TITLE: Standard Operating Procedure: Mouse Stereotaxic Intracranial Injection Surgery
AUTHORS: Hong-Yuan  Chu

[DESCRIPTION]
The intent of this Standard Operating Procedure (SOP) is to describe procedures for mouse stereotaxic intracranial injection surgery.

[GUIDELINES]
RESPONSIBILITY:
Principal investigator (PI), their staff, or any individual performing stereotaxic surgery on rodents or assisting in those procedures.


[STEPS]
SECTION: Surgical and Station Preparation:
1. Print the surgery documents and prepare them from Chu Lab SharePoint.
SECTION: Surgical and Station Preparation:
2. Prepare solutions needed for target injections according Chu Lab Inventory and SOPs from the Chu Lab SharePoint.  Place solutions on ice, if applicable.
SECTION: Surgical and Station Preparation:
3. Autoclave the necessary tools to prior to surgery.
SECTION: Surgical and Station Preparation:
4. Disinfection surgery station and place down a benchpad.
SECTION: Surgical and Station Preparation:
5. Setup the necessary equipment including:
Stereotaxic frame
Injector
Warming pad
Drill
Induction Chamber

SECTION: Surgical and Station Preparation:
6. Check the isoflurane levels. Levels visible on the front of the vaporizer.
SECTION: Surgical and Station Preparation:
7. Designate a sterile area on the working surface for the sterile material (instruments, suture material, gauze, etc.).
SECTION: Surgical and Station Preparation:
8. Clean the Hamilton syringe with sterile water, then 70% ethanol then sterile water again.
 

SECTION: Pre-operative Procedures:
9. 
 
Anesthetize the animal in induction chamber.
SECTION: Pre-operative Procedures:
9.1. Open valves to oxygen tank and adjust to a flow of 1 L per 1 min. Adjust the level of vaporizer to 3 percent isoflurane in oxygen and wait for the cessation of movement.
SECTION: Pre-operative Procedures:
10. Shave top of the head and remove hair.
 
Return animal to induction chamber if need before next step.

SECTION: Pre-operative Procedures:
11. Secure animal in the stereotaxic frame. Once nose cone is in place, close valve to chamber and open valve to nose cone and reduce vaporizer to 1.5-2 percent isoflurane in oxygen.
SECTION: Pre-operative Procedures:
12. Apply ophthalmic ointment in both eyes to prevent corneal desiccation. Reapply as needed.
SECTION: Pre-operative Procedures:
13. Place anal probe underneath body to regulate temperature. Heating system prove setpoint is 36.9 °C.
SECTION: Surgical Procedures:
14. Using 70% ethanol on a sterile cotton swab in a circular motion, sterilize surgery site from the center of the surgical site to the exterior.
SECTION: Surgical Procedures:
15. Expose the cranium by making a surgical anterior-posterior incision with a scalpel blade and reflect the skin.
SECTION: Surgical Procedures:
16. Use scope to locate bregma and lambda.
SECTION: Surgical Procedures:
17. Position needle directly over the intersection with the coronal and sagittal sutures to locate bregma.
SECTION: Surgical Procedures:
18. Repeat step 17 at the intersection of the lambdoid and sagittal sutures to locate Lambda.
SECTION: Surgical Procedures:
19. Adjust the level of the skull until there is less than a 50um D-V difference between Bregma and Lambda, and less than a 50um D-V difference between point +2 and -2 mm M-L to Bregma.
SECTION: Surgical Procedures:
20. Calculate target coordinates for injection site using leveled bregma coordinates.
SECTION: Surgical Procedures:
21. Move needle to target site and mark site using a pencil.
SECTION: Surgical Procedures:
22. Raise needle and, using the hand-held drill, make a single burr-hole in the skull at the injection site. Clean hole of any blood or debris using sterile saline and gelfoam spears.
SECTION: Surgical Procedures:
23. Draw up necessary volume of solution into the syringe.
 

SECTION: Surgical Procedures:
24. Remeasure the D-V coordinate at the dura and calculate the target D-V coordinate.
SECTION: Surgical Procedures:
25. Gradually lower the injection needle gradually  to target D-V and wait 1 min.
SECTION: Surgical Procedures:
26. Check that settings on the injector are correct:

Syringe Settings:
1700 Hmltn 10 µL 0.460mm.
Adjust volume and speed depending on injection.
Check injector is set to infusion.
SECTION: Surgical Procedures:
27. Press START button on injector to begin injection.
SECTION: Surgical Procedures:
28. Once injection is done, wait 3 min -5 min before retracting needle to avoid backflow.
For 1 mL of 6-OHDA, wait 10 min.
SECTION: Surgical Procedures:
29. Gradually withdraw needle 500 µm and wait 1 min.  Repeat. Continue to withdraw needle until completely removed from the brain.
SECTION: Surgical Procedures:
30. Repeat the procedure if more than one injection is required.
 

SECTION: Surgical Procedures:
31. Suture skin with sterile suture (polyamide-nylon, PDS, Vicryl; size: 3-0 or 5-0) and sterilize skin with betadine.
SECTION: Post-Operative Procedures:
32. Administer carprofen (5mg/kg, s.c.).
SECTION: Post-Operative Procedures:
33. Remove animal from stereotaxic frame and place animal into a cage on a warming pad.

SECTION: Post-Operative Procedures:
34. Document surgery on a surgical procedure cage card and return cage to appropriate room in the vivarium.

 

SECTION: Post-Operative Procedures:
35. Administer Carprofen (5mg/kg, s.c.) the next day.

 

SECTION: Post-Operative Procedures:
36. Continue daily observations for 3 days post-operation and record on the back of the surgical procedure cage card.

SECTION: Surgical and Station Preparation:
8.1. Check the needle is straight before beginning and replace if it appears bent.
SECTION: Surgical and Station Preparation:
8.2. Secure to needle to stereotaxic frame and adjust out of the way to avoid contact with needle.
SECTION: Surgical Procedures:
14.1. Then apply povidone-iodine solution using cotton swabs in a circular motion from the center of the surgical site to the exterior. Repeat this process for a second time.
SECTION: Surgical Procedures:
16.1. They will become more prominent over time and hydrogen peroxide solution can be applied to the skull to facilitate identification.
SECTION: Surgical Procedures:
17.1. Lower needle until it just touches the surface of the skull.
SECTION: Surgical Procedures:
17.2. Record the A-P, M-L, and D-V coordinates of Bregma.
 

SECTION: Surgical Procedures:
18.1. Record the A-P, M-L and D-V coordinates of Lambda.
SECTION: Post-Operative Procedures:
33.1. Observe until animal is awaking and regained righting reflexes.
SECTION: Post-Operative Procedures:
36.1. Extend observation period as necessary to ensure a healthy recovery.