TITLE: MicroCT Imaging of the Fascicular Structure in the Porcine Right and Left Cervical Vagus Nerve
AUTHORS: Bettina Kronsteiner, Max Haberbusch, Francesco Moscato

[DESCRIPTION]
This protocol outlines the procedure to dissect, prepare, and perform micro-CT imaging of the fascicular anatomy of the right and left vagus nerve in domestic pigs.

[STEPS]
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
1. Acquire fresh cadavers of male and/or female domestic pigs.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
2. Place cadavers in the dorsal recumbence position.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
3. Make a ventral skin incision (15–20 cm) on both sides next to the trachea starting at the level of the mandibles extending to the sternal notch.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
4. Dissect tissue and open the carotid sheath to expose the common carotid artery, internal jugular vein, the vagus nerve, and the sympathetic trunk.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
5. Begin nerve dissection at the level of the nodose ganglion (NG) and extend caudally to the superior cardiac branch, which originates approximately 2–5 cm cranial to the subclavian artery on both sides.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
6. Track branches emerging from and terminating in the vagus nerve (VN) to their origin structures.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
7. Identify both recurrent laryngeal nerves running close to the trachea looping back around the subclavian arteries.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
8. Open the chest by median sternotomy followed by gentle exposure of the heart.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
9. Continue surgical preparation of the VN towards the heart. Dissect the superior cardiac branch from the cardiac branching points at the VN towards the insertion points at the heart, ensuring not to cut any of the cross-connections between the VN and the sympathetic trunk (ST).
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
10. During dissection, moisten the nerves with isotonic sodium chloride solution to prevent drying.
SECTION: Surgical Dissection and Macroscopic Examination of Nerve Samples
11. Measure dimensions of the VN using a ruler and capture a photo for documentation.
SECTION: Vagal Nerve Preparation and Harvesting
12. Fix surgical sutures onto the epineurium of the VN below the nodose ganglion to maintain the anatomical in-situ orientation. Nerves should be harvested 1-2 hours post-mortem.  
SECTION: Vagal Nerve Preparation and Harvesting
13. After complete dissection, place and fix nerves onto a polystyrene plate, ensuring the anatomical in-situ orientation is maintained.
SECTION: Vagal Nerve Preparation and Harvesting
14. Wash nerve specimens in phosphate-buffered saline (PBS)
SECTION: Vagal Nerve Preparation and Harvesting
15. Fixate in 4% phosphate-buffered 4% Paraformaldehyde (PFA) at 4°C for 16–18 hours.
SECTION: Vagal Nerve Preparation and Harvesting
16. After fixation, wash the nerve samples again and store in freshly made PBS at 4°C until staining for micro-computed tomography imaging.
SECTION: Micro-computed Tomography
17. Stain nerves in 14 mL of 1% Lugol’s solution for 24 hours.

SECTION: Micro-computed Tomography
18. Place nerve specimens in 15 ml conical centrifuge tubes with residual staining solution and the polystyrene plate used for fixation.

SECTION: Micro-computed Tomography
19. Perform low-resolution scanning using the SCANCO μCT 50 scanner with 70 kVp (200μA, 0.5 mm Al filter, 550 projections, 100 ms integration time) at an isotropic resolution of 20 μm.

SECTION: Micro-computed Tomography
20. Divide the VNs into two nerve specimens for scanning, ensuring proper anatomical orientation for alignment.

SECTION: Micro-computed Tomography
21. Perform high-resolution scans at the cardiac branching point with 70 kVp (85 μA, 0.5 mm Al filter, 850 projections, 350 ms integration time) at an isotropic resolution of 8.9 μm over 1 cm of nerve length.

SECTION: Micro-computed Tomography
22. Re-stain nerves 24 hours prior to high-resolution scans to maintain full saturation with Lugol’s solution.

SECTION: Micro-computed Tomography
23. Analyze high-resolution data at the cardiac branching point.