The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Summary

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Abstract

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

Introduction

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the anterior portion of the optic nerve (ON)¹. NAION is the most common cause of sudden optic nerve related vision loss in individuals over the age of 50². The mechanism is believed to be a compartment syndrome that results in intraneural edema, and causes compression of the capillaries supplying the axons within the optic nerve³.

Since the ON is actually a central nervous system (CNS) tract, the rodent NAION (rNAION) model can be used to study the mechanisms and responses to isolated CNS white matter strokes. The rNAION model may therefore be useful in dissecting many problems associated with stroke related damage to white matter. It can be used to evaluate different neuroprotective strategies and agents in white matter stroke.

One of the most attractive features of the model is that it is a painless, noninvasive procedure. The laser power can be adjusted to produce various degree of ischemic damage. Another feature is that it relies on laser induced superoxide radicals to damage the capillary endothelium, producing a progressive capillary dysfunction. It is this dysfunction and progressive edema that is believed to be remarkably similar to the mechanism that causes NAION. Research has shown that it does not cause direct capillary clotting, but works through at least two mechanisms: superoxide induced death and stripping of some of the capillary endothelial cells⁴, and NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) associated inflammatory up regulation in remaining endothelium, with increased fluid transport across the cell membranes into the interstitium⁵ . The closure of the optic nerve capillaries and compression caused by interstitial fluid accumulation result in optic nerve head ischemia. A schematic picture is shown in Figure 2. The rNAION model can be used in both rat and mouse species^6,7, and can be varied in the level of its severity, from a mild lesion to a complete, but painless destruction of the optic nerve and the retina, such as central retinal artery occlusion(CRAO).

Protocol

This protocol was approved by the University of Maryland Institutional Animal Care and Utilization Committee (IACUC; Baltimore, MD, USA)

1. Experimental Set-up

1. Make a custom designed contact lens from a clear optical grade circular 7 mm in diameter Plexiglas, of 3 mm thickness. Cut the circular lenses with a drill press. Use a standard drill bit to make the inner curve, and finally polish the outer and inner curves using a contact lens polisher of ultrafine grit (1000/3000).
2. Prepare 2.5 mM Rose Bengal (RB) in pH 7.4 phosphate buffer saline (PBS) in advance, filter sterilize with a 0.45 micron syringe filter and store 1 ml aliquots in -20 °C in a light-tight container for up to 6 months.
   NOTE: The use of albino outbred male animals, such as Sprague Dawley minimizes the strain dependent response differences, enables increased ease of induction and reduces the variability that can occur with estrus cycling female.
3. Set up the frequency doubled Neodymium-yttrium aluminum garnet (Fd-YAG) ophthalmic medical laser, which generates a 532 nm laser light. Mount the laser on a Haag-Streit ophthalmic slit lamp using a standard ophthalmic laser adapter. This commercially available device enables simultaneous visualization of the animal's eye and laser spot application. The medical laser also has an aiming beam for proper focusing and centration using same spot size as laser induction. Laser power parameters are as follows:
   1. For rat rNAION induction: use 500 µm spot size/50 mW laser power/1,000 msec duration/1,000 msec interval.
   2. For mouse rNAION induction: change the spot size to 300 µm for smaller optic disc and leave other parameters the same as the rat setting.
   3. Routinely use a laser power meter to assure the laser power output.

