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. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: J Neuropathol Exp Neurol. 2012 Jun;71(6):520–530. doi: 10.1097/NEN.0b013e318258381b

Altered Pharyngeal Muscles in Parkinson Disease

Liancai Mu 1, Stanislaw Sobotka 1,2, Jingming Chen 1, Hungxi Su 1, Ira Sanders 3, Charles H Adler 4, Holly A Shill 5, John N Caviness 4, Johan E Samanta 6, Thomas G Beach 7; the Arizona Parkinson’s Disease Consortium
PMCID: PMC3358551  NIHMSID: NIHMS374446  PMID: 22588389

Abstract

Dysphagia (impaired swallowing) is common in Parkinson disease (PD) patients and is related to aspiration pneumonia, the primary cause of death in PD. Therapies that ameliorate the limb motor symptoms of PD are ineffective for dysphagia. This suggests that the pathophysiology of PD dysphagia may differ from that affecting limb muscles but little is known about potential neuromuscular abnormalities in the swallowing muscles in PD. This study examined the fiber histochemistry of pharyngeal constrictor (PC) and cricopharyngeal (CP) sphincter muscles in postmortem specimens from 8 PD and 4 age-matched control patients. Pharyngeal muscles in PD patients exhibited many atrophic fibers, fiber type grouping, and fast-to-slow myosin heavy chain transformation. These alterations indicate that the pharyngeal muscles experienced neural degeneration and regeneration over the course of PD. Notably, the PD patients with dysphagia had a higher percentage of atrophic myofibers vs. with those without dysphagia and controls. The fast-to-slow fiber type transition is consistent with abnormalities in swallowing, slow movement of food and increased tone in the CP sphincter in PD patients. The alterations in the pharyngeal muscles may play a pathogenic role in the development of dysphagia in PD patients.

Keywords: Dysphagia, Fiber types, Immunohistochemistry, Muscle fiber atrophy, Myosin heavy chain isoforms, Parkinson disease, Pharyngeal constrictor muscles, Swallowing, Upper esophageal sphincter

INTRODUCTION

Parkinson disease (PD) is a slowly progressive neurological movement disorder characterized by tremor at rest, bradykinesia, rigidity, and postural instability (1). These motor signs result primarily from a loss of dopaminergic neurons in the nigrostriatal pathway (2) and cause major disability and reduce the quality of life of PD patients. In the United States, PD is the fourth most common neurodegenerative disease in the elderly (3). It is estimated that approximately 1.5 million Americans suffer from PD, with approximately 40,000 new cases diagnosed every year (4, 5), and the total annual burden of PD in the US is estimated at 23 billion dollars (6).

Approximately 50% to 80% of PD patients develop dysphagia (7–12). Dysphagia impacts negatively on their psychological wellbeing and quality of life. More importantly, dysphagia may cause malnutrition and aspiration pneumonia, thus posing serious threats to health and longevity (13). Aspiration pneumonia is reported as the leading cause of death in PD patients (10, 14), with most cases likely secondary to dysphagia. The first symptom of dysphagia in PD patients is often excessive drooling that is believed to be caused by impaired swallowing (15, 16); PD patients with the most severe dysphagia usually have the most drooling (17). Tracheal penetration/aspiration has been noted in 25% to 50% of patients (18–20). Importantly, silent aspiration has been documented in as many as 15% of PD patients who do not complain of dysphagia (21).

The site of dysphagia is oropharyngeal in the majority of PD patients (10, 20, 21), but the anatomical or physiological factors responsible for the dysphagia in PD have never been determined. Histochemical studies on autopsied PD patient skeletal muscle from other sites have shown alterations in muscle fiber morphology and size, and fiber type grouping (22, 23), but histological and histochemical characteristics of the pharyngeal muscles in PD have not been reported.

The human pharynx is a muscular tubular passage of the digestive and respiratory tracts extending from the back of the nasal cavity and mouth to the esophagus. There are 3 pharyngeal constrictor (PC) muscles (i.e. superior [SPC], middle [MPC] and inferior [IPC]) that form the walls of the pharynx. The cricopharyngeus (CP) forms a sphincter at the transition from the pharynx to the esophagus. During swallowing, successive contraction of the PCs constricts the pharyngeal lumen to propel the bolus downward to the CP, which relaxes to allow the bolus to enter the esophagus (24). Identified swallowing abnormalities include a slowing of the peristalsis and a relative decrease in reflex CP relaxation. These motor abnormalities cause retained food or saliva in the pharynx after swallowing. Therefore, dysfunction of the pharyngeal muscles results in swallowing problems.

We hypothesized that alterations in the central or peripheral nervous system controlling the pharynx could result in changes in pharyngeal muscles in PD patients and that these neuromuscular alterations may be one of the risk factors leading to dysphagia. Here, we compared pharyngeal muscles from PD and control patients to identify specific changes that might suggest an underlying mechanism for neurogenic dysphagia in PD.

