HISTOLOGY OFNERVES AND MUSCLES IN ADDUCTOR SPASMODIC DYSPHONIA

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1 Ann Otol Rhinal Laryngol 112:2003 HISTOLOGY OFNERVES AND MUSCLES IN ADDUCTOR SPASMODIC DYSPHONIA DINESH K. CHHETRI, MD Los ANGELES, 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 with botulinum toxin. Histologic examination revealed average muscle fiber diameters ranging from 21 to 5711m. Botulinum toxin treatment-related muscle atrophy was observed up to 5 months after injection. Endomysial fibrosis was present in all samples. Histochemical analysis in8 patients revealed type 2 fiber predominance in7 patients and fiber type grouping in 2.Type-specific muscle fiber 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 small myelinated nerve fibers. Overall, the nerve fiber diameter was slightly larger inpatients than in controls. These findings may implicate thecentral nervous system in thepathophysiology of adductor spasmodic dysphonia. KEY WORDS - histology, muscle, nerve, pathophysiology, spasmodic dysphonia. INTRODUCTION The etiology and pathophysiology of spasmodic dysphonia (SD) remain elusive even after more than a century of discussion in the literature. Spasmodic dysphonia is classified into adductor and abductor types 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 associated with abnormal closure of the vocal cords during speech. There is a slight female predilection, and the disorder has a persistent course. 1Electromyographic (EMG) studies of laryngeal muscles ofpatients with ASD reveal pitch and phonatory breaks coincident with muscle spasms during vowels in connected speech. I On the basis of these EMG findings, ASD is currently classified as a focal dystonia affecting the larynx during speech.i-' Many etiologic theories of SD have been proposed. A long-standing psychogenic theory introduced by Traube in 1871 was replaced by a neurogenic theory when Aronson et all reported a high incidence of associated neurologic signs (mainly voice tremor) in patients with SD. Subsequent discussions have focused on whether the disorder lies in the central or the peripheral nervous system. Finitzo-Hieber et al 4 suggested a central causation based on abnormal wave V latencies from auditory brain stem reflex testing. Bielamowicz and Ludlow> reported improved EMG signals in contralateral thyroarytenoid (TA) muscles after unilateral botulinum toxin (BTX) injection, and they hypothesized that a central reduction in motoneuron activity secondary to reduced sensorimotor feedback could explain their findings. Ded0 6 commented that proprioceptive abnormalities may exist in patients and that these may be relieved after unilateral section ofthe recurrent laryngeal nerve (RLN). The most common treatment for ASD is injection of BTX type A (Allergan Inc, Irvine, California) into the TA muscle'? It causes flaccid paralysis by inhibiting the release of acetylcholine from nerve terminals." A variety of surgical therapies for ASD have been proposed. They include unilateral RLN section.f midline lateralization thyroplasty.? and selective bilateral laryngeal adductor denervation and reinnervation.!" Laryngeal denervation and reinnervation is the procedure of choice at our institution for a more permanent treatment of ASD. Lateral cricoarytenoid (LCA) muscle myotomy is also now concurrently performed. In this report we describe the histomorphology of the adductor branch of the RLN and the LCA 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, CORRESPONDENCE - Gerald S. Berke. MD CHS, UCLA Medical Center. Los Angeles, CA

2 Chhetri et al, Histology ofnerves & Muscles in Spasmodic Dysphonia 335 reviewed and approved by the Medical Institutional Review Board of the University of California, Los Angeles. Nine consecutive patients who received selective laryngeal adductor denervation, reinnervation, and LCA myotomy for ASD were enrolled in the study. All had received prior BTX therapy. The reason for surgery in all was either dissatisfaction with BTX therapy or loss ofbtx effectiveness, and a desire for permanent surgical cure. Laryngoscopic examination, acoustic analysis of voice, voice history typical for ASD, and response to previous BTX treatments were used to confirm the diagnosis ofasd. Five nerve specimens were analyzed as controls. The harvesting approach and location of