Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels May Contribute to Regional Anesthetic Effects of Lidocaine

Hyperpolarization-activated Cyclic Nucleotide-gated Channels May Contribute to Regional Anesthetic Effects of Lidocaine

Cheng Zhou, Ph.D., Bowen Ke, Ph.D., Yi Zhao, M.B., Peng Liang, M.D., Daqing Liao, M.B., Tao Li, Ph.D., Jin Liu, M.D., Xiangdong Chen, Ph.D., M.D.

ABSTRACT

Background: Local anesthetics (e.g., lidocaine) have been found to inhibit hyperpolarization-activated cyclic nucleotide-gated

(HCN) channels besides sodium channels. However, the exact role of HCN channels in regional anesthesia in vivo is still elusive. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/3/606/369991/20150300_0-00022.pdf by guest on 28 September 2021 Methods: Sciatic nerve block and intrathecal anesthesia were performed using lidocaine in wild-type and HCN1 channel −/− knockout (HCN1 ) mice. EC50 of lidocaine and durations of 1% lidocaine were determined. In electrophysiologic recordings, effects of lidocaine on HCN channel currents, voltage-gated sodium channel currents, and neural membrane properties were recorded on dorsal root ganglia neurons.

Results: In both sciatic nerve block and intrathecal anesthesia, EC50 of lidocaine for tactile sensory blockade (2 g von Frey −/− fiber) was significantly increased in HCN1 mice, whereas EC50 of lidocaine for pinprick blockade was unaffected. Durations of 1% lidocaine were significantly shorter in HCN1−/− mice for both sciatic nerve block and intrathecal anesthesia (n = 10). ZD7288 (HCN blocker) could significantly prolong durations of 1% lidocaine including pinprick blockade in sciatic nerve block (n = 10). Forskolin (raising cyclic adenosine monophosphate to enhance HCN2) could significantly shorten duration of pinprick blockade of 1% lidocaine in sciatic nerve block (n = 10). In electrophysiologic recordings, lidocaine could nonselectively inhibit HCN channel and sodium channel currents both in large and in small dorsal root ganglia neurons (n = 5 to 6). Meanwhile, lidocaine caused neural membrane hyperpolarization and increased input resistance of dorsal root ganglia neurons but not in large dorsal root ganglia neurons from HCN1−/− mice (n = 5–7). Conclusions: These data indicate that HCN channels may contribute to regional anesthetic effects of lidocaine. By inhibiting HCN channels, lidocaine could alter membrane properties of neurons. (Anesthesiology 2015; 122:606-18)

OLTAGE-GATED sodium channel is conventionally What We Already Know about This Topic V regarded as the main molecular target for local anesthetics.1 By blocking sodium channels, local anesthetics pro- • In addition to sodium ion channels, local anesthetics block additional channels such as nonselective hyperpolarization- duce nonselective sensory blockade, nociceptive blockade, activated, cyclic nucleotide-gated (HCN) channels 1 motor paralysis, and sympathetic nerve blockade. If local • It is unclear whether these interactions contribute to local an- anesthetics could specifically impair sensory or even nocicep- esthesia tive functions, they would be widely used in various medical What This Article Tells Us That Is New procedures, such as treatment of chronic pain. Besides voltage-gated sodium channel, increasing evi- • The efficacy and duration of anesthesia after sciatic nerve block and intrathecal administration of lidocaine are altered in dences have indicated that local anesthetics at clinical relevant HCN1 knockout mice concentrations could interact with a large spectrum of recep- • Lidocaine inhibits HCN currents in dorsal root ganglion neu- tors and/or ion channels, such as potassium channels,2 cal- rons in vitro cium channels,3 and transient receptor potential vanilloid-1 • Local anesthetic blockade of HCN channels may contribute to channels.4 For recent years, hyperpolarization-activated cyclic the intensity and duration of local anesthesia nucleotide-gated cation (HCN) channel has been identified as an underlying target of local anesthetics.5–7 Previous studies regional anesthetic adjuvants, such as clonidine,8,10 dexme- demonstrated that lidocaine could inhibit HCN channel cur- detomidine9 and ketamine,11,12 have been found as HCN rents6,7 and ZD7288 (HCN channel blocker) could enhance channels inhibitors at their regional anesthetic relevant con- the effects of lidocaine and ropivacaine.8,9 Interestingly, many centrations (the concentrations used in regional anesthesia

Drs. Zhou and Ke contributed equally to this study. Submitted for publication May 21, 2014. Accepted for publication November 6, 2014. From the Laboratory of Anesthesia & Critical Care Medicine, Translational Neuroscience Center, West China Hospital of Sichuan University, Chengdu, China (C.Z., B.K., D.L., T.L., J.L., X.C.); Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu, China (Y.Z., P.L., J.L.); and Department of Anesthesiol- ogy, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (X.C.). Copyright © 2014, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. All Rights Reserved. Anesthesiology 2015; 122:606-18

Anesthesiology, V 122 • No 3 606 March 2015 Perioperative Medicine

rather than systemic administration). Thus, recent studies and HCN1 knockout (HCN1−/−) mice were used. The demonstrated that the enhancement of clonidine and dexme- mice were housed in cages on a 12-h light-dark cycle with detomidine in regional anesthesia resulted from their modu- free access to food and water. HCN1−/− mice were bred, lation on HCN channels rather than adrenergic receptors.8,9 as previously described.11 HCN1−/− mice were normal in However, the exact role of HCN channels in regional anes- body size and longevity, and no obvious behavioral abnor- thesia in vivo is still elusive. malities were found.13 All the study mice were placed on HCN channel (consists of four subtypes, known as the experimental platform 1 h/day for consecutive 3 days HCN1–4)13–16 is widely expressed in various tissues, (acclimation to the experimental environment) before for- including central nervous system, heart, and peripheral mal experiments and were randomly assigned into each nervous system13,17 from both animals and human beings. group based on a SPSS-generated random number (SPSS

