Structure–Function Changes of the Porcine Distal Outflow Tract in Response to Nitric Oxide

Abstract

PurposeTo correlate outflow function and outflow tract vessel diameter changes induced by nitric oxide (NO).MethodsIn a porcine anterior segment perfusion model, the effects of a nitric oxide donor (100 μM DETA-NO) on outflow facility were compared with controls (n = 8 per group) with trabecular meshwork (TM) and after circumferential ab interno trabeculectomy (AIT). Outflow structures were assessed with spectral-domain optical coherence tomography (SD-OCT) before and after NO, or an NO synthase inhibitor (100 μM L-NAME) and the vasoconstrictor, endothelin-1 (100 pg/mL ET-1). Scans were processed with a custom macroscript and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA).ResultsThe facility increased after DETA-NO (Δ of 0.189 ± 0.081 μL/min·mm Hg, P = 0.034) and AIT (Δ of 0.251 ± 0.094 μL/min·mm Hg, P = 0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (Δ of 0.190 ± 0.074 μL/min·mm Hg, P = 0.023) and CSA by 13.9% (P < 0.001). L-NAME + ET-1 decreased CSA by −8.6% (P < 0.001). NO increased the diameter of focal constrictions 5.0 ± 3.8-fold.ConclusionsNO can dilate vessels of the distal outflow tract and increase outflow facility in a TM-independent fashion. There are short, focally constricting vessel sections that display large diameter changes and may have a substantial impact on outflow.

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract

“…Pre-treatment was compared to post-treatment with a one-sample t-test in Python 3.6 [18] . SD-OCT images were processed in ImageJ [19] (Version 1.50i, National Institute of Health, Bethesda, Maryland, United States) and Amira Aviso (version 9.1, FEI, ThermoScientific, Waltham, Massachusetts) as done previously [17] to remove noise, align pre-and post-treatment outflow tract signal voids in a three-dimensional space (3D), and to allow automated, quantitative measurement of cross-sectional areas (CSA). Cross-section areas of pre-and post-treatment were compared with a student's t-test.…”

“…Recent evidence from clinical [20][21][22][23] and laboratory [10,17] TM ablation studies demonstrated a significant post-TM outflow resistance that might be caused by a downstream regulatory mechanism [10,17] . Only 0.3% of patients undergoing trabecular ablation in AIT achieve the predicted decrement in IOP to 8 mmHg akin to the level of episcleral veins [23] .…”

“…We studied the outflow facility responses of anterior segment organ cultures to 0.1, 1, and 10 µM netarsudil. To establish the contribution by the TM to these responses, treatment groups were created for each concentration with and without circumferential ab interno trabeculectomy (AIT) using a trabectome as described before [17] . Eight eyes were randomly assigned to each treatment group and perfused for at least 48 hours to establish a stable baseline IOP before treatment.…”

“…Anterior segments were cultured at 37° C and perfused at 4 µl/min with media (Dulbecco's ADInstruments) [17] . Baseline IOP was achieved after 48 hours of perfusion.…”

“…Each porcine eye was positioned with the optic nerve remnant secured in a low-compression mount (CryoElite Cryogenic Vial #W985100; Wheaton Science Products, Millville, NJ, USA) and kept damp with phosphate-buffered saline. Anterior chambers were perfused at a constant pressure of 15 mmHg with perfusion media as done previously [17] . The eyes were placed under the sample arm of an SD-OCT equipped with a 10 mm telecentric lens (Envisu R2210, Leica, Bioptigen, Morrisville, NC, USA).…”