Concentration-Dependent Neurotoxicity of Articaine: An … · 2011. 12. 5. · and Thomas Alexander Gerds, Dr.Rer.Nat. BACKGROUND: We performed this study to quantify the detrimental

Concentration-Dependent Neurotoxicity of Articaine:An Electrophysiological and Stereological Study ofthe Rat Sciatic NerveSøren Hillerup, DDS, PhD, Dr.Odont.,*† Merete Bakke, DDS, PhD, Dr.Odont.,‡Jytte Overgaard Larsen, MD, PhD,§ Carsten Eckhart Thomsen, MScEE, PhD,‡and Thomas Alexander Gerds, Dr.Rer.Nat.�

BACKGROUND: We performed this study to quantify the detrimental effect of intraneuralinjection of 50 �L of saline, articaine 2%, or articaine 4% in the rat sciatic nerve.METHODS: Lumbar-evoked electrospinograms from stimulation of the sciatic nerve were re-corded before and immediately after injection and again after 3 weeks. Test substance wasinjected into the right sciatic nerve, and the untreated left sciatic nerve served as control. Theanimals were killed after the 3-week follow-up, and cross-sections of the sciatic nerve wereexamined stereologically.RESULTS: The evoked spinal cord field potential in the articaine groups faded away immediatelyafter injection and was concentration-dependently, significantly more reduced at the 3-weekfollow-up in comparison with the saline group. The response from the control sides wasunaffected in all groups. The number of myelinated axons was unaffected by the treatment. Themean cross-sectional axon area and the mean myelin sheath thickness were significantlyreduced in animals injected with articaine 4%.CONCLUSIONS: These observations indicate concentration-dependent neurotoxic injuries afterinjection of articaine with a significant difference between 2% and 4% formulations. Themechanical injury of needle penetration with saline injection had no significant effect on nerveconduction or histomorphology. (Anesth Analg 2011;112:1330–8)

Injection of local anesthetic is routinely given for paincontrol and is generally considered safe. Neurologicalside effects do occasionally develop. Reported adverse

drug reactions include prolonged anesthesia and tempo-rary or permanent neurosensory disturbances—such asanesthesia, hypoesthesia, paresthesia, dysesthesia, allo-dynia, ageusia, hypogeusia, and dysgeusia—indicating me-chanical or toxic nerve injury.1–3 Postmarketing reportsindicate a higher risk of permanent neurosensory distur-bance after injection of 4% formulations in comparison withalternative weaker formulations in current use. 1,3–7 Bothtype and concentration of anesthetic solution seem toinfluence the occurrence of sensory disturbances in hu-mans3,7 and the magnitude of tissue reactions in animalsand in vitro experiments.8–11

Kalichman et al.8 compared the neurotoxic potentials ofvarying concentrations of local anesthetics of both amidetype and ester type after perineural injection in the ratsciatic nerve. Nerve damage was assessed at the light

microscopic level by estimating the degree of nerve damage(i.e., axonal or myelin degeneration) and edema. Degenera-tive features increased with increased concentration of localanesthetic. Kroin et al.9 studied the neurotoxicity of lido-caine in concentrations 1%, 2%, and 4%. Infusion of 4%lidocaine led to loss of all conductive function and consid-erable nerve fiber degeneration, whereas infusion of 1%lidocaine did not elicit any neurotoxic reaction. In agree-ment with these studies, Cornelius10 and Cornelius et al.12

demonstrated that the neurotoxic injury in the rat sciaticnerve was more severe after intraneural injection of artic-aine 4% than after articaine 2% and lidocaine 2%. Theeffects of injection after 3 weeks were evaluated by meansof electrophysiology and histology. Preservatives, vasocon-strictors, and mechanical needle injury did not produce anydiscernible changes in nerve function or nerve structure.Unfortunately, this study,10 an approved German PhDthesis, was never published in full in a journal.

