Dexmedetomidine, a highly selective agonist of α2-adrenoceptors, is a potent sedative drug for critically ill patients in intensive care unit settings.1 However, dexmedetomidine not only has sedative potency but other actions have also been found. It was reported that α2-adrenoceptor agonists, including norepinephrine and clonidine, have a potent anti-inflammatory effect,2–5 inhibiting the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α),3–5 interleukin-1β,4 interleukin-1 receptor antagonist,2 and interleukin-6.3–5 It has been shown that generally administered dexmedetomidine had anti-inflammatory effects in several animal models,6,7 cultured cells,8,9 and in patients.10
Regarding the local injection of dexmedetomidine, 1 study11 showed that epidurally administered dexmedetomidine reversed inflammation induced by carrageenin. Furthermore, our previous study12 demonstrated the usefulness of dexmedetomidine as an addition to local anesthetics for enhancing the local anesthetic potency of lidocaine in guinea pigs. These findings suggest the usefulness of locally administered dexmedetomidine. Thus, the purpose of the present study was to evaluate the inhibitory effect of locally injected dexmedetomidine on local inflammatory responses, including edema, the accumulation of leukocytes, and production of TNF-α and cyclooxygenase-2 (COX-2), induced by an intraplantar injection of carrageenin into the paws of mice.
Carrageenin was purchased from Nacalai Tesque (Kyoto, Japan) and used as a 5% (weight/volume) solution, diluted by physiological saline. Dexmedetomidine (Precedex®) was purchased from Maruishi Pharmaceutical (Osaka, Japan). Yohimbine, a full antagonist of the α2-adrenoceptor, and phenylephrine, an agonist of the α1-adrenoceptor, were obtained from Sigma-Aldrich (St. Louis, MO).
The protocol of the present study was approved by the Animal Care and Use Committee of our institute. Male Crlj:CD1(ICR) mice (Japan SLC, Hamamatsu, Japan), aged 6 to 8 weeks, were obtained from Charles River Laboratories, Osaka, Japan. The animals were housed in steel cages in a room kept at 24°C with 50% ± 10% relative humidity under a 12-hour cycle of light and dark and fed a laboratory diet (CE-II, CLEA, Tokyo, Japan). Water was freely available. This study was done in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Science Research Center.
Injection of Tested Agents into the Hindpaws of Mice
Local inflammation was induced in the hindpaws of mice by intraplantar injection of 5% carrageenin at a volume of 20 μL with a 32-gauge needle. Dexmedetomidine was injected at final concentrations of 0.01, 0.1, 1, and 10 μM, combined with carrageenin. To ensure that the action of dexmedetomidine was via α2-adrenoceptors, we injected yohimbine at a final concentration of 10 μM, combined with carrageenin and dexmedetomidine. Furthermore, to evaluate the influence of the α1-adrenoceptor possibly being induced by dexmedetomidine, we also evaluated the effect of phenylephrine at a final concentration of 1 μM, using the same method as for dexmedetomidine.
To exclude the central effect of the agents, they were injected into the left and right paws in the following pairs: control solution (physiological saline) and carrageenin; carrageenin and carrageenin + dexmedetomidine; carrageenin and carrageenin + phenylephrine; carrageenin and carrageenin + yohimbine; carrageenin + dexmedetomidine and carrageenin + dexmedetomidine + yohimbine, respectively. The investigator who injected the agents was blinded to the injected agent, and the investigator who measured paw volume was also blinded.
Evaluation of Paw Edema
Paw volume was measured before and every 1 hour after the injection of agents for up to 6 hours, using water displacement plethysmometry (7140, Ugo Basile SRL, Comerio, Italy). The swelling value (percent edema) was the increase in paw volume expressed as a percentage of volume before carrageenin injection.
Furthermore, we compared the effects of dexmedetomidine among different concentrations. The ratio of the increasing volume of the paw injected with carrageenin + dexmedetomidine to that of the contralateral paw injected with only carrageenin was assessed in each mouse.
