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. 2020 Jul 13;10:11521. doi: 10.1038/s41598-020-67876-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effect of 4-MU treatment on inhibitory neurotransmission in the rat small intestine. (A) 10 Hz EFS-evoked NANC on-relaxation responses of the rat small intestine longitudinal muscle in CTR, sham-operated and I/R (blank columns) and in the same groups after 4-MU treatment (black columns) in the absence and presence L-NAME and 1400 W (N = 5 rats per group). Data are reported as mean ± SEM. Statistical significance: *P < 0.05, ***P < 0.001 vs. CTR; ^çP < 0.05, ^ççP < 0.01 vs. CTR 4-MU; ^##P < 0.01 vs. sham-operated; ^&&P < 0.01 vs. sham-operated 4-MU; ^§§P < 0.01 vs. I/R; ^@P < 0.05 vs. I/R 4-MU; °P < 0.05 and °°P < 0.01 vs. 1400 W-treated CTR, by one-way ANOVA followed by Tuckey’s post hoc test. (B) Representative confocal photomicrographs showing the distribution of nNOS immunoreactive myenteric neurons (red) and their co-localization with pan neuronal marker HuC/D (green). (C) percentage of HuC/D⁺-nNOS⁺ neurons with respect to total HuC/D⁺ neurons in small intestine LMMP whole-mount preparations in the different experimental groups, (N = 5 rats per group). (D,E) Morphological analyses of nNOS immunoreactive myenteric neurons in rat small intestine of the different experimental groups. (D) Ratios of total cell areas, including dendrites, to cell body area and (E) nNOS⁺ cell profile total areas, including dendrites. Values are expressed as means ± SEM. ***P < 0.001 vs. control, ^##P < 0.01 and ^###P < 0.001 vs. sham-operated by one way ANOVA followed by Tukey’s test. (F) Percentage of HuC/D⁺-iNOS⁺ neurons with respect to total HuC/D⁺ neurons in ileal LMMP whole-mount preparations in the different experimental groups, (N = 5 rats per group). Values are expressed as means ± SEM. ***P < 0.001 vs. control, ^###P < 0.001 vs. sham-operated and ^§P < 0.05 vs. I/R by one-way ANOVA followed by Tukey’s test. (G) Representative confocal photomicrographs showing the distribution of iNOS immunoreactive myenteric neurons (red) and their co-localization with pan neuronal marker HuC/D (green).