Ann Otol Rhinal Laryngol 112:2003

HISTOLOGY OFNERVES AND MUSCLES INADDUCTOR SPASMODICDYSPHONIA

DINESH K. CHHETRI, MD

LosANGELES, CALIFORNIA

HARRY V. VINTERS, MD

LosANGELES, CALIFORNIA

JOEL H. BLUMIN, MD

PHILADELPHIA, PENNSYLVANIA

GERALD S. BERKE, MD

LosANGELES, CALIFORNIA

To elucidate the etiology and pathophysiology ofspasmodic dysphonia, weexamined the adductor branch ofthe recurrent laryn­geal nerve and the lateral cricoarytenoid muscle from 9 consecutive patients with this disorder who were previously treated withbotulinum toxin. Histologic examination revealed average muscle fiber diameters ranging from 21 to5711m. Botulinum toxin treat­ment-related muscle atrophy was observed up to 5 months after injection. Endomysial fibrosis was present in all samples. His­tochemical analysis in8patients revealed type 2 fiber predominance in7 patients and fiber type grouping in2.Type-specific musclefiber size changes were notpresent. Nerve samples were examined in plastic sections. In 8 patients the nerves contained homoge­neous, large-diameter myelinated nerve fibers and sparse small fibers. One patient had a relatively increased proportion of smallmyelinated nerve fibers. Overall, the nerve fiber diameter was slightly larger inpatients than incontrols. These findings may impli­cate thecentral nervous system inthepathophysiology of adductor spasmodic dysphonia.

KEY WORDS - histology, muscle, nerve, pathophysiology, spasmodic dysphonia.

INTRODUCTION

The etiology and pathophysiology of spasmodicdysphonia (SD) remain elusive even after more thana century of discussion in the literature. Spasmodicdysphonia is classified into adductor and abductortypes based on the laryngeal muscle groups affected.Adductor spasmodic dysphonia (ASD) is an adult­onset voice disorder characterized by a "strain-stran­gle" voice quality and abrupt vocal stops associatedwith abnormal closure of the vocal cords duringspeech. There is a slight female predilection, and thedisorder has a persistent course. 1 Electromyographic(EMG) studies of laryngeal muscles of patients withASD reveal pitch and phonatory breaks coincidentwith muscle spasms during vowels in connectedspeech. I On the basis of these EMG findings, ASDis currently classified as a focal dystonia affectingthe larynx during speech.I-'

Many etiologic theories of SD have been proposed.A long-standing psychogenic theory introduced byTraube in 1871 was replaced by a neurogenic theorywhen Aronson et all reported a high incidence of as­sociated neurologic signs (mainly voice tremor) inpatients with SD. Subsequent discussions have fo­cused on whether the disorder lies in the central orthe peripheral nervous system. Finitzo-Hieber et al4

suggested a central causation based on abnormal waveV latencies from auditory brain stem reflex testing.

Bielamowicz and Ludlow> reported improved EMGsignals in contralateral thyroarytenoid (TA) musclesafter unilateral botulinum toxin (BTX) injection, andthey hypothesized that a central reduction in moto­neuron activity secondary to reduced sensorimotorfeedback could explain their findings. Ded0 6 com­mented that proprioceptive abnormalities may existin patients and that these may be relieved after uni­lateral section ofthe recurrent laryngeal nerve (RLN).

The most common treatment for ASD is injectionof BTX type A (Allergan Inc, Irvine, California) intothe TA muscle'? It causes flaccid paralysis by inhib­iting the release of acetylcholine from nerve termi­nals." A variety of surgical therapies for ASD havebeen proposed. They include unilateral RLN section.fmidline lateralization thyroplasty.? and selective bi­lateral laryngeal adductor denervation and reinnerva­tion.!" Laryngeal denervation and reinnervation is theprocedure of choice at our institution for a more per­manent treatment of ASD. Lateral cricoarytenoid(LCA) muscle myotomy is also now concurrentlyperformed. In this report we describe the histomor­phology of the adductor branch of the RLN and theLCA muscle from patients who received this surgi­cal therapy for ASD.

MATERIALS AND METHODS

Patients and Controls. This research protocol was

From the Department ofSurgery, Division ofHead and Neck Surgery (Chhetri, Blumin, Berke), and the Department ofPathology and Labora­tory Medicine, Section ofNeuropathology (Vinters), University ofCalifornia, Los Angeles, California.Presented at the meeting ofthe American Laryngological Association. Boca Raton. Florida, May 10-11, 2002.

