Macro-System Model for Hydrogen Energy Systems Analysis in Transportation: Preprint

J Physiol 00.00 (2014) pp 1–16 1

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Neuroscience Transient impairment of the axolemma following regional

anaesthesia by lidocaine in humans

Mihai Moldovan1, Kai Henrik Wiborg Lange2, Niels Jacob Aachmann-Andersen1, Troels Wesenberg Kjær3,Niels Vidiendal Olsen1,4 and Christian Krarup1,3

1Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark2Department of Anesthesia, Nordsjællands Hospital and University of Copenhagen, Copenhagen, Denmark3Department of Clinical Neurophysiology, The Neuroscience Center, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark4Department of Neuroanesthesia, The Neuroscience Center, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark

Key points

� We tested the recovery of motor axon conduction and multiple measures of excitabilityby ‘threshold-tracking’ after ultrasound-guided distal median nerve regional anaesthesia bylidocaine.

� Lidocaine caused a transient conduction failure that recovered completely by 3 h, whereasexcitability recovered only partially by 6 h and fully by 24 h.

� The up to 7-fold increase in threshold after complete recovery of conduction was associatedwith excitability changes that could only partially be explained by block of the voltage-gatedNa+ channel (VGSC).

� Mathematical modelling indicated that, apart from a reduction in the number of functioningVGSCs, lidocaine also caused a decrease of passive membrane resistance and an increase ofcapacitance.

� Our data suggest that lidocaine, even at clinical ‘sub-blocking’ concentrations, could cause areversible structural impairment of the axolemma.

Abstract The local anaesthetic lidocaine is known to block voltage-gated Na+ channels (VGSCs),although at high concentration it was also reported to block other ion channel currents as wellas to alter lipid membranes. The aim of this study was to investigate whether the clinical regionalanaesthetic action of lidocaine could be accounted for solely by the block of VGSCs or whetherother mechanisms are also relevant. We tested the recovery of motor axon conduction andmultiple measures of excitability by ‘threshold-tracking’ after ultrasound-guided distal mediannerve regional anaesthesia in 13 healthy volunteers. Lidocaine caused rapid complete motor axonconduction block localized at the wrist. Within 3 h, the force of the abductor pollicis brevis muscleand median motor nerve conduction studies returned to normal. In contrast, the excitability ofthe motor axons at the wrist remained markedly impaired as indicated by a 7-fold shift ofthe stimulus–response curves to higher currents with partial recovery by 6 h and full recoveryby 24 h. The strength–duration properties were abnormal with markedly increased rheobaseand reduced strength–duration time constant. The changes in threshold during electrotonus,especially during depolarization, were markedly reduced. The recovery cycle showed increasedrefractoriness and reduced superexcitability. The excitability changes were only partly similarto those previously observed after poisoning with the VGSC blocker tetrodotoxin. Assuming anunaltered ion-channel gating, modelling indicated that, apart from up to a 4-fold reduction in thenumber of functioning VGSCs, lidocaine also caused a decrease of passive membrane resistanceand an increase of capacitance. Our data suggest that the lidocaine effects, even at clinical‘sub-blocking’ concentrations, could reflect, at least in part, a reversible structural impairment ofthe axolemma.

C© 2014 The Authors. The Journal of Physiology C© 2014 The Physiological Society DOI: 10.1113/jphysiol.2014.270827

2 M. Moldovan and others J Physiol 00.00

(Received 8 January 2014; accepted after revision 2 April 2014; first published online 7 April 2014)Corresponding author C. Krarup: Department of Clinical Neurophysiology NF3063, Rigshospitalet, 9 Blegdamsvej,2100 Copenhagen, Denmark. Email: [email protected]

Abbreviations ADQ, abductor digiti quinti; APB, abductor pollicis brevis; CMAP, compound muscle actionpotential; CNAP, compound nerve action potential; FD, flexor digitorum; SDTC, strength–duration time constant;TTX, tetrodotoxin; VGSC, voltage-gated Na+ channel.

Introduction

Axons are complex structures specialized in reliableconduction of action potentials. Various hydrophilicnatural toxins and pharmaceutical compounds with highaffinity for specific ion channel proteins in the axonalmembranes have been found to selectively decrease thevoltage-dependent Na+ currents, increase the K+ currentsor reduce the inward rectifier currents (Huang 1997).As such, they can reduce the axonal ‘safety factor’for conduction (Tasaki, 1953) precipitating conductionfailure (Hodgkin & Huxley, 1952).

