Lidocaine, when present in the systemic circulation, produces well-known concentration-dependent central nervous system (CNS) toxicity whose manifestation initially reflects inhibitory symptoms such as sedation, drowsiness, and alterations in sensorium, before progressing to excitatory phenomena including generalized seizures, and, in the most severe cases, coma and death.1,2 That lidocaine affects the CNS is not surprising, because it readily passes the blood–brain barrier.3 However, in contrast to lidocaine’s peripheral local anesthetic (LA) effects, the mechanisms and sites of its central actions have received comparatively little study because previous work has emphasized the spinal cord, leaving supraspinal actions relatively unexplored.4 Accordingly, there are no specific mechanism-based therapies for LA CNS toxicity.
Exploring the thalamus as a central site implicated in systemic LA actions, we previously found in rat thalamocortical (TC) neurons that lidocaine at clinically neurotoxic concentrations increases excitability mediated by Na+-independent, high-threshold (HT) action potential spikes.5 Although the ionic basis of these action potentials is unknown, TC neurons–central to the generation of conscious states, absence epilepsy, as well as drug-induced sedation, anesthesia, and analgesia6–9–express high-voltage–activated (HVA) Ca2+ currents that generate HT potentials, similar to those in our investigations.10–12 Hence, we conducted an in vitro mechanism of action study with the goal to characterize these HT potentials and determine their specific underlying ionic conductances, testing the hypothesis that they are generated by HT Ca2+ currents, previously implicated in neurotoxicity.13,14 We also sought to identify and isolate the specific underlying subtype of HT Ca2+ current and investigate the mechanisms that mediate the observed increase in excitability.
Preparation of Brain Slices
All animal experiments were approved by the Committee on Animal Care of the University of British Columbia. Our experimental procedures were similar to those described previously.5 Sprague-Dawley rats, aged P13 to P16, were decapitated under deep isoflurane (Abbott Laboratories, Montreal, Canada) anesthesia. The cerebrum was rapidly removed and placed in oxygenated (5% CO2/95% O2), cold (1°C–4°C), artificial cerebrospinal fluid (ACSF) of the following composition (mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 10 dextrose (pH, 7.3–7.4; 290 mOsm). The brain was dissected into 1 block and mounted on a tissue slicer with cyanoacrylate adhesive. Coronal slices (thickness, 250–300 μm) containing the ventrobasal thalamic nuclei were cut on a Leica VT1200S vibratome (Leica Biosystems, Nussloch, Germany), and the block was submerged in oxygenated cold ACSF. After cutting, the slices were incubated for >1 hour at room temperature (22°C to 24°C) in oxygenated ACSF.
Whole-cell patch-clamp recordings were performed in the current-clamp mode with a MultiClamp 700B amplifier (Molecular Devices, LLC, Sunnyvale, CA). The patch pipettes for recording were prepared with a Narishige PP-83 2-stage electrode puller (Narishige Scientific Instrument Lab, Tokyo, Japan) using thin-walled borosilicate glass (World Precision Instruments, Inc., Sarasota, FL) and filled with a solution containing the following (in mM): 139 K-gluconate, 10 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 6 KCl, 4 NaCl, 3 MgCl2, 10 HEPES, 0.5 CaCl2, 3 adenosine 5′-triphosphate (disodium salt, Na2ATP), 0.3 guanosine-5′-triphosphate (sodium salt, NaGTP), titrated to pH 7.3 to 7.4 with 10% gluconate. The range of the measured electrode resistances was 5 to 6 MΩ, and access resistance ranged from 10 to 25 MΩ.
For recording, the slices were transferred into a Perspex submersion chamber (volume, 1.5 mL), where they were immobilized with nylon mesh and continuously perfused by gravity with oxygenated ACSF at a flow rate of 2.5 mL/min controlled by a FR-50 flow valve (Harvard Apparatus, Holliston, MA). Individual neurons were visualized with the aid of differential interference contrast infrared videomicroscopy. For this purpose, an Axio Examiner.D1 fixed-stage microscope (Carl Zeiss Microimaging GmbH, Göttingen, Germany) was equipped with an apochromatically corrected water immersion objective (W Plan-APOCHROMAT 40×/1.0 DIC M27; working distance, 2.5 mm), differential interference as well as Dodt contrast optics, and a 0.9 condenser. Infrared light was obtained by placing a 770-nm polarizer in the light path. The images were recorded with an IR-1000 infrared CCD video camera system (DAGE-MTI, Michigan City, IN) and displayed on a Triview TBM-1503 black and white monitor (Tatung Company of America, Inc., Long Beach, CA).
