Lidocaine, which is widely used as a local anesthetic and antiarrhythmic drug, inhibits the voltage-gated sodium channels by stabilizing their inactivation, thereby blocking neurotransmission. Lidocaine passes through the cell membrane because of its lipid solubility and binds to Na channel proteins from the inside.1 Lidocaine also crosses the blood–brain barrier.2,3 Although the precise mechanisms behind the effects of lidocaine remain unclear, the systemic administration of low-dose lidocaine through continuous IV infusion has been shown to relieve cancer pain, chronic pain, pain because of adiposis dolorosa, and pain after surgery.4,5
In many types of central neurons, including the cerebellar Purkinje neurons, the voltage-dependent Na+ channels, the kinetics of which differ from those of the fast Na+ channels, have been reported to produce inward currents, called persistent Na+ currents, and maintain long-lasting cellular depolarization.6,7 The persistent Na+ currents have various physiologic functions, such as burst generation in many types of neurons (including respiratory burst generation).8,9 The currents have also been suggested to play an important role in Na+ channel-related diseases such as epilepsy.10,11 Lidocaine has been shown to suppress persistent Na+ current.12
Although it has been reported that the IV administration of lidocaine induced the depression of respiratory activity,13,14 the central mechanisms are unknown. Interestingly, Onizuka et al.15 reported that lidocaine induced the excitation of respiratory pacemaker neurons in invertebrates through disinhibition. However, there are no reports concerning the effects of lidocaine on rhythm-generating neurons in the respiratory center of vertebrates. We hypothesized that, at certain doses, the central administration of lidocaine could induce antinociceptive effects without inducing respiratory depression. In this study, we examined the effects of lidocaine on respiratory rhythm generation in brainstem–spinal cord preparations from newborn rats, an in vitro model that has been used to analyze respiratory control.16 To assess the antinociceptive effects of drugs, it has been established that the slow ventral root potential induced by ipsilateral dorsal root stimulation in the isolated (typically lumbar) spinal cord of newborn rats reflects the nociceptive reflex. This in vitro experimental model is useful for assessing the actions of analgesics.17,18 We also examined the effects of lidocaine on reflex responses in the spinal cord, which are presumed to indicate a nociceptive response.19
Preparation and Solutions
Brainstem–spinal cord preparations from Wistar rats (postnatal day 0–3) were isolated under deep isoflurane anesthesia.20,21 The experimental protocols were approved by the Animal Research Committee of Showa University, which operates in accordance with Law No. 105 for the care and use of laboratory animals of the Japanese Government. The preparations were cut transversely at a level just rostral to the anterior inferior cerebellar artery. Preparations were superfused continuously at 2.5 to 3 mL/min in a 2-mL chamber with artificial cerebrospinal fluid,22 which is composed of (in millimolar) 124 NaCl, 5.0 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 30 glucose; equilibrated with 95% O2 and 5% CO2 at a pH of 7.4; and maintained at a temperature of 25°C to 26°C. Inspiratory activity corresponding to phrenic nerve activity was monitored from the fourth cervical ventral root (C4). Lidocaine (Sigma-Aldrich, Tokyo, Japan) was stocked as a 100 mM solution in dimethyl sulfoxide and was kept at 4°C. Lidocaine was dissolved with the aforementioned artificial cerebrospinal fluid and bath applied. To assess the effects of lidocaine on C4 activity, the burst rate (bursts per minute) was calculated from the mean rate for 3 to 5 minutes.
To simultaneously evaluate the effects of lidocaine on respiratory activity and putative nociceptive responses,19 the ipsilateral C7/C8 dorsal root was stimulated using a glass suction electrode, and the induced reflex response was recorded from C4/C5 together with rhythmic inspiratory activity through a 0.5-Hz high-pass filter. The dorsal root was stimulated every 10 seconds with a 5 to 20 V, 200 μs square pulse. Dorsal root stimulation within the interburst interval induced premature C4 inspiratory bursts if the stimulus intensity was high. Therefore, in the simultaneous recordings of C4 inspiratory activity and reflex responses in the intact brainstem–spinal cord preparation, we carefully adjusted the stimulus intensity for the induction of reflex responses such that the amplitude of the reflex responses was large enough to detect but not so high that it induced premature C4 inspiratory bursts. In some experiments, spinal cord preparations of the C4-Th2 level were isolated (thus, they did not include the medulla) and used to test the aforementioned reflex response. We measured the peak amplitude of the reflex response, which corresponded to a short latency oligosynaptic response.19 With regard to the effects of the lidocaine on the reflex response, there was no significant difference between the preparations that included and did not include the medulla. Therefore, we accumulated and analyzed data from both preparations. However, because it was difficult to completely exclude the effects of dorsal root stimulation on C4 inspiratory burst rate in the experiments in which the reflex responses were tested, the results of these experiments were excluded from the group data on the dose-dependent effects on the C4 respiratory rate.