The local anaesthetic lidocaine, synthesized under thename xylocaine by the Swedish chemist Nils Lofgrenin 1943, is thought to cause axonal conduction failure(Nathan & Sears, 1961) by impairing the function ofthe voltage-gated Na+ channel (VGSC) (Sheets & Hanck,2007). Although various VGSC mutations were foundto alter the susceptibility to local anaesthetics (Onizukaet al. 2012), lidocaine was also reported to affect othermembrane currents, such as voltage-gated K+ currents(Komai & McDowell, 2001; Panigel & Cook, 2011) and theinward rectifier currents (Raymond, 1992). This apparentlack of specificity of lidocaine, in contrast to, for example,the VGSC block caused by toxins (Stevens et al. 2011),questions its direct actions on ion channels altogether.

Lidocaine, like other ‘caine’-type local anaestheticsconsisting of a benzene ring linked to an amine group,dissolves in the lipids of the axolemma (Gokin et al.2001; Scholz, 2002). If the Meyer–Overton law describedover a century ago for general anaesthetics is alsoapplicable to local anaesthetics, anaesthesia could occurwhen a critical lidocaine concentration is reached in theaxolemma (Strichartz, 1973; Butterworth & Strichartz1990). Such a ‘membrane fluidization’ by lidocainewould not only directly alter its electrical properties(Tabatabai & Booth, 1990; Nau & Wang, 2004) but alsocould lead to conformational changes in the membraneion channel proteins indirectly affecting their function(Courtney, 1975). Disentangling the relative importanceof effects of lidocaine on the function of voltage-gatedion channels from structural changes of the axolemma ofthe clinically relevant local anaesthetic actions remains,however, methodologically challenging.

Clues about the membrane biophysical propertiesof peripheral myelinated axons can be obtained usingexcitability testing by a computer-controlled ‘threshold

tracking’ technique, in which the threshold, based onthe stimulus–response curve, is defined as the currentrequired to activate 40–50% of the maximal response(Bostock et al. 1998). A standardized protocol investigatingin sequence the threshold changes associated withstimulus duration, electrotonic polarization and after aconditioning response (Kiernan et al. 2000) has been usedduring the last decade in a wide range of physiological andpathological conditions (Burke et al. 2001; Nodera & Kaji,2006; Krarup & Moldovan, 2009) allowing interpretationby comparison. Furthermore, a space-clamped ionchannel-based mathematical membrane model, referredto hereafter as the ‘Bostock’ model (Bostock et al. 1991),was able to predict the changes in multiple measuresof excitability following accidental tetrodotoxin (TTX)poisoning in humans (Kiernan et al. 2005a) and inmutations of VGSC genes (Kiernan et al. 2005b), and cantherefore be of aid in interpretation.

The aim of this study was to investigate bynerve excitability testing whether the clinical regionalanaesthetic action of lidocaine could be accounted foronly by block of VGSC or whether other mechanisms arealso relevant. Specifically, we investigated the recovery ofconduction and multiple measures of excitability (Bostocket al. 1998) of the motor axons to the abductor pollicisbrevis muscle (APB) following ultrasound-guided distalmedian nerve regional anaesthesia.

Methods

Subjects and experimental design

The study was conducted according to the Helsinkideclaration using a protocol approved by the RegionalEthical Committee of the Capital Region of Denmark(protocol no. H-4-2011-075). After informed consent,the non-dominant hand was investigated in 13 healthyvolunteers (seven men), 28 ± 3 (mean ± SEM) years old,182±6 cm tall and 78±6 kg in body weight. The first threesubjects were used as methodological controls. Exclusioncriteria were a history of peripheral nerve disease includingcarpal tunnel syndrome and polyneuropathy, diabetes, ora history of adverse reaction to anaesthetics.

Our experimental setup is illustrated in Fig. 1. Afterbaseline median nerve motor conduction and excitabilitytests prior to application of lidocaine, we carried out adistal ulnar nerve block, followed by a median nerve block

C© 2014 The Authors. The Journal of Physiology C© 2014 The Physiological Society

J Physiol 00.00 Axon membrane impairment after lidocaine 3

at the wrist. The ulnar nerve block was performed to ensurethat strong stimulation currents at the wrist would notevoke responses in ulnar nerve innervated muscles andthus contaminate the compound muscle action potential(CMAP) recorded from the APB. The lidocaine injectedaround the ulnar nerve did not appear to significantlyinfluence the measurements from the median nerve atthe wrist through a possible ‘spill-over’ effect (data notshown). Electrophysiological recovery was monitored for6 h after the median nerve block. Clinical recovery ofmuscle strength was evaluated using the Medical ResearchCouncil scale for muscle strength. Four subjects were alsoexamined 24 h after the block to ascertain the completenessof recovery and reproducibility of findings. Two subjectswere recalled for an additional control experiment usingonly lidocaine vehicle (protocol no. H-2-2014-006).