MATERIALS AND METHODS

Tissues

An autopsy-based study of the human pharyngeal muscles was conducted on 8 patients with clinically diagnosed and neuropathologically confirmed PD (Table 1), and 4 age-matched controls. The PD pharynges were provided by the Brain and Body Donation Program (25) at Banner Sun Health Research Institute (Banner SHRI), which is part of Banner Health, a regional non-profit health care provider centered in metropolitan Phoenix, Arizona. BSHRI and the Mayo Clinic Arizona are the principal institutional members of the Arizona Parkinson’s Disease Consortium, which conducts a longitudinal clinicopathological study of PD and normal aging subjects with annual examinations from entry until death and autopsy. The healthy autopsied pharynges without known systemic neuromuscular disorders were obtained from Department of Pathology at Mount Sinai Medical Center in New York City.

Table 1.

Summary of Main Demographic Features and Clinical Data of Parkinson Disease Cases

Case Sex Age at death (y) Age at PD onset (y) PD duration (y) H&Y Motor UPDRS Dysphagia
1 M 75 55 20 2 17 Yes
2 M 73 62 11 3 18 No
3 M 78 59 19 4 51 Yes
4 F 84 64 20 3 29 No
5 M 80 69 11 4 53 No
6 M 81 70 11 4 43 Yes
7 F 79 68 11 4 47 No
8 M 75 45 30 4 66 Yes
Mean 78 62 17 3.5 41
(Range) (73–84) (45–70) (11–30) (2–4) (17–66)
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F, female; H&Y, Hoehn-Yahr clinical rating scale (score range 1–5); M, male; PD, Parkinson disease; UPDRS, Unified Parkinson’s Disease Rating Scale.

Clinical Assessment

BSHRI provided clinical information for each PD patient. Clinical assessment was performed by movement disorder specialists, who rated the clinical severity of PD using the Unified Parkinson’s Disease Rating Scale (UPDRS) (26), and the Hoehn and Yahr scale (27). Dysphagia was assessed subjectively using one of the questions in the UPDRS.

Muscle Sample Preparation

The pharyngeal muscles were obtained from 1 to 3 days after death (average, 44 hours); this postmortem interval does not hamper reliable histochemical analysis of autopsied muscles (28, 29). The PC and CP muscles were sampled as shown in Figure 1. The muscle samples were prepared for histology and immunohistochemistry. Each sample was placed in parallel and snap-frozen together in isopentane cooled by dry ice. Serial cross sections (10 μm thick) were cut on a cryostat (Reichert-Jung 1800; Mannheim, Germany) at −25°C and stored at −80°C until staining was performed.

Figure 1.

Figure 1

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(A, B) Posterior view of the human pharynx as shown by schematic diagram (A) and photograph from Parkinson disease (PD) pharynx (B) showing the anatomical locations of the pharyngeal muscles and sampling sites (enclosed regions in B) for histological and immunohistochemical studies. CA, carotid artery; CP, cricopharyngeus; IPC, inferior pharyngeal constrictor; MPC, middle pharyngeal constrictor; SB, skull base; SP, stylopharyngeus muscle; SPC, superior pharyngeal constrictor; UE, upper esophagus. The vertical dotted line in (B) indicates the midline.

Staining Methods

Serial cross-sections from each muscle sample were mounted on sets of 8 slides. For each set, the 8 serial sections were subjected to routine histological and enzyme-histochemical (sections #1–3) and immunohistochemical (sections #4–8) staining in the following order: 1) hematoxylin & eosin staining to show muscle structure; 2) reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), which is rich in mitochondria and used as a marker for aerobic metabolism, to demonstrate muscle fiber oxidative capacity; 3) α-glycerophosphate dehydrogenase (GPD) stain to examine anaerobic enzyme activity of the muscle fibers; 4) immunostaining for neural cell adhesion molecule (N-CAM) to detect denervated myofibers; and 5–8) immunostaining with type-specific anti-myosin heavy chain (MyHC) antibodies to investigate alterations in fiber type and MyHC composition.

Immunohistochemistry

N-CAM is abundant on the surface of early embryonic myotubes, declines in level as development proceeds, nearly disappears in the adult muscle, reappears when adult muscles are denervated, and is lost after reinnervation (30–32). N-CAM immunoreactivity on muscle fiber membranes in cross-sections provides a sensitive method for detecting denervated myofibers (32–35). Immunofluorescent labeling of N-CAM was performed as described (35). Briefly, cross-sections were fixed with methanol, incubated in 5% donkey serum (Sigma, St. Louis, MO), incubated in affinity-purified, polyclonal, rabbit anti-rat N-CAM antibody (Chemicon, Temecula, CA), and then incubated in CY3-conjugated AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). Control sections were stained as above except that the incubation with the primary antibody was omitted.