The present experimental study was conducted in thesame manner as the study performed by Cornelius10 tosubstantiate the relative roles of needle penetration withintraneural injection of saline versus articaine in 2% and 4%concentrations.

METHODSAnimals and Test SubstancesThirty female Wistar rats (median weight 253 g, range 232to 279 g; Taconic, Lille Skensved, Denmark) were used inthe study. Each rat received an intraneural injection of 50�L of the test substance (saline, articaine 2%, or articaine4%) in the right sciatic nerve. The left sciatic nerve served ascontrol. Each test substance was aspirated into 10 syringes,and the resulting 30 syringes were randomly numbered 1

From the *Department of Oral and Maxillofacial Surgery, Rigshospitalet,†School of Dentistry, ‡Department of Oral Medicine (Clinical Oral Physiol-ogy), School of Dentistry, §Department of Neuroscience and Pharmacology,and �Department of Biostatistics, Faculty of Health Sciences, University ofCopenhagen, Denmark.

Accepted for publication February 10, 2011.

Funding: This study was funded by the Danish Dental Association’sResearch Foundation.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Soren Hillerup, DDS, PhD, Dr.Odont., Depart-ment of Oral and Maxillofacial Surgery, Rigshospitalet, and School ofDentistry, Faculty of Health Sciences, University of Copenhagen, 20 NørreAlle, DK-2200 Copenhagen N Denmark. Address e-mail to [email protected].

Copyright © 2011 International Anesthesia Research SocietyDOI: 10.1213/ANE.0b013e3182172a2e

1330 www.anesthesia-analgesia.org June 2011 • Volume 112 • Number 6

through 30 in a sequence unknown to the investigators. Thetest substances used were Ultracaine d-S 1:200.000 (AventisPharma, Frankfurt am Main, Germany), containing artic-aine hydrochloride 40 mg/mL and epinephrine 0.006mg/mL (articaine 4%); Ultracaine Suprarenin (AventisPharma, Frankfurt am Main, Germany), containing artic-aine hydrochloride 20 mg/mL and epinephrine 0.006mg/mL (articaine 2%); and physiological saline (Nycomed,Roskilde, Denmark), pH 4.5 to 7.5. The study was approvedby the Animal Experiments Inspectorate, Denmark, No.2006/561 to 1148, and veterinary supervision was providedby the Department of Experimental Medicine, University ofCopenhagen. All experimental procedures in this studywere practiced and tested on 14 pilot animals before the 30test animals were included.

Anesthesia, Surgical Intervention and InjectionThe animals were anesthetized with a subcutaneous injec-tion of fentanyl/fluanisone-midazolam (0.3 mL per 100 gbody weight of a mixture of Hypnorm [Vetapharma, Leeds,UK], and Dormicum [Roche, Basel, Switzerland]; Hayton etal.13). The right thigh was shaved and disinfected withiodine, and the sciatic nerve was exposed through a 2-cmskin incision after separation of the biceps femoris muscle.The nerve was exposed by blunt dissection as far proximalas feasible. The injection site was at the medial intersectionof the nerve with the ileo-femoral ligament (Fig. 1). A 1-mLsyringe fitted with a simple, fine-threaded screw arrange-ment and a 30-gauge needle delivered a precise dose of thetest substance. A pilot test of the precision of the injectedvolume of 10 consecutive trial boluses weighed on aprecision scale showed a mean weight of 49.8 mg � �L(SD � 0.58 and coefficient of variation (SD/mean) � 0.012).After injection, the wound was closed in layers with Vicryl6 to 0 (Ethicon, Johnson & Johnson, St.-Stevens-Woluwe,Belgium). Postsurgical pain control was obtained withRimadyl Vet (50 mg/mL, Pfizer, NY) with a dose of 0.1mL/kg.