Ultrasound-guided median and ulnar nerve blocks

We used an ultrasound system with a high frequencylinear ultrasound transducer (probe 8870, 1202 FlexFocus 500, BK Medical ApS, Herlev, Denmark) to ensuregood nerve visualization during the injection of lidocaineclose to the ulnar and median nerves. Concentrationsof lidocaine similar to those used in routine peripheralnerve blocks were used. Continuous monitoring includedelectrocardiography and pulse oximetry. An intravenouscatheter was inserted into a superficial cubital vein of thecontralateral arm.

Ulnar nerve block. We localized the ulnar nerve incross-section as close to the wrist as possible asa typical hyper-echoic nerve structure running justulnar to the ulnar artery, and after disinfection withethanol/chlorhexidine (83 and 0.5%, respectively) weinserted a 35 mm insulated nerve stimulation needle(Stimuplex D 25 G 35 mm, 15O; B.Braun Melsungen AG,Melsungen, Germany) using an in-plane technique, andplaced the needle tip as close to the nerve as possible. Weconfirmed ulnar nerve identity by visible contractions ofhypothenar muscles in response to 2–3 electric impulses(2 Hz, 1.5 mA, 0.1 ms) generated by a nerve stimulator(Stimuplex HNS 12 Peripheral Nerve Stimulator, B.BraunMelsungen AG). After confirmation we aspirated ensuringa non-intravascular needle tip position and injected 1.5 mlof lidocaine 20 mg ml−1 with adrenaline 5 μg ml−1

(Lidokain-Adrenalin ‘SAD’, SAD, Copenhagen, Denmark)and observed perineurial spread of the local anaesthetic onthe ultrasound system screen.

Median nerve block. Median nerve block was performeddistally in the forearm at two distinct injection sites, eachseparated by a distance of approximately 5 cm. First, weidentified the median nerve in cross-section by performing

a dynamic ultrasound scan in proximal–distal directionof the forearm. We performed the distal median nerveblock as close to the wrist as possible using a similarultrasound-guided technique as described above. Mediannerve identity was confirmed by eliciting visible contra-ctions of the APB. The time of the distal median blockwas considered time zero of the experiment. Thereafter,we performed a median nerve block 5 cm proximalto the distal block. At each site we injected 5–6 mlof lidocaine 13 mg ml−1 (obtained by diluting 6.7 mlLidokain 20 mg ml−1 ‘SAD’, SAD, Copenhagen, Denmarkwith 3.3 ml isotonic saline) (Fig. 1C).

Vehicle control. To control for the effect of the injectionfluid itself, we obtained a lidocaine vehicle solution forhuman use from the Pharmacy of the Capital Region(Dansk Region Hovedstadens Apotek). Care was taken thatthe solution was titrated to the same pH as the standardlidocaine solution (Maurer et al. 2012). Serial ultrasoundscans (see Fig. 6) were captured from four consecutiveanatomical levels �2 cm apart starting distal to thestimulation site at wrist (Fig. 6B). Hourly measurementswere carried out from all levels up to 3 h after the injection(Fig. 6C).

Electrophysiological setup and conduction studies

The subjects rested comfortably with the arm extendedon a stand covered by hydrophobic cotton (Fig. 1A)under a custom-made rectangular heating lamp thermo-statically controlled to maintain the temperature of thestimulated site at 37–38°C, minimizing the effect oftemperature changes induced by the regional anaestheticblock (Lange et al. 2011). CMAPs were recorded from themedian nerve innervated APB, the ulnar nerve innervatedabductor digiti quinti (ADQ) as well as from the mediannerve innervated forearm flexor digitorum (FD) muscles(Fig. 1A, B). The CMAPs were recorded through surfaceelectrodes in a tendon–belly configuration (20 Hz–10 kHz,EMG amplifier type 15 C 01, DISA-Dantec, CopenhagenDenmark) using a custom-made data acquisition system(Nikolic & Krarup, 2011). The CMAP amplitude at sub-maximal and supramaximal stimulation was measuredpeak-to-peak and the latency of the fastest fibres wasmeasured at the first deflection from baseline (Fig. 2A).The median nerve was stimulated at wrist and elbowwhereas the ulnar nerve was stimulated only at elbow(proximal to the epicondylus). Stimulation was carriedout via single-use pre-gelled, non-polarizable Ag/AgClelectrodes (Blue Sensor, Ambu, Ballerup, Denmark) withthe cathode placed over the nerve at the wrist or elbow andthe anode at a distance of 10 cm over the radial forearm orupper arm. A single-use ground electrode was placed onthe dorsum of the hand.



During threshold tracking, current stimuli weredelivered via two linear constant current stimulatorseach of 50 mA maximal output (DS5, Digitimer Ltd,Welwyn Garden City, UK), connected in parallel. Thissetup was designed to ensure that adequate stimulationwas obtained during high threshold conditions. In controlexperiments, the two-stimulator setup was found toperform indistinguishably from the single stimulator setup(data not shown). For conduction studies we used negativerectangular current pulses with duration up to 1 ms,repeated with a frequency of 2 Hz or less.

