J Neuropathol Exp Neurol Vol. 71, No.

6
Copyright Ó 2012 by the American Association of Neuropathologists, Inc. June 2012
pp. 520Y530

ORIGINAL ARTICLE

Altered Pharyngeal Muscles in Parkinson Disease

Liancai Mu, MD, PhD, Stanislaw Sobotka, PhD, Jingming Chen, MD, Hungxi Su, MD, Ira Sanders, MD,
Charles H. Adler, MD, PhD, Holly A. Shill, MD, John N. Caviness, MD, Johan E. Samanta, MD,
Thomas G. Beach, MD, PhD, and the Arizona Parkinson’s Disease Consortium

INTRODUCTION
Abstract
Parkinson disease (PD) is a slowly progressive neuro-
Dysphagia (impaired swallowing) is common in patients with
logic movement disorder characterized by tremor at rest, bra-
Parkinson disease (PD) and is related to aspiration pneumonia, the
dykinesia, rigidity, and postural instability (1). These motor
primary cause of death in PD. Therapies that ameliorate the limb
signs result primarily from a loss of dopaminergic neurons in
motor symptoms of PD are ineffective for dysphagia. This suggests
the nigrostriatal pathway (2) and cause major disability and
that the pathophysiology of PD dysphagia may differ from that

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affecting limb muscles, but little is known about potential neuro-
muscular abnormalities in the swallowing muscles in PD. This study
examined the fiber histochemistry of pharyngeal constrictor and cri-
copharyngeal sphincter muscles in postmortem specimens from 8
subjects with PD and 4 age-matched control subjects. Pharyngeal
muscles in subjects with PD 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,
subjects with PD with dysphagia had a higher percentage of atrophic
myofibers versus 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 cri-
copharyngeal sphincter in subjects with PD. The alterations in the
pharyngeal muscles may play a pathogenic role in the development
of dysphagia in subjects with PD.
Key Words: Dysphagia, Fiber types, Immunohistochemistry, Muscle
fiber atrophy, Myosin heavy chain isoforms, Parkinson disease, Pha-
ryngeal constrictor muscles, Swallowing, Upper esophageal sphincter.
From the Upper Airway Research Laboratory (LM, SS, JC, HS), Department
of Research and Alice and David Jurist Institute for Biomedical Research
(IS), Hackensack University Medical Center, Hackensack, New Jersey;
Department of Neurosurgery (SS), Mount Sinai School of Medicine, New
York, New York; Parkinson’s Disease and Movement Disorders Center,
Mayo Clinic Arizona (CHA, JNC), Scottsdale; Cleo Roberts Center for
Clinical Research (HAS) and Civin Laboratory for Neuropathology
(TGB), Banner Sun Health Research Institute, Sun City; and Banner Good
Samaritan Medical Center (JES), Phoenix, Arizona.
Send correspondence and reprint requests to: Liancai Mu, MD, PhD,
Department of Research, Hackensack University Medical Center, Hack-
ensack, NJ 07601; E-mail: lmu@humed.com
This research is supported by National Institutes of Health Grant 5 R01
DC004728 from the National Institute on Deafness and Other Commu-
nication Disorders (to Dr Mu). 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 Con-
sortium) and the Michael J. Fox Foundation for Parkinson’s Research.
reduce the quality of life of patients with PD. 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 have PD, with approximately 40,000 new cases
diagnosed every year (4, 5), and the total annual burden of PD
in the United States is estimated at US $23 billion (6).
Approximately 50% to 80% of patients with PD de-
velop dysphagia (7Y12). Dysphagia negatively affects their
psychologic well-being and quality of life. More importantly,
dysphagia may cause malnutrition and aspiration pneumonia,
thus posing serious threats to health and longevity (13). As-
piration pneumonia is reported as the leading cause of death in
patients with PD (10, 14), with most cases likely secondary to
dysphagia. The first symptom of dysphagia in patients with PD
is often excessive drooling that is believed to be caused by
impaired swallowing (15, 16); patients with PD who have the
most severe dysphagia usually have the most drooling (17).
Tracheal penetration/aspiration has been noted in 25% to 50%
of patients (18Y20). Importantly, silent aspiration has been
documented in as many as 15% of patients with PD who do
not complain of dysphagia (21).
The site of dysphagia is oropharyngeal in most patients
with PD (10, 20, 21), but the anatomic or physiological factors
responsible for the dysphagia in PD have never been deter-
mined. Histochemical studies on skeletal muscle from other
sites of autopsied subjects with PD have shown alterations in
muscle fiber morphology and size and fiber type grouping
(22, 23), but histologic 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], mid-
dle [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 swal-
lowing, successive contraction of the PCs constricts the pha-
ryngeal lumen to propel the bolus downward to the CP, which
relaxes to allow the bolus to enter the esophagus (24). Iden-
tified swallowing abnormalities include a slowing of the