Preparation of Samples
Animals were euthanized 2, 4, and 6 hours after carrageenin injection by cervical dislocation. Hindpaws were excised and excess tissue was removed. Samples were fixed in 10% neutral buffered formalin. Afterward, the samples were embedded in paraffin, and sections were cut at a thickness of 5 μm for hematoxylin–eosin staining.
Regarding histological evaluation of the number of leukocytes, we used the same methods as described in a previous study.13 The evaluation was performed with a light binocular microscope and included a description of the observed tissue response. Additionally, 5 fields around each osseous defect were randomly captured using the ×200 magnification of a binocular microscope fixed with a charged coupled device camera to evaluate the number of leukocytes in the histologic sections. Images with 680 × 512 pixels, which corresponded to an area of 321.8 × 430.1 μm (138,406.18 μm2), were recorded in TIFF. The number of leukocytes was counted on ImageJ software (National Institute of Mental Health, Bethesda, MD). Images were converted to the grayscale and submitted to the binarization process in which nuclei were easily identified after being marked in black. Binarized images allowed the identification of the number of nuclei per field. We differentiated leukocytes from the other cells by the morphologic parameters of nuclei. We defined a foliaceous nucleus as a neutrophil, a rotund nucleus as a lymphocyte, and counted them as leukocytes. The cell that had a spindle-shaped nucleus was defined as a fibroblast, and it was excluded from the cell count. We also double-checked the morphology of nuclei and cells by using original hematoxylin–eosin staining images. For each image captured, the microscope, camera, and computer were calibrated according to a standardized procedure.
Similar to the comparisons of paw volume, we compared the effects of dexmedetomidine on the accumulation of leukocytes among different concentrations.
Immunohistochemistry for TNF-α and COX-2
For evaluation of expression of TNF-α and COX-2, we used paw samples 4 hours after injection of carrageenin, carrageenin + dexmedetomidine or carrageenin + dexmedetomidine + yohimbine. Sections (3 μm) were collected from the obtained samples and mounted on salinized slides for immunohistochemistry. In brief, sections were deparaffinized in a series of xylene for 15 minutes and rehydrated in graded ethanol solution. Endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 in methanol for 30 minutes. Antigen retrieval was achieved by heat treatment using 10-mM citrate buffer solution at pH 6.0. After treatment with normal serum, the sections were incubated with the primary antibodies for TNF-α and COX-2 (Abcam plc., Cambridge, United Kingdom) at 4°C. Tagging of the primary antibody was achieved by the subsequent application of envision peroxidase detecting reagent (Dako, Carpinteria, CA). Visualization of immunoreactivity was performed by developing the enzyme complex with diaminobenzidine/H2O2 solution (Histofine DAB substrate, Nichirei, Tokyo, Japan) and counterstained with Mayer hematoxylin. For the purpose of indicating how the used antibodies worked specifically, we performed duplicate tests by using the different antibodies for TNF-α (LifeSpan BioSciences, Inc., Seattle, WA) and COX-2 (Novus Biologicals, Littleton, CO).
Western Blotting Analysis for TNF-α and COX-2
Paw samples obtained 4 hours after injection of physiological saline, carrageenin, carrageenin + dexmedetomidine, or carrageenin + dexmedetomidine + yohimbine were pooled and homogenized in complete Lysis-M (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined using Coomassie PLUS™ (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using 10% Bis-Tris gels (Novex®, NuPAGE system, Carlsbad, CA) and antibodies against TNF-α (Cell signaling, Danvers, MA), COX-2 (BD Biosciences, San Jose, CA) and β-actin (Abcam plc.), a house-keeping protein. Horseradish peroxidase–conjugated secondary antibodies were used (GE Healthcare, Uppsala, Sweden) before detection with a chemiluminescent reagent (Amersham ECL Prime Western blotting detection system, GE Healthcare) and exposure to X-ray film. The results of Western blot analysis were scanned as TIFF files and quantified by measuring the relative intensity using the Analyze function of ImageJ software and were normalized to the band density of internal control (β-actin).