Multiple measures of excitability

Peripheral nerve excitability was assessed using QtracSstimulation software (version 23/10-2012, C© ProfessorHugh Bostock) and the TRONDNF multiple measuresof excitability protocol (Bostock et al. 1998; Kiernanet al. 2000; Tomlinson et al. 2010a). At a sampling rateof 10 kHz the amplified signal was digitized online bycomputer (PC) that was also used to control the stimulatorwith an analog-to-digital (A/D) board (NI-6221, NationalInstruments, Austin, TX, USA).

First, the stimulus–response curves were obtained usingtest stimuli of 1 ms duration to establish the maximalCMAP (100%) to supramaximal nerve stimulation(Fig. 3A, B). Then, the ‘threshold’ current necessary toevoke a submaximal target potential set to 40–50% ofthe maximum CMAP could be automatically tracked bytrial and error computer feedback. Although thresholdis a global index of excitability (i.e. an increase inthreshold corresponds to a decrease in excitability),the absolute value of threshold yields little informationabout the underlying membrane function (Bostock et al.1998). Instead, relative changes in threshold in variousexperimental settings were determined using a multipleexcitability sequence: strength–duration relationship,threshold electrotonus, current–threshold relationshipand recovery cycle. These excitability measures and theirderived excitability indices are illustrated in Figs. 3 and 4.

The strength–duration properties, reflecting primarilythe function of nodal membrane (Mogyoros et al. 1996,2000), were determined by measuring the thresholds fortest stimuli with duration of 0.2 to 1 ms. Rheobase(Fig. 3F) and the strength–duration time constant (SDTC,Fig. 4E) were derived from the corresponding linear charge(current strength × duration)–duration relationshipusing Weiss’s law (Bostock, 1983): rheobase from the slopeand SDTC from the negative intercept on the duration axis(Fig. 4D).

Accommodation to prolonged subthresholdpolarization was ascertained by measuring changesin threshold in response to subthreshold depolarizationand hyperpolarization during threshold electrotonus

(Fig. 4A) and current–threshold relationship (Fig. 4B).Threshold electrotonus was measured for 100 msdepolarizing and hyperpolarizing currents set to 40% ofthe control threshold current. The threshold reductionduring depolarization was quantified at 90–100 ms,referred to as TEd(90–100 ms) (Fig. 4A, F). The thresholdincrease (negative threshold reduction) during hyper-polarization was quantified at 20–40 ms, referred to asTEh(20–40 ms) (Fig. 4A, H). Accommodation to evenlonger polarization, reflecting inward rectification, wasinvestigated at the end of 200 ms current pulses from50% to −100% of the current–threshold relationship, athreshold analogue of the I–V relationship (Kiernan et al.2000).

Changes in threshold following a single supramaximalstimulus were tested at intervals from 1.5 to 200 msduring recovery cycle (Fig. 4C). In myelinated axons, theinitial increase in threshold a few milliseconds after theconditioning stimulus (relative refractoriness) is followedby a superexcitable (a smaller than normal threshold) anda late subexcitable (larger than normal threshold) period(Barrett & Barrett, 1982). Here we quantified the super-excitability after 5 ms (Fig. 4G).

Modelling of myelinated axons

Using Hodgkin–Huxley type differential equations(Hodgkin & Huxley, 1952), the ‘Bostock’ model (Bostocket al. 1991) accounts for the transient and persistentNa+ currents, the slow and fast K+ currents, the inwardrectifier current, the Na+/K+ pump current and thepassive leak currents distributed in distinct nodal andinternodal capacitive membrane compartments linked viaa Barrett–Barrett resistance (Barrett & Barrett, 1982). Herewe used the equations and parameters from a recentlyrevised version of the model that accounts also for thesubtle biophysical differences between myelinated motorand sensory axons (Howells et al. 2012).

The model optimization was provided by MEMFIT,part of QTRACP (version 15/4-2013, C© Professor HughBostock). The software allows for 1, 2 or 3 of 66 parametersto be automatically changed, simultaneously or in turn, sothat the discrepancy (measured in % change in error)between the model output and the measured multipleexcitability measures is reduced.

Data analysis

Quantitative changes are given as mean ± SEM. Analysisof multiple measures of nerve excitability was carried outusing QtracP software (version 15/4-2013, C© ProfessorHugh Bostock). Due to the relatively small number ofsamples per group, the normality of the distributions couldnot be reliably ascertained. As such, distribution-freenon-parametric statistical comparison tests were


J Physiol00.00 Axon membrane impairment after lidocaine 5

preferred. Difference levels of P < 0.05 were consideredsignificant.

