The overall goal of this procedure is to create experimental retinal detachments with a reproducible and sustained height of detachment without subretinal hemorrhage. This is accomplished by first inci, the temporal conjunctiva at the posterior limbus to separate the conjunctiva from the sclera. In the second step, the tip of a 30 gauge needle is used to make a self-sealing scleral incision and corneal puncture.
Next sodium urinate is injected into the subretinal space to detach the neurosensory retina from the underlying retinal pigment epithelium. In the final step, cyanoacrylate glue is placed on the scleral wound and the conjunctiva is reattached to its original position, reducing the risk of sodium hall urinate leakage. Ultimately, the fundus is examined using a cover glass to confirm the creation of a bolus retinal detachment without subretinal hemorrhage.
Existing methods for reducing retinal detachment in rodent can result in variability in height and persistence of retinal detachment leading to inconsistent cell death rates being measured. With this technique, a reproducible bullous and persistent retinal detachments can be created with a reduced risk of subretinal hemorrhoids, allowing for a reliable model of retinal detachment in rodents. Experimental retinal detachment in rodents is relatively simple to perform, has a reasonable time course, and is quite similar to the human disease.
So it's a great model for studying mechanisms of photoreceptor cell death and therapies for preventing vision loss. In retinal degenerative disorders In know that the variate photoreceptor cell death in the detached retina, it is advantageous to create reproducible detachment without subretinal hemorrhage. This is because the heightened duration of retinal detachment or the occurrence of subretinal hemorrhage can affect photoreceptor cell death.
After anesthetizing an eight week old mouse trim the animal's whiskers, then dilate the pupil with a solution containing 5%phenyl rine, and 0.5%tropic under a surgical microscope. Cut the animal clia, apply topical anesthesia, 0.5%propane hydrochloride ophthalmic solution. Place the mouse in a lateral position with the nose toward the surgeon, and then inci the temporal conjunctiva at the posterior limbus, separating the conjunctiva from the sclera using a pair of forceps.
Grasp the conjunctiva at the limbus to control the eye. Then holding a 30 gauge needle with a bevel pointed up. Use the tip of the needle to make a tunnel through the sclera, penetrating the sclera into the choroid without penetrating the retina.
Next, holding the 30 gauge needle parallel to the iris, puncture the cornea to reduce the intraocular pressure. Now pointing the bevel down. Insert a 33 gauge needle connected to a Hamilton 10 microliter syringe into the subretinal space, and inject 3.5 microliters of sodium urinate.
This will detach approximately 50%of the neurosensory retina from the underlying retinal pigment epithelium. Using forceps, push the cornea around the corneal puncture to let the aqueous humor flow out of the corneal puncture, and then use a surgical cellulose spear to confirm the absence of leakage from the scleral wound to reduce the risk of sodium urinate leakage. Place cyanoacrylate glue on the scleral wound and then rea attach the conjunctiva to its original position.
Use a cover glass to check the fundus and to confirm the creation of a bolus retinal detachment without subretinal hemorrhage. Finally, apply antibiotic ointment to the eye to reduce the risk of infection. Keep the mouse on a heating pad to prevent low body temperature.
Subsequent to low blood pressure. Return the animal back to its cage after it awakens from anesthesia. To assess the persistence of the retinal detachment made by this protocol, cryo sections were made on days three, seven, and 14 after induction of the detachment.
Six eyes were used for each time point, hematin and eosin. Staining was used to visualize the sections which each showed a bolus. Retinal detachment approaching the lens.
No eyes showed any signs of infection or lens injury. Once mastered, this surgery can be performed in about five minutes While attempting this procedure, it's important to perform each step gently to avoid causing leakage of sodium, high illuminate subretinal hemorrhage, or lens injury, which can all affect what receptor cell death in the detached retina. After watching this video, you should have a good understanding of how to create an experimental retinal detachment in rodents with a reproducible and sustained height of detachment without subretinal hemorrhage.
