Systemic administration of lidocaine has been used to relieve neuropathic pain, including that from malignant and nonmalignant disorders.1,2 Abnormal expression of voltage-gated sodium channels in both injured and neighboring areas correlates with ectopic activity3 that has been proposed as a mechanism underlying neuropathic pain,4 suggesting the role of blockade of specific sodium channels by intravenous lidocaine to produce neuropathic pain relief.5 However, lidocaine has other several beneficial effects in some clinical situations such as postoperative pain relief by epidural administration, stimulation of bowel function after colon surgery,6 and topical anesthesia in addition to properties including antithrombotic7 and anti-inflammatory actions,8 which are potentially important during the perioperative period. These multiple effects of lidocaine indicate that there may be mechanisms other than sodium channel blockade. Moreover, the difference of effective concentrations between intravenous and epidural administration also supports this possibility. Indeed, several reports demonstrated that lidocaine affects other pain signaling pathways,9 receptors, and ion channels including Gq protein,10 potassium channels,11 and calcium channels.12
Extracellular adenosine triphosphate (ATP) is a neurotransmitter acting through ATP receptors, which are classified as ligand-gated ion channels (P2X receptors) and G-protein–coupled receptors (P2Y receptors). Seven P2X (P2X1–7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–14) subunits have been identified.13 They are distributed in multiple organs and have important roles in various physiologic functions.14,15 Recently, specific receptor subunits have been shown to be involved in various pathologic conditions, including brain ischemia, pain, inflammation, osteoporosis, spinal cord injury, and bladder dysfunction; therefore, these receptors are considered to be potential therapeutic targets.16–19 It is especially suggested that several subunits including P2X3, P2X4, and P2X7 receptors, which are distributed in pain pathways within the nervous system, are involved in chronic pain mechanisms.20
P2X3 receptors, which are mainly distributed in sensory neurons such as dorsal root ganglia, have been shown to be involved in the mechanism of neuropathic pain by demonstrating that intrathecal treatment with P2X3 antisense oligonucleotide decreased nociceptive behavior in a model of chronic inflammation and reduced mechanical allodynia in a rat model of neuropathic pain.21 P2X4 receptors are widely expressed in the brain, spinal cord, autonomic and sensory ganglia, and microglia. Several reports demonstrated that upregulation of P2X4 receptors in activated microglia located in the dorsal horn of the spinal cord contributes to neuropathic pain.22 P2X7 receptors are expressed on cells of the immune system as well as glial cells. In addition, inflammatory and neuropathic hypersensitivities in response to mechanical and thermal stimuli were completely absent in mice lacking P2X7 receptors,23 suggesting a role of P2X7 receptors in pain modulation.
Although previous reports have demonstrated the effects of some general anesthetics, ethanol, and antidepressants on P2X receptors,24–28 no studies have investigated whether local anesthetics act on these receptors. Therefore, we investigated the effects of lidocaine and other local anesthetics on P2X3, P2X4, and P2X7 receptors to explore the mechanisms underlying the pain-relieving effects of lidocaine.
This study was approved by the Animal Research Committee of the University of Occupational and Environmental Health, Kitakyushu, Japan.
All chemicals, including ATP disodium salt, lidocaine hydrochloride, ropivacaine hydrochloride, bupivacaine hydrochloride, benzocaine, N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314), AZ11645373, and Brilliant Blue G (BBG), were purchased from Sigma-Aldrich (St. Louis, MO).
All plasmids including human P2X3, P2X4, and P2X7 receptor complementary DNA (cDNA) were purchased from OriGene Technologies (Rockville, MD).
cRNA Preparation and Oocyte Injection
After double digestion of cDNA with SacI and SmaI (P2X3 receptor) or linearization with SacI (P2X4 and P2X7 receptors), complementary RNAs (cRNA) were transcribed using T7 RNA polymerase using an mMESSAGE mMACHINE kit (Ambion, Austin, TX). Adult female Xenopus laevis frogs were obtained from Kyudo (Saga, Japan). X. laevis oocytes and cRNA microinjection were prepared and performed as described previously.29,30 In brief, stage IV to VI oocytes were manually isolated from a removed portion of ovary. Next, oocytes were treated with collagenase (0.5 mg/mL) for 10 minutes and placed in modified Barth solution (88 mmol/L NaCl, 1 mmol/L KCl, 2.4 mmol/L NaHCO3, 10 mmol/L HEPES, 0.82 mmol/L MgSO4, 0.33 mmol/L Ca[NO3]2, and 0.91 mmol/L CaCl2, adjusted to pH 7.5), supplemented with 10,000 U penicillin, 50 mg gentamicin, 90 mg theophylline, and 220 mg/L sodium pyruvate (incubation medium). cRNAs of P2X receptors were injected (total volume was 0.5–20 ng/50 nL) into Xenopus oocytes. Injected oocytes were incubated at 19°C in incubation medium, and 2 to 6 days after injection, the cells were used for electrophysiologic recordings.
