Mechanisms of Axonal Dysfunction in Facial Nerve Disorders

Mechanisms of axonal dysfunction in facial nerve disorders

Timothy James Eviston

thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy

Faculty of Medicine University of New South Wales 2016 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Eviston

First name: Timothy Other name/s: James MDCN9001 Abbreviation for degree as given in the University calendar:

School: Prince of wales clinical school Faculty: Medicine

Title: Mechanisms of axonal dysfunction in facial nerve disorders

Abstract 350 words maximum: (PLEASE TYPE)

Facial palsy is a debilitating condition which has a dramatic impact on aesthetic appearance, quality of life and social

interaction. Significant uncertainty still exists for clinicians around how to diagnose a cause, determine an accurate

prognosis, when to time interventions and how best to optimise recovery. The underlying pathophysiology of nerve

dysfunction and associated phenomena such as synkinesis (mass movement of muscle groups) and hypertonicity is also poorly understood. This thesis explores the development and application of new neurophysiology techniques to advance the understanding of how ion channel activity and functional axonal properties change in the context of

disease states. These findings are then analysed in the context of recent advances in the understanding of axonal

neurobiology and cell degeneration pathways to enable new perspectives on this important condition.

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Acknowledgments

Four years ago I met Professor Arun Krishnan to discuss the possibility of pursuing a PhD under his supervision. My thinking at the time was that, as someone with a surgical background, I needed a supervisor with expertise in clinical neurology and neuroscience if I were going to successfully translate nerve injury expertise into the surgical context and contribute a new perspective to the treatment of facial nerve disorders into the future. My criteria for choosing a supervisor were that they have integrity, be world-class, and be patient focused. Professor Krishnan was the perfect supervisor. I was given freedom to explore, experiment and pursue my areas of interest while having the right amount of guidance to keep me heading in the right direction. Thank you Arun for your leadership, judgement, kindness and mentorship.

To Natalie Kwai, Ria Arnold, Jenna Murray and William Huynh, thank you for welcoming me as one of the team. I felt at home from day one and I am constantly inspired by your devotion and passion for research.

To Jonathan Clark, John McGuinness and Bruce Ashford, your mentorship over the years has inspired me to be a better person, to put patients and the community first, and to work hard to be a better surgeon.

Glen Croxson, Sue Coulson, Lauren Chong and the Sydney Facial Nerve team, it has been a privilege working with you as we see our fledgling service grow. I am deeply thankful for your support and friendship and I hope many more PhD’s come out of our clinic in the future.

I am eternally grateful to my patients and the volunteers who took part. The sacrifices you have made to be involved in this research endeavor are greatly appreciated.

I am grateful to the National Health and Medical Research Council and to UNSW for supporting me while I undertake this research endeavor.

To my family and friends, thank you for your enduring support.

Finally, to my wife, Grace, thank you for your love, patience and support. This PhD was made possible because of you.

Abstract

This thesis investigated the underlying physiology of the human facial nerve in health and disease. This involved the translation and optimisation of techniques for determining axonal excitability measures in the human facial nerve. The techniques have proven to be valuable in determining pathophysiology in neuropathies and it was hypothesised that the information they provide may be helpful in understanding facial nerv disorders. The initial studies developed the technique in healthy controls and established normative data (Chapter 1). The initial study in a disease grou (Chapter 2) was targeted at a broad cross-section of facial palsy patients to explore the utility of the technique. Findings consistent with membrane hyperpolarisation were demonstrated. Subsequent studies examined specific disease groups including Bell’s palsy (Chapter 3), facial synkinesis (Chapter 4), and assessment of facial nerve function of the contralateral face to facial palsy (Chapter 5). Changes consistent with reduced inward sodium conductances were noted in Bell’s palsy and these occurred in a pattern that has been noted with tetrodotoxin ingestion. In patients with facial synkinesis, changes in excitability were observed that would be consistent with axonal membrane depolarisation and which may predispose to ectopic motor activity. Altered facial nerve function was also noted in recordings from the contralateral face in patients who had experienced facial palsy and the pattern of change varied accordin to the severity of facial palsy. These changes provide physiological support for treatment of the contralateral face with chemodenervation in patients with facial palsy. 4

Table of Contents

Acknowledgments 2

Abstract 3

Publications and presentations 5

Abbreviations 8

Literature review 9

Chapter 1: Assessment of axonal excitability properties in two

branches of the human facial nerve 58

Chapter 2: Altered axonal excitability in chronic facial palsy 77

Chapter 3: Evidence of sodium channel dysfunction in Bell’s palsy 89

Chapter 4: Axonal dysfunction in facial synkinesis 108

Chapter 5: Axonal abnormalities in the contralateral face in the

setting of facial palsy 120

Summary and Conclusions 135

References 138

Publications and presentations

Articles submitted for review

Chapter 2: Eviston TJ, Chong L, Clark JR, Krishnan AV. Altered axonal excitability properties i facial palsy. Muscle and Nerve, submitted August 29 2016

Chapter 3: Eviston TJ, Krishnan AV, Evidence of sodium channel dysfunction in Bell’s palsy, Brain, submitted August 27 2016

Chapter 4: Eviston TJ, Chong L, Clark JR, Krishnan AV. Axonal dysfunction in facial synkinesis. Movement Disorders, submitted August 31

Published works

Literature review: Eviston TJ, Croxson GR, Kennedy PGE, Hadlock T, Krishnan AV. (2015). Bell's palsy: aetiology, clinical features and multidisciplinary care. Journal of Neurology, Neurosurgery, and Psychiatry, 86(12), 1356–1361.

Chapter 1: Eviston TJ, Krishnan AV. (2016) Assessment of axonal excitability properties in two branches of the human facial nerve. Journal of Neuroscience Methods, 274, 53-60.

Associated publications

Eviston TJ, Yabe TE, Gupta R, Ebrahimi A, Clark JR. (2016). Parotidectomy: surgery in evolution. ANZ Journal of Surgery, 86(3), 193–199. 6

Pham M, Eviston TJ, and Clark JR. (2016), Reconstruction of limited parotidectomy defects using the dermofat graft. ANZ Journal of Surgery. (in press)

Luk, PP, Weston JD, Yu B, Selinger CI, Ekmejian R, Eviston TJ, Lum T, Gao K, Boyer M, O'Toole SA, Clark JR, and Gupta R (2016), Salivary duct carcinoma: Clinicopathologic features, morphologic spectrum, and somatic mutations. Head Neck, 38: E1838–E1847.

Hasmat S, Lovell NH, Eviston TJ, Ekmejian, Suaning GJ and J. Clark, (2015) Creating eye closure in patients with facial nerve paralysis using an implantable solenoid actuator, 37th Annual International Conference of the IEEE Engineering i Medicine and Biology Society (EMBC), Milan, 2015, pp. 1357-1360.

Luk PP, Selinger CI, Eviston TJ, Lum T, Yu B, O’Toole SA, Clark JR, Gupta R (2015). Mammary analogue secretory carcinoma: an evaluation of its clinicopathological and genetic characteristics. Pathology, 47(7), 659–666.

Weston J, Luk P, Selinger C, Ekmejian R, Eviston T, Lum T, et al. (2015). Salivary duct carcinoma: clinicopathologic features, morphologic spectrum and genetic changes. Int J Oral Maxillofac Surg, 44, e165.

Select Conference Presentations:

Eviston TJ, Croxson GR, Multidisciplinary facial nerve care in the 21st century. Presented at the Australian Society of Otolaryngology Head and Neck Surgery Annual Scientific Meeting, Brisbane, 29 March – 1 April, 2014 (invited speaker)

Eviston TJ, Croxson GR, Krishnan AV. Neurophysiological assessment of the facial nerve: past, present, and future. Presented at 12th International Facial Nerve Symposium, Boston, June 28 – July 2, 2013 7

Eviston TJ, McGuinness AJ. Objective facial nerve grading in the 21st century: a review of video and motion based facial nerve grading tools. Presented at 12th International Facial Nerve Symposium, Boston, June 28 – July 2, 2013

Poster presentations and publications:

Eviston TJ, Ashford BA, Ebrahimi A, Clark JR, Innervated vastus lateralis for facial reanimation in radical parotidectomy defects, Presented at Royal Australasian College of Surgeons Annual Scientific Congress, Singapore, May 5-9, 2014

Eviston TJ, Gao K, Gupta R, Clark JR, Outcomes of parotid malignancy subtypes in an Australian setting, Presented at Royal Australasian College of Surgeons Annual Scientific Congress, Singapore, May 5-9, 2014

Eviston TJ, Yabe T, Ebrahimi A, Clark JR, Parotidectomy: Surgery in Evolution, Presented at Royal Australasian College of Surgeons Annual Scientific Congress, Singapore, May 5-9, 2014

Eviston TJ, Lin C S-Y, Krishnan AV. A protocol for the assessment of facial nerve excitability properties. Presented at ANZAN clinical neurophysiology workshop, Gold Coast, Sept 29 – Oct 2 2013

Abbreviations

Na+ Sodium K+ Potassium Ca2+ Calcium PNS Peripheral nervous system CNS Central nervous system DRG Dorsal root ganglion NCS Nerve conduction study EMG Electromyography CMAP Compound muscle action potential TE Threshold electrotonus TEd Depolarising threshold electrotonus TEh Hyperpolarising threshold electrotonus RC Recovery cycle I/V Current-threshold RRP Relative refractory period τSD Strength-duration time constant TTX Tetrodotoxin HB House-Brackmann BP Bell’s palsy HZO Herpes zoster oticus SEM Standard error of the mean NS Not significant (p value >0.05)

Literature review

The nervous system: a brief introduction

The human nervous system comprises a network of a hundred billion cells

(neurons) functionally arranged to enable the simultaneous processing and transmission of information(Kandel et al. 2012). Anatomically, the nervous system can be divided into the central nervous system (CNS) comprising brain, brainstem and spinal cord, and the peripheral nervous system (PNS), which is made up of peripheral nerves and clusters of neuronal cell bodies

(ganglia)(Catala and Kubis 2013).