CORRESPONDENCE - Gerald S. Berke. MD. 62-132 CHS, UCLA Medical Center. Los Angeles, CA 90095.

334

Chhetri et al, Histology ofNerves & Muscles in Spasmodic Dysphonia 335

reviewed and approved by the Medical InstitutionalReview Board of the University of California, LosAngeles. Nine consecutive patients who received se­lective laryngeal adductor denervation, reinnervation,and LCA myotomy for ASD were enrolled in thestudy. All had received prior BTX therapy. The rea­son for surgery in all was either dissatisfaction withBTX therapy or loss of BTX effectiveness, and a de­sire for permanent surgical cure. Laryngoscopic ex­amination, acoustic analysis of voice, voice historytypical for ASD, and response to previous BTX treat­ments were used to confirm the diagnosis of ASD.

Five nerve specimens were analyzed as controls.The harvesting approach and location of controlnerves were identical to those of the patient nerves.Two were from autopsies of adult patients who diedof cardiovascular disease. Other than short-term in­tubation during intensive care, neither had a historyoflaryngeal abnormalities. Two specimens were fromlaryngectomies. The first was from a 47-year-old manwith a large supraglottic squamous cell carcinomawho had received no prior therapy. The right vocalcord had impaired mobility, and therefore only theleft nerve branch was analyzed. The second was froma 73-year-old man with a history of radiotherapy forT3NOsupraglottic carcinoma who required laryngec­tomy for epiglottic recurrence. The vocal cords weremobile bilaterally. The fifth control specimen wasfrom a 48-year-old woman with acute recurrent la­ryngospasm (laryngeal adductory dystonia) who wastreated with laryngeal denervation, reinnervation, andLCA myotomy.

Nerve and Muscle Biopsy. Berke et apa have re­ported the operative procedure for selective laryn­geal adductor denervation and reinnervation. The in­tralaryngeal course of the adductor branch of the RLNhas been reported by others. I I,12 A cartilage windowwas created in the posterior inferior portion of thethyroid lamina. The anterior adductor branch of theRLN was located, and a nerve stimulator was usedto confirm the identification of the nerve. The nervebranch was followed to the TA muscle and cut 5 mmfrom its muscular insertion. A l-mm section was cutfrom the distal nerve stump and immediately im­mersed in 2% glutaraldehyde solution. The LCAmuscle was then identified and cut in its midsectionwith fine scissors. An approximately 5-mm-long sam­ple of muscle was removed from the myotomy siteand immediately transported to the laboratory forquick freezing. The muscle samples were always fro­zen within 20 minutes of biopsy and kept in deepfreeze until sectioning.

Muscle Processing. The muscle samples were cutat ltl-um thickness or less in a cryostat, picked up

on a coverslip, and histologically and histochemicallyprocessed by routine procedures used almost daily inthe University of California muscle diagnostic histo­chemistry laboratory. The slides were observed andphotographed with a microscope equipped with a digi­tal camera system (Olympus BX40). Digital imageswere taken at 200x to 400x and printed on a laserprinter. Muscle diameter was measured directly onthe printed images by the "lesser fiber diameter" tech­nique described by Dubowitz and Brooke.l-' In thistechnique the maximum diameter across the lesseraspect of the muscle is measured, in order to over­come the distortion that occurs when muscle fibersare cut obliquely. At least 200 muscle fibers werecounted per muscle biopsy sample. Most of the his­tologic information was obtained from sections thathad been stained with the modified Gomori trichrome,because this stain demonstrates morphological fea­tures of the muscle fibers well. The adenosine triphos­phatase histochemical reaction was used to assessfiber type. In this histochemical reaction, type 2 (fast)muscle fibers stain dark and type 1 (slow) musclefibers stain light when samples are preincubated atpH 9.6. 13 This staining pattern is reversed with prein­cubation at pH 4.2 to 4.6. In some samples the wholespecimen did not undergo acid reversal; in thesecases, areas that reversed well were selected for anal­ysis. More than 75% prevalence of one fiber typewas considered type predominance.