Electrophysiological RecordingsLumbar-evoked electrospinograms, i.e., spinal cord fieldpotentials, were elicited from stimulation of the experimen-tal leg and the control leg. In the first session the immediate

pharmacological effect of the injection on the evoked elec-trospinograms was examined after 1, 2, and 5 minutes. Therats were reexamined after 3 weeks to assess a possibleeffect of the injections. The anesthetized rats were placed inprone position and strapped with moderately stretchedhindlegs (Fig. 1). A stimulus (0.1-ms square wave) to thedistal segments of the sciatic nerve was delivered at 3-Hzrepetition rate (120 to 140 stimulations) with a medianintensity of 1.0 mA (range 0.5 to 5.8 mA; KeypointEMG/NCS/EP workstation version 3.26; Dantec Medical,Skovlunde, Denmark) with 2 electrodes (disposable sub-dermal needle electrodes, model 017K025, length 12 mmand diameter 0.3 mm; Viasys Healthcare, Madison, WI)placed between the ankle joint and the Achilles’ tendonwith a 5-mm interelectrode distance. Potentials were ac-quired with needle electrodes (disposable subdermalneedle electrodes, model 017K025, length 12 mm anddiameter 0.3 mm; Viasys Healthcare, Madison, WI) with therecording electrode placed subcutaneously above the spi-nous process of the L5 and the reference electrode con-tralateral to the stimulation side in the gluteus maximusmuscle close to the hip bone. The ground electrode (model9013S0735, 19.5 cm; Alpine Biomed, Skovlunde, Denmark)was, for practical reasons, placed under the head andforelegs, not to interfere with the surgical, injection, andrecording procedures. The stimulation and recordingset-up was according to Cornelius.10 When shifting record-ing from one side to the other, the recording electrode andthe ground electrode stayed in place and the placement ofthe reference electrode and the stimulation electrodes werechanged. The electrospinogram was averaged (120 to 130individual responses) and displayed (10-ms window, 1 to 5�V/Division), and the intensity of the stimuli was in-creased until maximal amplitude of the evoked responsewas obtained with only weak movements of the hindpawof the stimulated side. The amplitude was calculated usingthe integrated algorithms of the Keypoint workstation. Toavoid disturbance from the stimulus artifact, we measuredthe amplitude (in microvolts) from the most negativecomponent of the response after the stimulus to the follow-ing most positive component or, if this was not welldefined, to the baseline in the last 5 ms of the window (Fig. 2).Both in the first session and at the 3-week follow-up, the

Figure 1. A, Schematic drawing (adaptedfrom and printed with permission by Cor-nelius10) and B, dissected specimenshowing rat sciatic nerve anatomy. Arrowon injection site at the intersection pointbetween the nerve and the medial aspectof the ileo-femoral ligament.

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recordings were obtained from both the right, experimen-tal, leg and the left, control, leg. In the first session thespinal cord field potentials were obtained from the experi-mental side in the rats before and 1, 2, and 5 minutes afterthe injection.

Fixation and Preparation of Tissues,Stereological Methods, and Statistical AnalysisThe animals were killed by perfusion fixation immediatelyafter the recordings of lumbar-evoked electrospinograms atthe 3-week follow-up. After a 15-second initial perfusionwith 0.2 M phosphate buffer through the ascending aorta,the animals were perfused for 10 minutes with phosphate-buffered 4% glutaraldehyde while their legs were stretched.Perfusion was made by a Masterflex easy-load pump(Cole-Parmer Instrument Company, London, UK) with aflow of 56 mL/min. The torso with hindlegs was stored inthe same fixative at 4°C until a 2- to 4-mm long segmentfrom the injection site (medial intersection with the ileo-femoral ligament; Fig. 2) was dissected from both sciaticnerves. After 3 rinses in 0.15 M phosphate buffer (pH 7.4),the specimens were postfixed in 2% OsO4 in 0.12 M sodiumcacodylate buffer (pH 7.2) for 2 hours. The specimens weresubsequently dehydrated in ethanol, transferred to propyl-ene oxide, and embedded in EPON resin according tostandard procedures. Semithin (1 �m) sections were cutperpendicular to the long axis of the nerve segment with aReichert Ultracut S microtome (Leica, Herlev, Denmark),stained with toluidine blue and coverslipped with Pertexmounting medium. One nerve segment was excluded fromthe study for technical reasons.

