All animal procedures were approved by the Animal Care Committee of Asahikawa Medical University and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Porcine eyes were enucleated immediately from pigs of either sex (age range, 16–24 weeks; weight range, 10–20 kg) after they were killed at a local abattoir and transported to the laboratory in a moist chamber on ice. The techniques to identify and isolate the retinal microvessels have been described previously.
10–12 Briefly, the anterior segment and vitreous body were removed carefully under a dissecting microscope. The posterior segment was placed in a cooled dissection chamber (∼8°C) containing a physiologic salt solution (PSS—NaCl, 145.0 mM; KCl, 4.7 mM; CaCl
2, 2.0 mM; MgSO
4, 1.17 mM; NaH
2PO
4, 1.2 mM; glucose, 5.0 mM; pyruvate, 2.0 mM; EDTA, 0.02 mM; and MOPS, 3.0 mM) with 1% albumin. Single second-order retinal arterioles (0.6–1.0 mm in length) were carefully dissected with the aid of a stereomicroscope (Model SZX12; Olympus, Tokyo, Japan). After careful removal of any remaining neural/connective tissues, the arterioles then were transferred for cannulation to a polymethylmethacrylate vessel chamber containing PSS-albumin solution equilibrated with room air at ambient temperature. One end of the arteriole was cannulated with a glass micropipette filled with PSS-albumin solution, and the outside of the arteriole was tied securely to the pipette with an 11-0 ophthalmic suture (MANI, Tochigi, Japan). The other end of the vessel was cannulated with a second micropipette and secured with a suture. After cannulation, the vessel and pipettes were transferred to the stage of an inverted microscope (Model CKX41; Olympus) coupled to a video camera (WAT-902B; Watec Co., Ltd., Yamagata, Japan), a video micrometer (V-94; Living Systems Instrumentation, Burlington, VT), and a data acquisition system (PowerLab; ADInstruments, Colorado Springs, CO) for continuous measurement and recording of the internal diameter throughout the experiment.
10 The vessel was pressurized to an intraluminal pressure of 55 cmH
2O (∼40 mm Hg) without flow, using two independent pressure reservoirs. This level of pressure was used based on previously documented pressure ranges in the retinal arterioles in vivo.
13 To examine the role of the endothelium, we intraluminally administered a pharmacologic inhibitor as described previously.
14 Briefly, a tube filled with the drug was inserted into the sidearm of the micropipette holder, and the micropipette was replaced with this drug. Flow was started into the vessel from the drug-filled micropipette by creating a pressure gradient (∼5 cmH
2O) across two pressure reservoirs. After perfusing the vessels with the drug-containing solution for 10 minutes, the flow was stopped and the vessels were incubated with an inhibitor for at least 30 minutes before agonist stimulation.