TITLE: AAV injection in the nodose ganglia in mouse
AUTHORS: Santiago Unda, Michael G. Kaplitt

[DESCRIPTION]
The gut-brain axis links the visceral organs to the medulla oblongata via the vagus nerve. Accessing to the afferent vagal pathway is important to dissect the role of cell populations in the bidirectional communication between brain and body. The jugular-nodose ganglion (JNG) complex contains the cell bodies of many heterogenous neural subpopulations responsible for sensing the physiological and pathological conditions of the thoracic and abdominal organs. However, the study of these ganglia is challenging in small animals due to size and location. Hence, in this protocol we describe a practical surgical approach to the vagal trunk and the JNG complex in mice. 

[BEFORE_START]
Disinfect the surgical work surface with 70% ethanol and prepare in advance sterile instruments (e.g., fine scissors, forceps, and retractor), gauzes, staples, and swabs by autoclaving. For multiple surgeries, clean and re-sterilize instruments with 70% ethanol or a dry bead sterilizer between mice. A surgical mask, clean lab coat, hair bonnet and sterile gloves should be worn. These ganglia are approximately 1mm-wide, and it is located deep in the cervical carotid triangle. Therefore, a surgical microscope will be needed for entirety of the procedure. 

[STEPS]
SECTION: Preparation of the surgical setup
1. Turn on the heating pad to 37 °C  

SECTION: Preparation of the surgical setup
2. Position the surgical microscope to be ready when the animal is under anesthesia.
SECTION: Preparation of the surgical setup
3. Thaw the aliquot of AAV to be used, mix it well, and keep it on ice. 

N.B: For intraneural injections (vagus trunk) based on our experience we recommend using the mosaic AAVrg/rh10 to maximize efficiency. This serotype selectively transduces afferent neurons, so none or minimal transduction of efferent vagal pathway should be expected. Titer should be in a range between 1-3x1012vg/ml with an injection volume of 4-6ul. For intraganglionic injections, based on our experience we recommend using AAV9. Titer should be in a range between 1-3x1012vg/ml with an injection volume of 2-3ul.
SECTION: Preparation of the surgical setup
4. Withdraw the AAV with a 10ul 33G syringe.
SECTION: Preparation of the surgical setup
5. Remove locking cap and gasket from the 10ul syringe.
SECTION: Preparation of the surgical setup
6. Connect the NanoFil sub-microliter injection system.
SECTION: Preparation of the surgical setup
7. Attach the SilFlex tubing to the 10ul syringe and the other end to the Neuros Syringe.
SECTION: Preparation of the surgical setup
8. Secure the 36G beveled NanoFil needle to the injection holder.
SECTION: Preparation of the surgical setup
9. De-gas the NanoFil system by slowly pushing the plunger.
SECTION: Surgery
10. Anesthetize mice using a mixture of ketamine (110mg/kg) and xylazine (8 mg/kg) or isoflurane.
SECTION: Surgery
11. Shave the whole anterior cervical area of the mouse with an electric razor or shaving cream.
SECTION: Surgery
12. Lay the mouse flat on supine position on a heating pad.
SECTION: Surgery
13. Apply ophthalmic ointment of the mouse eyes.
SECTION: Surgery
14. Sterilize surgical area skin with 70% alcohol, complex iodine, and 70% alcohol again. Place a surgical gauze in the sterile area. Make a cut/small opening in the gauze at the level of the surgical area.
SECTION: Surgery
15. Make sure the mouse is completely anesthetized by applying a strong pinch on the forepaw of the animal with a forceps. Make a small incision in the skin in the middle of the neck with straight thin scissors or a scalpel.
SECTION: Surgery
16. Retract the submandibular glands laterally to expose the cervical musculature.
SECTION: Surgery
17. Dissect the sternocleidomastoid muscle to the laterally and the omohyoid muscle medially.
SECTION: Surgery
18. Identify the carotid bifurcation and gently dissect the connective tissue surrounding the area.
SECTION: Surgery
19. Carefully remove the connective tissue surrounding the vagus trunk above the carotid bifurcation. (If intra neural injections are required, proceed with injection.)

N.B: It is crucial that the carotid is treated carefully, otherwise major bleeding could be caused. It is also crucial to avoid excessively disturbing the vagus trunk as this could cause autonomic dysfunction after surgery.
SECTION: Surgery
20. Identify the temporal bone underneath the posterior belly of the digastric muscle.
SECTION: Surgery
21. Dissect the muscle fibers, and next to the mastoid notch of the temporal bone the JNG swelling will appear. 

N.B: Muscle fibers should be gently opened in the same orientation of the fibers to avoid damage of the occipital and auricular artery that irrigates the posterior belly of the digastric muscle. 

 

SECTION: Surgery
22. If the JNG can be fully visualized, proceed with intraganglionic injection. If needed open completely the posterior belly of the digastric muscle to gain a clear view of the cervical portion of the vagus trunk arriving to the JNG, and then proceed with injection. 

N.B: Intraganglionic injections can be done with the help of a micropositioner or free-handed, based on operator preference. 10ul syringe should inserted in a pump set up at a infusion rate of 2nl/s, final volume of 500nl. 

SECTION: Surgery
23. Close the skin with a sterile suture and apply povidone-iodine.
SECTION: Surgery
24. Apply antibiotic ointment to the wound. Carprofen (5mg/kg) as analgesic is injected subcutaneously by body weight of the mice (100ul of Carprofen for each 10 g of mouse).
SECTION: Surgery
25. After the surgery, place mouse under a heat lamp until fully awake; return the mouse back in the cage when full motility is restored.