Anterior segment organ culture perfusions

The anterior segment perfusion model^48,49 was used to study the eNOS-GFP and eNOS-SEAP adenoviruses. A syringe pump was used to generate a constant inflow rate for human eyes (PHD 2000 Programmable Syringe Pump, Harvard Apparatus, Holliston, MA). A wet-wet differential pressure transducer (0–50 mmHg; Omega Engineering, USA) continuously measured intrachamber pressure. Customized iPerfusion software⁵⁰ recorded IOP measurements throughout the perfusion (multifunction DAQ, National Instruments, USA). A reservoir was connected between the perfusion chambers and the pressure transducer to facilitate media exchanges (DMEM with 4.5 g/L glucose, supplemented with 1% FBS, 1% PSG and 0.025% BSA) of perfusion chambers and to control intrachamber pressures for retroperfusion. When a stable baseline pressure was achieved at a constant inflow rate of 2.5 µl/min, the anterior segments were retroperfused with adenovirus. After the retroperfusion steps were completed, 2 protocols were followed after restarting forward perfusion. In all perfusions, paired anterior segments were initially perfused for 24–48 hours to achieve an initial stable baseline. In cases in which both segments achieved a stable baseline pressure (n = 5) forward perfusion was restarted as follows; in control anterior segments, a constant flow rate of 2.5 µl/min was maintained while in experimental anterior segments we increased the flow rate to 5 µl/min 24 hours post-retroperfusion. In cases where one of the anterior segments of a pair failed to establish a stable baseline pressure (n = 5), only the segment with a stable baseline was used for the experiment. In these cases, perfusion was initially restarted at 2.5 µl/min for 24 hours, before increasing the flow rate to 5 µl/min for 24 hours. Conditioned media was collected every 24 hours after retroperfusion and used for analysis of nitrite and SEAP content.

Once the perfusion was completed, the trabecular meshwork was removed to gain better visualisation of the outer wall of Schlemm’s canal, and the anterior segments were then fixed overnight in 3% PFA before being cut into quadrants for further analysis.