Anterior Segment Perfusion Culture

Anterior segment perfusion culture is an established technique to study outflow facility ex vivo.³⁰ Use of human donor eye tissue was approved by the Oregon Health and Science University Institutional Review Board and experiments were conducted in accordance with the tenets of the Declaration of Helsinki for the use of human tissue. Human eye tissue was obtained postmortem from Lions VisionGift (Portland, OR, USA), and we obtained no information that could lead to identification of a tissue donor. Length of time from death to stationary culture was less than 48 hours and anterior segments initially were placed into serum-free stationary organ culture for 5 to 7 days to facilitate postmortem recovery.³¹ The age range was 52 to 95 years and average age of the donors' eyes for all experiments in this study was 77.3 ± 8.7 years. Donor demographics are included in Supplementary Table S1. After stationary culture, human anterior segments were perfused with serum-free Dulbecco's modified Eagle's medium (DMEM; a 1:1 mixture of high and low glucose DMEM) containing 1% penicillin/streptomycin/Fungizone, at constant pressure (8.8 mm Hg) with an average flow rate of 1 to 9 μL/min, which is similar to normal physiologic rate and pressures (minus episcleral venous pressure) in vivo. Anterior segments were perfused continuously at 8.8 mm Hg pressure (or “1×”). In some cases anterior segments were elevated to 17.6 mm Hg (or “2×”) perfusion pressure to produce a pressure challenge to trigger intraocular homeostatic responses⁶ as noted in the Figure legends. During the final stage of perfusion, fluorescently-labeled amine-modified 200 nm Fluospheres or Cell Mask Orange (Thermo Fisher Scientific, Waltham, MA, USA) were diluted 1:1000 into PBS, vortexed vigorously, and 200 μL of that mixture was injected as a bolus directly in-line into the anterior segment organ culture and perfused for 1 hour. This time was adjusted to accommodate different flow rates such that approximately equal volumes of the tracer were perfused. Perfusion was stopped and anterior segments were imaged en face using a Leica DM500 microscope (Leica Microsystems, Buffalo Grove, IL, USA) before cutting into radial wedges of HF or LF regions. HF and LF regions were separated routinely based on relative fluorescence of the TM on a per eye basis. A representative image is shown in our previous study indicating HF and LF regions.²⁵ In the same study, relative fluorescence intensity was mapped across flow regions of the TM and plotted versus distance using ImageJ software.²⁵ For all eyes in this study, we measured the circumference of the TM using a protractor overlay of the whole eye en face image. The same overlay application was used to determine the number of degrees of that circumference that were HF or LF with the remainder considered intermediate flow (IF). Most normal eyes have approximately a third HF, a third LF, and a third IF regions, or rather, the intensity of HF regions represents the upper 30th percentile, the intensity of LF regions represents the lower 30th percentile, and the IF regions were everything in between. IF regions were not included. TM tissues then were dissected and used for subsequent analysis by AFM or quantitative proteomics. Data from individual eyes were combined where possible and representative images were used. The number of eyes used for each treatment is noted in the Figure legend and is included in Supplementary Table S1.