Type-specific anti-MyHC monoclonal antibodies (mAbs) were used for immunohistochemistry (Table 2). The avidin-biotin complex (ABC) method was used to identify the major MyHC-containing fibers, as described (36–39). Briefly, the sections were fixed in 4% paraformaldehyde for 10 minutes, blocked in 2% bovine serum albumin (BSA) with 0.1% Triton X-100 for 20 minutes, incubated with primary mAbs for 1 hour at room temperature (RT; mAb NOQ7-5-4D) or overnight at 4°C (mAbs MY-32 and SC-71), incubated with an anti-mouse IgG (ATCC; Rockville, Maryland) for 1 hour and reacted in ABC reagent (Vector Laboratories, Burlingame, CA) for 1 hour at RT, reacted for 10 minutes at RT with a solution containing 3,3′-diaminobenzidine (DAB) as chromogen to localize peroxidase for primary antibodies according to a DAB substrate kit (SK-4100, Vector Laboratories), and dehydrated in graded concentrations of ethanol, cleared in xylene, and mounted with Permount.

Table 2.

Monoclonal Antibody Specificities

Antibody MyHC Isoforms Source Working
I IIa IIx MyHc-emb Solution
NOQ7-5-4D + − − − Sigma, (St. Louis, MO) 1:1,000
MY-32 − + + − Sigma 1:50
SC-71 − + − − ATCC, (Rockville, MD) supernatant
F1.652 − − − + ATCC supernatant
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*

+, positive reaction between mAb and MHC; −, negative reaction between antibody and MHC; emb, MHC-emb.

ATCC, hybridoma cells purchased from the American Type Culture Collection. The hybridoma cell culture supernatants of mAbs SC-71 and F1.652 were used.

MnHC, myosin heavy chain, embryonic MyHC (MyHC-emb).

Indirect immunofluorescent staining was also used to detect embryonic MyHC (MyHC-emb) isoforms in the muscle fibers as described (36–39). The sections were fixed in 4% paraformaldehyde for 10 minutes, blocked in 2% BSA with 0.1% Triton X-100 for 20 minutes, incubated with primary mAb F1.652 at 4°C for 12 hours, blocked again with 1% BSA for 5 minutes, incubated in a fluorescein isothiocyanate-labeled goat anti-mouse IgG (FITC-GAM; 1:50 dilution; Sigma) for 1 hour, mounted with Vectashield mounting medium (Vector Laboratories) and kept in the dark at 4°C. Control sections were stained as above except that the primary antibody incubation was omitted.

Muscle Fiber Typing

Fibers previously classified as type IIB in human muscle have been renamed type IIX or type IID fibers based on their MyHC complement, which resembles the type IIx MyHC isoform of rat muscle (40, 41). At present there is no mAb specific to type IIx MyHC isoform. In this study, the type II fibers (i.e. MY-32-positive and NOQ7-5-4D-negative) that failed to react with the mAb specific to type IIa MyHC isoform (i.e. SC-71-negative) were identified as type IIX fibers.

The stained muscle sections were examined under a Zeiss photomicroscope (Axiophot-2; Carl Zeiss, Gottingen, Germany) equipped with epifluorescence optics and differential interference contrast microscopy and photographed using a digital camera (Spot-32, Diagnostic Instruments; Keene, NH) that was attached to the photomicroscope and connected to a personal computer with image analysis software. All muscle sections stained with the 4 anti-MyHC mAb were analyzed on an individual fiber basis. The fibers that expressed only a single MyHC isoform were classified as pure fibers; fibers that were positively stained with more than one anti-MyHC mAb were classified as hybrid fibers. The positive and negative fibers reacted to a specific mAb were manually identified and marked using a computerized image analysis system (SigmaScan; Jandel Scientific, San Rafael, CA). Three major MyHC-based fiber types were classified as type I (NOQ7-5-4D-positive MY-32-negative fibers), fast type IIA (SC-71-positive, MY-32-positive, NOQ7-5-4D-negative fibers), fast type IIX (MY-32-positive, SC-71-negative, NOQ7-5-4D-negative fibers), and embryonic (F1.652-positive) MyHC-containing fibers. Relative proportions and distribution patterns of the MyHC-based fiber types were computed.

Fiber Diameter Measurements

The muscle sections stained with mAb NOQ7-5-4D for type I MyHC fibers were used for measuring the least fiber diameter (the maximum diameter across the least aspect of the muscle fiber) using a previously established method (42). In each muscle, at least 100 fibers were randomly selected to measure the least fiber diameter. Myofibers with diameters of ≤20 μm were considered to be atrophic.

