Morphologic Evaluation of the Fetal Recurrent

Laryngeal Nerve and Motor Units in the

Thyroarytenoid Muscle

*,†,‡Joel Henrique Ellwanger, *Jo~ao Paulo da Costa Rosa, *Iuri Pereira dos Santos,

*,†Helen Tais da Rosa, §,jj,{Geraldo Pereira Jotz, #L�eder Leal Xavier, and*,†,§Deivis de Campos, *ySanta Cruz do Sul and zxjj{#Porto Alegre, Rio Grande do Sul, Brazil

Summary: This study is a morphologic description of the recurrent laryngeal nerve (RLN) and of the number and size

AccepFrom t

Universide CiencCruz doem BioloVale, Porciencias,Sul, PortInstitutoAlegre, Rversidadedo Sul, BCienciasCat�olicaAddre

tologia eSul (UNIdcamposJourna0892-1� 201http://d

of motor units (MUs) in the thyroarytenoid (TA) muscle bilaterally of a human fetus aged 25 weeks. A quantitative anal-ysis of RLN and MUs is presented to investigate similarities with equivalent structures in adults. In the fetus used in ourstudy, the morphologic organization of the RLNwas similar to that commonly described in the adult RLN. Moreover, asis observed in adult TA, the TA of the analyzed fetus, particularly the right TA, showed MUs typical of muscles withgreat motor accuracy. These results may be used to increase our knowledge of the features of the voice in adults andnewborns.Key Words: Microanatomy–Recurrent laryngeal nerve–Thyroarytenoid muscle–Motor units–Fetus.

INTRODUCTION

The cry of the newborn is rich in sounds and acoustic propertiesthat have great clinical importance at birth. This complex pat-tern of vocalization in both the newborn and the adult dependson an action that involves nerve and muscle structures such asthe recurrent laryngeal nerve (RLN) and the thyroarytenoid(TA) muscle.1–3

The RLN, a branch of the vagus nerve, extends through thechest cavity and neck. The left and right RLNs follow differentpaths to the larynx. The left RLN arises from the vagus nerve,then bends under the aortic arch, and ascends along the trache-oesophageal groove until the larynx. The right RLN arises fromthe vagus nerve, curves under the subclavian artery, and ascendstoward the larynx.4 This difference between the paths promotesasymmetry between the lengths of nerves in adults with the leftRLN exhibiting an average length of 43 cm and the right RLNof 32 cm.5

Functionally, the RLN is associated with motor innervationof the intrinsic muscles of the larynx,6 especially the TA, alsoreferred to as the vocal muscle.7,8 The TA is responsible forselective adjustments in different parts of the vocal fold.7–9

Injuries to the RLN, both arising from accidental trauma orsurgical procedures, can lead to unilateral or bilateral

ted for publication July 10, 2013.he *Laborat�orio de Histologia e Patologia, Departamento de Biologia e Farm�acia,dade de Santa Cruz do Sul, Santa Cruz do Sul, Rio Grande do Sul, Brazil;yCursoias Biol�ogicas, Departamento de Biologia e Farm�acia, Universidade de SantaSul, Santa Cruz do Sul, Rio Grande do Sul, Brazil;zPrograma de P�os-Graduac~aogia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Campus doto Alegre, Rio Grande do Sul, Brazil; xPrograma de P�os-Graduac~ao em Neuro-Instituto de Ciencias B�asicas da Sa�ude, Universidade Federal do Rio Grande doo Alegre, Rio Grande do Sul, Brazil; jjDepartamento de Ciencias Morfol�ogicas,de Ciencias B�asicas da Sa�ude, Universidade Federal do Rio Grande do Sul, Portoio Grande do Sul, Brazil; {Departamento de Ciencias B�asicas da Sa�ude, Uni-Federal de Ciencias da Sa�ude de Porto Alegre, Porto Alegre, Rio Grande

razil; and the #Laborat�orio de Biologia Celular e Tecidual, Departamento deMorfofisiol�ogicas, Faculdade de Biociencias, Pontif�ıcia Universidade

do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.ss correspondence and reprint requests to Deivis de Campos, Laborat�orio de His-Patologia, Departamento de Biologia e Farm�acia, Universidade de Santa Cruz doSC), Av. Independencia, 2293, Santa Cruz do Sul-RS, 96815-900, Brazil. E-mail:@unisc.brl of Voice, Vol. 27, No. 6, pp. 668-673997/$36.003 The Voice Foundationx.doi.org/10.1016/j.jvoice.2013.07.004

paralysis of the vocal folds and result in changes in voice,dysphagia, and dyspnea.10,11