All electrical recordings were performed at room temperature (20–23°C). Oocytes were placed in a 100-μL recording chamber and perfused at 2 mL/min with extracellular Ringer’s solution (110 mmol/L NaCl, 2.5 mmol/L KCl, 10 mmol/L HEPES, 1.8 mmol/L BaCl2, pH 7.5) using a peristaltic pump (World Precision Instruments, Sarasota, FL). Ca2+ in the solution was replaced with Ba2+ to prevent the activation of Ca2+-dependent Cl− channels.31,32 Recording electrodes were prepared with borosilicate glass capillary tubing using a puller (PP-830; Narishige, Tokyo, Japan); microelectrodes had a resistance of 1 to 3 MΩ when filled with 3 mol/L KCl. Whole-cell voltage clamp was achieved using these two electrodes with a Warner Instruments model OC-725C system (Warner, Hamden, CT) at −70 mV. Local anesthetics, BBG, and ATP disodium salt stocks were prepared and diluted before adding to the bath solution. AZ11645373 stocks were prepared in dimethylsulfoxide and diluted in bath solution to a final dimethylsulfoxide concentration not exceeding 0.05%. We measured the peak of the transient inward current in response to ATP that was applied for 20 seconds. Local anesthetics were preapplied for 2 minutes to allow a complete change of bath solution.
To characterize the inhibitory effect of lidocaine on P2X7 receptors, we measured ATP-induced currents at 10 μmol/L to 5 mmol/L in the presence or absence of 300 μmol/L lidocaine. The quaternary local anesthetic QX-314, which is 99.9% permanently charged and does not penetrate the cell membrane, was either injected directly into oocytes or applied outside the cell to identify whether it acts intracellularly or extracellularly. QX-314 (50 nL of 5 mmol/L diluted in 150 mmol/L KCl) was injected into oocytes to result in an intracellular concentration of approximately 500 μmol/L although, in practice, the intracellular concentration in each oocyte may be variable because the oocytes are heavily compartmentalized. Control cells were injected with 150 mmol/L KCl. Recordings were performed 10 minutes after injection. To investigate the potential use-dependent effects of lidocaine, ATP was applied at 5-minute intervals in the continuous presence of 100 μmol/L lidocaine for 30 minutes. The results are expressed as percentages of control responses.
GraphPad Prism software (GraphPad Software, San Diego, CA) was used to conduct the statistical analysis. All values are presented as means ± SEM. The n values refer to the number of oocytes examined. Each experiment was performed with oocytes from at least two frogs.
The concentration–response curves for the ATP-induced peak current were fitted using the logistic function: I = I min + (I max − I min)/[1 + (EC50/A)n], where I is the response induced by ATP concentration, I min is the minimal response, I max is the maximal response, EC50 is the half maximal effective concentration, A is the concentration of ATP, and n is the Hill coefficient. Differences were evaluated statistically using unpaired, two-tailed t test and one-way analysis of variance followed by Dunnett multiple comparison test, where the objective is to identify groups whose means are significantly different from the mean of a selected “reference group,” in our case, no treatment with local anesthetics, or Tukey multiple comparison test, where the mean of each group is compared with the mean of every other group. Hill coefficient, half maximal inhibitory concentration (IC50), and EC50 values were also calculated. Values of P < 0.01 were taken as showing a significant difference.