The patch pipettes were mounted on the headstage of the amplifier and advanced with an MP-285 motorized precision micromanipulation system (Sutter Instrument, Novato, CA). Signals were low-pass filtered at a frequency of 3 kHz and digitized at 10 kHz with a Digidata 1440A 16-bit data acquisition system (Molecular Devices, LLC) controlled using pCLAMP software version 10 (Molecular Devices, LLC). Membrane potentials were corrected offline for a liquid junction potential of −8 mV.15 No leak subtraction was performed.
Lidocaine HCl, Cd2+, and nifedipine were obtained from Sigma-Aldrich Canada Ltd. (Mississauga, ON, Canada); ω-conotoxin GVIA and tetrodotoxin (TTX) were obtained from Alomone Labs, Ltd. (Jerusalem, Israel). To prepare concentrated drug stock solutions, lidocaine and Cd2+ were dissolved in ACSF; TTX and ω-conotoxin GVIA were dissolved in distilled water; and nifedipine was dissolved in dimethyl sulfoxide. Before application, required aliquots of the stock solutions (stored at 4°C [lidocaine, nifedipine] and −20°C [Cd2+, TTX, ω-conotoxin GVIA], respectively) were dissolved in fresh ACSF to obtain the respective concentrations. All drugs were applied to the bath by switching from the control perfusate to ACSF containing a desired drug concentration with the aid of a VC-6 perfusion valve control system (Warner Instruments, LLC, Hamden, CT). Recordings were conducted after 6 minutes of perfusion of the slices with a test solution. All results reported reflect steady-state responses.
Data are presented as mean ± SD unless mentioned otherwise. Multigroup dose-response comparisons were conducted with the use of 1-way analysis of variance with the Bonferroni post test as appropriate. Testing for differences of normalized data from baseline (i.e., a hypothetical mean of 1.0) was performed with a 1-sample t test. Comparisons between 2 groups were conducted with the use of a paired Student t test. We quantified the degree to which 2 variables are related by computing the Pearson correlation coefficient. Statistical tests were 2 tailed. Sample sizes were based on previous experiments,5,16 and, to be conservative, results of statistical comparisons were considered significant at α = 0.01. Configuration variables (amplitudes and dV/dtmax) of individual spikes in action potential trains are given descriptively and expressed as ranges. Data were analyzed with the use of ORIGIN (OriginLab Corporation, Northampton, MA) and Prism (GraphPad, La Jolla, CA) software.
All neurons used in this study exhibited the characteristic electrophysiologic properties of TC relay neurons, firing repetitive tonic spikes when depolarized by direct current injection from membrane potentials near rest, and a low-threshold Ca2+ potential triggering a spike burst on depolarization from hyperpolarized potentials or on rebound from hyperpolarizing pulses.5,10,16 They were characterized by an average resting membrane potential of −64.6 ± 4.8 mV, input resistance of 315 ± 131 MΩ, and membrane capacitance of 188 ± 62 pF (n = 46).
