Local anesthetics (LAs) such as bupivacaine and lidocaine are used clinically to reversibly block nerve conduction by inhibiting voltage-gated Na+ (NaV) channels.1 Furthermore, LAs also inhibit a variety of ion channels, including voltage-gated K+ channels,2–4 2-pore domain K+ channels,5 G-protein–gated inward rectifier K+ channels,6 and hyperpolarization-activated cation (I h) channels,7,8 with 50% inhibitory concentrations (IC50) mostly in the tens of micromolar range. Because many of these channels are expressed in neuronal and cardiac tissues, the influence of LAs on their activity may be related to the regional and systemic side effects that occur after LA administration.
Strong inward rectifier K+ channels composed of subunits in the Kir2 subfamily are widely but differentially expressed in the central nervous system, including the spinal cord as well as in the peripheral nervous system.9–13 The channels expressed in neurons and glial cells are thought to play important roles in controlling neuronal excitability by regulating the resting membrane potential and extracellular K+ concentration, respectively.10 Previous studies demonstrated that LAs do not affect heterologously expressed Kir2 channels or native inward rectifier K+ channels that are considered to be composed of Kir2 subunits.2,6,14,15 However, most studies used low LA concentrations of ≤1 mM. During regional anesthesia, extremely large concentrations of LAs are administered in close proximity to neurons. For example, in Japan, 0.5% (approximately 15 mM) bupivacaine and 3% (approximately 110 mM) lidocaine are used in spinal anesthesia, during which LAs are injected into the small lumbar subarachnoid space where the spinal cord and spinal nerve roots are exposed. Bupivacaine is commonly used because of its beneficial long-lasting action with fewer side effects, whereas lidocaine is associated with rare but serious neurotoxic complications such as transient neurologic symptoms characterized by pain in the lower extremities.16,17 Mechanisms underlying the neuronal damage caused by LAs have been extensively studied but remain to be fully understood.18
In this study, we examined the effects of clinical concentrations of the amide-type LAs bupivacaine and lidocaine on human Kir2.1, Kir2.2, and Kir2.3 channels stably expressed in a human cell line. Both LAs inhibited Kir2 channels, an effect that may affect the excitability of neuronal cells. The results implied that the LAs disrupt the Kir2 channel function from the cytoplasmic side. Inhibition of Kir2 channels under clinical usage may be much stronger with lidocaine than with bupivacaine, given the slow onset of the latter.
Overview of the Experimental System
In the first 3 sections of the Results, we describe studying the effects of bupivacaine and lidocaine on human Kir2.1, Kir2.2, and Kir2.3 channels stably expressed in human embryonic kidney 293 (HEK293) cells by measuring whole-cell currents using a conventional patch-clamp method. The last section of the Results describes recordings of mouse Kir2.1 channels transiently expressed in HEK293T cells that were also made. The effects of the LAs in the cytoplasmic solution were examined by measuring the currents from excised inside-out patch membranes in this expression system. The mouse clone of Kir2.1 that gives a high expression level was used for inside-out experiments to allow macroscopic currents to be recorded from a small patch of membrane.19 It was verified in the whole-cell recordings that the species difference of Kir2.1 (human versus mouse) did not notably influence the susceptibility to the LAs (Supplemental Digital Content, Supplemental Table 1, http://links.lww.com/AA/B350).
