Determining the Fascicular Anatomy of the Porcine Vagus Nerve with MicroCT
Authors: Thompson Nicole , Ravagli Enrico , Mastitskaya Svetlana , Iacoviello Francesco , Aristovich Kirill , Perkins Justin , Shearing Paul R. , Holder David 
Published: April 2020

Vagus nerve stimulation (VNS) is a promising therapy for the treatment of conditions resistant to standard therapeutics. However, due to the lack of understanding of the functional fascicular organisation of the cervical vagus nerve, VNS leads to unwanted off‐target effects. Contrast‐enhanced micro‐computed tomography (microCT) was optimised to provide rapid visualisation of fascicles within the nerve in 3D with little distortion of the sample and minimal artefacts in rat sciatic nerves. The purpose of this work was to trace the functional anatomy of fascicles in autonomic nerves using microCT in the pig cervical vagus nerve as a prelude to human studies. Specific aims were to: 1) Determine if the microCT pre‐processing and scanning protocol works to trace fascicles in pig vagus nerve, 2) Determine the fascicular representation in the cervical vagus nerve of the recurrent laryngeal, cardiac and pulmonary branches.The vagus nerves of 6 domestic female 60–70 kg pigs were dissected from the cervical region to the branches of the target organs, fixed in neutral buffered formalin for a minimum of 24 hours, sectioned into 4 cm lengths with appropriate landmarks for registration left in place and soaked in Lugol’s solution (1% total iodine) for 5 days for sufficient contrast staining. On each of day of scanning, the sections of nerves were blotted dry, wrapped in cling film and a layer of sponge, and placed in a 3D printed mount within the microCT scanner (Nikon XT H 225, Nikon Metrology, Tring, UK). A molybdenum target was used and the scanning parameters, for all automated and overlapping scans, were set to the following: 4 μm voxel size, 35 kVp energy, 114 mA current, 4 W power, 0.25 fps in 4 s exposure time, and 3176 projections. Scans were reconstructed in CT Pro 3D (Nikon Metrology, Tring, UK), analysed in ImageJ and fascicles segmented with a custom algorithm in Matlab (Mathworks, Natick, Massachusetts, USA) and Seg3D (NIH Centre for Integrative Biomedical Computing, SCI Institute, University of Utah, USA).The eventual goal was to trace up to 20 cm of nerve from the entry point of the branch to the mid‐cervical level of the vagus nerve. Until now, it has been possible to image and segment 15–20 fascicles over 4 cm of cervical vagus with an accuracy of <2.5% difference in fascicles diameter compared to histology with H&E staining (n=3 1 cm segments). Work in progress is to extend this to trace fascicular anatomy from both cervical vagus nerves to the branch entry points for the above 3 nerves in the pig. The resulting segmentation map of the functional anatomical organisation of the vagus nerve will enable selective VNS ultimately allowing for the avoidance of off‐target effects and improving its therapeutic efficacy. The imaging technique used can be employed to assist in the studies of other peripheral nerves involving peripheral nerve repair, microsurgery and improving the implementation of nerve guidance conduits. We plan to extend the approach to delineating the functional anatomy of the cervical vagus nerve in humans.Support or Funding InformationThis work is supported by the Medical Research Council (MR/R01213X/1).