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J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012 Pharyngeal Muscles in PD

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 pha-
ryngeal muscles results in swallowing problems.
We hypothesized that alterations in the central or pe-
ripheral nervous system controlling the pharynx could result
in changes in pharyngeal muscles in subjects with PD and that
these neuromuscular alterations may be one of the risk factors
leading to dysphagia. Here, we compared pharyngeal muscles
from subjects with PD and control subjects to identify specific
changes that might suggest an underlying mechanism for neu-
rogenic dysphagia in PD.

MATERIALS AND METHODS

Tissues FIGURE 1. (A, B) Posterior view of the human pharynx as
An autopsy-based study of the human pharyngeal mus- shown by schematic diagram (A) and photograph from Par-
cles was conducted on 8 subjects with clinically diagnosed and kinson disease (PD) pharynx (B) showing the anatomic loca-
neuropathologically confirmed PD (Table 1) and 4 age-matched tions of the pharyngeal muscles and sampling sites (enclosed

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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 nonprofit health care provider centered in metropolitan
Phoenix, Ariz. Banner SHRI and the Mayo Clinic Arizona are
the principal institutional members of the Arizona Parkinson’s
Disease Consortium, which conducts a longitudinal clinico-
pathologic study of PD and normal aging subjects with annual
examinations from entry until death and autopsy. The healthy
autopsied pharynges without known systemic neuromuscu-
lar disorders were obtained from Department of Pathology at
Mount Sinai Medical Center in New York City.
Clinical Assessment
Banner SHRI provided clinical information for each sub-
ject with PD. Clinical assessment was performed by move-
ment 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 as-
sessed subjectively using one of the questions in the UPDRS.
Muscle Sample Preparation
The pharyngeal muscles were obtained from 1 to 3 days
after death (mean, 44 hours); this postmortem interval does not
regions in B) for histologic and immunohistochemical studies.
CA, carotid artery; CP, cricopharyngeus; IPC, inferior pha-
ryngeal constrictor; MPC, middle pharyngeal constrictor; SB,
skull base; SP, stylopharyngeus muscle; SPC, superior pharyn-
geal constrictor; UE, upper esophagus. The vertical dotted line
in B indicates the midline.
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 Km thick) were cut on a cryostat
(Reichert-Jung 1800; Mannheim, Germany) at j25-C and
stored at j80-C until staining was performed.
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 histologic and enzyme-histochemical
(Sections 1Y3) and immunohistochemical (Sections 4Y8) stain-
ing in the following order: 1) hematoxylin and eosin staining
to show muscle structure; 2) reduced nicotinamide adenine
dinucleotide tetrazolium reductase (NADH-TR), which is rich

TABLE 1. Summary of Main Demographic Features and Clinical Data of Cases With Parkinson Disease
Case No. 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 (range) 78 (73Y84) 62 (45Y70) 17 (11Y30) 3.5 (2Y4) 41 (17Y66)
F, female; H&Y, Hoehn-Yahr clinical rating scale (scores range 1Y5); M, male; PD, Parkinson disease; UPDRS, Unified Parkinson’s Disease Rating Scale.