The stereological methods used in the present studyhave been described in detail by Larsen.14 Briefly, to obtainunbiased number and size estimates from a sample ofaxons, all axons regardless of size, shape, orientation, andlocation must have an equal probability of being sampled.The unbiased counting frame ensures that all axons have anequal probability of being sampled, and systematic, uni-form random sampling of frames ensures that all locations

within the nerve cross-section are equally represented.Estimates of the total number of myelinated axons wereobtained by the fractionator technique. With this techniquethe myelinated axons in a known fraction, F, of the nervecross-section are counted with systematic, randomlysampled unbiased counting frames. Estimates of the totalnumber of myelinated axons, N, are obtained as the number

of axons counted, �Q, divided by the sampling fraction, F:

N: � 1/F��Q.

Size estimates were performed on the nerve fibersuniformly sampled by the 2-dimensional (2D)-fractionator.The areas of myelinated axons, a, were estimated withthe 2D-nucleator, where the distances from an approxi-mately central point in the axon to the profile boundary,�, are measured in a predetermined number of isotropicdirections:

a: � ��2.

A square root transformation of myelinated axon areaswas performed to correct for right-skewed size distribu-

tions. The transformation was chosen as 2 � �a

�because it

may be regarded as the equivalent circle diameter, i.e., thediameter a circle of equal area would have, and is thuscomparable to studies that report axon diameter.

Myelin thickness was measured at a boundary-weightedrandom point. The random point was found by placing agrid of parallel 2D IUR (isotropic uniform random) linesrandomly on the axon profile. The myelin thickness wasmeasured at the lower-most right intersection point be-tween the line grid and the axon profile. The g rate (i.e., theaxon diameter divided by the fiber diameter) was calcu-lated from the equivalent circle diameter and the measuredmyelin thickness. The cross-sectional area of the nervetrunk was estimated by averaging 5 estimates obtained bythe 4-way 2D nucleator. The practical analysis of myelin-ated axons was performed on video images of microscopicfields merged with a graphic representation of an unbiasedcounting frame and a 2D IUR line grid using the C.A.S.T.-GRIDsoftware (Olympus Denmark, Ballerup, Denmark). Thestage of the microscope was controlled by stepping motorsthat moved the section in precise steps of length dx and dyalong the X-Y coordinates. Myelinated axons were countedand measured at a final magnification of �5125 using a�100 UPLAN oil immersion objective (NA � 1.35). Thea(frame) was 88.7 �m2, dx and dy were 65 �m, i.e., the

sampling fraction was88.7

65.65�

1

48� 0.021.

Four isotropic directions were chosen for the nucleatormeasurements. On average, 187 (range 161 to 233) myelin-ated axons were counted and measured per nerve cross-section. Assuming a Poisson distribution of the axons, thecoefficient of error on the number estimates is the inverse ofthe square root of the number of axons counted:

CE: �1

��Q� 0.073.

Figure 2. Lumbar-evoked electrospinogram (spinal cord field poten-tial) with definition of amplitude measure. The averaged responsefrom 120 to 140 stimuli is shown in red with the initial stimulusartifact blanked (dots). The amplitude (�V) of the response wasmeasured from the most negative component of the response afterthe stimulus (minus sign) to the following most positive component(plus sign) (� Amp) or, if this was not well defined, to the baseline inthe last 5 ms of the window (B) (� Amp�).

