Rat Anesthesia and Preparation Protocol for Ophthalmic Research

All animal experiments were performed with IACUC approval and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animal preparation was similar to that reported previously.^{14 (link),15 (link)} Adult male Sprague–Dawley rats (n = 26, 250–300g) were anesthetized with 2% isoflurane, intubated and ventilated (Harvard apparatus, Model 683, South Natick, MA). The respiratory rate of the ventilator was set between 57 and 60 stroke/min. End-tidal CO2 was continuously monitored by a capnometer (Surgivet V9004, Waukesha, WI) and kept within normal range (3–3.5%). A regulated heating pad was used to maintain body temperature at 37°C. The right femoral artery was cannulated with PE50 tubing for mean arterial blood pressure (MABP) and blood-gas measurement. MABP was continuously monitored via the arterial line by a BIOPAC system (Acknowledge, Santa Barbara, CA) and was maintained between 90 and 110 mmHg as needed using hetastarch (0.5–1.5 ml/animal, i.v.). The left femoral artery was cannulated with a PE10 tubing for collecting the reference blood sample of circulating microspheres during the BF measurement. The right femoral vein was also catheterized for drug administration (e.g. heparin, pancuronium bromide and hetastarch). After surgery, the isoflurane level was reduced to 1.2–1.5% and the animal was then positioned in a custom-built head holder. Atropine (1%, topical, Bausch & Lomb, Tampa, FL) was applied to dilate the pupil. Pancuronium bromide (3 mg/kg, i.v.) was administered to paralyze the animals. Before the BF measurements, arterial PaCO₂ was measured (IRMA, Series 2000, DiaMedic, St. Paul, MN) and maintained within normal physiological ranges by adjusting the tidal volume. Arterial PaO₂ was not recorded. A previous study with similar ventilation parameters and identical anesthesia resulted in a PaO₂ of 100–105 mmHg.^{15 (link)}