Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Abstract

Source: Bunker, S., et al. Experimental glaucoma induced by ocular injection of magnetic microspheres. J. Vis. Exp. (2015)

In this video, an anesthetized rat undergoes an ocular procedure where magnetic beads are injected into the anterior chamber under a magnetic field to block the iridocorneal angle. This blockage obstructs the trabecular meshwork, preventing aqueous humor drainage and leading to increased intraocular pressure, damaging the retinal ganglion cells and optic nerve. This develops a glaucoma-like condition.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.    

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. Other pigmented rats may be suitable, although the user would need to validate them first.
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 IOP, with access to food and water.
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 the depth of anesthesia by testing the 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 settle better into the iridocorneal angle and prevent binding to the lens. Apply ocular ointment to prevent corneal drying on the unoperated contralateral eye.
5. Wash the operative eye with 5% povidone-iodine in water 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 them to a warm box and supply them 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 eye.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
250-300g female Brown Norway ex-breeder rats	Harlan UK	203
Tonolab Rebound Tonometer	Tiolat	TV02
Ketaset (Ketamine)	Fort Dodge Animal health	BN1000118	37.5 mg/kg
Domitor (medetomidine hydrochloride)	Orion Pharma	140-999	0.25 mg/kg
Povidone iodine	Ecolab	BN4369LE10	5% in H2O
Minim's Saline Solution	Bausch and Lomb	PL00033/5017
Toroidal magnet	Supermagnete	R-10-07-03-N
Magnetic Microspheres	Bangs Laboratories	UMC4N/9692
HBSS	Invitrogen	14025
33-guage bevelled needle	Hamilton	7747-01	Custom needle
Luer tip syringe	Hamilton	80601
Antisedan (atipemezole hydrochloride )	Orion Pharma	141-003	0.25 mg/kg
Chloramphenicol ointment	Medicom	18956-0005