Thirteen male Long-Evans pigmented rats (450–650 g) were used. The animals were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were anesthetized using ketamine (85 mg/kg IP) and xylazine (3.5 mg/kg IP). An oxygen-sensitive molecular probe
40,41 (Oxyphor R2; Oxygen Enterprises, Ltd., Philadelphia, PA) was dissolved in saline and 3 μL (0.5 mM) was injected intravitreally. The presence of the probe in the vitreous was confirmed immediately after injection, by imaging the vitreous cavity with a slit lamp biomicroscope to visualize the bolus. The animals were imaged 24 hours after injection, an experimentally determined duration for the probe to diffuse from the vitreous into the retina. For monitoring systemic arterial PO
2, a catheter was placed in the femoral artery to obtain blood samples. Blood pressure and heart rate were monitored with a pressure transducer attached to the catheter and linked to a data-acquisition system (Biopac Systems Inc., Goleta, CA). Before imaging, the pupils were dilated with 2.5% phenylephrine and 1% tropicamide. Hydroxypropyl methylcellulose (1%) was applied to the cornea, and a glass coverslip was placed on the cornea to eliminate its refractive power and to prevent corneal dehydration. Normal body temperature was maintained with an animal holder connected to a water heater composed of copper tubing. FiO
2 was varied via a high-flow face mask system. Gas mixtures containing 10%, 21% (room air), and 50% oxygen were administered to the rats 10 minutes before and during imaging. Concurrent with imaging, arterial blood was drawn through the catheter without exposure to air, and systemic arterial PO
2 was measured with a blood gas analyzer (Radiometer, Westlake, OH). The rat was placed in front of the imaging instrument. The laser power was adjusted to 100 μW at the cornea. Because of pupil size, 40 μW entered the eye, yielding a retinal irradiance of approximately 30 mW/cm
2, which is safe for continuous viewing for 3600 seconds, according to the American National Standard Institute for Safety Standards.
42 Three images were acquired at each location within 2 disc diameters of the optic nerve head, over a period of approximately 60 seconds. Of the 13 rats, 3 were imaged in all three FiO
2 conditions, 5 in two, and 5 in one. In total, in each of the three FiO
2 conditions, data from eight rats were available, yielding a total of 24 data points.