The histologic and anatomic differences between right andleft RLNs have been well described.3,12 However, there is nodata in the literature about these parameters in newborns orfetuses. In addition, gathering data on the morphometricfeatures of the RLN is certainly very important forunderstanding and improving the techniques used for vocalfold reinnervation.13–17 It is therefore important to understandtheir anatomy in terms of their ability to resist damage.Moreover, to our knowledge, there is no evidence in thecurrent literature showing the connective and adipose tissuessurrounding the laryngeal nerves in the fetal period. However,it is described that information regarding the quantity andcomposition of these tissues in laryngeal nerves is importantfor determining their protective characteristics and thisinformation could also contribute to surgical approaches suchthat nerve damage may be prevented.18

Another clinically relevant aspect is the number and size ofthe motor units (MUs) in the TA. A few studies have attemptedto provide a morphometric estimation of MUs in human intrin-sic laryngeal muscles, although they did not consider the age ofthe subjects or the side of the muscles. Therefore, additionaldata regarding the number and size of MUs in human intrinsiclaryngeal muscles are of interest to both anatomists and clini-cians.19 Nevertheless, there is no data in the literature aboutthe number and size of the MUs in fetal or newborn TA.Thus, the aim of this study was to describe the MUs of the TA

bilaterally in fetal period by quantifying histomorphometricdata from the RLN (fiber density, intraperineural area, intraper-ineural diameter, total number of fibers, fiber area, and fiber di-ameter) and from the TA (fiber density, fiber area, fiberdiameter, total number of fibers, cross-sections of the area,and diameter).

MATERIALS AND METHODS

Human tissue

For this analysis, we decided to use the same fetus recently usedin another study,20 a 25-week female fetus, with cause of death

Joel Henrique Ellwanger, et al Fetal RLN and MUs in the TA Muscle 669

unknown (obtained from the collection of the Laboratory ofHistology and Pathology at Universidade de Santa Cruz doSul, Rio Grande do Sul, Brazil). This decision was made dueto the great difficulty of obtaining fetal tissue for this type ofresearch.

Data acquisition

The digitized images of the RLN and TAwere captured using anOlympus BX 50 microscope (34, 310, and 3100) (Olympus,Tokyo, Japan) coupled to a video camera (Leica DC 300F) in-terfaced by Leica Image 50 (IM50) software (Leica, Wetzlar,Germany).

Histologic and morphometric measurements

of the RLN and its branch to the TA

Based on previous studies,3 the dissection of the RLN extendeddeeply toward the point where it entered the larynx (cricothy-roid joint). In our study, this region was chosen because the to-pography of the RLN is more uniform near the insertion point ofthe nerve into the larynx.21 Thus, the segments of RLN used inour histologic analysis were obtained 1 and 2 mm below the cri-cothyroid joint (Figure 1).

To estimate the number and size of the MUs in the TA, it wasnecessary to dissect a branch of the RLN to the TA muscle.Therefore, a midline incision was made to divide the larynxinto two hemi-larynges. With the aid of a magnifying glass(ALLZWECK—LUPENLEUCHTE [31.75/2.25 magnifica-tion]), the branch of the RLN to each muscle was exposedand sectioned (2–3 mm) by two experts in the anatomy and his-tology of the larynx.

These segments were fixed in buffered formalin 10%, em-bedded in paraffin wax and sectioned using amicrotome (Leica,Nussloch, Germany). The sections were then mounted ontoslides, deparaffinized with xylene, rehydrated, stained withhematoxylin-eosin, washed in water, dehydrated in a graded se-

FIGURE 1. Path (indicated by a head arrow) of the right (A) and left

(B) RLN. The lines of intersection (indicated by the dotted line) in the

nerves show the regions analyzed. TG, thyroid gland; T, trachea.

ries of ethanol, cleared with xylene, and covered with Entellan(Merck, Darmstadt, Germany) and coverslips. At least four sec-tions (7 mm) were obtained from each segment and analyzed.

