The LaryngoscopeVC 2010 The American Laryngological,Rhinological and Otological Society, Inc.

Electromyographic and Histologic Evolution of the RecurrentLaryngeal Nerve From Transection and Anastomosis toMature Reinnervation

Michael J. Pitman, MD; Philip Weissbrod, MD; Rick Roark, PhD; Sansar Sharma, PhD;

Steven D. Schaefer, MD

Objectives: To describe the natural evolution of recurrent laryngeal nerve (RLN) reinnervation in an animal model.Study Design: Twenty Sprague Dawley rats underwent unilateral RLN transection and anastomosis. Animals were sacri-

ficed at 4, 8, 12, 16, and 20 weeks. Prior to sacrifice, each rat underwent electromyography (EMG) and visual grading of vocalfold motion. Bilateral RLNs were harvested and evaluated histologically.

Results: EMG revealed synkinetic reinnervation at all time periods except at 4 weeks. EMG evolution plateaued at 16weeks. Vocal fold motion was slight in three rats at 4 weeks but was otherwise absent except for one rat at 12 weeks. Histo-logic changes of the axons and their myelin sheaths were consistent at each time period. At 16 weeks, histologic changesplateaued.

Conclusions: Consistent EMG, histologic, and vocal fold motion changes occur at specific time periods during RLN rein-nervation after transection and anastomosis in a rat model. Reinnervation is mature at 16 weeks. Findings corroborate theo-ries of preferential and synkinetic reinnervation after RLN transection. Use of a rat model to investigate the effect of interven-tions on RLN reinnervation requires a minimum of 16 weeks between transection and investigation to allow for maturationof reinnervation.

Key Words: Recurrent laryngeal nerve, rat model, vocal fold paralysis, nerve reinnervation.Level of Evidence: N/A.

Laryngoscope, 121:325–331, 2011

INTRODUCTIONDue to the morbidity of recurrent laryngeal nerve

(RLN) paralysis and the suboptimal treatments thatexist, reasonable efforts have been made to investigateRLN reinnervation. Many researchers have utilized therat as an experimental model to study the patterns oflaryngeal muscle innervation as well as reinnervationfollowing RLN injury and treatment with neurotrophicfactors.1–11 The rat has been one of the mammals ofchoice due to its relatively low cost, simplicity of care,the ease of surgery, and their tolerance of intervention.To accurately evaluate the effects of interventions onRLN reinnervation, an understanding of the naturalevolution of RLN reinnervation from transection andanastomosis to mature reinnervation is essential. In the

present study, electromyographic, histologic, and kinesio-logic observations were used to investigate chronologicchanges in these parameters during the natural evolu-tion of RLN reinnervation following transection andanastomosis. This will advance our understanding of therat model of RLN injury, increase our knowledge of RLNreinnervation in general, delineate the best timing ofinterventions for successful and proper reinnervation,and enhance our ability to evaluate the effect of theseinterventions.

MATERIALS AND METHODS

Experimental AnimalsTwenty female Sprague Dawley rats were used to evaluate

the time required for complete RLN regeneration after transec-tion and anastomosis. Prior to surgical intervention, rats weresedated with isoflurane, anesthetized with an intramuscularinjection of a mixture of 70 mg/kg of ketamine and 7 mg/kgxylazine, and secured to a modified stereotactic surgical table.One percent lidocaine was injected into the site of the skin inci-sion. This study was performed in accordance with the PublicHealth Service (PHS) Policy on Humane Care and Use of Labo-ratory Animals, the National Institutes of Health (NIH) Guidefor the Care and Use of Laboratory Animals, and the AnimalWelfare Act (7 U.S.C. et seq.); the animal use protocol wasapproved by the Institutional Animal Care and Use Committee(IACUC) of New York Medical College. Humane care was pro-vided for these animals and all institutional and nationalguidelines were observed.

From the Department of Otolaryngology (M.J.P., P.W., S.D.S.), The NewYork Eye and Ear Infirmary, New York, New York, and the Departments ofOtolaryngology (R.R.) and Cell Biology (S.S.), The New York MedicalCollege, Valhalla, New York, U.S.A.

Editor’s Note: This Manuscript was accepted for publication June29, 2010.

Presented at the Triological Society Annual Meeting, Las Vegas,Nevada, U.S.A., April 30, 2010.

This work was supported by a Triological Society Research CareerDevelopment Award.