Neurotoxicity of Articaine

1332 www.anesthesia-analgesia.org ANESTHESIA & ANALGESIA

Statistical AnalysesElectrophysiology. Differences in amplitudes of the elec-trospinogram at the 3-week follow-up after the injections ofsaline and 2% and 4% articaine were investigated usingordinary least square regression and analysis of variance(ANOVA). The analysis included the body weight in thefirst session before intervention, and the amplitudes of thelumbar electrospinograms in the first session before inter-vention as explanatory factors.

ANOVA P values were reported, and if the overall effectof the treatment group was significant, post hoc compari-sons were reported between articaine 2% and saline andbetween articaine 4% and saline.Stereology. One-way ANOVA was used to compare the 3groups, and in case of significance, post hoc unpaired t testswere also used for saline-treated versus 2% articaine–treatedgroups, saline-treated versus 4% articaine-treated groups, and2% articaine–treated versus 4% articaine–treated groups. Theanalyses were performed with Microsoft Office Excel 2003and R (R Foundation for Statistical Computing, Vienna,Austria). Statistical significance was considered at P � 0.05.

RESULTSIn general the clinical course was uneventful, and mostanimals gained weight during the observation period.One animal from the 2% articaine group had a superficialwound infection that responded favorably to antibiotictreatment. As a clinical effect, the affected hindleg inanimals with a severe depression of electrospinogram

amplitude appeared atrophic and less forceful. Thisfeature was not foreseen, and therefore not registeredsystematically.

Lumbar-Evoked ElectrospinogramsThe immediate pharmacological effect in each animal isillustrated in Fig. 3 in which the evoked lumbar responses1 to 5 minutes after the injection clearly differed betweenarticaine and saline groups. Marked effects of the injectionsafter 3 weeks were also present. At the follow-up the leastsquare regression analysis of the evoked-lumbar electrospi-nogram in the experimental side showed a significanttreatment-group effect on the amplitudes of the lumbarresponses (ANOVA P value � 0.0022). The effects ofbaseline body weight and baseline amplitude (recorded inthe first session) were not statistically significant. Between-groups comparison showed that injection of articaine 2%(P value � 0.0295) and injection of articaine 4% (P value �0.0006) both significantly decreased the amplitude in com-parison with injection of saline (Table 1 and Fig. 4). Asimilar analysis of the control side amplitudes (left legs)showed no significant treatment-group effect (ANOVA Pvalue � 0.6924).

Qualitative and Quantitative Structural ChangesMicrographs of nerve cross-sections are shown in Fig. 5,and the quantitative histological data on the myelinatedaxons and the cross-sectional areas of the nerve trunks arepresented in Fig. 6, and corresponding P values are given in

Figure 3. The immediate pharmacological effect of the injections into the right sciatic nerve after 1, 2, and 5 minutes. The individual amplitudesof the lumbar evoked electrospinograms are shown separately for each group. The effect of saline was insignificant, whereas articaine 2% andarticaine 4% produced significant depression of amplitudes. The dotted black lines indicate that 2 amplitudes were not available 1 minute afterinjection of saline and articaine 4%, respectively.

Table 1. Three-Week Follow-Up of Amplitude in Comparison with Preinjection Baseline

FactorAverage change of

amplitude per factor unitStandard

errorConfidenceinterval 95% P value

Experimental side (right leg)Amplitude first session before intervention (�V) �0.03 0.25 �0.54–0.49 0.9174Body weight first session before intervention (g) 0.04 0.02 0–0.08 0.0781Saline group 0 — — —Articaine 2% group �1.24 0.54 �2.35–�0.13 0.0295Articaine 4% group �2.09 0.53 �3.13–�1.0 0.0006

Control side (left leg)Amplitude session (�V) 0.28 0.18 �0.1–0.65 0.1403Body weight first session before intervention (g) �0.04 0.02 �0.09–0.01 0.1179Saline group 0 — — —Articaine 2% group �0.58 0.67 �1.97–0.81 0.3984Articaine 4% group �0.34 0.68 �1.73–1.06 0.6234

Analysis of least-squares regression of amplitudes of lumbar-evoked electrospinograms from stimulation of the sciatic nerve (10 rats in each treatment group).


