TITLE: Vagotomy and Tamoxifen treatment
AUTHORS: arpine.sokratian, andrew.west

[DESCRIPTION]
This protocol provides the methodology of vagotomy procedure and tamoxifen injections in a murine model, investigating the pathological role of alpha-synuclein in the gastrointestinal (GI) system. The mouse model includes features of exclusive expression of alpha-synuclein in cholecystokinin (CCK) cells within the gut.
The protocol intricately details the surgical procedure, including a dissection of the vagus node to ensure the mice not only survive but thrive post-surgery. Subsequent tamoxifen injections, utilizing a tamoxifen-inducible promoter, aim to induce the expression of alpha-synuclein.
The significance of this protocol lies in presenting a unified and standardized procedure for both vagotomy and tamoxifen injections. This streamlined approach is crucial for investigating the role of alpha-synuclein in the GI system.

[STEPS]
SECTION: Vagotomy and Tamoxifen treatment
1. Perform surgical subdiaphragmatic vagotomy in 1-month-old male and female SNCAbow;Vil-CreERT2 mice.
SECTION: Vagotomy and Tamoxifen treatment
2. Prior to surgery, sterilize all surgical instruments. Use syringe, needles, sutures, and scalpels from sterile packs, open it at the time of the operation.
SECTION: Vagotomy and Tamoxifen treatment
3. Sterilize dissecting instruments before using on the next mouse.
SECTION: Vagotomy and Tamoxifen treatment
4. Surgeon washed their hands thoroughly for 5 min before donning sterile gloves.

SECTION: Vagotomy and Tamoxifen treatment
5. Surgeon maintained a sterile operating field during the operation.
SECTION: Vagotomy and Tamoxifen treatment
6. Apply lubricant eye ointment to mouse eyes to prevent corneal drying.
SECTION: Vagotomy and Tamoxifen treatment
7. Anesthetize mice with ketamine/xylazine at a dose of 87/13 mg/kg by intraperitoneal injection. Determine adequate anesthesia by lack of movement, lack of response to tail pinch, and lack of whisker twitching.
SECTION: Vagotomy and Tamoxifen treatment
8. Administer buprenorphine 0.05 mg/kg subcutaneously before the surgery.

SECTION: Vagotomy and Tamoxifen treatment
9. After initial anesthesia, place the mouse on top of a heating pad lined with a silicone pad within the stereotactic apparatus field. 
 

SECTION: Vagotomy and Tamoxifen treatment
10. Make a mid-line incision with scissors to expose the abdominal contents.
SECTION: Vagotomy and Tamoxifen treatment
11. Immediately below the diaphragm, identify the vagus nerve and isolate it from surrounding connective tissue and vessels.
SECTION: Vagotomy and Tamoxifen treatment
12.  Excise 2 mm section of the vagus nerve.
SECTION: Vagotomy and Tamoxifen treatment
13. Monitor mice for intra-operatively signs of arousal. Continuously monitor mouse breathing throughout the surgery by observing chest wall movement.
SECTION: Vagotomy and Tamoxifen treatment
14. Close the laparotomy in two layers with suturing and surgical clips. Suture the inner layer of skin with a continuous suture pattern using 5-0 monofilament absorbable suture and close the outer skin with 9 mm stainless steel wound clips (MikRon Precision Inc).
SECTION: Vagotomy and Tamoxifen treatment
15. Apply analgesic bupivacaine (1-2 drops) to the incision site after suturing and allow the animals to awaken.
SECTION: Vagotomy and Tamoxifen treatment
16. Following surgery, assess mice every 30 min until they return to baseline level of activity with no signs of breathing complications or lasting motor deficits.

SECTION: Vagotomy and Tamoxifen treatment
17. Record the well-being of the mice in the post-surgery monitoring log. Postoperatively, give mice analgesics.
SECTION: Vagotomy and Tamoxifen treatment
18. Administer mice analgesics (buprenorphine hydrochloride at a dose of 0.05 mg/kg) and observe daily for 5 days for any signs of infection, distress, or changes in behavior.

SECTION: Vagotomy and Tamoxifen treatment
19. Give mice free access to food and water.
SECTION: Vagotomy and Tamoxifen treatment
20. In sham-operated animals, perform abdominal laparotomy, and expose the vagus nerve but do not excise it.
SECTION: Vagotomy and Tamoxifen treatment
21. Weight loss of ~15% was noted in mice undergoing vagotomy compared to sham surgery.
SECTION: Vagotomy and Tamoxifen treatment
22. One week after surgery, treat mice with tamoxifen dissolved in corn oil (50 mg/kg) or vehicle administer by intraperitoneal injection daily for five days.