Send correspondence to Dr. Michael J. Pitman, 310 E. 14th Street,6th Floor, New York Eye and Ear Infirmary, New York, NY 10003.E-mail: mpitman@nyee.edu

DOI: 10.1002/lary.21290

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SurgeryAll surgeries were performed using an operating microscope

(Zeiss, Hennigsdorf, Germany). A vertical midline cervical inci-sion was made. The right RLN was exposed in thetracheoesophageal groove and sharply transected with an iridec-tomy scissor at the seventh tracheal ring. The nerve ends wereanastomosed using a common suture free technique utilizing sa-line hydrated gel foam (Pharmacia & Upjohn, New York, NY) toprevent further nerve trauma from suture anastomosis.12 Gelfoam was placed beneath the nerve prior to transection. Whenthe nerve was transected, there was minimal separation of thenerve ends secondary to their adherence to the underlying gelfoam. The gel foam was then wrapped around the nerve ends,creating a firm connection. The superior laryngeal nerve (SLN)provides collateral innervation to the endolaryngeal musculaturein both rats and humans.3,13 These collateral innervations mayinterfere with RLN reinnervation, confounding this and futureinvestigations. To prevent interference, the SLN was exposed byretraction of the omohyoid, ligated proximally and distally usingsmall vessel clips (Ethicon, Somerville, NJ), and then transectedbetween the clips. A 0! 4-mm endoscope (Karl Storz, Tuttlingen,Germany) fitted with an epiglottis retractor was inserted transor-ally to visualize the larynx and confirm right vocal fold paralysis.

Four, 8, 12, 16, and 20 weeks following initial surgery,animals were sedated as described above, transoral laryngealelectromyography (EMG) was performed, and vocal foldmotion was evaluated as detailed below and previously.14 Ratswere then sacrificed via an overdose of isoflurane. RLN nervesections were then harvested proximal and distal to the site oftransection. A corresponding section of the left RLN was har-vested as a control in each rat. Nerves were subsequentlyprocessed as described previously and below.14 Ultimately, EMG,histologic, and kinesiologic findings were corroborated to evalu-ate the chronologic evolution of reinnervation and the timeneeded to allow for maturation of reinnervation.

KinesologyA 0! 4-mm endoscope (Karl Storz) fitted with an epiglottis

retractor was inserted transorally to visualize the larynx.14 Acolor LCD camera (Richard Wolf, ) was attached to the endo-scope and its signal routed to a color video monitor that waswithin view of all investigators. Bilateral vocal fold motion wasvisualized and graded on a scale of 0 to 4, 0 being absence ofmovement, 1 consistent but minimal motion, 2 purposefulmotion with significant paresis, 3 purposeful motion with mini-

mal paresis, and 4 being normal motion compared with thecontralateral vocal fold used as a control. Abduction reliablyoccurs during phasic ventilation, cycling every 600 to 800 ms.Adduction spontaneously occurs during swallow, cough, andreflexive adduction. The scoring was performed by three separateobservers. The mode score of observers was used as a final score.

Transoral ElectromyographyAnimals were placed supine on a modified stereotactic

operating table in a Faraday cage recording room. The tonguewas retracted using a 3-0 silk suture placed midline in the ante-rior tongue and suspended. A 0! 4-mm endoscope (Karl Storz)fitted with an epiglottis retractor was inserted transorally tovisualize the larynx. Transoral EMG was performed (Figs. 1and 2). EMG of the left thyroarytenoid and lateral cricoaryte-noid muscles (TA/LCA) was undertaken to confirm the absenceof motor unit action potentials (MUAPs) during ventilation andits presence during adduction. EMG of the left posterior

Fig. 1. Transoral electromyography (EMG) using a 0! operating endoscope (A) and an artist rendition of the procedure (B). [Color figure canbe viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 2. Electromyography (EMG) electrode insertion points formeasuring consistent strong signals from the posterior cricoaryte-noid ( , red) and thyroarytenoid ( , green) muscles of the rat lar-ynx. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