Ivabradine, an HCN blocker, has been used in the clinical Inc., Chicago, IL). For behavioral assessment of mice, Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/3/606/369991/20150300_0-00022.pdf by guest on 28 September 2021 setting for the treatment of heart failure.18–21 For peripheral observers were blinded to the treatment and group assign- nervous system, HCN1 and HCN2 are the most common ment of mice. subtypes,13 which have been found in dorsal root ganglia (DRG)22–25 and nerve fibers.26,27 Previous studies indi- Chemicals cated that the functions of nerve axon were modulated by Lidocaine was purchased from Fortune Zhaohui Pharma- HCN channels,28,29 which might be involved in peripheral ceutical Co. Ltd. (Shanghai, China). ZD7288 was pur- nerve block. Interestingly, HCN1 subtype is predominated chased from Tocris Bioscience (Ellisville, MO). Lidocaine in large diameter DRG neurons, whereas HCN2 subtype and ZD7288 were prepared with normal saline. Stock solu- mainly in small ones.22–24,30 Because small-diameter nerve tion of ZD7288 was 20 mM. Forskolin was purchased from fibers are widely recognized for high-threshold sensory Sigma-Aldrich Co. Ltd. (Shanghai, China) and diluted with transmission,31,32 HCN2 subtype might contribute to noci- dimethyl sulfoxide to stock solution at concentration of ceptive sensation. 5 mg/ml and then prepared with normal saline to final con- In the current study, we hypothesized that in addition to centration. Isoflurane was provided by Abbott Pharmaceuti- voltage-gated sodium channel, HCN channels contribute to cal Co. Ltd. (Shanghai, China). All the study solutions were regional anesthetic effects of lidocaine in vivo. prepared immediately before injection.

Materials and Methods Western Blotting Analysis DRG and sciatic nerves from wild-type and HCN1−/− knockout Animals mice were homogenized, and BCA kit (Pierce, Rockford, IL) With the approval of the Institutional Animal Experimen- was used to determine protein concentrations of the superna- tal Ethics Committee of Sichuan University (Chengdu, tant. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis Sichuan, China), male adult wild-type C57BL/6J mice was applied to separate the proteins of supernatant and then

Fig. 1. Western blotting analysis of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels expression in dorsal root ganglia (DRG) and sciatic nerves. (A) Both HCN1 and HCN2 channel protein with expected molecular weight (approximately 100 kDa) were detected in DRG and/or sciatic nerves from wild-type mice (n = 4), and no HCN1 protein (with expected molecular weight) was found in DRG or sciatic nerves from HCN1−/− mice (n = 3). (B) Quantitative analysis was determined by the ratio between HCN1 or HCN2 and β-actin immunoreactivity. KO = HCN1−/− knockout; WT = wild type.

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Fig. 2. The role of HCN1 subtype in physiological sensation. (A) Thresholds of tactile sensation in wild-type and HCN1−/− mice were similar (evaluated by von Frey fibers). (B) Thermal sensory thresholds in wild-type and HCN1−/− mice were determined by foot-flick latency (heat stimuli), and no difference was found between these two genotypes. (C) For sciatic nerve block model in wild-type mice, ZD7288 (5.5 mM) alone did not induce any tactile sensory blockade. (D) For sciatic nerve block model in wild- type mice, ZD7288 (5.5 mM) alone did not induce thermal sensory blockade. (E) For intrathecal anesthesia model in wild-type mice, ZD7288 (5.5 mM) alone did not induce any tactile sensory blockade on foot. (F) For intrathecal anesthesia model in wild- type mice, ZD7288 (5.5 mM) alone did not induce thermal sensory blockade on tails of mice. HCN = hyperpolarization-activated cyclic nucleotide-gated channels. transferred to a nitrocellulose membrane. Primary antibod- of mice to heat stimulus increased by more than 50% as pre- ies of HCN1 (#ab65706, Abcam, Cambridge, MA), HCN2 viously described33; pinprick sensory blockade was evaluated (#ab65704, Abcam), and β-actin (#ab1801, Abcam) were by a 24G needle (Terumo Medical Corp., Tokyo, Japan), used. After incubation with primary antibody, the membrane and pinprick blockade was defined as no aversive responses was incubated with horseradish peroxidase–conjugated anti- to pinprick. Motor function of mice was scored according rabbits secondary antibody (1:10,000; Pierce) for 1 h. Super- to a 4-point scale34: 1 = normal; 2 = intact dorsiflexion of signal chemiluminescence detection kit (Pierce) was used for foot with impaired ability to splay toes when elevated by the the development of blot. Immunoblotting was visualized by tail; 3 = toes and foot plantar flexed with no splaying ability; Kodak X-ray Processor 102 (Eastman Kodak, Rochester, NY) 4 = loss of dorsiflexion, flexion of toes, and impairment of and analyzed by the software Quantity One (Bio-Rad Labora- gait. Score of motor function up to 2 (≥2) was defined as tories, Hercules, CA). motor blockade.