Table 2. Both the cross-sectional area of the nerve trunk andthe total number of myelinated axons were comparable inall treatment groups, though the ANOVA P value was intrend (P value � 0.065) for an increased number of myelin-ated axons in the groups treated with articaine. The meanaxon area, the mean equivalent circle diameter, and themean myelin sheath thickness were significantly reduced inthe animals that received articaine 4% when compared withthe saline-treated animals, and reduced, but not signifi-cantly, in the animals that received articaine 2% whencompared with the saline-treated animals. The g ratio wasunaffected by either of the treatments. The mean axon areaand the mean equivalent circle diameter were both signifi-cantly smaller in the 4% articaine–treated animals whencompared with the 2% articaine–treated animals. The indi-vidual size distributions (for which size is chosen as theequivalent circle diameter) of the myelinated axons areshown in Fig. 7. It is clear that the large myelinated axonsdisappear in almost all animals that were treated witharticaine 4% and to a lesser extent in the animals that weretreated with articaine 2%. The 2 articaine-treated groupsinstead had supernumerary small myelinated axons.

DISCUSSIONOpinions on the causative mechanism leading to neurosen-sory disturbance in humans have been divided, needlepenetration trauma and neurotoxic reaction to the injected

substance being potential options. The neurotoxicity ofarticaine 4% has been denied as a potential mechanismbehind an apparent overrepresentation of 4% formulationsof local anesthetic-associated neurosensory disturbance inhumans.15 A recent combined clinical and registry study ondata from the Danish Medicines Agency’s national data-base on adverse drug reactions indicated that neurotoxicityof the injected substance is the causative factor rather thanthe needle penetration.16 Thus, an experimental study toexamine the effects of needle penetration with injection ofsaline solution versus needle penetration with injection of alocal anesthetic in different concentrations was deemedappropriate. A group with needle penetration alone (withoutinjection) was not included because it would be incompat-ible with the blinding. Thus, saline without preservativesand without vasoconstrictors was chosen as the mostprobable inert substance for the control group. Therefore,the mechanical trauma of needle penetration and theinfluence of injection of an equal volume of a commerciallyavailable formulation of 2% and 4% articaine (includingpreservatives) were tested against saline.

The rat sciatic nerve model has proved valid in anumber of previous studies similar to ours,9,12,17,18 someof which with slightly different aims. The sensory axonsconstitute about 70% of the sciatic nerve fibers, andthe sensory neurons are located in lumbar gangliaL3 to 6.18

Figure 4. Box plot of amplitudes (median values, interquartile range, extreme values) of the lumbar evoked electrospinograms at the 3-weekfollow-up after intraneural injection of test substance. Control side (untreated) and experimental side showing the 3 injection groups (saline,articaine 2%, articaine 4%) indicating significant concentration-dependent depression of amplitudes in the articaine 2% group (P � 0.03) and4% group (P � 0.0006, Table 1).

Figure 5. Micrographs of nerve cross-sections from a saline-treated rat (left) and arat injected with articaine 4% (right). Thenumber of myelinated axons is the same inthe 2 treatment groups, but in the groupinjected with articaine 4%, the axons aremuch smaller and the endoneurial connec-tive tissue increased. Few remnants ofdegenerating nerve fibers are seen, andseveral of the smallest myelinated axonsare surrounded by a nucleated Schwanncell, indicating regeneration. Magnificationbar shown in the picture is 1000 �m. Stain:osmium tetroxide and toluidine blue.