Expression of Kir2.x Genes
HEK293 and HEK293T cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (200 U/mL), and streptomycin (200 mg/mL). Complementary DNA (cDNA) of human Kir2.1,20 human Kir2.2,20 and human Kir2.321 (all subcloned into the mammalian expression vector pCXN222) were individually transfected into HEK293 cells using Effectene (Qiagen, Venlo, The Netherlands), and the cell lines stably and functionally expressing the human Kir2 subtypes were selected from G418 (500 µg/mL)-resistant cell clones using an electrophysiologic method (see below). Mouse Kir2.1 cDNA23 (also subcloned into pCXN2 vector) was transiently transfected into HEK293T cells (which allow high-level recombinant protein expression because of stably expressed SV40 large T-antigen) using Effectene, together with enhanced green fluorescent protein cDNA to visualize the cells expressing exogenous proteins, as described before.19 cDNAs for human Kir2.3 cDNA and mouse Kir2.1 were kindly provided by Prof. C. Vandenberg (University of California, Santa Barbara, CA) and Prof. L. Y. Jan (University of California, San Francisco, CA), respectively.19
Patch-Clamp Recordings and Data Analysis
Preparation of Cells
To record whole-cell currents, HEK293 cells stably expressing human Kir2.1, Kir2.2, or Kir2.3 channels were treated with 0.25% trypsin-EDTA solution on the day of the experiments, and spherical single cells detached from the culture dish were used. For inside-out patch recordings, HEK293T cells transfected with mouse Kir2.1 and enhanced green fluorescent protein (EGFP) cDNAs were seeded onto polylysine-coated glass coverslips and cells exhibiting green fluorescence 24 to 48 hours after transfection were used.24
Membrane currents were recorded using a standard patch-clamp method25 in whole-cell mode and inside-out patch mode with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The recording chamber (area, 5 mm × 20 mm; volume, approximately 0.5 mL) mounted on the stage of an inverted microscope (TE-200U; Nikon, Tokyo, Japan) was continuously perfused with the bath solution at a rate of approximately 3 mL/min. Patch pipettes, when filled with the pipette solutions, had a resistance of 3.0 to 4.5 MΩ and 1.8 to 2.0 MΩ for whole-cell and inside-out recordings, respectively. Voltage stimulation and data acquisitions were performed through an analog-to-digital converter (Digidata 1322A or 1440A; Molecular Devices) using a personal computer and pCLAMP software (v8 or 9; Molecular Devices). All experiments were conducted at room temperature (22–24°C).
To record whole-cell currents, the extracellular (bath) solution was a modified Tyrode solution containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.33 mM NaH2PO4, 5.5 mM glucose, and 5 mM HEPES (pH adjusted to 7.4 with NaOH) or piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES; pH adjusted to 6.6 with NaOH); the intracellular (pipette) solution contained 85 mM K-aspartate, 2 mM K2ATP, 2 mM K2EDTA, 10 mM KH2PO4, 5 mM MgCl2, 0.3 mM Na2GTP, and 5 mM HEPES (pH adjusted to 7.2 with KOH). To record currents from inside-out patch membranes, the extracellular (pipette) solution contained 145 mM KCl, 1 mM CaCl2, and 5 mM HEPES (pH adjusted to 7.4 with KOH); the cytoplasmic (bath) solution contained 120 mM KCl, 4 mM K2EDTA, 7.2 mM K2HPO4, and 2.8 mM KH2PO4 (pH adjusted to 7.4, 7.2, or 6.6 with KOH or HCl). To maintain a near-physiologic degree of inward rectification of Kir2.1 currents during the inside-out patch recordings, phosphate buffer was used to adjust the pH of the cytoplasmic solution to avoid HEPES-related rectification,26 and 1 µM spermine-4Cl (Nacalai Tesque, Kyoto, Japan) was added to the cytoplasmic bath solution.19
Bupivacaine and lidocaine are weak bases (pKa 7.9 and 8.1, respectively) existing in physiological solutions as a mixture of positively charged (water soluble) and uncharged neutral (lipid soluble) forms. To prepare the bath solutions (i.e., extracellular solution for the whole-cell and cytoplasmic solution for the inside-out patch) containing the desired concentrations of bupivacaine and lidocaine, the LAs were dissolved at the highest concentration in an acidic Tyrode solution of pH <6 where the LAs exist almost exclusively in water-soluble forms, and the solutions were then serially diluted in the bath solution with the pH readjusted afterward if necessary (usually unnecessary for 1 mM concentrations or less).
To change the ratio between the charged and neutral forms of lidocaine and bupivacaine in the bath solutions, the pH of the bath solutions was made acidic in some experiments. The charged/neutral ratios of lidocaine and bupivacaine at the pH values used in the experiments were 0.76/0.24 (pH 7.4), 0.83/0.17 (pH 7.2), and 0.95/0.05 (pH 6.6) for lidocaine and 0.86/0.14 (pH 7.4), 0.91/0.09 (pH 7.2), and 0.98/0.025 (pH 6.6) for bupivacaine. Bupivacaine HCl, lidocaine HCl, and QX-314 (lidocaine N-ethyl bromide) were obtained from Sigma-Aldrich (St. Louis, MO).