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cricoarytenoid muscle (PCA) was then performed to confirm thepresence of MUAPs during phasic ventilation and absence dur-ing adduction. An identical procedure was used for evaluationof the right TA/LCA and PCA. Synkinesis was considered pres-ent when consistent MUAPs were observed in the TA/LCAduring abduction and in the PCA during adduction. Myoelectricsignals were detected using a 25-gauge 45-mm quadrifilar nee-dle electrode (Delsys, Boston, MA). The quadrifilar electrodehas four 50-lm-diameter selective surfaces located along can-nula side ports that are spaced 200 lm apart in a squarepattern.15 Myoelectric activity was monitored visually using adigital real-time oscilloscope (Tektronix TDS210, ) and audiblyby means of an amplifier/speaker unit connected in parallel tothe EMG data recording system. Bipolar EMG signals wererecorded from two of the four electrode detectors. Signals were

amplified using a preamplifier situated at the needle head andthen fed into high-gain differential amplifiers located nearby(in-house construction). Myoelectric signals were bandpass fil-tered from 15 Hz to 1,000 Hz (Krohn-Hite Model 3360,Brockton, MA), digitized (Data Translation Model DT2821G,Marlboro, MA), and stored in real time (Dolch Computer Sys-tems Model LPAC-PT, Freemont, CA). MUAPs and EMGsignals were analyzed using Matab software by three separateobservers (The Mathworks, Release 2007b, Natick, MA).

HistologyIsolated RLNs were harvested and all specimens were im-

mediately immersed in a mixture of 1% glutaraldehyde and 2%paraformaldehyde in 0.1 M pH 7.2 phosphate buffer. They were

Fig. 3. Evolution of electromyography (EMG) signals from weeks 4 (A), 8 (B), 12 (C), 16 (D), and 20 (E) at a 20-millisecond sweep speed.[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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postfixed for 1 hour in 1% osmium tetroxide in 0.1 M phosphatebuffer, dehydrated in graded ethanol, and then embedded in ep-oxy resin. Thin 1-lm sections were cut with glass knives on aSorvall ultramicrotome and stained with aqueous 1% toluidineblue. Nerve sections were then evaluated under light micros-copy. Qualitative evaluation of axon presence and size as wellas myelin thickness was performed by two observers using a40" objective. All nerve sections were compared to determinethe maturation of axon and myelin morphology.

RESULTS

KinesologyOf the 20 rats in five surgical groups, four rats pre-

sented volitional motion of the experimental vocal foldprior to sacrifice. Three of these rats were sacrificed 4weeks postsurgery. One displayed grade 2 vocal foldmotion, meaning purposeful movement with severelyimpaired velocity and range of motion. Two of these fourrats had grade 1 motion, meaning a perceptible and con-sistent twitching motion in phase with the oppositevocal fold. One of these further displayed a continuoustremor of the right vocal fold. One of the four rats sacri-ficed at 12 weeks postsurgery displayed grade 3 vocalfold motion, meaning a slight decrease in velocity andrange of motion. Only abduction was observed in theseanimals. All other rats displayed no observable volitionalmotion of the experimental vocal fold on either abduc-tion or adduction, regardless of the time period.

Transoral ElectromyographyIn all animals, electric silence was never observed.

The left vocal fold, serving as a control, showed normal

EMG morphology and absence of synkinesis. On the ex-perimental side, there was a clear evolution of the EMGsignal observed from week 4 until week 16 (Fig. 3). Atweek 4, none of the rats showed electromyographic evi-dence of synkinesis. MUAPs were consistent withcomplex recovery potentials and significant polyphasicresponses. Synkinesis was first observed 8 weeks follow-ing surgery (Fig. 4). EMGs revealed MUAPs withcontinued polyphasic patterns but with the presence ofmore discrete and narrow units. At week 12, three offour rats displayed synkinesis in the experimental PCAand TA/LCA. Wide polyphasic MUAPs were still presentbut narrowed compared with week 8. In addition, occa-sional MUAPs with normal morphology were observedin these three rats. One 12-week rat displayed synkine-sis in the PCA only. MUAPs were seen in the TA/LCAwhen the control vocal fold adducted. This same animaldisplayed grade 3 vocal fold motion. The morphology ofthe MUAPs of this animal was similar to the MUAPmorphology of the other 12-week animals. By week 16,MUAPs were narrow and mature. Rare polyphasicpotentials were observed in one of four animals. Therewas also a significant increase in recruitment with mul-tiple MUAPs during all recordings. EMGs at week 20were similar to week 16 with narrow, mature MUAPsand recruitment of multiple MUAPs. Polyphasic MUAPswere not observed in any of the rats at 20 weeks.

HistologyAxon and myelin sheath regeneration plateaued at

16 weeks (Fig. 5). At 4 weeks, there was a significantloss of axons and myelin degeneration with evidence of

Fig. 4. Sample of synkinesis fromthe posterior cricoarytenoid (PCA)muscle of a 16-week rat. Note thebursts of motor unit action poten-tials (MUAPs) on phasic respirations.The nonphasic recruitment of high-amplitude MUAPs is secondary tosynkinetic electromyographic activ-ity on adduction during a swallow(2-second sweep speed). [Color fig-ure can be viewed in the onlineissue, which is available atwileyonlinelibrary.com.]