Ó 2012 American Association of Neuropathologists, Inc. 521

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Mu et al
in mitochondria and used as a marker for aerobic metabo-
lism, to demonstrate muscle fiber oxidative capacity; 3) >-
glycerophosphate dehydrogenase (GPD) stain to examine
anaerobic enzyme activity of the muscle fibers; 4) immuno-
staining for neural cell adhesion molecule (N-CAM) to detect
denervated myofibers; and 5Y8) immunostaining with type-
specific antiYmyosin heavy chain (MyHC) antibodies to in-
vestigate alterations in fiber type and MyHC composition.
Immunohistochemistry
Neural CAM is abundant on the surface of early em-
bryonic myotubes, declines in level as development proceeds,
nearly disappears in the adult muscle, reappears when adult
muscles are denervated, and is lost after reinnervation (30Y32).
Neural CAM immunoreactivity on muscle fiber membranes in
cross sections provides a sensitive method for detecting dener-
vated myofibers (32Y35). 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, poly-
clonal, rabbit anti-rat N-CAM antibody (Chemicon, Temecula,
CA) and then incubated in CY3-conjugated AffiniPure don-
key anti-rabbit immunoglobulin G (IgG; Jackson Immuno-
Research, West Grove, PA). Control sections were stained as
previously mentioned except that the incubation with the pri-
mary antibody was omitted.
Type-specific anti-MyHC monoclonal antibodies (mAbs)
were used for immunohistochemistry (Table 2). The avidin-
biotin complex method was used to identify the major MyHC-
containing fibers, as described (36Y39). 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, MD) for 1 hour and reacted in avidin-
biotin complex reagent (Vector Laboratories, Burlingame,
CA) for 1 hour at RT, reacted for 10 minutes at RT with a
solution containing 3,3¶-diaminobenzidine as chromogen to
localize peroxidase for primary antibodies according to a 3,3¶-
diaminobenzidine substrate kit (SK-4100; Vector Laborato-
ries), and dehydrated in graded concentrations of ethanol,
cleared in xylene, and mounted with Permount (Fischer Sci-
entific, Fair Lawn, NJ).
Indirect immunofluorescent staining was also used to
detect embryonic MyHC (MyHC-emb) isoforms in the muscle
TABLE 2. Monoclonal Antibody Specificities
MyHC Isoforms
Antibody I IIa IIx MyHC-emb Source
NOQ7-5-4D + j j j Sigma
MY-32 j + + j Sigma
SC-71 j + j j ATCC
F1.652 j j j + ATCC
+, positive reaction between mAb and MyHC; j, negative reaction between antibody
and MyHC.
ATCC: hybridoma cells were purchased from the American Type Culture Collection.
The hybridoma cell culture supernatants of mAbs SC-71 and F1.652 were used.
MyHC, myosin heavy chain; MyHC-emb, embryonic MyHC.
522
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J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012
fibers as described (36Y39). 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 isothiocyanateY
labeled goat anti-mouse IgG (1:50 dilution; Sigma) for 1 hour,
mounted with Vectashield mounting medium (Vector Labora-
tories), and kept in the dark at 4-C. Control sections were
stained as previously mentioned except that the primary anti-
body incubation was omitted.
Muscle Fiber Typing
Fibers previously classified as Type IIB in human mus-
cle 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-32Ypositive and NOQ7-5-4DYnegative) that
failed to react with the mAb specific to Type IIa MyHC iso-
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form (i.e. SC-71Ynegative) 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 differ-
ential 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 mAbs 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 1 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 (NOQ7-5-4DYpositive
MY-32Ynegative fibers), fast Type IIA (SC-71Ypositive, MY-
32Ypositive, NOQ7-5-4DYnegative fibers), fast Type IIX (MY-
32Ypositive, SC-71Ynegative, NOQ7-5-4DYnegative fibers), and
embryonic (F1.652-positive) MyHC-containing fibers. Rela-
tive 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).
Working In each muscle, at least 100 fibers were randomly selected to
Solution measure the least fiber diameter. Myofibers with diameters of
1:1000 20 Km or less were considered to be atrophic.
1:50
Supernatant Data Analysis
Supernatant Histologic and immunohistochemical properties of the
PC and CP muscles in subjects with PD and control subjects
were compared. Relative proportions of denervated and atro-
phic 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
Ó 2012 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012 Pharyngeal Muscles in PD

were reported. Data were analyzed with 3-way repeated-measure diameters and angular small fibers in the muscles of subjects
analysis of variance (ANOVA; repeated factor: fiber type). with PD (Fig. 2).
Subsequently, data from each main fiber type were analyzed Levels of NADH-TR and GPD activities as indicated
with 2-way ANOVA followed by post hoc analysis with Tukey- by reaction product densities varied across fiber types. In the
Kramer adjustment for multiple comparisons. The main fac- muscles of control subjects, Type I fibers had high NADH-TR
tors were group (PD and control) and muscle type (CP and but low GPD, whereas Type II fibers had high GPD but low
IPC). The influence of dysphagia on percentages of atrophic NADH-TR. The reaction product densities were ranked I 9
muscle fibers was evaluated in the PD group with 3-way
repeated-measure ANOVA (main effects: presence of dyspha-
gia, 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 G 0.05.

