Mechanisms of Swallowing, Speech and Voice Disorders in Parkinson's Disease: Literature Review with Our First Evidence for the Periperal Nervous System Involvement.

The majority of patients with Parkinson's disease (PD) develop swallowing, speech, and voice (SSV) disorders. Importantly, swallowing difficulty or dysphagia and related aspiration are life-threatening conditions for PD patients. Although PD treatments have significant therapeutic effects on limb motor function, their effects on SSV disorders are less impressive. A large gap in our knowledge is that the mechanisms of SSV disorders in PD are poorly understood. PD was long considered to be a central nervous system disorder caused by the death of dopaminergic neurons in the basal ganglia. Aggregates of phosphorylated α-synuclein (PAS) underlie PD pathology. SSV disorders were thought to be caused by the same dopaminergic problem as those causing impaired limb movement; however, there is little evidence to support this. The pharynx, larynx, and tongue play a critical role in performing upper airway (UA) motor tasks and their dysfunction results in disordered SSV. This review aims to provide an overview on the neuromuscular organization patterns, functions of the UA structures, clinical features of SSV disorders, and gaps in knowledge regarding the pathophysiology underlying SSV disorders in PD, and evidence supporting the hypothesis that SSV disorders in PD could be associated, at least in part, with PAS damage to the peripheral nervous system controlling the UA structures. Determining the presence and distribution of PAS lesions in the pharynx, larynx, and tongue will facilitate the identification of peripheral therapeutic targets and set a foundation for the development of new therapies to treat SSV disorders in PD.

Focal Application of Neurotrophic Factors Augments Outcomes of Nerve-Muscle-Endplate Grafting Technique for Limb Muscle Reinnervation.

BACKGROUND: We have developed a novel muscle reinnervation technique called "nerve-muscle-endplate grafting (NMEG) in the native motor zone (NMZ)." This study aimed to augment the outcomes of the NMEG-NMZ (NN) by focal application of exogenous neurotrophic factors (ENFs) for limb reinnervation. METHODS: Adult rats were used to conduct NN plus ENF (NN/ENF) and autologous nerve grafting (ANG, technique control). The nerve innervating the left tibialis anterior (TA) muscle was resected and the denervated TA was immediately treated with NN/ENF or ANG. For NN procedure, an NMEG pedicle was taken from the lateral gastrocnemius muscle and transferred to the NMZ of the denervated TA. For ANG, the nerve gap was bridged with sural nerve. Three months after treatment, the extent of functional and neuromuscular recovery was assessed by measuring static toe spread, maximal muscle force, wet muscle weight, regenerated axons, and innervated motor endplates (MEPs). RESULTS: NN/ENF resulted in 90% muscle force recovery of the treated TA, which is far superior to ANG (46%) and NN alone (79%) as reported elsewhere. Toe spread recovered up to 89 and 49% of the control for the NN/ENF and ANG groups, respectively. The average wet muscle weight was 87 and 52% of the control for muscles treated with NN/ENF and ANG, respectively. The mean number of the regenerated axons was 88% of the control for the muscles treated with NN/ENF, which was significantly larger than that for the ANG-repaired muscles (39%). The average percentage of the innervated MEPs in the NN/ENF-treated TA (89%) was higher compared with that in the ANG-repaired TA (48%). CONCLUSION: ENF enhances nerve regeneration and MEP reinnervation that further augment outcomes of NN. The NN technique could be an alternative option to treat denervated or paralyzed limb muscles caused by traumatic nerve injuries or lesions.

Neuromuscular Specializations of the Human Hypopharyngeal Muscles.

The hypopharyngeal muscles in humans play a vital role in swallowing, speech, and respiration. Increasing evidence indicates that these muscles are specialized to perform life-sustaining upper aerodigestive functions. This review aims to provide current knowledge regarding the key structural, physiological, and biochemical features of the hypopharyngeal muscles, including innervation, contractile properties, histochemistry, biochemical properties, myosin heavy chain (MyHC) expression and regulation, and age-related alterations. These would clarify the unique neuromuscular specializations of the human hypopharyngeal muscles for a better understanding of the functions and pathological conditions of the pharynx and for the development of novel therapies to treat related upper airway disorders.