Effects of Lidocaine on Peak ATP-Induced Inward Currents in the P2X3, P2X4, and P2X7 Receptors
We determined the ATP concentration–response relation under our experimental conditions for P2X3, P2X4, and P2X7 receptors. Nonlinear regression analyses of the curves yielded the EC50 for ATP and slope variables (Hill coefficient) of 2.3 ± 0.8 µmol/L and 0.74 ± 0.1 in oocytes expressing P2X3 receptors, 10.8 ± 3.3 µmol/L and 0.69 ± 0.2 in oocytes expressing P2X4 receptors, and 1.2 ± 0.1 mmol/L and 3.7 ± 0.9 in oocytes expressing P2X7 receptors, respectively (Fig. 1). Based on these results, the effects of lidocaine on ATP-induced currents were examined at an ATP concentration of 2 μmol/L for P2X3 receptor, 10 μmol/L for P2X4 receptor, and 1 mmol/L for P2X7 receptor. Figure 2B shows concentration–response relations of lidocaine-mediated inhibition on ATP-induced currents in three receptors. Lidocaine inhibited the currents in a concentration-dependent manner in oocytes expressing P2X7 receptor; the IC50 value of lidocaine for ATP-induced currents and the slope were 282 ± 45 µmol/L and 0.72 ± 0.07, respectively (Table 1). These inhibitory effects were significant at concentrations of lidocaine ≥30 μmol/L (Fig. 2B). By contrast, the inhibitory effects of lidocaine on the P2X3 and P2X4 receptors were limited. Specifically, only a high concentration of 10 mmol/L lidocaine significantly suppressed ATP-induced currents to 68 ± 6% and 71 ± 6% of the control in oocytes expressing P2X3 and P2X4 receptors, respectively (Fig. 2B).
Effects of Other Local Anesthetics on the P2X7 Receptor
We next examined the effects of other local anesthetics including mepivacaine, ropivacaine, and bupivacaine on ATP-induced currents in oocytes expressing P2X7 receptor because lidocaine inhibited this receptor most strongly. Although mepivacaine suppressed ATP-induced currents in a concentration-dependent manner, the inhibitory effects were less than those of lidocaine with the IC50 value and slope variable of 6.0 ± 0.06 mmol/L and 0.62 ± 0.06, respectively. This suggests that the inhibitory potency of mepivacaine was one-twentieth of that of lidocaine (Fig. 3, Table 1). By contrast, ropivacaine and bupivacaine had little effect on ATP-induced currents (Fig. 3).
Characterization of the Inhibitory Effects of Lidocaine on the P2X7 Receptor
To determine whether lidocaine competes with ATP for the P2X7 receptor, we next investigated the effects of 300 μmol/L lidocaine on the ATP concentration–response curve. ATP-induced currents at concentrations of 10 μmol/L to 5 mmol/L were measured in the absence and presence of lidocaine. As shown in Figure 4, lidocaine-mediated inhibition was not overcome by increasing the ATP concentration. In addition, lidocaine significantly reduced the E max value (maximal response) of the ATP concentration–response curve to 49% ± 4% (P < 0.0001; 99% confidence interval [CI], −65.7 to −35.1). The EC50 and slope variables in the absence and presence of lidocaine were 1.2 ± 0.1 mmol/L and 3.7 ± 0.9, and 1.4 ± 0.2 mmol/L and 4.8 ± 1.1, respectively. Thus, lidocaine significantly suppressed E max without significantly changing EC50 (P = 0.385; 99% CI, −0.279 to 0.665) and slope variables (P = 0.483; 99% CI, −2.165 to 4.268), suggesting noncompetitive inhibition.
Effects of Charged and Uncharged Local Anesthetics on the P2X7 Receptor
We next assessed the effects of QX-314, which is a permanently charged and non–membrane-permeable lidocaine analog, on the P2X7 receptor. When applied extracellularly, QX-314 inhibited ATP-induced currents in a concentration-dependent manner; the IC50 value and slope variable were 500 ± 75 μmol/L and 0.36 ± 0.03, respectively (Fig. 5B, Table 1). Moreover, intracellular QX-314 injection also significantly suppressed ATP-activated currents to 51 ± 9% of the control, whereas injected 150 mmol/L KCl had no effect (112 ± 11% of the control) (Fig. 5C). These results suggest that QX-314 can act on the P2X7 receptor both extracellularly and intracellularly, and the charged lidocaine can suppress the function of P2X7. Therefore, we investigated whether charge is required for the inhibitory actions of local anesthetics on the P2X7 receptor by measuring the effects of benzocaine, a local anesthetic that is almost completely uncharged and highly membrane permeable. Benzocaine suppressed the response to ATP in a concentration-dependent manner with the IC50 value and the slope variable of 1.6 ± 0.04 mmol/L and 0.88 ± 0.07, respectively (Fig. 5B, Table 1). These data suggest that both charged and uncharged local anesthetics can suppress P2X7 receptor function although it remains unclear whether benzocaine acts intracellularly or extracellularly.
