This article details a procedure for inducing glaucoma in anesthetized rats by injecting hypertonic saline into the episcleral vein. The method aims to study the effects of increased intraocular pressure on retinal ganglion cells, which are crucial for visual information transmission.
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'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.
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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