Nerve growth factor and basic fibroblast growth factor promote reinnervation by nerve-muscle-endplate grafting.

INTRODUCTION: This study was designed to test whether exogenous application of nerve growth factor (NGF) and basic fibroblast growth factor (FGF-2) to muscles reinnervated with nerve-muscle-endplate band grafting (NMEG) could promote specific outcomes. METHODS: The right sternomastoid muscle in adult rats was experimentally denervated and immediately reinnervated by implanting an NMEG pedicle from the ipsilateral sternohyoid muscle. A fibrin sealant containing NGF and FGF-2 was focally applied to the implantation site. Maximal tetanic force, muscle weight, regenerated axons, and motor endplates were analyzed 3 months after treatment. RESULTS: Mean tetanic force, wet muscle weight, and number of regenerated axons in the treated muscles were 91%, 92%, and 84% of the contralateral controls, respectively. The majority of endplates (86%) in the treated muscles were reinnervated by regenerated axons. DISCUSSION: Focal administration of NGF and FGF-2 promotes efficacy of the NMEG technique. Muscle Nerve 57: 449-459, 2018.

Immunohistochemical Detection of Motor Endplates in the Long-Term Denervated Muscle.

BACKGROUND: We have demonstrated that the native motor zone (NMZ) within a muscle is an ideal target for performing nerve-muscle-endplate band grafting (NMEG) to restore motor function of a denervated muscle. This study was designed to determine spatiotemporal alterations of the myofibers, motor endplates (MEPs), and axons in the NMZ of long-term denervated muscles for exploring if NMEG-NMZ technique would have the potential for delayed reinnervation. METHODS: Sternomastoid (SM) muscles of adult female Sprague-Dawley rats (n = 21) were experimentally denervated and denervation-induced changes in muscle weight, myofiber size, MEPs, and intramuscular nerve axons were evaluated histomorphometrically and immunohistochemically at the end of 3, 6, and 9 months after denervation. The values obtained from the ipsilateral normal side served as control. RESULTS: The denervated SM muscles exhibited a progressive reduction in muscle weight (38%, 31%, and 19% of the control) and fiber diameter (52%, 40%, and 28% of the control) for 3-, 6-, and 9-month denervation, respectively. The denervated MEPs were still detectable even 9 months after denervation. The mean number of the denervated MEPs was 79%, 65%, and 43% of the control in the 3-, 6-, and 9-month denervated SM, respectively. Degenerated axons in the denervated muscles became fragmented. CONCLUSIONS: Persistence of MEPs in the long-term denervated SM suggests that some surgeries targeting the MEPs such as NMEG-NMZ technique should be effective for delayed reinnervation. However, more work is needed to develop strategies for preservation of muscle mass and MEPs after denervation.

Outcomes of Muscle Reinnervation with Direct Nerve Implantation into the Native Motor Zone of the Target Muscle.

Background Our recent work has demonstrated that the native motor zone (NMZ) within a given skeletal muscle is the best site for muscle reinnervation. This study was designed to explore the outcomes of direct nerve implantation (DNI) into the NMZ of denervated sternomastoid (SM) muscle in a rat model. Methods The right SM muscle was experimentally denervated by transecting its innervating nerve. The proximal stump of the severed SM nerve was immediately implanted into a small muscle slit made in the NMZ of the muscle where denervated motor endplates were concentrated. The outcomes of DNI-NMZ reinnervation were evaluated 3 months after surgery. Specifically, the degree of functional recovery was examined with muscle force measurement. The extent of nerve regeneration and endplate reinnervation was assessed using histological and immunohistochemical methods. Results This study showed that the mean muscle force of the treated muscles was 64% of the contralateral control. Reinnervated SM muscles weighed 71% of the weight of the control muscles. Abundant regenerated axons were identified in the NMZ of the target muscle. The mean number and area of the regenerated axons in the treated muscles was computed to be 62% and 51% of the control muscles, respectively. On average, 66% of the denervated endplates in the treated muscles were reinnervated by regenerated axons. Conclusion Our results suggest that the NMZ within a muscle is an ideal site for endplate reinnervation and satisfactory functional recovery. Further studies are needed to promote the efficacy of DNI-NMZ technique for muscle reinnervation.