Analyzing the Use Dependency of Lidocaine-Mediated Inhibition of the P2X7 Receptor
We assessed whether the effects of lidocaine on the P2X7 receptor were use-dependent because local anesthetics exhibit use-dependent block of voltage-gated sodium channel function, ATP was applied to oocytes at 5-minute intervals in the absence or continuous presence of 100 μmol/L lidocaine for 30 minutes (Fig. 6A). In the continuous presence of lidocaine, the response to the second application of ATP (after 5 minutes) was significantly reduced to 66 ± 3% of the current induced by the first application (0 minute), and the inhibitory effect of the seventh application (30 minutes) was similar to that of the second application (5 minutes) (Fig. 6, B and C). Therefore, these results revealed the effectiveness of lidocaine as it reached steady state in the second application of ATP (5 minutes), suggesting that lidocaine-mediated inhibition of the P2X7 receptor is use-dependent. More potent inhibition might be observed in the second application compared with the first because lidocaine would be able to access its site of action sufficiently during a 5-minute treatment, whereas the first application in which lidocaine and ATP were simultaneously applied (no lidocaine pretreatment) would not be sufficient for lidocaine to access its site of action.
Effects of Selective P2X7 Receptor Antagonists on the Inhibitory Actions of Lidocaine
We next investigated the action of lidocaine in the absence and presence of selective antagonists of the P2X7 receptor, BBG, or AZ11645373 to determine whether these compounds modulate the inhibitory actions of lidocaine on the P2X7 receptor. Oocytes were pretreated with 1 μmol/L BBG or 300 nmol/L AZ11645373 2 minutes before coapplication of 10 μmol/L to 3 mmol/L lidocaine for 2 minutes (Fig. 7, A and B). Figure 7, C and D, shows normalized inhibition curves for lidocaine in the absence and presence of preapplied and coapplied BBG or AZ11645373. The IC50 values and slope variables of the lidocaine concentration–response curves with BBG or AZ11645373 were 315 ± 56 μmol/L and 0.92 ± 0.14, or 258 ± 52 μmol/L and 0.93 ± 0.18, respectively. This suggests that neither BBG nor AZ11645373 modulated the effects of lidocaine, which exhibited an IC50 value and slope variable of 282 ± 45 μmol/L and 0.72 ± 0.07, respectively (P = 0.658 and 0.237, or P = 0.736 and 0.309, respectively, for the IC50 values and the slope variables of the lidocaine concentration–response curves with BBG or AZ11645373, 99% CI, −132.7 to 198.7 and −0.161 to 0.561, or −134.6 to 182.6 and −0.235 to 0.655). Overall, these data suggest that lidocaine interacts with a different site on the P2X7 receptor from the sites of action of either BBG or AZ11645373, which are noncompetitive antagonists of the P2X7 receptor.
In the present study, we demonstrated that lidocaine selectively inhibited ATP-induced inward currents of the P2X7 receptor in a concentration-dependent manner. To our knowledge, this study is the first direct evidence that lidocaine suppresses the P2X7 receptor. Substantial pain relief is achieved at plasma concentrations of 2 to 5 μg/mL (7–20 μmol/L) by continuous infusion of lidocaine in cancer patients with neuropathic pain.33 In the present study, the IC50 value of lidocaine-mediated P2X7 inhibition was 282 ± 45 μmol/L. Lidocaine tended to suppress ATP-induced currents at concentrations ≥10 μmol/L, and these inhibitory effects were significant at concentrations ≥30 μmol/L (P < 0.001). Although it is not proven whether a small inhibitory effect (7% inhibition) of lidocaine at 10 μmol/L produces pain relief in systemic administration, lidocaine might suppress P2X7 function at least when it is administered locally such as epidural administration because P2X7 receptors are expressed on glial cells in spinal neurons.