We compared TNF-α and COX-2 production in the paw samples receiving saline and carrageenin, carrageenin and carrageenin + dexmedetomidine, or carrageenin + dexmedetomidine and carrageenin + dexmedetomidine + yohimbine, respectively.
Sample size was defined by power analyses before the experiment started. Statistical analyses were performed using statistical analysis software (GraphPad Prism version 6, GraphPad Software, Inc., La Jolla, CA). We used 2-way analysis of variance (ANOVA) with agents as the main factor, time as repeated measure, and a post hoc Bonferroni test for comparisons of paw volume or the accumulation of leukocytes at each time point. We used 1-way ANOVA with a post hoc Bonferroni test for comparisons of paw volume and the accumulation of leukocytes at different concentrations, and the Mann-Whitney U test for comparisons of the quantity by Western blotting analysis. Significance was defined as P < 0.05.
Paw Edema in Mice
We first examined whether locally injected dexmedetomidine inhibits carrageenin-induced paw edema in mice. Paw volume significantly increased after the injection of carrageenin compared with physiological saline at 2 to 6 hours after injection (2-way ANOVA with post hoc Bonferroni test, the interaction of main factor and time was significant). Paw volume increased by about 80% of the baseline volume before the injection (Fig. 1A). Dexmedetomidine at 1 μM significantly inhibited the carrageenin-induced increase in paw volume at 3 to 6 hours after injection (2-way ANOVA with post hoc Bonferroni test, the interaction of main factor and time was also significant; Fig. 1B). We examined whether the inhibitory effect of dexmedetomidine depends on the dexmedetomidine concentration. Dexmedetomidine at 10 μM significantly inhibited the increase in paw volume compared with the low concentration (P = 0.0022, 1-way ANOVA with Bonferroni test; Fig. 2). Moreover, we examined whether phenylephrine, an agonist of the α1-adrenoceptor, or yohimbine, an antagonist of the α2-adrenoceptor, influences carrageenin-induced paw edema. Additionally, we examined whether yohimbine antagonizes the inhibitory effect of dexmedetomidine on carrageenin-induced edema. Phenylephrine at 1 μM had no significant effect on the carrageenin-induced increase in paw volume (Fig. 1C). Yohimbine at 10 μM significantly antagonized the inhibitory effect of dexmedetomidine at 2 to 6 hours after injection (2-way ANOVA with post hoc Bonferroni test, the interaction of main factor and time was significant; Fig. 1D); however, only yohimbine did not significantly influence carrageenin-induced paw edema (Fig. 1E).
Accumulation of Leukocytes
Next, we analyzed the effect of dexmedetomidine on leukocyte infiltration. We examined whether locally injected dexmedetomidine decreases the level of leukocytes induced by carrageenin injection.
Leukocytes were not found in the injected region of physiological saline-injected samples (data not shown), but accumulated in carrageenin-injected samples. Dexmedetomidine at 1 μM significantly inhibited the accumulation of leukocytes at 4 and 6 hours after injection (2-way ANOVA with post hoc Bonferroni test, the interaction of main factor and time was significant; Fig. 3A). Furthermore, we examined whether the inhibitory effect of dexmedetomidine on leukocyte infiltration depends on the dexmedetomidine concentration. Dexmedetomidine at 0.1, 1, and 10 μM inhibited the accumulation of leukocytes in the injected paw compared with the low concentration, respectively (P < 0.0001, 1-way ANOVA with Bonferroni test; Fig. 4). As in the paw edema experiment, we examined whether phenylephrine or yohimbine influenced leukocyte infiltration, and also examined whether yohimbine antagonized the inhibitory effect of dexmedetomidine on leukocyte infiltration. Phenylephrine at 1 μM had no significant effect on the accumulation of leukocytes (Fig. 3B). Yohimbine at 10 μM significantly antagonized the inhibitory effect of dexmedetomidine on the accumulation of leukocytes at 4 and 6 hours after injection (2-way ANOVA with post hoc Bonferroni test, the interaction of main factor and time was significant; Fig. 3C), however, only yohimbine had no significant effect on the accumulation of leukocytes (Fig. 3D).
