Human organ donor-derived vagus nerve biopsies allow for well-preserved ultrastructure and high-resolution mapping of myelinated and unmyelinated fibers

Leif A Havton,Esther Stern,Terry L Powley,Elizabeth Baronowsky,John M Wo,Deborah M Jaffey,Anita Gupta,Chandrashekhar A Kubal,Natalia P Biscola,Matthew P Ward,Plamen V Mihaylov

doi:10.1038/s41598-021-03248-1

Abstract

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.

Highlights

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression
Immediate immersion fixation of the samples in a mixed aldehyde solution followed by tissue processing and embedding in a plastic resin allowed for light microscopic (LM) studies of toluidine blue-stained sections from all subjects (n = 27)
Prior ultrastructural investigations of autopsy-derived human vagus nerves have identified an early post-mortem loss of nerve fiber integrity and that unmyelinated fibers are vulnerable to pre-fixation autolysis[24]

Summary

Introduction

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. In order to improve on current electrode designs and refine stimulation strategies for optimized and expanded VNS applications to treat medical conditions, this rapidly expanding research field urgently needs a better understanding of the normal vagus nerve morphology, including the distribution and packaging of different myelinated and unmyelinated fiber types within individual nerve fascicles. Earlier studies across multiple species, including rodents, pigs, and humans, have shown extensive variability in the size and fascicular organization of the vagus nerve[15,16] These species differences are of important consideration for the development of optimized electrical stimulation strategies, as thresholds for electrical activation and blocking of fibers in the vagus nerve are influenced by, for instance, the number and size of nerve fascicles as well as perineurium thickness and the areal distribution of the e ndoneurium[17]

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Conclusion