Data Analysis

Histological and immunohistochemical properties of the PC and CP muscles in PD and control patients were compared. Relative proportions of denervated and atrophic muscle fibers and various MyHC-containing fibers in each of the studied muscles were determined by averaging overall samples. Means and data ranges in both PD and control groups were reported. Data were analyzed with 3-way repeated measure ANOVA (repeated factor - fiber type). Subsequently, data from each main fiber type were analyzed with 2-way ANOVA followed by post-hoc analysis with Tukey-Kramer adjustment for multiple comparisons. The main factors were group (PD and control) and muscle type (CP and IPC). The influence of dysphagia on percentages of atrophic muscle fibers was evaluated in the PD group with 3-way repeated measure ANOVA (main effects: presence of dysphagia, muscle type and fiber type). Statistical computations were performed using the Statistical Analysis System 9.2 (SAS, Inc., Cary, NC). Statistical significance was set at p < 0.05.

RESULTS

Demographic Data

In the PD patient group, there were 6 men and 2 women, mean age was 78.1 years (range, 73–84 years), the mean age at onset of PD was 61.5 years (range, 45–70 years); the mean duration of PD at time of death was 16.6 years (range, 11–30 years) (Table 1). In this group, 6 had developed dementia. All patients were Caucasian. The mean Hoehn & Yahr stage was 3.5; 1 patient was stage 2; 2 patients were stage 3; 5 patients were stage 4. The mean score for the motor UPDRS (UPDRS: Part III) was 41.0 points, the mean time since last examination was 11.4 months, and the mean time since last dose of medication was 5.1 hours. Based on the UPDRS part II scale, dysphagia occurred in 4 (50%) out of the 8 patients (Table 1). The 4 age-matched controls included 2 men and 2 women without neuromuscular disorders. The mean age of the healthy subjects was 77.5 years (range, 76–78 years).

Neuropathological Findings in PD Brains

All cases of PD included in this study met neuropathological criteria for PD based on examination by TGB (Table 1). Microscopic examinations revealed that the substantia nigra and locus ceruleus showed moderate to marked depletion or loss of pigmented neurons and there were several Lewy bodies in each region (data not shown). Immunohistochemical staining for phosphorylated α-synuclein showed frequent immunoreactive neuronal inclusions and related neurites in the olfactory bulb, brainstem, amygdala, transentorhinal area and cingulate gyrus, with variable densities in the 3 neocortical regions examined (temporal, frontal, parietal). The major spinal cord subdivisions were examined in 7 cases; 6 of these had positive Lewy-type synucleinopathy in the spinal cord. Using the Unified Staging System for Lewy Body Disorders (43), 6 cases were classified as “Neocortical stage” and 2 were “Brainstem and Limbic stage.” Five cases were demented and met consensus clinicopathological criteria for Alzheimer disease (AD) (44). One other case had dementia on the basis of progression of PD without concurrent AD.

Histopathological and Enzyme-Histochemical Features of PD Patient Muscles

The PC and CP muscles in PD cases showed altered fiber morphology and enzyme-histochemical activities. Central nuclei also were identified mainly in the PD muscles. There were considerable variations in fiber diameters and angular small fibers in the PD muscles (Fig. 2).

Figure 2.

Figure 2

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(A–D) Serial cross sections of cricopharyngeus (CP) muscles from a Parkinson disease (PD) patient (PD #1, 75-year-old male) and an age-matched control (76-year-old male) stained for NOQ7-5-4D (A, A′), NADH-TR (B, B′), MY-32 (C, C′), and α-glycerophosphate dehydrogenase (GPD) (D, D′) showing the levels of NADH-TR and GPD activities and the patterns of the reaction product densities for slow type I (labeled with asterisks), fast type II (labeled with red dots), and type I/II hybrid (labeled with yellow dots) fibers. In the control CP (A′–D′), type I fibers have high NADH-TR but low GPD, whereas type II fibers have high GPD but low NADH-TR. In contrast, type I/II hybrid fibers in the control CP have high NADH-TR and GPD. In the PD CP (A–D), grouped atrophic type I fibers are shown by circles. Type I fibers without significant atrophy have high NADH-TR and intermediate to high GPD, whereas atrophied type I fibers (enclosed small fibers) have high NADH-TR but low GPD. Type II fibers have high GPD but low NADH-TR, whereas type I/II hybrid fibers have intermediate NADH-TR and GPD in the PD CP. Scale bar: D′ = 100 μm.

Levels of NADH-TR and GPD activities as indicated by reaction product densities varied across fiber types. In control muscles, type I fibers had high NADH-TR but low GPD, whereas type II fibers had high GPD but low NADH-TR. The reaction product densities were ranked I > I/IIa > IIa > IIx for NADH-TR and I < I/IIa < IIa < IIx for GPD. Pharyngeal muscles in PD cases mostly showed preserved common enzyme-histochemical features as seen in the control muscles (Fig. 2). However, in the PD muscles, type I fibers without significant fiber atrophy had high NADH-TR and intermediate to high GPD, whereas atrophic type I fibers had high NADH-TR but low GPD (Fig. 2).

