TITLE: General Setup and Takedown Procedures for Rodent Neurosurgery
AUTHORS: Avalon Amaya, Jackie Swapp, Ali Williford, Robert E Howard

[DESCRIPTION]
This protocol describes the pre-operative setup and post-operative take-down procedures utilized for rodent stereotaxic neurosurgical procedures.

[BEFORE_START]
Notice:
Refer to sections via table of contents to view surgery specific setup. Skip any section titled with "Setup specific..." if not applicable.

[GUIDELINES]
Only perform this procedure in accordance with IACUC and veterinary requirements.

[STEPS]
SECTION: Prepare Surgical Station for Surgery (all procedures)
1. Disinfect the surgical area.

SECTION: Prepare Surgical Station for Surgery (all procedures)
1.1. Spray area for the surgical drape with PREempt and let sit for at least 5 min.

SECTION: Prepare Surgical Station for Surgery (all procedures)
1.2. Spray all other surfaces - surgical rig, induction chamber, station tools, knobs buttons, and switches you touch during the procedure with 70% Ethanol reapplying after 5 min, so you have a minimum contact time of 10 min.

SECTION: Prepare Surgical Station for Surgery (all procedures)
1.3. Using a non-sterile Kimwipe wipe up any residual PREempt and 70% Ethanol.
SECTION: Prepare Surgical Station for Surgery (all procedures)
2. Cover heating pad on surgical rig with a layer of press ‘n’ seal.
SECTION: Prepare Surgical Station for Surgery (all procedures)
3. Prepare the surgical drape with supplies.
SECTION: Prepare Surgical Station for Surgery (all procedures)
3.1. Open a fresh sterile drape, touching only the blue side to ensure sterility, place it white side up and blue side down, on the area disinfected with PREempt.
SECTION: Prepare Surgical Station for Surgery (all procedures)
3.2. Open sterile packages surgical supplies (Cotton swabs, kimwipes, gauze, and sugi absorbent spears), pouring the items onto the surgical drape to preserve sterility.
SECTION: Prepare Surgical Station for Surgery (all procedures)
3.3. Fill a 10mL or 20mL syringe with ACSF (Artificial Cerebrospinal Fluid), then attach 0.2um syringe filter and 25G 5/8” needle.
SECTION: Prepare Surgical Station for Surgery (all procedures)
3.4. Prepare peri-operative drugs.

 

SECTION: Prepare Surgical Station for Surgery (all procedures)
3.5. Remove autoclaved surgery tools from the sterilization tray and place on the sterile drape, taking care to not touch the instrument tips.
SECTION: Prepare Surgical Station for Surgery (all procedures)
3.6. Obtain titanium headpost with associated well, spray with 70% ethanol and place on drape with the side that will interface with the skull up. 
SECTION: Prepare Surgical Station for Surgery (all procedures)
3.7. Prepare additional 0.3ml insulin syringe for vetbond application.

 

SECTION: Prepare Surgical Station for Surgery (all procedures)
3.8. Obtain #10 Scalpel blade for skin incision.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
25.3. Ensure that all tubes are connected securely and that you can feel the vacuum suction in the scoop underneath the nose cone. 

SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
25.4. Ensure the Isoflurane line to the induction chamber is open and the line to the nose cone is closed via their designated stopcocks.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
26.1. Remove the animal from its experimental cage, obtain a preoperative weight.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
27. Place the mouse into the induction chamber, open the vacuum valve (stopcock) and isoflurane line (stopcock) for the chamber, and then turn on the isoflurane vaporizer to 5%.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
27.1. Once the mouse is fully unconscious, turn off the isoflurane and wait at least 10 seconds with the vacuum on to allow the chamber to clear before removing the animal.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
27.2. Position mouse on surgical rig by placing maxillary incisors in the hole on the bite bar and securing head with ear bars.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
27.3. Secure the nose cone over the mouse’s snout. Make sure the body of the mouse is on top of the heading pad, resting comfortably. Redirect the gas flow from the induction chamber to the surgical rig via line stopcocks.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
27.4. Set the isoflurane to ~1.5-2%, turn off the vacuum line to the induction chamber and close the lid.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
27.5. Monitor the mouse’s breathing throughout the process and adjust gas levels as necessary. 
SECTION: Prepare the Mouse for Surgery (all procedures)
28. Apply Systane to the end of a cotton swab and use to push the whiskers away from the surgical field. 
SECTION: Prepare the Mouse for Surgery (all procedures)
29. Cover the mouse’s eyes with a generous amount of Systane. Additional Systane should be added as needed to prevent eye dryness and protection from the scope light. 
SECTION: Prepare the Mouse for Surgery (all procedures)
30. Administer any drugs that have a timing after induction or prior to incision.
SECTION: Prepare the Mouse for Surgery (all procedures)
31. Use black handled scissors to shorten hair from top of the head. Use caution when trimming hair around the eyes. Avoid cutting off whiskers.