Figure 1. A typical neuron

The typical neuron is a polarised structure. In a peripheral nerve, the myelin sheath is provided by Schwann cells. These are specialised glial cells.

Source: Shutterstock

The nervous system has two cell types: neurons and glia. Neurons are characterised by their functional and structural asymmetry and their ability to communicate across long distances(Kandel et al. 2012). They are electrically and chemically excitable and are organized into domains that include a cell body, dendrites, and an axon . Glia are supportive cells which facilitate neuronal cell function through metabolic, paracrine and immunological activity and are responsible for the myelination of axons.

The typical human peripheral nerve is composed of axons intermixed with connective tissue in a typical arrangement. The myelin and connective tissue immediately encasing each individual axon is termed endoneurium. Adjacent axons are held together into fascicles by perineurium. Fascicles bundled into a peripheral nerve are bounded circumferentially by epineurium.

Structurally the myelinated axon of the peripheral nervous system (PNS) can be divided into nodal, paranodal, juxtaparanodal and internodal segments by schwann cells (Salzer 2008). Cytoskeletal proteins maintain the integrity of these domains. These domains have characteristic ion channel constituents and are thereby responsible for different ion conductance currents at rest and in response to depolarising and hyperpolarising stimuli (Krishnan 2009).

Figure 2. Axonal domains

Depicted in this image are the functional regions of a myelinated axon. The axon is divided into nodal (blue), paranodal (green), juxtaparanodal (red) and internodal (orange) segments by the encasing Schwann cell. These domains have distinct molecular compositions and their integrity is critical to enabling salutatory conduction (Hille 2001)

Axonal physiology

At rest the potential difference across the intracellular and extracellular compartments of an axon is maintained by the activity of the sodium/potassium

ATPase pump. As a consequence, the resting axon has a high intracellular concentration of potassium (K+) and a low concentration of sodium (Na+) compared to the extracellular compartment resulting in an electrical gradient of approximately -70mV. When the resting membrane potential rises, voltage- gated Na+ channels are activated resulting in a rapid influx of Na+ ions and an increase in membrane potential to +40mV(Kandel 2012). This rapid depolarisation of the axon is termed an action potential. These voltage-gated sodium channels (Nav) are in their highest density at the nodes of Ranvier.

As membrane starts to depolarise towards threshold, a ‘regenerative’ process of

Na+ channel recruitment occurs where an increase in sodium channel conductance results in more channels opening which results in increased sodium conductance (Barnett and Larkman 2007). This ensures that the action potential is an ‘all-or-none’ response. The point at which voltage-gated channels are activated to produce an action potential in an axon is termed the threshold. In a healthy axon, the depolarisation of an axon at one node of Ranvier by an action potential depolarises subsequent adjacent nodes of Ranvier, resulting in the propagation of signal along the length of an axon.

nFollowing a action potential transient, Na+ channels become inactive, allowing for the membrane to repolarise (Schwarz 1995). This inactivation is a reflection of their channel kinetics which determine that they only remain open for 1ms 14

(Kiss 2008). Repolarisation is a result of current leak into the internodal segments and sodium channel inactivation. Fast K+ channels, located in the paranodal region (Salzer 2008) contribute to an afterpolarisation effect which limits the re-excitation of an axon after impulse conduction (Barrett and Barrett

1982)

Na+ channels are the most important contributor to the action potential. Nine isoforms of the Nav channel have been characterised with the predominant isoform involved in healthy peripheral nerve axons being Nav1.6 (Goldin 2001).

Altered transcription resulting in other sodium channel isoforms in the setting of injury and disease has been described (Waxman 2001) and likely represents a cause for abnormal nerve function in disease states.

Axonal excitability studies

Axonal excitability studies involve the application of non-invasive neurophysiological techniques to explore axonal physiology. These techniques are increasingly being used for studying the pathophysiological changes in membrane function which occur in health and disease (Krarup 2009).

Motor excitability recordings are obtained by surface stimulation of the nerve of interest and recording the compound muscle action potential (CMAP) from an innervated muscle(Krishnan 2009). Most studies have undertaken excitability recordings of the median nerve, stimulated at the wrist, with the CMAP recorded from the abductor pollicis brevis muscle(Burke, Anderson and Bostock 2000).

Detailed information regarding the function of a nerve can be determined by using computer software (QTRAC, ©Institute of Neurology, London) to ‘track’ 15 the current stimulus required to achieve a predefined submaximal CMAP response (‘threshold’) while applying a defined protocol (Kiernan et al. 2000) of conditioning and polarising currents. By monitoring the change in threshold to these conditioning currents a profile is obtained across a number of parameters including stimulus-response, recovery cycle, strength-duration and current threshold relationship. The interpretation of change amongst these parameters i the setting of disease or physiological change gives insight into axonal biophysical properties (Krarup and Moldovan 2009).

The interpretation of axonal excitability studies is complex. Morphology, structural change, resting membrane potential, technical factors such as electrode properties and configuration, and individual variability in properties such as skin resistance, may all contribute to the axonal excitability properties generated during a recording. For contextualising and interpreting the results of a study, the clinician or researcher will refer to comparable conditions and disease models, animal models, and patterns of change to build a profile of indirect evidence for a pathophysiological process, with mean group data used in preference to individual recordings. Recently, mathematical modeling of axonal function has emerged as a tool to further assist the interpretation of axonal excitability results (Howells et al. 2012).

Stimulus-response (SR)

The stimulus response curve is generated by gradually increasing the stimulus current until a maximal CMAP is reached (Figure 3) (Bostock, Cikurel, and Burke

1998). A submaximal ‘threshold’ is then determined at a steep part of the curve

(usually 40% of the maximal CMAP). Once a target CMAP has been calculated, strength-duration, TE, RC and I/V sequences are performed.

Stimulus-response curve

Figure 3. Stimulus response curve.

A plot of the relationship between stimulus intensity and the corresponding

CMAP response. The plot above is taken from mean recordings from a cohort of normal facial nerve recordings. A ‘threshold’ is determined by the stimulus required to achieve a target submaximal response of 40% maximal

CMAP(Bostock, Cikurel, and Burke 1998). This is represented by the line and circle in the above figure. The change in this threshold stimulus is tracked during axonal excitability studies as conditioning stimuli and polarising subthreshold currents are applied. 17

Strength-duration properties

As stimulus duration increases, the threshold current required to achieve a target response decreases. Weiss’ law (Weiss 1901) describes the linear relationship between charge (current x duration) and stimulus duration.

According to Weiss’ law, this relationship can be used to calculate the strength- duration time constant (τSD), a measure of the rate at which the target potential declines as stimulus increases and a reflection of passive nodal membrane properties, and in particular persistent Na+ channels which do not inactivate with changes in membrane potential (Mogyoros et al. 1998)

(Bostock and Rothwell 1997). τSD is sometimes referred to as chronaxie(Bostock and Rothwell 1997) and it can also be calculated using latent addition (Mogyoros et al. 1998).

Rheobase is also a reflection of nodal membrane properties (Bostock, Cikurel, and Burke 1998) and it is defined as the stimulus current required to produce a threshold response for a stimulus duration which is infinitely long. It can be calculated as the slope of the regression line (Bostock, Cikurel, and Burke 1998) as demonstrated in Figure 4.

Strength-duration properties

Figure 4. Strength-duration properties.

The stimulus-duration relationship for a normal facial nerve recording is demonstrated. The inset demonstrates the change in stimulus intensity and duration used to achieve the target potential. Stimulus durations of 0.2ms, 0.4ms,

0.8ms and 1ms were used. Using Weiss’ law the x-intercept is the strength- duration time constant and the slope of the line is the rheobase. Both rheobase and τSD will change in response to nodal Na+ conductances (Kiernan et al. 2005) and to membrane potential changes(Kiernan et al. 2000).

Threshold Electrotonus (TE)

TE measures the change in threshold to conditioning subthreshold depolarising and hyperpolarising currents of up to 100ms duration. Threshold is tracked before, during and after these conditioning currents. Examining the threshold change in response to the application of these subthreshold currents gives insight into nodal and internodal conductances. Polarising currents initially result in current spread into the internodal region and a further increase in magnitude of the threshold change (S1 phase).

In response to depolarising conditioning, slow K+ channels open, which acts as a rectifying channel. This limits the accumulation of depolarising charge in the internodal segment and is seen in the TE curve as an accommodative response

(S2 accommodation). These channels are slow to deactivate when the conditioning current is removed, this results in ‘undershoot’.

For hyperpolarising conditioning currents, slow K+ channels are inactive so a greater S1 phase is seen. The rectification of this charge eventually occurs through hyperpolarisation activated rectification currents (Ih)(Krishnan 2009).

With the removal of the hyperpolarising conditioning currents the continued activity of these rectification currents and the reactivation of slow K+ channels results in ‘overshoot’ of the threshold change.

TE is sensitive to membrane potential changes (Kiernan 2000). Membrane depolarisation which results in a decreased potential difference across the axonal membrane can be seen as a ‘fanning in’ of the TE curve with decreased 20 magnitude threshold changes to both depolarising and hyperpolarising conditioning currents. An increase in the resting membrane potential can conversely be seen as a ‘fanning out’(Kiernan and Kaji 2013) of the TE curve with increased magnitude changes in threshold to both depolarising and hyperpolarising condition currents.

Threshold Electrotonus

Figure 5. TE profile of a normal facial nerve.

The inset diagram represents the ±40% subthreshold condition currents applied prior to testing threshold change at different timepoints. Values used for analysis, which are extracted from specific time points, are demonstrated.

Recovery cycle

The excitability of an axon changes in the time period immediately following impulse conduction. By using a supramaximal stimulus and then testing threshold change at multiple time points between 2-200ms after the impulse, these excitability changes can be determined(Burke, Kiernan, and Bostock 2001).

For the first 0.5-1ms after the conduction of an impulse an axon is inexcitable.

This is attributable to the inactivation kinetics of transient Na+ channels

(Hodgkin and Huxley 1952). Once this inactivation recovers the axon then become ‘superexcitable’ due to capacitive charge stored in the internodal segment of the axon (Barrett and Barrett 1982). This effect peaks at 5-7ms after impulse conduction. After the period of increased membrane excitability there is a period of ‘subexcitability’. This is due to the slow inactivation kinetics of nodal slow K+ channels (Schwarz 1995).