Production of TNF-α and COX-2
Finally, we examined whether dexmedetomidine inhibits the production of TNF-α and COX-2 in the injected region, and yohimbine antagonizes this inhibitory effect of dexmedetomidine. TNF-α and COX-2 production was immunohistochemically identified in the subcutaneous region of the hindpaw receiving carrageenin 4 hours after injection (Fig. 5A). In the carrageenin + dexmedetomidine-injected sample, the production of TNF-α and COX-2 was lower compared with that in the carrageenin-injected sample (Fig. 5B). However, in the carrageenin + dexmedetomidine + yohimbine-injected sample, the expression of TNF-α and COX-2 retuned to the same level as in the carrageenin-injected sample (Fig. 5C). Duplicate tests yielded similar findings (data not shown).
In Western blotting analysis, TNF-α (trimeric form) was found to be induced by carrageenin compared with saline (P = 0.0079, Mann-Whitney U test; Fig. 6B) and was significantly inhibited by dexmedetomidine (P = 0.035, Mann-Whitney U test; Fig. 6B). Furthermore, yohimbine significantly antagonized the inhibitory effect of dexmedetomidine on the production of TNF-α (P = 0.028, Mann-Whitney U test; Fig. 6B).
COX-2 was also induced by carrageenin (P = 0.015, Mann-Whitney U test; Fig. 6D), and was significantly inhibited by dexmedetomidine (P = 0.015, Mann-Whitney U test; Fig. 6D). Furthermore, yohimbine also significantly antagonized the inhibitory effect of dexmedetomidine on COX-2 production (P = 0.031, Mann-Whitney U test; Fig. 6D).
Dexmedetomidine has been reported to reduce brain dysfunction and the mortality rate in septic patients.14 The authors of that study suggested that the effect of dexmedetomidine on brain dysfunction and mortality is due to its anti-inflammatory action. Similarly, dexmedetomidine has been reported to reduce inflammatory responses in septic patients.15–17 Thus, dexmedetomidine is gaining attention as a candidate for anti-inflammatory therapy in the clinical setting.
How does the local injection of dexmedetomidine have anti-inflammatory effects? It has been shown that some α2-adrenoceptor agonists including dexmedetomidine modulate the production of cytokines by macrophages, monocytes, or primary microglia.2,8,9 Other possible mechanisms are the inhibition of apoptotic cell death,18 enhancement of the phagocytic ability of macrophages,19,20 and stimulation of the cholinergic anti-inflammatory pathway was also discussed.4,10 However, the anti-inflammatory mechanism of dexmedetomidine remains unclear. Because dexmedetomidine is well known to have effects on the central nervous system (CNS), it is possible that its anti-inflammatory effect occurs via the CNS. The central effect of the α2 agonist via its central sympatholytic action on peripheral inflammation has been reported.21,22 Thus, dexmedetomidine is thought to have direct and/or indirect effects on inflammatory responses via the CNS. In the present study, dexmedetomidine + carrageenin and only carrageenin were injected into the left and right paws of a mouse, respectively, and the effects were compared. Even if dexmedetomidine had central effects, these effects were equally visible in paws on both sides. Thus, in the present study, the effect of dexmedetomidine via the CNS was excluded. However, it had already been shown that local inflammation had bilateral consequences, and that local treatment could have systemic and/or contralateral effects.23–25 Therefore, the anti-inflammatory effect of dexmedetomidine that we noted possibly included an effect via the peripheral nervous system, and/or other systemic factors, not only local effects. However, it was clear that dexmedetomidine had an anti-inflammatory effect mediated by α2-adrenoceptors, because the effect was clearly inhibited by yohimbine, a full antagonist of α2-adrenoceptors.
