Under general anesthesia, both femoral arteries and a vein were cannulated with polyethylene tubes (PE50) for BP monitoring, reference blood sample collection (see the following text), and administration of drugs, respectively. The left ventricle was cannulated via the right brachial artery with the same type of polyethylene tubing. Once arterial BP and end-tidal PCO2 were stabilized within normal ranges, 60 million fluorescent microspheres (PolySciences, Inc., Warrington, PA) with a 10-μm diameter, suspended in a solution of 5 mL of 0.15 M NaCl and 0.05% Tween 20, were injected into the left ventricle over a period of 25 to 45 seconds after heparinizing the blood (500 IU/kg, IV). Note that in two male monkeys with larger body weight, 100 million microspheres were injected. A reference blood sample was drawn from one of the cannulated arteries, starting from the onset of microsphere injection, for 1 or 2 minutes. Animals were then euthanized by overdose of intravenous pentobarbital (Euthasol; Delmarva Laboratories, Inc., Midlothian, VA), then both eyes were enucleated and postfixed in 4% paraformaldehyde for 48 hours, after which they were embedded in optimal cutting temperature (O.C.T.) compound (Tissue-Tek O.C.T. Compound; Sakura Finetek USA, Inc., Torrance, CA).
For each embedded eye, consecutive serial longitudinal frozen sections were obtained by a cryostat with 25 μm thickness. These sections were collected on a series of numbered glass slides and air dried. The microsphere concentration in the reference blood was determined in a hemacytometer (Fuchs–Rosenthal Counting Chamber; Electron Microscopy Sciences, Hatfield, PA). Each ONH section was photographed with a 10× lens under a fluorescent microscope equipped with an automated imaging system. Montages of the photographs for each ONH section were created digitally. The total microspheres within a given region of ONH (see the following text) were counted. With the microsphere counts in both reference blood and ONH, total BF in a given defined region (μL/min) was calculated as: N tissue/C ref, where N tissue was the microsphere count in the tissue of interest and C ref is the microsphere number per μL reference blood per minute.
The ONH BF was determined for two layers (depth regions) divided by the posterior border of the lamina cribrosa: the first layer included the superficial NFL, prelaminar tissue, and lamina cribrosa (anterior ONH); the second layer included the first 1 mm of the retrolaminar optic nerve (posterior ONH). BF in the peripapillary retina was also measured in seven pairs of the eyes from an annulus of tissue obtained by two trephine cuts (6 and 2.5 mm) centered on the optic disc. The number of microspheres within this 23.4 mm2 peripapillary annulus of retina was counted and BF was calculated using the same method as described earlier for ONH BF.