The RC is sensitive to membrane potential changes (Krishnan 2009; Kiernan

2000). Membrane hyperpolarisation causes an increase in superexcitability and a decrease in refractoriness. Membrane depolarisation causes a decrease in superexcitability and an increase in refractoriness. Refractoriness is also sensitive to nodal Na+ conductance changes (Kiernan et al 2005; Kuwabara et al.

2005), temperature change (Kiernan, Cikurel, and Bostock 2001) and in the setting of demyelination (Kuwabara et al. 2002).

Recovery Cycle

Figure 6. A recovery cycle plot of a human facial nerve.

The inset demonstrates the supramaximal stimulus and points of threshold tracking used to determine the RC. Parameters determined include refractoriness, superexcitability and subexcitability.

Current-Threshold Relationship (I/V) Rectification properties of an axon can be examined by using prolonged 200ms currents prior to tracking the threshold change to a 1ms impulse. The conditioning currents range between +50% and -100% of the threshold current i 10% steps (Kiernan et al. 2000). The slope of this curve gives insight into resting membrane potential and rectifying currents. Outward rectification due to fast and slow K+ conductances causes an increased slope in the top right.

Hyperpolarising I/V slope reflects Ih rectification currents (Krishnan 2009). 25

Current-threshold (I/V)

Figure 7. I/V plot of a normal human facial nerve.

Inset image (top left) represent the long polarising currents used to condition the nerve prior to examining the changes in threshold. Variables analysed include resting I/V slope and hyperpolarising I/V slope, which are demonstrated.

Th facial nerve: structure and function

Anatomy

The human facial nerve is the seventh cranial nerve (CNVII) and comprises motor, sensory and parasympathetic components. Its function is responsible for voluntary and mimetic facial movement, taste to the anterior two thirds of the tongue, and control of salivary gland and lacrimal gland secretions.

The facial nerve receives axons from the superior part of the solitary nucleus and superior salivary nucleus which form the nervus intermedius component

(sensory and parasympathetic axons) and motor efferent fibers from the facial nucleus which receives synaptic input from the contralateral motor cortex for all facial movements except the forehead which has bicortical input.

The path of the facial nerve has intracranial, intratemporal and extratemporal components. Its intracranial course runs from the ponto-medullary angle to the internal acoustic meatus where it is accompanied by the vestibulo-cochlear nerve (CNVIII). The intratemporal course of the facial nerve is long and tortuous.

During its intratemporal course the nerve encounters the geniculate ganglion and gives rise to the superior petrosal nerve, the nerve to stapedius, and chorda typani nerve branches before exiting the skull base through the styloid foramen.

The extratemporal facial nerve courses through the substance of parotid gland dividing it into deep and superficial lobes. It gives off the posterior auricular nerve and nerve to the posterior belly of digastric before dividing into its terminal facial branches. There is significant variation in the branching pattern 27 of the terminal facial branches which are traditionally conceptualised into temporal, zygomatic, buccal, marginal mandibular and cervical branches. These terminal motor branches are responsible for all facial expression and functional tasks such as eye and mouth closure and nasal patency during inspiration.

Throughout its course the facial nerve forms multiple communications between its own branches and with adjacent cranial nerves(Diamond et al. 2011).

Facial palsy: overview

Facial palsy is a severely debilitating condition that is a consequence of facial nerve injury or dysfunction. It has has dramatic psychological, physical and functional consequences for the sufferer(VanSwearingen and Brach 1996). Facial palsy can be congenital or acquired and a large number of pathologies are described(Fattah et al. 2012). The acquired palsies can be broadly divided into idiopathic, infective/inflammatory, traumatic and malignant categories. Detailed discussion of all pathologies is beyond the scope of this review.

Bell’s Palsy

BP is an acute onset peripheral facial neuropathy and is the most common cause of lower motor neuron facial palsy(Peitersen 2002). The clinical presentation of the disorder is a rapid onset, unilateral, lower motor neuron type facial weakness with accompanying symptoms of post auricular pain, dysguesia, subjective change in facial sensation and hyperacusis. This clinical presentation can be explained by the anatomical construct of the human facial nerve, specifically its mixed nerve profile containing motor, sensory and 28 parasympathetic fibers. The propensity for the facial nerve to form numerous connections with adjacent cranial nerves(Diamond et al. 2011) may also explain the occasionally observed features of altered facial sensation (cranial nerve V), vestibular dysfunction (cranial nerve VIII) or pharyngeal symptoms (cranial nerves IX and X)(Adour, Hilsinger, and Callen 1985; Adour et al. 1978). Reduced lacrimation and salivation secondary to parasympathetic effects may also occur(Royal and Vargas 2014; Adour et al. 1978). Maximal disability occurs within the first 48-72 hours and the severity of the palsy correlates with the duration of facial dysfunction, the extent of facial recovery and impairment of quality of life.

Despite extensive study of the condition, the exact pathogenesis of BP is still controversial (Kennedy 2010). Infection (herpes simplex type-1) (McCormick

1972; Baringer 1996; Hato, Murakami, and Gyo 2008), nerve compression(Fisch

1981; Gantz et al. 1999) and autoimmunity(Greco et al. 2012) may all play a role, yet the exact sequence and magnitude of these influences remains unclear.

History

Sir Charles Bell (1774-1842) was fascinated by the nervous system. As an accomplished anatomist, artist, surgeon and teacher, his work on the characterisation of the peripheral nervous system through anatomical study, vivisection, and clinical correlation, provided a significant contribution to medical knowledge of his time. In particular, he was fascinated with the separation of sensory (trigeminal nerve) and motor supply (facial nerve) to the 29 face. It was his eloquent and logical descriptions which elevated his status among his contemporaries and ushered the “post-Bell’s” era which saw a rapid increase in the number of publications relating to acute idiopathic facial palsy – “Bell’s

Palsy”.

Although Bell’s descriptions were admirable in their ability to inspire his contemporaries to document and study the disorder, myriad other detailed descriptions exist in Greek, Persian and European medical texts as far back as the

5th century BCE(Sajadi, Sajadi, and Tabatabaie 2011) and there is prehistoric ceramic art depicting facial palsy identified in ancient Peruvian culture(Canalis and Cino 2003). Other clinicians who recognised the entity of acute idiopathic facial paralysis before Bell (and perhaps may have inspired some of his observations) include Sydenham, Stalpart van der Wiel, Douglas, Friedreich, and

Thomassen a` Thuessink(van de Graaf and Nicolai 2005; van de Graaf et al. 2009;

Sajadi, Sajadi, and Tabatabaie 2011).

Approximately 1000 years before Bell, the persian physician and scholar, Razi

(865E) -925 C described facial palsy at length in his seminal 9th century text “al-

Hawi”(Sajadi, Sajadi, and Tabatabaie 2011). This remarkable description referenced the contributions of Galen and Celsus, among others and included a diagnostic algorithm delineating peripheral facial palsy from more sinister central causes which were accompanied by delirium, coma, hemiplegia, blindness, or deafness and tended to have a poor prognosis.

Epidemiology

BP is a common cranial mononeuropathy. It affects males and females equally and has a slightly higher incidence in mid- and later life but certainly occurs across all age ranges. The described population incidence rates range from 11.5 to 40.2/100000(De Diego et al. 1999) with specific studies demonstrating similar annual incidences between the UK(20.2/100000), Japan (30/100000), and the United States 25-30/100000 (Hauser, Karnes, and Annis 1971).

Clustering and epidemic phenomena are not demonstrated in the majority of studies. An exception to this is the recent occurrence of an increase in incidence of BP during a trial of intranasal vaccine delivery. This was possibly due to the immune effects of the detoxified Escherichia coli Heat Labile Toxin adjuvant used in this form of vaccine delivery(Lewis et al. 2009). The incidence is higher in pregnancy, following viral upper respiratory tract infection, in the immunocompromised setting, and with diabetes mellitus and hypertension(Peitersen 2002). There is no distinct latitudinal variation for incidence, nor is there a racial or ethnic predilection. Some epidemiological data demonstrate seasonal variation, with a slightly higher incidence in cold months vs warm months(De Diego et al. 1999), and a slight preponderance to arid over non-arid climates(Campbell and Brundage 2002).

Aetiology

There is a diverse body of evidence implicating immune, infective and ischaemic mechanisms as potential contributors to the development of BP but the cause of classical BP remains unclear. One possible cause that has been suggested is that 31 of a reactivated herpes simplex virus (HSV)-1 infection centered around the geniculate ganglion, a theory first outlined by McCormick(McCormick 1972). The association with HSV-1 is supported by the presence of HSV-1 in intratemporal facial nerve endoneural fluid(Murakami et al. 1996) harvested during nerve decompression and the ability to incite facial palsy in an animal model through primary infection(Sugita et al. 1995) and reactivation induced by immune modulation(Esaki et al. 2014). Despite this evidence, the behavior of BP is unusual compared to other diseases more commonly caused by HSV such as herpes labialis (cold sores) and genital herpes (Steiner and Mattan 1999;

Kennedy 2010). Further, it is not justifiable to assume a cause and effect relationship between the presence of HSV-1 in the patients' endoneural fluid and the development of BP.

HSV-1 is one of several human herpes viruses known to have a neurotrophic capacity for peripheral nerves, and other viruses in this category include Herpes

Simplex type 2 (HSV-2) and Varicella Zoster Virus (VZV). They enter the body through mucocutaneous exposure and establish their presence in latent form with highly restricted gene transcription in multiple ganglia thoughout the neuroaxis for the entire life of the host, including in the cranial, dorsal root and autonomic ganglia (Steiner and Kennedy 1993; Mitchell et al. 2003). This latent presence in ganglia in the absence of active viral replication and assembly is characteristic, well described and widely distributed through normal and diseased populations. HSV has a global distribution and is a fundamentally resilient virus. HSV and VZV can both reactivate in an immunocompetent host 32 and in the presence of circulating antibodies, although reactivation is more likely i the setting of immunodeficiency, especially in the case of VZV.

