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....

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 needl....

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