Results

The transient conduction failure

In all subjects, lidocaine caused complete conductionfailure and APB paralysis when tested at 30 min (Fig. 2).No measurable APB CMAP could be recorded whenstimulating the median nerve at the wrist (anaesthetizedregion) up to the maximum stimulator output of80–90 mA at a stimulus duration of 1 ms (Fig. 2A, B).In addition, CMAP could also not be recorded from theAPB when stimulating the median nerve at the elbow

(un-anaesthetized region) at an intensity that evoked amaximal CMAP at the FD (Fig. 2A, B).

From 2 h after lidocaine, a CMAP at the APB couldagain be evoked by stimulating the median nerve at thewrist, whereas the recovery from the elbow was �1 hfurther delayed (Fig. 2A, B). Of note, the early submaximalAPB responses that could be evoked from the elbow hadprolongation of latency (conduction slowing) of morethan 25% (Fig. 2A, C).

Recovery of threshold

After 2–3 h, the CMAP amplitude and latency andforce of the APB were normal. The excitability of the

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Figure 1. Experimental setupA, photograph of the experimental setup.Compound muscle action potentials(CMAP) were recorded from the abductorpollicis brevis (APB), the abductor digitiquinti (ADQ) and the flexor digitorum (FD)muscles. The median nerve was stimulatedat the wrist and elbow whereas the ulnarnerve was stimulated only at the elbow(proximal to the medial epicondyle). B,diagram of the experimental setupindicating the ulnar nerve anaesthesiafollowed by the median nerve anaesthesia.C, ultrasound image during lidocaineinjection (arrow) near the median nerve.The spread of lidocaine (arrow) over severalcentimetres along the nerve is depicted inthe three-dimensional reconstruction to theright.

C� 2014 The Authors. The Journal of Physiology C� 2014 The Physiological Society

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C� 2014 The Authors. The Journal of PhysiologyC� 2014 The Physiological Society


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median nerve at the wrist remained, however, markedlyimpaired as indicated by the shift of the stimulus–responsecurves to higher currents (Fig. 3A, B). Note thatthe stimulus–response curves (Fig. 3A) and excitabilitymeasures (data not shown) from the elbow were normal.At the wrist, the stimulus threshold (for a 1 ms pulse)was increased from 8.6 ± 2 to 36.7 ± 3 mA (Fig. 3E;P < 0.01) measured as a 7-fold increase in rheobase(Fig. 3F; P < 0.01). These changes in threshold recoveredslowly during the next few hours (Fig. 3B, E, F), whereasthe peak response (Fig. 3C) and threshold latency (Fig. 3D)remained unchanged.

Recovery of the multiple measures of excitability

The deviations in excitability were largest at 2 hfollowing lidocaine (Fig. 4). Recording of some excitabilitymeasures, typically the current–threshold relationship,had to be skipped at 2 h to avoid discomfort athigh stimulus currents. At 3 h, there was a shorteningof the SDTC from 415 ± 30 to 145 ± 8 μs(Fig. 4D, E; P < 0.01). The changes during electro-tonic depolarization (Fig. 4A) were markedly abnormal.After 90 ms of 40% subthreshold depolarization, thethreshold reduction normally accommodated to plateauof TEd(90–100 ms) �40% of threshold (Fig. 4A, F). At3 h after lidocaine, TEd(90–100 ms) dropped to nearly0 (0.9 ± 5% of threshold, P < 0.01). In parallel, thechanges during electrotonic hyperpolarization, measuredat TEh(20–40 ms), indicated a 37% smaller than normalthreshold increase upon hyperpolarization after 30 ms(Fig. 4A, H; P < 0.01). The recovery cycle was alsoabnormal, indicating a larger increase in threshold atshort inter-stimulus intervals (Fig. 4C). This ‘increasedrefractoriness’ occurred at the expense of the super-excitable period, as indicated by a decrease in super-excitability at 5 ms inter-stimulus interval by 22% ofthreshold (Fig. 4G; P < 0.01).

The excitability measures recovered slowly and were thesame at 24 h as before the injection of lidocaine (Fig. 4).The longest lasting abnormality was the change duringelectrotonic depolarization, as seen as an inward shift onthe depolarizing threshold electrotonus (Fig. 4A) and a leftshift of the depolarizing (upper half) current–threshold

relationship. Thus, at 6 h after lidocaine, TEd(90–100 ms)remained significantly reduced to 35.1 ± 3 % of threshold(Fig. 4F; P < 0.05).

Mathematical electrochemical modelling of thedeviations in excitability measures

The mean of the control measurements (PRE)corresponded to the amended ‘Bostock’ model (Howellset al. 2012) after increasing the temperature (Tabs) by 3°C(Fig. 5A) in accordance with our temperature control at37°C (see Methods).