Muscle reinnervation with nerve-muscle-endplate band grafting technique: correlation between force recovery and axonal regeneration.

BACKGROUND: This study was designed to determine the correlation between functional recovery and the extent of axonal regeneration after muscle reinnervation with our recently developed nerve-muscle-endplate band grafting (NMEG) technique in a rat model. MATERIALS AND METHODS: The right experimentally paralyzed sternomastoid (SM) muscle by nerve transection was immediately reinnervated with an NMEG pedicle harvested from a neighboring sternohyoid muscle. The NMEG pedicle contained a muscle block (6 × 6 × 3 mm), a donor nerve branch with nerve terminals, and a motor endplate band. Three months after surgery, the tetanic force of the SM muscle was measured and the regenerated axons in the muscle were detected using neurofilament immunohistochemistry. RESULTS: The results showed that the maximal tetanic force (a measure of muscle functional recovery) of the NMEG-reinnervated SM muscle reached up to 66.0% of the normal control. The wet weight of the reinnervated SM muscle (a measure of muscle mass recovery) was 87.2% of the control. The area fraction of the regenerating axons visualized with neurofilament staining within the NMEG-reinnervated SM muscle (a measure of muscle reinnervation) was 42.3%. A positive correlation was revealed between the extent of muscle reinnervation and maximal muscle force. CONCLUSIONS: Our newly developed NMEG technique results in satisfactory functional outcomes and nerve regeneration. Further improvement in the functional recovery after NMEG reinnervation could be achieved by refining the surgical procedure and creating an ideal environment that favors axon-endplate connections and accelerates axonal growth and sprouting.

Alpha-Synuclein Pathology in Sensory Nerve Terminals of the Upper Aerodigestive Tract of Parkinson's Disease Patients.

Dysphagia is common in Parkinson's disease (PD) and causes significant morbidity and mortality. PD dysphagia has usually been explained as dysfunction of central motor control, much like other motor symptoms that are characteristic of the disease. However, PD dysphagia does not correlate with severity of motor symptoms nor does it respond to motor therapies. It is known that PD patients have sensory deficits in the pharynx, and that impaired sensation may contribute to dysphagia. However, the underlying cause of the pharyngeal sensory deficits in PD is not known. We hypothesized that PD dysphagia with sensory deficits may be due to degeneration of the sensory nerve terminals in the upper aerodigestive tract (UAT). We have previously shown that Lewy-type synucleinopathy (LTS) is present in the main pharyngeal sensory nerves of PD patients, but not in controls. In this study, the sensory terminals in UAT mucosa were studied to discern the presence and distribution of LTS. Whole-mount specimens (tongue-pharynx-larynx-upper esophagus) were obtained from 10 deceased human subjects with clinically diagnosed and neuropathologically confirmed PD (five with dysphagia and five without) and four age-matched healthy controls. Samples were taken from six sites and immunostained for phosphorylated α-synuclein (PAS). The results showed the presence of PAS-immunoreactive (PAS-ir) axons in all the PD subjects and in none of the controls. Notably, PD patients with dysphagia had more PAS-ir axons in the regions that are critical for initiating the swallowing reflex. These findings suggest that Lewy pathology affects mucosal sensory axons in specific regions of the UAT and may be related to PD dysphagia.

Comparison of muscle force after immediate and delayed reinnervation using nerve-muscle-endplate band grafting.

