Methylene blue induces an analgesic effect by significantly decreasing neural firing rates and improves pain behaviors in rats

https://doi.org/10.1016/j.bbrc.2021.01.008Get rights and content

Highlights

  • Methylene blue, a dye, attenuated significantly neural firing rates compared to lidocaine.

  • The dye also markedly delayed hind paw withdrawal reflexes elicited by mechanical and thermal stimuli.

  • Methylene blue lessens pain by dampening neural excitability.

  • Methylene blue may be developed as a therapeutic agent for pain patients.

Abstract

Methylene blue (MB) is a blue cationic thiazine dye and currently used in different medical settings. Notably, there have been several attempts to introduce MB for attenuating pain in the last decade. Some clinical studies reported remarkable results, which, however, have been much debated. In addition, accumulating evidence have revealed that MB diminishes voltage-gated sodium channel currents. Accordingly, in the present study, we conducted in vivo experiments, including in vivo single nerve recording and behavioral test, to investigate whether MB dampens neural firing rates and ultimately contributes to pain relief. As a result, neural firing rates significantly decreased and finally converged to zero after MB administration. This event lasted longer than that of lidocaine and was dose-dependently modulated. Furthermore, there was a marked improvement in pain behaviors. The withdrawal threshold and latency of hind paws significantly rose post-MB administration. Therefore, these results demonstrate that MB lessens pain by significantly weakening neural excitability, which implies a strong possibility that this dye may be developed as a pain-relieving medication in the future. This is the first in vivo study to elucidate the effect of MB on nerves and pain relief.

Introduction

Methylene blue (MB) is a blue cationic thiazine dye, which was synthesized by German chemist Heinrich Caro in 1876 and applied at first for textile industry [1,2]. Shortly afterwards, MB began to unveil its medical talent and has been introduced as a medication to treat different medical disorders, including malaria [3], methemoglobinemia [4,5], vasoplegic syndrome or abnormal vasodilation [6,7], and even chronic itching and pain [8,9].

In 1968, a Russian surgeon, Rygick AN, introduced MB to patients with chronic pruritus ani (PA), who significantly improved after intradermal MB administration [10]. The doctor believed that this improvement was attributable to death of nerve terminals connected to the skin. An electron microscopic investigation indicated that there were no distinct nerve endings in the perianal skin of PA patents treated with subcutaneous MB [11].

More recently, a growing number of studies have revealed that MB is deeply associated with pain relief. MB has a critical role in abating inflammation by negatively regulating pro-inflammatory mediators in inflamed tissues and ultimately contributes to pain relief [[12], [13], [14], [15]]. Of note, Peng and collaborators reported for the first time that intradiscal MB application served to lessen pain in patients with chronic discogenic low back pain (CDLBP) [16]. After three years, they performed a randomized placebo-controlled trial in 72 eligible participants with the almost same methodology and reported also dramatic improvements [17].

However, this clinical study brought immediate refutations and controversies from other researchers regarding the efficacy and safety of MB [[18], [19], [20]]. Notably, Kallewaard and collaborators conducted a clinical trial almost identical to that of Peng et al., 2010 and directly refuted their outcomes [21]. Conversely, some studies reported the short-term effect of MB on CDLBP for three [22] or six [23] months post-treatment. Until now, the contribution of MB to pain reduction is controversial, further, basic investigations for the contribution have rarely been conducted.

Accordingly, in this study, we carried out in vivo experiments to get closer to clinical situations, and tried to demonstrate whether MB affects neural discharge, which is one of the most crucial factors for pain transmission, and ultimately alleviates pain. In vivo single nerve recordings were performed aimed at the saphenous nerve of rats, which is the largest cutaneous branch of the femoral nerve and is very useful for recording the activity of peripheral nerves, to measure changes in neural firing rates by time after MB administration. In addition, behavioral experiments were conducted to verify that MB actually reduces pain by examining the paw withdrawal responses of rats to noxious stimuli post-MB administration. Of note, MB was administered subcutaneously to the plantar area in both electrophysiological and behavioral experiments to explore the relation between neural firing patterns and pain behaviors.

Section snippets

Animals

Adult male rats (Sprague Dawley; 230–250 g; Orient Bio, South Korea) were used in all experiments and accommodated in an animal room under the controlled conditions (22–25 °C; 12-h dark/light cycle). Food and water were provided ad libitum to the animals. All experiments were conducted complying with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Korea University College of Medicine and all procedures related to the experiments were approved by the Committee

MB suppresses significantly neural firing and this effect lasts longer than that of lidocaine

In the first step, the maximum number of spikes per second (neural firing rate) evoked by mechanical stimulus, a 10-g von-Frey filament, was measured in MSAs at each time point after administration of saline (vehicle, 40 μl), lidocaine (0.02%, 40 μl), and MB (0.02%, 40 μl) to the receptive field adjacent to the right plantar surface (Fig. 1A and B).

There were little changes in neural firing rates in the saline (SAL) group (Fig. 1, Fig. 2A). On the contrary, in the lidocaine (LIDO) group, no

Discussion

The present study showed that neural discharge in MASs was significantly abated and ultimately eliminated after MB administration, displaying anesthetic-like firing patterns. Of note, the dye significantly suppressed AP generation longer than lidocaine. Moreover, pain behaviors were also markedly ameliorated following MB administration. In effect, the HPWT and HPWL significantly increased in hind paw-incised rats and the HPWL significantly increased even in naïve rats. Thus, our results

Declaration of competing interest

The authors declare that there are no competing interests or personal relationships that could have influenced the work reported in this paper.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number: NRF-2019R1I1A1A01063196).

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