The work contained in this paper has been supported by the Dr. Douglas H. Johnson Award for Glaucoma Research from the American Health Assistance Foundation (XL), the William &amp; Mary Greve Special Scholar Award from the Research to Prevent Blindness (XL), the Illinois Society for the Prevention of Blindness (HC) and NIH grant R01EY019034 (XL).

Procedures
C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) are raised at Northwestern University's Animal Care Facility. All animals are used in accordance with protocols approved by Northwestern University Institutional Animal Care and Use Committee and conformed to the guidelines on the Use of Animals in Neuroscience Research from the NIH.
1. Laser Photocoagulation
The procedure of laser photocoagulation is modified from previously published protocols 5-7.
<ol>
	<li>Anesthetize a 40-60 day old mouse by an intraperitoneal injection of ketamine (100 mg/kg, Butler Schein Animal Health, OH) and xylazine (10 mg/kg, Lloyd Inc. of Iowa, Shenandoah, IA).</li>
	<li>Dilate the pupil of the right eye of the experimental animal by topical treatment with one or two drops of 1% atropine sulfate solution (Alcon Labs, Inc., Fort Worth, TX).</li>
	<li>After mydriasis, flatten the anterior chamber to enhance laser induction 6. Insert a glass micropipette with sharp tip (World Precision Instruments Inc, Sarasota, FL) into the anterior space under the slit lamp (SL-3E, Topcon, Oakland, NJ) to drain out the fluid in the anterior chamber.</li>
	<li>Restrain the mouse in a plastic cone holder (Braintree Sci Inc., MA) and tied up on a homemade platform (See Figure 1A). Hold the mouse with restrainer and exposes the right eye of the mouse to the light source behind the slit lamp. Align the right eye of the anesthetized mouse under the slit lamp.</li>
	<li>While holding the mouse restrainer with both hands, apply the laser illumination to the corneal limbus using an Argon laser (Ultima 2000SE, Coherent, Santa Clara, CA). Deliver about 80-100 laser spots (514 nm, 100 mW, 50 msec pulse, and 200 &mu;m spot) perpendicularly around the circumference of the trabecular meshwork. The C57BL/6 mice have pigmented iris which serves as a barrier for any potential stray energy 7.</li>
	<li>Instill topical 0.5% moxifloxacin (Alcon Labs, Inc., Fort Worth, TX) on the ocular surface to disinfect the laser-treated area and 0.5% Proparacaine (Bausch &amp; Lomb, Rochester, NY) to relieve pain.</li>
	<li>Keep the animal on a heating pad (Sunbeam Products Inc, Boca Raton, FL) for recovery for about an hour until it is fully awake.</li>
	<li>The left eye is untreated to serve as a control.</li>
</ol>
2. IOP Measurements
<ol>
	<li>Place the awake mouse into a tube to load into the plastic cone holder and then restrain it on the platform (See Figure 2A).</li>
	<li>Allow five to ten minutes to let the mouse get adapted to the holder position. Approach the rebound tonometer (TonoLab, Colonial Medical Supply, Franconia, NH) to the mouse eye until the probe tip is 2-3 mm away from the surface of cornea 14.</li>
	<li>Press the measurement button to let the probe tip hit the center surface of cornea gently. Three consecutive sets of six-measurements of IOP of the same eye are acquired and averaged as the IOP of the eye. The untreated control eye is always measured first to get a baseline reading for the laser-treated eye that is measured next.</li>
</ol>
3. Optomotor Test
Visual acuity and contrast sensitivity are tested 14,15 . The two eyes of individual mice are examined separately by reversing the drifting grating direction; i.e. a clockwise drifting grating is used to identify the visual function of the left eye and a counter-clockwise drifting grating for the right eye 16. Each test takes about 15 min and is repeated by two observers independently.
<ol>
	<li>Place the mouse and allow the mouse to move freely on an elevated platform surrounded by four computer monitors (Figure 3A-B).</li>
	<li>Set up the monitors so that they display horizontally drifting sinusoidal gratings as visual stimuli with mean luminance of 39 cd/m2. The moving direction of the grating should alternate consecutively between clockwise and counterclockwise.</li>
	<li>Analyze the animal's movements. The animal's movements in-concert with the drifting gratings are considered "positive" within 15 sec after the visual stimulus is on and then gradually increased. The highest response-eliciting visual stimulus is defined as the animal's visual acuity 17.</li>
	<li>Examine the contrast sensitivity at three pre-selected spatial frequencies: 0.075, 0.16, and 0.3 cycles per degree (cpd). The contrast threshold for each eye is defined as the lowest contrast that elicits visual responses at the pre-fixed frequency. The contrast sensitivity is the reciprocal of the threshold 17.</li>
</ol>

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The authors declare that they have no competing financial interests.
	The authors are full-time employees of Northwestern University.
	The authors received NO funding that was provided by companies which produce reagents and instruments used in this article.

We report above that sustained ocular hypertension can be induced by laser illumination in mouse eyes. Compared to the saline injection model 18 and the vein cautery model 11 both of which require extensive microsurgical skills, the laser illumination is relatively simple and easy to perform. Usually we can perform the laser illumination for 4-6 mice in 2-3 hr. The critical steps to achieve sustained IOP elevation are the anterior chamber flattening before laser and the parameters for laser illumina...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>moxifloxacin</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 0065-4013-03</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Proparacaine Hydrochloride</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Ophthalmic Solution USP</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>.5 %, Rx only</td>
 </tr>
 <tr>
 <td>ketamine</td>
 <td>Butler Schein Animal Health</td>
 <td>NDC 11695-0550-1</td>
 <td>100 mg / kg </td>
 </tr>
 <tr>
 <td>xylazine</td>
 <td>LLOYD Inc. of Iowa</td>
 <td>NADA 139-236</td>
 <td>10 mg / kg </td>
 </tr>
 <tr>
 <td>atropine sulfate solution</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 61314-303-02</td>
 <td>1 %, Rx only</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Slit Lamp, TOPCON </td>
 <td>Visual Systems Inc</td>
 <td>SL-3E</td>
 <td>powered by PS-30A</td>
 </tr>
 <tr>
 <td>OptoMotry 1.8.0 virtual</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>opto-kinetic testing system</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tonometer, TonoLab, for mice</td>
 <td>Colonial Medical Supply</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>Sunbeam Products Inc</td>
 <td>722-810</td>
 <td></td>
 </tr>
 <tr>
 <td>Argon laser </td>
 <td>Coherent Inc</td>
 <td>Ultima 2000SE</td>
 <td></td>
 </tr>
 <tr>
 <td>DECAPICONE Plastic cone holder </td>
 <td>Braintree Sci Inc.</td>
 <td>MDC-200</td>
 <td>for mouse</td>
 </tr>
 </tbody>
 </table>

a laser-induced mouse model of chronic ocular hypertension to characterize visual defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

As described in the Procedures, laser illumination is aimed at the trabecular meshwork in the limbal region to photocoagulate the aqueous outflow, inducing angle closure (Figure 1). Most lasered eyes exhibited no significant physical damage, pigment detachment or infection, consistent with previous findings 6. When a small group of mice (less than 5% of all lasered animals) exhibited physical signs of severe damage such as deflated eye balls, severe cataract, significant pigment detachment, or...

Watch this Scientific Journal Video about A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects at JoVE.com

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

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12.9K Views.  Northwestern University. Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.