BACKGROUND: Because of poor functional outcomes of currently used reinnervation methods, we developed novel treatment strategy for the restoration of paralyzed muscles-the nerve-muscle-endplate band grafting (NMEG) technique. The graft was obtained from the sternohyoid muscle (donor) and implanted into the ipsilateral paralyzed sternomastoid (SM) muscle (recipient). METHODS: Rats were subjected to immediate or delayed (1 or 3 mo) reinnervation of the experimentally paralyzed SM muscles using the NMEG technique or the conventionally used nerve end-to-end anastomosis. The SM muscle at the opposite side served as a normal control. RESULTS: NMEG produced better recovery of muscle force as compared with end-to-end anastomosis. A larger force produced by NMEG was most evident for small stimulation currents. CONCLUSIONS: The NMEG technique holds great potential for successful muscle reinnervation. We hypothesize that even better muscle reinnervation and functional recovery could be achieved with further improvement of the environment that favors axon-end plate connections and accelerates axonal growth and sprouting.

Force recovery and axonal regeneration of the sternomastoid muscle reinnervated with the end-to-end nerve anastomosis.

BACKGROUND: End-to-end nerve anastomosis (EEA) is a commonly used nerve repair technique. However, this method generally results in poor functional recovery. This study was designed to determine the correlation of functional recovery to the extent of axonal reinnervation after EEA procedure in a rat model. MATERIALS AND METHODS: Seven adult rats were subjected to the immediate reinnervation of an experimentally paralyzed sternomastoid (SM) muscle. The SM nerve was transected and immediately repaired with EEA. The SM muscle at the opposite side, without nerve transection, served as a control. Three months after EEA nerve repair, the muscle force of the SM muscle was measured and the regenerated axons in the muscle were detected using neurofilament immunohistochemistry. RESULTS: Three months after surgery, the reinnervated SM muscle produced limited anatomical and functional recovery (calculated as the percentage of the control). Specifically, the wet weight of the operated SM muscle (a measure of muscle mass recovery) was 78.0% of the control. The maximal tetanic force (a measure of muscle functional recovery) was 56.7% of the control. The area fraction of the neurofilament stained intramuscular axons (a measure of axonal regeneration and muscle reinnervation) was measured to be only 13.4% of the control. A positive correlation was revealed between the extent of muscle reinnervation and maximal muscle force. CONCLUSIONS: The EEA reinnervated SM muscle in the rat yielded unsatisfactory muscle force recovery as a result of mild to moderate nerve regeneration. Further work is needed to improve the surgical procedure, enhance axonal regeneration, and/or develop novel treatment strategies for better functional recovery.

Reinnervation of Paralyzed Limb Muscle by Nerve-Muscle-Endplate Grafting Technique.

BACKGROUND: We have developed a novel reinnervation technique called nerve-muscle-endplate grafting in the native motor zone (NMEG-NMZ). However, it remains unknown whether the NMEG-NMZ is effective for limb reinnervation. OBJECTIVE: To evaluate the efficacy of the NMEG-NMZ in limb muscle reinnervation. METHODS: Forty-five adult rats were divided into 3 groups: NMEG, end-to-end anastomosis (EEA, technique control), and denervation control (DC). The left tibialis anterior muscle was denervated by resecting its nerve. For NMEG-NMZ, the denervated tibialis anterior was reinnervated by transferring a NMEG pedicle from the lateral gastrocnemius muscle. Three months after surgery, static toe spread analysis was performed for all rats and muscle force was measured for the rats treated with NMEG and EEA. Muscle weight, myofiber morphology, regenerated axons, and reinnervated motor endplates in the treated muscles were also quantified and compared with those in the DC group. RESULTS: NMEG-NMZ technique resulted in better muscle force recovery (79% of the control) compared with EEA (51% of the control, P = .048). Toe spread analysis in NMEG-NMZ reinnervated muscles showed static sciatic index = -16.8, whereas -41.4 in EEA, P < .0001). The average weight of the NMEG-NMZ reinnervated muscles (86%) was greater than those of the EEA treated (71%) and DC (26%) muscles (all P < .0001). The mean count of the regenerated axons in the muscles with NMEG-NMZ was 76% of the control, which was larger than that in the muscles with EEA (46%), P < .0001. CONCLUSION: NMEG-NMZ technique has unique advantages and is superior to EEA for muscle reinnervation and functional recovery.