A possible cause of neural dysfunction due to HSV-1 is the activation of intra- axonal degradation and apoptotic pathways driven by local direct and indirect responses of the axon to the virus itself in a susceptible phenotype. Although not previously associated specifically with the pathogenesis of BP, the emergence of literature pertaining to the role of intra-axonal signal molecules (eg

SARM1)(Wang et al. 2012), mitochondrial permeabilization(Galluzzi, Blomgren, and Kroemer 2009) and the molecular mechanisms underpinning Wallerian degeneration(Conforti, Gilley, and Coleman 2014) suggests that acute axonal degeneration in the context of viral infection may be an evolutionarily conserved innate immune response to prevent virus transport to the central nervous system(Conforti, Gilley, and Coleman 2014).

Recent in vitro work has demonstrated local mRNA transcription in peripheral nerve axons(Koyuncu, Perlman, and Enquist 2013) incited by the presence of alpha-herpes virus particles. In this compartmentalised model, protein and signal transduction changes were not reliant on nuclear machinery, i.e. when a virus enters an axon, the axon responds locally. Earlier work examining cellular physiology in the setting of herpes infection demonstrated an acute decrease in sodium conductivity in the setting of HSV-1(Storey 2002). Changes in sodium conductance can result in a reversed sodium-calcium exchange (NCX)(Persson et al. 2013) current and the accumulation of intracellular calcium. This aberration in calcium homeostasis leads to protease activation and intra-axonal 33 degeneration. These processes of axonal degeneration would drive the abrupt onset of BP and explain the lack of a pronounced immune response. This would not necessarily discount the role of compression to the pathogenesis, but rather, may answer the question as to why the nerve swells, leading to impingement in the first place.

Another possible contributor to the pathogenesis of BP implicates the role of a cell mediated immune response against myelin, akin to a mononeuropathic form of Guillaine-Barre Syndrome (GBS). The evidence for this stems from the indirect laboratory finding of GBS like changes in peripheral blood percentages of T and B lymphocytes, elevated chemokine concentrations and in vitro reactivity to myelin protein (P1L) in blood samples taken from BP patients (Greco 2012).

Diagnosis

BP is a clinical diagnosis. The characteristic findings are acute onset of unilateral lower motor neuron facial paralysis that affects muscles of both the upper and lower face and which reaches its peak by 72 hours. These findings are frequently accompanied by symptoms of neck, mastoid, or ear pain, dysgeusia, hyperacusis or altered facial sensation. These associated symptoms are present in 50-

60%(Croxson 1990) and are reassuring for the diagnosis of BP.

The involvement of posterior auricular, petrosal, chorda tympani and stapedius nerves implicates the site of dysfunction being within the temporal bone.

Localisation to the intratemporal facial nerve is further supported by unilateral enhancement of the geniculate, labyrinthine and meatal segments of the facial 34 nerve on contrast enhanced MRI studies. This imaging finding is hypothesized to represent disruption of the blood-brain barrier and vascular congestion of the facial nerve. Attempts to determine surrogate diagnosis through polymerase chain reaction techniques with VZV and HSV primers applied to posterior auricular, tear or facial muscle specimens have failed to demonstrate any consistent correlation between viral load and clinical features(Stjernquist-

Desatnik, Skoog, and Aurelius 2006); these tests are therefore of limited value as diagnostic tools.

Differential diagnosis

The differential diagnosis for facial palsy is broad(Hohman and Hadlock 2014), and misdiagnosis is not uncommon. Causes of facial palsy may be divided into congenital and acquired aetiologies. Congenital causes include genetic syndromes, birth related trauma and isolated disorders of development (eg. developmental hypoplasia of facial muscles). Acquired causes include infective

(VZV, lyme disease, mycobacterium tuberculosis, HIV), traumatic (iatrogenic or head trauma), inflammatory (vasculitis, sarcoidosis, autoimmune disease), neoplastic (benign or malignant) and cerebrovascular causes, among others. In the experience of an expert referral setting, the rate of misdiagnosis of BP by the initial consulting clinician is 10.8%(Croxson, Coulson, and Mukherjee 2013).

Missed diagnoses include tumours (e.g facial nerve schwannoma, parotid malignancy and rarely acoustic neuroma), herpes zoster oticus and granulomatous diseases such sarcoidosis and granulomatosis with polyangiitis

(Wegener’s granulomatosis). 35

Figure 8. Patterns of facial palsy(Eviston et al. 2015).

A structured clinical approach which considers the pattern of facial palsy (Figure

8) along with patient characteristics and a thorough physical examination will generally provide evidence for an alternative diagnosis, and prompt appropriate investigation(Hohman and Hadlock 2014). Particular patterns of facial palsy which require thoughtful consideration include: (i) Fluctuant, step-wise or slowly progressive (beyond 72hours) facial palsy (ii) Bilateral palsy (GBS, carcinomatosis, lymphoma) (iii) Recurrent facial palsy (facial nerve neuroma)

(iv) Prolonged complete palsy (>4 months) (v) Sudden complete facial palsy

(haemorrhage into a tumour). These patterns should prompt a detailed search for an underlying cause. Likewise, the presence of a mass in the parotid region, a history of cutaneous malignancy, or segmental facial nerve weakness should raise suspicion for a tumour. A history of trauma, ear symptoms such as ipsilateral deafness, tinnitus, fullness or discharge, or systemic symptoms such as fever are also red flags warranting further investigation and specialist otological consultation.

The consideration of cerebrovascular disease as a cause of facial palsy is important with this being the main concern for many patients and clinicians, often prompting expert neurology consultation. The preservation of upper facial movement (frontalis contraction) is a discriminator between cortical (central) and peripheral facial nerve weakness. Rarely, ipsilateral pontine pathology may result in lower motor neuron pattern facial weakness due to direct compromise of the facial nucleus. This will be accompanied by other cranial nerve and long tract symptoms and signs. Ipsilateral abducens nerve (CNVI) dysfunction (lateral gaze palsy) is a particularly useful sign. 37

Grading

The most widely used systems for recording the severity of BP are the House-

Brackmann(House and Brackmann 1985) (HB) scale or the Facial Nerve Grading

Scale(ROSS, Fradet, and NEDZELSKI 1996) (also known as the Sunnybrook system). The subjective nature of these scales makes them prone to some misinterpretation and inter-observer variability, however, their ease of use has cemented their role in clinical practice for communicating the overall degree of dysfunction, for monitoring outcomes and for the presentation of group data in a research or audit setting. In a recent survey of facial nerve specialists(Fattah et al. 2014), photography and videography were ubiquitous amongst respondants and indeed, the use and importance of video recording of standardised facial movements (eye brow raise, gentle eye closure, tight eye closure, snarl, open and closed lip smiles, lip depression and lip puckering) is emphasised for the accurate outcome assessment of interventions such chemodenervation, physiotherapy or surgery which may be considered in those patients who have an incomplete recovery.

Neurophysiology

There have been a number of studies exploring the potential utility of neurophysiological assessment for treatment selection and prognosis. In the past, nerve conduction studies of the facial nerve, also referred to as

Electroneuronography (ENoG) in the surgical literature, have been suggested in some studies to be useful in the selection of patients who may require surgical decompression of the facial nerve. The demonstration of a greater than 90% 38 reduction in compound muscle action potential (CMAP) in the first 10 days of onset compared to the unaffected side is associated with a 50% chance of incomplete recovery(Gantz et al. 1999) and was a trigger for operative intervention in some centers.

In contemporary practice, facial nerve decompression has increasingly fallen out of the normal domain due to cost, risk and lack of efficacy(Adour 2002; Baugh et al. 2013). With this trend away from surgery, the time and cost of neurophysiology assessment outweighs the benefit for the vast majority of patients. An exception to this is the clinical setting of complete paralysis. Here neurophysiology provides useful information with the presence of a residual response on neurophysiology suggesting a predominantly neuropraxic injury with the prospect of a good recovery (HB I or II) are high. The absence of a neurophysiological response is suggestive of complete degeneration and a prolonged paralysis with incomplete recovery which may be complicated by synkinesis. The knowledge of a prolonged recovery may sway the clinician and patient towards surgical eye protection procedures and the proactive involvement of a facial therapist early in the course of recovery.

Acute treatment

Both the American Academy of Neurology(Gronseth, Paduga, American Academy of Neurology 2012) (AAN) and the American Academy of Otolaryngology – Head and Neck Surgery Foundation(Baugh et al. 2013) (AAO-HNSF) have recently published guidelines for the treatment of BP. Although the structure and scope of 39 these guidelines were different, they are essentially complimentary documents which reinforce the role of corticosteroids in the treatment of BP and argue against the routine use of antiviral therapy. Furthermore, the AAO-HNSF guidelines discourage routine laboratory, imaging or neurophysiological testing at the first presentation of typical BP. The dose of oral steroids should be commenced in the first 72 hours of onset and a regime mirroring either of the

Scottish(Sullivan et al. 2007) or European(Engström et al. 2008) randomised controlled trials (RCTs) should be used. This is either 50mg prednisone for 10 days or 60 mg for the first 5 days then reducing by 10mg each day for the next 5 days. Both seem effective. It has been argued that the lack of significance demonstrated by combined corticosteroid and antiviral therapy over corticosteroids alone in double blind RCTs represents a dilution effect of mild and moderate palsies, which have a high rate of spontaneous recovery, masking any demonstrable benefit for the severe palsy subgroup. Supporting this is the positive findings in favour of combined therapy in non-double blinded studies

(de Ru and van Benthem 2011; Minnerop et al. 2008; Hato et al. 2007).

The key rationale for giving patients with BP antiviral treatment with the anti- herpes drug aciclovir is the possible role of HSV-1, based on the current circumstantial evidence. Another reason given for using antiviral therapy in the setting of clinical equipoise is that a portion of those given a provisional diagnosis of BP will have zoster sine herpete i.e symptomatic VZV reactivation without the typical vesicular eruption pathognomonic of a typical VZV infection

(Ramsay-Hunt syndrome).