Mice Sciatic Nerve Block Model Mice Intrathecal Anesthesia Model Under inhalation of 2% isoflurane, 50 μl study solution was Under inhalation of 2% isoflurane, study solutions (saline or injected into popliteal fossa of mice. All the mice received ZD7288 or lidocaine) at 5 μl were intrathecally injected at two injections with an interval of 20 min. For instance, L5–L6 of mice by microinjector, as previously described.35–37 ZD7288 (5.5 mM9) or forskolin (62.5 μg/ml8) or normal After injection, tactile sensory blockade, pinprick sensory saline was injected 20 min before injection of lidocaine. After blockade, and motor blockade of mice foot were evaluated injection of lidocaine, tactile sensation of injected foot was as described in sciatic nerve block model. Left or right foot evaluated by von Frey fibers (37450-277; Ugo Basile, Come- was randomly chosen for each mouse. For thermal sensory rio, Italy), and tactile blockade was defined as no aversive blockade, heat stimulus was applied to tails of mice with reactions to 4 g von Frey fiber; thermal sensation was deter- tail-flick unit (7360; Ugo Basile). Thermal sensory blockade mined by heat stimulus (Plantar test 37370; Ugo Basile), was defined as reaction latency of mice tail to heat stimulus and thermal sensory blockade was defined as reaction latency increased by more than 50%.

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Fig. 3. Concentration sequences of up-and-down test for determination of lidocaine (Lido) EC50 (median effective concentration). (A) EC50 of lidocaine for pinprick blockade was determined in sciatic nerve model of mice. (B) EC50 of lidocaine for tactile blockade (evaluated by 2 g von Frey fibers) was determined in sciatic nerve model of mice. (C) EC50 of lidocaine for pinprick blockade was determined in intrathecal anesthetic model of mice. (D) EC50 of lidocaine for tactile blockade (evaluated by 2 g von Frey fibers) was determined in intrathecal anesthetic model of mice. HCN = hyperpolarization-activated cyclic nucleotide-gated channels; WT = wild type.

Determination of Lidocaine EC50 by the Up-and-Down 2 g von Frey fiber and pinprick, respectively. Based on our Method preliminary experiments, initial concentration of lidocaine

Estimated EC50 (median effective concentration) of lido- was 0.4% for the first mouse. After each successful block- caine in both sciatic nerve block and intrathecal anesthe- ade, lidocaine concentration was decreased by 0.8-fold (e.g., sia was determined by the up-and-down method.38 Briefly, 0.32%); after each failure, it was increased by 0.8-fold (e.g., 0.5%). The up-and-down procedure was repeated until six EC50 of lidocaine for tactile blockade and pinprick blockade were determined, respectively. Tactile blockade and crossovers points appeared. pinprick blockade were defined as no aversive responses to Electrophysiologic Recordings Acute isolated DRG neurons were used for electrophysi- Table 1. EC50 of Lidocaine in Sciatic Nerve Block and Intrathecal Anesthesia ologic recordings. The isolation procedure of DRG neurons was modified from the method as previously described.39 Tactile Blockade Briefly, adult mice were anesthetized by ketamine/xylazine Groups Pinprick Blockade (2 g von Frey) (40/15 mg/kg), and DRG was removed and digested in WT sciatic 0.35 ± 0.05% 0.14 ± 0.03% papain (1 mg/ml) for 40 to 50 min and in collagenase I (1 mg/ WT sciatic + 0.23 ± 0.04%* 0.11 ± 0.02%* ml) for 30 to 40 min. Large DRG neurons (>30 μm) and ZD7288 small DRG neurons (<20 μm) were recorded, respectively. HCN1−/− sciatic 0.36 ± 0.07% 0.22 ± 0.04%* For whole-cell voltage clamp recording of HCN channel WT intrathecal 0.45 ± 0.05% 0.28 ± 0.06% currents, pipettes (3 to 5 MΩ) were filled with KCl 130 mM, −/− HCN1 intrathecal 0.47 ± 0.07% 0.38 ± 0.08%† NaCl 4 mM, MgCl2 1 mM, CaCl2 0.5 mM, HEPES 10 mM, EGTA 10 mM, MgATP 3 mM, and GTP-Tris 0.3 mM (pH EC50 of lidocaine were determined by up-and-down method and expressed as mean ± SD. For example, “WT sciatic” group indicates sciatic nerve 7.3, osmolarity 310 to 315 mOsmol/l). Standard bath solu- block in wild-type mice; “WT intrathecal” group indicates intrathecal anes- tion contained NaCl 140 mM, KCl 3 mM, HEPES 10 mM, thesia in wild-type mice. CaCl 2 mM, MgCl 2 mM, and glucose 10 mM (pH 7.4, *P < 0.05 vs. WT sciatic group; †P < 0.05 vs. WT intrathecal group. 2 2 osmolarity 300 to 310 mOsmol/l). Tetrodotoxin (0.5 μM; EC50 = median effective concentration; HCN = hyperpolarization-activated cyclic nucleotide-gated channels; WT = wild type. Alomone Labs, Jerusalem, Israel) was added to bath solution