Membrane currents and potentials were obtained at the end of 200-millisecond step pulses. Membrane potentials were not corrected for the liquid junction potential between the bath solution and the pipette solution, which was calculated to be approximately −10 mV for whole-cell recordings and to be negligibly small (<0.4 mV) for the inside-out recordings. To evaluate the effects of bupivacaine and lidocaine on Kir2 currents, fractions of uninhibited currents (I/I 0; I 0 being the control currents) were obtained. IC50 values were determined by fitting the mean I/I 0 values obtained at different concentrations of the LAs ([LA]) with the Hill equation, I/I 0 = 1 / (1 + ([LA] / IC50)H), where H is the Hill coefficient. Data were analyzed using pCLAMP and Origin software (version 9; OriginLab Corp., Northampton, MA).
Values were expressed as the means ± SD, and n represents the number of data. The error bars in the figures indicate SD with n given in parentheses. Statistical comparisons between 2 groups of data were conducted with Student unpaired t test and those among 3 groups were performed using Tukey method after 1-way analysis of variance. Values of P <0.01 were judged to be statistically significant and P value >0.15 to be not significant; actual P values are provided in Results and figure legends. The 95% confidence intervals (CIs) of differences measured between groups are also reported where appropriate. Statistical analysis was performed using Origin software (version 9; OriginLab Corp.).
Inhibition of Human Kir2.x Currents with Bupivacaine
After application of bupivacaine-containing extracellular solution, the large inward currents of Kir2.1 channels observed at voltages more negative than −70 mV decreased gradually in the presence of 1 or 10 mM bupivacaine, and the degree of inhibition was larger at 10 mM than at 1 mM (Fig. 1, A and B). Notably, removal of bupivacaine from the extracellular solution did not restore Kir2.1 currents. Current-voltage (I-V) relations obtained in the absence and presence of bupivacaine intersected close to −70 mV, which was in agreement with the reversal potential for K+ currents under the experimental condition, and the relatively small outward currents observed at voltages ranging between −70 and −20 mV were suppressed concurrently with the inward currents (Fig. 1C). HEK293 cells exhibited a negligible level of endogenous currents at voltages below −20 mV that were not affected by bupivacaine (Supplemental Digital Content, Supplemental Figure 1, A and B, http://links.lww.com/AA/B350). Thus, the above findings indicate that bupivacaine inhibited Kir2.1 inward rectifier K+ currents in a voltage-independent manner. Endogenous outward currents of HEK293 cells that were activated during depolarization were more strongly inhibited by bupivacaine within several dozen seconds after its application and restored quickly upon its removal (Supplemental Digital Content, Supplemental Figure 1, B and C, http://links.lww.com/AA/B350). The effects of bupivacaine were also observed with Kir2.2 and Kir2.3. Figure 1D compares the concentration dependence of inhibition of Kir2.1, Kir2.2, and Kir2.3 currents caused by bupivacaine. Because current inhibition by bupivacaine did not reach a clear steady-state level (Fig. 1A), comparisons were made between fractions of uninhibited currents (I/I 0) after approximately 5 minutes of bupivacaine exposure. I/I 0 values obtained with 1 mM bupivacaine were 0.92 ± 0.07 (n = 7), 0.87 ± 0.22 (n = 3), and 0.33 ± 0.11 (n = 3) for Kir2.1, Kir2.2, and Kir2.3, respectively. Inhibition induced by bupivacaine of Kir2.3 was stronger than that of Kir2.1 and Kir2.2 at 1 mM (P = 0.82, 95% CI, −0.28 to 0.18 for Kir2.2 versus Kir2.1; P = 0.00012, 95% CI, −0.81 to −0.35 for Kir2.3 versus Kir2.1; P = 0.00095, 95% CI, −0.81 to −0.25 for Kir2.3 versus Kir2.2). IC50 values for the inhibition caused by approximately 5-minute exposure of bupivacaine were 9.9, 8.2, and 0.60 mM for Kir2.1, Kir2.2, and Kir2.3, respectively.