VZV is an important differential diagnosis in all acute lower motor neuron facial palsies. Ramsay-Hunt syndrome has a worse prognosis than BP and, on average, presents as a more severe palsy. It is more responsive to combined antiviral and steroid therapy and the rate of complications from antiviral therapy is low. Since

VZV is known to cause facial palsy, the use of antivirals in Ramsay-Hunt syndrome has a clear evidence base and is justified and rational(Kennedy 2010).

A typical regime to adequately cover VZV would be 3000mg/day (1000mg

Valacyclovir three times per day) for 7 days. Valacyclovir has a higher bioavailability than acyclovir.

At the present time the use of combined aciclovir and corticosteroids in treating classical BP remains controversial with conflicting data emerging from different trials and, indeed, from different meta-analyses(Kennedy 2010). Based on current evidence, particularly the extensive Scottish BP study(Sullivan et al.

2007) of 551 patients in a double-blind, placebo-controlled, randomised study, it seems reasonable to treat classic BP with oral corticosteroids alone without aciclovir. However, combined aciclovir and corticosteroids may possibly have a beneficial role in cases of severe BP, and this issue needs to be resolved in a large prospective clinical trial. In cases where the patient is severely immunocompromised consideration may be given to intravenous regimes of acyclovir to prevent possible central nervous system complications.

Eye care

The institution of an eye protection strategy for each patient with incomplete closure is of paramount importance. Lacrimation occurs at the lateral aspect of the conjunctival membrane and is swept lateral to medial as a film by the action of the orbicularis oculi during blink and effective eye closure. Loss of this mechanism results in epiphora (tearing), due to an ineffective pump mechanism to spread the tear film, and exposure and irritation of the eye itself. Prolonged drying and irritation will progress to keratitis and ulceration, and eventually can threaten sight. At the first consultation the clinician must enact a strategy to avoid ocular exposure, and refer any cases of concern to an ophthalmologist.

Effective eye protection uses barrier protection (eg wrapped sunglasses), lubrication (artificial tears during the day, ointment at night) and taped closure at night. Particular environments which may present a challenge for the patient include showering and swimming, dusty and windy environments; these situations are best avoided.

The early use of an eyelid weight should be considered in the elderly, diabetics, those with pre-existing eye disease, complete facial palsy with no response on neurophysiology, and the presence of ongoing irritation despite the use of the eye-protective therapies described above(Hohman and Hadlock 2014).

Oral care

Loss of the sphincter function of the orbicularis oris confers the social inconvenience of oral incontinence and predisposes the lip and inner cheek to 42 abrasion during mastication and subsequent ulceration. Strategic eating may lessen the impact of these in the setting of flaccid facial paralysis. Using a straw for liquids and tending towards soft foods when ordering are often helpful. The inability to lower and evert the lower lip precludes eating certain foods.

Temporary dental “spacers” adhered to the lateral aspect of the molar teeth may be used to prevent chewing of the buccal mucosa.

Physiotherapy

From an evidence based perspective this diverse modality of treatment which broadly encompasses heat therapy, electrostimulation, massage, mime therapy, and biofeedback(Baugh et al. 2013) is hard to analyse as a whole. There is a plethora of treatment regimes and their timing and variability in implementation makes their broader assessment of utility complex. Although not recommended for all suffers of BP(Baugh et al. 2013), there are subgroups of patients where there is evidence supporting the use of physiotherapy (Robinson and Hadlock

2010; Nicastri et al. 2013). This includes patients who have incomplete recovery and who have developed hypertonia, hyperkinesis or synkinesis and in these groups neuromuscular retraining is trialed before consideration of chemodenervation(Hohman and Hadlock 2014). Physiotherapy and chemodenervation are complimentary in the treatment of synkinesis.

Prognosis

Natural history studies have demonstrated that ~85% of patients experience some recovery in first three weeks(Devriese et al. 1990; Peitersen 2002; 43

Devriese and Moesker 1988). Predictors of incomplete recovery include severe facial palsy, the length of time prior to onset of recovery, and persistent pain.

Patients with complete facial palsy (House Brackman grade 5-6) who have not experienced some recovery in the first 3 to 4 months after onset are more likely to have incomplete recovery of facial function, with or without spasm and synkinesis. Prolonged pain is also a predictor of worse outcome.

The natural history of BP has been elucidated through a number of large studies

(Peitersen 2002). Based on conclusions drawn from these large studies, clinicians can expect that without intervention, approximately 70% of patients will experience full recovery. Of those who don’t recover fully, half will have mild sequelae and the remainder moderate to severe sequelae. In the setting of acute steroid use the rate of full recovery is over 90%(Sullivan et al. 2007).

Managing incomplete recovery

Chronic facial palsy is a disabling condition which has a dramatic impact on social function, emotional expression, and quality of life. Aesthetic, functional

(nasal patency, eye closure, speech and swallowing) and psychological considerations need to be addressed by the treating team. Over the last three decades, the treatment of incomplete recovery of facial palsy has evolved from static techniques aimed at the suspension of the oral commissure and eye closure, into a multimodal(Hadlock et al. 2006), zonal based approach which utilises the complementary aspects of physiotherapy, chemodenervation and selective surgical procedures to maximise the cosmetic and functional outcome 44 needs of each patient. Increasingly, multidisciplinary collaboration between interested clinicians from a wide variety of subspecialties has proven effective.

In outlining treatment modalities and their appropriateness, one must consider incomplete recovery of facial function as a heterogeneous entity which encompasses different degrees of flaccidity, hypertonicity, and synkinesis. Each of these issues can range in severity from absent to severe. Generally, functional issues such as brow ptosis, nasal valvular collapse, and eye closure are addressed through directed structural interventions including nasal valvular suspension, brow ptosis correction, platinum weight insertion into the upper eyelid, lower lid suspension, or tarsorrhaphy to improve eye closure.

Figure 9. Botulinum toxin injection sites for the treatment of synkinesis.

Blue (affected side) and green (unaffected side) dots represent planned point of injection for 1.5-3 IU of Onabotulinum A. The pre-application of topical local anaesthetic cream and the use of a fine bore needle size (25-30gauge) are used to reduce the pain of injection. Subcutaneous injection is as effective as intramuscular.

Synkinesis

Synkinesis refers to abnormal facial muscular contraction during voluntary facial movements and has been traditionally attributed to aberrant re-innervation of facial musculature following nerve injury. It can be seen as involuntary eye- closure during midface movement such as eating or smiling (oro-ocular synkinesis); as lip excursion during eye closure (oculo-oral synkinesis); or as chin dimpling or muscular neck cords during midface movement due to involuntary mentalis or platysma activation. The treatment of synkinesis centers around physiotherapy, with a particular focus on biofeedback exercises to retrain facial symmetry, and selective chemodenervation using directed botulinum toxin to problem areas (Figure 9). Most patients express a high degree of satisfaction with this approach, and objective improvement is seen in the majority(Hadlock et al. 2006).

Treatment of synkinesis with botulinum toxin is tailored according to the individual patient. Injection points focused o orbicularis occuli and platysma relieve spasm and synkinesis whilst selective use of contralateral (unaffected side) brow and depressor angularis oris injection points enhances facial symmetry and cosmesis. Injection of the weak/synkinetic zygomaticus muscles is best avoided due to the debilitating effect of loosing smile function from an already weakened face. Some patient have a reduced benefit with repeated injections while for others the requirement for recurrent injections three to four times per year is unsatisfactory. In selected cases, a more permanent solution can be achieved through surgical interventions such as selective myectomy. 47

Recently, selective neurectomy has also proven to be very effective(Hohman,

Lee, and Hadlock 2013).

Facial reanimation

In the setting of incomplete recovery with ineffective smile, nerve-to-nerve transfer, and regional and free tissue transfer techniques offer the opportunity to restore midface movement. A regional tendon transfer technique which relocates the temporalis muscle tendon to the oral commissure has been popularised by the French surgeon, Labbe(Labbé, Bénateau, and Bardot 2002; Nduka, Hallam, and Labbé 2012). Alternatively, innervated free muscle transfer techniques which utilise the gracilis muscle can be inserted into the face with nerve coaptation from the contralateral facial nerve and/or from the ipsilateral mandibular branch of trigeminal nerve. Nerve-to-nerve transfers are also becoming increasingly popular for those with auto-preservation of facial musculature but a frozen oral commissure. Donor nerves include the masseteric branch of the trigeminal nerve, and cross face nerve grafting. These complex reconstructive procedures offer an opportunity for smile reanimation and the subsequent benefits in social function and quality of life.

Methodology

Subjects

Participants were recruited from research sites at Prince of Wales Hospital, Chris

O’Brien Lifehouse and Liverpool Hospital, Sydney, Australia. Recruitment referral networks included neurology, otolaryngology, head and neck surgery, and plastic surgery professionals from participating institutions.

All studies were approved by institutional ethics committees and written informed consent was obtained from all participants. All participants were screened prior to undergoing study procedures. Screening consisted of clinical assessment and specific questioning for present or past disorders of the facial nerve or neurological system including conditions and treatments known to alter axonal excitability properties (e.g. Diabetes mellitus, renal disease, active malignancy, previous chemotherapy, hereditary neuropathy, undiagnosed peripheral neuropathy symptoms, pregnancy, medications). All participants were greater than 18 years of age.

Grading

After being clinically assessed, facial movements were videotaped using a standardised process for facial palsy grading (Figure 10). HB and Sunnybrook scores, measures of facial palsy severity, were assigned by two clinicians who independently graded the videotapes (House and Brackmann 1985). HB (Figure

11) is a widely used facial palsy assessment tool with six levels of severity. It is a global assessment scale which is not sensitive to synkinesis or facial sub- regions(Lee 2012). For this reason the Sunnybrook facial nerve grading scale

(Ross et al. 1996) was also included. 50

Figure 10. Video Analysis Protocol

Rationale

Videoanalysis is the gold standard for objectively grading facial palsy and for evaluating surgical reanimation procedures (Terzis 2012). It is the standard from which subjective scales such as the House-Brackmann scale are designed

(Lee et al. 2012).

Prerequisite setup

1. Video camera on Tripod.

2. Alignment with nose. 90-100cm from subject’s face.

3. Well lit room

Patient directions

“When instructed please perform the following facial movements.”

