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. 2021 May 6;11:9735. doi: 10.1038/s41598-021-89122-6

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Comparison of key, pain-related brainstem nuclei and lumbar spinal cord laminae activated by hind paw stimulation with low intensity mechanical force (0.16 g) at chronic stages of neuropathic pain (43 days post-nerve injury) in SNI mice receiving repetitive M1 tDCS or 0 mA sham stimulation over 35–39 days post-nerve injury. The control group received no mechanical stimulation (control). (A) Typical examples of immunohistochemistry for the neuronal activity marker protein Fos with nuclear counterstaining with DAPI (scale bar = 100 µm) over ventrolateral periaqueductal grey (vlPAG) and lateral PAG (LPAG). (B,C) Quantitative analysis of Fos-expressing cells over LPAG (B) and vlPAG (C); n = 12 sections from 4 mice in the repetitive M1 tDCS group (with mechanical stimulation) and the sham treatment group (with or without mechanical stimulation), n = 9 sections from 3 mice in repetitive M1 tDCS group (without mechanical stimulation). (D,E) Typical examples of Fos immunohistochemistry with DAPI counterstaining (D; scale bar = 200 µm) or quantitative summary (E) over superficial laminae (I and II) in L3–L5 lumbar spinal cord segments; n = 6–12 sections were processed from each of 4 mice per group. Images were acquired with a confocal laser-scanning microscope (Leica LAS X, version 3.3.0), imported to Fiji-Image J software (version 1.50b). In all panels, ANOVA for random measures was performed, followed by Sidak’s test for multiple comparisons. *Indicates p < 0.05 as compared to the corresponding sham treatment group, # indicates p < 0.05 as compared to control in the sham treatment group and the repetitive M1 tDCS group, respectively. Data are represented as mean ± S.E.M.