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Fig. 4. For sciatic nerve block model, durations of 1% lidocaine (Lido) were shortened in HCN1−/− mice. (A) Duration of tactile blockade was significantly shortened in HCN1−/− mice, evaluated by von Frey fibers. (B) Duration of motor blockade was significantly shortened in HCN1−/− mice. The inset figure was average motor function scores at each time point. C( ) Duration of thermal sensory blockade was significantly shortened in HCN1−/− mice than wild-type mice, determined by foot-flick latency (heat stimuli). (D) Duration of pinprick blockade by 1% lidocaine was unchanged between wild-type and HCN1−/− mice. *P < 0.05, versus wild-type saline/lido group. HCN = hyperpolarization-activated cyclic nucleotide-gated channels; WT = wild type. routinely for inhibition of action potential. Recordings were voltage, and fitted with Boltzmann curves for derivation of obtained by Axopatch 200B amplifier (Axon Instruments, half-activation voltage (V1/2a) as: G/Gmax = 1/(1 + e(V1/2a − Inc., Sunnyvale, CA) at room temperature (approximately V)/k), G/Gmax = normalized conductance; Gmax = maximum 25°C). HCN channel currents in mice DRG neurons were conductance; V1/2a = voltage activation of half-maximum; recorded by a series of hyperpolarizing step voltage pulses and k = slope factor. Concentration–response relationship (−60 to −140 mV with 10 mV increments) from a hold- for lidocaine on maximal current amplitude was fitted to ing potential of −60 mV, followed by a voltage step of −100 the Hill equation: Y = 1/[1 + 10(logIC50 − X)×h], where mV to record tail currents. Tail currents were normalized, Y = inhibitory effect; X = concentration of lidocaine; h = Hill plotted as a function of the preceding hyperpolarization step coefficient.

Table 2. Durations of 1% Lidocaine in Sciatic Nerve Block Model

Durations of Blockade (min)

Groups Tactile Motor Thermal Pinprick

WT saline/saline N/A N/A N/A N/A WT saline/ZD7288 N/A N/A N/A N/A WT saline/lido 15.0 (6.3)* 17.5 (11.3)* 10.0 (5.0)* 6.0 (6.3) HCN1−/− saline/lido 9.0 (5.0)† 10.0 (6.3)† 5.0 (6.0)† 6.0 (5.0) WT ZD7288/lido 20.0 (1.3)† 25.0 (10.0)† 15.0 (5.0)† 12.5 (11.3)† HCN1−/− ZD7288/lido 12.0 (5.0)* 15.0 (6.3)* 10.0 (5.0)* 10.0 (11.3)* WT forskolin/lido 12.5 (3.0) 15.0 (11.3) 7.5 (6.3) 3.5 (2.0)† HCN1−/− forskolin/lido 7.5 (5.5) 10.0 (6.3) 5.0 (3.3) 3.0 (2.0)*

The data were expressed as median (IQR, interquartile range) and n = 10 for each group; e.g., the group WT saline/saline indicated preinjection of saline and injection of saline in wild-type mice; group HCN1−/− ZD7288/lido indicated preinjection of ZD7288 and injection of lidocaine in HCN1−/− mice. *P<0.05 vs. HCN1−/− saline/lido group; †P < 0.05 vs. WT saline/lido group. HCN = hyperpolarization-activated cyclic nucleotide-gated channels; lido = 1% lidocaine; N/A = no action; WT = wild type.

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Fig. 5. For intrathecal anesthesia in mice, durations of 1% lidocaine (Lido) were shortened in HCN1−/− mice. (A) Duration of tactile blockade on foot was significantly shortened in HCN1−/− mice, evaluated by von Frey fibers. (B) Duration of motor function blockade was significantly shortened in HCN1−/− mice. The inset figure was average motor function scores at each time point. (C) Duration of thermal sensory blockade was not significantly shortened in HCN1−/− mice, determined by tail-flick latency (heat stimuli). (D) Duration of pinprick blockade by 1% lidocaine was unchanged between wild-type (WT) and HCN1−/− mice. *P < 0.05, versus WT lido group. HCN = hyperpolarization-activated cyclic nucleotide-gated channels.

For whole-cell voltage clamp recording of voltage-gated equation: G/Gmax = 1/(1 + e (V1/2a − V)/k), G/Gmax = nor- sodium channel currents, holding potential was −90 mV. The malized conductance; Gmax = maximum conductance; V1/2a extracellular solution contained NaCl 110 mM, NaHCO3 = voltage activation of half-maximum; and k = slope fac- 26 mM, KCl 5.6 mM, NaH2PO4 1 mM, CaCl2 0.1 mM, tor. Sodium channel conductance (GNa) was calculated as: MgCl2 5 mM, tetraethylammonium 20 mM, and glucose GNa= INa/(Vt − Vr), INa = peak sodium current; Vt = test poten- 11 mM, pH 7.4, and osmolarity 300 to 310 mOsmol/L. tial; and Vr = sodium channel reversal potential. Concen- The pipette electrode solution contained CsCl 140 mM, tration–response curves of lidocaine were calculated by Hill

EGTA 5 mM, MgCl2 1 mM, HEPES 10 mM, and MgATP equation: Y = 1/[1 − 10(logIC50 − X)×h], where Y = inhibitory 3 mM, pH 7.3, and osmolarity 310 to 315 mOsmol/L. Cur- effect; X = concentration of lidocaine; h = Hill coefficient. rents were sampled at 20 kHz and filtered at 1 to 3 kHz and Neural membrane properties of DRG neurons includ- recorded using a series of depolarizing step voltage pulses ing resting membrane potential (RMP) and input resistance from −60 to 40 mV with 10 mV increments. Activation (Rin) were measured by current clamp recording, from voltage curves of sodium channel currents were fit to the Boltzmann responses to hyperpolarizing current injection. RMP was the