In our study, we used carrageenin to induce the local inflammation. Carrageenin-induced acute inflammation is a very good model to investigate the inflammatory phase associated with the infiltration of phagocytes and overproduction of free radicals, as well as the release of inflammatory mediators such as TNF-α, COX-2, and inducible nitric oxide synthase.26 Carrageenin-induced acute inflammation reaches a peak 2 to 3 hours after injection, and the inflammation resolves within 24 to 74 hours.27–29 This carrageenin-induced inflammation has been described as a biphasic event in which various mediators play roles to produce an inflammatory response.30,31 The initial phase of the inflammation (0–1 hours) has been attributed to the release of histamine, 5-hydroxytryptamine, and bradykinin.32 However, the delayed phase has been related to the increased production of prostaglandins and COX-2,32,33 and it is expected that TNF-α is also associated with delayed phase inflammation.26,31 Our results indicate that dexmedetomidine had a potent anti-inflammatory effect in the delayed phase, because it significantly inhibited the carrageenin-induced increase in paw volume 3 to 6 hours after injection, and not 1 to 2 hours after injection. Moreover, we also showed that dexmedetomidine inhibited the production of COX-2 and TNF-α (Figs. 5 and 6). From these findings, we speculate that dexmedetomidine inhibited some factors that were related to the production of COX-2 and TNF-α and produced an anti-inflammatory effect. However, the exact mechanism of the anti-inflammatory effect of dexmedetomidine was not investigated in our study.
Dexmedetomidine causes clinical sedation when its plasma concentrations reach approximately 0.005 μM (1 ng/mL).34 In the present study, dexmedetomidine at a concentration >0.1 μM was effective against acute inflammatory responses induced by carrageenin (Figs. 2 and 4). These concentrations are higher than clinical plasma concentrations. In our models, we injected dexmedetomidine into paws subcutaneously. In these models, it is very difficult to compare our effective dose with the clinical plasma concentration, because the final concentration of the locally injected dexmedetomidine in tissue depends on several factors that relate to its absorption. In some studies8,9 using animal cells, dexmedetomidine at a clinical dosage had no significant effect on lipopolysaccharide-induced inflammatory cytokine production, including TNF-α, interleukin-1β, interleukin-6, inducible nitric oxide synthase, and prostaglandin E2. These findings suggest that a relatively high concentration is necessary for inhibitory effects on the production of mediators associated with inflammation.
In conclusion, the present results indicate that locally injected dexmedetomidine at a relatively high concentration inhibits local acute inflammatory responses, including edema, the accumulation of leukocytes, and production of TNF-α and COX-2, induced by an intraplantar injection of carrageenin into the paws of mice. The findings suggest that the local injection of dexmedetomidine has a potent anti-inflammatory effect on local acute inflammatory responses, mediated by α2-adrenoceptors. Furthermore, dexmedetomidine can be a useful candidate additive to a local anesthetic for not only enhancing anesthetic potency,12 but also inducing anti-inflammatory potency.
Name: Shintaro Sukegawa, DDS.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Shintaro Sukegawa has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is responsible for archiving the study files.
Name: Hitoshi Higuchi, DDS, PhD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Hitoshi Higuchi has seen the original study data and approved the final manuscript.
Name: Miho Inoue, PhD.
Contribution: This author helped conduct the study.
Attestation: Miho Inoue has seen the original study data and approved the final manuscript.
Name: Hitoshi Nagatsuka, DDS, PhD.
Contribution: This author helped histological evaluation pathologically.
Attestation: Hitoshi Nagatsuka has seen the original study data and approved the final manuscript.
Name: Shigeru Maeda, DDS, PhD.
Contribution: This author helped analyze the data.
Attestation: Shigeru Maeda has seen the original study data and approved the final manuscript.
Name: Takuya Miyawaki, DDS, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Takuya Miyawaki has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is responsible for archiving the study files.
This manuscript was handled by: Martin S. Angst, MD.
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