Standard Operating Procedure: Mouse Stereotaxic Intracranial Injection Surgery

Print the surgery documents and prepare them from Chu Lab SharePoint.

Prepare solutions needed for target injections according Chu Lab Inventory and SOPs from the Chu Lab SharePoint. Place solutions on ice, if applicable.

Autoclave the necessary tools to prior to surgery.

Disinfection surgery station and place down a benchpad.

Setup the necessary equipment including:

• Stereotaxic frame
• Injector
• Warming pad
• Drill
• Induction Chamber

Check the isoflurane levels. Levels visible on the front of the vaporizer.

Designate a sterile area on the working surface for the sterile material (instruments, suture material, gauze, etc.).

Clean the Hamilton syringe with sterile water, then 70% ethanol then sterile water again.

Check the needle is straight before beginning and replace if it appears bent.

Secure to needle to stereotaxic frame and adjust out of the way to avoid contact with needle.

Anesthetize the animal in induction chamber.

Open valves to oxygen tank and adjust to a flow of 1L per 0h 1m 0s. Adjust the level of vaporizer to 3 percent isoflurane in oxygen and wait for the cessation of movement.

Shave top of the head and remove hair.

Return animal to induction chamber if need before next step.

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.

Apply ophthalmic ointment in both eyes to prevent corneal desiccation. Reapply as needed.

Place anal probe underneath body to regulate temperature. Heating system prove setpoint is 36.9°C.

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.

Expose the cranium by making a surgical anterior-posterior incision with a scalpel blade and reflect the skin.

Use scope to locate bregma and lambda.

They will become more prominent over time and hydrogen peroxide solution can be applied to the skull to facilitate identification.

Position needle directly over the intersection with the coronal and sagittal sutures to locate bregma.

Lower needle until it just touches the surface of the skull.

Record the A-P, M-L, and D-V coordinates of Bregma.

Repeat step 17 at the intersection of the lambdoid and sagittal sutures to locate Lambda.

Record the A-P, M-L and D-V coordinates of Lambda.

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.

Calculate target coordinates for injection site using leveled bregma coordinates.

Move needle to target site and mark site using a pencil.

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.

Draw up necessary volume of solution into the syringe.

Remeasure the D-V coordinate at the dura and calculate the target D-V coordinate.

Gradually lower the injection needle gradually to target D-V and wait 0h 1m 0s.

Check that settings on the injector are correct:

Syringe Settings:

• 1700 Hmltn 10µL 0.460mm.
• Adjust volume and speed depending on injection.
• Check injector is set to infusion.

Press START button on injector to begin injection.

Once injection is done, wait 0h 3m 0s -0h 5m 0s before retracting needle to avoid backflow.

• For 1mL of 6-OHDA, wait 0h 10m 0s.

Gradually withdraw needle 500 µm and wait 0h 1m 0s. Repeat. Continue to withdraw needle until completely removed from the brain.

Repeat the procedure if more than one injection is required.

Suture skin with sterile suture (polyamide-nylon, PDS, Vicryl; size: 3-0 or 5-0) and sterilize skin with betadine.

Administer carprofen (5mg/kg, s.c.).

Remove animal from stereotaxic frame and place animal into a cage on a warming pad.

Observe until animal is awaking and regained righting reflexes.

Document surgery on a surgical procedure cage card and return cage to appropriate room in the vivarium.

Administer Carprofen (5mg/kg, s.c.) the next day.

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.