Sep 27, 2023

Public workspaceMicroCT Imaging of the Fascicular Structure in the Porcine Right and Left Cervical Vagus Nerve

DOI
dx.doi.org/10.17504/protocols.io.ewov1qd2ygr2/v1
  • 1Center of Anatomy and Cell Biology, Medical University of Vienna;
  • 2Center for Medical Physics and Biomedical Engineering, Medical University of Vienna
Max Haberbusch
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DOI: dx.doi.org/10.17504/protocols.io.ewov1qd2ygr2/v1
Protocol Citation: Bettina Kronsteiner, Max Haberbusch, Francesco Moscato 2023. MicroCT Imaging of the Fascicular Structure in the Porcine Right and Left Cervical Vagus Nerve. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qd2ygr2/v1
Manuscript citation:
Kronsteiner, Bettina, et al. "Mapping the functional anatomy and topography of the cardiac autonomic innervation for selective cardiac neuromodulation using MicroCT." Frontiers in Cell and Developmental Biology 10 (2022): 968870.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 14, 2023
Last Modified: September 27, 2023
Protocol Integer ID: 87769
Funders Acknowledgement:
European Project H2020
Grant ID: 824071
Austrian Research Promotion Agency R&D Infrastructure Project
Grant ID: 858060
Abstract
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.
Materials
  • Fresh cadavers of pigs (domestic pigs)
  • Isotonic sodium chloride solution (NaCl, 0.9%)
  • Surgical sutures (5–0 silk suture, Silkam‱B-Braun Aesculap, Tutlingen, Germany)
  • Polystyrene plate with V-shaped incisions
  • PBS (Phosphate-Buffered Saline)
  • 4% phosphate-buffered 4% Paraformaldehyde (Roti-Histofix, Roth, Karlsruhe, Germany)
  • 1% Lugol’s solution
  • GIMP open-source software (GIMP 2.10.24, revision 2)
  • SCANCO μCT 50 (SCANCO Medical AG, Brutistellen, Switzerland) specimen μCT scanner
Surgical Dissection and Macroscopic Examination of Nerve Samples
Surgical Dissection and Macroscopic Examination of Nerve Samples
Acquire fresh cadavers of male and/or female domestic pigs.
Place cadavers in the dorsal recumbence position.
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.
Dissect tissue and open the carotid sheath to expose the common carotid artery, internal jugular vein, the vagus nerve, and the sympathetic trunk.
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.
Track branches emerging from and terminating in the vagus nerve (VN) to their origin structures.
Identify both recurrent laryngeal nerves running close to the trachea looping back around the subclavian arteries.
Open the chest by median sternotomy followed by gentle exposure of the heart.
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).
During dissection, moisten the nerves with isotonic sodium chloride solution to prevent drying.
Measure dimensions of the VN using a ruler and capture a photo for documentation.
Vagal Nerve Preparation and Harvesting
Vagal Nerve Preparation and Harvesting
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.
After complete dissection, place and fix nerves onto a polystyrene plate, ensuring the anatomical in-situ orientation is maintained.
Wash nerve specimens in phosphate-buffered saline (PBS)
Fixate in 4% phosphate-buffered 4% Paraformaldehyde (PFA) at 4°C for 16–18 hours.
After fixation, wash the nerve samples again and store in freshly made PBS at 4°C until staining for micro-computed tomography imaging.
Micro-computed Tomography
Micro-computed Tomography
Stain nerves in 14 mL of 1% Lugol’s solution for 24 hours.

Place nerve specimens in 15 ml conical centrifuge tubes with residual staining solution and the polystyrene plate used for fixation.

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.

Divide the VNs into two nerve specimens for scanning, ensuring proper anatomical orientation for alignment.

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.

Re-stain nerves 24 hours prior to high-resolution scans to maintain full saturation with Lugol’s solution.

Analyze high-resolution data at the cardiac branching point.
Protocol references
Kronsteiner, Bettina, et al. "Mapping the functional anatomy and topography of the cardiac autonomic innervation for selective cardiac neuromodulation using MicroCT." Frontiers in Cell and Developmental Biology 10 (2022): 968870. doi: https://doi.org/10.3389/fcell.2022.968870