Lidocaine Unmasks High-Threshold Action Potentials
Consistent with its well-known LA properties, bath application of lidocaine at 100 μM to 1 mM abolished Na+-dependent tonic firing evoked by injection of depolarizing current pulses into cells current-clamped at approximately −58 mV in all neurons (Fig. 1A, left and center panels). However, in 39 of 46 (85%) neurons, lidocaine unmasked a distinct repetitive firing pattern evoked by injection of current pulses of increased amplitude and characterized by a higher activation threshold and a different spike configuration (Fig. 1A, right panel). The average activation threshold for these HT potentials, defined as the lowest membrane voltage at which these potentials were activated after depolarizing injection of increasing step current pulses from a holding potential of approximately −58 to −60 mV, was −21 ± 8 mV (n = 12), compared with −44 ± 3 mV for control action potentials (P < 0.001). The average lowest current pulse evoking HT potentials (“current threshold”) exceeded that for control action potentials approximately 4-fold (251 ± 158 vs 60 ± 42 pA; n = 12; P < 0.001). The individual HT action potentials themselves had lower amplitudes (range, 25−37 vs 60−75 mV in control) but were followed by fast afterhyperpolarizations (AHPs) characterized by higher amplitudes (range, 8−16 vs 3−11 mV in control). The HT potentials also exhibited markedly slower depolarization and repolarization phases (maximum rates of increase and decrease, dV/dtmax), which ranged between 0.23 to 0.55 and 2.5 to 4.4 mV/ms, respectively, compared with 13 to 48 and 7 to 23 mV/ms for control action potentials at baseline (Fig. 1B). Of note, the first 1 to 3 spikes in a train of HT action potentials unmasked by lidocaine were fast and had similar morphology to those in control (Fig. 1A, right panel). TTX application to neurons (600 nM) also suppressed baseline tonic firing and unmasked the tonic firing of HT potentials (Fig. 1C). As opposed to lidocaine, TTX completely inhibited all fast action potentials preceding the HT potentials. Compared with the 39 neurons in which lidocaine unmasked HT potentials, the 7 neurons that did not exhibit lidocaine-induced HT potentials had similar resting membrane potentials and input resistances (P = 0.88 and 0.43, respectively); the average membrane capacitance in the latter was 136 ± 51 vs 197 ± 60 pF in the former (P = 0.015; 95% confidence interval of difference between means, 12−109).
Ionic Basis of High-Threshold Action Potentials Unmasked by Lidocaine
Coapplication of TTX (600 nM) with lidocaine did not block the unmasked HT potentials, supporting the hypothesis that the underlying current was not primarily carried by Na+ ions but mediated by Ca2+. When neurons in which HT potentials were evoked in the presence of lidocaine and TTX were perfused with an extracellular solution deprived of Ca2+, the potentials disappeared completely in all neurons tested (n = 4, Fig. 2A). This effect was fully reversible, with the HT potentials reappearing instantly when the superfusing medium was changed back to the Ca2+-containing control solution. Application of the nonselective HVA Ca2+ channel blocker, Cd2+ (50 μM),12 completely blocked these HT potentials in a reversible manner in all neurons tested (n = 4; Fig. 2B). Collectively, these results indicated that the HT action potentials unmasked by lidocaine in TC neurons are driven by a HVA Ca2+ current.
The L-Type Ca2+ Current Mediates High-Threshold Action Potentials Unmasked by Lidocaine
Ventrobasal TC neurons express a variety of HVA Ca2+ currents that potentially could underlie the HT potentials, including L, N, P/Q, and R types.17 Of these, N-type, L-type, and residual “R”-type currents have been shown to mediate HT potentials in TC neurons in a composite manner, with the latter R-type current previously reported to represent the major component.12,18 In the present experiments, the observed sustained nature of the HT tonic firing and depolarized holding potential implied that inactivation properties (voltage dependence and rate of inactivation) of Ca2+ currents are critical for determining their suitability for mediating this firing. Characterized by the slowest inactivation rate and most depolarized voltage dependence of inactivation among HVA Ca2+ currents is the “long-lasting” L type.19,20 To test the hypothesis that this current mediates the HT tonic firing unmasked by lidocaine, we used the selective L-type Ca2+ channel blocker, nifedipine. In all neurons tested, application of 1 μM nifedipine decreased the amplitude of HT potentials (paired t test, P = 0.0001, n = 4) from 34 ± 4 to 25 ± 4 mV (mean ± SD of pairwise difference, −8 ± 1 mV) and increased (depolarized) their activation threshold (paired t test, P = 0.009, n = 4) from −24 ± 9 to −17 ± 10 mV (mean ± SD of pairwise difference, 7 ± 2 mV. At 5 μM, nifedipine completely blocked the HT potentials and produced low-amplitude (~2–5 mV) irregular membrane potential oscillations (Fig. 3A). Of note, the fast action potentials at the beginning of the current pulse were not affected by nifedipine. The above effects of nifedipine were reversible. Application of the selective N-type Ca2+ channel blocker, ω-conotoxin GVIA (1 μM), had little effect on the HT firing in 2 of 4 tested neurons (data not shown) and decreased (hyperpolarized) its activation threshold without affecting the amplitude of potentials in the other 2 neurons (ΔV, −7 and −8 mV, respectively; Fig. 3B). Collectively, these results implied that the L-type Ca2+ current, and not N- or R-type currents, mediates the HT action potentials unmasked by lidocaine in TC neurons.