RESULTS
Demographic Data
In the group of subjects with PD , there were 6 men and
2 women, mean age was 78.1 years (range, 73Y84 years), the
mean age at onset of PD was 61.5 years (range, 45Y70 years),

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and the mean duration of PD at time of death was 16.6 years
(range, 11Y30 years; Table 1). In this group, 6 had developed
dementia. All subjects were white. The mean Hoehn and Yahr
stage was 3.5; 1 subject was stage 2; 2 subjects were stage 3;
and 5 subjects 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. On the basis of
the UPDRS Part II scale, dysphagia occurred in 4 (50%) of the
8 subjects (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, 76Y78 years).

Neuropathologic Findings in Brains With PD

All cases of PD included in this study met neuro-
pathologic criteria for PD based on the examination by T.G.B.
(Table 1). Microscopic examinations revealed that the sub-
stantia 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). Immunohisto-
chemical staining for phosphorylated >-synuclein showed fre-
quent immunoreactive neuronal inclusions and related neurites
in the olfactory bulb, brainstem, amygdala, transentorhinal area,
and cingulate gyrus, with variable densities in the 3 neocor-
tical regions examined (temporal, frontal, and 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 Dis-
orders (43), 6 cases were classified as ‘‘neocortical stage’’ and
2 were ‘‘brainstem and limbic stage.’’ Five cases were de-
mented and met consensus clinicopathologic criteria for Alz-
heimer disease (AD) (44). One other case had dementia on the
basis of progression of PD without concurrent AD.
Histopathologic and Enzyme-Histochemical
Features of Muscles of Subjects With PD
The PC and CP muscles in PD cases showed altered
fiber morphology and enzyme-histochemical activities. Cen-
tral nuclei also were identified mainly in the muscles of sub-
jects with PD. There were considerable variations in fiber
FIGURE 2. (AYD) Serial cross sections of cricopharyngeus (CP)
muscles from a subject with Parkinson disease (PD 1, 75-year-old
male) and an age-matched control (76-year-old male) stained
for NOQ7-5-4D (A, A¶), nicotinamide adenine dinucleotide
tetrazolium reductase (NADH-TR) (B, B¶), MY-32 (C, C¶), and >-
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¶YD¶), 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
(AYD), 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 Km.

