TITLE: Standard Operating Procedure: Mouse Spinal Cord Injection Surgery
AUTHORS: Chu

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

[GUIDELINES]
RESPONSIBILITY

Principal investigator (PI), their staff, or any individual performing surgery on rodents or assisting in those procedures. 

[STEPS]
SECTION: Surgical and Station Preparation:
1. Print and prepare spinal cord surgery forms from Chu Lab SharePoint.
SECTION: Surgical and Station Preparation:
2. Prepare solutions (e.g., PBS, AAV virus, and retrograde beads) needed for spinal cord surgery following Chu Lab inventory and SOPs.
SECTION: Surgical and Station Preparation:
3. Sterilize the necessary surgical tools and supplies, including scalpel, blunt forceps, dumont forceps, fine scissors, and retractor hooks in an autoclave.
SECTION: Surgical and Station Preparation:
4. Disinfection surgery station and place down a bench pad.
SECTION: Surgical and Station Preparation:
5. Setup necessary equipment including:

a.	Stereotaxic frame
b.	WPI glass pipette injector and controller
c.	Warming pad
d.	Drill
e.	Induction Chamber
SECTION: Surgical and Station Preparation:
6. Check the isoflurane levels, make sure the isoflurane is enough to complete surgery.
SECTION: Surgical and Station Preparation:
7. Put a metal plate under the surgery frame to allow the magnetic retractors stand.
SECTION: Surgical and Station Preparation:
8. Disinfect an area on the working bench for the sterile surgical tools and supplies.
SECTION: Pre-operative Procedures:
9. 
 

Weigh the mouse and then put it into an induction chamber for anesthesia.
SECTION: Pre-operative Procedures:
9.1. Open valves to oxygen tank and adjust to a flow of 1 liter per minute .  Adjust the level of vaporizer to 3 percent isoflurane in oxygen and wait for the cessation of movement.

SECTION: Pre-operative Procedures:
10. Shave the neck and the upper back of mouse for cervical injection.
SECTION: Pre-operative Procedures:
11. Place mouse in the stereotaxic frame. Once nose cone is in place, close value 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. Administer carprofen (5 3483, s.c.).
SECTION: Surgical Procedures:
15. 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. 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. Tape the limbs and tail of mouse to the surgery frame, make sure its body is steady during surgery.
SECTION: Surgical Procedures:
17. Place a suitable wedge under the lower neck/upper back of mouse to lift the cervical spinal cord.
SECTION: Surgical Procedures:
18. Apply ophthalmic ointment to the eyes to protect them during the surgery.
SECTION: Surgical Procedures:
19. Make a 2 cm long incision using a scalpel starting at the base of the mouse head and extending through the middle of the back.
SECTION: Surgical Procedures:
20. Use three retractors to retract the skin and create a triangular surgical window.
SECTION: Surgical Procedures:
21. Using micro scissors to cut the fascia that might hold the trapezius muscle and adipose tissue. Adjust the three retractors to tuck away the musculature and adipose tissue.
SECTION: Surgical Procedures:
22. Locate the second thoracic vertebra (T2) and snip the muscles that are attached to T2.
SECTION: Surgical Procedures:
23. Use another two retractors to retract the midline muscles to fully expose the T2.
SECTION: Surgical Procedures:
24. Use T2 as a landmark to locate the appropriate cervical segments for injection.
SECTION: Surgical Procedures:
25. Gently clean the exposed spinal segments using swabs and gelfoam spears to obtain a clear view of the segments.
SECTION: Surgical Procedures:
26. Carefully peel the meninges to make sure that glass pipette can puncture into the spinal cord.
SECTION: Surgical Procedures:
27. Fill the glass pipette with miner oil and mount it to the WPI injector.
SECTION: Surgical Procedures:
28. Draw up the necessary volume of solution (e.g., retrograde beads, AAV virus, and PBS) into the glass pipette and WPI injector.
SECTION: Surgical Procedures:
29. Move the glass pipette tip to the surface of target site using micro-manipulator. The tip should be 500 3190 away from the midline.
SECTION: Surgical Procedures:
30. Gradually lower the glass pipette tip to target D-V using the micro-manipulator and wait 1 min.

SECTION: Surgical Procedures:
31. Set the volume and rate of release of solution on the WPI injector controller. Typically, 300 3260 of the AAVs solution is injected at a rate of 50 nl/min.
SECTION: Surgical Procedures:
32. Once injection is done, wait 3 min-5 min before retracting glass pipette to avoid backflow.
SECTION: Surgical Procedures:
33. Gradually withdraw glass pipette until completely removed from the spinal cord.
SECTION: Surgical Procedures:
34. Repeat the procedure if more than one injection is required.
SECTION: Surgical Procedures:
35. Suture muscles and skin with sterile suture (polyamide-nylon, PDS, Vicryl; size: 3-0 or 5-0) and sterilize skin with betadine.
SECTION: Post-Operative Procedures:
36. Remove animal from stereotaxic frame and place animal into a cage on a warming pad. Observe until animal is awaking and regained righting reflexes.
SECTION: Post-Operative Procedures:
37. Document surgery on a surgical procedure cage card and return cage to appropriate room in the vivarium.
SECTION: Post-Operative Procedures:
38. Administer Carprofen (5 3483, s.c.) the next day.
SECTION: Post-Operative Procedures:
39. Continue daily observations for 3 days post-operation and record on the back of the surgical procedure cage card. Extend observation period as necessary to ensure a healthy recovery.