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early regeneration as exhibited by thinly myelinatedaxons of small diameter. The process of myelinationoccurred quickly. Minimal myelin density changes wereobserved after week 8. In contrast, as reinnervation pro-gressed, axons increased significantly in diameter. At 16and 20 weeks, axons resembled the morphologic appear-ance of the normal control nerve with varying diameteraxons. Minimal differences were observed betweennerves at weeks 16 and 20. Similar changes occurred inthe RLN proximal and distal to the nerve transection.

DISCUSSIONRecurrent laryngeal nerve injury occurs at a rela-

tively steady rate during cervical and cardiothoracicsurgery. Permanent unilateral vocal fold paralysis occurs

in approximately 2% of thyroidectomies, carotid endar-terectomies, and open-heart surgeries. The percentagesof paralysis in thoracic and skull base surgeries aremuch higher.16 Our current treatment methods, whilequite helpful, are suboptimal. Optimal treatment of arecurrent laryngeal nerve injury should result in resto-ration of patient’s dynamic preinjury voice, swallowing,and breathing. Because of the complexity of vocal foldmotion and mass control needed to achieve normal la-ryngeal function, mechanical correction of paralysis orsynkinetic reinnervation is unlikely to result in normalfunctional recovery. Such recovery can only occur if RLNinjury and reinnervation can be artificially influenced tooccur in an ordered fashion that results in establishmentof preinjury innervation patterns. To this end, muchresearch has been focused on investigating the influenceof neurotrophic factors on reinnervation and vocal foldfunction.1–11 An appropriate and well-described animalmodel is integral to conducting this research. The rat isone mammal of choice due to its relatively low cost, sim-plicity of care, ease of surgery, and high tolerance tointervention. Relatively few investigations have beenperformed to analyze the natural history of the rat RLNafter injury.14,17 Because so little is known about recov-ery after this injury, it is difficult to draw validconclusions about the effect of interventions, such as theapplication of neurotrophic factors or antibodies, on theinjured nerve.

With the aim of improving our knowledge of RLNinjury, this investigation was performed to elucidate thenatural evolution of rat RLN transection and anastomo-sis via evaluation of EMG, kinesiologic, and histologicobservations at 4, 8, 12, 16, and 20 weeks posttransec-tion and anastomosis. In addition, the time point atwhich reinnervation matures was identified to optimizefuture experimental procedures by minimizing risk ofearly animal sacrifice, reducing false-negative results,and limiting cost of prolonged animal maintenance.

In the current report, vocal fold motion was absenton the experimental side in 16 of 20 animals followingtransection. At 4 weeks, three of four animals displayedparetic vocal fold motion. In addition, at 12 weeks, oneof four animals in this group displayed grade 3 abduc-tion, no adduction, and synkinesis in the PCA with anabsence of synkinesis in the TA/LCA. This phenomenonadds credence to the hypothesis of preferential reinner-vation as suggested by Woodson in the cat.18 One maysurmise that contrary to the cat, a rat will preferentiallyreinnervate its PCA musculature because the primaryfunction of the rat larynx is respiration as opposed tophonation. In addition, while inconclusive, it appearsthat more rat laryngeal motor neurons innervate thePCA than the TA/LCA, wheras the reverse is likely truein the cat.19,20 As such in the rat, preferential PCA rein-nervation would be expected. Preferential PCAinnervation was observed in the 4-week rats as the PCAwas reinnervated predominately by abductor LMNs asevidenced by a lack of EMG synkinesis in the PCA. Incontrast, minimal reinnervation of any kind was notedin the TA/LCA. This early innervation pattern resultedin PCA abduction, without antagonistic TA/LCA

Fig. 5. Left control recurrent laryngeal nerve (RLN) (A). Histologicprogression of right RLN reinnervation from weeks 4 (B), 8 (C), 12(D), 16 (E), and 20 (F) at 40" magnification. [Color figure can beviewed in the online issue, which is available at wileyonlinelibrary.com.]

