C. Linn is supported by an NIH grant (NIH NEI EY022795).

All procedures using animal subjects have been in accordance with the standards of the Institute of Animal Care and Use Committee (IACUC) at Western Michigan University.
1. Animals
<ol>
	<li>Use male and female rats 3 months of age in this study.</li>
	<li>Keep animals in a 12 hr light/dark cycle with free access to food and water.</li>
</ol>
2. Preparation of KAX Cocktail for Animal Anesthesia
<ol>
	<li>Dissolve 50 mg of xylazine (20 mg/ml) in 5 ml ketamine (100 mg...

The authors have no conflicting or competing interests to disclose.

This protocol describes a method of inducing glaucoma-like conditions in an in vivo rat model. This procedure uses an injection of hypertonic saline to induce scarring in the trabecular meshwork 29, 32. Developing scar tissue occludes the outflow of aqueous humor which increases pressure in the anterior chamber. With decreased outflow and pressure build up, the lens suspended by elastic ligaments pushes back into the vitreous chamber. Vitreous humor then applies pressure onto the retina damaging the f...

Glaucoma is a group of eye diseases affecting neurons in the retina, specifically, the retinal ganglion cells1-2. The axons of these cells converge to become the optic nerve carrying visual information to the brain where vision is perceived. Damage to RGCs and their axons therefore causes visual defects.
The primary characteristics associated with glaucoma disorders are RGC degeneration and death, increased intraocular pressure (IOP), and optic disk cupping and atrophy. These features lead to visual field loss or complete, irreversible blindness. Currently, glaucoma has caused blindness in 70 million people worldwide 3. As such, it is the world&#39;s third largest cause of blindness 4.
The exact mechanism of RGC death in glaucoma remains unknown. Much research has been done to unlock the mystery. It is known, however, that the primary risk factor of glaucoma is an increase in intraocular pressure due to irregular circulation of aqueous humor (AH) in the anterior chamber of the eye. AH acts as a transparent and colorless replacement for blood in the avascular anterior chamber of the eye. It nourishes the surrounding cells, removes secreted waste products from metabolic processes, transports neurotransmitters, and permits the circulation of drugs and inflammatory cells within the eye during pathological states 1.
The maintenance of aqueous humor circulation involves the ciliary body and the trabecular meshwork. Aqueous humor is produced by the ciliary body. It then flows into the anterior chamber to maintain the overall health of the ocular tissue. 75 - 80% of aqueous humor outflow is actively secreted through non-pigmentary ciliary epithelium when the fluid is filtered through three layers of spongy tissue in the ciliary muscle. The fluid exits through the trabecular meshwork and through Schlemm&#39;s Canal which empties into the blood system 5.The remaining 20 - 25% of outflow bypasses the trabecular meshwork and is passively secreted by ultrafiltration and diffusion through the uveo-scleral pathway. This pathway appears to be relatively independent of intraocular pressure 1.
When aqueous humor production and outflow are out of balance, pressure builds within the eye. As stated, this increase in intraocular pressure is the primary risk factor in the development of glaucoma. Such pressure causes damage to the intricate layers of neurons in the retina at the back of the eye. Damage to the retinal ganglion cell axons of the optic nerve causes the brain to no longer receive accurate visual information. As a result, the perception of vision is lost and complete blindness can occur.
To date, there is no cure for glaucoma. Different treatment methods exist that primarily aim to reduce intraocular pressure. These include topical medication classes such as beta1-adrenergic receptor blockers, or topical prostaglandin analogues. Beta blockers reduce the intraocular pressure by decreasing the production of aqueous humor 7. Prostaglandins function to reduce IOP by increasing the outflow of aqueous humor 8-14. Alpha adrenergic agonists and carbonic anhydrase inhibitors are also used as secondary methods of treatment. Alpha adrenergic agonists increase outflow through the uveoscleral pathway 15-17. Carbonic anhydrase inhibitors reduce the production of AH by enzymatic inhibition 18. Much more invasive procedures are also being used to treat glaucoma. Laser trabeculoplasty is used to increase the outflow of aqueous humor 19. Another surgical therapy, called trabeculectomy, creates an alternative drainage site to filter AH when the traditional trabecular pathway is blocked 20-21.
These treatment options have been known to effectively reduce IOP. However, up to 40% of glaucoma patients show normal IOP levels indicating a need for more complete therapeutic methods.22,23 Additionally, retinal ganglion cell death seen in glaucoma is irreversible once it begins and current treatments do not stop the progression of the disease 24-28. This has highlighted the need for effective neuroprotective therapies that target the survival of the neurons themselves. Development of glaucoma models is crucial for this development.
In this study we are demonstrating a method of inducing glaucoma-like effects in adult Long Evans rats using a modified procedure originally outlined by Morrison29. In this procedure, injections of 2 M hypertonic saline into the episcleral venous plexus induces glaucoma-like conditions by scarring tissue to reduce aqueous humor outflow in the trabecular meshwork leading to an increase in intraocular pressure and a significant loss of RGCs within one month of the procedure 30-31. Glaucoma-inducing procedures, such as the one described here, may be the key to unlocking new developments in glaucoma treatments.

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			<td height="20" style="height:21px;width:299px;">Xylazine hydrochloride, Minimum 99%</td>
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			<td>S7653</td>
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			<td height="20" style="height:21px;">Heavy Polished Standard and Thin Walled Borosilicate Tubing&#160;</td>
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			<td>without filament, 0.86 mm</td>
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			<td height="20" style="height:21px;">Microfil syringe needle for filling micropipettes</td>
			<td>World Precision Instruments, Inc</td>
			<td>MF28G</td>
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			<td height="20" style="height:21px;">18 gauge Luer-Lock needle</td>
			<td>Fisher Scientific</td>
			<td>1130421</td>
			<td>Syringe needle</td>
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			<td height="20" style="height:21px;">Flexible Polyethylene Tubing</td>
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			<td>22046941</td>
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			<td>NDC 17478-263-12</td>
			<td>15 mL&#160; sterile bottle&#160;</td>
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			<td>14061-11</td>
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			<td height="20" style="height:21px;">Hemostat Clamp&#160;</td>
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			<td height="20" style="height:21px;">Triple Antibiotic Ointment&#160;</td>
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			<td>NC0664481</td>
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			<td>Fine Science Tools&#160;</td>
			<td>10004-13</td>
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			<td height="20" style="height:21px;">Scalpel blade # 11</td>
			<td>Fine Science Tools&#160;</td>
			<td>10011-00</td>
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			<td height="20" style="height:21px;">60 mm x 15 mm Disposable Petri Dish</td>
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			<td>351007</td>
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			<td height="20" style="height:21px;">Phosphate Buffered Saline 10x Concentrate</td>
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			<td>11251-30</td>
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			<td>BD Biosciences</td>
			<td>357524 or 52947-948</td>
			<td>1 and 2 mL graduations</td>
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			<td height="20" style="height:21px;">35 mm x 10 mm Easy Grip Petri Dish&#160;</td>
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			<td>351008</td>
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			<td>N/A</td>
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			<td>Cat 554892</td>
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			<td height="20" style="height:21px;">Alexa Fluor 594 goat anti-mouse&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>A11005</td>
			<td>1:300 dilution&#160;</td>
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			<td>Corning&#160;</td>
			<td>2948-75x25</td>
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			<td height="20" style="height:21px;">Glycerol&#160;</td>
			<td>Sigma&#160;</td>
			<td>G5516-100 mL&#160;</td>
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			<td height="20" style="height:21px;">Coverglass&#160;</td>
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			<td>Nikon&#160;</td>
			<td>C2 Eclipse Ti</td>
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glaucoma-inducing procedure in an in vivo rat model and whole-mount retina preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

This section illustrates the apparatus components and procedure used to induce glaucoma-like conditions in an in vivo rat glaucoma model. We show the individual tools and equipment used to perform a hypertonic saline injection which causes an increase in intraocular pressure. We show the injection into the episcleral venous plexus with its characteristic blanching effect and the cloudy appearance of the anterior chamber that results. We also describe the process of retina removal...

Watch this Scientific Journal Video about Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation at JoVE.com