control nerves were identical to those of the patient nerves. Two were from autopsies of adult patients who died of cardiovascular disease. Other than short-term intubation during intensive care, neither had a history oflaryngeal abnormalities. Two specimens were from laryngectomies. The first was from a 47-year-old man with a large supraglottic squamous cell carcinoma who had received no prior therapy. The right vocal cord had impaired mobility, and therefore only the left nerve branch was analyzed. The second was from a 73-year-old man with a history of radiotherapy for T3NOsupraglottic carcinoma who required laryngectomy for epiglottic recurrence. The vocal cords were mobile bilaterally. The fifth control specimen was from a 48-year-old woman with acute recurrent laryngospasm (laryngeal adductory dystonia) who was treated with laryngeal denervation, reinnervation, and LCA 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 adductorbranch of the RLN has been reported by others. I I,12 A cartilage window was created in the posterior inferior portion of the thyroid lamina. The anterior adductor branch of the RLN was located, and a nerve stimulator was used to confirm the identification of the nerve. The nerve branch was followed to the TA muscle and cut 5 mm from its muscular insertion. A l-mm section was cut from the distal nerve stump and immediately immersed in 2% glutaraldehyde solution. The LCA muscle was then identified and cut in its midsection with fine scissors. An approximately 5-mm-long sample of muscle was removed from the myotomy site and immediately transported to the laboratory for quick freezing. The muscle samples were always frozen within 20 minutes of biopsy and kept in deep freeze until sectioning. Muscle Processing. The muscle samples were cut at ltl-um thickness or less in a cryostat, picked up on a coverslip, and histologically and histochemically processed by routine procedures used almost daily in the University of California muscle diagnostic histochemistry laboratory. The slides were observed and photographed with a microscopeequippedwith a digital camera system (Olympus BX40). Digital images were taken at 200x to 400x and printed on a laser printer. Muscle diameter was measured directly on the printed images by the "lesserfiber diameter" technique described by Dubowitz and Brooke.l-' In this technique the maximum diameter across the lesser aspect of the muscle is measured, in order to overcome the distortion that occurs when muscle fibers are cut obliquely. At least 200 muscle fibers were counted per muscle biopsy sample. Most of the histologic information was obtained from sections that had been stainedwith the modifiedgomori trichrome, because this stain demonstrates morphological features of the muscle fibers well. The adenosinetriphosphatase histochemical reaction was used to assess fiber type. In this histochemical reaction, type 2 (fast) muscle fibers stain dark and type 1 (slow) muscle fibers stain light when samples are preincubated at ph This staining pattern is reversed with preincubation at ph 4.2 to 4.6. In some samples the whole specimen did not undergo acid reversal; in these cases, areas that reversed well were selected for analysis. More than 75% prevalence of one fiber type was considered type predominance. Nerve Processing. The nerve biopsy specimens were immediately immersion-fixed in 2% glutaraldehyde for at least 2 hours and post-fixed for 1 hour in 1% osmium tetraoxide. The tissue was further processedfor electronmicroscopyby standardtechniques and embedded in Epon. Sections I to 211mthick were cut and stained with toluidine blue for light microscopy. For electron microscopy, 50-nm ultrathin sections were cut, stained with uranyl acetate followed by lead citrate, and examined and photographed at 1,900x with a transmissionelectronmicroscope (JEOL JEM-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 the photographs. The maximumdiameteracross the lesser aspect of the fiber was measured by a technique similar to that described above for measurement of muscle diameter. Nerve density analysis was performed from an area 150 x m2 at 400x magnification. The total number of nerve fibers within this areawas manuallycounted, and the numberof smalldiameter «6 11m) fibers was divided by the total number to generate a percentage of small fibers.