1. RAISE YOUR EYEBROWS

2. GENTLY CLOSE YOUR EYES

3. TIGHTLY CLOSE YOUR EYES

4. SNARL

5. SMILE AS WIDELY AS POSSIBLE

6. PUCKER YOUR LIPS

7. SHOWE M THE BOTTOM ROW OF YOUR TEETH

8. ETENSE TH MUSCLES AT THE FRONT OF YOUR NECK

Description Characteristics Grade I Normal Normal facial function in all areas II Mild dysfunction Gross: slight weakness noticeable on close inspection; may have very slight synkinesis. At rest: normal symmetry and tone Motion: Forehead: moderate to good function Eye: complete closure with minimum effort Mouth: slight asymmetry III Moderate Gross: obvious but not disfiguring difference dysfunction between two sides; noticeable but not severe synkinesis, contracture, and / or hemifacial spasm At rest: normal symmetry and tone Motion: Forehead: slight to moderate movement Eye: complete closure with effort Mouth: slightly weak with maximum effort IV Moderately severe Gross: obvious weakness and / or disfigurin dysfunction asymmetry At rest: normal symmetry and tone Motion: Forehead: none Eye: incomplete closure Mouth: asymmetric with maximal effort V Severe dysfunction Gross: only barely perceptible motion At rest: asymmetry Motion: Forehead: none Eye: incomplete closure Mouth: slight movement

VI Total paralysis No movement

Figure11. House Brackmann grading scale (House and Brackmann 1985) 52

Figure 12. Sunnybrook Facial Grading System (Ross et al. 1996) 53

Axonal excitability

Facial nerve excitability properties were acquired using QTRAC software

(©Institute of Neurology, London). The software’s TROND protocol enables the automated acquisition of multiple excitability parameters. Initially a stimulus response curve was generated using 1ms test impulses until a maximal compound muscle action potential (CMAP) was reached. A target response of

40% of maximal CMAP amplitude was then tracked during the course of a defined protocol of conditioning currents. This protocol included an automated sequence of four testing phases to determine the properties of strength-duration time constant (τSD), threshold electrotonus (TE), recovery cycle (RC) and current-threshold relationship (I/V) (see Literature Review).

To attain facial nerv recordings, participants were seated upright for the duration of assessment. Skin preparation was performed routinely and makeup was removed where required. Skin temperature was maintained above 32˚C for the duration of testing. Non-polarisable, solid gel surface electrodes

(WhiteSensor® WS, Ambu A/S, Ballerup, Denmark) were placed according to pre-determined anatomically optimised sites facial nerve recordings (Chapter 1).

An example is shown in Figure 13. 54

Figure 13. Facial nerve – Nasalis recording setup.

This is a typical electrode configuration with stimulation (black), stimulation reference (red), earth (green), recording (yellow) and recording reference (blue)

(located on the forehead). The white dressing is from a surgical procedure and is not related to the recording setup.

Data analysis

The analyses of excitability indices were performed using QTRAC-P (©Institute of Neurology, London). Statistical analysis of branch data was performed using student’s t-test. Results are presented as mean (±SEM). To complement this dataset, the correlation between variables and additional statistical analysis was performed using SPSS Version 22 (©IBM Corp, Armonk, NY). A p value of <0.05 was predetermined as the level of significance.

Technical setup

Equipment and Materials

• Surface EMG recording electrodes (WhiteSensor® WS, Ambu A/S, Ballerup, Denmark) • High Performance AC Amplifier (LP511 AC amplifier, Grass Technologies, West Warwick, US) • Data acquisition device (DAQ USB 6221 National Instruments, Austin, USA) • Isolated linear bipolar constant current stimulator (DS5, Digitimer, Welwyn Garden City, UK) • Hum Bug 50/60 Hz Noise Eliminator, (Quest Scientific Instruments, North Vancouver, Canada) • QTRAC Software with TROND-NF excitability protocol (©Institute of Neurology, Queen Square, London) • Red Dot Trace prep (2236, 3M Canada) • Thermistor thermometer (5831-A, Omega Engineering, Manchester, UK).

Figure 14. Technical flowchart.

This flow chart demonstrates the relationships between the stimulation and recording hardware and the patient. 56

Figure 15. Equipment

The hardware required for performing axonal excitability testing. Anticlockwise from top right, Grass LP511 amplifier, Hum Bug noise eliminator, Digitimer DS5 constant current stimulator, NI USB-6221 D/A data acquisition device, Windows computer (out of picture).

Figure 16. QTRAC.

An example screenshot demonstrating QTRAC software running through a

TROND protocol. Top line demonstrates the stimulation output. The second line demonstrates the CMAP, the third line demonstrates the changes in threshold.

Chapter 1:

Assessment of axonal excitability properties in two branches o the human facial nerve.

Summary

To date, experience with attaining axonal excitability measures in the facial nerve has been limited. In order expand excitability techniques into the study of facial neuropathy it was necessary for the technique to be optimised and refined in a robust group of normal controls. This chapter demonstrates the feasibility of applying two standardised techniques for acquiring facial nerve exitability measures: zygomatic nerve to nasalis, and marginal mandibular to depressor angularis oris. Recordings were attained across a broad cross-section of ages.

Facial nerve recordings were found to be similar for both branches.

Introduction

To date the application of this technique to the study of the facial nerve has been limited to a single small study (Krishnan, Hayes, and Kiernan 2007). The facial nerve presents a number of unique challenges for translating this technique due to its complex anatomical arrangement, with many nerve branches intercommunicating and subdividing to form a highly variable network in the confined space of the face. Using anatomical information gleaned from the surgical literature (Captier et al. 2005; Dorafshar et al. 2013; May 2000) the present study was designed to assess facial nerve excitability properties in two different braches of the nerve, namely the marginal mandibular branch and zygomatic branches and in a wide age range of participants. As many facial nerve pathologies, including congenital, infectious, traumatic and malignant causes, may selectively affect individual branches(Croxson 1990), a method of testing more than one branch may increase the utility of this technique in studies of facial nerve injury.

Methods

27 participants (11 M : 16 F; age range 20-64 years; mean 36.1 years) underwent facial nerve excitability studies of the zygomatic branch. Of this group, 19 had studies performed in two branches (zygomatic and marginal mandibular branches) in the same sitting. Factors limiting marginal mandibular testing in some subjects included the presence of facial hair and participant preference. A full set of excitability parameters was obtained in each recording.

Median nerve studies with abductor pollicus brevis (APB) recordings from 29 61 control subjects were used for comparison. The anatomical setups for the recording protocols are as follows:

Technique 1: Zygomatic-Nasalis (Zyg-Nas)

The zygomatic branch (figure 1.1) supplying the nasalis muscle was assessed using a stimulating electrode placed over Zuker’s point(Dorafshar et al. 2013), a surface anatomical landmark for the middle division of the facial nerve located

5cm along the line from the tragus to the corner of the mouth. The reference stimulation electrode was located over the angle of the mandible. The primary recording electrode was located over the nasalis muscle and the recording reference was located over the midline lower forehead. The ground was located between the stimulation electrode and the recording electrode on the subjects face. Zuker’s point was chosen to optimise the selective activation of the branch to nasalis while minimising volume conduction effects due to stimulation of adjacent cranial nerves. Due to the short distance between stimulation and recording electrodes clamping was performed following the stimulation artifact and prior to the onset of the tracked CMAP recording.

Technique 2. Marginal Mandibular-Depressor Angularis Oris (MM-DAO)

For recording the properties of the marginal mandibular nerve to the depressor angularis oris (DAO) the stimulation electrode was placed 1cm below and anterior to the angle of the mandible (figure 1.1). For subjects with more extensive skin laxity or poor jaw definition the head was turned 45 degrees to the contralateral side. All recordings were made with the patient sitting upright. 62

The recording electrode was located 2cm below and 1cm lateral to the corner of the mouth on the ipsilateral side.

DAO

Nasalis

Zuker’s point

Figure 1.1. Stimulation sites.

Anatomical representations of marginal mandibular (A) and zygomatic branch

(B) landmarks used for recording facial nerve excitability properties. 64

Results

The techniques for facial nerve excitability described were well tolerated across the cohort of participants. All subjects completed the testing protocol. Table 1.1 illustrates the multiple excitability measures for both the zygomatic and marginal branch methods. The excitability parameters were found to be similar between the two facial nerve branches with only differences in latency and refractoriness reaching levels significance. These changes may reflect technical differences.

A comparison between nasalis facial nerve recordings and median nerve recordings (Figure 1.2 and Figure 1.3) demonstrates significant differences between the two nerves. Stimulus response properties show a decreased peak response (nasalis 2.3±1.1mV; median nerve 8.6±1.1mV, p<0.001), a right shift in stimulus current for 50% maximal response (nasalis 3.9±1.1mA; median nerve

3.0±1.1mA, p<0.05), and a decrease in the slope of the . The strength-duration time constant was not significantly altered between the two nerves. Comparison of TE results between the facial and median nerve recordings demonstrates widespread reductions in the magnitude of response to both hyperpolarising and depolarising currents resulting in a ‘fanned-in’ appearance (Figure 1.2C).

Changes to RC include a decrease in magnitude of superexcitability(%) (nasalis

10.3±1.3; median nerve -25.4±1.1, p<0.001), a trend towards increase in the relative refractory period (RRP) (nasalis 3.3±1.0; median nerve 3.1±1.0, p=0.08).

Overall the pattern of change, and in particular the fanning in of TE, the trend to increased refractoriness and decrease in magnitude of superexcitability between 65 the facial nerve and median nerve are suggestive of a relative membrane depolarisation. These results are consistent with the findings of a previous study comparing facial nerve recordings with median and tibial nerve excitability parameters(Krishnan, Hayes, and Kiernan 2007).

Zygomatic branch Vs Marginal mandibular branch

27 zygomatic branch (Nasalis) recordings were compared to 19 marginal mandibular branch (depressor angularis oris) recordings (Table 1.1). In comparing peak response, nasalis (2.3mV) and depressor angularis oris (2.3mV) were found to be similar. The demonstrable difference in latency (nasalis 7.1+/-

0.2ms; DAO 6.0+/-0.3ms, p<0.005) is not unexpected given the shorter distance between the marginal mandibular stimulation and recording sites. A difference in early refractoriness at 2.5ms as represented by percentage threshold change was also seen. This is likely a reflection of artifact created by facial movement in closely paire pulses.