Inhibition of Human Kir2.x Currents with Lidocaine
The influence of lidocaine on Kir2.1, Kir2.2, and Kir2.3 currents was also examined (Figs. 2 and 3). Similar to bupivacaine, lidocaine inhibited both the inward and outward currents in a concentration-dependent manner (Figs. 2B and 3B). However, lidocaine acted very rapidly, reaching steady state within approximately 1 minute, and inhibition was almost completely reversed by its removal (Fig. 2, A–C). I/I 0 values obtained with lidocaine, analyzed at a voltage of −110 mV, were not significantly different; the I/I 0 values obtained with 1 mM lidocaine were 0.70 ± 0.078 (n = 7), 0.76 ± 0.060 (n = 4), and 0.65 ± 0.17 (n = 3) for Kir2.1, Kir2.2, and Kir2.3, respectively (P = 0.54, 95% CI, −0.10 to 0.23 for Kir2.2 versus Kir2.1; P = 0.78, 95% CI, −0.23 to 0.14 for Kir2.3 versus Kir2.1; P = 0.33, 95% CI, −0.32 to 0.090 for Kir2.3 versus Kir2.2). IC50 values were 2.2, 2.7, and 1.5 mM for Kir2.1, Kir2.2, and Kir2.3 currents, respectively (Figs. 2D and 3C).
Charged Form of the LAs Does Not Inhibit Kir2.x Channels from the Extracellular Side
The effects of the LAs on Kir2.1 currents were examined under an acidic extracellular pH (pHo) of 6.6, which increases their charged/neutral ratios. Inhibitory effects induced by the LAs were attenuated at pH 6.6, suggesting that charged forms acting from the extracellular side were not responsible for their effects (Fig. 4). Supporting this notion, the addition of 10 mM QX-314, a membrane-impermeable, permanently cationic derivative of lidocaine, to the extracellular solution did not alter Kir2.x currents (n = 5; Supplemental Digital Content, Supplemental Figure 2A, http://links.lww.com/AA/B350). In contrast, inclusion of 1 mM QX-314 in the internal (pipette) solution induced a decrease in Kir2.1 currents (n = 4; Supplemental Digital Content, Supplemental Figure 2B, http://links.lww.com/AA/B350), suggesting that charged LAs in the cytoplasm may act on Kir2 channels.
Charged Form of the LAs Inhibits Kir2.x Channels from the Cytoplasmic Side
In inside-out patch recordings, the inhibitory effects on Kir2.1 caused by lidocaine and bupivacaine in the cytoplasmic solution were similar to those observed after external application during the whole-cell recordings, in terms of the time course and the reversibility of the effects. Namely, inhibition of Kir2.1 with cytoplasmic lidocaine was rapid and reversible, whereas that induced by bupivacaine was slow and irreversible (Figs. 1, 2, and 5). Addition of 1 mM QX-314 to the cytoplasmic solution also induced rapid and reversible inhibition similar to that caused by lidocaine (I/I 0 values at −40 mV were 0.34 ± 0.007, n = 3). When the cytoplasmic pH (pHi) was decreased from 7.2 to 6.6, inhibition with lidocaine was augmented, though only modestly, whereas inhibition with bupivacaine was not notably changed (Fig. 6A). These findings were explained by the analysis of dose-inhibition relations; the relations obtained at different pHi were well approximated by a single relation if plotted against the concentration of the charged form, but not if plotted against that of the neutral form (Fig. 6, B and C).
In clinical practice, large concentrations of LAs are administered in close proximity to nerves to produce analgesia,27 and a variety of ion channels and receptors are inhibited by LAs. We found that human inward rectifier K+ channels of the Kir2.x subfamily, Kir2.1, Kir2.2, and Kir2.3, are inhibited by 2 amide-type LAs, bupivacaine and lidocaine. Inhibition with bupivacaine was slow, continuous, and irreversible; Kir2.3 was more strongly inhibited by bupivacaine than Kir2.1 and Kir2.2 with IC50 values after approximately 5 minutes application of 8, 10, and 0.8 mM for Kir2.1, Kir2.2, and Kir2.3, respectively (Fig. 1). Inhibition caused by lidocaine was fast and reversible, and the degree of inhibition was similar among the 3 Kir2 channel subtypes (IC50s, 1.5–2.7 mM; Figs. 2 and 3).