membrane potential at I = 0, and Rin was calculated with V/I Table 3. Durations of 1% Lidocaine in Intrathecal Anesthesia relationship by the Ohm law (R = U/I) based on hyperpolarizing current injection (from 20 to −100 pA with 20 pA increments) Durations of Blockade, min and amplitudes of voltages were the stable value for each cur- Groups Tactile Motor Thermal Pinprick rent injection on hyperpolarization state. Pipette solution contained KCl 17.5 mM, potassium gluconate 122.5 mM, HEPES Wild-type saline N/A N/A N/A N/A 10 mM, EGTA 0.2 mM, NaCl 9 mM, MgCl 1 mM, MgATP Wild-type ZD7288 N/A N/A N/A N/A 2 Wild-type lido 5.0 (0.5) 12.0 (7.5) 5.0 (7.0) 3.0 (2.0) 3 mM, and GTP-Tris 0.3 mM (pH 7.3). Standard bath solution HCN1−/− lido 3.5 (2.0)* 9.0 (5.0)* 4.0 (2.0) 3.0 (3.0) contained NaCl 140 mM, KCl 3 mM, HEPES 10 mM, CaCl2 2 mM, MgCl2 2 mM, and glucose 10 mM (pH 7.4). The data were expressed as median (IQR, interquartile range) and n = 10 for each group; e.g., the group wild-type saline indicated intrathecal injection of saline in wild-type mice; group HCN1−/− lido indicated intrathecal Statistical Analysis −/− injection of 1% lidocaine in HCN1 mice. SPSS version 16.0 (SPSS Inc.) was used for all the statistical *P < 0.05 vs. wild-type lido group. HCN = hyperpolarization-activated cyclic nucleotide-gated channels; analysis, except where noted. By preliminary test (n = 4) on lido = 1% lidocaine; N/A = no action. difference of pinprick blockade durations between wild-type

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Fig. 6. In sciatic nerve block model of mice, preinjection of ZD7288 (5.5 mM) could prolong durations of 1% lidocaine (Lido) in wild- type (WT) and HCN1−/− mice. (A) Duration of tactile blockade was significantly prolonged by preinjection of ZD7288, evaluated by von Frey fibers. (B) Duration of motor function blockade was significantly prolonged by adding ZD7288. The inset figure was average motor function scores at each time point. (C) Duration of thermal sensory blockade was significantly prolonged by preinjection of ZD7288, evaluated by foot-flick latency (heat stimuli). (D) Blockade of pinprick was significantly prolonged by ZD7288. *P < 0.05, versus WT saline/lido group; #P < 0.05, versus HCN1−/− ZD7288/lido group. HCN = hyperpolarization-activated cyclic nucleotide-gated channels. saline/lidocaine and wild-type ZD7288/lidocaine (7.5 ± 2.9 Results vs. 12.5 ± 2.9 min) groups, calculated minimal sample size HCN1 and HCN2 Expressed on DRG and Sciatic Nerves was 7.0 (α = 0.05; β = 0.10). Therefore, sample size of 10 of Mice was chosen for in vivo assay. Of note, data of the preliminary test were not included in the final data; therefore, the power Both HCN1 and HCN2 channels were detected in DRG and analysis based on preliminary test was not an interim analy- sciatic nerves (fig. 1A). Generally, expression levels of HCN1 sis for final analysis and P values were not adjusted. Data and/or HCN2 channels were higher in DRG than sciatic nerves, were presented as mean ± SD or median (interquartile range) and expression level of HCN1 was higher than that HCN2 in for normal distribution data and skewed data, respectively. DRG (fig. 1B). No expression of HCN1 channel protein (with −/− Latency data (e.g., durations) were compared by the Krus- expected molecular weight) was found in HCN1 mice. kal–Wallis test followed by post hoc of the Dunn test (software Prism 6.0; GraphPad, San Diego, CA). For “survival In Vivo Animal Model Assay curves” of pinprick blockade and motor blockade, Kaplan– Deletion of HCN1 Subtype Did Not Affect Physiological Meier method with log-rank test was used for comparison Sensation. Tactile sensation (by von Frey fibers, fig. 2A) among groups. EC of lidocaine was calculated by averaging and thermal sensation (by foot-flick test, fig. 2B) were 50 −/− concentration points of crossovers (12 points) and compared similar between wild-type and HCN1 mice (n = 15 and with one-way ANOVA with post hoc of Games–Howell. For 18 for each genotype). For sciatic nerve block model in comparison of HCN and sodium channel currents, two- wild-type mice, ZD7288 alone did not produce any anes- way ANOVA (drugs and genotypes as principal factors) was thetic relevant actions: tactile (n = 10, fig. 2C) and thermal performed, with the Tukey correction of the t test for post (n = 10, fig. 2D) sensations were unchanged after injection hoc pairwise comparisons. Activation curves of HCN and of 5.5 mM ZD7288, which was similar to the effect of saline sodium channel currents were fit to the Boltzmann equa- (n = 10 and 10). For intrathecal anesthesia, ZD7288 alone tion. Two-way ANOVA (drugs and genotypes as principal did not produce tactile (n = 10, fig. 2E) or thermal (n = 10, factors) was performed for the comparison of membrane fig. 2F) sensory blockade. EC of Lidocaine for Tactile Blockade Increased in HCN1−/− properties (RMP and Rin). For all the cases, P < 0.05 was 50 considered as statistically significant. Mice. EC50 (median effective concentration) of lidocaine for