Nerve Processing. The nerve biopsy specimenswere immediately immersion-fixed in 2% glutaral­dehyde for at least 2 hours and post-fixed for 1 hourin 1% osmium tetraoxide. The tissue was further pro­cessed for electron microscopy by standard techniquesand embedded in Epon. Sections I to 211mthick werecut and stained with toluidine blue for light micros­copy. For electron microscopy, 50-nm ultrathin sec­tions were cut, stained with uranyl acetate followedby lead citrate, and examined and photographed at1,900x with a transmissionelectronmicroscope (JEOLJEM-lOOCXII electron microscope). At least 3 pho­tographs were taken per nerve sample. Each photo­graph contained about 10 to 15 nerve fibers cut trans­versely.

Nerve fiber diameter was measured directly on thephotographs. The maximum diameter across the less­er aspect of the fiber was measured by a techniquesimilar to that described above for measurement ofmuscle diameter. Nerve density analysis was per­formed from an area 150 x 150 11m2 at 400x magni­fication. The total number of nerve fibers within thisarea was manually counted, and the number of small­diameter «6 11m) fibers was divided by the total num­ber to generate a percentage of small fibers.

336 Chhetri et al, Histology ofNerves & Muscles in Spasmodic Dysphonia

Statistical Analysis. A 2-tailed Student's r-test forindependent samples was used to compare means be­tween patient and control samples.

TABLE 1. DEMOGRAPHIC DATA

Patient AgeNo. (y)

I 642 48

3 664 39

5 40

6 53

7 67

8 339 59

Duration ofSpasmodic

Sex Dysphonia (y)

M 4

F 6M 20

F 17

F 17

F 14

M 18

F 20

F 4

Duration ofBotulinum

ToxinTherapy (y)

32

6

2

4

10

10

7

5

Fig 1. Lateral cricoarytenoid muscle from patient withspasmodic dysphonia shows loosely arranged muscle fi­bers and moderate endomysial fibrosis (original x200).

RESULTS position between individual muscle fibers, was pres­ent in 5 patients (Fig 1). One patient had minimal

Nine consecutive patients were enrolled into this endomysial fibrosis. The average muscle fiber diam-study (Table 1).There were 6 women and 3 men. The eter was between 21 and 57 11m. No statistical dif-average age was 45 years (range, 33 to 59 years) in ference in muscle fiber diameter was found betweenwomen and 66 years (range, 64 to 67 years) in men. sides (p =.8), because fiber diameters were relatedThe average age at onset of SO was 32 years in wom- to recent treatment with BTX. The average muscleen (range, 13 to 55 years) and 52 years (range, 46 to fiber diameter in the BTX-treated side in the 2 pa-60 years) in men. A tendency for younger age attreat- tients (Nos. 3 and 6) who received unilateral BTXment was found for women (p =.09). The age at on- treatment was half that of the untreated side (Tableset of SO was significantly lower in women (p =.01). 2). Of note, 1 patient (No.6) had her last BTX treat-The duration of SO and the length of BTX therapy ment 5 months before surgical therapy. When mus-were comparable in both sexes. All but 2 patients had cle samples were grouped by exposure to BTX lessreceived bilateral BTX therapy (Table 2). Of the 2 than or more than 5 months before surgical therapy,patients receiving unilateral BTX injections, 1 (No. a tendency toward significantly reduced muscle fi-3) received alternating unilateral injections and an- ber diameter in the more recently treated group wasother (No.6) always received BTX injections on the found (p =.08).

right side of the larynx. Muscle histochemistry was analyzed from bothHistologic analysis was performed in muscle sam- LCA muscles in 5 patients and a single LCA muscle

pies from 6 patients (Table 2). Results were obtained in 3 patients (Table 3). At pH 9.6 it was difficult tofrom both LCA muscles in all but 1patient. The mus- differentiate between type 1 and type 2 fibers. How-cle's morphological pattern in all patients consisted ever, acid reversal was almost complete at pH 4.6 inof a loosely arranged fiber pattern with transverse the great majority of samples and was complete atand slightly longitudinal fibers present in the same pH 4.2 in all samples, and type 1 and type 2 fibershistologic sections (Fig 1). Moderate endomysial fi- could be easily differentiated. Type 2 fiber predom-brosis, characterized by excess connective tissue de- inance was present in 7 patients (Fig 2). Of the 13

TABLE 2. HISTOLOGY OF LATERAL CRICOARYTENOID MUSCLE

Last Botulinum Toxin Injection Fiber Diameter (pm)*

Patient No. Sex Months Ago Side Left Right Fibrosis

I M II Bilateral 42 ± 12.5 44± 13.1 Moderate

3 M 2 Left 21 ±9.5 40 ± 14.2 Moderate

4 F 4 Bilateral 23 ± 6.7 34 ± 16.5 Moderate

5 F 6 Bilateral 26 ± 8.9 21 ± 6.6 Moderate

6 F 5 Right 57 ± 20.2 26 ± 8.9 Moderate

7 M 6 Bilateral 40 ± 18.3 NA MinimalNA - not available.

*Mean + standard deviation.

