TITLE: Mouse Stereotaxic Surgery for Alpha Synuclein Pre-formed Fibrils (PFFs) Injection in the Dorsal Striatum
AUTHORS: 

[DESCRIPTION]
This protocol describes the steps for performing stereotaxic surgery in mice. It is applicable to intracranial injections of PFFs into the dorsal striatum of the mouse brain.

[STEPS]
2. Prepare drugs and sonicated PFF
1. Surgery room setup
3. Mouse preparation with anesthesia. 
4. Surgery
5. Drilling and skull preparation. 
6. Inject PFF using Nanoject II Auto-Nanoliter Injector at the desired rate and desired volume. We inject 2 µL   5 µg/µL at the rate of  2 µL 

None. Closing/Suturing
After completion of all injections and implants, the scalp can be sutured. 
For non-implant surgery, this typically involves 3-4 sutures along the length of the incision. 
For implant surgery, this sometimes involves 1 suture (most often posterior), but sometimes no sutures are required as the scalp fits just around the implant. You do not want the scalp to be too tight around the implant. 
None. Monitoring and cleanup 
None. Postoperative Care 
2.1. Draw up in syringes all drugs that you will need for the surgery (Lidocaine below 7 mg/kg and Bupivicaine below 8 mg/kg, Preoperative analgesics Caprofen 5mg/kg SC and Ethiqa XR 3.25 mg/kg SC, sterile normal saline,).

Sonicate PFF and make it ready for injection 
1.1. Turn on the bead sterilizer and light. 
1.2. Turn on the injection device you will be using (Nanoject II Auto-Nanoliter Injector, Drummond). 
1.3. Set a clean, empty cage halfway onto the heating pad on the prep area, and turn on the heating pad to medium. 
1.4. Place your sterile surgical tools (forceps, scalpel, small scissors, hemostat, and surgical clips) on one side of the stereotax (KOPF, Model 940). 
1.5. Dispense a small amount of Nair hair remover into a weigh boat and place to one side. 
1.6. Dispense a small amount of Betadine into a weigh boat and place to one side. 
1.7. Place a packet of sterile swabs nearby. 
1.8. Make sure the small heating pad on the stereotax is on, and cover with folded napkins and tape down.
3.1. Transfer the mouse into an anesthesia induction chamber and induce anesthesia using 4% isoflurane with an oxygen flow of 1 L/min (Harvard Apparatus Mobile Anesthesia Systems, catalog # 750239). Maintain this setup until the mouse is completely anesthetized, which typically takes around 2.5 to 5 minutes (confirmed by lack of response to nociceptive stimuli such as pinching the toes and observing a normal breathing rate after full anesthesia is achieved).

Swiftly remove the mouse from the chamber and position it in a stereotaxic frame by gently opening its mouth and putting its teeth through the bite bar, and slipping the nose cone over the snout. 

Continue administering anesthesia using 1–1.5% isoflurane at an oxygen flow of 0.5–1 L/min. To sustain the mouse's body temperature around 37-38 degrees celcius during the surgery, employ a warming pad equipped with temperature sensor probes (RightTemp® Jr, Kent) underneath the mouse.





4.1. Apply lubricant to the eyes bilaterally to prevent corneal damage and dryness. 
4.2. For the remainder of the procedure, check the breathing rate (by eye) and responsiveness (by toe pinch) at least every 15 minutes. Adjust the anesthesia accordingly (typically between 1.5-1.0% for maintenance). 
4.3. Adjust the ear bars so that the animal’s head is symmetrically held in the stereotax. You can use the forceps to exert gentle downward pressure on the head and verify it does not move. If it moves, then adjust the ear bars. 
4.4. Prep the scalp as follows:
Use a swab to apply hair remover to a strip down the middle of the mouse’s scalp, working it down to the scalp, and leave for 2-3 minutes. 
Wipe away hair remover and hair with a fresh swab. Once all hair remover has been wiped off, you should have a strip of bare scalp. 
To this area, apply alternating swabs of betadine and 80% ethanol x 3. Inject a mixture of lidocaine and bupivacaine at the scan as local anesthesia.   

4.5. Confirm there is no toe pinch response.  
Use the scalpel to cut a midline incision down the long axis of the scalp. Use the small scissors to enlarge the incision to the desired length. Use the surgical clips to grab the midpoint of the skin on each side of the incision to enlarge the surgical field.
5.1. Take the scalpel and use the rounded edge to scrape the surface of the skull, removing any muscle or other tissue. 


5.2. Drill hole at desired coordinates:
Place the Nanoject II Auto-Nanoliter Injector fitted with a capillary needle at Bregma, lift it up, and move it in the AP and ML directions to position the desired coordinates for injection. For dorso-striatal injection of PFF, we are using +0.2 mm anterior, +/-2.0 mm lateral, and +2.6 mm ventral to Bregma coordinates. 
Using foot pedal, drill through the skull at this location, preferably without piercing the dura or damaging the underlying brain. 

5.3. Remove the drill from the stereotax.
6.1. After the injection has been completed, leave in place for 10-15 minutes. 
6.2. Slowly withdraw the needle while watching for fluid coming out of the hole upon withdrawal (which would indicate a failed injection, possibly due to a clog in the needle).
7. Close the opened skin using the appropriate suture. We are using PGA+ absorbable suture (Covetrus, SLU 057463). Transfer the mouse from the stereotaxic frame to another mouse cage provided with a heating pad for recovery. Once the mouse has recovered, please return it to its home cage.
    
8. Provide postoperative treatment and care for a minimum of 72 hours after the surgery.