Whole-Cell Patch-Clamp Recording and Histologic Analysis
The membrane potentials of the preinspiratory (Pre-I) and inspiratory neurons in the rostral ventrolateral medulla corresponding to the caudal part of the parafacial respiratory group (pFRG), in which respiratory neurons have been recorded in a number of previous studies,21,23 were recorded by a blind whole-cell patch-clamp method with a high input impedance-DC amplifier (CEZ-3100; Nihon Koden, Tokyo, Japan).20,24 The electrodes, which had an inner tip diameter of 1.2 to 2.0 μm and a resistance of 4 to 8 MΩ, were filled with the following pipette solution (in millimolar) composed of 130 K-gluconate, 10 EGTA, 10 HEPES, 2 Na2-ATP, 1 CaCl2, and 1 MgCl2, with pH 7.2 to 7.3 adjusted with KOH. We analyzed the membrane potential, input resistance, burst duration, and drive potential of the Pre-I and inspiratory neurons.23 The magnitude of the drive potential was determined as the voltage difference between the resting membrane potential in the interburst phase and the peak of the depolarization plateau during the burst phase (Tables 1 and 2). In the control solution, current pulses (amplitude, 10–100 pA; duration, 0.5 seconds) were injected around the middle of the interburst period to examine the firing properties of neurons in response to a depolarizing pulse and to estimate the input resistance with a hyperpolarizing pulse.
Under the blockade of the potassium and calcium channels, the detection of negative slope conductance is thought to be an indicator of the presence of persistent Na+ current.25–27 In some experiments, we analyzed the negative slope conductance in response to depolarizing voltage-ramp stimulation under voltage clamp conditions using a continuous voltage-clamp amplifier (current-voltage converter type; Axopatch 1D, Axon Inc., Foster City, CA). In this experiment, electrodes were filled with the following (potassium channel blockade) pipette solution (in millimolar): 100 CsCl, 20 TEA-Cl, 11 K-BAPTA, 4 Na2-ATP, 1 CaCl2, 2 MgCl2, 10 HEPES, and 0.5% Lucifer Yellow (lithium salt), with a pH of 7.2 to 7.3 adjusted with NaOH.28 After the establishment of whole-cell recordings, we added 0.1 mM CdCl2 into the external solution to block the calcium channels. C4 activity disappeared within 10 minutes. The cell was then clamped at −70 mV. To detect negative slope current, we tested the slope of ramp stimulation in the range of 10 to 50 mV/s. Na+ current contamination, i.e., transient, unclamped action potential-generating Na+ current that appeared as downward spikes in the current trace (see Results),26 was observed in most cases under all of the tested slope conditions (under our experimental conditions). This was presumably because the space clamp was insufficient for the large dendritic field of these respiratory neurons.9 In most cases, we used 46.7 mV/s ramp stimulation because the negative slope component was clearly detectable, despite the contamination of the fast Na+ current.
For histologic analysis of the recorded cells, the electrode tips were filled with 0.5% Lucifer Yellow (lithium salt). After the experiments, preparations were fixed overnight at 4°C in 4% paraformaldehyde in 0.1M phosphate buffer solution, transferred into 18% sucrose/phosphate buffer solution, and cut into 50-μm thick transverse sections. Neurons were visualized with Lucifer Yellow staining under a fluorescence microscope (BX60, Olympus Optical, Tokyo, Japan) and photographed. We confirmed that most of the neurons in the intracellular recordings were located in the caudal part of the pFRG, which corresponded to the level within ±100 μM rostrocaudal to the caudal end of the facial nucleus.23
The sample size was determined based on the general consensus of in vitro studies for basic science in which statistical significance could be achieved with the sample size >5 in every experiment,19,21,24 but all efforts were made to minimize the number of animals. All of the data analyses were performed using the LabChart 7 Pro software program (ADInstruments, Castle Hill, Australia). Data are presented as the mean and SD for all preparations. The significance of the values was analyzed by paired t test for effects on C4 burst rate, membrane parameters, and the peak current in the negative slope (considering statistically significant with a 2-tailed P value <0.05) or a 1-way analysis of variance followed by a Tukey–Kramer multiple comparisons test for spinal reflex response (GraphPad InStat; GraphPad Software Inc., La Jolla, CA) at a confidence level of P < 0.05.