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contractions. As reinnervation progressed beyond fourweeks, TA/LCA innervation increased. In addition, aber-rant mixed reinnervation of both the TA/LCA and PCAmuscles became evident. This synkinetic innervation pat-tern resulted in antagonistic adducting and abductingmuscle forces that produced an immobile vocal fold. Theevolution of EMG signals during this study supports thishypothesis as all rats after 4 weeks evidenced significantEMG sykinetic reinnervation of the TA/LCA and PCA aswell as an immobile vocal fold. However, one 12-week ani-mal showed near normal motion on abduction with anabsence of adduction. On adduction, EMG showed normalTA/LCA MUAPs in addition to synkinetic PCA MUAPs.This electromyographic activity pattern results in antago-nistic muscle activity on attempted adduction, as both thePCA and TA/LCA are stimulated with the resulting lackof vocal fold. In contrast, on abduction the PCA evidencednormal MUAPs and the TA/LCA was silent. Due to thelack of antagonistic muscle activity, grade 3 abductionoccurred. These findings support the hypothesis of prefer-ential PCA reinnervation. In addition, they corroborateFlint’s observation of synkinetic reinnervation after RLNtransection and anastomosis in a rat as well as Crumley’sclinical observations that vocal fold immobility in humansis due to synkinetic reinnervation, not insufficient rein-nervation.20,21 This concept of vocal fold immobilityfollowing RLN injury is further supported by a recentreport highlighting the presence of significant EMG syn-kinesis as the most accurate indicator of persistent vocalfold immobility after a RLN injury.22 To summarize, theseobservations suggest that during early reinnervation, theaxons reaching the PCA are predominately abductors andas a whole outnumber axons reaching the TA/LCA. As aresult, vocal fold motion is possible due to PCA abductionthat is unopposed by the weakly reinnervated TA/LCA.As reinnervation proceeds beyond the initial phase andmore aberrant axons reach the TA/LCA and PCA, thePCA is no longer able to abduct the vocal fold due toantagonistic muscle contraction. What allowed one ani-mal to develop motion at 12 weeks is unclear, butfortuitous reinnervation following RLN transection,though rare, is not unheard of in the human.23

In addition to observations of synkinesis, EMGstudies allowed evaluation of the morphologic evolutionof MUAPs. The evolution observed in this study corre-lates well with what is seen following RLN injuryclinically in humans and experimentally in rats.14,17,24

These EMG findings also correlate well with histologicevolution of nerve recovery. At 4 weeks, early reinnerva-tion results in significant polyphasic MUAPs that maybe referred to as complex recovery potentials due totheir greater width and more turns than usual polypha-sic potentials. Histologically these MUAPs correlate withthe immature reinnervation and continuing axonal deg-radation. Over time the MUAPs began to narrow andturns decreased as the myelin thickened. Between 4 and8 weeks, the complex reinnervation potentials evolvedinto the usual morphology of polyphasic MUAPs. At 12weeks, although polyphasic MUAPs predominate theEMG, the MUAPs have narrowed further and the turnshave continued to decrease. Occasionally normal MUAPs

were observed. Histologically this is seen as a generalincrease in myelin thickness and axon diameter. By 16weeks, histologically the nerve appears mature.Although myelin thickness plateaus at 12 weeks, thereis clearly a significant and startling increase in the di-ameter of many of the axons between 12 and 16 weeks.Correspondingly, at 16 weeks EMG reveals normalMUAPs with occasional giant waves. In one of four rats,only a rare polyphasic potential was observed. Between16 and 20 weeks, there was minimal evolution in theEMG or histology. Based on these findings, the reinner-vation of a rat RLN after transection and anastomosistakes 16 weeks to mature.

CONCLUSIONSThe goal of this study was to evaluate and describe

the histologic, electromyographic, kinesologic, and tempo-ral progression of reinnervation after transection andanastomosis of the recurrent laryngeal nerve in a ratmodel. Findings in this investigation support previousevidence of preferential reinnervation in the larynx of oneantagonistic muscle over the other. In the rat, this prefer-ence was demonstrated to be for the PCA. Findings alsosupport the hypothesis that vocal fold immobility afterRLN injury is due to aberrant reinnervation, not insuffi-cient reinnervation. Histologic and EMG evolutioncorrelate well over the course of reinnervation. Histologicand electromyographic changes plateaued at 16 weeks,suggesting that it takes 16 weeks for maturation of ratRLN reinnervation after transection and anastomosis.

These histologic, electromyographic, kinesiologic,and temporal observations should be considered whenevaluating the efficacy of various interventions torestore normal vocal fold mobility following RLN injury.Based on the present observations, final outcomes of theintervention should not be measured until 16 weeks af-ter transection and anastomosis so that reinnervationhas had a chance to mature.

AcknowledgmentsThe authors would like to thank Jason Reid for his

significant contribution in the preparation of the histologicsections.

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