Chhetri et al, Histology ofNerves & Muscles in Spasmodic Dysphonia

TABLE 3. HISTOCHEMISTRY OF LATERAL CRICOARYTENOID MUSCLE

337

Last Botulinum Toxin Injection % Type 2 Fibers Fiber Type Type 2 FiberPatient No. Months Ago Side Left Right Grouping Predominance

1 11 Bilateral 80 92 No Yes

3 2 Left NA 78 Right Yes

4 4 Bilateral NA 84 No Yes

5 6 Bilateral 66 57 No No

6 5 Right 87 49 Right Left

7 6 Bilateral 82 NA No Yes

8 2 Bilateral 95 95 No Yes9 7 Bilateral >95 >95 No Yes

separate LCA samples examined, 10 had type 2 fi­ber predominance (Table 3). Fiber type grouping waspresent in 2 patients. Of these 2 patients, 1 (No.3)was on an alternating unilateral BTX treatment regi­men and had overall type 2 fiber predominance (78%)mixed with some discrete areas of type 1 fiber group­ing. The other patient (No.6) always received right­side BTX treatments and had fiber type grouping inthe BTX-treated right LCA (Fig 3) and type 2 fiberpredominance in the never-treated contralateral mus­cle. It was not possible to differentiate different sub­types of type 2 fibers. Type-specific alterations in mus­cle diameter or other histologic and histochemicalabnormalities were not observed in any patient mus­cle samples.

The RLN adductor branch morphological charac­teristics were analyzed in 9 patients and 5 controls(Table 4). Nerve samples for study were availablefrom both sides in only 3 patients. Nerve density wasmeasured in 3 patients (Nos. 7, 8, and 9) and 3 con­trols (Table 5). The nerve diameter ranged from 220to 500 urn in patients and from 180 to 450 urn in con­trols. In the first 8 patients, the nerves were com­posed almost entirely of uniformly distributed mye­linated large nerve fibers (Fig 4). Small-diameter fi­bers «6 urn) were sparsely interspersed and repre-

sented 8% of the total fibers. Higher magnificationunder light and electron microscopy confirmed thisfinding (Fig 5). Unmyelinated nerve fibers were onlyoccasionally seen by electron microscopy and wereevenly distributed. Although the mean fiber sizes weredifferent between patients, they were similar betweensides in the same patient (Table 4).

Nerve samples from 1 patient (No.9) were nota­bly different (Table 5). The left branch had a nervedensity similar to that of other patient nerves, but theproportion of small fibers was increased to 31%. Theright branch had slightly increased nerve density(+18%) and an increased proportion of small fibers(to 19%). The density of nerve fibers and proportionof small fibers were similar in controls. When nervefibers from all patients and all controls were pooledtogether, the average fiber diameter was 8.3!lm (stan­dard deviation, 2.8 urn) in patients with SD and 7.1urn (standard deviation, 2.2 urn) in controls (p <.0001). The relative distribution of nerve fiber diam­eter in patients and controls is illustrated in Fig 6.

DISCUSSION

Both the TA and LCA muscles act synchronouslyto adduct the vocal folds and close the larynx. In acomprehensive fine-wire EMG study of laryngeal

Fig 2. Lateral cricoarytenoid muscle from patient 7 stained with adenosine triphosphatase at A) pH 9.6 and B) pH 4.2 (originalx100). At pH 4.2, type 1 (dark) and type 2 (light) fibers are clearly delineated.