The Effects of Lidocaine on C4 Inspiratory Activity
We first examined the effects of lidocaine (2–400 μM, n = 58) on the C4 burst rate. The 15-min bath application of lidocaine (100–400 μM) induced a dose-dependent decrease in the C4 burst rate (Fig. 1, A–D). C4 bursts were completely blocked by the 15-min application of 400 μM of lidocaine (Fig. 1G). In contrast, the application of lower concentrations of lidocaine (10–20 μM) tended to increase the C4 burst rate (Fig. 1, E–G). After lidocaine washout (100–200 μM) in 63% preparation, the burst rate showed a partial recovery and the bursts changed into an episodic pattern which consisted of multiple (3–6) short discharges (Fig. 2).
The Effects of Lidocaine on the Membrane Potentials
We examined the effects of lidocaine (100–400 μM) on the membrane potentials and burst activity of 8 Pre-I and 14 inspiratory neurons in the caudal pFRG (Tables 1 and 2). The burst duration of the Pre-I and postinspiratory phases of Pre-I neurons decreased in a dose-dependent manner after the application of 100–400 µM lidocaine (Fig. 3 and Tables 1 and 2). The burst activity vanished irreversibly after the application of 400 μM lidocaine (Fig. 3). The burst duration of inspiratory neurons tended to decrease after the application of lidocaine (>100 μM); however, these changes did not reach statistical significance due to both the large degree of variation and the small sample size (Tables 1 and 2). The application of 400 μM lidocaine resulted in the irreversible cessation of inspiratory burst generation (Fig. 4). The driving potentials of both neurons also tended to decrease in a dose-dependent manner, but the change did not reach statistical significance (Tables 1 and 2). We did not find significant change in the membrane potentials and the input resistances of neurons in response to lidocaine treatment.
After the cessation of C4, Pre-I (n = 5) and inspiratory (n = 8) neuron burst activities in response to the application of 400 μM of lidocaine, action potentials continued to be induced during membrane depolarization by current pulse injection (500 milliseconds). However, the number of induced action potentials was decreased in comparison to control, because the induction of action potentials was limited to the initial part of the depolarization by the stimulation pulse (Fig. 5A).
Because lidocaine reduced the burst duration of Pre-I neurons (as shown by the above results), we examined the effects of lidocaine on negative slope conductance under voltage clamp conditions in Pre-I neurons (n = 5). A representative trace from a Pre-I neuron is illustrated in Figure 5B. The activation of persistent Na+ current was reflected by negative slope conductance on the current-voltage plot. The average peak inward current, – 43.8 ± 11.4 pA (n = 5), was obtained by subtracting the linear leak current from the total current. The application of lidocaine (100 μM) for 15 minutes resulted in a considerable reduction of the inward deflection in comparison to control (51.1% ± 11.0%, P = 0.00074 by paired t test), indicating that the lidocaine-sensitivity of the Na+ current might underlie the conductance.
The Effects of Lidocaine on Spinal Reflex Responses
The dorsal root stimulation of C7/C8 induced reflex responses in the C4/C5 ventral root, which typically lasted for 0.5–1 second and which were presumed to be oligosynaptic responses (Fig. 6).19 The effects of lidocaine on C4/C5 reflex responses that were induced by ipsilateral C7/C8 dorsal root stimulation were examined in 11 preparations at concentrations of 5 or 20 μM. The amplitude of the C4/C5 inspiratory nerve activity did not change in response to the application of lidocaine at these ranges of concentration. However, as shown in Figure 6, the amplitude of the C4/C5 reflex responses decreased after the application of 20 μM lidocaine. The depressing effects partially recovered 20 to 30 minutes after washout (Table 3).
Low doses of lidocaine (10–20 μM) tended to increase the C4 burst rate, whereas high doses of lidocaine (100–400 μM) decreased the C4 burst rate in a dose-dependent manner and then blocked the burst activity completely. The burst duration of Pre-I neurons decreased in a dose-dependent manner after the application of high doses of lidocaine (100–400 μM). After the cessation of Pre-I and inspiratory neuron burst generation was induced by a high dose of lidocaine, depolarizing current stimulation continued to induce action potentials, whereas the induction of the spike train was depressed because of strong adaptation. The application of lidocaine (100 μM) reduced the negative slope conductance, suggesting the partial blockade of persistent Na+ current. After the washout of lidocaine, the C4 inspiratory burst structure gradually transformed into an episodic burst pattern in which 1 burst was composed of multiple short discharges. A low dose of lidocaine (20 μM) had inhibitory effects on the C4/C5 spinal reflex response induced by the ipsilateral dorsal root stimulation of C7/C8, which was presumed to reflect, at least in part, a nociceptive response.