A limitation of the current software implementationof the Bostock model is that the stimulus charge fora 1 ms stimulus is pre-scaled to 1 for all investigatedconditions (Fig. 5, right column). We therefore weightedthe model optimization on threshold changes duringelectrotonic depolarization TEd(90–100 ms) that showedthe largest relative change after lidocaine (Fig. 4F). Wefirst attempted to fit the model to the recordings at3 h (mean and SD) by changing a single parameter(data not shown). This pointed towards a reduction inVGSC permeability (PNaN); however, the reduction indiscrepancy remained unsatisfactory. Varying the gatingproperties of the modelled VGSC in addition to PNaNdid not add a significant improvement either (data notshown). We therefore attempted to improve the fit byoptimizing three parameters (two in addition to PNaN)while leaving the PNaN gating unmodified.

Modelling indicated that at 3 h after lidocaine (Fig. 5B)there was a 74% reduction in VGSC permeability (PNaN),which recovered to about half by 5 h (Fig. 5C). At 6 h, allexcitability deviations could be reasonably explained by a15% reduction in PNaN (Fig. 5D). Nevertheless, at earliertime points (less lidocaine wash-out), modelling indicatedan additional alteration in passive electrical membraneproperties: an increase in axonal conductance (GLkRel)by up to 50% and an increase in axonal capacitance(CAX) by up to 83% (Fig. 5B, C). Note that the model,although predicting the measured reduction in super-excitability, underestimated the extent of the progressiveincrease in threshold at short inter-stimulus intervals(Fig. 5B, C).

Figure 3. Recovery of motor response threshold from 2 h (2H) to 24 h (24H) after lidocaine as comparedto measures prior to lidocaine (PRE)A, stimulus–response relationships from the wrist (filled circles) and elbow (Elb, triangles) to APB are presented fora single subject before lidocaine injection and at 2 h. Note that in spite of the large right shift in threshold at thewrist, measurements from the elbow remained unchanged. B, mean stimulus–response relationships at the wristare shown for all subjects at the different time points before and after lidocaine injection. The stimulus currentaxis is presented on a log scale to facilitate the display of the large changes in threshold. Error bars represent SEM.C–F, dot plots presenting the changes over time of the peak CMAP amplitude (C), threshold latency (D), thresholdstimulus (E) and rheobase (F). Hourly means are indicated.



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No confounding effect of injection ÔßuidÕ

The fact that the modelling indicated a change in thepassive electrical properties in addition to a reduction inVGSC permeability raised the possibility that such changescould re�ect electrical alterations in the near-nerveenvironment rather than true axonal changes. To test thispossibility directly we carried out an additional set ofcontrol experiments, monitoring the effect of lidocainevehicle (Fig. 6). We found that at 3 h, when the describedeffects of lidocaine were maximal, the excitability aftervehicle was unchanged (Fig. 6A) and no �uid aroundthe nerve could be detected by ultrasound (Fig. 6C).Furthermore, in the �rst hour after injection when the�uid around the stimulation site was maximal (Fig. 6B,C), excitability measures remained unchanged (Fig. 6A).We therefore concluded that the injection �uid did notconfound the effects of lidocaine.

Discussion

Longitudinal studies in normal subjects of motor axonfunction during and after regional lidocaine anaesthesiashowed a marked discrepancy between the completerecovery of conduction and force while recovery of motoraxon excitability lagged hours behind the amplitudeand conduction velocity of the CMAP. Assuming anunaltered ion-channel gating, modelling indicated thatthe lidocaine effects were not limited to reduction of theVGSC currents but that lidocaine also increased passivemembrane conductance and capacitance. This indicatesthat effects of lidocaine, even at clinical ‘sub-blocking’concentrations, may re�ect, at least in part, a transientstructural impairment of the axolemma.

Transient conduction block after lidocaine

With increasing concentrations of anaesthetics, pre-vious electrophysiological studies of ‘-caines’ (Gasser& Erlanger, 1929; Gasser & Grundfest, 1939) found aprogressive decrease in amplitude, slowing of conductionand increase in threshold of the compound nerve actionpotential (CNAP). As CNAPs re�ect the summatedelectrical �eldofaxonswithdifferentconductionvelocitiesand stimulation thresholds (Gasser & Erlanger, 1927), itremained possible that the progressive conduction failure

re�ected merely the progressive inability of the stimulationto overcome the anaesthetic-induced increase in threshold(Nathan&Sears,1962;Kassahunetal.2010).Nevertheless,experimental studies of single �bre preparations indicatedthat there is in fact a ‘critical dose’ of local anaesthetic forwhich all or none action potential generation is no longerpossible, the susceptibility to anaesthetic conductionblock being different between neuronal populationsaccording to their functional specializations (Butterworth& Strichartz, 1990; Parket al. 2012) consistent withdifferences in their ‘safety factor’ for conduction (Tasaki,1939; Franz & Perry, 1974; Gordh, 2010; Moldovanet al.2013).