Na+ Channel–Independent Actions of Lidocaine Contribute to the Unmasking of High-Threshold Action Potentials
In addition to voltage-gated Na+ channels, a variety of other ion channels and receptors have been implicated in the actions of lidocaine.5,21–24 To better understand whether such Na+ channel–independent actions contribute to the unmasking of HT spikes in TC neurons, we investigated lidocaine’s actions under conditions of robust Na+ channel blockade in neurons pretreated with TTX (600 nM).
Lidocaine concentration dependently decreased the current pulse magnitude required to trigger HT potentials in neurons (“current threshold”; Fig. 4A). This effect correlated strongly with a concomitant concentration-dependent increase in neuronal input resistance (Fig. 4B).5 At the same time, lidocaine did not affect HT potential spikes per se. The voltage threshold for activation of the HT potential spikes remained unaffected by 600 μM lidocaine in 3 of 5 neurons (Fig. 4A); in 2 neurons, the threshold decreased from approximately −19 and −30 to −29 and −37 mV, respectively (data not shown). We observed no significant shift in the voltage threshold of HT potentials with higher (1 mM) and lower (100 and 300 μM) concentrations; as illustrated in Figure 4A, lidocaine also did not significantly affect HT potential amplitudes over the concentration range (each concentration, n = 3−5; data not shown).
At the same time, however, lidocaine blocked the slow AHPs after cessation of depolarizing current pulse injections, known to be attributable to deactivation of the hyperpolarization-activated mixed cationic current, Ih25 (Fig. 5). Under control conditions (TTX), injection in 15-second intervals of step current pulses of a successively increased magnitude (20–40 pA increments) into neurons produced successively larger slow AHPs that gradually hyperpolarized the resting potential at the onset of the next depolarizing pulse (Fig. 5, A and C). This AHP increase was most pronounced (maximum increase, 10 mV) at depolarized voltages after the activation of tonic HT potential firing and associated with a successive decrease in their frequency (Fig. 5, B and D). Application of lidocaine at 100 μM substantially (Fig. 5, A and B) and at 600 μM completely (Fig. 5, C and D) inhibited the AHPs and facilitated the HT firing, such that neurons responded to increased current amplitudes with higher discharge frequencies in a quasilinear manner (Fig. 5D).
In the present in vitro mechanism of action study, we found that lidocaine, in the clinically CNS-toxic and convulsive concentration range, reversibly unmasked evoked HT action potentials mediated by the L-type Ca2+ current in rat TC neurons, while blocking Na+-dependent excitability as expected. The higher thresholds, slower rates of rise and fall, and lower amplitudes of the unmasked Ca2+-dependent action potentials compared with those driven by Na+ are explained by the differences between the activation and inactivation properties of voltage-gated Ca2+ versus Na+ channels.19,26,27
These findings in TC neurons, implicated in the systemic CNS actions of LAs,5,16 are in contrast to previous observations in other tissue preparations that lidocaine blocks various Ca2+ conductances.28–31 Conversely, our results are remarkably consistent with those of Mulle et al.,32 who reported an unmasking of fast prepotentials in TC neurons by intracellular injection of QX-314 (0.1 M in the pipette), a relatively impermeable experimental quaternary lidocaine derivative. The authors concluded that these potentials represent dendritic Ca2+ spikes. Somewhat similarly, Liu et al.33 found in dorsal root ganglion neurons from newborn rats that ropivacaine at 10 and 30 μM markedly increases a HVA Ca2+ current (but decreases it at ≥50 μM).