According to previous protocols3,22 of planar morphometry,the fiber density (number of fibers per square millimeter),intraperineural area (square millimeter), intraperineuraldiameter (millimeter), total number of fibers/nerve, totalnumber of fibers/branch, fiber area (square micrometer), andfiber diameter (micrometer) were estimated using Image Pro-Plus Software [Image Pro-Plus 6.0; Media Cybernetics, SilverSpring, MD] (IPP 6.0) (Figure 2).

Histologic and morphometric measurements

of the TA

We analyzed five sections (7 mm) of the central portion ofthe TA obtained at 100 mm intervals. Based on previousprotocols20,23 of planar morphometry, the muscle fiberdensity (number of muscle fibers per square millimeter),muscle fiber area (square micrometer), muscle fiber diameter(micrometer), total number of muscle fibers, cross-sections ofthe muscular area (square millimeter), and muscular diameter(millimeter) were estimated using the IPP 6.0.

Unlike earlier studies,24,25 we decided to specify the regionin the TA muscle in which the fibers count was performed(Figure 3). This region was previously defined by two expertsin the anatomy and histology of the larynx. Every care wastaken to ensure that no tissue other than TAwas collected.

Estimation of the number and size of MUs in the TA

Based on other studies,26,27 it was assumed that 60% of all largemyelinated nerve fibers were alpha motor nerve fibers andcorresponded to the number of MUs. MU size was estimatedby dividing the total number of muscle fibers by the totalnumber of alpha motor nerve fibers in the RLN to the TAmuscle.

Data normalization

The analysis of the RLN and TAwas based on a pilot study per-formed by two blinded researchers (histology specialists). Pear-son’s correlation coefficients (r) were calculated to determinethe relationship between the results obtained by the tworesearchers.

RESULTS

The length of the left RLN from its origin to its entry point intothe larynx (cricothyroid joint) was 90.1 mm, whereas that of theright RLN from its origin to the cricothyroid joint was75.25 mm.

The qualitative description of the TA in this fetus was previ-ously described in our previous study.20 In relation to the RLN,the histologic sections of the nerves showed collagen fibers sur-rounding the nerves, adipose tissue, and blood vessels in the ad-jacencies (Figure 2A and B).

Furthermore, regarding the RLN, it is evident that the intra-perineural area and diameter of the right RLN (Figure 2A) islarger than that of the nerve on the left side (Figure 2B). Thisdifference is confirmed by the total number of fibers in the right

FIGURE 2. Digitized images of the sections showing the comparison of the intraperineural area (black delineation) of the RLN between the right

side (A) and left side (B) presenting evident differences. Images demonstrating the subtle differences between the calibers of the fibers (asterisk) of

the right (C) and left (D) nerves. CF, collagen fibrils; AT, adipose tissue; BV, blood vessel (hematoxylin-eosin stain).

Journal of Voice, Vol. 27, No. 6, 2013670

nerve and its branches which is greater than the left nerve(Table 1). However, this morphologic difference is compen-sated by the area and diameter of nerve fibers, which is smallerin the right RLN (Figure 2C) than in the left nerve (Figure 2D)(Table 1).

The values obtained by the correlation test were 0.8696 and0.9570 for the RLN and TA, respectively. These values demon-strate the high level of reliability of the observations made bythe blinded researchers.

In our study, the tissue shrinkage factor generated by the fix-ation and paraffin wax embedding procedures could not be cal-culated.3 Thus, all the quantified parameters have beenexpressed in micrometers or millimeters and percentages. Forthe RLN, TA, and MUs, the results are also expressed as a per-centage of the right side that was considered to be 100%.

The results obtained in the morphometric evaluation of theRLN, TA, and MUs are shown in Table 1 and will be discussedin the next section.

DISCUSSION

In relation to the environment of the RLN in the fetus, as inadults, considerable amounts of connective and adipose tissueswere found.3 It is proposed that in adults, this extra padding sur-rounding the RLN could help to prevent damage resulting from,for example, blows to the neck, compression and elongation re-lated to movements of the head, such as flexion, extension, androtational movements.3 Thus, it is probable that this ‘‘defensemechanism’’ is already formed by the 25th gestational week.