Marginal Mandibular - Zygomatic – Nasalis DAO p value Number of subjects 27 19

M:F 11:16

Age (years) 36.1 (2.9) 30.3 (2.7) NS

Stimulus response behaviour

Peak response\(mv) 2.6 (0.2) 2.6 (0.3) NS Latency (ms) 7.1 (0.2) 6.0 (0.3) **

Strength-duration property

Stimulus (mA) for 50% max 4.1 (0.3) 4.2 (0.3) NS response Strength-duration time constant 0.4 (0.0) 0.4 (0.0) NS (ms) Rheobase (mA) 2.6 (0.2) 2.8 (0.2) NS

Threshold electrotonus

TEd(10-20ms) (%) 59.4 (1.0) 58.9 (1.1) NS TEd(90-100ms) (%) 39.4 (0.9) 40.9 (0.9) NS TEh(90-100ms) (%) -111.8 (4.3) -111.7 (6.4) NS

Recovery Cycle

RRP (ms) 3.3 (0.1) 3.3 (0.1) NS Refractoriness at 2.5ms (%) 34.1 (3.9) 22.4 (3.6) * Superexcitability (%) -10.3 (1.3) -7.3 (1.4) NS Subexcitability (%) 14.1 (1.2) 13.9 (1.2) NS

I/V parameters

Resting I/V slope 0.7 (0.0) 0.6 (0.0) NS Minimum I/V slope 0.2 (0.0) 0.2 (0.0) NS Hyperpol. I/V slope 0.3 (0.0) 0.3 (0.0) NS

Table 1.1. Nerve excitability results for two branches of the facial nerve

Data are given as mean (±SEM), p values were determined using paired t-test for

the 19 paired recordings performed in the same subjects during the same sitting. 67

A B

0 5 d charge d Current

(mA.ms) (mA.ms) (% threshold) threshold) (% Threshol

-100 0 -500 0 -1 0 1 Threshold reduction (%) Stimulus width (ms)

100 100 C D

0 (%) (%) (%) (%) Threshold change Threshold

Threshold reduction Threshold -100 0

-200 0 100 200 10 100 Delay (ms) Interstimulus interval (ms)

Figure 1.2. Facial nerve Vs median nerve.

Facial nerve-nasalis (red, filled circles; n=27) excitability measures are compared with age matched median nerve (blue, hollow circles; n=29) axonal excitability recordings. Figures demonstrate mean recordings (filled line) with standard error of mean (dotted lines). Panels include current-threshold (A), strength-duration (B), threshold electrotonus (C) and RC (D) waveforms.

100 A B 100

10 (mv) (mv) TEd(10-20ms) TEd(10-20ms) Peak response Peak

1 50

Median Nasalis DAO Median Nasalis DAO

-50 40 C D

20 S2 accommodation accommodation S2 Superexcitability (%) (%) Superexcitability 10

0 0

Median Nasalis DAO Median Nasalis DAO

Figure 1.3. Distribution of excitability variables.

(A) peak response, (B) TEd(peak) (C) Superexcitability (%) and (D) S2 accommodation are demonstrated for median nerve (n=29), nasalis (n=27) and

DAO (n=19) recordings. Straight line demonstrates mean recordings, dotted lines represent the SEM 69

Age

Facial nerve recordings (zygomatic branch to nasalis) were subgrouped into 3 age groups for comparison: under 30 (n=15); 31-50 (n=5); 51 and over (n=5). No difference between excitability properties such as TE, RC and strength-duration time constant (τSD) were found across age groups. Changes in stimulus response properties (figure 1.4) were demonstrated to have statistically significant results included a decrease in peak response (p<0.01), an increase in the stimulus (mA) required to achieve a 50% maximal response (p,0.05), and rheobase (p<0.05).

These findings are typical of ageing and have been demonstrated in other excitability studies in other muscles(Jankelowitz, McNulty, and Burke 2007; Bae et al. 2008). These changes represent a higher stimulation current requirement possibly due to physical changes in current conductance or global changes in nerve current response as opposed to a biophysical or pathological alteration in resting membrane potential or specific channel conductivity.

Figure 1.4. Nasalis stimulus response changes with age.

Mean responses for three age groups are demonstrated, less than 30years (pink),

30-50years (blue) and greater than 50 years (green). CMAP responses were attained for a 1-ms duration stimulus of gradually increasing amplitude until maximum CMAP response was achieved. 71

Gender

Nasalis recordings between male (n=11) and female (n=16) participants were compared and analysed for statistical differences. Females had a lower

TEh(overshoot) 8.45 vs 11.62 (p<0.01) and the resting I/V slope was lower

0.643 vs 0.735 (p<0.05). In isolation these findings are of uncertain significance.

The pattern and overlay of excitability properties is demonstrated in Figure 1.5.

There was no demonstrable difference in CMAP size between male and female subjects (2.3mV Vs 2.4mV). Gender based subgrouping of DAO recordings are shown in Figure 1.6. There were no demonstrable changes between males and females in the studied cohort.

A B

0 5 d charge d Current (mA.ms) (mA.ms) (% threshold) threshold) (% Threshol

-100 0 -500 0 -1 0 1 Threshold reduction (%) Stimulus width (ms)

100 100 C D

0 (%) (%) (%) (%) Threshold change Threshold

Threshold reduction Threshold -100 0

-200 0 100 200 10 100 Delay (ms) Interstimulus interval (ms)

Figure 1.5. Gender based excitability measures for nasalis recordings.

Mean group data is presented for males (green, empty triangles) and females

(blue, filled diamonds). Current-threshold (A), strength-duration (B), threshold electrontonus (C) and RC (D) plots are demonstrated. Statistically significant difference was noted in TEh overshoot (Males 11.6+/-0.8; Females 8.6+/-0.7, p<0.01) and a flattening in IV 73

A B

0 5 d charge d Current

(mA.ms) (mA.ms) (% threshold) threshold) (% Threshol

-100 0 -500 0 -1 0 1 Threshold reduction (%) Stimulus width (ms)

100 100 C D

0 (%) (%) (%) (%) Threshold change Threshold

Threshold reduction Threshold -100 0

-200 0 100 200 10 100 Delay (ms) Interstimulus interval (ms)

Figure 1.6. Gender based excitability measures for mean DAO recordings.

Males are presented as green, empty circles (n=6), females are presented as blue, filled circles (n=13). There were no statistically significant differences between sexes. Excitability measures presented are current-threshold (A), strength- duration (B), threshold electrontonus (C) and RC (D).

Discussion

This study demonstrates that excitability properties can be measured consistently across a range of age groups, in both sexes and in both the zygomatic and marginal mandibular branches of the facial nerve. Although a number of observations were made regarding minor excitability differences with age and gender, a limitation of the study is that it may not be adequately powered to demonstrate all age and gender differences which have been shown i larger studies in other nerves(McHugh, Reilly, and Connolly 2011).

Comparison of facial nerve recordings with median nerve has demonstrated a number of differences including a decrease in the magnitude of superexcitability, fanning-in of threshold electrotonus and a right shift of the stimulus-response curve with a lower maximal CMAP.

During this study, a number of challenges arose which are peculiar to facial nerve excitability techniques. There is considerable variation in face size and skin texture. This can make electrode choice and placement difficult. The liberal use of skin preparation and specialised medical tape was useful in offsetting electrode displacement during the test. The location of the stimulating electrode at Zuker’s point occasionally resulted in direct masseteric and/or nerve to masseter stimulation in a number of patients. A number of subjects observed a metallic taste possibly due to parotid glandular stimulation.

The extra-cranial facial nerve is anatomically deep at its exit from the styloid foramen and is covered by the sternocleidomastoid, the digastric muscle and the parotid gland. After exiting the styloid foramen and entering the parotid gland, 75 where the nerve courses to become more superficial, the facial nerve divides at the pes anserinus into upper and lower divisions; the upper giving rise to the temporal and zygomatic branches and the lower giving rise to the marginal mandibular, cervical branches. The buccal branch may come from the upper or lower division. The subsequent branching pattern of the facial nerve resembles a fishing net with considerable intercommunication and variability. The extensive peripheral arborisation results in traditional named branches in reality representing multiple small branches supplying specific functions(Diamond et al.

2011; May 2000).

This anatomical diversity of facial nerve branching patterns(Diamond et al.

2011) presents a challenge for standardising stimulation and recordings sites for axonal excitability testing. To consolidate the anatomical diversity into a reproducible techniques, regions of branch anatomical consistency was sought from the literature. Two such points of anatomical consistency were Zuker’s point(Dorafshar et al. 2013), a location 5cm along the line between the tragus and the corner of the mouth which overlies the facial nerve branch supplying the midface musculature (and nasalis), and the marginal mandibular branch just below the angle of the mandible(May 2000).

Traditionally facial motor nerve conduction studies stimulate the facial nerve trunk at the stylomastoid foramen(Kennelly 2012). Although this may be feasible with axonal excitability testing, there are potential disadvantages including retroauricular pain due to post-auricular nerve activation, accessory nerve 76 stimulation and sternocleidomastoid stimulation resulting in discomfort and involuntary neck, shoulder and head movement at higher stimulation currents.

This study lays important ground work for the future study of facial nerve disorders. Axonal excitability measures provide valuable insight into the biophysical properties of peripheral nerves in health and disease and the development of this technique for application into the study of facial nerve disorders is an important step towards understanding the pathophysiology of common causes of facial neuropathy.