What possible functional implications do the present results have, and how do they change our views about LA neurotoxicity and its mechanisms? The overall physiologic role of HVA Ca2+ currents in TC neurons, while incompletely defined, includes regulation of tonic repetitive firing by triggering Ca2+-induced Ca2+ release from intracellular stores with subsequent activation of Ca2+-dependent K+ currents (IK(Ca))11,17,18,34,35 and generation of high-frequency oscillatory activity.36 They often complement the firing on top of the low-threshold Ca2+ potentials mediated by T-type Ca2+ currents,37 providing a range of burst firing patterns, and have been implicated in activity-dependent synaptic plasticity.38 In addition to these putative physiologic functions, however, HVA Ca2+ channels play an important role in neurotoxicity and cell death in the CNS. The pathogenesis of neurotoxicity involves several mechanisms that collectively lead to an increase in intracellular Ca2+ concentrations ([Ca2+]i). HVA Ca2+ channels participate in this process both directly and indirectly. They directly facilitate Ca2+ flux intracellularly, which, partially because of their slow inactivation, may produce substantial increases in [Ca2+]i.11,34 Alternatively, HVA Ca2+ channels, particularly those of the N type, may increase [Ca2+]i indirectly by mediating excitatory amino acid release12,39 and subsequent excitatory amino acid–induced excitotoxicity.40 In this regard, the present observations that lidocaine also unmasked the HT Ca2+ spikes under conditions of presynaptic transmitter release blockade by TTX and N-type Ca2+ channel blockade render direct increases in [Ca2+]i from extracellular sources more likely to be relevant for lidocaine’s neurotoxicity than indirect actions such as those mediated by N-type channels.
Although more is known in the literature about the processes that govern excitotoxicity in general (cf. above) than those involved in systemic LA neurotoxicity specifically, the present findings add to the growing body of evidence indicating that impaired Ca2+ homeostasis similarly contributes to the complex and multifaceted mechanisms of the latter. For example, a previous study found that IV lidocaine increases [Ca2+]i in the cortex of anesthetized rats.41 In vitro, an increase in [Ca2+]i in cultured neurons has been reported to play a critical role in the pathogenesis of lidocaine toxicity.23,42 However, it is noteworthy that the effective millimolar concentrations of lidocaine in the latter in vitro studies were higher than the micromolar concentrations corresponding to systemic CNS-toxic blood plasma levels in vivo and are, therefore, perhaps more applicable to spinal and regional anesthesia, where such high perineural concentrations of lidocaine are normally encountered.43 These considerations notwithstanding, there has been comparatively little research on the specific molecular and cellular mechanisms that govern lidocaine-induced increases in [Ca2+]i; both the release of Ca2+ from internal stores and the influx of extracellular Ca2+ have been implicated.23 Hence, the present finding of an unmasking of an L-type channel–mediated HVA Ca2+ current may help bridge the gap in explaining the mechanistic basis of CNS toxicity produced by high lidocaine concentrations in vivo.
Clustered at the somatodendritic junction, L-type Ca2+ channels in TC relay neurons are coupled to intracellular Ca2+ stores through ryanodine receptors and modulate Na+-dependent tonic firing by triggering Ca2+-induced Ca2+ release (cf. above).35,44,45 Therefore, a lidocaine-induced unmasking of L-type Ca2+ current-driven firing can lead to considerable Ca2+ influx and increases in [Ca2+]i in these neurons. The roles of L-type Ca2+ channels in the mediation of neurodegeneration and cell death, on one hand, and neuroprotective attenuation by pharmacologic L-type Ca2+ channel blockade, on the other hand, are well known from studies in other neuronal tissues.13,14,46,47 For LA neurotoxicity, the observation in the peripheral nervous system by Bainton and Strichartz48 that nifedipine renders frog sciatic nerves more resistant to lidocaine-induced degradation supports this hypothesis. Given the previous findings on the role of [Ca2+]i increases in the pathogenesis of LA neurotoxicity (mentioned earlier),23,42 the present results are consistent with the literature and, hence, represent a plausible contributing mechanism for lidocaine’s supraspinal toxic effects. However, the net level of [Ca2+]i increases in ventrobasal TC relay neurons in humans in vivo is unknown and will also depend on whether lidocaine affects other regulatory mechanisms of Ca2+ homeostasis, such as Ca2+-induced Ca2+ release, Ca2+-dependent inactivation, and functioning of the Na+–Ca2+ exchanger.35,49–51 For example, Xu et al.22 reported that lidocaine inhibits KCl- and carbachol-evoked intracellular Ca2+ transients in a concentration range similar to that in our study (0.1–2.3 mM), albeit in a neuronal cell culture. Clearly, further research, including studies using Ca2+ imaging, will be helpful and required to elucidate the net effect of lidocaine on Ca2+ homeostasis in the thalamus.