Hydrophilic charged forms of LAs cannot easily penetrate the cell membrane, whereas neutral forms diffuse into the membrane and then into the cytoplasm where they re-equilibrate between the charged and neutral forms. Because ambient pH changes the charged/neutral form ratio of LAs, a low-pH extracellular solution was used to examine which of the 2 forms of bupivacaine and lidocaine were acting and where the site(s) of their action was. Kir2.1 was studied to gain an insight into the mechanism of inhibition. Kir2.2 is functionally very similar to Kir2.1, whereas Kir2.3 exhibits strong sensitivity to extracellular pH,28,29 which could interfere with the analysis. Acidification of pHo from 7.4 to 6.6, which increases the amount of charged LAs, attenuated Kir2.1 inhibition (Fig. 4). Furthermore, for lidocaine, a decrease of approximately 5-fold in the fraction of the neutral form (from 24% to 5%) was reflected by an increase in the IC50 of approximately the same magnitude (from 2.2 mM, Fig. 2D, to approximately 10 mM, Fig. 4A). However, these findings did not necessarily imply that the neutral form acted on Kir2.1 because the amount of the charged form in the cytoplasmic solution would be reduced by about one-fifth together with that of the neutral form, as shown in Figure 7.30 To better understand the pathway of Kir2 inhibition, the effects of the LAs in the cytoplasmic solution were studied using inside-out patches, which enables direct control of the solution facing the cytoplasmic side of the membrane. Inhibition of Kir2.1 induced by lidocaine and bupivacaine in the cytoplasmic solution was similar to that observed after external application in terms of the time course and the reversibility of the effects (Fig. 5), indicating that the slow inhibition observed with bupivacaine in the whole-cell recordings was not related to the time required for its neutral form to diffuse across the membrane into the intracellular milieu. QX-314, a membrane-impermeable cationic derivative of lidocaine, did not affect Kir2 currents from the extracellular side during the whole-cell recordings but exerted inhibitory effects on Kir2.1 from the cytoplasmic side. Furthermore, dose-inhibition relations obtained at different pHi could be approximated by a single relation if plotted against the theoretical concentration of the charged LA, but not if plotted against that of the neutral LA (Fig. 6), as has been demonstrated with NaV channels.31 Thus, it was concluded that the effects of bupivacaine and lidocaine on Kir2 channels are attributable to their charged forms acting either directly or indirectly from the cytoplasmic side; that is, the neutral forms of the LAs diffuse into the cells, and the charged forms converted from the neutral forms act on Kir2 channels. It should be noted that IC50s of Kir2.1 inhibition obtained in the whole-cell recordings at the pHo/pHi values of 7.4/7.2 were 9.9 mM for bupivacaine (Fig. 1D) and 2.2 mM for lidocaine (Fig. 2), and these values theoretically correspond to concentrations of 13.8 and 2.6 mM of the charged forms in the cytoplasmic solution, respectively. IC50s of the cytoplasmic charged LAs obtained using cell-free inside-out patches (1.5 mM for bupivacaine and 0.37 mM for lidocaine; Fig. 6) were lower than the above values. Because it was confirmed that this discrepancy was not because of the species difference of the Kir2.1 clones used in the whole-cell and inside-out experiments (human versus mouse), it may be speculated that the absence of binding of LAs to cellular components enhanced their inhibitory effects under the cell-free configuration.
Mechanisms of LA Action on Kir2 Channels
The hydrophilic pathway from the cytoplasmic side is also known for LA-mediated inhibition of NaV channels, which is voltage dependent, not only because the channels are voltage gated but also because the binding site(s) for LAs are located in the channel pore, i.e., within the electrical field.32 In contrast, inhibition of Kir2.x depended little on voltage (Figs. 1–3 and 5). Although the voltage dependence of Kir2 currents, i.e., their inward rectification, arises from cytoplasmic organic polyamines, plugging the channel pore,24,33 the voltage independence of the effects of LA on Kir2 channels implied that the channels were not inhibited by charged LAs plugging the channel pore, but rather through a mechanical disruption. An example is positively charged LAs interrupting the interaction between Kir2 and the negatively charged membrane lipid phosphatidylinositol 4, 5-biphosphate (PIP2), which is required for Kir2 activity.34 Indeed, such a mechanism has been proposed for the inhibitory action of bupivacaine on G-protein–gated Kir3 channels that exhibit a weaker interaction with PIP2 than Kir2,6 as well as those of cationic amphiphilic drugs (e.g., quinacrine and carvedilol) on Kir2 channels.35–37 The finding that Kir2.3 displayed greater sensitivity to bupivacaine than Kir2.1 or Kir2.2 (Fig. 1D) is in accordance with the evidence that the channel-PIP2 interaction is weaker for Kir2.3 than for Kir2.1 or Kir2.2.38 However, sensitivity to lidocaine was not significantly different among Kir2.1, Kir2.2, and Kir2.3 (Figs. 2 and 3). The mechanism underlying Kir2 inhibition may not be common between the 2 LAs. For the bacterial KcsA channel, it has been demonstrated that binding of lidocaine to the inner cavity of the channel pore reduces the tetramer stability of the channel.39 Further studies are required to address the molecular mechanisms of Kir2 inhibition caused by the LAs.