338 Chhetri et ai, Histology ofNerves & Muscles in Spasmodic Dysphonia

TABLE 5. FIBER DENSITY AND DISTRIBUTIONIN ADDUCTOR BRANCH OF RECURRENT

LARYNGEAL NERVE

% Small FibersTotal Fibers*SideSubject

Patient 7Patient 8Patient 9

L 110 8L 89 8L 111 31R 131 19

C~~crl 1M 8Laryngectomy 2 104 4Laryngospasm 109 9

*Total fibers were counted from area 150 x 150 11m at400x.

closely correlates with the contraction speed of a~uscle.15 This histochemical marker clearly differen­nates the slow type I fibers from the fast type 2 fi­bers (Figs 2 and 3).13,15 The functional requirementof human skeletal muscle is clearly reflected in thedistribution of muscle fiber types, and laryngeal mus­cles follow this pattern as well. Teig et aI16 reportedtha~ the TA muscle has the highest percentage of type2 fibers (mean ± standard deviation, 65% ± 11.6%)and that the posterior cricoarytenoid (PCA) musclehas the highest percentage of type I fibers (67% ±8.6%). The LCA muscle was intermediate, with about60% ±5.2% type 2 muscle fibers. Other studies havecorroborated these findings and report the range oftype 2 fibers in the LCA muscle as between 50%and 64%.17,18 The muscles with the highest propor­tion of type 2 fibers (TA, LCA, interarytenoid) allcontribute to the sphincter function of the larynx. TheP~A muscle, which is tonically active during the in­spiratory phase of respiration, has the least amountof type 2 fibers.

A significant proportion of our patients with ASOhad type 2 fiber predominance in their LCA muscles.

Fi~ 3. L~teral cricoarytenoid muscle from patient 6stained With adenosine triphosphatase at pH 4.2 (origi­nal x200). Fiber type grouping is present.

muscle activity, Hillel l4 found that the LCA muscletends to act similarly to the TA muscle in both nor­mal subjects and patients with SO. No consistentEMG pattern differentiates between LCA and TAfunction. When BTX therapy is employed for SO,the typical treatment target is the TA muscle, but thetoxin easily diffuses into and paralyzes the ipsilater­al LCA muscle. Clear evidence for this phenomenonwas seen in the 2 patients who had unilateral BTXtherapy (Nos. 3 and 6). In these patients the ipsilat­eral LCA muscles were atrophied. Their TA muscles~ere injected 2 and 5 months before surgery, respec­tively, The LCA muscle is thus intimately involvedin the pathophysiology and treatment of SO.

Actomyosin adenosine triphosphatase activity

TABLE 4. MORPHOLOGY OF ADDUCTOR BRANCH OFRECURRENT LARYNGEAL NERVE

Cadaver 1Cadaver 2Laryngectomy 1Laryngectomy 2Laryngospasm

*Mean ± standard deviation.tp < .0001.

Subject Side

Patient I LR

Patient 2 LPatient 3 LPatient 4 LPatient 5 RPatient 6 LPatient 7 L

RPatient 8 LPatient 9 L

R

Nerve Size(pm)

450450290220300350350350500450420420425180375450400

Fiber Size (um)"

11.1 ± 3.210.2 ± 3.28.3 ± 1.67.8 ± 2.17.8 ± 2.5

11.4 ± 3.29.7 ± 1.47.4 ± 2.17.4 ± 2.78.6 ± 1.5

5.7t ± 2.38.2t ± 2.4

7.5 ± 1.66.7 ± 1.55.4 ± 2.77.8 ±2.27.7 ± 2.5

Fig 4. Light micrograph of adductor branch of left recur­rent laryngeal nerve in patient 7 shows homogeneouslydistributed large-diameter myelinated fibers and fewsmall-diameter myelinated fibers (original x200).

Chhetri et al, Histology ofNerves & Muscles in Spasmodic Dysphonia 339

Fig 5. A) Light micrograph (original x400) and B) electron micrograph (original xl ,900) of adductor branch of left recurrentlaryngeal nerve from patient 8 show that nerve is composed of homogeneous distribution of mostly large-diameter fibers.

This finding is not explained by selective type I mus­cle fiber loss, because evidence of muscle necrosisor type-specific atrophy was not present. Selectivedegeneration of motoneurons innervating type I mus­cle fibers could also lead to type 2 muscle fiber pre­dominance, but there is also no evidence of nerve fi­ber or density abnormalities. A potentially confound­ing variable is prior BTX treatment. For instance, ourfindings could be explained if BTX treatment inducedhistochemical conversion of muscle toward type 2fibers. However, current data do not support this con­tention. Several studies on the effects of BTX on theorbicularis oculi muscle have revealed only dener­vation atrophy and no fiber type-specific alter­ations. 19-21 In addition, Duchen22-24 reported in sev­eral well-designed studies using a mouse model thattype 1 muscle fibers recover faster histologically andhistochemically than type 2 muscle fibers after in­tramuscular injection of a sublethal dose of BTX. Intype 1 (soleus) muscle, nerve recovery began within