Denervated and Atrophic Fibers

Denervated and atrophic myofibers in the PD and control muscles were immunopositive for N-CAM protein. N-CAM-positive fibers were identified more frequently in the PD CP and IPC muscles than in the control muscles. Most of the N-CAM-positive fibers in the PD muscles were atrophied (Fig. 3).

Figure 3.

Figure 3

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Photomicrographs of cross-sections of the cricopharyngeus (CP) muscles from a Parkinson disease (PD) patient (PD #3, 78-year-old male; A, B) and a normal control (78-year-old male; A′ B′). (A, A′) Sections immunostained for N-CAM to label denervated muscle fibers. There are more N-CAM-positive fibers (arrows) in the PD CP muscle vs. the control muscle. Most of the N-CAM-positive fibers in the PD muscle are atrophic. (B, B′) Sections stained with anti- myosin heavy chain (MyHC) monoclonal antibody (mAb) NOQ7-5-4D that is specific for slow type I MyHC isoform. There is fiber type grouping in the slow type I muscle fiber population in the PD CP muscle (B). Scale bars: A′ = 50 μm; B′ = 100 μm.

Fiber type grouping and myofiber atrophy were more predominant in the PD than the control muscles (Figs. 3–8). On average, muscle fibers in the control CP were 37 μm in diameter for slow type I and 39 μm in diameter for fast type II fibers. Diameters of IPC muscle fibers were 38 μm for type I and 40 μm for type II fibers. Atrophied fibers (≤20 μm in diameter) in the control CP and IPC accounted for 9% and 6% of the total fiber population, respectively (Fig. 7).

Figure 8.

Figure 8

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Percentages of atrophic type I and type II fibers in the cricopharyngeus (CP) and inferior pharyngeal constrictor (IPC) muscles in Parkinson disease (PD) patients with dysphagia (D) and without dysphagia (N).

Figure 7.

Figure 7

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Relative proportions of atrophic type I and type II fibers in the Parkinson disease (PD) and control cricopharyngeus (CP) and inferior pharyngeal constrictor (IPC) muscles.

The diameters of the PD muscle fibers were reduced vs. those of the controls (Fig. 6). On average, type I and type II fibers were 23 μm and 36 μm for PD CP muscle, and 33 μm and 35 μm for PD IPC muscle, respectively. CP had more atrophied fibers (28% of the total fiber population), either clustered in groups or scattered, as compared with the IPC (15% of the total fiber population; p < 0.001; Fig. 7). Comparisons between the PD and control groups showed that the former had significantly more atrophied myofibers than the latter (F = 30.02, p < 0.0001, df = 1/20). In PD muscles, the majority of the atrophied myofibers were mainly type I fibers (approximately 80% of the atrophied fibers; Figs. 2A, 4C, 5A), and less in the type II fiber population (p < 0.0001; Figs. 2C, 4D, 5B, C, 7). Interestingly, the atrophied myofibers in PD muscles were more frequent in the outer layer than in the inner layer of the CP and IPC muscles (Fig. 4C). The pharyngeal muscles in the PD patients with dysphagia contained higher percentages of atrophied myofibers (27.6% of the total fiber population) vs. those in PD patients without dysphagia (16% of the total fiber population; F = 59.92, p < 0.0001, df = 1/12; Fig. 8). Thus, there was significant fiber atrophy of pharyngeal muscles in PD patients, especially in those with dysphagia.

Figure 6.

Figure 6

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(A, B) Bar graphs showing diameter distribution of the type I and type II fibers in the Parkinson disease (PD) and control cricopharyngeus (CP; A) and inferior pharyngeal constrictor (IPC; B) muscles.

Figure 4.

Figure 4

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Photomicrographs of cross-sections of the inferior pharyngeal constrictor (IPC) muscles from normal subjects (A, B) and Parkinson disease (PD) patients (C, D). The sections were stained with anti-MyHC monoclonal antibody (mAb) NOQ7-5-4D specific for slow type I (A–C) and mAb MY-32 for all fast type II (D) MyHC isoforms. The normal IPC muscles are from a relatively young (61-year-old; A) and an older (76-year-old; B) male subject. The diseased IPC muscle (C, D) is from a 78-year-old male PD patient (PD #3). The IPC muscle in the young subject (A) is composed of a slow inner layer (SIL) and a fast outer layer (FOL). In the 76-year-old normal patient (B) and PD patient (C, D), the fiber layers in the IPC muscle are not evident. Also note that there are numerous atrophic myofibers in the PD IPC (C, D). The majority of the atrophied fibers are type I fibers and are mainly in the outer layer (C). Scale bar: D = 100 μm.

Figure 5.