Although we have found no studies in the literature thatshowed some data that could be compared with our resultsfrom fetal tissues, there is evidence suggesting that the left

RLN generally contains relatively less epineurium at its originfrom the vagus nerve. According to Barkmeier and Luschei,18

as the left RLN ascended toward the larynx, the relative amountof epineurial tissue increased until the nerve began to branchnear the larynx. This pattern of nerve morphology indicatesthat the left RLN may not be as well protected from damageto deforming forces associated with compression near its origin.These authors also described that as the nerve travels along thetracheo-esophageal groove, it acquires increasing amounts ofprotective supportive tissue until entering the larynx.According to the descriptions performed by Sarr et al,28 in

the postnatal environment, expansion of adipose tissue occursmainly after birth through increases in adipocyte size and en-largement of adipose capillaries under the actions of enzymessuch as lipoprotein lipase, a regulator of adipocyte lipid filling.Adipocyte hyperplasia following birth appears limited; how-ever, studies do report its activation for the renewal of adipo-cytes suggesting that brown adipose tissue (BAT) and whiteadipose tissue (WAT) in humans still contain precursor cells ca-pable of differentiating into adipocytes at adulthood.It was also described by the same authors that in the human

fetus and newborn, BAT is located mainly in the cervical, axil-lary, perirenal, and periadrenal depots and plays an importantrole in nonshivering heat production during neonatal life andthus provides protection against lethal cold exposure (hypother-mia). In humans, WAT is distributed unevenly through the bodyand fat lobules appear first in the face, neck, breast, and abdom-inal wall at 14 weeks’ gestation. By 15 weeks, they are also ev-ident over the back and shoulders and further development ofwhite fat lobules in the upper and lower extremities and anteriorchest begins around this time. After the 23rd week of gestation,the total number of fat lobules remains approximately constant,

FIGURE 3. Digitized images of histologic cross-sections of the TA

muscle (black delineation) on the right side (A) and left side (B). Im-

ages demonstrating the differences between the fibers (asterisk) of the

right (C) and left (D) TA in terms of fiber area/diameter. VF, vocal fold;

V, ventricle; LC, lateral cricoarytenoid muscle (hematoxylin-eosin

stain).

Joel Henrique Ellwanger, et al Fetal RLN and MUs in the TA Muscle 671

whereas from the 23rd to 29th week, the growth of adipose tis-sue is determined mainly by an increase in size of the fatlobules.28,29

Regarding the differences between nerves, the results pre-sented in our study showed asymmetry between the right andleft nerves, both in terms of the histomorphometric levels andthe length of the nerves. Of particular importance, we foundthat the fibers in the left nerve have a larger area/diameterthan those in the right nerve. This morphometric parameter isvery important in assessing the electrical impulse conductionalong the nerve fibers30 because the conduction velocity ofthe electrical impulse is directly proportional to the area/diam-eter of the nerve fibers.31,32

Therefore, we infer that at least in the fetus used in our study,there is morphologic evidence to suggest that the nerve fibers inthe left present a higher speed when compared with the rightnerve. In this context, a classic study carried out in 1981 by Har-rison12 showed that, in humans, larger diameter myelin fiberspredominate in the left RLN which is not the case in the rightRLN. This may be related to the need for the nerve impulseto be conducted faster along the left RLN because the path islonger. These morphologic features may ensure that the nerveimpulses from both RLN arrive simultaneously at the vocal

fold. On the other hand, recently Jotz et al3 showed that thearea of the right RLN fibers in adults is higher when comparedwith RLN left.

Previous investigations into fiber density and diameter ofRLNs, in both humans and animals, have produced varying re-sults.21 These results may indicate differences between individ-uals within species or, more probably, a failure to section nervesat a specific position. It has been demonstrated that the numbersof both bundles and individual fibers depend on the level atwhich sectioning is carried out.21 However, this informationis lacking in many reports12,33,34 and that may explain thedifferent results found between the authors.

