Observational Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 7, 2022; 28(29): 3994-4006
Published online Aug 7, 2022. doi: 10.3748/wjg.v28.i29.3994
Structure of the myenteric plexus in normal and diseased human ileum analyzed by X-ray virtual histology slices
Bela Veress, Niccolò Peruzzi, Marina Eckermann, Jasper Frohn, Tim Salditt, Martin Bech, Bodil Ohlsson
Bela Veress, Department of Pathology, Skåne Universiity Hospital, Malmö 205 02, Sweden
Niccolò Peruzzi, Martin Bech, Medical Radiation Physics, Department of Clinical Sciences, Lund University, Lund 221 00, Sweden
Marina Eckermann, Jasper Frohn, Tim Salditt, Institute for X-Ray Physics, University of Göttingen, Göttingen 37077, Germany
Marina Eckermann, Tim Salditt, Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen 37077, Germany
Marina Eckermann, ESRF, The European Synchrotron, Grenoble 38043, France
Bodil Ohlsson, Department of Internal Medicine, Skåne University Hospital, Lund University, Malmö S-205 02, Sweden
Author contributions: Veress B performed the histochemical and pathological analyses of the X-ray data and wrote the initial draft together with Ohlsson B; Peruzzi N, Eckermann M, and Frohn J scanned the samples; Peruzzi N analyzed the technical data; all authors planned and designed the study, contributed to intellectual analysis of the results, and approved the final version of the manuscript.
Supported by the Development Foundation of Region Skåne, No. REGSKANE-818781 and No. 2018-Projekt0024; and the Foundation Skåne University Hospital, No. 2020-0000028.
Institutional review board statement: The study was reviewed and approved by the Regional Ethics Review Board at Lund University (2009/209, Date of approval 28/04/2009; 2012/527, Date of approval 16/10/2012 and 2018/132, Date of approval 02/08/2018) and the Swedish Biobank Act.
Informed consent statement: All study participants provided informed written consent prior to study enrollment.
Conflict-of-interest statement: There are no conflicts of interest to report.
Data sharing statement: No additional data is available.
STROBE statement: The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Bodil Ohlsson, MD, PhD, Full Professor, Department of Internal Medicine, Skåne University Hospital, Lund University, 15 Jan Waldenströms Street, Malmö S-205 02, Sweden. bodil.ohlsson@med.lu.se
Received: April 28, 2022
Peer-review started: April 28, 2022
First decision: May 11, 2022
Revised: May 18, 2022
Accepted: July 11, 2022
Article in press: July 11, 2022
Published online: August 7, 2022
Processing time: 96 Days and 16.8 Hours
Abstract
BACKGROUND

The enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm.

AIM

To examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS.

METHODS

Six patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples.

RESULTS

In the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings.

CONCLUSION

The analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed.

Keywords: Enteric nervous system; Immunohistochemistry; Neuropathy; Synchrotron; Virtual histology; X-ray phase-contrast nanotomography

Core Tip: Full-thickness biopsies of 1 mm diameter and up to 1 cm length from the human ileum were scanned using two laboratory-based µ-computed tomography setups to study the architecture of the enteric nervous system (ENS) and further scanned by a synchrotron-based end-station for histopathological studies, without any staining. Several pathological neuronal changes, such as vacuolar degeneration, autophagic activity, appearance of sequestosomes, chromatolysis, and apoptosis, were identified in diseased patients. Phenomena that were undetectable by light microscopy were observed. The relationships among various tissue components could be followed in all directions. Thus, this method provides a unique analysis of the ENS.