Search

Methylene blue (MB) has been shown to reduce mortality and morbidity in vasoplegic patients after cardiac surgery. Though MB is considered to be safe, extravasation of MB leading to cutaneous toxicity has been reported. In this study, we sought to characterize MB-induced cutaneous toxicity and investigate the underlying mechanisms. To induce MB-induced cutaneous toxicity, we injected 64 adult male Sprague-Dawley rates with 200 µL saline (vehicle) or 1%, 0.1%, or 0.01% MB in the plantar hind paws. Paw swelling, skin histologic changes, and heat and mechanical hyperalgesia were measured. Injection of 1%, but not 0.1% or 0.01% MB, produced significant paw swelling compared to saline. Injection of 1% MB produced heat hyperalgesia but not mechanical hyperalgesia. Pain behaviors were unchanged following injections of 0.1% or 0.01% MB. Global transcriptomic analysis by RNAseq identified 117 differentially expressed genes (111 upregulated, 6 downregulated). Ingenuity Pathway Analysis showed an increased quantity of leukocytes, increased lipids, and decreased apoptosis of myeloid cells and phagocytes with activation of IL-1β and Fos as the two major regulatory hubs. qPCR showed a 16-fold increase in IL-6 mRNA. Thus, using a novel rat model of MB-induced cutaneous toxicity, we show that infiltration of 1% MB into cutaneous tissue causes a dose-dependent pro-inflammatory response, highlighting potential roles of IL-6, IL-1β, and Fos. Thus, anesthesiologists should administer dilute MB intravenously through peripheral venous catheters. Higher concentrations of MB (1%) should be administered through a central venous catheter to minimize the risk of cutaneous toxicity., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dr Banik has received book honorarium and not related to this specific topic. The authors declare no other conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Bradykinin (BK), which has potent algesic and sensitizing effect on nociceptors, is of current interest in understanding the mechanisms of chronic pain. BK response is mediated by B2 receptor in normal conditions; however, findings that B1 receptor blockade alleviated hyperalgesia in inflammation have been highlighting the role of B1 receptor in pathological conditions. It has not yet been clear whether nociceptor activities are modified by B1 receptor agonists or antagonists during inflammation. In addition, previous studies reported the change in BK sensitivity of nociceptors during short-lasting inflammation, and data in persistent inflammation are lacking. Therefore we investigated whether an experimentally induced persistent inflammatory state modulates the BK sensitivity of nociceptors and which receptor subtype plays a more important role in this condition. Complete Freund's adjuvant was injected into the rat-tail and after 2-3 wk, persistent inflammation developed, which was prominent in the ankle joint. Using an in vitro skin-saphenous nerve preparation, single-fiber recordings were made from mechano-heat sensitive C-fiber nociceptors innervating rat hairy hindpaw skin, and their responses were compared with those obtained from C-fibers tested similarly in normal animals. BK at 10(-8) M excited none of the 10 C-fibers in normal animals while it excited 5 of 11 (45%) C-fibers of inflamed animals, and at 10(-6) M BK excited all of the 11 inflamed C-fibers (or 94% of 36 tested C-fibers) but only 4 of 10 (or 45% of 58 tested C-fibers) in normal animals. Thus the concentration-response curves based on the incidence of BK induced excitation, and the total number of impulses evoked in response to BK were significantly shifted to the left. Moreover, an increased percentage of the inflamed C-fibers responded to 10(-6) M BK with bursting or high-frequency discharges. Thirty-percent of inflamed C-fibers had spontaneous activity, and these fibers showed comparatively less tachyphylaxis to consecutive second and third 10(-6) M BK stimulation. A B2 receptor antagonist (D-Arg-[Hyp3, Thi5,8,D-phe7]-BK) completely eliminated BK responses in inflamed rats, while B1 receptor antagonists (B 9958 and Des-Arg9-[Leu8]-BK) had no effect. Selective B1 receptor agonist (Des-Arg10-Kallidin) excited 46% (n = 13) of inflamed C-fibers at 10(-5) M concentration, which is 1,000 times higher than that of BK needed to excite the same percentage of inflamed C-fibers. We conclude that in chronically inflamed tissue, sensitivity of C-fiber nociceptors to BK, which is B2 receptor mediated, is strongly increased and that B1 receptor may not be important to a persistent inflammatory state, at least at the primary afferent level.

We investigated whether there are any differences between the Lewis and Sprague-Dawley (SD) rats in chronic inflammation-induced norepinephrine (NE) sensitivity of nociceptors. Activities of C-fiber nociceptors innervating rat hairy hindpaw skin were recorded in an in vitro skin-nerve preparation. Sixty-five percent of C-fibers from inflamed Lewis rats were excited by NE (10 microM), against only 38% of C-fibers from inflamed SD rats. The average of the total impulses evoked in response to NE was also significantly higher in Lewis rats. The alpha2-adrenoceptor antagonist CH 38083 (10 microM) and yohimbine (10 microM) consistently blocked the NE-excitation of both strains. These results show that after chronic inflammation, C-fiber nociceptors of Lewis strain rats have a stronger sensitivity to NE, and that alpha2-adrenoceptors are predominately involved in the NE-sensitivity of inflamed rats in both strains.