Ventrobasal thalamic neurons functionally express 2 L-type Ca2+ channel isoforms that are distinguished biophysically and pharmacologically, Cav1.2 and Cav1.3.51–53 Cav1.3 channels, which are less sensitive to dihydropyridines, are blocked by 5 to 10 μM nifedipine, whereas Cav1.2 channels are already significantly (>90%) inhibited by nifedipine concentrations as low as 1 μM.52 Therefore, our observations imply that Cav1.3, rather than Cav1.2, likely drives the observed HT action potentials. Furthermore, the more hyperpolarized (~20–25 mV) activation range of Cav1.3 channels compared with Cav1.2 would render them more suitable for mediating the tonic HT firing. The biophysical properties of a fast rate of inactivation, hyperpolarized inactivation range, and depolarized activation threshold collectively appear to be the key factors preventing other HVA Ca2+ currents expressed in TC neurons from mediating the HT firing. In this regard, our findings also indicate that an R-type Ca2+ current, contrary to what might have been expected based on older studies12,18 (cf. Results, fourth parapgraph) does not significantly contribute to the HT potentials unmasked by lidocaine. This in turn is consistent with reports that the R-type channel, Cav2.3, is not densely expressed in TC neurons.54,55
In this study, TTX also was able to unmask tonic HT firing while completely inhibiting Na+-dependent action potentials. However, the actions of lidocaine, which unlike TTX readily crosses the blood–brain barrier,3 differed from those of TTX in a number of ways because its unmasking effect was more pronounced than TTX. First, the threshold amplitudes of injected current evoking Ca2+-dependent action potentials were lower in lidocaine-treated neurons. It is noteworthy in this context that the HT potentials recorded in the presence of TTX had voltage thresholds consistent with those previously observed in ventrobasal thalamus by Ries and Puil (circa ≥−30 mV)56 but higher than those reported in rat auditory thalamus (ventral partition of the medial geniculate body; approximately ≥−40 mV) by Tennigkeit et al.37 Although these discrepancies remain unclear because neither of these 2 studies isolated the specific underlying HVA Ca2+ currents, it is known that Ca2+ channels, and specifically those of the L type, are subject to differential distribution and expression in different thalamic neurons.45 Second, as opposed to TTX, lidocaine-treated neurons, because of the concentration-dependent inhibition of the slow AHPs, were able to respond to increased current amplitudes by increased HT firing frequency. These effects of lidocaine were likely because of its robust inhibition of Ih in these neurons, which we have previously shown to result in an increase in input resistance and AHP inhibition.5 Third, the activation threshold of the HT potentials, although not in all neurons, was higher in the presence of TTX. A direct action of lidocaine on the L-type Ca2+ current producing a hyperpolarization of the activation threshold may account for this effect. In addition, we have previously demonstrated that inhibition of Ih and a K+ conductance (other than the inward rectifier, IKir) by lidocaine in the high micromolar range produces depolarization of TC neurons operating in the tonic firing mode.5 The latter is consistent with recent evidence that LAs block various members of the 2-pore domain (“tandem”) K+ channel family that underlies “leak” K+ currents (IKL) in a range of other tissues/preparations.57–59 Combined with a concomitant increase in input resistance, a lidocaine-induced K+ conductance blockade explains the reduction of the amplitudes of injected current required for triggering Ca2+-dependent potentials to the levels necessary for evoking Na+-dependent action potentials under control conditions. In this regard, our findings support the 3-decade-old hypothesis of Mulle et al.32 that the occurrence of dendritic HT spikes in TC neurons in the presence of QX-314 (cf. above) results from an increase in input resistance because of inhibition of persistent Na+ and/or K+ conductances. Given the all-or-none nature of Ca2+-driven action potentials, we postulate that the above Na+ channel–independent actions of lidocaine collectively account for the unmasking of HT action potentials driven by the L-type Ca2+ current.
