All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Cannulation and injection

NOTE: This step requires at least two people and is carried out with the animal's head elevated and neck flexed.

1. Begin with an anesthetized pig, pre-operated with surgically accessible cisterna magna.
2. Ensure that one person elevates and flexes the head and neck of the animal whilst the other palpates for the cisterna magna making a note of its anatomical location.
3. Slowly and carefully introduce a 22 G cannula through the dura and into the cisterna magna at an oblique angle to the longitudinal axis.
   NOTE: Do not insert the cannula too deep, as this can cause damage to the brain. Knowing how far to insert the cannula comes with experience in understanding how it feels for the cannula to pierce the dura. Essentially, just as the dura has been pierced, the cannula is then deep enough for a successful tracer injection. This depth is approximately 3-5 mm but will differ based on the size or age of the animal. Successful cannulation should be immediately evident through the visualization of clear, pulsatile CSF ascending the cannula. For the best outcome, it is recommended to practice several cannulations beforehand in euthanized animals to get one's understanding of dural piercing.
4. Retract the needle from the cannula and place a cap on the lock.
5. First, apply superglue and an accelerator where the cannula enters the tissue, followed by the application of the dental cement. Wait for 5 min for the cement to harden.
6. Carefully remove the cap from the cannula and attach the male end of the IV line tap with 10 cm extension, with the tracer, to the cannula.
7. Slowly inject the tracer by hand or using a micro-infusion pump at a rate of 100 μL/min. Remove the 3-ways IV line tap with 10 cm extension and replace it with the cap. Tracer should now be visible pulsating at the base of the cannula.
   NOTE: If injecting by hand, do this until the tracer is just still visible in the cannula shaft, approximately 1-2 mm above where the dental cement is covering the shaft.
8. Following injection, place sandbags under the neck to maintain some flexion. The head may then be released, and the animal is left in a resting prone position.
9. Release the self-retaining retractors and place muscles as they lay before. Bring the skin together over the muscles using surgical towel clamps.
10. Cover towel clamps and incision with gauze and then a blanket to limit heat loss.
11. Allow the tracer to circulate for the desired time before euthanizing the animal by i.v. Pentobarbital injection (140 mg/kg). Confirm euthanasia by the absence of heart sounds upon auscultation with a stethoscope.