25

a week and continued for another 3 to 4 weeks. By 6weeks, normal histochemical and morphologicalcharacteristics were almost completely restored. Intype 2 (gastrocnemius) muscle, nerve recovery be­gan after 3 to 4 weeks and continued for 6 to 8 weeks.Some histochemical and morphological abnormali­ties were still present at 3 months and sometimeseven at 6 months after injection. These results sup­port the notion that if muscle fiber predominancewere to be present after intramuscular BTX injec­tion, it would be a predominance of type 1 fibers­a result opposite to our actual findings. A study oflong-term changes in myosin heavy chain isoformpatterns in extraocular muscles of rats after BTX in­jection also showed that the profile was shifted to­ward slower isoforms even at 8 months after injec­tion.25 It should also be noted that in 1 patient (No.6), type 2 muscle fiber predominance was present inthe left LeA muscle, which was never treated withBTX. However, a histomorphological study of mus-

20

15

IFig 6. Fiber analysis of adductor nerves in pa- :.tients with spasmodic dysphonia (black bars) 10

and controls (gray bars).

1111 I. .8 9 10 11 12 13 14 15 16 17 18 19 20

F_ DIllmele< (mlcrome.....)

340 Chhetri et al, Histology ofNerves & Muscles in Spasmodic Dysphonia

cle samples from patients never treated with BTXwould be required to remove any possible confound­ing influence of previous treatment. Because BTXremains the mainstay of initial treatment for ASD,we must await enrollment of previously untreatedASD patients for selective adductor denervation-rein­nervation surgery.

It has been shown previously that muscle fibertypes reflect functional differences in the innervat­ing motoneurons. Cross-reinnervation of fast andslow muscles reverses their enzyme profiles and char­acteristics of contraction.26,27 It is possible that achange in the properties of laryngeal motoneuronsfrom tonic (slower discharge frequency) to phasic(faster discharge frequency) leads to histochemicalchanges in the muscle. Telerman-Toppet et aF8 havefound type 2 fiber predominance in adult patientswith muscle cramps and exertional myalgia. Theypropose that abnormal stimulation of muscle fiberscould be responsible for the conversion from type 1to type 2 through a modification in the pattern ofmuscle fiber activation. The mechanisms leading tochanges in motoneuron properties in patients withSD are unknown, but may involve central nervoussystem interneurons. The hallmark of SD is suddenand quick movements of laryngeal muscles, and type2 muscle fiber predominance is consistent with thisclinical manifestation.

Our other histologic findings are in agreement withpublished reports. 16-18 Unlike limb skeletal muscles,laryngeal muscles tend to be arranged loosely, withlongitudinal extensions of fibers interspersed withfibers cut transversely. Individual muscle fibers rarelytouch each other because of an increase in endomysialconnective tissue (Fig 1). In limb muscles, endomy­sial fibrosis is more commonly seen in myopathiessuch as Duchenne dystrophy than in neuropathies,although it has also been reported to be present inneurogenic atrophies. 13 It seems that some degree ofendomysial fibrosis may be normal in laryngeal mus­cles. Because we used previously published controldata to compare with our study data, a graded com­parison of endomysial fibrosis is not possible.

Muscle fiber type grouping is a result of neuromus­cular degeneration in which denervated muscle fi­bers are reinnervated by nerve sprouting from adja­cent intact motor units.I? Type grouping has also beenobserved in the PCA muscle, and it has been theo­rized that terminal nerve branches in the PCA musclemight be prone to damage when large lumps of foodare swallowed.v-" Our 2 cases of type grouping like­ly arose from BTX-related denervation followed byreinnervation.

Prior studies of nerve histology from patients with

SD have examined the RLN and have found no ap­parent morphometric differences between nerves frompatients and controls. Initial studies found distinctareas within the nerve that consisted of sheets of un­myelinated axons,30 but further studies concluded thatRLNs from both patients and controls contain small«6/-lm), medium (6 to l l um), and large (11 to 18urn) myelinated fibers, as well as unmyelinated fi­bers.U As the RLN courses toward the larynx, mye­linated and unmyelinated fibers, most likely contain­ing sensory or autonomic fibers to tracheal and esoph­ageal mucosa, exit the nerve.U A section of the RLNat a more proximal location contains an increasednumber of small myelinated fibers. Thus, the uni­form, large-diameter nerve fibers found in the ad­ductor branch of the RLN in both patients and con­trols are consistent with this paradigm. At this loca­tion the majority of nerve fibers are expected to be­long to large u-motoneurons. In contrast to otherRLN studies, in the adductor branch in both patientsand controls we found only occasional unmyelinatedfibers randomly placed throughout the nerve, and noareas resembling bundles of unmyelinated fibers asdescribed by Dedo et apo or areas occupied mainlyby unmyelinated fibers as described by Carlsoo et al.3!