2. Experimental Procedure

1. Turn on the laser power and set up the appropriate laser parameter. Warm the laser for at least five minutes before use.
2. Weigh the animal to determine the appropriate dose for ketamine/xylazine and RB dye. Anesthetize the animal by intraperitoneal injection of a 1 ml/kg mixture of 80 mg/ml ketamine and 4 mg/ml xylazine.
3. Leave the animal in a heated cage until fully anesthetized. Check for no response to aversive stimuli (tail or toe pinch). Check animals for depth of anesthesia every 10 min.
4. Dilate the animal's pupils with 1% tropicamide and anesthetize the surface of the eye with 0.5% proparacaine. If using pigmented animals, such as Long Evans, a 2.5% neosynephrine ocular drops will increase pupil dilation.
5. Use scissors to cut the whiskers close to the muzzle on the side to be induced to avoid blocking the view.
6. Put a drop of 1% methylcellulose or other ophthalmic coupling drop to the inside of the custom-made contact lens, and then apply the lens onto the rat eye.
7. Place the animal on a platform adjusted to the height of the slit lamp. Set the animal's head at a 45° angle so that the eye is perpendicular to the slit lamp and laser beam.
8. Visualize the eye through the ophthalmic slit lamp. Make sure the aiming beam is the right size as well as focused and centered directly on the visualized optic nerve. Photograph the retinal fundus using a digital camera with high ASA (1,200 - 2,000) speed mounted on one of the eye pieces of the slit lamp with a custom made adapter.
   NOTE: The ability to see the choroidal vessels reveals the transparency of the retina. This is an important sign in order to be able to detect later retinal ischemia, which can confound the interpretation if it occurs. rNAION is optic nerve ischemia, which results in isolated RGC loss, while retinal ischemia results in retinal damage affecting all of the cells of the inner retinal layers.
9. Optionally, further image the retina and optic nerve using a spectral domain-optical coherence tomograph (SD-OCT⁸) to evaluate the un-induced eye. Scan en face (Figure 3B) as well as 7 cross-sectional scans through the retina (Figure 3C). The SD-OCT imaging uses the same contact lens used for laser induction.
10. Inject 1 ml/kg RB intravenously through tail vein, and wait for 30 sec, then activate the laser power. This time delay enables the RB to distribute evenly throughout the circulation. We use a 50 mW laser pulse at one sec/pulse.  Greater energies (≥ 60 mW) can damage the retina or cause retinal vascular ischemia.
    Caution: Make sure everyone has a pair of laser safety filtering glasses of the appropriate blocking wavelength to prevent stray laser light entering investigator's eye.
    NOTE: The laser administration must be given rapidly after the IV RB injection since the dye is eliminated quickly from the circulation. The longer the laser induction, the more severe the optic nerve ischemia. Generally, animals are given 7 - 12 sec pulses in a rapid succession. The contact of the laser light with the circulating dye gives the optic nerve vessels a beautiful golden glow that can be seen through the slit lamp (Figure 4B). This proves the dye was injected systemically and distributed into the blood stream. If the glow is faint, or none at all (Figure 4A), the dye was not injected intravenously. In this case, do not give a second injection immediately, since the animal will need to recover for at least two days before reinjection.
11. Immediately after induction, remove the contact lens. Cover both eyes with ophthalmic triple antibiotic (Neosporin/polymyxin/bacitracin) ointment with dexamethasone, and place the animal on a 37 °C warming pad in a single housed cage under close observation until full recovery.
    1. Clean the contact lens with distilled water and wipe dry with a non-abrasive cleaning cloth for future use.
    2. Two days after the induction evaluate the optic nerve edema by both fundus color photography and SD-OCT analysis⁸.
       NOTE: Comparing the degree of optic nerve edema gives an estimate of the severity of the optic nerve ischemia. At two days, the optic nerve disk margin is blurred, and the retinal veins are slightly dilated, compared with the contralateral (un-induced) eye. Perform electroretinography (ERG) and flash visual evoked potential (VEP¹¹) at two and four weeks post induction for electrophysiology analysis.

Results

The contact lens enabled central retina visualization (Figure 1). The focal laser spot illuminates the optic disc at the back of the retina (Figure 2). The normal un-induced retina is shown imaged by slit lamp bio-microscope (Figure 3A) and by SD-OCT (Figure 3B and 3C). During laser induction, when no dye is present in the circulation, laser light does not result in vessel and disk fluorescence (Figure 4A...

Discussion

While there are a number of models of optic nerve damage (optic nerve crush¹², optic nerve transection¹³, and PION¹⁴), the rNAION model is humane, adaptable to both rats and mice. It more closely resembles the human clinical condition of NAION. This condition includes progressive anterior optic nerve edema, an anterior optic nerve compartment syndrome, focal axonal ischemia, isolated retinal ganglion cell axonal damage and loss over a prolonged time course. The current report gives the ap...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mW 532 nm laser	Iridex		Standard Ophthalmic Laser
0 - 100 mW 532 nm laser	Laserglow technologies		Substitute for iridex
Laser slit lamp adapter	Iridex		SMA coupled adapter for laser output
Coherent Fieldmate laser meter with thermopile sensor	Coherent		others also appropriate
Ophthalmic Examing Slit lamp biomicroscope	Various		Haag-Streit is the best; cheaper versions available on ebay
Rose Bengal	Sigma	330000-1G	Photoinducing agent
Fundus Contact lens or glass cover slip	custom/Cantor and Nissel (UK)		Custom designed planoconvex plastic lens for eye exam and induction
Tropicamide 1%
Tamiya polishing compound	Tamiya, INC	87068	polishing contact lens
2.5% Hypromellose (Goniovisc)/1% Methycellulose	HUB Pharmaceuticals		contact lens coupling agent
2.5% Neosynephrine Ophthalmic drops	Alcon labs		pupil dilating agent
Tropicamide 1%	Alcon labs		pupil dilating agent
0.5% Proparacaine	Alcon labs		topical Anesthetic
30 G fused needle insulin syringe	Various	Various	for intravenous injection of rose bengal
Ophthamic Antibiotic ointment with dexamethasone added (Triple antibiotic ointment)	Various	Various	Apply after induciton to minimize corneal scarring
Heidelberg Corporation Spectral domain-Optical Coherence Tomograph	Heidelberg Corporation		For Optical coherence measurements baseline and post-induction; not essential for induction