Minimally-invasive Technique for Injection into Rat Optic Nerve

NOTE: All animal procedures were approved by the Johns Hopkins Animal Care and Use Committee. Anesthesia machines require yearly inspection and calibration as necessary.

1. Anesthesia and Positioning

1. Anesthesia.
   1. Perform all surgical procedures under anesthesia with 2-3% isofluorane. Confirm appropriate level of anesthesia by toe pinch and breathing rate. Check that the rat does not flinch in response to a toe pinch.
      NOTE: A flinch indicates anesthesia that is too late and may require longer anesthesia before beginning or a higher isofluorane concentration. A breathing rate less than 1 breath every 2 sec is too slow indicating the anesthesia is too high and may require lightening of the isofluorane concentration.
   2. Secure the animal as necessary to avoid head movement during the procedure. Place a drop of lidocaine on the surgical eye. Use artificial tears every 10 min to prevent dryness while under anesthesia. Give an injection of buprenorphine of 0.01 mg/kg SC pre-operatively and then every 6-8 hr as needed. 
2. Positioning.
   1. Place rats into a stereotaxic frame and keep warm with a heating pad. Wet the scalp fur with alcohol taking care to avoid exposure on the eyes. Use sterile tools and sterile technique to minimize risk of post-operative infections.

2. Eye Control

1. Place a 4-0 suture in the lateral conjunctiva and tie it with enough suture to permit gentle traction.

3. Dissection

1. Initial Dissection.
   1. Make a ~1 inch incision in the skin overlying the orbital ridge using a size 10 scalpel as shown in Figure 1. Retract the skin and the underlying fascia and carefully dissect away the fascia. To prevent excessive bleeding into the surgical, avoid cutting blood vessels while dissecting the fascia. Use strategically placed cotton tips to provide hemostasis.
2. Deeper Dissection.
   NOTE: 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, this muscle must be cut and the retro-orbital fat removed. 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 (Figure 1).
   1. Make a small incision in the dura using the scalpel or a 31 gauge beveled needle to make piercing less traumatic.

4. Pipette Injector

1. Pull a glass micropipette to a diameter of 50-100 µm. To provide stability, mount the micropipette on a micromanipulator and attach to a Hamilton syringe connected to an infusion pump.
2. Pull up the beads (or stem cells) reconstituted in a 0.5-1.0 µl volume into the micropipette retrograde along with 0.5 µl volume of methyl blue solution before and after.
   NOTE: An infusion rate set to 0.5-2 µl per min prevents trauma to the optic nerve.

5. Injection

1. Lower the tip of the micropipette onto the optic nerve just above the nicked dura.
   NOTE: A tiny, but brisk movement of the glass tip into the optic nerve results in the least damage. As the infusion pump begins, the methyl 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.
2. Follow the second band of methyl blue to determine when the stem cell injection is complete and turn the infusion pump off as the second dose of methyl blue is injected. Keep the micropipette still within the optic nerve for 2 min for each 1 µl volume injected into to prevent high pressure ejection upon withdrawal of the micropipette.
   NOTE: An alternative approach is to connect the pipette tip to an air filled syringe that can be manipulated manually. Either way, avoid excessive force to minimize damage to the optic nerves. We recommend injecting no more than 2 µl of volume in each optic nerve.

6. Follow up

1. Immediate.
   1. When the injection is complete, remove the micropipette along with any cotton tips used to provide hemostasis. Suture the skin with 3-0 silk and remove the conjunctival suture.
   2. 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 recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.
   3. If the animal is exhibiting signs of pain including excessive lethargy, ataxia, or labored breathing, administer appropriate analgesia with intramuscular buprenorphine (0.05 mg/kg) up to three times daily.
2. Long term.
   1. For the next 24-48 hr, observe the rat for complications of surgery including swelling or discharge from the wound or other signs of pain such as vocalizing, hunched appearance, non-grooming, or not eating. Consider consultation with a veterinarian or ethical euthanasia as necessary.