Lidocaine had little effect on the P2X3 and P2X4 receptors, but selectively suppressed the function of P2X7 receptors. The P2X7 receptor is structurally distinct from other P2X receptors; it also has different gating properties although these are only poorly understood.34 The P2X7 receptor is permeable not only to small cations (sodium, potassium, and calcium) as similar to other subunits but also to larger cations such as N-methyl-D-glucamine and nanometer-sized dyes,35 probably through the progressive dilation of the pore or the opening of a distinct accessory channel.36 Therefore, it is of great interest to explore how lidocaine inhibits the function of the P2X7 receptor alone. Lidocaine would not affect three ATP-binding sites that are shown to exist in the extracellular region of P2X receptors37 because lidocaine-mediated inhibition was found to be noncompetitive in this study. Moreover, lidocaine-mediated inhibition of the P2X7 receptor was use-dependent. Taken together, these findings suggest that lidocaine exerts its effects by affecting the site in the ion channel pore. By contrast, the inhibitory effects of QX-314 and benzocaine suggest that both charged and uncharged local anesthetics can also suppress P2X7 function. Moreover, intracellular injection of the charged local anesthetic QX-314 also inhibited P2X7 function. These results suggest that lidocaine acts on both intracellular and extracellular sites of the P2X7 receptor and that both charged and uncharged lidocaine could modulate this receptor.
We demonstrated that other local anesthetics including mepivacaine, ropivacaine, and bupivacaine have only limited effects on P2X7 receptor function. All these three compounds, but not lidocaine, contain a piperidine ring (Fig. 8), to which is attached a carbon chain of different lengths; one carbon in mepivacaine, three in ropivacaine, and four in bupivacaine. Therefore, it is possible that the piperidine ring is an obstacle to the action of local anesthetics because the inhibitory potency of mepivacaine was one-twentieth of that of lidocaine. Moreover, longer carbon chains connected to the piperidine ring may further hinder potential effects because both ropivacaine and bupivacaine had little effect on the P2X7 receptor. Therefore, it is possible that lidocaine suppresses P2X7 receptor function by acting on a binding site in the ion channel. Recent X-ray crystal structural analyses of P2X4 receptor in zebrafish revealed that P2X receptors exhibit homotrimeric architecture and that each subunit consists of a large hydrophilic extracellular domain and a transmembrane domain composed of two α-helices, which resemble the shape of a dolphin.38,39 These analyses also revealed that binding of ATP to the ATP-binding pocket within an intersubunit cleft rotates each subunit, resulting in promotion of the ion channel pore opening. P2X3, P2X4, and P2X7 are 40% to 50% identical in amino acid sequence, and each subunit of the P2X7 receptor has a longer amino acid sequence (595) than P2X3 (397) and P2X4 (388) because only P2X7 has a long intracellular C-terminus. Therefore, lidocaine might bind to the binding pocket for local anesthetics that exists mainly in the P2X7 receptor because of the differences of amino acid sequence to prevent the subunits from rotating that leads to channel opening.
Many P2X7 receptor antagonists have been reported in addition to antagonists of other P2X receptor subunits although they are not used clinically.34 Some antagonists, including pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid and periodate-oxidized ATP, were demonstrated to interact at the ATP-binding sites, showing that they were competitive antagonists.40 By contrast, many of them inhibit P2X7-mediated responses by acting in a noncompetitive manner. We examined whether BBG and AZ11645373 interact with the inhibitory action of lidocaine on P2X7 receptors because these two compounds are selective P2X7 inhibitors and act in a similar noncompetitive manner to lidocaine. BBG was shown to produce a noncompetitive inhibition of rat P2X7 receptors more potently than human P2X7 receptors; IC50 values were 10 and 200 nmol/L, respectively,41 whereas it was reported that AZ11645373 is a highly selective and potent antagonist at human, but not at rat, P2X7 receptors.42 Our data suggested that lidocaine acts on a different site of the P2X7 receptor than both BBG and AZ11645373.
