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.
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