STZ (Sigma-Aldrich Corp, St. Louis, MO, USA) with the dosage of 50 mg/kg body weight dissolved in 10 mM of citrate buffer (pH 4.5) after overnight fasting. The blood glucose was measured with a glucose analyzer (Beckman Instruments, Zürich, Switzerland), 3 days after the STZ injection and weekly thereafter. Only the animals with blood glucose higher than 350 mg/dL were used as diabetic models in this study. At 3 weeks after STZ injection, animals were treated with fenofibrate eye drop or vehicle eye drop twice daily for 7 days [23 (link)].
In all procedures, rodents were anesthetized with an intraperitoneal injection of a mixture of ketamine (50 mg/kg, Zetamine, MWI, Boise, ID, USA) and xylazine (5 mg/kg, AnaSed LA, MWI, Boise, ID, USA), and pupils were dilated with topical administration of 1% tropicamide ophthalmic solution (Bausch + Lomb, a division of Bausch Health US, LLC, Bridgewater, NJ, USA).

In vivo image acquisition was performed using a confocal scanning laser ophthalmoscope (CSLO) (Retinal Angiograph II, Heidelberg, Germany), designed for clinical retinal imaging, with a 55° field of view lens attachment. The distribution of Alexa-labeled TTc was imaged with a baseline prior to injection followed by an imaging time point 3 h after an intravitreal injection of Alexa-488-TTc. The rats were positioned on the CSLO patient chin rest fitted with a level surface fitted with anesthesia. Prior to imaging, the pupils were dilated with 1% Tropicamide ophthalmic solution (Bausch and Lomb, Tampa, FL, USA). The corneal surface was protected during imaging with a custom made hard polymethyl methacrylate contact lens (Cantor and Nissel Limited, Brackley, UK). During CSLO imaging acquisition, the infrared reflection mode (IR, diode laser at 820 nm) was used to center the eye and focus on the retinal nerve fiber layer while the distribution of TTc-488 was captured in fluorescent angiograph mode with a blue solid laser at 488 nm with a 500-nm barrier filter. The images for quantitation were acquired as 25 aligned frames (5 images/s) at a video sensitivity of 80. ImageJ 1.52 (https://imagej.nih.gov/ij/download.html) [29 (link)] was used to average video clips to obtain a single low-noise high contrast image. Auto contrast mode was used for morphological characterization.