The mice were anesthetized by intraperitoneal injection of ketamine, xylazine, and acepromazine (50, 10, and 2 mg/kg, respectively). One eye was proptosed, and a glass cannula with tip diameter of 50 μm was inserted into the inferior portion of the anterior chamber, with injection of 2 μL of 6-μm beads (Polybead Microspheres; Polysciences, Inc., Warrington, PA), followed by 3 μL of viscoelastic solution (10 mg/mL hyaluronate sodium; Healon; Advanced Medical Optics Inc., Santa Ana, CA) through a syringe (Hamilton, Inc., Reno, NV). The beads were sterilized before injection by placing them in 100% ethanol in 0.5-mL tubes (Eppendorf, Fremont, CA). They were then centrifuged, resuspended in alcohol, and washed twice in sterile phosphate-buffered saline with recentrifugation. The final pellet was aspirated directly into the glass micropipette used for injection (at 3 × 106 beads per microliter). The needle was left in place for 2 minutes, to minimize efflux of injected material.
During the development of induced glaucoma from bead injection, IOP measurements were made in both eyes with the TonoLab tonometer (TioLat), with mice under both topical anesthesia with 0.5% proparacaine hydrochloride eyedrops and general anesthesia (as described earlier). IOP was measured before bead injection, 3 days after injection, and weekly thereafter, until the terminal calibration experiments at 3, 4, 5, or 6 weeks after injection. The TonoLab acquires six valid rebounds of the device from the eye and takes the mean of the middle four readings to make one summary measurement. The instrument indicates the reliability of the reading by the position of a bar next to the reported value. A single set of readings was collected with the best reproducibility indicator (no bar) at each time point during the periodic IOP measurements before calibration studies.