Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Ethics statement: All animal experiments have been conducted in accordance with the Institutional Animal Care and Use Committee (IACUC), and were approved in agreement with United Kingdom Home Office guidelines (http://goo.gl/FLkirW, last accessed 10^th June, 2014) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (http://goo.gl/4LFOjD, last accessed 10^th June, 2014).

1. Ocular Hypertension Induction

1. Induce experimental glaucoma by elevating the intraocular pressure (IOP) via unilateral injection of paramagnetic microspheres into the anterior chamber of Brown Norway rats, based on the method of Samsel et al.³ Other pigmented rats may be suitable, although these would need to be validated first by the user.
2. House 250-300 g female ex-breeder Brown Norway rats in a constant low-light environment (40-60 lux) to minimize diurnal fluctuations in IOP⁷, with access to food and water ad libitum.
3. Take baseline IOP measurements in awake animals⁸ prior to anesthesia and bead injection, using a rebound tonometer calibrated for use in the rat eye⁹. IOP is taken as the mean of five readings.
4. Anaesthetize rats with 37.5 mg/kg ketamine, and 0.25 mg/kg medetomidine hydrochloride delivered intraperitoneally. Confirm depth of anesthesia by testing animal’s rear foot reflexes, prior to povidone iodine application (see step 1.5), and bead injection (see step 1.8). Administer 0.5% proparacaine hydrochloride for analgesia.
   NOTE: Do not dilate the pupil at any stage. This will help the beads to settle better into the iridocorneal angle, and prevent binding to the lens. Apply ocular ointment to prevent corneal drying on un-operated contralateral eye.
5. Wash the operative eye with 5% povidone iodine in water¹⁰ 5 min prior to injection.
6. After 5 min, wick the povidone iodine off using sterile gauze, and wash the eye with 0.9% sterile saline solution. Keep the eye moist during anesthesia with regular application of sterile saline.
7. Place a toroidal magnet around the eye.
8. Inject 25 μl of a solution containing 30 mg/ml of gamma-irradiation sterilized 8 μm magnetic microspheres in Hank’s Balanced Salt Solution (HBSS) into the anterior chamber, using a 33 G beveled needle.
   1. To prepare beads, wash by re-suspending, then centrifuging 3 times at 10,000 x g for 5 min with 1 ml HBSS, before making the final 30 mg/ml solution. Maintain sterile conditions throughout.
   2. For injection, be careful to avoid inserting the needle to the iris, to minimize the risk of iris trauma. This can be prevented by orientating the needle tangential to the corneal surface, as parallel to the iris as possible. This will also help to minimize bead loss from the injection site.
      NOTE: Inject beads at a rapid rate to ensure an even distribution around the iridocorneal angle, which is critical to raising IOP. Additionally, store beads, needles and magnetic rings separately so that the beads do not form clusters, making them difficult to load into the syringe and inject, and the needle does not become magnetized.
9. Leave the needle in place for 1 min post-injection to ensure that beads settle into the iridocorneal angle to impede aqueous drainage from the trabecular meshwork. Slightly angle the needle after the beads have initially settled to allow some leakage of aqueous, to minimize transient increases in IOP. At the end of the surgery flush the needle through with first phosphate buffered saline (PBS), then 70% ethanol, followed by distilled water, to ensure continued use of the needle in separate procedures. Alternatively, one could use disposable needles if the correct gauge and syringe combination is available. Optional: needles may be sharpened using a beveller to prolong their use.
10. At this stage if necessary, remove the magnet, and use it to draw beads into areas of incomplete coverage.
11. Leave the magnet in place around the eye for a further 10 min post-injection to ensure beads settle well into the iridocorneal angle.
12. Reverse anesthesia using 0.25 mg/kg atipemezole hydrochloride.
13. Administer chloramphenicol, or other antibiotic ointment, for example gentamycin or terramycin topically to prevent infection, and 0.5% proparacaine hydrochloride for analgesia. Leave animals to recover on a heat mat until they regain movement, then transfer to a warm box and supply with additional nutrition, such as a food supplement or moistened regular diet until recovery is complete. Systemic or local analgesia should be given to animals displaying signs of pain 24 hr after surgery. If these symptoms persist despite treatment, animals should be humanely culled.
14. Use the contralateral eye as an unoperated control.
15. Take IOP measurements every 2-3 days following bead administration, and every 2-3 days thereafter using a rebound tonometer calibrated for use in the rat eye⁹.
16. The criteria for including eyes in studies can be: if 1) the IOP is elevated above the contralateral control pressure by 5 mmHg, and 2) does not exceed 60 mmHg.
17. Eyes where the pressure returns to baseline (usually by 1 week post-injection) should not be included in the studies, however it is possible to re-inject beads in eyes that fail to develop high IOP if desired.
18. Euthanize animals by CO₂ asphyxiation at the end of the experiment.
19. Dissect eyes and optic nerves, and fix in 4% paraformaldehyde (PFA) overnight for further histological analysis.

2. Assessing Retinal Neuron Damage Using TUNEL Staining

1. To quantify apoptotic cells in whole mount retinasuse the terminal deoxynucleotidyl transferase-mediated dUTP nick-labelling (TUNEL) assay, according to the manufacturer’s instructions.
2. Dissect the retina from the eye cup, wash for 3 x 5 min in 0.3% Triton X-100 in phosphate buffered saline (T-PBS).
3. Permeabilize the tissue in 3% T-PBS for 2 hr.
4. Pre-equilibrate retinas in Equilibration Buffer for 10 min, before incubation in TUNEL reaction solution for 1 hr at 37 °C.
5. Wash the tissue for 3 x 5 min in 0.3% T-PBS, rinse in 0.3% T-PBS containing 5 µM DAPI, and flat-mount in mounting media.
6. To quantify TUNEL positive nuclei use a confocal microscope to take 10 µm z-stacks through the ganglion cell layer at 20X magnification. Take 3 images on each of the 4 petals, at sites close to the optic nerve, in the mid-periphery, and at the far periphery of the retina, giving a total of 12 images per whole mount, sampling approximately 7,000 cells. Morphological criteria discriminated non-neuronal (endothelial and glial) cells from neuronal cells.
7. Select areas for imaging using only the DAPI channel, and mask investigators to treatment groups.

3. Assessing Optic Nerve Damage Using Toluidine Blue Staining

1. Fix optic nerves overnight in Karnovsky’s solution at 4 °C.
2. Treat specimens for 2 hr in 1% (w/v) osmium tetroxide and then dehydrate in 100% ethanol.
3. Incubate optic nerves in propylene oxide for 30 min, and place in a 50:50 mixture of propylene oxide:araldite overnight.
4. Change this solution to 100% araldite, followed by incubation overnight at 60 °C.
5. Cut semithin sections (0.75 mm thick) and stain with 1% toluidine blue/ borax (TB) in 50% ethanol before examination by light microscopy.

4. Statistical Analysis

1. Carry out statistical analyses using an appropriate statistics software package. A two-way ANOVA with Newman-Keul’s post-hoc test may be used to calculate statistical significance for IOP changes over time.
2. A p value of less than 0.05 may be considered significant.