Medicine
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A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice
Chapters
Summary November 15th, 2019
Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.
Transcript
Glaucoma is the leading cause of irreversible blindness, characterized by retinal ganglion cell death and the opto-neurodegeneration. Elevation of intraocular pressure, also called ocular hypertension, is the most recognized risk factor for glaucoma. A simple but effective ocular hypertension model in experimental animals such as mice is important for investigating glaucometers in neurodegeneration and testing treatment for neuroprotection.
Thus, with results, we develop a mouse ocular hypertension model that closely mimics the secondary glaucoma observed in human patients. This model faithfully replicate the secondary glaucoma caused by silicone oil-induced for pupilar blocking. The procedure is simple the IOP elevation is stable, and if the neurodegeneration associated with ocular hypertension is severe in mouse eyes, more importantly, the ocular hypertension is reversible because the silicone oil can be easily removed from the mouse eyes.
Demonstrating the procedure will be Dr.Fang Fang, a post-op fellow from my laboratory. To begin, make a glass micropipette for intracameral silicone oil injection by pulling a glass capillary with a pipette puller. Cut an opening at the tip of the micropipette and sharpen the tip using a micro-grinder beveling machine to make a 35 to 40-degree bevel.
Polish the edges of the bevel and wash it with water to remove all debris. Autoclave the micropipette before use. To prepare the paracentesis needle for the corneal entry, attach a 32 gauge needle to a five-milliliter syringe on a lure lock and further secure it with tape.
Bend the needle bevel tip, face-up, at 30 degrees. To prepare the silicone oil injector, attach and secure a blunt end 18 gauge needle on a 10-milliliter syringe. Then attach a plastic tube with the 18 gauge needle on one end and fill up with silicone oil as needed through the other end.
After anesthetizing a nine to 10 week old C57 black 6J mouse, check for the lack of response to the toe pinch with the lack of movement of the whiskers or the tail. Place the mouse in a lateral position on a surgery platform. Apply one drop of 5%proparacaine hydrochloride to the cornea before the injection to reduce its sensitivity during the procedure.
Use the 32 gauge paracentesis needle to make an entry incision at the super temporal quadrant about 5 millimeters from the limbus. Tunnel through the layers of the cornea for about 3 millimeters before piercing into the anterior chamber, being careful not to touch the lens or iris. Withdraw the needle slowly to release about one to two microliters of aqueous humor from the anterior chamber through the tunnel and wait for about eight minutes to further decrease the intraocular pressure.
Measure the contralateral control eye to determine the pressure. Insert the glass micropipette preloaded with silicone oil through the corneal tunnel into the anterior chamber with the bevel facing down into the iris surface. Push the syringe plunger slowly to inject silicone oil into the anterior chamber until the oil droplet covers most of the iris surface, approximately 2.3 to 2.4 millimeters in diameter.
Leave the micropipette in the anterior chamber for an additional 10 seconds before withdrawing it slowly. Gently push the upper eyelid to close the cornea incision to minimize silicone oil leakage. And then apply antibiotic ointment to the eye surface.
Keep the mouse on the heating pad until fully recovered from anesthesia. To remove silicone oil from the anterior chamber, anesthetize the mouse and then check for the lack of response to the toe pinch to determine the anesthetic strength and the lack of movement of the whiskers or the tail. Place the mouse on a surgery platform and secure it in the lateral position with tape.
Apply one drop of 5%proparacaine hydrochloride to the cornea to reduce its sensitivity. Using the pre-made 32 gauge paracentesis needle, make two incisions in the temporal quadrant of the cornea between approximately two and five o'clock at the edge of the silicone oil droplet. Insert a 33 gauge irrigation needle connected to irrigating solution through one corneal incision at maximum speed.
Insert another 33 gauge drainage needle attached to the syringe without a plunger through the other corneal incision to allow the silicone oil droplet to exit the anterior chamber while irrigating with irrigating solution. Withdraw the drainage needle then the irrigation needle. Use a glass micropipette connected to a Hamilton syringe to inject an air bubble into the anterior chamber to maintain its normal depth and press to close the corneal incision.
Apply antibiotic ointment to both eyes and keep the mouse on the heating recovery pad until fully recovered from the anesthesia. Place the mouse into an induction chamber. Anesthetize it as described in the manuscript and keep its cornea moist by applying artificial tears throughout the procedure.
Wait about 15 minutes to allow the pupil to fully dilate. Measure the IOP of both eyes using a tonometer according to product instructions. Bring the tonometer near the mouse eye, keeping the distance from the tip of the probe to the mouse cornea at about three to four millimeters.
Press the measuring button six times to generate one reading and obtain three machine-generated readings from each eye to acquire the mean IOP. Perform immunohistochemistry of whole-mount retina retinal ganglia cell counting optic nerve semi-thin sections and quantification of surviving axons on the animals sacrificed at eight weeks after silicone oil injection. Intraocular pressure of mouse eyes with silicone oil droplets larger than 1.6 millimeters was measured once a week for eight weeks, after a single silicone oil injection.
The pressure of the eye receiving the oil remained high, mostly double the pressure of the contralateral control eye indicating effective pupil blocking. In another group of mice, silicone oil was flushed from the anterior chamber two weeks after the injection. After waiting a week to allow the cornea to recover, intraocular pressure returned to normal, the same as the control eye.
To determine the effects of silicone oil-induced ocular hypotension on retinal ganglion cells, the surviving somata in the peripheral regions of the retinal whole mounts was quantified by RBPMS staining showing dramatic retinal ganglion cell death after silicone oil injection. The surviving axons in the optic nerve semi-thin cross-sections were quantified by PBD staining eight weeks after the injection. There was a great axon degeneration in silicone oil-induced ocular hypertension.
The animals can be imaged with SLOOCT to determine the change in the retina. The visual function of the animals can be assayed by pattern ERG and OKR, as well. This ocular hypertension model will facilitate the investigation of the high IOP induced glaucometers neurodegeneration.
It can also be used to test promising neuroprotective therapies preclinically.
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