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Mu et al
I/IIa 9 IIa 9 IIx for NADH-TR and I G I/IIa G IIa G IIx for
GPD. Pharyngeal muscles in PD cases mostly showed pre-
served common enzyme-histochemical features as seen in
the muscles of control subjects (Fig. 2). However, in the
muscles of subjects with PD, Type I fibers without sig-
nificant 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 muscles of
subjects with PD and of control subjects were immunopositive
for N-CAM protein. Neural CAMYpositive fibers were identi-
fied more frequently in the CP and IPC muscles of subjects
with PD than in those of control subjects. Most of the N-
CAMYpositive fibers in the muscles of subjects with PD were
atrophied (Fig. 3).
Fiber type grouping and myofiber atrophy were more
predominant in the muscles of subjects with PD than in those
of control subjects (Figs. 3Y8). On average, muscle fibers in
the control CP were 37 Km in diameter for slow Type I and
39 Km in diameter for fast Type II fibers. Diameters of IPC
muscle fibers were 38 Km for Type I and 40 Km for Type II
fibers. Atrophied fibers (e20 Km in diameter) in the CP and
IPC muscles of control subjects accounted for 9% and 6% of
the total fiber population, respectively (Fig. 7).
FIGURE 3. Photomicrographs of cross sections of the crico-
pharyngeus (CP) muscles from a subject with Parkinson disease
(PD 3, 78-year-old male; A, B) and a control subject (78-year-
old male; A¶ B¶). (A, A¶) Sections immunostained for neural cell
adhesion molecule (N-CAM) to label denervated muscle fibers.
There are more N-CAMYpositive fibers (arrows) in the CP
muscle of the subject with PD versus that of the control subject.
Most of the N-CAMYpositive fibers in the muscle of the subject
with PD are atrophic. (B, B¶) Sections stained with antiYmyosin
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 CP
muscle of the subject with PD (B). Scale bars = (A¶) 50 Km;
(B¶) 100 Km.
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J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012
FIGURE 4. Photomicrographs of cross sections of the inferior
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pharyngeal constrictor (IPC) muscles from control subjects
(A, B) and subjects with Parkinson disease (PD) (C, D). The
sections were stained with anti-MyHC monoclonal antibody
(mAb) NOQ7-5-4D specific for slow Type I (AYC) 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 subject with PD (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
control subject (B) and subject with PD (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). Most of the
atrophied fibers are Type I fibers and are mainly in the outer
layer (C). Scale bar = (D) 100 Km.
The diameters of the muscle fibers of subjects with PD
were reduced versus those of the controls (Fig. 6). On aver-
age, Type I and Type II fibers were 23 and 36 Km for CP
muscle of subjects with PD and 33 and 35 Km for IPC muscle
of subjects with PD , respectively. The 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 G 0.001; Fig. 7). Comparisons be-
tween the PD and control groups showed that the former had
significantly more atrophied myofibers than the latter (F1,20 =
30.02, p G 0.0001). In the muscles of subjects with PD, most
of the atrophied myofibers were mainly Type I fibers (ap-
proximately 80% of the atrophied fibers; Figs. 2A, 4C, and 5A)
and less in the Type II fiber population (p G 0.0001; Figs. 2C,
4D, 5B, C, and 7). Interestingly, the atrophied myofibers in
the muscles of subjects with PD were more frequent in the
outer layer than in the inner layer of the CP and IPC muscles
(Fig. 4C). The pharyngeal muscles in subjects with PD with
dysphagia contained higher percentages of atrophied myofi-
bers (27.6% of the total fiber population) versus those in
subjects with PD without dysphagia (16% of the total fiber
population; F1,12 = 59.92, p G 0.0001; Fig. 8). Thus, there was
significant fiber atrophy of pharyngeal muscles in subjects
with PD, especially in those with dysphagia.
Ó 2012 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012
FIGURE 5. Immunohistochemistry on serial cross sections of
the cricopharyngeus (CP) muscles from a subject with Parkin-
son disease (PD 8, 75-year-old male; AYD) and a control sub-
ject (76-year-old male; A¶YD¶). Sections were stained with
antiYmyosin heavy chain (MyHC) monoclonal antibodies spe-
cific 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 (AYC, A¶YC¶)
show the major MyHC-containing fibers; sections stained by
indirect immunofluorescence (D, D¶) show embryonic MyHC-
containing fibers. The numbers in the photomicrographs
(AYC¶) 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 Km.
MyHC Transformation and Fiber Type
Distribution in the Muscles of Subjects With PD
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 his-
tochemically 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
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Pharyngeal Muscles in PD
FIGURE 6. (A, B) Bar graphs showing diameter distribution of
the Type I and Type II fibers in the cricopharyngeus (CP; A)
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and inferior pharyngeal constrictor (IPC; B) muscles of subjects
with Parkinson disease (PD) and control subjects.
Type II fibers (38), whereas IPC contained 54% Type I and
46% Type II fibers (45) in normal middle-aged humans.