 

SECTION: Prepare the Mouse for Surgery (all procedures)
32. Apply Nair with the pointed end of a non-sterile cotton swab, gently swirling it down to the interface of the skin and hair.
SECTION: Prepare the Mouse for Surgery (all procedures)
33. Remove all Nair with several alcohol swabs. 
SECTION: Prepare the Mouse for Surgery (all procedures)
34. Disinfect the surgical site with 3 rounds of alternating Betadine-soaked sterile swabs and alcohol wipes. The last application of Betadine should not be wiped off.
SECTION: Prepare the Mouse for Surgery (all procedures)
35. Place Saran Wrap over the trunk of the animal and change into new gloves.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
7. Prepare the 24-well plate:
 



SECTION: Take Down Steps When the Procedure is Complete
36. Dispose of used syringes, blades, drill bits, swabs and needles or anything that could puncture a plastic bag in the biohazard sharps container.
SECTION: Take Down Steps When the Procedure is Complete
37. Dispose of all disposable materials that came into contact with blood in the biohazard waste container.
SECTION: Take Down Steps When the Procedure is Complete
38. Spray down surgical tools with pH neutral surgical tool cleaner and wipe with a kimwipe or alcohol swab. Be sure to wipe any blood, skin, or cement residue off the tools. Use caution when wiping down Dumont’s.
SECTION: Take Down Steps When the Procedure is Complete
39. Place tools into tool kit. Place tool kit into sterilization pouch and write your name, department initials and the date on the pack. Bring tool kit to the designated location to be autoclaved.
SECTION: Take Down Steps When the Procedure is Complete
39.1. If the tools are to be used again the same day, sterilize using the hot bead dry sterilizer.
SECTION: Take Down Steps When the Procedure is Complete
40. Disinfect ear bars with alcohol swab, then place back on the surgical rig.
SECTION: Take Down Steps When the Procedure is Complete
41. Turn off the vacuum and oxygen sources by switching the stopcocks to the off position.
SECTION: Take Down Steps When the Procedure is Complete
42. Release the air from the drill lines by pressing down on the pedals until the pressure reads 0 psi. If the air source is connected to a wall valve, then turn the valve to the off position.
SECTION: Take Down Steps When the Procedure is Complete
43. Turn off heating pad, bead sterilizer, stereotax, and scope light.
SECTION: Take Down Steps When the Procedure is Complete
44. Turn off compressed air, vacuum, and oxygen concentrator.
SECTION: Take Down Steps When the Procedure is Complete
45. Ensure Isoflurane is turned off.
SECTION: Take Down Steps When the Procedure is Complete
46. Spray down station with 70% cleaning up any debris and detritus from the surgery paying close attention to the nose cone, ear bars, vacuum scoop, and Induction chamber.
SECTION: Setup procedures specific to Nanoject III injection (with or without headpost)
None. Press and hold the ‘FILL’ button and draw up the desired amount of virus. Release the button when finished.
 

SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
25. Prepare the anesthesia system.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
26. Anesthetize the mouse.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
8. For 5mm or 3mm craniotomy procedures: use forceps to place a stacked coverslip in one of the three 70% ethanol wells in the well plate.

 

SECTION: Setup Specific to Headpost and Craniotomy Procedures
9. Place the craniotomy tracer in 70% Ethanol well.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
10. Place stylus in one of 70% ethanol wells.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
7.4. Soak three cotton swabs in the Betadine well for betadine application to incision site.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
7.2. Fill three to four wells with ACSF.

 

SECTION: Setup Specific to Headpost and Craniotomy Procedures
7.3. Fill three wells with 70% ethanol.

 

SECTION: Setup Specific to Headpost and Craniotomy Procedures
7.5. Use sterile forceps to tear off pieces of sterile surgifoam and place in well with ACSF to soak.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
9.1. For 5mm or 3mm craniotomies: place glass coverslip in well. 
SECTION: Setup Specific to Headpost and Craniotomy Procedures
9.2. For whole hemisphere craniotomies: place the WHC tracer in the well, ensuring the portion that will come in contact with the skull is submerged.
SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
5. 
Prepare the 24-well plate with supplies.
 


SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
5.4. Soak three cotton swabs in the Betadine well for betadine application to incision site.
SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
5.2. Fill three wells with ACSF.

 

SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
5.3. Fill two wells with 70% ethanol.