Only I report has previously examined nerve fi­bers from the adductor branch of the RLN. Kosakiet aP2 examined adductor nerve samples from 2 pa­tients with SD by electron microscopy and found anincreased ratio of thin fibers (5 to 10 urn) as com­pared to normal controls. However, no quantitativedata were presented. We doubt that the pathophysi­ology of SD lies entirely in changes in the numberof small myelinated fibers, because 8 of 9 patientnerve samples displayed uniform-diameter nerve fi­bers with sparse small-diameter fibers. However,their finding may represent the clinical heterogene­ity of this disorder, as I of our patients (No.9) alsodemonstrated fiber density abnormalities and an in­creased proportion of small-diameter nerve fibers.Finally, Kosaki et al reported an average of 55.7 to­tal nerve fibers in the adductor nerve branch. Our re­sults differ here as well. All nerve fibers were countedin nerve samples from 2 patients (Nos. 7 and 8), andtotals of 400 and 570 fibers were counted, respec­tively.

CONCLUSIONS

We report histomorphological findings in the LCAmuscle and the adductor branch of the RLN in a smallseries of patients with ASD. The type 2 muscle pre­dominance seen in the majority of patients could beexplained by changes in neuromuscular activationpatterns. The mechanisms leading to these changesin activation patterns may lie in the central nervous

Chhetri et al, Histology ofNerves & Muscles in Spasmodic Dysphonia 341

system. However, BTX-related effects cannot be ex­cluded entirely at this time, and a follow-up studyon patients never treated with BTX is needed. Themorphological characteristics of the nerve fibers were

similar in patients with SD and controls except for 1patient, who had a relatively increased ratio of smallfibers. The findings in this patient may represent theclinical heterogeneity of this disorder.

------::\CKNOWLEDGMENTS - The authors acknowledge Birgitta Sjostrand for help with electron microscopic techniques and Ivan Lopez forguidance in tissue processing and digital photography.

REFERENCES

1. Aronson AE, Brown JR, Litin EM, Pearson JS. Spasticdysphonia. II. Comparison with essential (voice) tremor andother neurologic and psychogenic dysphonias. J Speech HearDisord 1968;33:219-31.

2. Blitzer A, Lovelace RE, Brin MF, Fahn S, Fink ME. E1ec­tromyographic findings in focal laryngeal dystonia (spastic dys­phonia). Ann Otol Rhinol LaryngoI1985;94:591-4.

3. Blitzer A, Brin MF, Fahn S, Lovelace RE. Clinical andlaboratory characteristics of focal laryngeal dystonia: study of110 cases. Laryngoscope 1988;98:636-40.

4. Finitzo-Hieber T, Freeman FJ, Gerling 11, Dobson L,Schaefer SD. Auditory brainstem response abnormalities in ad­ductor spasmodic dysphonia. Am J Otolaryngol 1982;3:26-30.

5. Bielamowicz S, Ludlow CL. Effects of botulinum toxinon pathophysiology in spasmodic dysphonia. Ann Otol RhinolLaryngoI2000;109:194-203.

6. Dedo HH. Recurrent laryngeal nerve section for spasticdysphonia. Ann Otol Rhinol LaryngoI1976;85:451-9.

7. Ludlow CL. Treatment of speech and voice disorders withbotulinum toxin. JAMA 1990;264:2671-5.

8. Blitzer A, Sulica L. Botulinum toxin: basic science andclinical uses in otolaryngology. Laryngoscope 2001;111:218­26.

9. Isshiki N, Tsuji DH, Yamamoto Y, Iizuka Y. Midline lat­eralization thyroplasty for adductor spasmodic dysphonia. AnnOtol Rhinol LaryngoI2000;109:187-93.

10. Berke GS, Blackwell KE, Gerratt BR, Vemeil A, Jack­son KS, Sercarz JA. Selective laryngeal adductor denervation­reinnervation: a new surgical treatment for adductor spasmodicdysphonia. Ann Otol Rhinol Laryngol 1999;108:227 -31.