In the present study we found a significant concentration-related suppression of the amplitude in the lumbar-evokedelectrospinogram in the articaine groups 3 weeks afterinjections. Because the amplitudes of the electrospinograms inthe articaine groups were significantly lower than those in thesaline group, the suppression could not have been caused bydamage from the needle penetration alone but rather by the

injected substances. Electrophysiological findings with suchreduction in the amplitude of the evoked responses as seen inthe articaine groups might be associated with axonal loss inhumans.19 In addition, it was not possible to elicit alumbar-evoked response in one third of the animals in thegroup treated with articaine 4%, whereas responses wereelicited in every animal in the articaine 2% and the saline

Figure 6. A–E, Quantitative data on myelinatedaxons. The saline-treated animals are indicated bycircles, where the open circles indicate the un-treated left nerve and the solid circles indicate thetreated right nerve. The animals treated with artic-aine 2% are shown by triangles and those treatedwith articaine 4% are shown by squares. For botharticaine-treated groups the untreated nerve wasomitted from the examination. The horizontal linesindicate the group means. A, The cross-sectionalarea of the nerve. B, The total numbers of axons. C,The mean equivalent circle diameters. D, The meanmyelin thickness. E, The mean g ratio. Significantconcentration-dependant differences were found inmean equivalent circle diameter and mean myelinthickness (Table 2).


Table 2. P Values for the Statistical Tests Performed on the Stereological Data

ANOVAPaired t test,saline treated

Unpaired t test

Saline treatedvs. articaine 2%

Saline treatedvs. articaine 4%

Articaine 2%vs. articaine 4%

Number of myelinated axons 0.065 n.s. — — —Mean axon area 0.00021 n.s. 0.095 0.00020 0.010Mean equivalent circle diameter 0.00018 n.s. 0.08 0.00018 0.010Mean myelin thickness 0.0051 0.032 0.22 0.0010 0.057

ANOVA � analysis of variance; n.s. � not significant.

Figure 7. Individual absolute size distributionsof myelinated axons on a linear scale ofequivalent circle diameter. Most rats injectedwith articaine 4% have very few large myelin-ated axons and an increased number of smalldiameter axons when compared with thesaline-treated animals.



group. These results compare favorably with the in vitrostudy by Werdehausen et al.11 and the experimental studiesof Cornelius et al.12 Werdehausen et al.11 found that alllocal anesthetics were neurotoxic in a concentration-dependent manner and even in clinically used concentrations.In a study similar to ours, Cornelius et al.12 demonstratedextinction of somatosensory evoked-lumbar responses in 9of 10 animals with 4% articaine and 1 of 10 with 2%articaine. Likewise, Kroin et al.9 demonstrated severe neu-rotoxic reaction to lidocaine 4% in the rat tibial nerve.

Lumbar-evoked responses have proven suitable as amethod to express variations of the chemical action ofdifferent concentrations and formulations of local anesthet-ics.8,12 Considering the limited number of animals in ourstudy, a dependable baseline response level was essential.This was achieved through averaging of a large number ofrecordings of evoked lumbar responses from the sciaticnerve, as well as the randomization of the test substancesand blinding of the investigators. An anesthetic effect wasquickly established after injections of the test substances inthe first session, shown as reduced amplitude in thelumbar-evoked electrospinogram in the articaine groups.The quick onset after 1 minute was precipitated by theintraneural injection of the drug. Delivery of a precisevolume of test substances was attempted. However, in anumber of cases some spillage was inevitable, which mayaccount for some variability. The technique may also havecaused minor trauma, resulting in a missing response at 1minute in 1 saline rat and in 1 articaine 2% rat20,21 (Fig. 3).Fried et al.17 showed limited histological damage in theclose vicinity of the needle lesion when using a microneu-rography electrode in the rat sciatic nerve. These lesionsunderwent spontaneous healing during the subsequentweeks.

