A previously established mouse eye perfusion protocol was modified slightly for the present study.
12,23,24 Briefly, paired eyes from C57BL/6 mice were enucleated and randomized to drug (IWP-953 or DETA-NO) or vehicle perfusions. Working solutions of drug and vehicle were prepared from stock solutions (20 mM) diluted in Dulbecco's phosphate-buffered saline (PBS) plus 5.5 mM D-glucose (DBG) just prior to use. Four different concentrations of IWP-953 (10, 30, 60, and 100 μM) were tested and compared to a positive control, DETA-NO (100 μM). IWP-953 was dissolved in DMSO (final concentration of 0.5%) while DETA-NO was dissolved in DBG. The contralateral eye was stored in DBG at 4°C during the first perfusion (approximately 3 hours).
Enucleated eyes were affixed to a post in the perfusion chamber using cyanoacrylate gel and were then cannulated using a 33-gauge needle (World Precision Instruments, Sarasota, FL, USA), guided by a micro manipulator and stereo microscope. During the cannulation procedure, eyes were held stable using a 0.5-mm curret. The inserted needle was connected by pressure tubing to a 50-μL glass syringe (Hamilton Robotics, Reno, NV, USA), which was mounted in a Harvard minipump (model 33; Harvard Apparatus, Holliston, MA, USA) and controlled by custom software written in Labview (National Instruments, Newbury, UK). The software monitored pressure readings from a Honeywell transducer (model 142PC01G; Honeywell, Fort Washington, PA, USA) that was in communication with eye interior via a three-way stop cock connecting tubing between syringe pump and eye. Desired pressure steps were maintained by the computer controlled pump, adjusting flow rate. After a 45-minute equilibration period at 8 mm Hg to allow drug/vehicle to access outflow tissues, eyes were perfused for approximately 20 minutes at each of four pressure steps (4, 8, 12, and 16 mm Hg). To calculate flow rate at each pressure step, we used a minimum of 10 minutes of stable data. Stable flow rate for at least three of the four pressures was a requirement in order to be included in the final analysis.