 

SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
5.5. Use sterile forceps to tear off pieces of sterile surgifoam and place in well with ACSF to soak.
SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
6. Place stylus in one of 70% ethanol wells.
SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
11. Prepare 24-well plate:

  


SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
12. Prepare procedure-specific additional supplies:
5-0 Monofilament suture
Parafilm square
Bulldog clamp
Silver wire
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
15. Prepare 24-well plate:
  




SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
16. Prepare procedure-specific additional supplies:
5-0 Monofilament suture
Parafilm square
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
15.4. Soak three cotton swabs in the Betadine well for application to incision site.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
17. Remove one aliquot of virus from -80ºC freezer, thaw at Room temperature, and spin down in the mini centrifuge.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
18. Obtain Nanoject-specific pipette.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
19. Prepare nanoject-specific pipette.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20. Load the virus into the pipette
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
19.1. Using a 30g, 2” backfilling Hamilton syringe filled with mineral oil, backfill the pipette.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
19.2. Insert the tip of the Hamilton syringe into the pulled pipette, all the way to the shoulder, and slowly depress the plunger on the Hamilton syringe, filling the pipette with oil. Be sure not to introduce bubbles into the system or the injection may not be successful.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
19.3. Once the pipette is backfilled with oil, loosen the collet on the end of the injector. The wire plunger should be 2-3mm past the collet.

 

SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
19.4. Gently slide the pipette over the wire plunger and push it through the chuck and seating it in the green rubber seal.  

 

SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
19.5. Tighten the collet.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20.1. Once the pipette is secured to the collet, press, and hold the ‘EMPTY’ button on the control box until an audible beep is heard. This will drive the wire plunger out forcing oil to the tip of the pipette. Any excess oil will be expelled. Expel about 4-5 drops and work out any air bubbles.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20.2. Take virus aliquot and spin down for 10-15 seconds.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20.3. Use the P20 micropipette with a microfil tip to draw up ~2 µl of virus.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20.4. Using the surgical bed as a platform, aspirate the virus sample onto a clean piece of Parafilm.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20.5. Using the stereotaxic apparatus, lower the tip of the pipette (secured in the Nanoject) into the sample. Be careful to not “bottom out”.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
20.6. Press the ‘FILL’ button and draw up the desired amount of virus (button will change to red). Press the button again to stop the filling of the pipette when finished.

 


SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
21. Carefully set injector aside where pipette will not be disturbed.
SECTION: Setup Specific to Optic Fiber Implants
22. Prepare 24-well plate:


  


SECTION: Setup Specific to Optic Fiber Implants
22.4. Soak three cotton swabs in the Betadine well for application to incision site.
SECTION: Setup Specific to Headpost Only Procedures (no craniotomy)
5.1. Fill one well with Betadine and one well with Nair.
SECTION: Setup Specific to Headpost and Craniotomy Procedures
7.1. Fill one well with Betadine and one well with Nair.
SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
11.1. Fill one well with Betadine and one well with Nair.
SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
11.3. Fill 1-2 wells with 70% Ethanol.

 

SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
15.2. Fill three wells with ACSF.

 

SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
15.1. Fill one well with Betadine and one well with Nair.
SECTION: Setup Specific to Nanoject III Injection (with or without headpost)
15.3. Fill two wells with 70% ethanol.

 

SECTION: Setup Specific to Optic Fiber Implants
22.1. Fill one well with Betadine and one well with Nair.
SECTION: Setup Specific to Optic Fiber Implants
22.2. Fill 3-5 wells with ACSF.

 

SECTION: Setup Specific to Optic Fiber Implants
22.3. Fill three wells with 70% ethanol.

 

SECTION: Setup Specific to Optic Fiber Implants
22.5. Use sterile forceps to tear off pieces of sterile surgifoam and place in well with ACSF to soak.
SECTION: Setup Specific to Optic Fiber Implants
23. Gather fiber optic implants and soak them in the 70% ethanol wells.
SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
13. Remove one aliquot of virus from -80ºC freezer, thaw at Room temperature , and spin down in the mini centrifuge.
SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
11.2. Fill three wells with ACSF.

 

SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
11.4. Soak three cotton swabs in the Betadine well for application to incision site.
SECTION: Setup Specific to Iontophoretic Injections (with or without headpost)
14. Obtain Iontophoretic-specific pipette.
SECTION: Setup Specific to Optic Fiber Implants
24. If using a headframe that is not already sterilized, soak headframe in 70% ethanol.
SECTION: Prepare Surgical Station for Surgery (all procedures)
4. Proceed to desired procedure "Specific Setup" section.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
25.1. Follow anesthesia system's manufacturer guidelines and your facilities environmental health and safety committee regarding the use of an anesthesia system for rodent neurosurgery.
SECTION: Prepare the Anesthesia System and Anesthetize the Mouse (all procedures)
25.2. Turn on the oxygenator and ensure your facilities vacuum lines are functioning properly (i.e vacuum lines are on and gauge displays psi).