The CP and IPC muscles of subjects with PD exhibited
marked alterations in the MyHC composition and fiber type
distribution. The inner and outer muscle fiber layers were not
observed in the muscles of PD subjects and senescent control
subjects (Figs. 4BYD) because the fast outer layer had an in-
crease 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 muscles of PD subjects
and control subjects are summarized in Table 3. On average,
the CP muscle of subjects with PD was slower (82% Type I)
than the control (73% Type I; p G 0.014). Similarly, the IPC
muscle of subjects with PD also contained more slow Type I
fibers (72%) compared with the control (61% Type I; p G
0.0036). An increase in percentage of the Type I fibers in the
CP and IPC muscles of subjects with PD accompanied with a
concomitant decrease in percentages of both the Type I/IIA and
FIGURE 7. Relative proportions of atrophic Type I and Type II
fibers in the cricopharyngeus (CP) and inferior pharyngeal con-
strictor (IPC) muscles of subjects with Parkinson disease (PD) and
control subjects.
525
Mu et al
FIGURE 8. Percentages of atrophic Type I and Type II fibers in
the cricopharyngeus (CP) and inferior pharyngeal constrictor
(IPC) muscles in subjects with Parkinson disease (PD) with
dysphagia (D) and without dysphagia (N).
IIA fibers (Table 3), indicating that PD induced fast-to-slow
MyHC isoform transformation in these muscles.
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 pathologic changes, including the pres-
ence of considerable denervated and atrophied fibers, angular
small fibers, centrally placed nuclei, and large variations in
fiber size. Second, fiber type grouping and atrophied myofi-
bers were mainly Type I fibers. Third, PD was associated with
fast-to-slow MyHC transformation and altered fiber type dis-
tribution. Specifically, the pharyngeal muscles in PD had a slow
phenotype compared with the controls. Finally, pharyngeal
muscles in subjects with PD with dysphagia contained more
atrophied myofibers than those in subjects with PD without
dysphagia.
Dysphagia resulting from neurologic disorders includ-
ing 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 patients with PD include inefficient mastica-
tion, impaired bolus formation, and delayed initiation of swal-
lowing (11, 20). Impaired mastication and oral preparatory
lingual movements are most commonly seen during dynamic
videofluoroscopy (11) and may be caused by PD-related aki-
TABLE 3. Percent Composition of Major Myosin Heavy ChainYBased Fiber Types in Muscles of Subjects With Parkinson Disease
and Control Subjects
Cricopharyngeal Sphincter Muscles
MyHC I IIA I/IIA
PD 82 (77Y86) 11 (8Y16) 5 (3Y8)
Control 73 (63Y78) 12 (8Y15) 14 (10Y16)
Values are percent (range).
MyHC, myosin heavy chain; PD, Parkinson disease.
526
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012
nesia, bradykinesia, and rigidity (48). Impaired pharyngeal
bolus transport is the major determinant of dysphagia and
pharyngeal phase abnormalities in PD include delayed phar-
yngeal swallowing reflex, vallecula and pyriform sinus pool-
ing, 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 patients with PD with dysphagia and, thus,
a major factor in the pathogenesis of dysphagia in some patients
with PD (51, 52).
The precise mechanisms of dysphagia in PD remain
unclear. There is a general belief that dysphagia in PD is as-
sociated with akinesia and rigidity of oropharyngeal muscles
by impaired basal ganglia motor control (11, 15, 53). Dysfunc-
tion 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). Pre-
vious studies using simultaneous videoradiography and phar-
yngeal manometry found no correlation between overall muscle
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rigidity score and abnormal pharyngeal wall motion in patients
with PD (49). Importantly, anti-PD drugs and surgical inter-
ventions (pallidotomy, thalamotomy, and deep brain stimula-
tion), 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 treat-
ment 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 investiga-
tors suggested that other neurotransmitter systems (8, 50, 56)
or some other nondopaminergic mechanisms (8) may also be
involved. In addition to degeneration of the nigrostriatal dopa-
minergic pathway, a variety of neuronal systems are likely
involved in PD (57), causing multiple neuromediator dys-
functions that account for the complex patterns of functional
deficits.
Previous histochemical studies have demonstrated atro-
phic and hypertrophic fibers and fiber type grouping in limb
muscles in patients with PD (22, 23). However, changes in the
muscle fibers seem 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
patients with PD with very severe akinesia and long disease
duration. Normal or slightly hypertrophic Type I fibers were
identified in patients with PD with marked rigidity, whereas
atrophic Type I fibers were found in patients with PD with
prevalent akinesia and slight rigidity (22). These findings
Inferior Pharyngeal Constrictor Muscles
IIX I IIA I/IIA IIX
2 (0Y4) 72 (65Y76) 18 (14Y25) 6 (2Y8) 4 (1Y7)
1 (0Y3) 61 (57Y66) 32 (26Y35) 4 (1Y7) 3 (0Y6)
Ó 2012 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol  Volume 71, Number 6, June 2012
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 re-
main more inactive, an effect due to the rigidity and akinesia