Clinical Relevance of LA Effects on Kir2
When using LAs for regional anesthesia, adverse systemic effects such as convulsions and cardiac arrhythmia may occur,40 not only through effects on NaV channels but also through effects on other ion channels expressed in the central nervous and cardiovascular systems. However, the effects of bupivacaine and lidocaine on Kir2 found in the present study may not contribute to the systemic side effects, even in the case of accidental venous injection. This is because the highest anesthetic concentrations reached after IV administration appear to be submillimolar,41 which is at the lower limit of the effective concentration range for inhibiting Kir2 channels.
However, the inhibitory effects of bupivacaine and lidocaine on Kir2 may contribute to local manifestations associated with administration of LAs. During spinal anesthesia, drug spread in the cerebrospinal fluid (CSF) shows a high interpatient variability because of a variety of factors,42,43 and it has been reported that bupivacaine concentrations in the lumbar CSF after intrathecal injection of 0.5% (approximately 15 mM) plain bupivacaine (20 mg for men/17.5 mg for women) declined with an estimated half-life of approximately 50 minutes, occasionally remaining in the millimolar range within 45 minutes.44,45 Similarly, lidocaine concentrations in the lumbar CSF after 5% (approximately 180 mM) lidocaine injection for spinal anesthesia (150–200 mg) sometimes remained even in the tens of millimolar range after 30 minutes.46 Thus, although our evaluation of the effects on Kir2 was conducted approximately 5 minutes after its application of bupivacaine, its CSF concentrations may remain high enough for >5 minutes in some clinical cases to irreversibly influence Kir2 channels in neuronal tissues. Furthermore, given the fast onset of inhibition with lidocaine, Kir2 inhibition in clinical use may be much stronger with lidocaine than with bupivacaine (Fig. 2C).
Neurotoxic side effects such as transient neurologic symptoms and the more severe cauda equina syndrome remain matters of concern. The mechanisms underlying these manifestations are not fully understood because neuronal damage may be caused by diverse actions of LAs, including increases in intracellular Ca2+ levels, mitochondrial dysfunction, and amphiphilic interactions between LAs and membrane lipids.18,47–49 LAs (including bupivacaine and lidocaine) display significant cytotoxicity against cultured neuronal and glial cells at millimolar concentrations,50,51 and this may be related to their effects on ion channels.27 Depolarization of the resting membrane potential induced by clinical concentrations of lidocaine has been demonstrated in isolated nerve axons, which may be attributable to inhibition of K+ conductance52,53 Because inhibition of inward rectifier K+ conductance indeed depolarizes the cell membrane (as shown by the changes in the zero-current potential in Figs. 2B and 3B), our findings suggest that the use of bupivacaine and lidocaine in regional anesthesia may depolarize neuronal cells through inhibition of Kir2 channels in some clinical cases, thereby by affecting neuronal excitability.54,55
Name: Kei Nakahira, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Kei Nakahira has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Kensuke Oshita, MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Kensuke Oshita reviewed the analysis of the data and approved the final manuscript.
Name: Masayuki Itoh, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Masayuki Itoh has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Makoto Takano, MD, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Makoto Takano reviewed the analysis of the data and approved the final manuscript.
Name: Yoshiro Sakaguchi, MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Yoshiro Sakaguchi approved the final manuscript.
Name: Keiko Ishihara, MD, PhD
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Keiko Ishihara has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
The authors thank Hideko Yoshitake and Akemi Sakamoto for secretarial support.
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