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Fig. 7. In sciatic nerve block model of mice, preinjection of forskolin (62.5 μg/ml) could shorten pinprick blockade of 1% lidocaine (Lido) in wild-type (WT) and HCN1−/− mice. (A) Duration of tactile blockade was not significantly changed, evaluated by von Frey fibers. (B) Duration of motor blockade was similar with or without preinjection of forskolin. The inset figure was average motor function scores at each time point. (C) Duration of thermal sensory blockade was unchanged by preinjection of forskolin, evaluated by foot-flick latency (heat stimuli). (D) Duration of pinprick blockade was significantly shortened by adding forskolin in both HCN1−/− and WT mice. HCN = hyperpolarization-activated cyclic nucleotide-gated channels. tactile sensory blockade (2 g von Frey fiber) and pinprick sen- P = 0.721) between the two genotypes. All the results were sory blockade were determined by the up-and-down method list in table 2. In intrathecal anesthesia, durations of 1% lido- −/− (fig. 3 and table 1). In sciatic nerve block, EC50 of lidocaine caine were also decreased in HCN1 mice (n = 10) than in for tactile blockade was significantly increased in HCN1−/− wild-type mice (n = 10) (fig. 5). Durations of tactile sensory mice than wild-type mice (0.22 ± 0.04% vs. 0.14 ± 0.03%, blockade and motor blockade were significantly shortened in −/− P < 0.001). EC50 of lidocaine for pinprick blockade was simi- HCN1 mice (P = 0.022 and 0.039, respectively), whereas lar between HCN1−/− and wild-type mice (0.36 ± 0.07% vs. durations of thermal sensory blockade and pinprick sensory

0.35 ± 0.05%, P = 0.807). Meanwhile, EC50 of lidocaine was blockade were similar (P = 0.490 and 0.898, respectively). significantly decreased both in tactile blockade (P = 0.033 vs. All the results were list in table 3. lidocaine alone) and in pinprick blockade (P<0.001 vs. lido- HCN Channel Blocker ZD7288 Prolonged Durations of caine alone) with preinjection of 5.5 mM ZD7288 (table 1). In Lidocaine. ZD7288 (HCN channel blocker, nonselectively intrathecal anesthesia, EC50 of lidocaine for tactile blockade was inhibits HCN1 and HCN2 subtypes) could prolong dura- significantly increased in HCN1−/− mice than wild-type mice tions of 1% lidocaine. In wild-type mice (n = 10), durations

(0.38 ± 0.08% vs. 0.28 ± 0.06%, P = 0.008), whereas EC50 of of tactile sensory blockade increased from 15.0 (6.3) to 20.0 lidocaine for pinprick blockade was similar between HCN1−/− (1.3) min (fig. 6A, P = 0.011); durations of motor block- and wild-type mice (0.47 ± 0.07% vs. 0.45 ± 0.05%, P = 0.878). ade increased from 17.5 (11.3) to 25.0 (10.0) min (fig. 6B, Durations of Lidocaine Were Shortened in HCN1−/− Mice. P = 0.024); durations of thermal sensory blockade increased In sciatic nerve block model, durations of 1% lidocaine from 10.0 (5.0) to 15.0 (5.0) min (fig. 6C, P = 0.016); and were shortened in HCN1−/− mice (n = 10) than that in durations of pinprick sensory blockade increased from 6.0 wild-type mice (n = 10), while maximal effects of lidocaine (6.3) to 12.5 (11.3) min (fig. 6D, P = 0.018). In HCN1−/− were unchanged (fig. 4). Durations of tactile sensory block- mice (n = 10), durations of tactile sensory blockade increased ade decreased from 15.0 (6.3) to 9.0 (5.0) min (fig. 4A, from 9.0 (5.0) to 12.0 (5.0) min (fig. 6A, P = 0.035); dura- P = 0.008); durations of motor blockade decreased from tions of motor blockade increased from 10.0 (6.3) to 15.0 17.5 (11.3) to 10.0 (6.3) min (fig. 4B, P = 0.021); dura- (6.3) min (fig. 6B, P = 0.006); durations of thermal sensory tions of thermal sensory blockade decreased from 10.0 blockade increased from 5.0 (6.0) to 10.0 (5.0) min (fig. 6C, (5.0) to 5.0 (6.0) min (fig. 4C, P < 0.001). However, dura- P = 0.038); and durations of pinprick sensory block- tions of pinprick sensory blockade were similar (fig. 4D, ade increased from 6.0 (5.0) to 10.0 (11.3) min (fig. 6D,

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Fig. 8. Lidocaine nonselectively inhibited hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) in both −/− large and small dorsal root ganglia (DRG) neurons. (A) Ih were recorded in large and/or small DRG neurons. For HCN1 mice, Ih was almost eliminated in large neurons while the currents were preserved in small neurons. Lidocaine at 100 μM could inhibit Ih in both large and small DRG neurons. (B) Activation curves of HCN channel were recorded. In large DRG neurons of HCN1−/− mice, activation curve of HCN channel was diminished, whereas in small neurons, activation curve of HCN channel was unaffected by HCN1 deletion. (C) Lidocaine reversibly inhibited HCN channel currents without selectivity between large (n = 5) and small (n = 6) neurons (estimated IC50 = 82 ± 10 and 88 ± 9 μM, respectively. (D) Lidocaine at 100 μM significantly hyperpolarized half-maximal activation potential of Ih in large neurons (top), while without significant effect in small neurons (bottom).