3 336 Chhetri et al, Histology ofnerves & Muscles in Spasmodic Dysphonia Patient No. TABLE 1. DEMOGRAPHIC DATA Age (y) I Sex Duration of Spasmodic Dysphonia (y) M 4 F 6 M 20 F 17 F 17 F 14 M 18 F 20 F 4 Duration of Botulinum Toxin Therapy (y) Statistical Analysis. A 2-tailed Student's r-test for independent samples was used to compare means between patient and control samples Fig 1. Lateral cricoarytenoid muscle from patient with spasmodic 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 diamstudy (Table 1).There were 6 women and 3 men. The eter was between 21 and 57 11m. No statistical difaverage age was 45 years (range, 33 to 59 years) in ference in muscle fiber diameter was found between women and 66 years (range, 64 to 67 years) in men. sides (p =.8), because fiber diameters were related The average age at onset of SO was 32 years in wom- to recent treatment with BTX. The average muscle en (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 BTX ment was found for women (p =.09). The age at on- treatment was half that of the untreated side (Table set 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 muswere comparable in both sexes. All but 2 patients had cle samples were grouped by exposure to BTX less received 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 was other (No.6) always received BTX injections on the found (p =.08). right side of the larynx. Muscle histochemistry was analyzed from both Histologic 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 to from both LCA muscles in all but 1patient. The mus- differentiate between type 1 and type 2 fibers. Howcle's morphological pattern in all patients consisted ever, acid reversal was almost complete at ph 4.6 in of a loosely arranged fiber pattern with transverse the great majority of samples and was complete at and slightly longitudinal fibers present in the same ph 4.2 in all samples, and type 1 and type 2 fibers histologic sections (Fig 1). Moderate endomysial fi- could be easily differentiated. Type 2 fiber predombrosis, 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 ± ± 13.1 Moderate 3 M 2 Left 21 ± ± 14.2 Moderate 4 F 4 Bilateral 23 ± ± 16.5 Moderate 5 F 6 Bilateral 26 ± ± 6.6 Moderate 6 F 5 Right 57 ± ± 8.9 Moderate 7 M 6 Bilateral 40 ± 18.3 NA Minimal NA - not available. *Mean + standard deviation.

4 Chhetri et al, Histology ofnerves & Muscles in Spasmodic Dysphonia 337 TABLE 3. HISTOCHEMISTRY OF LATERAL CRICOARYTENOID MUSCLE Last Botulinum Toxin Injection % Type 2 Fibers Fiber Type Type 2 Fiber Patient No. Months Ago Side Left Right Grouping Predominance 1 11 Bilateral No Yes 3 2 Left NA 78 Right Yes 4 4 Bilateral NA 84 No Yes 5 6 Bilateral No No 6 5 Right Right Left 7 6 Bilateral 82 NA No Yes 8 2 Bilateral No Yes 9 7 Bilateral >95 >95 No Yes separate LCA samples examined, 10 had type 2 fiber predominance (Table 3). Fibertype grouping was present 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 in the BTX-treated right LCA (Fig 3) and type 2 fiber predominance 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 histochemical abnormalities were not observedin any patientmuscle samples. The RLN adductor branch morphological characteristics were analyzed in 9 patients and 5 controls (Table 4). Nerve samples for study were available from both sides in only 3 patients. Nerve density was measured in 3 patients (Nos. 7, 8, and 9) and 3 controls (Table 5). The nerve diameter ranged from 220 to 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 represented 8% of the total fibers. Higher magnification under light and electron microscopy confirmed this finding (Fig 5). Unmyelinated nerve fibers were only occasionally seen by electron microscopy and were evenly distributed. Although the mean fiber sizes were different between patients, they were similar between sides in the same patient (Table 4). Nerve samples from 1 patient (No.9) were notably different (Table 5). The left branch had a nerve densitysimilarto that of otherpatientnerves, but the proportion of small fibers was increased to 31%. The right branch had slightly increased nerve density (+18%) and an increased proportion of small fibers (to 19%). The density ofnerve fibers and proportion of small fibers were similar in controls. When nerve fibers from all patients and all controls were pooled together, the average fiber diameterwas 8.3!lm (standard deviation, 2.8 urn) in patients with SD and 7.1 urn (standard deviation, 2.2 urn) in controls (p <.0001). The relative distribution ofnerve fiber diameter in patients and controls is illustrated in Fig 6. DISCUSSION Both the TA and LCA muscles act synchronously to adduct the vocal folds and close the larynx. In a comprehensive 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 (original x100). At ph 4.2, type 1 (dark) and type 2 (light) fibers are clearly delineated.