In our study we tracked the effect of lidocaine on theCMAP of the APB because: (1) the CMAP size dependsexclusively on summation of all or none activated singlemotor units (the motor �bres innervated by a single motoraxons); (2) the investigated �bre population consists ofonly a few hundred functionally homogeneous myelinatedaxons (Neuwirthet al.2011); and (3) the ‘Bostock’ modelcould reliably be used to aid interpretation (Bostocket al.1991; Howellset al.2012).

No CMAP could be recorded from the APB by electricalstimulation of the median nerve at the wrist (anaesthetizedregion) or elbow (un-anaesthetized region) within 30 minfollowing lidocaine, whereas the forearm FD CMAPevoked at the elbow remained maximal, consistent withan anaesthetic conduction block (Fig. 2). Moreover, therecovery of APB response from the elbow was delayedas compared to the wrist (Fig. 2B), probably due to thespatial spread of lidocaine along the nerve (Fig. 1C).The action potentials originating at the elbow had topropagate through a longer region with reduced ‘safetyfactor’ of conduction than from the wrist (Nakamuraet al.2003), as indicated by the greater increase in latency(Fig. 2C).

Impaired excitability after lidocaine in theÔsub-blockingÕ range

Within 3 h after lidocaine, the APB force as well asmedian nerve conduction studies returned to normal.In contrast, even at these ‘sub-blocking’ concentrations,median nerve excitability at the wrist (the region exposeddirectly to lidocaine) remained markedly abnormal, thenrecovered incompletely by 6 h and completely by 24 h.

Figure 4. Recovery of motor axon excitability measures after lidocaine blockA–D, mean changes in threshold electrotonus (A), current–threshold relationship (B), recovery cycle (C) andcharge–duration relationship (D). Error bars represent SEM. Measurements are presented prior to lidocaine (PRE,grey line), and at 2 h (2H, open circles), 4 h (4H, open triangles) and 24 h (24H, �lled circles).E–H, dot plots pre-senting the changes over time after lidocaine injection of the corresponding measurements in strength–durationtime constant, SDTC (E), threshold reduction during depolarizing threshold electrotonus, TEd(90–100 ms) (F), super-excitability of the recovery cycle at 5 ms (G) and threshold reduction during hyperpolarizing threshold electrotonus,TEh(20–40 ms) (H). Hourly means are indicated.

C� 2014 The Authors. The Journal of PhysiologyC� 2014 The P


(Howells et al. 2012) did not improve the modelprediction beyond the simple PNaN reduction (data notshown). Although the software model implementation didnot allow accurate estimation of the magnitude of therheobase changes, it was also theoretically unlikely thata reduction in VGSC currents alone, whatever the gatingalterations, could lead to the measured 7-fold change inthreshold (Noble, 1966). Thus, modelling indicated that,with increasing lidocaine concentrations (less washout),mechanisms other than reduced VGSC currents werephysiologically important.

Assuming unaltered ion-channel gating properties,the discrepancy between the modelled and recordedexcitability changes at high lidocaine concentrations

could be greatly reduced by changing, in addition toPNaN, the passive (non-voltage-dependent) propertiesof the axolemma, namely an increase in the membraneleak conductance (GLkRel) and an increase in axonalcapacitance (CAX). These passive changes could not beattributed to changes in the near nerve environment,i.e. due to the ‘fluid accumulation’ around the nerve(Fig. 6). Furthermore, a change in the passive electricalproperties of the axonal membrane was not experimentallyunrealistic as a reduced membrane resistance (increasein conductance) of similar magnitude was previouslymeasured after lidocaine (Tabatabai & Booth, 1990; Nau& Wang, 2004), as well as an increased membranecapacitance (Tabatabai & Booth, 1990). The change in

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Figure 6. Effect of lidocaine vehicleThe effect of lidocaine vehicle (Veh.) was compared tothe effect of lidocaine (Lid. + Veh.) in two subjectsre-examined for this control experiment. A, meanexcitability measures (charge–duration (upper left),recovery cycle (upper right), threshold electrotonus(lower left) and the current–threshold relationships(lower right)) were measured before injection (PRE, fulllines), immediately after injection (0H, open circles)and at 3 h (3H, filled circles). The vehiclemeasurements are shown in grey. Note that themeasurements at immediately after injection couldonly be obtained for the vehicle injection. B,photograph showing four consecutive anatomicallevels of injection from distal to proximal from thestimulation site. C, corresponding ultrasound scans(approx. 2 cm in width/1.5 cm in height) for onesubject. White arrows indicate the fluid volumearound the median nerve just after injection.



three parameters from the 66 ‘Bostock’ model parameters(including various ion channel gating properties) couldalso not be regarded as simple ‘parameterization’. Infact, our data indicate that changes in voltage-gated K+currents (Komai & McDowell, 2001; Panigel & Cook,2011), inward rectifier currents (Raymond, 1992) andmembrane potential (Tabatabai & Booth, 1990), pre-viously reported to be altered with high concentrationsof lidocaine, did not appear to contribute decisively tothe alterations in excitability in the investigated clinicalsituation.