Morphometric and Immunohistochemical Characteristics of the Adult Human Soft Palate Muscles.

The soft palate is the only structure that reversibly separates the respiratory and gastrointestinal systems. Most species can eat and breathe at the same time. Humans cannot do this and malfunction of the soft palate may allow food to enter the lungs and cause fatal aspiration pneumonia. Speech is the most defining characteristic of humans and the soft palate, along with the larynx and tongue, plays the key roles. In addition, palatal muscles are involved in snoring and obstructive sleep apnea. Considering the significance of the soft palate, its function is insufficiently understood. The objectives of this study were to document morphometric and immunohistochemical characteristics of adult human soft palate muscles, including fiber size, the fiber type, and myosin heavy chain (MyHC) composition for better understanding muscle functions. In this study, 15 soft palates were obtained from human autopsies. The palatal muscles were separated, cryosectioned, and stained using histological and immunohistochemical techniques. The results showed that there was a fast type II predominance in the musculus uvulae and palatopharyngeus and a slow type I predominance in the levator veli palatine. Approximately equal proportions of type I and type II fibers existed in both the palatoglossus and tensor veli palatine. Soft palate muscles also contained hybrid fibers and some specialized myofibers expressing slow-tonic and embryonic MyHC isoforms. These findings would help better understand muscle functions.

Force characteristics of the rat sternomastoid muscle reinnervated with end-to-end nerve repair.

The goal of this study was to establish force data for the rat sternomastoid (SM) muscle after reinnervation with nerve end-to-end anastomosis (EEA), which could be used as a baseline for evaluating the efficacy of new reinnervation techniques. The SM muscle on one side was paralyzed by transecting its nerve and then EEA was performed at different time points: immediate EEA, 1-month and 3-month delay EEA. At the end of 3-month recovery period, the magnitude of functional recovery of the reinnervated SM muscle was evaluated by measuring muscle force and comparing with the force of the contralateral control muscle. Our results demonstrated that the immediately reinnervated SM produced approximately 60% of the maximal tetanic force of the control. The SM with delayed nerve repair yielded approximately 40% of the maximal force. Suboptimal recovery of muscle force after EEA demonstrates the importance of developing alternative surgical techniques to treat muscle paralysis.

Limb Muscle Reinnervation with the Nerve-Muscle-Endplate Grafting Technique: An Anatomical Feasibility Study.

BACKGROUND: Peroneal nerve injuries results in tibialis anterior (TA) muscle paralysis. TA paralysis could cause "foot drop," a disabling condition that can make walking difficult. As current treatment methods result in poor functional recovery, novel treatment approaches need to be studied. The aim of this study was to explore anatomical feasibility of limb reinnervation with our recently developed nerve-muscle-endplate grafting (NMEG) in the native motor zone (NMZ). METHODS: As the NMEG-NMZ technique involves in nerves and motor endplates (MEPs), the nerve supply patterns and locations of the MEP bands within the gastrocnemius (GM) and TA muscles of rats were investigated using Sihler's stain and whole-mount acetylcholinesterase (AChE) staining, respectively. Five adult rats underwent TA nerve transaction. The denervated TA was reinnervated by transferring an NMEG pedicle from the ipsilateral lateral GM. At the end of a 3-month recovery period, maximal muscle force was measured to document functional recovery. RESULTS: The results showed that the TA was innervated by the deep peroneal nerve. A single MEP band was located obliquely in the middle of the TA. The GM was composed of two neuromuscular compartments, lateral (GM-l) and medial (GM-m), each of which was innervated by a separate nerve branch derived from the tibial nerve and had a vertically positioned MEP band. The locations of MEP bands in the GM and TA muscles and nerve supply patterns demonstrated that an NMEG pedicle can be harvested from the GM-l and implanted into the NMZ within the TA muscle. The NMEG-NMZ pilot study showed that this technique resulted in optimal muscle force recovery. CONCLUSION: NMEG-NMZ surgery is feasible for limb reinnervation. Specifically, the denervated TA caused by peroneal nerve injuries can be reinnervated with a NMEG from the GM-l.