5 338 Chhetri et ai, Histology ofnerves & Muscles in Spasmodic Dysphonia TABLE 5. FIBER DENSITY AND DISTRIBUTION IN ADDUCTOR BRANCH OF RECURRENT LARYNGEAL NERVE Subject Side Total Fibers* % Small Fibers Patient 7 L Patient 8 L 89 8 Patient 9 L R C~~crl 1M 8 Laryngectomy Laryngospasm *Total fibers were counted from area 150 x m at400x. Fi~ 3. L~teral cricoarytenoid muscle from patient 6 stained With adenosine triphosphatase at ph 4.2 (original x200). Fiber type grouping is present. muscle activity, Hillel l4 found that the LCA muscle tends to act similarly to the TA muscle in both normal subjects andpatients with SO. No consistent EMG pattern differentiates between LCA and TA function. When BTX therapy is employed for SO, the typical treatment target is the TA muscle, but the toxin easily diffuses into and paralyzes the ipsilateral LCA muscle. Clear evidence for this phenomenon was seen in the 2 patients who had unilateral BTX therapy (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 involved in the pathophysiology and treatment of SO. Actomyosin adenosine triphosphatase activity TABLE 4. MORPHOLOGY OF ADDUCTOR BRANCH OF RECURRENT LARYNGEAL NERVE Nerve Size Subject Side (pm) Fiber Size (um)" Patient I L R Patient 2 L Patient 3 L Patient 4 L Patient 5 R Patient 6 L Patient 7 L R Patient 8 L Patient 9 L R Cadaver 1 Cadaver 2 Laryngectomy 1 Laryngectomy 2 Laryngospasm *Mean ± standard deviation. tp < ± ± ± ± ± ± ± ± ± ± t ± t ± ± ± ± ± ± 2.5 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 requirement of human skeletal muscle is clearly reflected in the distribution of muscle fiber types, and laryngeal muscles follow this pattern as well. Teig et ai16 reported tha~ the TA muscle has the highest percentage of type 2 fibers (mean ± standard deviation, 65% ± 11.6%) and that the posterior cricoarytenoid (PCA) muscle has the highest percentage of type I fibers (67% ± 8.6%). The LCA muscle was intermediate, with about 60% ± 5.2% type 2 muscle fibers. Other studies have corroborated these findings and report the range of type 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) all contribute to the sphincter function of the larynx. The P~A muscle, which is tonically active during the inspiratory phase of respiration, has the least amount of type 2 fibers. A significant proportion of our patients with ASO had type 2 fiber predominance in their LCA muscles. Fig 4. Light micrograph of adductor branch of left recurrent laryngeal nerve in patient 7 shows homogeneously distributed large-diameter myelinated fibers and few small-diameter myelinated fibers (original x200).

6 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 recurrent laryngeal 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 necrosis or type-specific atrophy was not present. Selective degeneration 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, our findings could be explained if BTX treatment induced histochemical conversion of muscle toward type 2 fibers. However, current data do not support this contention. Several studies on the effects of BTX on the orbicularis oculi muscle have revealed only denervation atrophy and no fiber type-specific alterations In addition, Duchen reported in several well-designed studies using a mouse model that type 1 muscle fibers recover faster histologically and histochemically than type 2 muscle fibers after intramuscularinjection of a sublethal dose of BTX. In type 1 (soleus) muscle, nerve recovery began within a week and continued for another 3 to 4 weeks. By 6 weeks, normal histochemical and morphological characteristics were almost completely restored. In type 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 sometimes even at 6 months after injection. These results support the notion that if muscle fiber predominance were to be present after intramuscular BTX injection, it would be a predominance of type 1 fibersa result opposite to our actual findings. A study of long-term changes in myosin heavy chain isoform patterns in extraocularmuscles 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 in the left LeA muscle, which was never treated with BTX. However, a histomorphological study of mus I Fig 6. Fiber analysis of adductor nerves in pa- :. tients with spasmodic dysphonia (black bars) 10 and controls (gray bars) I F_ DIllmele< (mlcrome...)

7 340 Chhetri et al, Histology ofnerves & Muscles in Spasmodic Dysphonia cle samples from patients never treated with BTX would be required to remove any possible confounding influence of previous treatment. Because BTX remains the mainstay of initial treatment for ASD, we must await enrollment of previously untreated ASD patients for selective adductor denervation-reinnervation surgery. It has been shown previously that muscle fiber types reflect functional differences in the innervating motoneurons. Cross-reinnervation of fast and slow muscles reverses their enzyme profiles and characteristics of contraction. 26,27 It is possible that a change in the properties of laryngeal motoneurons from tonic (slower discharge frequency) to phasic (faster discharge frequency) leads to histochemical changes in the muscle. Telerman-Toppet et af8 have found type 2 fiber predominance in adult patients with muscle cramps and exertional myalgia. They propose that abnormal stimulation of muscle fibers could be responsible for the conversion from type 1 to type 2 through a modification in the pattern of muscle fiber activation. The mechanisms leading to changes in motoneuron properties in patients with SD are unknown, but may involve central nervous system interneurons. The hallmark of SD is sudden and quick movements oflaryngeal muscles, and type 2 muscle fiber predominance is consistent with this clinical manifestation. Ourother histologic findings are in agreement with published reports Unlike limb skeletal muscles, laryngeal muscles tend to be arranged loosely, with longitudinal extensions of fibers interspersed with fibers cut transversely. Individual muscle fibers rarely touch each other because of an increase in endomysial connective tissue (Fig 1). In limb muscles, endomysial fibrosis is more commonly seen in myopathies such as Duchenne dystrophy than in neuropathies, although it has also been reported to be present in neurogenic atrophies. 13 It seems that some degree of endomysial fibrosis may be normal in laryngeal muscles. Because we used previously published control data to compare with our study data, a graded comparison of endomysial fibrosis is not possible. Muscle fiber type grouping is a result ofneuromuscular degeneration in which denervated muscle fibers are reinnervated by nerve sprouting from adjacent intact motor units.i? Type grouping has also been observed in the PCA muscle, and it has been theorized that terminal nerve branches in the PCA muscle might be prone to damage when large lumps of food are swallowed.v-" Our2 cases oftype grouping likely arose from BTX-related denervation followed by reinnervation. Prior studies ofnerve histology from patients with SD have examined the RLN and have found no apparentmorphometric differences betweennerves from patients and controls. Initial studies found distinct areas within the nerve that consisted of sheets of unmyelinated axons,30 but further studies concluded that RLNs from both patients and controls contain small «6/-lm), medium (6 to l l um), and large (11 to 18 urn) 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 ofthe RLN at a more proximal location contains an increased number 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 other RLN studies, in the adductor branch in both patients and controls we found only occasional unmyelinated fibers randomly placed throughout the nerve, and no areas resembling bundles of unmyelinated fibers as described by Dedo et apo or areas occupied mainly by unmyelinated fibers as described by Carlsooet al.3! Only I report has previously examined nerve fibers from the adductor branch of the RLN. Kosaki et ap2 examined adductor nerve samples from 2 patients with SD by electron microscopy and found an increased ratio of thin fibers (5 to 10 urn) as compared to normal controls. However, no quantitative data were presented. We doubt that the pathophysiology of SD lies entirely in changes in the number of small myelinated fibers, because 8 of 9 patient nerve 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) also demonstrated 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 counted in nerve samples from 2 patients (Nos. 7 and 8), and totals of 400 and 570 fibers were counted, respectively. CONCLUSIONS We report histomorphological findings in the LCA muscle and the adductor branch ofthe RLN in a small series of patients with ASD. The type 2 muscle predominance seen in the majority ofpatients could be explained by changes in neuromuscular activation patterns. The mechanisms leading to these changes in activation patterns may lie in the central nervous

8 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 study on patients never treated with BTX is needed. The morphological characteristicsof the nerve fibers were similar in patients with SD and controls except for 1 patient, who had a relatively increased ratio of small fibers. The findings in this patient may represent the clinical heterogeneity of this disorder ::\CKNOWLEDGMENTS - The authors acknowledge Birgitta Sjostrand for help with electron microscopic techniques and Ivan Lopez for guidance in tissue processing and digital photography. 1. Aronson AE, Brown JR, Litin EM, Pearson JS. Spastic dysphonia. II. Comparison with essential (voice) tremor and other neurologic and psychogenic dysphonias. J Speech Hear Disord 1968;33: Blitzer A, Lovelace RE, Brin MF, Fahn S, Fink ME. E1ectromyographic findings in focal laryngeal dystonia (spastic dysphonia). 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