The overall goal of this procedure is to provide for a minimally invasive approach to inject drugs or stem cells in the optic nerve of a live rat. This is accomplished by first anesthetizing the rat, positioning it on the dissection stand, and inserting a suture into the conjunctiva for manipulation of the eye. Later on, the second step is to dissect down the orbital ridge behind the eye to expose the optic nerve.
Next, the glass pipette is inserted into the optic nerve, and the drug or stem cells are injected before closing of the surgical site and reviving the rat from anesthesia. Ultimately, after a certain period of time, the rat is sacrificed, the optic nerve dissected and the effects of the surgical injection or transplantation can be visualized by microscopy. The main advantage of this technique over existing methods is that this approach is minimally invasive, and that allows the rat to survive as long as you need.
Study the effects of surgical injection or transplantation. Demonstrating the procedure will be Kate Schwartz, a student technician from my laboratory. Perform all surgical procedures under anesthesia with two to 3%isof fluorine.
Confirm appropriate level of anesthesia of by toe pinch and breathing rate. Check that the rat does not flinch in response to a toe.Pinch. Secure the animal as necessary to avoid head movement during the procedure.
Then place a drop of lidocaine on the surgical eye. Use artificial tears every 10 minutes to prevent dryness while under anesthesia. Give a preoperative injection of buprenorphine at 0.01 milligrams per kilogram subcutaneously, and then every six to eight hours as needed.
Keep the animal warm with a heating pad. Wet the scalp fur with alcohol. Taking care to avoid exposure on the eyes.
Be sure to use sterile tools and sterile technique to minimize risk of postoperative infections. Next place a four oh suture in the lateral conjunctiva and tie it with enough suture to permit gentle traction. Make an approximate one inch incision in the skin overlying the orbital ridge.
Using a size 10 scalpel. Retract the skin and the underlying fascia and carefully dissect away the fascia. Place one suture in the retracted skin to allow exposure of the superior orbital sac to prevent excessive bleeding into the surgical site.
Avoid cutting blood vessels while dissecting the fascia. With gentle traction on the conjunctiva, pulling the eye down and out of the socket, the superior orbital muscle will come into view. In order to expose the optic nerve, the retroorbital fat must be removed, and this muscle cut.
The fat can be discarded and should not be replaced after injection. From this point, the optic nerve fascia should be visible as a bundle of the optic nerve itself, along with blood vessels wrapped in dura. To make the deeper dissection, make a small incision in the DRA using the scalpel or a 31 gauge beveled needle to make piercing less traumatic, have prepared a pulled glass micro pipette with a diameter of 50 to 100 microns to provide stability.
Mount the micro pipette on a micro manipulator and attach to a 20 cc plastic syringe. Pull up beads or stem cells reconstituted in 0.5 to one microliters into the micropipet retrograde, along with 0.5 microliters of methyl blue solution before and after the glass pipette is loaded with dye. Using a one cc syringe and a 30 gauge needle, lower the tip of the micro pipette onto the optic nerve just above the nicked dura.
A tiny but brisk movement of the glass tip into the optic nerve results in the least damage while applying slow and constant pressure on the plunger. The Evans blue dye should highlight the area of the injected optic nerve. The dye should remain localized within the optic nerve without leaking out in the subarachnoid space.
Follow the second band of Evan's Blue Dye to determine when the stem cell injection is complete. When the necessary volume is injected into the optic nerve, let go of the plunger. Keep the micro pipette still within the optic nerve for five minutes to prevent high pressure ejection upon withdrawal of the micro pipette when the injection is complete.
Remove the micro pipette along with any cotton tips used to provide hemostasis. Suture the skin with 3.0 silk and remove the conjunctival suture. Keep rats warm on a heating pad until they emerge from anesthesia.
Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbent. If the animal is exhibiting signs of pain, including excessive lethargy, ataxia, or labored breathing, administer appropriate analgesia with intramuscular buprenorphine up to three times daily. After the experiment, rats were sacrificed and perfused with paraldehyde.
The optic nerves were carefully dissected and mounted for cryostat sectioning shown here is an example of a rat hole optic nerve at lower power in which Evan's blue dye was injected. In order to visualize the site, the arrow identifies the precise location of the injection. This dissection was done within a few days of the injection as indicated by the restricted diffusion of the dye down the nerve After its development.
This technique paved the way for researchers in the field of regenerative neurology to explore new ways to regenerate the optic nerve and potentially restore lost vision in this rat model of demyelinating disease.