Innervation of human soft palate muscles.

Our objective was to determine the branching and distribution of the motor nerves supplying the human soft palate muscles. Six adult specimens of the soft palate in continuity with the pharynx, larynx, and tongue were processed with Sihler's stain, a technique that can render large specimens transparent while counterstaining their nerves. The cranial nerves were identified and dissection followed their branches as they divided into smaller divisions toward their terminations in individual muscles. The results showed that both the glossopharyngeal (IX) and vagus (X) nerves have three distinct branches, superior, middle, and inferior. Only the middle branches of each nerve contributed to the pharyngeal plexus to which the facial nerve also contributed. The pharyngeal plexus was divided into two parts, a superior innervating the palatal and neighboring muscles and an inferior innervating pharyngeal constrictors. The superior branches of the IX and X nerves contributed innervation to the palatoglossus, whereas their middle branches innervated the palatopharyngeus. The palatoglossus and palatopharyngeus muscles appeared to be composed of at least two neuromuscular compartments. The lesser palatine nerve not only supplied the palatal mucosa and palatine glandular tissue but also innervated the musculus uvulae, palatopharyngeus, and levator veli palatine. The latter muscle also received its innervation from the superior branch of X nerve. The findings would be useful for better understanding the neural control of the soft palate and for developing novel neuromodulation therapies to treat certain upper airway disorders such as obstructive sleep apnea.

Characteristics of tetanic force produced by the sternomastoid muscle of the rat.

The sternomastoid (SM) muscle plays an important role in supporting breathing. It also has unique anatomical advantages that allow its wide use in head and neck tissue reconstruction and muscle reinnervation. However, little is known about its contractile properties. The experiments were run on rats and designed to determine in vivo the relationship between muscle force (active muscle contraction to electrical stimulation) with passive tension (passive force changing muscle length) and two parameters (intensity and frequency) of electrical stimulation. The threshold current for initiating noticeable muscle contraction was 0.03 mA. Maximal muscle force (0.94 N) was produced by using moderate muscle length/tension (28 mm/0.08 N), 0.2 mA stimulation current, and 150 Hz stimulation frequency. These data are important not only to better understand the contractile properties of the rat SM muscle, but also to provide normative values which are critical to reliably assess the extent of functional recovery following muscle reinnervation.

Human tongue neuroanatomy: Nerve supply and motor endplates.

The human tongue has a critical role in speech, swallowing, and respiration, however, its motor control is poorly understood. Fundamental gaps include detailed information on the course of the hypoglossal (XII) nerve within the tongue, the branches of the XII nerve within each tongue muscle, and the type and arrangement of motor endplates (MEP) within each muscle. In this study, five adult human tongues were processed with Sihler's stain, a whole-mount nerve staining technique, to map out the entire intra-lingual course of the XII nerve and its branches. An additional five specimens were microdissected into individual muscles and stained with acetylcholinesterase and silver staining to study their MEP morphology and banding patterns. Using these techniques the course of the entire XII nerve was mapped from the main nerve to the smallest intramuscular branches. It was found that the human tongue innervation is extremely dense and complex. Although the basic mammalian pattern of XII is conserved in humans, there are notable differences. In addition, many muscle fibers contained multiple en grappe MEP, suggesting that they are some variant of the highly specialized slow tonic muscle fiber type. The transverse muscle group that comprises the core of the tongue appears to have the most complex innervation and has the highest percentage of en grappe MEP. In summary, the innervation of the human tongue has specializations not reported in other mammalian tongues, including nonhuman primates. These specializations appear to allow for fine motor control of tongue shape.

Sensory Innervation of the Human Soft Palate.

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc.