a hypertonic saline injection technique for inducing glaucoma in rats

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

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 micro-needle 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 micro-needle.
Slowly inject 50 microliters of saline into the vein. The veins will 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 in 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.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 (Ketamine + Xylazine + Acepromazine) Cocktail for Animal Anesthesia
<ol>
	<li>Dissolve 50 mg of xylazine (20 mg/ml) in 5 ml ketamine (100 mg/ml) with 1 ml acepromazine (10 mg/ml) and 3 ml of distilled water. Mix thoroughly.</li>
	<li>Sterilize with a syringe filter and store this solution into a 10 ml serum bottle.</li>
</ol>
3. KAX Injection
<ol>
	<li>Weigh animal (g) and return to cage until ready for injection.</li>
	<li>Inject 0.1 ml&#160;KAX/100 g animal body weight intraperitoneally, using a 1 ml insulin syringe with a 28 G&#160;needle.</li>
	<li>Allow for animal to become unconscious. Check reflexes by pinching the feet and tail.</li>
	<li>Keep all animals safely in the lab for the duration of surgery.</li>
	<li>Post-surgery, replace animals to their cages and keep comfortable in room temperature (RT)&#160;until consciousness is regained. Only return animals to the animal facility when the animals awaken and resume normal behavior.</li>
</ol>
4. Preparation for Surgery and Microneedle Assembly
<ol>
	<li>Make a sterile 2 M NaCl solution.</li>
	<li>Use a microelectrode puller (Figure 1C) to pull one 0.86 mm inner diameter heavy polished standard and thin-walled borosilicate tube into two finely tapered glass microneedles (Figure 1D, Figure 1E).</li>
	<li>Backfill one microneedle from the previous step with 2 M saline using a backfilling syringe needle and a 1 ml syringe (Figure 1B). Tap out air bubbles from the tip of the electrode.</li>
	<li>Fill a second 1 ml syringe with 2 M NaCl. Connect an 18 G&#160;needle and then attach a length (approximately 10 inches) of polyethylene tubing (Figure 1A). Use the syringe plunger to fill the polyethylene tubing with saline through the needle.</li>
	<li>When both the microneedle and tubing are filled with saline, carefully connect the two. Eliminate any air in the connection between them (Figure 2).</li>
	<li>Finely bevel the tip of the microneedle by scraping it very lightly against the grain of a course paper towel.</li>
	<li>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 mm.</li>
</ol>
5. Preparation of Animal
<ol>
	<li>Apply 1 - 2 drops topical anesthetic to cornea (Proparacaine Hydrochloride Ophthalmic Solution, USP, 0.5%). Wait until no ocular reflex occurs.</li>
	<li>Trim whiskers with scissors.</li>
	<li>Saturate a cotton tip applicator with betadine solution and swab area around the experimental eye.</li>
	<li>Using a microscope, attach a hemostat to clamp the bottom eyelid to bulge the eye, expose the episcleral vein and restrict eye movement. (Figure 3, arrowhead)</li>
</ol>
6. Glaucoma-inducing Saline Injection
<ol>
	<li>When the microneedle assembly and the animal are prepared, begin injections.</li>
	<li>When the animal is confirmed to be unresponsive to feet/tail pinch, carefully pierce the episcleral vein with the microneedle by coming at a low angle between 10 and 20 degrees to the vein (Figure 3, white arrow). A successful puncture into the vein is apparent when blood enters the tip of the microneedle (Figure 3, black arrow).</li>
	<li>Slowly and manually inject approximately 50 &#181;l saline into the vein. This should take approximately 10 sec. The veins will blanch white as the salt circulates through the vasculature (Figure 4, arrowhead). Some regions may maintain a blood red appearance (Figure 4, arrow).
	<ol>
		<li>Perform a second injection into the vein, opposite to the site of the first, to ensure thorough retinal damage to the complete retinal ganglion cell layer. 
		Note: Within minutes, one should see a distinct cloudy appearance through the iris of the eye as the salt circulates through the vascular system.</li>
	</ol>
	</li>
	<li>Leave the opposite eye untreated for use as an internal control.</li>
</ol>

Source: Gossman, C. A. et al. <a href="https://app.jove.com/v/53831">Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation.</a> J. Vis. Exp. (2016)
This video demonstrates glaucoma induction in an anesthetized rat by injecting hypertonic saline into the episcleral vein. The saline enters the trabecular meshwork via Schlemm&#39;s canal, causing tissue scarring, which blocks aqueous humor outflow and raises intraocular pressure. The elevated pressure damages retinal ganglion cells, leading to glaucoma.

<img alt="Figure 1" src="/files/ftp_upload/22971/22971_Figure1.jpg" /> 
Figure 1. Microneedle Components. (A)&#160;Syringe with polyethylene tubing used for saline injection.&#160;(B)&#160;Backfill syringe used to backfill the borosilicate microneedle.&#160;(C)&#160;Narishige electrode puller used to make borosilicate microneedles.&#160;(D)&#160;Borosilicate glass electrodes before and after being pulled in the elect...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take an anesthetized rat and apply an anesthetic to its eye to numb reflexes.
Trim whiskers and disinfect the area around the eye to prevent contamination.
Under a microscope, clamp the lower eyelid to bulge the eye, exposing the episcleral vein.
This vein connects to the anterior chamber via Schlemm&#39;s canal and trabecular meshwork for the outflow of aqueous humor.
Insert a microneedle with hypertonic saline into the exposed vein.
Inject the saline, displacing blood. Perform a second injection opposite the first for a uniform effect across the eye.
The saline enters the trabecular meshwork.
The high salt concentration causes osmotic stress, leading to cell death and tissue scarring. This scarring blocks aqueous humor outflow through the meshwork, increasing intraocular pressure.
The elevated pressure damages the retinal ganglion cells, which are neurons carrying visual information to the brain. The damage leads to glaucoma, a condition that causes vision loss.

12 Views. A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

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