Figure 5

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Immunohistochemistry on serial cross-sections of the cricopharyngeus (CP) muscles from a Parkinson disease (PD) patient (PD #8, 75-year-old man; A–D) and a normal subject (76-year-old man; A′-D′). Sections were stained with anti-myosin heavy chain (MyHC) monoclonal antibodies specific for slow type I (A, A′), all fast type II (B, B′), fast type IIa (C, C′), and embryonic MyHC isoforms (D, D′). Atrophic fibers in the PD case are mainly type I fibers. Sections stained using the avidin-biotin immunoperoxidase procedure (A–C, A′–C′) show the major MyHC-containing fibers; sections stained by indirect immunofluorescence (D, D′) show embryonic MyHC-containing fibers. The numbers in the photomicrographs (A–C′) represent the classified fiber types identified by fiber-to-fiber comparison: fiber 1 = type I; fiber 2 = type IIA; fiber 3 = type I/IIA; fiber 4 = type IIx. Scale bar: D′ = 100 μm.

MyHC Transformation and Fiber Type Distribution in the PD Muscles

Immunohistochemically stained cross sections showed MyHC-based fiber types and their distribution patterns in the muscles. As in our previous studies (38, 45), the CP and PCs in the normal middle-aged humans were composed of 2 histochemically well-defined fiber layers, a slow inner layer and a fast outer layer (Fig. 4A). The slow inner layer contained predominantly type I MyHC-containing fibers, whereas the fast outer layer contained mainly type II MyHC-containing fibers. As a whole, CP contained 64% of type I and 36% of type II fibers (38), whereas IPC contained 54% type I and 46% type II fibers (45) in normal middle-aged humans.

The PD CP and IPC muscles exhibited marked alterations in the MyHC composition and fiber type distribution. The inner and outer muscle fiber layers were not observed in the PD and senescent control muscles (Figs. 4B–D) because the fast outer layer had an increase in the type I fibers with a concomitant decrease in the type II fibers. Relative percentages of the MyHC-based major fiber types and their distribution in the PD and control muscles are summarized in Table 3. On average, the PD CP muscle was slower (82% type I) than the control (73% type I; p < 0.014). Similarly, the PD IPC muscle also contained more slow type I fibers (72%) as compared with the control (61% type I; p < 0.0036). An increase in percentage of the type I fibers in the PD CP and IPC muscles accompanied with a concomitant decrease in percentages of both the type I/IIA and IIA fibers (Table 3), indicating that PD induced fast-to-slow MyHC isoform transformation in these muscles.

Table 3.

Percent Composition of Major Myosin Heavy Chain-Based Fiber Types in Parkinson Disease and Control Muscles

MyHC: Cricopharyngeal Sphincter Muscles Inferior Pharyngeal Constrictor Muscles
I IIA I/IIA IIX I IIA I/IIA IIX
% (range) % (range)
PD 82 (77–86) 11 (8–16) 5 (3–8) 2 (0–4) 72 (65–76) 18 (14–25) 6 (2–8) 4 (1–7)
Control 73 (63–78) 12 (8–15) 14 (10–16) 1 (0–3) 61 (57–66) 32 (26–35) 4 (1–7) 3 (0–6)
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MyHC, myosin heavy chain; PD, Parkinson disease.

DISCUSSION

To our knowledge, this is the first study to examine whether PD is associated with changes in pharyngeal muscle fiber morphology, enzyme histochemistry, MyHC composition and fiber type distribution. There are several notable findings. First, PD pharyngeal muscles, particularly the CP and IPC, exhibited pronounced pathological changes, including the presence of considerable denervated and atrophied fibers, angular small fibers, centrally placed nuclei, and large variations in fiber size. Second, fiber type grouping and atrophied myofibers were mainly type I fibers. Third, PD was associated with fast-to-slow MyHC transformation and altered fiber type distribution. Specifically, the pharyngeal muscles in PD had a slow phenotype as compared to the controls. Finally, pharyngeal muscles in PD patients with dysphagia contained more atrophied myofibers than those in PD patients without dysphagia.

Dysphagia resulting from neurological disorders including PD affects approximately 300,000 to 600,000 people every year (46). It can lead to malnutrition, weight loss, choking, aspiration, pneumonia, and death (47). Swallowing consists of 3 stages: oral, pharyngeal and esophageal phases). Oral phase abnormalities in PD patients include inefficient mastication, impaired bolus formation, and delayed initiation of swallowing (11, 20). Impaired mastication and oral preparatory lingual movements are most commonly seen during dynamic videofluoroscopy (11) and may be caused by PD-related akinesia, bradykinesia, and rigidity (48). Impaired pharyngeal bolus transport is the major determinant of dysphagia and pharyngeal phase abnormalities in PD include delayed pharyngeal swallowing reflex, vallecula and pyriform sinus pooling, reduced pharyngeal peristalsis/constrictor function, and incomplete upper esophageal sphincter relaxation (8, 9, 21, 49, 50). Cricopharyngeal achalasia has been reported to be present in 30% of PD patients with dysphagia and, thus, a major factor in the pathogenesis of dysphagia in some PD patients (51, 52).

