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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.