11. Schweizer V, Dorfl J. The anatomy of the inferior laryn­geal nerve. Clin OtolaryngoI1997;22:362-9.

12. Nguyen M, Junien-Lavillauroy C. Anatomy of the lar­ynx, study of the anterior branch of the recurrent laryngeal nerve.Rev Laryngol Otol Rhinol (Bord) 1990;111:153-5.

13. Dubowitz V, Brooke MH. Muscle biopsy: a modem ap­proach. Philadelphia, Pa: WB Saunders, 1973.

14. Hillel AD. The study of laryngeal muscle activity in nor­mal human subjects and in patients with laryngeal dystonia us­ing multiple fine-wire electromyography. Laryngoscope 2001;111(suppl 97).

15. Barany M. ATPase activity of myosin correlated withspeed of muscle shortening. J Gen Physiol 1967;50(suppl): 197­218.

16. Teig E, Dahl HA, Thorkelsen H. Actomyosin ATPase ac­tivity of human laryngeal muscles. Acta Otolaryngol (Stockh)1978;85:272-81.

17. Malmgren LT, Gacek RR. Histochemical characteristicsof muscle fiber types in the posterior cricoarytenoid muscle.Ann Otol Rhinol Laryngol 1981;90:423-9.

18. Rosenfield DB, Miller RH, Sessions RB, Patten BM.Morphologic and histochemical characteristics of laryngeal mus­cle. Arch Otolaryngol 1982;108:662-6.

19. Porter JD, Strebeck S, Capra NF. Botulinum-inducedchanges in monkey eyelid muscle. Comparison with changesseen in extraocular muscle. Arch Ophthalmol 1991;109:396­404.

20. Hom AK, Porter JD, Evinger C. Botulinum toxin paraly­sis of the orbicularis oculi muscle. Types and time course ofalterations in muscle structure, physiology and lid kinematics.Exp Brain Res 1993;96:39-53.

21. Harris CP, Alderson K, Nebeker J, Holds JB, AndersonRL. Histologic features of human orbicularis oculi treated withbotulinum A toxin. Arch Ophthalmol 1991;109:393-5.

22. Duchen LW. Effects of botulinum toxin on the distribu­tion of succinate dehydrogenase and phosphorylase in fast andslow skeletal muscles of the mouse. J Neurol Neurosurg Psychia­try 1970;33:580-5.

23. Duchen LW. An electron microscopic study of changesinduced by botulinum toxin in the motor end-plates of slowand fast skeletal muscle fibres of the mouse. J Neurol Sci 1971;14:47-60.

24. Duchen LW. Changes in the electron microscopic struc­ture of slow and fast skeletal muscle fibres of the mouse afterthe local injection of botulinum toxin. J Neurol Sci 1971;14:61­74.

25. Kranjc BS, Sketelj J, D'Albis A, Erzen I. Long-termchanges in myosin heavy chain composition after botulinumtoxin A injection into rat medial rectus muscle. Invest Ophthal­mol Vis Sci 2001;42:3158-64.

26. Romanul FC, Van der Meulen JP. Reversal of the en­zyme profiles of muscle fibres in fast and slow muscles by cross­innervation. Nature 1966;212:1369-70.

27. Yellin H. Neural regulation of enzymes in muscle fibersof red and white muscle. Exp Neurol 1967;19:92-103.

28. Telerman-Toppet N, Bacq M, Khoubesserian P, Coers C.Type 2 fiber predominance in muscle cramp and exertional my­algia. Muscle Nerve 1985;8:563-7.

29. Edstrom L, Kugelberg E. Histochemical mapping of mo­tor units in experimentally re-innervated skeletal muscle. Ex­perientia 1969;25: 1044-5.

30. Dedo HH, Izdebski K, Townsend 11. Recurrent laryn­geal nerve histopathology in spastic dysphonia: a preliminarystudy. Ann Otol Rhinol Laryngol 1977;86:806-12.

31. Carlsoo B, Izdebski K, Dahlqvist A, Domeij S, Dedo HH.The recurrent laryngeal nerve in spastic dysphonia. A light andelectron microscopic study. Acta Otolaryngol (Stockh) 1987;103:96-104.

32. Kosaki H, Iwamura S, Yamazaki I. Histologic study ofthe recurrent laryngeal nerve in spasmodic dysphonia. Otolaryn­gol Head Neck Surg 1999;120:129-33.