The precise mechanisms of dysphagia in PD remain unclear. There is a general belief that dysphagia in PD is associated with akinesia and rigidity of oropharyngeal muscles by impaired basal ganglia motor control (11, 15, 53). Dysfunction of the upper esophageal sphincter has been hypothesized to be the result of a lack of dopaminergic stimulation at the supramedullary level causing skeletal muscle rigidity (54). Previous studies using simultaneous videoradiography and pharyngeal manometry found no correlation between overall muscle rigidity score and abnormal pharyngeal wall motion in PD patients (49). Importantly, anti-PD drugs and surgical interventions (pallidotomy, thalamotomy, and deep brain stimulation), which have been shown to be efficacious for the treatment of the primary clinical features affecting the limb function in PD, do not produce consistent or positive effects in the treatment of the dysphagia (12, 50, 55). These findings suggest that oropharyngeal dysphagia in PD may not be linked solely to a reduction in basal ganglia dopamine activity. Some investigators suggested that other neurotransmitter systems (8, 50, 56) or some other non-dopaminergic mechanisms (8) may also be involved. In addition to degeneration of the nigrostriatal dopaminergic pathway, a variety of neuronal systems are likely involved in PD (57), causing multiple neuromediator dysfunctions that account for the complex patterns of functional deficits.

Previous histochemical studies have demonstrated atrophic and hypertrophic fibers and fiber type grouping in limb muscles in PD patients (22, 23). However, changes in the muscle fibers appear to be muscle-specific and related to PD subtypes. For example, type II fibers in the biceps brachii were atrophic (22), whereas those in the tibialis anterior were normal or hypertrophic (23). The biceps brachii exhibited up to 60% atrophic type II fibers, which were found mainly in PD patients with very severe akinesia and long disease duration. Normal or slightly hypertrophic type I fibers were identified in PD patients with marked rigidity, whereas atrophic type I fibers were found in PD patients with prevalent akinesia and slight rigidity (22). These findings indicate that muscle fiber changes in PD limb muscles are associated with the duration, severity, and subtypes of the disease. Some investigators believe that muscle fiber changes in PD may be caused by the selective use of low threshold tonic motor units, whereas the high threshold motor units remain more inactive, an effect due to the rigidity and akinesia (58, 59). However, others have speculated that the muscle fiber changes may have resulted from hypomobilization, which provokes disuse myofiber atrophy, rather than a consequence of the modified pattern of motor unit activation (60, 61).

Pharyngeal muscles have the capacity to alter their morphological profiles, enzyme levels, fiber type and MyHC composition, and contractile properties in response to various physiological and pathophysiological stimuli. However, unlike in limb muscles, we found that the majority of the atrophied fibers in the pharyngeal muscles were type I fibers. This suggests that muscle fiber alterations in PD may occur in a muscle-specific manner. Moreover, the pharyngeal muscles exhibited a number of morphological and immunohistochemical changes associated with chronic denervation and reinnervation, suggesting that disuse atrophy does not explain the observed findings and that denervation of pharyngeal muscles may play a pathogenic role in the development of dysphagia in PD.

PD is a multi-system disease (62). Sensory and autonomic disturbances are part of the clinical picture of PD and electrophysiological studies have shown that peripheral neuropathy is present in large proportions of PD patients (63–66). The cause(s) of peripheral neuropathy in PD is (are) unknown. It may be a complication of levodopa (L-dopa) therapy or a primary peripheral nervous system manifestation of PD (66, 67). Recent studies have shown that in PD patients, α-synuclein is deposited in the peripheral autonomic nervous system, including in the cardiac plexus (68–70), enteric nervous system of the alimentary tract (71–73), and skin (74–78). Peripheral sensory nerve involvement in PD has also been demonstrated by functional and histochemical assessments of cutaneous sensory nerve endings. The cutaneous denervation and the sensory impairment in PD were indicated by significant increase in tactile and thermal thresholds, a reduction in mechanical pain perception, and significant epidermal nerve fiber loss (75, 79).