P = 0.039). Of note, durations of 1% lidocaine in HCN1−/− further determined the effects of lidocaine on HCN chan- ZD7288/lido group were similar to wild-type saline/lido nel currents in both large and small DRG neurons from group (fig. 6). Both HCN1 and HCN2 subtypes contrib- wild-type mice. Lidocaine reversibly inhibited HCN chan- uted to tactile sensory blockade, thermal sensory blockade, nel currents without selectivity between large and small neu- and motor blockade of lidocaine, whereas HCN2 subtype rons (estimated IC50 = 82 ± 10 and 88 ± 9 μM, respectively). contributed to pinprick sensory blockade. All the results Maximal inhibition of lidocaine on HCN channel currents were list in table 2. was 53 ± 9% and 48 ± 10%, respectively, for large and small Forskolin Shortened Durations of Lidocaine. In wild-type neurons (fig. 8C). At a concentration of 100 μM, lidocaine and HCN1−/− mice (n = 10 and 10), pinprick blockade significantly hyperpolarized the activation potential of HCN durations of 1% lidocaine were significantly shortened by channel currents in large neurons but not in small neurons forskolin (fig. 7 and table 2). Although durations of tactile (fig. 8D). sensory blockade, thermal sensory blockade, and motor Deletion of HCN1 Subtype Did Not Affect Sodium Channel blockade were shorter than that of 1% lidocaine alone, no Currents. Sodium channel currents and inhibition of lido- significance was found. caine on sodium channel currents were unaffected by HCN1 deletion. Sodium channel currents were similar between In Vitro Electrophysiologic Recordings wild-type and HCN1−/− mice (fig. 9, A and B). Lidocaine Lidocaine Inhibited HCN Channel Currents in DRG Neurons. could reversibly inhibit sodium channel currents with- For wild-type mice, activation kinetics of HCN channel cur- out selectivity between large and small neurons (fig. 9, C rents were slower in small neurons than large neurons (time and D). For sodium channel currents in large DRG neu- constant at −120 mV: 62 ± 11 vs. 382 ± 46 ms, P < 0.001). rons, estimated IC50 of lidocaine were 86 ± 6 and 99 ± 6 μM For HCN1−/− mice, HCN channel currents were almost (P = 0.092), respectively, for wild-type and HCN1−/− mice diminished in large neurons, whereas HCN channel currents (fig. 9D). For sodium channel currents in small DRG neu- in small neurons were unaffected (fig. 8A). I-V (current-volt- rons, estimated IC50 of lidocaine were 103 ± 7 and 111 ± 7 age) curves of HCN channel currents in small neurons were μM (P = 0.098), respectively, for wild-type and HCN1−/− similar between wild-type and HCN1−/− mice (fig. 8B). We mice (fig. 9D).

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Fig. 9. Lidocaine inhibited sodium channel currents (INa) both in large and/or in small dorsal root ganglia (DRG) neurons (n = 5–6). (A) INa was recorded in both large and small DRG neurons and was not modulated by HCN1 deletion. Kinetics of INa was fast in −/− large neurons and slow in small neurons. (B) I-V curves and activation curves of INa were similar between wild-type and HCN1 mice. (C) INa traces indicated significant inhibition by 100 μM lidocaine in both large and small neurons. (D) Lidocaine could inhibit −/− INa with similar effects in large and small neurons from wild-type and HCN1 mice. HCN = hyperpolarization-activated cyclic nucleotide-gated channels.

Lidocaine Affected Neural Membrane Properties via HCN local anesthetics could inhibit propagation of action poten- Channels. As shown in figure 10, RMP of large neurons was tial.1 Besides sodium channel, previous studies have demon- significantly hyperpolarized by HCN1 deletion (−67 ± 10 vs. strated that lidocaine could also inhibit HCN channels,6,7 and

−56 ± 9 mV, P < 0.001) and Rin of large neurons was signifi- enhancement of clonidine and dexmedetomidine on regional cantly increased (72 ± 17 vs. 140 ± 18 MΩ, P < 0.001). For anesthesia was achieved by inhibiting HCN channels.8,9 large neurons from wild-type mice, both lidocaine 100 μM The current study indicates that HCN channel contributes and ZD7288 100 μM could hyperpolarize RMP of neurons to regional anesthetic effects of lidocaine. This finding not only provides an innovative regional anesthetic mechanism and increase their Rin. The modulation of lidocaine on RMP −/− but also indicates a molecular target for designing novel local and Rin was diminished in large neurons by HCN1 dele- anesthetics. tion (fig. 10, A and C). However, RMP and Rin of small neurons were unaffected by HCN1−/− deletion (fig. 10, B and HCN channel is widely expressed on various tissues in D). For small neurons from wild-type mice and HCN1−/− both animals and human beings.13–17 For peripheral ner- mice, lidocaine 100 μM and ZD7288 100 μM could signifi- vous system, HCN1 and HCN2 subtypes have been found in DRG22–25 and nerve fibers.26,27 Previous study indicated cantly hyperpolarize RMP of neurons and increase their Rin (fig. 10, B and D). All the membrane properties data were that function of nerve axon was modulated by HCN chan- 28,29 listed in table 4. nels, which might contribute to peripheral nerve block. In addition, it has been demonstrated that HCN1 subtype is predominant in large diameter DRG neurons, whereas Discussion HCN2 is predominant in small ones.22–24 This differen- It is widely believed that sodium channel is the main tar- tial distribution between HCN1 and HCN2 subtypes has get for local anesthetics, and by blocking sodium channels, also been confirmed by electrophysiologic recordings.22,25

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Fig. 10. Neural membrane properties of dorsal root ganglia (DRG) neurons were recorded. (A) In large neurons, resting membrane potential (RMP) was hyperpolarized by HCN1 deletion (n = 6). Lidocaine at 100 μM significantly hyperpolarized RMP of neurons in wild-type mice (n = 5), and this effect was diminished by HCN1 deletion (n = 6). (B) In small neurons, RMP of neurons was unaffected by HCN1 deletion (n = 7) and lidocaine could hyperpolarize RMP of neurons in both HCN1−/− (n = 7) and wild- type mice (n = 6). (C) In large neurons, input resistance (Rin) of neurons increased by HCN1 deletion (n = 6). Lidocaine at 100 μM −/− significantly increased Rin of the neurons in wild-type mice (n = 5) but not in HCN1 mice (n = 6). (D) In small neurons, Rin of −/− neurons was unchanged by HCN1 deletion (n = 7) and 100 μM lidocaine significantly increased inR of neurons in both HCN1 (n = 7) and wild-type mice (n = 6). *P < 0.05 versus control. HCN = hyperpolarization-activated cyclic nucleotide-gated channels.