Our findings suggest that the respiratory depression that is induced by high doses of lidocaine is because of the inhibitory effect on the burst generation of Pre-I and inspiratory neurons. In contrast, the detailed mechanisms behind the slight facilitation of respiratory rhythm by low doses of lidocaine are not clear. Onizuka et al.15 reported that lidocaine facilitated molluscan respiratory rhythm through the inhibition of the γ-aminobutyric acid system. Lidocaine has also been reported to cause the inhibition of the mammalian central inhibitory system.29,30 This mechanism might partially explain the facilitatory effects that were induced by low doses of lidocaine in this study.
Lidocaine blocks the voltage-gated Na+ channels. It is thought that lidocaine penetrates the cell membrane and then binds to the cytoplasmic side of the channel.1 Studies in the dorsal root ganglions have suggested that persistent Na+ current is blocked by a low concentration of lidocaine (10 μM) and that transient Na+ current is blocked by high concentrations of lidocaine (5 mM).12 In this study, we confirmed that lidocaine (100 μM) depressed persistent Na+ current and decreased the burst duration of Pre-I neurons. These inhibitory effects may be the main reason for the inhibition of respiratory rhythm by lidocaine.
After the washout of bath-applied lidocaine (100–200 μM), we found that the structure of C4 inspiratory bursts gradually transformed into an episodic burst pattern in which 1 burst was composed of 3 to 6 short discharges (Fig. 3). A similar cluster-type burst pattern was also induced by treatment with riluzole.9 Both lidocaine and riluzole decreased the repetitive firing of action potentials during depolarizing stimulation in Pre-I and inspiratory neurons. We hypothesize that the depression of repetitive firing by lidocaine may cause the induction of the cluster-type burst pattern, whereas it is not clear how blockade of persistent Na+ current contributes to the alteration of the C4 burst pattern and the blockade of repetitive firing. It is known that the effects of lidocaine are short lived under the in vivo condition.31 Slow recovery from the effects of lidocaine on respiratory rhythm and reflex responses after washout that were observed in this study may imply that the removal of intracellular lidocaine is difficult under the present experimental conditions.
The clinical use of systemically administered lidocaine for pain treatment (postoperative pain relief) was first introduced in 1961.4 Since then, the systemic administration of lidocaine has been increasingly reported for the management of neuropathic pain. Clinical and experimental evidence indicates that the effective plasma concentration of lidocaine for the management of chronic pain is in the range from 1 to 2 µg/mL, which is equivalent to 3.5 to 7.0 μM/L.30 Lidocaine produces analgesia through the blockade of the peripheral and central Na+ channels. Studies in animal preparations have suggested a link between spontaneous ectopic discharges of the injured nerve and the peripheral mechanisms of neuropathic pain and indicate that such spontaneous discharges can be suppressed by the IV administration of lidocaine at concentrations well below those that are necessary to produce conduction blockade in nerves.30 Our findings indicated that the concentration of lidocaine that was required to depress nociceptive-related responses (20 μM) was lower than that which induced respiratory depression. This concentration was slightly higher than the plasma concentration required for the effective management of chronic pain. Although the results from experiments with in vitro preparations cannot simply be expanded to understand the effects of drugs in human clinical use or in adult in vivo preparations, our findings provide the basic neuronal mechanisms that support the clinical use of lidocaine, which shows antinociceptive effects with minimal side effects on breathing.
Name: Tomoharu Shakuo, MD.
Contribution: This author helped design the study, perform the experiments, analyze the data, and prepare the manuscript.
Attestation: Tomoharu Shakuo approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Shih-Tien Lin.
Contribution: This author helped perform the experiments, analyze the data, and prepare the manuscript.
Attestation: Shih-Tien Lin approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Hiroshi Onimaru, PhD.
Contribution: This author helped design the study, perform the experiments, analyze the data, and prepare the manuscript.
Attestation: Hiroshi Onimaru approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript and is the archival author.
This manuscript was handled by: Gregory Crosby, MD.
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