Disability in PD is mostly due to motor impairment (80, 81). Electrophysiological abnormalities such as prolonged latencies and smaller nerve action potentials in both sensory and motor nerves of arms and legs were detected in some PD patients (66). Electromyographic recordings showed denervation signs in some limb muscles in PD (82). Pharynx and larynx play an important role in swallowing and airway protection. The thyroarytenoid muscles in the vocal cords are active not only during phonation, but also during swallowing to protect the airway (83). It has been reported that approximately 80% of PD patients have vocal difficulties (84), which are most likely associated with incomplete closure of the vocal cords during phonation caused by vocal cord atrophy as demonstrated by laryngoscopy (85, 86). Vocal cord (85, 86) and pharyngeal muscle atrophy (this study) suggest motor impairment in PD. Neurogenic myofiber atrophy may be due to a PD-related loss of functioning motoneurons and/or degenerative alterations in the peripheral nerves innervating the muscles. Pharyngeal and laryngeal muscles receive their motor innervation from CN X and several studies have demonstrated α-synuclein in fibers in the cervical peripheral X nerve in PD (73, 87–89). We have similar findings (data not shown). Beach et al (89) have shown that occasional large anterior horn neurons as well as some fibers within the CN X and sciatic nerves in PD subjects were immunoreactive for phosphorylated α-synuclein, suggesting a possible mechanism for muscle denervation (89).

Further evidence for limited muscle denervation in PD has come from electromyographic studies (90–94). We have demonstrated that normal adult human CP and PC muscles are composed of a slow inner layer and a fast outer layer that receive their motor innervation from CNS IX and CN X, respectively (45). The results from the present study showed that PD-induced fast-to-slow MyHC transformation and muscle fiber atrophy occurred mainly in the outer layer of the pharyngeal muscles, suggesting that the neuromuscular system controlled by CN X is significantly affected by the neuropathological process of PD. Lewy neurites have been identified in CN X in PD (73, 87–89). Because CN X contains sympathetic, parasympathetic, sensory and motor axons, it is not known whether the α-synuclein pathology in this nerve occurs in the pure motor fibers, intramuscular motor terminals or neuromuscular junctions in the skeletal muscles it innervates.

Recent studies have demonstrated that limb muscle strength is reduced in patients with PD (95), but it is not known whether muscle weakness is of central or peripheral origin or whether it is intrinsic to the disease or a secondary phenomenon. In the PD pharyngeal muscles, decrease in muscle fiber size, reduction in the number of fast type II fibers, and increase in slow type I fibers could cause functional changes in the intrinsic force-generating capacity of the muscles. As a result, strength and contraction speed of pharyngeal muscles during swallowing could be reduced. Muscle fiber atrophy and fast-to-slow MyHC transformation could account for a large portion of the reduction in force.

Motor innervation is one of the most important factors influencing the fiber type and MyHC composition as well as morphological profile of muscle fibers. In general, decreased neuromuscular activation, which is usually caused by neurological diseases, denervation or disuse, results in muscle fiber atrophy and fiber type and MyHC transformation. MyHC conversion could lead to fast-to-slow shift in the fast-twitch muscle (96) or fast muscle region such as fast outer layer in the pharyngeal muscles as demonstrated by this study. These neuromuscular alterations in the PD pharynx are most likely caused by motor abnormalities at the levels of central motoneurons and peripheral nerves and lead to dysphagia. Thus, oropharyngeal dysphagia in PD patients could at least in part be explained by peripheral mechanisms related to the disease-induced neuromuscular alterations.

The definitive cause of the dysphagia in PD remains unclear. Because PD-related dysphagia does not positively respond to anti-PD drugs, more work is needed to elucidate its possible pathophysiological mechanisms for the development of effective therapies. Relatively few studies focus on exploring PD related pathophysiological, immunohistochemical, and biochemical changes at the peripheral level and oropharyngeal dysphagia in PD is most likely multifactorial. In view of studies indicating the presence of Lewy neurites in CN X (73, 87–89), as well as in sciatic nerve (89) in PD patients, further elucidation and characterization of peripheral nervous system lesions will have implications for the diagnosis and treatment of the disease.

In addition, oropharyngeal sensory afferents also play a fundamental role in the coordination of the swallowing act by triggering and modulating inputs to the medullary central pattern generators, which in turn determine the timely relaxation of the upper esophageal sphincter (97, 98). Relaxation abnormalities of the upper esophageal sphincter in PD patients (49, 99) are caused by reflex neural dysfunctions. PD patients with dysphagia have impaired voluntary cough (100) and reflex sensitivity (14) that contributes to an even higher potential level of aspiration risk. Because the mechanism of oropharyngeal swallowing involves a complex integration of neuromuscular and sensory modalities, many of which may be compromised in PD, further studies are needed to determine the PD-induced alterations in the peripheral motor and sensory nervous systems and their contribution to oropharyngeal dysphagia.

Acknowledgments

This research is supported by National Institutes of Health Grant 5 R01 DC004728 from the National Institute on Deafness and Other Communication Disorders (to Dr. Liancai Mu). We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program and the Arizona Parkinson’s Disease Consortium for the provision of whole-mount tongue-pharynx-larynx specimens and associated clinical and neuropathological data from PD subjects. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer’s Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinson’s Disease Consortium) and the Michael J. Fox Foundation for Parkinson’s Research.

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