A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Summary

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Abstract

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Introduction

The progressive loss of retinal ganglion cells (RGCs) and their axons is the hallmark of glaucoma, a common neurodegenerative disease in the retina¹. It will affect more than 100 million individuals 40−80 years old by 2040². IOP remains the only modifiable risk factor in the development and progression of glaucoma. In order to explore the pathogenesis, progression, and potential treatments of glaucoma, a reliable, reproducible, and inducible experimental ocular hypertension/glaucoma model that replicates key features of human patients is imperative.

IOP depends on aqueous humor inflow to the anterior chamber from the ciliary body in the posterior chamber and outflow through the trabecular meshwork (TM) at the angle of the anterior chamber. Upon reaching a steady state, IOP is maintained. When the inflow exceeds or is less than the outflow, IOP rises or falls respectively. By decreasing the aqueous outflow either by occluding the angle of the anterior chamber or by damaging the TM, several glaucoma models have been established^{3,4,5,6,7,8,9,10}. These models are normally associated with irreversible ocular tissue damage, and the high IOP in the anterior chamber also causes unwanted complications such as corneal edema and intraocular inflammation, which make retinal imaging and visual function assays difficult to perform and interpret.

To develop a model that overcomes these shortcomings, we focused on the well-sudocumented secondary glaucoma caused by silicone oil (SO) that occurs as a postoperative complication of human vitreoretinal surgery^11,12. SO is used as a tamponade in retinal surgeries because of its high surface tension. However, SO can physically occlude the pupil because it is lighter than the aqueous and vitreous fluids, which prevents aqueous flow into the anterior chamber. The obstruction causes IOP elevation in the posterior chamber due to the aqueous humor accumulation. This motivated us to develop and characterize a novel ocular hypertension mouse model based on intracameral SO injection and pupillary block¹³, with key features of the secondary glaucoma: effective pupillary block, significant IOP elevation that can return to normal after SO removal, and glaucomatous neurodegeneration.

Here we present a detailed protocol for SO-induced ocular hypertension in the mouse eye, including SO injection and removal and IOP measurement.

Protocol

All procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University.

1. Ocular hypertension induction by intracameral injection of SO

1. Prepare a glass micropipette for intracameral SO injection by pulling a glass capillary with a pipette puller to generate a micropipette. Cut an opening at the tip of the micropipette and further sharpen the tip with a microgrinder-beveling machine to make a 35°−40° bevel.
2. Polish the edges of the bevel and remove all debris by washing with water. Autoclave the micropipette before use.
3. Prepare the paracentesis needle for the corneal entry. To do so, attach a 32 G needle to a 5 mL syringe on a Luer lock, and further secure it with tape. Bend the needle bevel tip face up at 30°.
4. Prepare the SO injector by attaching and securing a blunt end 18 G needle on a 10 mL syringe first. Then attach a plastic tube with the 18 G needle on one end and fill up with SO as needed through the other end.
5. Attach the sterilized micropipette to the plastic tube and push the syringe plunger to fill the entire micropipette with SO.

2. Intracameral SO injection for one eye

1. Place a 9−10-week-old male C57B6/J mouse into an induction chamber with 3% isoflurane mixed with oxygen at 2 L/min for 3 min.
2. Intraperitoneally inject 2,2,2-tribromoethanol at 0.3 mg/g body weight.
   NOTE: Unlike ketamine/xylazine, 2,2,2-tribromoethanol does not cause obvious pupil dilation.
3. Check for the lack of response to a toe pinch and the lack of movement of the whiskers or the tail to determine the anesthetic strength.
4. Place the mouse in a lateral position on a surgery platform. To reduce its sensitivity during the procedure, apply one drop of 0.5% proparacaine hydrochloride to the cornea before the injection.
5. Make an entry incision with the 32 G paracentesis needle at the superotemporal quadrant, about 0.5 mm from the limbus.
6. Tunnel through the layers of the cornea for about 0.3 mm before piercing into the anterior chamber. Be careful not to touch the lens or iris.
7. Withdraw the needle slowly to release some aqueous humor (about 1−2 µL) from the anterior chamber through the tunnel (paracentesis).
8. Wait ~8 min to further decrease the IOP. This can be determined by measuring the contralateral, control eye.
9. Insert the glass micropipette preloaded with SO through the corneal tunnel into the anterior chamber, with the bevel facing down to the iris surface.
10. Push the syringe plunger slowly to inject SO into the anterior chamber until the SO droplet covers most of the iris surface, ~2.3−2.4 mm in diameter.
11. Leave the micropipette in the anterior chamber for 10 s more before withdrawing it slowly.
12. Gently push the upper eyelid to close the cornea incision to minimize SO leakage.
13. Apply antibiotic ointment (bacitracin-neomycin-polymyxin) to the eye surface.
14. Throughout the procedure, frequently moisten the cornea with artificial tears.
15. Keep the mouse on the heating pad until fully recovered from anesthesia.

