Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Pathophysiol. Jun 20, 2020; 11(4): 84-103
Published online Jun 20, 2020. doi: 10.4291/wjgp.v11.i4.84
P2X7 receptor antagonist recovers ileum myenteric neurons after experimental ulcerative colitis
Roberta Figueiroa Souza, Mariá Munhoz Evangelinellis, Cristina Eusébio Mendes, Marta Righetti, Múcio Cevulla Silva Lourenço, Patricia Castelucci
Roberta Figueiroa Souza, Cristina Eusébio Mendes, Marta Righetti, Múcio Cevulla Silva Lourenço, Patricia Castelucci, Department of Anatomy, University of São Paulo, São Paulo 05508-900, Brazil
Mariá Munhoz Evangelinellis, Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo 05508-900, Brazil
Author contributions: Souza RF performed the immunohistochemistry experiments and analyzed the results; Evangelinellis MM and Mendes CE helped with inflammation and BBG protocols; Righetti M and Lourenço MCS performed the histological protocols; Castelucci P planned experiments, analyzed the results, and wrote and edited the manuscript.
Supported by Foundation São Paulo Research, No. 2014/25927-2 and No. 2018/07862-1; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
Institutional animal care and use committee statement: This study was approved by the Institute of Biomedical and Sciences/an Faculty of Veterinary Medicine and Animal Science, University of São Paulo, Protocol number # 1793240815. The animal experiments in this study were conducted according to the current regulations of the Ethics Committee on Animal Use of the Biomedical Science Institute of the University of São Paulo. Furthermore, all protocols were approved by the Ethics Committee on Animal Use of the Biomedical Science Institute of the University of São Paulo (Protocol 68/2016).
Conflict-of-interest statement: The authors have no conflicts of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
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: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Patricia Castelucci, PhD, Associate Professor, Department of Anatomy, University of São Paulo, Lineu Prestes, 2415, São Paulo 05508-900, Brazil. pcastel@usp.br
Received: January 7, 2020
Peer-review started: January 7, 2020
First decision: February 19, 2020
Revised: April 4, 2020
Accepted: April 18, 2020
Article in press: April 18, 2020
Published online: June 20, 2020
Processing time: 160 Days and 17.5 Hours
ARTICLE HIGHLIGHTS
Research background

The enteric nervous system performs functions in gastrointestinal tract such as motility, control of gastric acid secretion, regulation of fluid movement through the epithelium. This system has two ganglionic plexuses, the myenteric plexus and the submucosal plexus. Inflammatory bowel diseases (IBDs) are disorders that include ulcerative colitis and Crohn's disease. In experimental ulcerative colitis, there are changes in enteric neurons. The P2X7 receptor has been described in the ENS.

Research motivation

Studies have demonstrated that P2X7 antagonist, brilliant blue G (BBG) recovers neurons following injuries.

Research objectives

The topics of this work were to analyze the effects of experimental ulcerative colitis in enteric neurons and enteric glial cells in the ileum in animals treated with P2X7 antagonist (BBG).

Research methods

The rats were anesthetized with a mixture of xylazine (20 mg/kg) and ketamine (100 mg/kg) administered subcutaneously. Inflammation was induced through the intrarectal insertion of a polypropylene 8 cm cannula. 2,4,6-trinitrobenzene sulfonic acid (TNBS, Sigma, Saint Louis, United States) was injected at a dose of 30 mg/kg in 600 μL of 30% ethanol in the colon lumen (n = 5). Sham animals (n = 5) were injected with vehicle. BBG (50 mg/kg, Sigma Aldrich, United Kingdom, n = 5) or saline was injected 1 h following TNBS injection (n = 5). The survival time after colitis induction was 24 h. For immunohistochemistry, fresh segments of the ileum were dissected after fixed. Double labeling has been done of P2X7 receptor with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and HuC/D (a pan-neuronal marker) and enteric glial cells immunoreactive for glial fibrillary acidic protein (GFAP). The stained tissue specimens were examined using a Nikon 80i fluorescent and Confocal microscope. The counting of the neurons per area and glial cell were done in fluorescent microscope.

Research results

The numbers of nNOS-, ChAT-, HuC/D- immunoreactive (ir) neurons and GFAP-ir glial cells were decreased in the TNBS group and recovered in the BBG group. The neuronal profile area (μm2) demonstrated that nNOS-ir neurons decreased in the TNBS group and recovered in the BBG group. There were no differences in the profile areas of ChAT- and HuC/D-ir neurons. Our data conclude that ileum myenteric neurons and glial cells were affected by ulcerative colitis and that treatment with BBG had a neuroprotective effect. Thus, these results demonstrate that the P2X7 receptor may be an important target in therapeutic strategies.

Research conclusions

Ileum myenteric neurons and glial cells were affected by experimental ulcerative colitis and that treatment with P2X7 receptor antagonist, BBG had a neuroprotective effect. The results demonstrate that the P2X7 receptor may be an important target in therapeutic strategies. P2X7 receptor may be a possible therapeutic target in the treatment of the effects of experimental ulcerative colitis Ileum myenteric neurons and glial cells were affected by experimental ulcerative colitis and treatment with BBG may recover enteric neurons. P2X7 receptor may be a possible therapeutic target in the treatment of the experimental ulcerative colitis. Injection of BBG (50 mg/kg, Sigma Aldrich, United Kingdom) for experimental ulcerative colitis and effects in the distal ileum. Inflammation was induced through the intrarectal insertion of a polypropylene 8 cm cannula. 2,4,6-trinitrobenzene sulfonic acid (TNBS, Sigma, Saint Louis, United States) was injected at a dose of 30 mg/kg in 600 μL of 30% ethanol in the colon lumen. There was affected the distal ileum. Additionally, injection of BBG recover enteric neurons distal ileum. Studies show that BBG is a P2X7 antagonist, and its low toxicity and high selectivity make this compound an ideal candidate to block the adverse effects of P2X7 receptor activation. BBG treatment was shown to be effective in the recovery of ileum myenteric neurons, thus demonstrating that the P2X7 receptor may be a possible therapeutic target in the treatment of the effects of experimental ulcerative colitis.

Research perspectives

Study of effects of the experimental ulcerative colitis in the ileum and may use of the P2X7 receptor for therapeutic target. Additionally, study effects of BBG in the distal colon following experimental ulcerative colitis. The direction of the future research will be study effects of the experimental ulcerative colitis of myenteric neurons in the P2X7 receptor-deficient animals. The best method will be use P2X7 receptor-deficient animals.