All experimental procedures complied with Home Office (UK) regulations and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Sixty-one retired male breeder Brown Norway rats were housed in a constant low-light environment (40–60 lux) to minimize diurnal fluctuations in IOP, with food and water provided ad libitum. IOPs were measured three times before injection and daily or every 2 to 3 days after injection using a rebound tonometer (TonoLab; Tiolat, Oy, Finland) calibrated for use with the rat eye.
19 All measurements were made in awake animals in which the cornea was anesthetized using topical 0.4% oxybuprocaine hydrochloride eye drops (Chauvin, Kingston-Upon-Thames, UK). The IOP was taken as the mean and SD of five readings.
In each animal, the IOP was elevated in the left eye by injecting a sterile balanced salt solution (BSS; Alcon UK, Hemel Hempstead, UK) containing 30 mg/mL ferro-magnetic microspheres (Corpuscular Inc, Cold Spring, NY; bead diameter, 5 μm) into the anterior chamber of the left eye. Microspheres were sterilized by γ-irradiation (Gammacell 1000 Elite Caesium Source, 22 TBq; Nordon International, Inc., Ontario, Canada). All injections were made under isoflurane anesthesia, with topical chloramphenicol (0.5%) administered pre- and post-injection (Chauvin). Approximately 10–20 μL was injected into the anterior chamber of the left eye, delivering approximately 0.3–0.6 mg of beads. The right eye acted as an unoperated control. Injections were made using a 32- or 33-gauge needle (Hamilton Corporation, Bonaduz, Switzerland; and WPI Ltd, Stevenage, UK, respectively) inserted parallel to the iris. The 33-gauge needles were beveled on three sides to facilitate injection through the cornea and provided the easiest transit through the corneal stroma. A tunneled injection was made with the needle running parallel to the anterior surface of the iris to minimize the risk of iris trauma. Since the beads tended to settle in the syringe under the influence of gravity, the syringe and needle were agitated using a vortex stirrer immediately before injection to resuspend the beads in BSS. The minimum period between subsequent injections was 1 week.
Once the injection of beads was completed, the needle was kept in position to ensure maintenance of anterior chamber depth, and the beads then drawn away from the injection site using a small handheld magnet (0.45 Tesla), thereby minimizing the egress of beads from the injection track. The magnet was then used to distribute the microspheres around the iridocorneal angle to reduce the outflow of aqueous humor via the trabecular meshwork. Immediate increases in IOP could be moderated by allowing the leakage of aqueous around the injection cannula as it was withdrawn from the eye. Attempts to do this without redirection of beads resulted in the loss of beads via the injection site. Microsphere injections were performed up to three times in the left eye, depending on the required level and duration of the IOP elevation. For the purposes of control studies, five rats were injected with 20 μL of magnetic microspheres without magnetic direction to the iridocorneal angle. Five rats received a vehicle injection of 20 μL BSS (no beads) into the anterior chamber.
All rats were subsequently killed by an overdose of CO2 and selected retinas prepared as whole mounts for the quantification of cell loss in the retinal ganglion cell (RGC) layer. Anterior segment images of the beads distribution were taken as required with a camera (Nikon Coolpix 4500; Nikon, Tokyo, Japan) attached to a slitlamp (Haag Streit UK Ltd, Harlow, UK). Cryosections (10 μm) of the anterior chambers prepared on gelatin-coated slides were stained with hematoxylin and eosin for bright-field microscopic imaging or mounted under DPX (Raymond A. Lamb) for DIC imaging. Section images were taken using a bright-field microscope (Leica DMRA2; Leica Microsystems [UK] Ltd, Milton Keynes, UK) and associated software (Leica QWin v.3; Leica).