We also found that the total number of axons wereslightly higher (9% and 7%, respectively) in the groupstreated with articaine when than those in the saline-treatedgroup, but the difference did not reach significance. Afterthe code was broken the sections were inspected (but notblinded), and remnants of degenerating axons were presentin the rats treated with articaine 4%. It was also ourimpression that more small-diameter myelinated axonsthan usual were surrounded by a nucleated Schwann cell inthe 2 groups treated with articaine. These small-diameteraxons associated with the Schwann cell nucleus may rep-resent regenerated axons,10 which can explain the apparentparadox that the groups treated with articaine had (insig-nificantly) more axons than did the saline-treated animals.The mean axon area, the equivalent circle diameter, and themyelin sheath thickness were significantly smaller in theanimals treated with articaine 4% when compared withboth the saline-treated animals and the articaine2%–treated animals, but the g ratio was the same in allgroups. Size distributions showed that the animals treatedwith articaine had virtually no large myelinated axons butinstead had many small-diameter axons. This shift towardssmaller axon diameters may be reflective either of shrink-age of existing axons or of the combined effect of lostlarge-diameter axons and the appearance of new regener-ating axons with small diameter. A short-term study isneeded to establish whether articaine 4% injection can elicit

significant degeneration of large myelinated axons and asubsequent regeneration with supernumerary regeneratingaxons.

Despite species-related differences, our results are inaccord with epidemiologic studies on humans in demon-strating a significant difference between 2% and 4% formu-lations of local anesthetics.7,16 Considering the relativerobustness of the rat sciatic nerve, more dramatic changesmight affect humans and other higher species. A recentstudy on reports of paresthesia involving dental localanesthetics during the period from November 1997 throughAugust 2008 from the United States Food and DrugAdministration Adverse Event Reporting System7 dem-onstrated significant overrepresentation of neurosensorydisturbance associated with 4% solutions of prilocaine andarticaine used in dentistry. Also in Europe, clinical andregistry studies on data from the Danish Medicines Agen-cy’s national registry on adverse drug reactions associatedwith local anesthetics show overrepresentation of adversedrug reactions with articaine 4%.3,16,22

CONCLUSIONThe observed degenerative changes (stereology) and thereduced amplitude of the lumbar-evoked electrospi-nograms (electrophysiology) after 3 weeks indicate aconcentration-dependent neurotoxic effect of intraneuralinjection of 50 �L of commercially available 2% and 4%formulations of an articaine-based local anesthetic.Needle penetration with injection of 50 �L of salinehad no significant effect on nerve conduction andhistomorphology.

ACKNOWLEDGMENTSProf. C. P. Cornelius, MD, DDS, Lab technician J. S. Lykkeaa,Consultant T. Dalager, MD, Prof. R. H. Jensen, MD, Dr. C. J.Bundgaard, DVM, and Prof. M. Lauritzen, MD, are cordiallyacknowledged for their kind assistance.

DISCLOSURESName: Søren Hillerup, DDS, PhD, Dr. Odont.Contribution: This author helped design the study, conductthe study, analyze the data, and write the manuscript.Attestation: Søren Hillerup has seen the original study data,reviewed the analysis of the data, approved the final manu-script, and is the author responsible for archiving the studyfiles.Name: Merete Bakke, DDS, PhD, Dr.Odont.Contribution: This author helped conduct the study, analyzethe data, and write the manuscript.Attestation: Merete Bakke has seen the original study data,reviewed the analysis of the data, and approved the finalmanuscript.Name: Jytte Overgaard Larsen, MD, PhD.Contribution: This author helped conduct the study, analyzethe data, and write the manuscript.Attestation: Jytte Overgaard Larsen has seen the original studydata, reviewed the analysis of the data, and approved the finalmanuscript.Name: Carsten Eckhardt Thomsen, MScEE, PhD.Contribution: This author helped conduct the study, analyzethe data, and write the manuscript.


Attestation: Carsten Eckhardt Thomsen has seen the originalstudy data, reviewed the analysis of the data, and approvedthe final manuscript.Name: Thomas Alexander Gerds, Dr.Rer.Nat.Contribution: This author helped analyze the data and writethe manuscript.Attestation: Thomas Alexander Gerds has seen the originalstudy data, reviewed the analysis of the data, and approvedthe final manuscript.

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