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. 2019 Jan 24;9:569. doi: 10.1038/s41598-018-37090-9

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© The Author(s) 2019

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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Functional role of nitric oxide synthase (NOS) in the effects of local anaesthetics (LAs) on intracellular neutrophil nitric oxide (NO) generation, (A,B) overall and (C) in cord (n = 11) and adult blood neutrophils (n = 10) as shown by graphical representations of significant interactions determined by multifactorial ANOVA between the factors of ‘anaesthetic’ (type of anaesthetic), ‘concentration category’ (concentration categorized into three levels: lowest, middle and highest), ‘group’ (cord or adult blood neutrophils) and ‘NOS stimulation and inhibition’ (unstimulated neutrophils, phorbol myristate acetate (PMA)-stimulated neutrophils, PMA-stimulated neutrophils incubated with N^G-nitro-l-arginine methyl ester (l-NAME) or aminoguanidine (AG)). (A,B) Overall effect of NOS inhibition on intracellular NO generation in unstimulated and PMA-stimulated neutrophils exposed to: (A) the three LAs, showing a significant interaction between ‘anaesthetic’ and ‘NOS stimulation and inhibition’; and (B) lidocaine (left graph), and ropivacaine (right graph), showing a significant interaction between ‘concentration category’ and ‘NOS stimulation and inhibition’. (C) Differences in intracellular NO generation between unstimulated and PMA-stimulated adult and cord blood neutrophils exposed to bupivacaine, lidocaine and ropivacaine in the presence of NOS inhibitors showing a significant interaction between ‘group’ and ‘NOS stimulation and inhibition’. NO production was determined by flow cytometry and expressed as mean fluorescence intensity of triazolofluorescein (MFI DAF-2T); nitrite concentrations were measured using ozone-based chemiluminescence as described in the Materials and Methods. Data are displayed in box plots (median, interquartile range and min-max). Datasets are compared using abcd notation; means with the same letter are not significantly different from each other (P > 0.05; multifactorial ANOVA followed by Tukey test). Further details are summarized in Tables S5, S6.