3. SO removal

1. Prepare the irrigation system.
   1. Prepare the irrigating solution according to the manufacturer’s instructions and place it in the irrigation bottle. Elevate the irrigating solution bottle to 110−120 cm (81−88 mmHg) above the surgery platform.
   2. Attach an IV administration set to the irrigating solution bottle. Remove air bubbles from the IV tubing. Connect a 33 G needle bent to 20° face up to the IV tubing.
2. To prepare the drainage system, remove the plunger from a 1 mL syringe. Attach a 33 G needle to the syringe and bend the needle to 20°.
3. Remove SO from the anterior chamber.
   1. Intraperitoneally inject 2,2,2-tribromoethanol (0.3 mg/g body weight). Check for the lack of response to the toe pinch to determine the anesthetic strength and the lack of movement of the whiskers or the tail.
   2. Place the mouse on a surgery platform and secure it in the lateral position with tape. Apply one drop of 0.5% proparacaine hydrochloride to the cornea to reduce its sensitivity.
   3. Make two incisions in the temporal quadrant of the cornea between ~2 and 5 o’clock at the edge of the SO droplet using the premade 32 G paracentesis needle.
   4. Insert a 33 G irrigation needle connected to irrigating solution through one corneal incision, maximum speed.
   5. Insert another 33 G drainage needle attached to the syringe without a plunger through the other corneal incision to allow the SO droplet to exit the anterior chamber while irrigating with irrigating solution.
   6. Withdraw the drainage needle, then the irrigation needle.
   7. Inject an air bubble into the anterior chamber to maintain its normal depth and press to close the corneal incision.
   8. Apply antibiotic ointment to both eyes.
   9. Keep the mouse on the heating recovery pad until fully recovered from the anesthesia.

4. IOP measurement once a week

1. Place the mouse into an induction chamber perfused with 3% isoflurane mixed with oxygen at 2L/min for 3 min.
2. Intraperitoneally inject xylazine and ketamine (0.01 mg xylazine/g, 0.08 mg ketamine/g).
3. Keep the cornea moist by applying artificial tears throughout the procedure.
4. Wait about 15 min to allow the pupil to fully dilate.
5. Measure the IOP of both eyes using a tonometer according to product instructions. Bring the tonometer near the mouse eye. Keep the distance from the tip of the probe to the mouse cornea at about 3−4 mm. Press the measuring button 6x to generate one reading. Three machine-generated readings are obtained from each eye to acquire the mean IOP.
6. Sacrifice the animals at 8 weeks after SO injection and perform immunohistochemistry of whole-mount retina, RGC counting, optic nerve (ON) semi-thin sections, and quantification of surviving axons, which have been described before¹³.

Results

Soon after the injection we can easily identify mice that do not produce stable ocular hypertension because of the SO droplets being too small (≤1.5 mm)¹³. These animals are excluded from subsequent experiments. Following the injection procedures, more than 80% of SO injected mice end up with droplets larger than 1.6 mm. We measured the IOP of these mouse eyes once a week for 8 weeks after a single SO injection. The IOP of the eye receiving SO remained high, generally double the IOP of the c...

Discussion

Here we demonstrate a simple but effective procedure for inducing sustained IOP elevation in the mouse eye by intracameral injection of SO. This procedure can be learned quickly by anyone with experience in microdissection under a microscope. The primary potential risk of failure is the leakage of SO from the corneal incision. However, one of the advantages of using SO is that because the oil droplet is visible and measurable, we can easily identify mice that received droplets too small to induce stable ocular hypertensi...

Materials

Name	Company	Catalog Number	Comments
0.5% proparacaine hydrochloride	Akorn, Somerset
10mL syinge	BD		Luer-Lok Tip
18G needle	BD		with Regular Bevel, Needle Length:25.4 mm
2,2,2-Tribromoethanol (Avertin)	Fisher Scientific	CAS# 75-80-9	50g
32G nano	BD	320122	BD Nano Ultra Fine Pen Needle-32G 4mm
33G ophalmology needle	TSK/ VWR	TSK3313/ 10147-200
5mL syinge	BD		Luer-Lok Tip
AnaSed Injection (xylazine)	Butler Schein		100 mg/ml, 50 ml
artificial tears	Alcon Laboratories	300651431414	Systane Ultra Lubricant Eye Drops
BSS PLUS Irrigating solution	Alcon Laboratories	65080050
Dual-Stage Glass Micropipette Puller	NARISHIGE	PC-10
EZ-7000 Classic System	EZ system
Isoflurane	VetOne	502017	isoflurane, USP, 250ml/bottle
IV Administration sets	EXELint/ Fisher	29081
KETAMINE HYDROCHLORIDE INJECTION	VEDCO	50989-996-06	KETAVED 100mg/ml * 10ml
microgrind bevelling machine	NARISHIGE	EG-401
Miniature EVA Tubing	McMaster-Carr	1883T4	0.05" ID, 0.09" OD, 10 ft. Length
silicon oil (SILIKON)	Alcon Laboratories	8065601185	1,000 mPa.s
Standard Glass Capillaries	WPI/ Fisher	1B150-4	4 in. (100mm) OD 1.5mm ID 0.84mm
TonoLab tonometer	Colonial Medical Supply, Finland
veterinary antibiotic ointment	Dechra Veterinary	1223RX	BNP ophthalmic ointment, Vetropolycin