TITLE: Protocol for &#34;ventriculography-assisted stereotaxic surgery in non-human primates
AUTHORS: jlanciego

[DESCRIPTION]
An stept-by-step procedure describing ventriculography-assisted stereotaxic surgery in non-human primates is provided here, including (i) animal pre-surgical preparation, (ii) stereotaxic surgery and (iii) post-surgical care. This protocol is intended to be used for intraparenchymal deliveries of viral suspensions.

[STEPS]
SECTION: Animal preparation
1. Pre-surgical anesthesia: to be induced with ketamine (5 mg/Kg) and midazolam (0.5 mg/Kg), administered intramuscularly.
SECTION: Animal preparation
2. Surgical shaving: comprising left and right superior and inferior limbs and thorax for the placement of adhesive EKG electrodes.
SECTION: Animal preparation
3. Induction of initial anesthesia: 2% isofluorane prior to intubation of the animal followed by intravenous injection of propofol (1.5 mg/Kg), fentanest (0.01 mg/Kg) and anectine (1 mg/Kg).
SECTION: Animal preparation
4. Intubation: a size 3 pediatrics endotracheal tube to be used. Keep oxygen volume between 10-15 ml/Kg under a respiratory rate of 15-20 rpm.
SECTION: Animal preparation
5. Catheter placement in the saphenous vein: to secure intravenous access to deliver a continuous infusion of saline solution with 5% glucose.
SECTION: Animal preparation
6. Pre-surgical anti-inflammatory treatment: comprising the  subcutaneous delivery of carprofen (4 mg/Kg / 24h /3 consecutive days), methylprednisolone (10mg/Kg; intravenous, dexamethasone (10 mg/Kg; intravenous) and enrofoxacine (10 mg/Kg / 24h / 7 days; intramuscular).
SECTION: Animal preparation
7. Animal monitoring: comprising rectal temperature, oxygen saturation, expired CO2, EKG, respiration rate and blood pressure.
SECTION: Animal preparation
8. Surgical anesthesia: continuous intravenous infusion of propofol (6 mg/Kg/h)
SECTION: Animal preparation
9. Placement of the animal in the stereotaxic frame: macaque head fixed with elevated earbars, palate and orbital adaptors. Animal placed on top of an electric heat pad.
SECTION: Stereotaxic surgical procedure
10. Animal head cleaned with an iodine solution. Skin treated with local anesthesia (lidocaine spray).
SECTION: Stereotaxic surgical procedure
11. Preparation of the sterile surgical field and the sterile surgical instruments table.
SECTION: Stereotaxic surgical procedure
12. Perform a surgical medial incision of the skin through the entire rostrocaudal extent of the head, followed by removal of the periosteum.
SECTION: Stereotaxic surgical procedure
13. First craniotomy: drill a burr hole (4 mm in diameter) to expose the midsagittal venous sinus.
SECTION: Stereotaxic surgical procedure
14. Secure access to the CSF space: a pediatric needle is inserted in the right lateral ventricle in the frontal lobe. Under the control of a stereotaxic micromanipulator, a orthogonal trajectory 1.2 mm to the midsagittal sinus is used to place a CSF pediatric needle into the ventricle. Adequate placement of the needle is confirmed upon CSF aspiration.
SECTION: Stereotaxic surgical procedure
15. First ventriculography: Delivery of a bolus of 0.3 ml of X-ray contrast to properly delineate the ventricular system. The first ventriculography enables to calculate the location of the anterior and posterior commissures (ac and pc, respectively), as well as to measure the length of the ac-pc plane, required for proper calibration of the stereotaxic coordinates in keeping with the stereotaxic atlas of reference (see Figure 1, panels A & E)
SECTION: Stereotaxic surgical procedure
16. Second craniotomy: another burr hole is drilled to expose the brain surface at the level of the calculated coordinates for the putamen nucleus
SECTION: Stereotaxic surgical procedure
17. Fill the injection device (Hamilton syringe) with the viral suspension of AAV9-SynA53T.
SECTION: Stereotaxic surgical procedure
18. Second ventriculography: Once the injection needle is located in the pre-calculated place of delivery within the putamen nucleus, a second X-ray plate is taken ensuring the right location of the tip of the needle (to be repeated for each AAV delivery; see Figure 1, panels B-D & F-G).
SECTION: Stereotaxic surgical procedure
19. Delivery of the viral suspension: Pressure deliveries of AAV9-SynA53T were made in pulses of 5 ml/min for a total volume of 25 microliters each into three sites in the left putamen, each deposit spaced 1 mm in the rostrocaudal direction to obtain the highest possible transduction extent of the putamen. Once injections were completed, the needle was left in place for an additional time of 10 min before withdrawal to minimize AAV reflux through the injection tract.
SECTION: Stereotaxic surgical procedure
20. Stereotaxic coordinates: 1mm rostral to ac, 1 mm dorsal to the ac-pc plane and 11 mm lateral to midline (rostral injection). 0 mm to ac, 1 mm dorsal to the ac-pc plane and 11.5 mm lateral to midline (mid injection). 1 mm caudal to ac, 1.5 mm dorsal to the ac-pc plane and 12.5 mm lateral to midline (caudal injection).
SECTION: Stereotaxic surgical procedure
21. Once all AAVs were completed, the muscular plane was closed with absorbable suture, followed by surgical staples to close the skin and cleaned with an iodine solution.
SECTION: Post-surgical care
22. Animals were kept under constant monitoring in individual cages with a libitum access to food and water. Once animals showed a complete post-surgical recovery (24 h), they were returned to the animal vivarium and housed in groups.
SECTION: Post-surgical care
23. Post-surgical analgesia was achieved with a single intramuscular injection of flunixin meglumine (5 mg/Kg) delivered at the end of the surgical procedure and repeated 24 and 48 h post-surgery.
SECTION: Post-surgical care
24. A similar schedule was conducted for antibiotic coverage (ampicillin, 0.5 ml/day).