Regarding the clinical relevance of the present lidocaine concentrations, 100 μM lidocaine HCl converts to approximately 27 μg/mL (i.e., a 0.0027% solution). Lidocaine readily passes the blood–brain barrier; according to the classic study by Usubiaga et al.,3 CSF concentrations of lidocaine in humans after IV injection correlate to arterial blood levels with a factor between 0.73 and 0.83. In rabbits receiving a continuous IV lidocaine infusion, the correlation factor (with a 10-minute latency between arterial and CSF sampling) ranges between ~0.47 and 0.64.60 These data collectively imply that a 100 μM CSF concentration would correspond to arterial levels between ~33 and 57 μg/mL in vivo, which unequivocally is in the CNS-toxic range.a
By the nature of the experimental methodology, this study has inherent limitations and shortcomings. First, because the investigations were conducted in juvenile rat brain slices in vitro, the question arises as to what extent the results can be extrapolated to the human patient in vivo. We recognize that such extrapolations require caution. For example, because the experiments were performed at room temperature to facilitate neuronal viability and stable recording conditions, it is reasonable to assume that effective concentrations at 37°C in vivo would likely be lower. Consistent with this, Yokoyama et al.62 found the mean lidocaine plasma concentrations in adult rats at the onset of CNS toxicity (using convulsions as an end point) to be 10.8 μg/mL; similar observations were made by Spiegel et al.63 Although systemic CNS toxicity in humans has been observed at concentrations <5 μg/mL,64 plasma lidocaine levels associated with systemic analgesia in humans are remarkably similar to those effective in rats.65–67 Second, the present studies focused on TC neurons in the ventrobasal thalamus; although these neurons have characteristic electrophysiologic properties,5,10,16 it remains to be determined whether other types of neurons would exhibit similar responses to lidocaine. Third, we did not study lidocaine concentrations in excess of 1 mM. However, as discussed earlier, although LA systemic toxicity is not normally associated with millimolar concentrations in the CSF, Mulle et al.32 found that intracellular injection of TC neurons with quaternary lidocaine at a pipette concentration of 0.1 M unmasked fast dendritic Ca2+ spikes. Fourth, in part because of the well-known difficulties in attaining adequate space clamp of dendritically expressed currents in neurons with a large dendritic tree, we report no data from experiments where HVA Ca2+ currents of TC neurons in the slices were voltage clamped. Finally, it must be emphasized that it was not the goal of this electrophysiologic in vitro study in brain slices to examine histologic or behavioral end points of LA CNS toxicity or investigate lidocaine’s effects on [Ca2+]i; clearly, a body of such future work will be required to elucidate the myriad of concentration-dependent effects that LAs exert on the brain. However, despite unavoidable shortcomings, experiments with slices provide a unique opportunity to study neurons in a relatively intact physiologic tissue environment.
In summary, at high-micromolar and clinically CNS-toxic concentrations, lidocaine unmasked in rat TC neurons evoked HT action potentials mediated by the L-type Ca2+ current while substantially suppressing Na+-dependent excitability. The present findings add to our understanding of the complex myriad of LA actions. On the basis of the well-known role of an increase in [Ca2+]i in the pathogenesis of LA neurotoxicity, this novel action in the thalamus, implicated as a central supraspinal site for LAs to produce their systemic CNS effects, represents a plausible contributing candidate mechanism for lidocaine’s CNS toxicity in vivo.
Name: Igor Putrenko, PhD.
Contribution: This author helped conduct the study, collect the data, analyze the data, and prepare the manuscript.
Attestation: Igor Putrenko has reviewed the original study data and data analysis, attests to their integrity, and approved the final manuscript.
Name: Amer A. Ghavanini, MD, PhD, FRCPC.
Contribution: This author helped conduct the study, collect the data, analyze the data, and prepare the manuscript.
Attestation: Amer A. Ghavanini approved the final manuscript.
Name: Katrin S. Meyer Schöniger, MD.
Contribution: This author helped conduct the study and collect the data.
Attestation: Katrin S. Meyer Schöniger approved the final manuscript.
Name: Stephan K. W. Schwarz, MD, PhD, FRCPC.
Contribution: This author developed the study rationale and design; helped conduct the study, collect the data, analyze the data, and prepare the manuscript; and is the archival author.
Attestation: Stephan K. W. Schwarz has reviewed the original study data and data analysis, attests to their integrity, and approved the final manuscript.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
The authors thank Ernest Puil, PhD, for valuable discussions and insightful comments.
a In contrast to the aforementioned studies are the results of 1 investigation that found correlation factors between ~0.06 and 0.08 in humans.61 Although lidocaine’s well-known property to readily penetrate biological membranes at physiologic pH values raises questions about the plausibility of these results, whole brain lidocaine concentrations in the study (1) were significantly higher than those in the CSF or blood and (2) exceeded cortical concentrations, indicating that lidocaine accumulates in the cerebral tissue with a preference for subcortical structures, e.g., thalamic nuclei.
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