The systemic administration of lidocaine has been considered to reduce neuropathic pain via suppression of ectopic activity by inhibiting abnormally expressed voltage-gated sodium channels.5 One study demonstrated that the incidence of C-fibers with ongoing activity was significantly reduced by systemic administration of lidocaine resulting in low plasma concentrations in a chronic inflammatory model.43 However, it was also shown that this effect was only partially correlated with chronic pain relief compared with the complete inhibition of spontaneous discharge in C-fibers, suggesting that there may be mechanisms other than blocking sodium channel activity. Moreover, many reports demonstrate that lidocaine also affects other pain signaling pathways.6 It has been demonstrated that intravenous lidocaine infusion increased acetylcholine concentrations in cerebrospinal fluid, which exacerbated inhibitory descending pain pathways resulting in analgesia.44 Lidocaine has also been shown to produce central inhibitory effects via spinal strychnine-sensitive glycine receptors45 and to stimulate the release of endogenous opioids to promote its analgesic effect.46 Moreover, lidocaine has been reported to reduce the postsynaptic depolarization mediated by N-methyl-D-aspartate and neurokinin receptors,47 and a study in a recombinant model demonstrated that local anesthetics, including lidocaine, directly inhibited the activation of human N-methyl-D-aspartate receptors in a concentration-dependent manner.48
The P2X7 receptor is expressed predominantly on immune cells and has been shown to play an important role in the inflammatory response by demonstrating that activation of the P2X7 receptor leads to maturation and release of interleukin (IL)-1β and initiation of a cytokine cascade.49,50 Several reports also suggest a role for the P2X7 receptor in pain modulation because systemic administration of selective antagonists of P2X7 receptors produced antinociceptive effects,51 and hypersensitivity was not observed in P2X7-knockout mice52 in neuropathic and inflammatory pain models. In addition, some reports suggest that lidocaine exerts anti-inflammatory effects by suppressing cytokine-induced injury53 or attenuating the production of proinflammatory cytokines including tumor necrosis factor-α, IL-1β, and IL-6 induced by extracellular ATP in microglia.54 Taken together, these data indicate that P2X7 receptor antagonists might be beneficial for the treatment of neuropathic and inflammatory pain. Therefore, suppression of the P2X7 receptor might be a mechanism underlying the anti-inflammatory effects and chronic pain relief affected by lidocaine. Although our present results suggest that the P2X3 and P2X4 receptors are not involved in the mechanism of lidocaine-induced pain relief, further experiments are needed to investigate the effects on other subunits including P2X2/3 or P2Y12, which are also related to chronic pain. Although ropivacaine and bupivacaine can relieve neuropathic pain effectively by epidural administration, P2X7 signaling would not be involved in their mechanism underlying pain relief.
In conclusion, lidocaine selectively inhibited ATP-induced currents of P2X7 receptors expressed in Xenopus oocytes at clinically relevant concentrations when it was administered locally at least. The effect of lidocaine on P2X7 receptors was likely a result of noncompetitive inhibition at both extracellular and intracellular sites in the ion channel pore. To our knowledge, these results are the first evidence showing novel lidocaine-mediated effects on P2X receptors in a recombinant experimental system and might become the key to elucidate the mechanisms of pain relief by lidocaine. However, further studies are needed to clarify the relevance of P2X7 receptor inhibition of the analgesic effects of lidocaine.
Name: Dan Okura, MD.
Contribution: This author conducted data collection, data analysis, and manuscript preparation.
Attestation: Dan Okura approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Takafumi Horishita, MD, PhD.
Contribution: This author helped study design, data collection, data analysis, and manuscript preparation.
Attestation: Takafumi Horishita approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript and also is the archival author.
Name: Susumu Ueno, MD, PhD.
Contribution: This author helped design the study, and prepare the manuscript.
Attestation: Susumu Ueno approved the final manuscript.
Name: Nobuyuki Yanagihara, PhD.
Contribution: This author helped prepared the manuscript.
Attestation: Nobuyuki Yanagihara approved the final manuscript.
Name: Yuka Sudo, PhD.
Contribution: This author helped prepare the manuscript.
Attestation: Yuka Sudo approved the final manuscript.
Name: Yasuhito Uezono, MD, PhD.
Contribution: This author helped prepare the manuscript.
Attestation: Yasuhito Uezono approved the final manuscript.
Name: Tomoko Minami, MD.
Contribution: This author helped prepare the manuscript.
Attestation: Tomoko Minami approved the final manuscript.
Name: Takashi Kawasaki, MD, PhD.
Contribution: This author helped prepare the manuscript.
Attestation: Takashi Kawasaki approved the final manuscript.
Name: Takeyoshi Sata, MD, PhD.
Contribution: This author helped conduct the study and prepare the manuscript.
Attestation: Takeyoshi Sata approved the final manuscript.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
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