The difference in length between the right and left nervesarises during processing of the sixth pair of aortic arches.From the sixth week of gestation, both nerves that supply thesixth pairs of pharyngeal arches and curve around the sixthpair of aortic arches on their way to the site where the larynxwill develop. On the right, because of the degeneration of thedistal part of the sixth right aortic arch, the right RLN movesup and bends around the proximal part of the right subclavianartery, the derivative of the fourth aortic arch. On the left, theRLN bends around the arterial duct formed by the distal partof the sixth aortic arch. When this artery bypass involutes afterbirth, the nerve curves around the arterial ligament and aorticarch.29 In our study, the nerves were already asymmetric interms of the length and thickness of the fibers; however, inthe current literature, we found no study that showed asymme-try in relation to the histomorphometric parameters of the RLNin this embryonic period. Thus, we emphasize that although ourmorphologic investigation provides innovative and reliable re-sults about the fetal RLN, it is based on the bilateral analysisof only one subject. Therefore, to avoid an overestimation ofour results, we suggest that future studies with larger numbersof subjects will be needed to confirm our findings.

Additionally, our results could be used as a point of departurefor other studies into prenatal development to elucidate the pro-cesses of nerve conduction in the RLN during the period of or-ganizational differentiation. Moreover, this study is unique inthe literature in that it describes the morphometric measure-ments of the RLN in a human fetus using planar morphometry,which can also serve as a basis for improving anastomosis tech-niques35 involving portions of the RLN during the prenatal pe-riod because the success of such techniques also dependsheavily on the understanding of the microanatomy of thenerves.14,15

A recent study36 has demonstrated that larger muscle fibersrequire greater innervation, such as nerve fibers with largerareas/diameters. Thus, we assume that because the muscle fi-bers in the left TA are larger than those in the right TA, theywould undoubtedly need greater innervations, which wouldbe provided by larger caliber nerve fibers, as found in our study.

Regarding MUs, our findings demonstrate that the TA fibersfrom both the right and left sides already have a pattern indicat-ing a great capacity for fine control, especially the right TA thathas many relatively small-sized MUs. This pattern is similar tothat observed in adults and also in extraocular muscles.19 In-deed, intrinsic laryngeal muscles share many physiological

TABLE 1.

Comparison of All Morphometric Parameters Estimated in the Fetal RLN and TA Muscle

Morphometric Parameters RLN RLN Right NLR Left

Fiber density (fiber/mm2) 25 510 (100%) 25 241 (98.9%)

Intraperineural area (mm2) 0.483 (100%) 0.211 (43.6%)

Intraperineural diameter (mm) 0.784 (100%) 0.517 (65.9%)

Total number of fibers/nerve 8117 (100%) 5398 (66.5%)

Total number of fibers/branch 4666 (100%) 4000 (85.7%)

Fiber area (mm2) 5.41 (100%) 6.85 (126.6%)

Fiber diameter (mm) 2.61 (100%) 2.94 (112.6%)

Morphometric parameters TA TA Right TA Left

Fiber density (fiber/mm2) 46 365 (100%) 44 172 (95.2%)

Fiber area (mm2) 17.5 (100%) 24.5 (140%)

Fiber diameter (mm) 4.61 (100%) 5.56 (120.6%)

Total number of fibers 59 048 (100%) 65 723 (111.3%)

Cross-sections of the muscular area (mm2) 1.27 (100%) 1.48 (116.5%)

Muscular diameter (mm) 1.27 (100%) 1.38 (108.6%)

MU estimation in TA TA Right TA Left

MU number 2800 (100%) 2400 (85.7%)

MU size 21.0 (100%) 27.3 (130%)

Abbreviations: RLN, recurrent laryngeal nerve; TA, thyroarytenoid muscle.

Journal of Voice, Vol. 27, No. 6, 2013672

and anatomical properties with extraocular muscles such as in-nervation by cranial nerves.37,38

Because MU size is smaller in muscles that control finemovements than in those controlling gross movements, thepresent findings indicate a greater capacity of TA to regulatethe length of the vibrating part of the vocal folds, acting onthe control of pitch.39 These results should be interpreted care-fully because the muscles analyzed are still in a developmentalstage and may not match those found in newborns.

However, the results regarding MUs in fetal TA are relevantbecause there are few studies about the morphometric estima-tion of MUs in human intrinsic laryngeal muscles.19,24,25

Thus, we believe there is insufficient knowledge about theorganization of the MUs in the muscle studied here,especially during the fetal period. In this context, our resultsalthough obtained from a single individual provide innovativeinformation that will serve as a reference for future studiesinvolving a larger number of individuals. This limited samplesize is due to the fact that this material is extremely rare anddifficult to obtain; and so, at this moment, it would beimpossible to obtain more samples to extend our results.Therefore, we encourage other researchers who are able toaccess these tissues to perform a similar study with a largersample size.

CONCLUSIONS

Our findings demonstrated that there are morphologic asymme-tries between the left and right fetal RLN nerves. These mor-phologic findings are probably related to physiologicaldifferences between these two nerves that exist as from the fetalperiod. In addition, we observed that the TA of the fetus ana-

lyzed showed MUs typical of muscles with great motor accu-racy, especially the right TA. Thus, these results may be usedto increase our knowledge of the features of the voice in adultsand newborns and can be the starting point to understand thecomplete embryonic development of the RLN and TA.

REFERENCES1. Nicollas R, Giordano J, Perrier P, et al. Modelling sound production from an

aerodynamical model of the human newborn larynx. Biomed Signal Pro-

cess Control. 2006;1:102–106.

2. Nita LM, Battlehner CN, FerreiraMA, Imamura R, Sennes LU, Caldini EG,

Tsuji DH. The presence of a vocal ligament in fetuses: a histochemical and

ultrastructural study. J Anat. 2009;215:692–697.

3. Jotz JP, de Campos D, Rodrigues FR, Xavier LL. Histological asymmetry

of the human recurrent laryngeal nerve. J Voice. 2011;25:8–14.

4. Bowden REM. The surgical anatomy of the recurrent laryngeal nerve. Br J

Surg. 1955;43:153–163.

5. Shin T, Rabuzzi DD. Conduction studies of the canine recurrent laryngeal

nerve. Laryngoscope. 1971;81:586–596.

6. Nakai T, Goto N, Moriyama H, Shiraishi N, Nonakan N. The human recur-

rent laryngeal nerve during the aging process. Okajimas Folia Anat Jpn.

2000;76:363–367.

7. Standring S. Gray’s Anatomy—The Anatomical Basis of Clinical Practice.

40th ed. New York, NY: Churchill Livingstone; 2008.

8. Moore KL, Dalley AF, Agur AMR. Clinically Oriented Anatomy. 6th ed.

Philadelphia, PA: Lippincott Williams & Wilkins; 2009.

9. Sanders I, Han Y,Wang J, Biller H. Muscle spindles are concentrated in the

superior vocalis subcompartment of the human thyroarytenoid muscle.

J Voice. 1998;12:7–16.

10. Khan A, Pearlman RC, Bianchi DA, Hauck KW. Experience with two types

of electromyography monitoring electrodes during thyroid surgery. Am J

Otolaryngol. 1997;18:99–102.

11. Endo K, Okabe Y, Maruyama Y, Tsukatani T, Furukawa M. Bilateral vocal

cord paralysis caused by laryngeal mask airway. Am J Otolaryngol. 2007;

28:126–129.

12. Harrison DF. Fibre size frequency in the recurrent laryngeal nerves of man

and giraffe. Acta Otolaryngol. 1981;91:383–389.

Joel Henrique Ellwanger, et al Fetal RLN and MUs in the TA Muscle 673

13. Crumley RL. Unilateral recurrent laryngeal nerve paralysis. J Voice. 1994;

8:79–83.

14. Paniello RC. Laryngeal reinnervation. Otolaryngol Clin North Am. 2004;

37:161–181.

15. Aynehchi BB, McCoul ED, Sundaram K. Systematic review of laryngeal

reinnervation techniques. Otolaryngol Head Neck Surg. 2010;143:

749–759.

16. Asaoka K, Sawamura Y, NagashimaM, Fukushima T. Surgical anatomy for

direct hypoglossal-facial nerve side-to-end ‘‘anastomosis’’. J Neurosurg.

1999;91:268–275.

17. Vacher C, DaugeMC. Morphometric study of the cervical course of the hy-

poglossal nerve and its application to hypoglossal facial anastomosis. Surg

Radiol Anat. 2004;26:86–90.

18. Barkmeier JM, Luschei ES. Quantitative analysis of the anatomy of the

epineurium of the canine recurrent laryngeal nerve. J Anat. 2000;196:

85–101.

19. Santo Neto H, Marques MJ. Estimation of the number and size of motor

units in intrinsic laryngeal muscles using morphometric methods. Clin

Anat. 2008;21:301–306.

20. De Campos D, Ellwanger JH, da Costa Rosa JP, et al. Morphology of fetal

vocal fold and associated structures. J Voice. 2013;27:5–10.

21. Carlsoo B, Izdebski K, Dahlqvist A, Domeij S, Dedo HH. The recurrent la-

ryngeal nerve in spastic dysphonia. A light and electron microscopic study.

Acta Otolaryngol. 1987;103:96–104.

22. De Campos D, DoNascimento PS, Ellwanger JH, GehlenG, RodriguesMF,

Jotz GP, Xavier LL. Histological organization is similar in human vocal

muscle and tongue—a study of muscles and nerves. J Voice. 2012;26:

811.e19–26.

23. Ilha J, Araujo RT, Malysz T, Hermel EE, Rigon P, Xavier LL, Achaval M.

Endurance and resistance exercise training programs elicit specific effects

on sciatic nerve regeneration after experimental traumatic lesion in rats.

Neurorehabil Neural Repair. 2008;22:355–366.

24. Ruedi L. Some observations on the histology and function of the larynx.

J Laryngol Otol. 1959;73:1–20.

25. English DT, Blevins CE. Motor units of laryngeal muscles. Arch Otora-

lyngol. 1969;89:778–784.

26. Feinstein B, Lindegard B, Nyman E, Wohlfart G. Morphologic studies of

motor units in normal human muscles. Acta Anat (Basel). 1955;23:

127–142.

27. Santo Neto H, Meciano Filho J, Passini R Jr, MarquesMJ. Number and size

of motor units in thenar muscles. Clin Anat. 2004;17:308–311.

28. Sarr O, Yang K, Regnault TR. In utero programming of later adiposity: the

role of fetal growth restriction. J Pregnancy. 2012;2012:134758.

29. Moore KL, Persaud TVN. The Developing Human: Clinically Oriented

Embryology. 6th ed. Philadelphia, PA: W.B. Saunders Company; 2003.

30. Prades JM, Dubois MD, Dumollard JM, Tordella L, Rigail J,

Timoshenko AP, Peoc’h M. Morphological and functional asymmetry of

the human recurrent laryngeal nerve. Surg Radiol Anat. 2012;34:903–908.

31. Gasser HS, Grundfest H. Axon diameters in relation to the spike dimen-

sions and the conduction velocity in mammalian a fibers. Am J Physiol.

1939;127:393–414.

32. Schlaepfer WW, Myers FK. Relationship of myelin internode elongation

and growth in the rat sural nerve. J Comp Neurol. 1973;147:255–266.

33. Miyauchi Y, Moriyama H, Goto N, Goto J, Ezure H. Morphometric nerve

fiber analysis of the human inferior alveolar nerve: lateral asymmetry.Oka-

jimas Folia Anat Jpn. 2002;79:11–14.

34. Silva AP, Jord~ao CE, Fazan VP. Peripheral nerve morphometry: compari-

son betweenmanual and semi-automated methods in the analysis of a small

nerve. J Neurosci Methods. 2007;15:153–157.

35. Paniello RC. Laryngeal reinnervation with the hypoglossal nerve: II. Clin-

ical evaluation and early patient experience. Laryngoscope. 2000;110:

739–748.

36. De Campos D, Ellwanger JH, Do Nascimento PS, Da Rosa HT, Saur L,

Jotz GP, Xavier LL. Sexual dimorphism in the human vocal fold innerva-

tion. J Voice. 2013;27:267–272.

37. Hoh JF. Laryngeal muscle fibre types. Acta Physiol Scand. 2005;183:

133–149.

38. McLoon LK, Rowe J, Wirtschafter J, McCormick KM. Continuous myo-

fiber remodeling in uninjured extraocular myofibers: myonuclear turnover

and evidence for apoptosis. Muscle Nerve. 2004;29:707–715.

39. Ludlow CL. Central nervous system control of the laryngeal muscles in hu-

mans. Respir Physiol Neurobiol. 2005;147:205–222.