In conclusion, mathematical modelling suggestedthat the reduction of excitability after lidocaine, atconcentrations that do not block conduction of actionpotentials, could not be explained solely by a reduction inVGSC currents. In addition, there was a change of twovoltage-independent parameters (axonal resistance andcapacitance), both reflecting axolemma structure.

Relevance of effects of lidocaine on the axolemma

The current wisdom is that lidocaine impairs excitabilityto the extent of conduction failure by interacting with theVGSC and reducing the regenerative Na+ influx. Althoughthe local anaesthetic binding site has not been elucidatedcompletely, several lines of evidence suggest that it islocated on the inner pore of the VGSC, and that it ismore accessible in the open channel state (Fozzard et al.2011). To reach the binding site, however, lidocaine hasto diffuse through the axolemma in its uncharged (base)form and then concentrate inside the membrane as acharged (protonated) form (Sano et al. 1999). This longrecognized ‘membrane solubility’ of lidocaine (as wellas other local anaesthetics) (Ritchie & Greengard, 1966)fuelled the alternative view that the presence of lidocaine inthe axolemma could also indirectly affect the VGSC gating(Lee, 1976). More recent studies using X-ray scatteringtechniques confirmed that lidocaine and other related localanaesthetics alter the membrane structure by increasing itsthickness/stiffness (Mateu et al. 1997; Luzzati et al. 1999)to an extent that it can impair the VGSC function (Hendryet al. 1985).

The reduction in Na+ current predicted by ourstudy could not distinguish between a direct and anindirect effect of lidocaine on the VGSC. Nevertheless,it is tempting to suspect that the predicted increasein capacitance reflected the structural alteration of themembrane by lidocaine (Fernandez et al. 1983; Hendryet al. 1985). Furthermore, the structural membranealterations could also be responsible for the pre-dicted increase of the voltage-independent membraneconductance either directly by altering the electricalproperties of the axolemma or indirectly by increasing theconductance of some voltage-independent ‘leak’ channels,

such as the two-pore domain K+ channel family thatwas reported to be opened by anaesthetics (Patel et al.1999). Taken together, both the increase in capacitanceand the leakiness of the membrane induced by lidocaineaggravate the excitability impairment induced by VGSCblock, further reducing the safety factor for conductionand thus enhancing the anaesthetic effect.

Since the description of the cauda equina syndromeoccurring after a lidocaine overdose (Rigler et al. 1991),it has become apparent that, at high concentrations,lidocaine is neurotoxic, leading to irreversible conductionblock (Lambert et al. 1994) by exerting a detergent-likedisruption of the membrane structure (Kitagawa et al.2004). A similar structural disruption could also beresponsible for the reported antibacterial properties oflidocaine (Tustin et al. 2014). Nevertheless, the dogmaremained that, at low but clinically effective lidocaineconcentrations, the alterations in axolemma structureare of little consequence (Strichartz, 1973; Putrenko &Schwarz, 2011). In contrast, our data suggest that evenat concentrations that do not prevent conduction ofaction potentials, lidocaine causes reversible structuralaxolemma alterations that aggravate the excitabilityimpairment due to the reduction of Na+ currents.Recognizing the relevance of these structural effects atsub-anaesthetic concentrations could be important forunderstanding lidocaine actions beyond VGSC block, suchas the reported analgesic effectiveness of both systemiclidocaine (Mao & Chen, 2000) and topical lidocaine(Argoff, 2013) for the management of chronic pain.

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Additional information

Competing interests

None declared.

Author contributions

C.K., N.V.O. and M.M. designed the study. K.H.W.L.and N.J.A.A. carried out the ultrasound-guided lidocaineinjection. All authors contributed to the lidocaine and controlexperiments. M.M. analysed the data and wrote the first draftof the manuscript. All authors critically revised the manuscriptfor important intellectual content and approved the final sub-mission.

Funding

The project was supported by the Lundbeck Foundation andthe Danish Medical Research Council (C.K.) and The ResearchFoundation of University of Copenhagen (N.V.O.).

Acknowledgements

Brief reports of these studies were presented at the 26th NordicCongress of Clinical Neurophysiology, Lund, Sweden, in May2012 and at the 15th Clinical Neurophysiology Workshopof the Australian and New Zealand Association of Neuro-logists (Festschrift for David Burke)’, Gold Coast, Australia, inSeptember 2013.


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