TITLE: Stereotaxic Surgery 
AUTHORS: Beatriz E Nielsen

[DESCRIPTION]
This protocol describes the steps for performing mouse stereotaxic surgery. It is applicable to intracranial injections of viruses and drugs.

[STEPS]
SECTION: Before Surgery
1. Autoclave instruments (forceps, clamps, and scalpel holders; max 5 mice per tool pack) and cotton swabs.


SECTION: Before Surgery
2. Pull injector glass micropipettes.
SECTION: Before Surgery
3. Gather supplies needed for surgery (viruses, drugs, anesthetics, analgesics).

SECTION: Surgery Setup
4. Disinfect surgical station.
SECTION: Surgery Setup
5. Turn on injector (Nanoject III), glass beads sterilizer, digital readout system, lamp, and heating pad.
SECTION: Surgery Setup
6. Load injector micropipettes with injectable (virus, drugs).
SECTION: Surgery Setup
10. Set a clean, empty cage for recovery onto a heating pad.

SECTION: Surgery Setup
7. Set an area on one side of the stereotaxic frame to place sterile surgical tools, leveling tools and drill. 

SECTION: Surgery Setup
8. Set out cotton swabs, super glue, carprofen, bupivacaine, eye ointment, alcohol wipes and betadine.
SECTION: Surgery Setup
11. Start surgical log for the session.
SECTION: Mouse surgical preparation
12. Turn on the oxygen (1 L/min) and adjust the anesthesia machine vaporizer to 5% isoflurane.
SECTION: Surgery Setup
9. Place paper towels in the anesthesia chamber and on top of a heating pad in the stereotaxic frame.
SECTION: Mouse surgical preparation
15. Place mouse in anesthesia chamber until surgical plane of anesthesia is reached (check with toe pinch).

SECTION: Mouse surgical preparation
16. Shave scalp.
SECTION: Mouse surgical preparation
13. Weigh mouse.
SECTION: Mouse surgical preparation
18. Switch isoflurane flow from anesthesia chamber to stereotaxic frame nose cone and lower it to 1-2%.
SECTION: Mouse surgical preparation
19. Mount the ear bars to held the mouse’s head symmetrically held in the stereotaxic frame. Verify that the head is stable by lightly pushing the back.
SECTION: Mouse surgical preparation
17. Place mouse on the stereotaxic frame: scruff mouse, gently pull tongue out and to the side with forceps, place front teeth onto the hole in the nose clip, pull cone over nose/mouth.
SECTION: Mouse surgical preparation
20. Apply eye ointment bilaterally.
SECTION: Mouse surgical preparation
21. Prep incision site applying betadine with a cotton swab and ethanol sterile wipes.
SECTION: Surgery
22. For the remainder of the procedure, check the breathing rate and responsiveness for surgical plane of anesthesia with a toe pinch (this should be done every ~5 min).
SECTION: Surgery
23. Use the scalpel to cut an incision down midline of scalp. Enlarge the surgical field using surgical clips to grab the skin on each side of the incision.
SECTION: Surgery
25. Adjust lights and pre-level skull by eye.
SECTION: Surgery
26. Level the skull:
SECTION: Surgery
26.1. Using the microscope, lower the drill bit until it touches bregma. Lift the drill bit and move it posterior and lower it to lambda. Adjust the head's position so that bregma and lambda are aligned in the midline.
SECTION: Surgery
26.2. Position the drill in the middle between bregma and lambda and use the stereotaxic alignment indicator for leveling the head in X- and Y-directions.
SECTION: Surgery
27. Drill holes:
SECTION: Surgery
27.1. Re-zero drill tip at bregma.
SECTION: Surgery
27.2. Move to X and Y desired coordinates.
SECTION: Surgery
27.3. Drill through the skull without piercing the dura or damaging the brain. 
SECTION: Surgery
27.4. Remove the drill from the stereotaxic frame.
SECTION: Surgery
28. Injection:
SECTION: Surgery
28.1. Puncture the exposed dura using a bent 32G needle. Use cotton swabs to clear any debris and fluids.
SECTION: Surgery
28.2. Load injector onto the stereotaxic frame and drive it to center of the drilled hole and zero Z-axis at the dura level or the surface of brain. 
SECTION: Surgery
28.3. Select the appropriate program volume and rate (usually 2nL/s) for the virus/drug to inject and start the injection. Add saline to the skull with a syringe to prevent drying.
SECTION: Surgery
28.4. After the injection has been completed, leave in place another 5 minutes to allow difussion.
SECTION: Surgery
28.5. Slowly withdraw the injector.
SECTION: Surgery
28.6. Repeat for other holes if needed.
SECTION: Surgery
24. Apply a splash of bupivacaine (0.6 mg/mL bupivacaine) under the skull skin.
SECTION: Surgery
29. Close incision:
SECTION: Surgery
29.1. Remove ear bars and pull skin back together using cotton swabs.
SECTION: Mouse surgical preparation
14. Inject carprofen (5mg/kg) subcutaneously (s.c.).
SECTION: Surgery
29.2. Apply a thin layer of glue along incision and pinch skin together using forceps.
SECTION: Surgery
29.3. Loosen the mouse cone and remove mouse from stereotaxic frame.
SECTION: Surgery
29.4. Place mouse gently in the recovery cage until ambulation; then return mouse to home cage.
SECTION: Surgery
29.5. Complete surgical log.
SECTION: Between surgeries clean-up
30. If another surgery will be done, sterilize tips of instruments using the bead sterilizer. If surgical tools have been used 5 times, open a new pack of sterile tools and replace scalpel blade.
SECTION: Between surgeries clean-up
32. At end of day, turn off oxygen, anesthesia machine, injector, bead sterilizer, digital readout system, lamp, heating pads.
SECTION: Between surgeries clean-up
31. Disinfect stereotax with ethanol.
SECTION: Post-operative care
33. Administer a second dose of carprofen (5 mg/kg, s.c.) 24 hours after surgery.