Small DRG neurons are widely known as nociceptor, and blockade. Contrarily, forskolin, a cyclic adenosine mono- most HCN1-positive neurons are A-type neurons.31,32 We phosphate (cAMP) enhancer, could significantly shorten confirmed the differential distribution between HCN1 and duration of lidocaine for pinprick blockade but with no sig- HCN2 subtypes in this study because HCN channel cur- nificant effect on tactile sensory blockade, thermal sensory rents were almost diminished in large DRG neurons by blockade, and motor blockade. By previous studies, activa- HCN1 deletion, while these currents were unaffected in tion of HCN channels could be enhanced by cAMP, and small neurons. HCN2 subtype is more sensitive than HCN1 subtype.13,25 In the current study, durations of lidocaine were short- Comparing with HCN1 subtype, HCN2 subtype is acti- ened in HCN1−/− mice except for pinprick blockade. By vated at hyperpolarized potential.13,40 Thus, it has been dem- blocking HCN2 subtype in HCN1−/− mice, ZD7288 could onstrated that cAMP analogs could depolarize the activation prolong durations of lidocaine, including pinprick sensory potential of HCN channels in C-type neurons but not in

Table 4. Membrane Properties of Current Clamp Experiments

RMP (mV) Rin (MΩ)

Groups Control ZD7288 Lidocaine Control ZD7288 Lidocaine Cm (pF) Wild-type large neurons 56 ± 9 74 ± 11* 63 ± 10* 72 ± 17 160 ± 21* 100 ± 12* 49 ± 8 Wild-type small neurons 64 ± 7 80 ± 8* 69 ± 8* 40 ± 7 105 ± 12* 65 ± 10* 20 ± 3 HCN1−/− large neurons 67 ± 10 72 ± 9* 69 ± 8 140 ± 18 180 ± 19* 150 ± 15 47 ± 6 HCN1−/− small neurons 66 ± 7 78 ± 9* 72 ± 9* 42 ± 7 95 ± 12* 60 ± 10* 19 ± 2

The data were expressed as mean ± SD. For example, the group “wild-type large neurons” indicated the data recorded on large neurons of wild-type mice. *P < 0.05 vs. control group.

Cm = membrane capacitance. HCN = hyperpolarization-activated cyclic nucleotide-gated channels; Rin = input resistance; RMP = resting membrane potential.

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A-type.26 For this study, the effect of forskolin on pinprick This finding provides a molecular target for designing long- blockade indicates that HCN2 subtype contributes to high- lasting nociceptive-preferred local anesthetics. threshold sensory blockade. Interestingly, previous studies found that HCN1 subtype was more critical than HCN2 Acknowledgments for the function of motor axon, and the HCN channels This study was supported by the grant 81371249 (to Dr. expressed on motor axon were activated at the resting mem- Chen) from the National Natural Science Foundation of Chi- brane potential.29,41,42 Therefore, selectively inhibit HCN2 na (Beijing, China); the grant 81401139 (to Dr. Zhou) from subtype in regional anesthesia might induce a nociceptive- the National Natural Science Foundation of China (Beijing, China); and the grant 2014M552361 (to Dr. Zhou) from the preferred blockade. National Postdoctoral Foundation of China (Beijing, China). By electrophysiologic recordings, we found that HCN1 deletion did not affect the functions of sodium channels. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/3/606/369991/20150300_0-00022.pdf by guest on 28 September 2021 Competing Interests Lidocaine could inhibit sodium channel currents without selectivity between large and small DRG neurons. Mean- The authors declare no competing interests. while, lidocaine also inhibited HCN channel currents in Correspondence both large and small DRG neurons at relevant concentra- Address correspondence to Dr. Liu: Laboratory of Anesthesia tions. Of note, maximal inhibition of lidocaine on HCN & Critical Care Medicine, Translational Neuroscience Center channel currents was merely approximately 50% in the cur- and Department of Anesthesiology, West China Hospital, Si- rent study while previous study found that lidocaine could chuan University, Chengdu 610041, Sichuan, P.R. China. scu- almost completely inhibit HCN channel currents when [email protected]. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead concentration of lidocaine reached 600 μM (while IC of 50 page at the beginning of this issue. Anesthesiology’s articles lidocaine was 72 ± 7 μM, which was similar to the current are made freely accessible to all readers, for personal use study) in central nervous system.7 This discrepancy might only, 6 months from the cover date of the issue. result from different recording samples and protocols. We further investigated the effect of lidocaine on neural mem- References brane properties. Like ZD7288, lidocaine could hyperpolar- 1. Butterworth JF 4th, Strichartz GR: Molecular mechanisms of ize RMP and increase input resistance of neurons at regional local anesthesia: A review. Anesthesiology 1990; 72:711–34 anesthetic relevant concentrations. Therefore, HCN chan- 2. 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