(58, 59). However, others have speculated that the muscle fi-
ber 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 mor-
phologic profiles, enzyme levels, fiber type and MyHC com-
position, and contractile properties in response to various
physiological and pathophysiological stimuli. However, unlike
in limb muscles, we found that most 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 num-
ber of morphologic and immunohistochemical changes asso-
ciated 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.
Parkinson disease is a multisystem disease (62). Sen-
sory and autonomic disturbances are part of the clinical pic-
ture of PD and electrophysiological studies have shown that
peripheral neuropathy is present in large proportions of pa-
tients with PD (63Y66). 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 patients with PD, >-synuclein is deposited in the periph-
eral autonomic nervous system, including in the cardiac plexus
(68Y70), enteric nervous system of the alimentary tract (71Y73),
and skin (74Y78). Peripheral sensory nerve involvement in PD
has also been demonstrated by functional and histochemical
assessments of cutaneous sensory nerve endings. The cutane-
ous denervation and the sensory impairment in PD were indi-
cated by significant increase in tactile and thermal thresholds,
a reduction in mechanical pain perception, and significant epi-
dermal nerve fiber loss (75, 79).
Disability in PD is mostly due to motor impairment (80,
81). Electrophysiological abnormalities such as prolonged la-
tencies and smaller nerve action potentials in both sensory and
motor nerves of arms and legs were detected in some patients
with PD (66). Electromyographic recordings showed dener-
vation signs in some limb muscles in PD (82). Pharynx and
larynx play an important role in swallowing and airway pro-
tection. 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 approx-
imately 80% of patients with PD have vocal difficulties (84),
which are most likely associated with incomplete closure of
the vocal cords during phonation caused by vocal cord atro-
phy 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 innervat-
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Pharyngeal Muscles in PD
ing the muscles. Pharyngeal and laryngeal muscles receive
their motor innervation from CN X, and several studies have
demonstrated >-synuclein in fibers in the cervical peripheral X
nerve in PD (73, 87Y89). 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 subjects with PD were immunoreactive
for phosphorylated >-synuclein, suggesting a possible mech-
anism for muscle denervation.
Further evidence for limited muscle denervation in PD
has come from electromyographic studies (90Y94). We have
demonstrated that normal adult human CP and PC muscles are
composed of a slow inner layer and a fast outer layer that re-
ceive their motor innervation from CN IX and CN X, re-
spectively (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 pha-
ryngeal muscles, suggesting that the neuromuscular system
controlled by CN X is significantly affected by the neuro-
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pathologic process of PD. Lewy neurites have been identified
in CN X in PD (73, 87Y89). Because CN X contains sym-
pathetic, parasympathetic, sensory, and motor axons, it is not
known whether the >-synuclein pathology in this nerve oc-
curs 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 phenom-
enon. In the PD pharyngeal muscles, decrease in muscle fiber
size, reduction in the number of fast Type II fibers, and in-
crease 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 fac-
tors influencing the fiber type and MyHC composition as well
as morphologic profile of muscle fibers. In general, decreased
neuromuscular activation, which is usually caused by neuro-
logic diseases, denervation or disuse, results in muscle fiber
atrophy and fiber type and MyHC transformation. MyHC con-
version could lead to fast-to-slow shift in the fast-twitch mus-
cle (96) or fast muscle region such as fast outer layer in the
pharyngeal muscles as demonstrated by this study. These neu-
romuscular alterations in the PD pharynx are most likely
caused by motor abnormalities at the levels of central moto-
neurons and peripheral nerves and lead to dysphagia. Thus,
oropharyngeal dysphagia in patients with PD 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 re-
spond 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 explor-
ing PD-related pathophysiological, immunohistochemical, and
biochemical changes at the peripheral level and oropharyngeal
527
Mu et al
dysphagia in PD is most likely multifactorial. In view of
studies indicating the presence of Lewy neurites in CN X (73,
87Y89), as well as in sciatic nerve (89) in patients with PD,
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 relax-
ation of the upper esophageal sphincter (97, 98). Relaxation
abnormalities of the upper esophageal sphincter in patients
with PD (49, 99) are caused by reflex neural dysfunctions.
Patients with PD 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 mo-
tor and sensory nervous systems and their contribution to oro-
pharyngeal dysphagia.
ACKNOWLEDGMENTS
The authors thank 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 clin-
ical and neuropathologic data from PD subjects.
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