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

The overall goal of this procedure is to create significant loss of retinal ganglion cells in the rat retina by inducing glaucoma-like conditions. This method can help answer key questions in the fields of glaucoma and neural protection. The main advantage of this technique is that it causes a gradual loss of retinal ganglion cells, similar to that seen in human glaucoma.To begin this procedure, pull two finely-tapered glass microneedles from a heavy polished standard thin-walled Borosilicate Tube with a microelectrode puller. Then, backfill one microneedle with two molar saline, using a one milliliter syringe. Tap out the air bubbles from the tip of the electrode.Next, fill a second one milliliter syringe with two molar NaCl. Connect it to an 18 gauge needle, and then attach the needle to 10-inch polyethylene tubing. Use the syringe plunger to fill the polyethylene tubing with saline through the needle.When both the microneedle and tubing are filled with saline, carefully connect the two. Eliminate any air in the connection by tapping out the air bubbles. Then, scrape the microneedle tip lightly against a paper towel to finely bevel it.Check the resistance of the microneedle by gently pushing the plunger on the syringe until a fine stream of liquid can be seen on the paper towel. The stream of liquid should be no wider than 0.5 millimeters to ensure the appropriate integrity of the needle tip. In this procedure, after anesthetizing the animal with KAX cocktail, check the reflexes by giving pinches to its feet or tail to ensure the animal is unconscious.Then, trim the whiskers with scissors. Subsequently, saturate a cotton tip applicator with betadine solution and swab the area around the experimental eye. Under the microscope, use a hemostat to clamp the bottom eyelid, in order to bulge the eye, expose the episcleral vein, and restrict the eye movement.Next, carefully pierce the episcleral vein with the microneedle at an angle between 10 and 20 degrees to the vein. A puncture into the vein is successful when the blood is observed to enter the tip of the microneedle. Slowly inject 50 microliters of saline into the vein.The veins with blanch white as the salt circulates through the vasculature, but some regions may maintain a blood-red appearance. The single most critical step in this procedure occurs during the injection of the two molar saline. It is imperative that blanching in the vasculature occurs to ensure a successful outcome.Maintaining a steady needle and a 20 degree angle are crucial for this procedure. After that, perform a second injection into the vein to ensure thorough damage of the retinal ganglion cell layer. Within minutes, one should see a distinct cloudy appearance through the iris of the eye, as the salt circulates through the vascular system.Now, use a scalpel to cut the connective tissue in the orbital cavity surrounding the eye, being careful not to cut into the eyeball itself. Then, carefully cut the optic nerve and any remaining tissue to extract the intact eyeball with the curved-edge scissors. Place the extracted eyeball in a sterile petri dish containing fresh PBS.Next, make an eye cup from the eyeball by making a small incision with the scalpel just posterior to the border between the iris and the sclera. Continue the incision around the eye circumference to remove the corneal hemisphere from the eyeball. Holding the pigmented layer of the retina with forceps to stabilize the eye cup, use another pair of forceps to gently tease the pinkish-beige whole intact retina off from the back of the eye.Subsequently, cut or pinch off the area where the optic nerve is attached to the retina. Be sure to cut away any residual pigment epithelium or scleral tissue from the retina. Then, gently transfer the isolated retina to a clean Sylgard-coated petri dish containing fresh PBS.Once in the Sylgard dish, pin the retina in place. Keep the retinal ganglion cell layer facing up, and the optic nerve down. Next, cut the retina into four quadrants, making the shape of a four-leaf clover, radiating from the optic nerve head.Then, pin the quadrants of the retina with additional cactus needles to make the retina as flat as possible without stretching. Afterward, fix the pinned retina in the Sylgard dish with three milliliters of 4%paraformaldehyde over night at room temperature before conducting the standard staining procedures. Here is a comparison of the control untreated eye and the experimental eye, one month after receiving glaucoma-inducing saline injection.The retinal ganglion cells are labeled with an antibody against Thy 1.1. The procedure leads to a reduction in the number of RGCs, defasciculation of the axons off the main axon bundles, and distortion to the circularity of the remaining RGCs. These are the characteristic defasciculating axons resulting from the saline injection.A blood vessel is shown within the retina, and these are the axon bundles. Once mastered, this technique can be done in 30 minutes, if it is performed properly. While attempting this procedure, it's important to remember to always use a fresh, sharp, beveled microneedle.Following this procedure, other methods like immunocytochemistry, Western Blots, in situ hybridization, mRNA sequencing, or behavioral analysis can be performed in order to answer additional questions associated with glaucoma, such as how the loss of retinal ganglion cells affects animals'visual acuity. After its development, this technique paved the way for researchers in the field of glaucoma to explore neural protective strategies in rats. After watching this video, you should have a good understanding of how to induce a significant loss of retinal ganglion cells in the rat, using a hypertonic saline injection to the episcleral vein.

glaucoma-inducing-procedure-an-vivo-rat-model-whole-mount-retina

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Medicine

12.9K Views.  Western Michigan University. The overall goal of this procedure is to create significant loss of retinal ganglion cells in the rat retina by inducing glaucoma-like conditions. This method can help answer key questions in the fields of glaucoma and neural protection. The main advantage of this technique is that it causes a gradual loss of retinal ganglion cells, similar to that seen in human glaucoma.To begin this procedure, pull two finely-tapered glass microneedles from a heavy polished standard thin-walled Boros...