Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 14, 2015; 21(34): 9936-9944
Published online Sep 14, 2015. doi: 10.3748/wjg.v21.i34.9936
Neurochemical features of endomorphin-2-containing neurons in the submucosal plexus of the rat colon
Jun-Ping Li, Ting Zhang, Chang-Jun Gao, Zhen-Zhen Kou, Xu-Wen Jiao, Lian-Xiang Zhang, Zhen-Yu Wu, Zhong-Yi He, Yun-Qing Li
Jun-Ping Li, Ting Zhang, Zhen-Zhen Kou, Zhen-Yu Wu, Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
Jun-Ping Li, Xu-Wen Jiao, Lian-Xiang Zhang, Zhong-Yi He, Department of Anatomy, Histology and Embryology, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Nationality Autonomous Region, China
Chang-Jun Gao, Department of Anesthesiology, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, Shaanxi Province, China
Author contributions: Li JP, Zhang T and Gao CJ performed the majority of experiments and analyzed the data; Kou ZZ and Wu ZY participated in tissue preparation and whole-mount preparations; Jiao XW and Zhang LX performed immunofluorescence histochemical double-staining and cell quantification; He ZY and Li YQ designed and coordinated the research; Li JP wrote the paper.
Supported by Grants from the National Natural Science Foundation of China, No. 30971123 and No. 81371239.
Institutional review board statement: The study was reviewed and approved by the Fourth Military Medical University Institutional Review Board (Xi’an, China).
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (Xi’an, China).
Conflict-of-interest statement: The authors declare no competing financial interests.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (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: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Yun-Qing Li, Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, No. 169 Changle West Road, Xi’an 710032, Shaanxi Province, China. deptanat@fmmu.edu.cn
Telephone: +86-29-84774501 Fax: +86-29-83283229
Received: January 14, 2015
Peer-review started: January 15, 2015
First decision: March 26, 2015
Revised: April 10, 2015
Accepted: June 10, 2015
Article in press: June 10, 2015
Published online: September 14, 2015
Processing time: 243 Days and 6.6 Hours
Abstract

AIM: To investigate the distribution and neurochemical phenotype of endomorphin-2 (EM-2)-containing neurons in the submucosal plexus of the rat colon.

METHODS: The mid-colons between the right and left flexures were removed from rats, and transferred into Kreb’s solution. For whole-mount preparations, the mucosal, outer longitudinal muscle and inner circular muscle layers of the tissues were separated from the submucosal layer attached to the submucosal plexus. The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin-coated glass slides, and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), nitric oxide synthetase (NOS), neuron-specific enolase (NSE), substance P (SP) and vasoactive intestinal peptide (VIP). After staining, all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGRP, ChAT, NOS, NSE, SP and VIP, ganglia, which have a clear boundary and neuronal cell outline, were randomly selected from each specimen for this analysis.

RESULTS: In the submucosal plexus of the mid-colon, many EM-2-immunoreactive (IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM-2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for ChAT, SP, VIP or NOS. EM-2-IR nerve fibers coursed near ChAT-IR neurons, and some of these fibers were even distributed around ChAT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers, but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for ChAT, SP, VIP or NOS were approximately 91% ± 2.6%, 36% ± 2.4%, 44% ± 2.5% and 44% ± 4.7%, respectively, but EM-2 did not co-localize with CGRP.

CONCLUSION: EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.

Keywords: Endomorphin-2; Submucosal plexus; Enteric neuron; Colon; μ-opioid receptor

Core tip: Previous studies have showed that morphine can cause gastrointestinal disorders viaμ-opioid receptor (MOR). However, the regulatory mechanisms of MOR relating to gastrointestinal activity remain unclear. Endomorphin-2 (EM-2) is the endogenous ligand for the MOR. The distribution of EM-immunoreactive (IR) structures is generally similar to that of the MOR in the nervous system, but not the submucosal plexus of the enteric nervous system. This study demonstrates for the first time that EM-2-IR neurons are present in the submucosal plexus of the rat colon, and EM-2 co-localized with several neurochemical makers in distinct populations of submucosal neurons.