Chapter 2:

Altered axonal excitability in chronic facial palsy

Introduction

The present study aimed to establish the feasibility of axonal excitability techniques in the setting of established facial palsy. Axonal excitability measures are an established investigative modality which provide important insights into the behaviour of voltage-gated ion channels, pumps and exchangers that are involved in impulse conduction (Krishnan et al. 2009; Krarup and Moldovan

2009; Kiernan and Kaji 2013) . These techniques have demonstrated changes in numerous forms of peripheral nerve injury (Sawai et al. 2008; Arnold et al. 2013;

Lin et al. 2011). These techniques may fill an important need by providing insight into the underlying pathophysiology of axonal dysfunction or provide justification for new avenues of therapeutic intervention to optimise residual axonal function. In the present study, we recruited a group of patients with chronic facial palsy due to viral and non-viral aetiologies.

Methods

Inclusion criteria for this study were patients greater than 18 years of age who had facial palsy due to viral causes (BP or herpes zoster) which had not recovered after 6 months, facial palsy in the context of a parotid tumour

(primary or recurrent), or facial palsy secondary to traumatic injury. Exclusion criteria were pregnancy, pre-existing neurological disease, prior treatment with botulinum toxin injection and conditions known to alter axonal excitability properties such as diabetes, renal impairment and prior chemotherapy exposure. 79

Group characteristics

A broad cross-section of chronic facial palsy patients were represented with 30 patients being included in the study. Recordings were attained from 23 patients, this included 15 viral, five trauma and three tumour related facial palsy patients.

Seven patients did not have full excitability recordings performed. This was due to the facial nerve being inexcitable or because of technical limitations such as excessive signal artifact with small CMAP responses. Of the patients with full excitability recordings, the mean duration of facial palsy was 73 months (range:

2-283 months) and the mean of the House-Brackmann scores was 3.4 (range 2-

5). Patient groups were classified according to aetiology as follows:

1. Viral: unrecovered BP or HZO (Ramsay-Hunt Syndrome).

2. Trauma: all trauma related cases in this group were associated with

iatrogenic facial nerve injury from intracranial, skull-base or head and

neck surgery. All patients in this group had normal facial nerve function

prior to surgery.

3. Tumour: this group included patients with head and neck tumours, such

as parotid tumours, who also had demonstrable facial weakness most

likely due to malignant infiltration.

Normative data for comparison was from facial nerve excitability studies performed on 27 normal controls. The standardised method of facial nerve excitability testing used the zygomatic branch of the facial nerve recorded from 80 nasalis. A description of this setup is described elsewhere(Eviston, Lin, and

Krishnan 2014).

Results

Chronic facial palsy

30 patients with chronic facial palsy were enrolled in the study. Threshold tracking was found to be feasible in 23 subjects (13 females, 10 males; mean age

46.4; range 29-71). Four subjects had insufficient recordable CMAP to perform axonal excitability measures. Full recordings were achieved from participants with facial palsy severities between HB grade II-V.

Grouped facial palsy data were compared with normal controls (figure 2.1 and figure 2.2). Changes in excitability measures between facial palsy and normal controls included changes in stimulus-response properties, TE, RC and I/V.

Stimulus-response recordings demonstrated a decrease in peak response (facial palsy 0.7338±1.18mV; control 2.332±1.1 mV; p<0.0001), an increase in stimulation current for 50% maximal CMAP (facial palsy 6.59±1.1mA; control

3.86±1.07mA, p<0.001) and increased rheobase (facial palsy 4.59±1.11mA; control 2.44±1.08mA, p<0.001). The strength-duration time constant was not significantly altered (facial palsy 0.4156±0.0359; control 0.4361±0.0183, NS).

Figure 2.1. Excitability measures in chronic facial palsy

Chronic facial palsy (filled circles; n=23) are compared to normal control (hollow circles; n=27) facial nerve recordings. (A) demonstrates current-threshold (I/V) properties, (B) Strength-duration properties, (C) TE, (D) RC. 82

Figure 2.2 – Distribution of excitability variables.

for normal control (n=27) and diseased facial nerve (n-23) recordings. Mean

(solid line) and Standard Error of the Mean (dotted line) are displayed.

Statistically significant differences are seen in Rheobase (p<0.0001), peak response (p<0.0001) and resting I/V slope (p<0.0001). Superexcitability (%) changes did not reach significance (p=0.07).

TE recordings using polarising subthreshold currents ±40% of the unconditioned threshold current demonstrated an increase in magnitude to both hyperpolarising and depolarising threshold electrotonus values. The increased magnitude response to hyperpolarising subthreshold currents was found to be significant for TEh (20-40ms) (facial palsy -102 ± 5.23%; control -88.83 ± 1.97%, p<0.05), and TEh (90-100ms) (facial palsy -132.3 ± 8.42%; control 113.2 ±

4.23%, p<0.05). For depolarising subthreshold currents, the increase in magnitude reached significance at TEd (40-60ms) (facial palsy 46.46 ± 1.38%; control 43.2 ± 0.72%, p<0.05).

For the current-threshold relationship (I/V), a measure of threshold change to prolonged depolarising and hyperpolarising currents of different intensities, the resting I/V slope, a representation of membrane responses to stimuli of 200ms duration, was decreased (facial palsy 0.597 ± 0.029; control 0.689 ± 0.021, p<0.01) while hyperpolarising I/V slope was increased (facial palsy 0.389 ±

0.049; control 0.28 ± 0.016, p<0.05).

Assessment of RC parameters, a measure of threshold change to paired stimuli of various interstimulus intervals, demonstrated a decrease in refractoriness at

2.5ms (facial palsy 18.24 ± 5.03%; control 34.06 ± 3.95%, p<0.05), a decrease in the magnitude of subexcitability (facial palsy 9.603 ± 1.33%; control 13.3 ± 0.9%, p<0.01).

Subgroup analysis

15 patients with longstanding facial palsy attributed to a viral aetiology were compared to those with non-viral aetiologies (3 tumour and 5 trauma). The purpose of this analysis was to explore if there were distinct patterns of change exhibited by different aetiologies.

Comparing viral to non-viral aetiologies demonstrated distinct points of difference. In particular, the pattern of change in response to depolarising subthreshold currents in threshold electrotonus for virus-induced facial palsy was similar to normal controls whereas non-viral aetiologies demonstrated a

‘fanned out’ threshold electrotonus pattern with increased magnitude in the response to both depolarising and hyperpolarising 40% subthreshold polarising currents. The peak CMAP was similar between both groups (viral 0.84 ± 1.26mV; non-viral 0.56 ± 1.23mV, p=0.27). Specific variables which demonstrated significant changes between viral and traumatic groups were TEd (10-

20ms)(viral 59.2 ± 1.3%; trauma 67.8 ± 3.2%, p<0.01) and TEd (peak)(viral 58.4

± 1.2%; trauma 65.7 ± 3.1%, p<0.05). Variables that were approaching significance were TEd (40-60ms)(viral 45.2 ± 1.4%; trauma 51.1 ± 3.4%, p=0.07), TEd (90-100ms) (viral 40.9 ± 1.6%; trauma 47.9 ± 4.0%, p=0.07). RC, strength-duration and current-threshold comparisons between viral and non- viral aetiologies did not demonstrate any statistically significant differences. 85

Figure 2.3 – Threshold electrotonus.

Viral (filled black circle; n=15) and non-viral facial palsy (gray triangle; n=8) compared to normal controls (black hollow circle; n=27). Plot represents % changes in the stimulation required to achieve threshold before, during and after conditioning subthreshold currents ±40% of baseline threshold current up to a length of 100ms. By convention positive values represent a reduction in threshold.

Discussion

In the present study, we were able to attain excitability recordings in 23 participants with established facial palsy. Recordings were feasible in all House-

Brackmann grades, aside from Grade VI. The constellation of excitability changes of decreased refractoriness, increased superexcitability, exaggerated responses to hyperpolarising electrotonus and a decrease in resting I/V slope are consistent with experimental findings of hyperpolarisation of the resting membrane potential(Kiernan 2000) and are concordant with excitability changes noted in models of Wallerian degeneration(Sawai et al. 2008; Moldovan and Krarup 2004a; Moldovan, Alvarez, and Krarup 2009). Previous studies have shown that axonal excitability abnormalities in regenerating axons following

Wallerian degeneration may persist for a prolonged period following the initial insult (Moldovan and Krarup 2004a). These changes have been hypothesised as being due change in fibre diameter, altered resting membrane potential or altered rectification properties(Moldovan and Krarup 2004b). Such abnormalities were noted across all facial palsy groups in this study. The strength duration time constant (τSD), a marker of nodal sodium conductance(Krishnan et al. 2009), was not significantly different between normal controls and the disease groups in our study. This is a point of difference from previous studies of regenerated axons in the setting of vasculitis(Sawai et al. 2008) and animal studies of Wallerian degeneration(Moldovan, Alvarez, and

Krarup 2009) have demonstrated an increase in τSD.

On subgroup analysis, facial palsy due to viral aetiology had minimal changes in depolarising threshold electrotonus, which distinguished it from the non-viral facial palsy group. The ability of an axon to accommodate to a depolarising subthreshold current is determined by slow K+ conductances(Moldovan and

Krarup 2007) and this difference may reflect an aetiologically distinct pattern of axonal function or be representative of maturational differences between the disease groups.

A limitation of the subgroup analysis was the smaller number of subjects in the non-viral group and these findings will therefore need to be established in larger studies. Another confounding factor may be the duration of facial palsy. 50% of the non-viral palsy group had experienced facial palsy for less than 6 months duration whereas the entire viral group had facial palsy for longer than 6 months. This introduces a bias toward more immature axons in the non-viral group with the observed differences potentially reflecting changes in maturity of axons rather than distinct aetiological differences. Long-term studies of regenerated axons in a cat model demonstrated a tendency for depolarising threshold electrotonus to return towards normal while abnormalities in hyperpolarising threshold electrotonus persisted(Moldovan and Krarup 2004b).

Further exploration of this discrepancy has suggested that the combination of structural shortening of internodal segments and overactivity of outwardly rectifying fast K+ currents may be the underlying cause(Moldovan and Krarup

2007).

From a clinical perspective, the manifestations of chronic facial palsy encompass a varying degree of weakness, spasm, involuntary movement, synkinesis, parasympathetic dysfunction (excessive or reduced tearing/salivation) and sensory symptoms such as pain, tightness and fatigue(Hadlock et al. 2006;

Eviston et al. 2015; VanSwearingen and Brach 1996). Given this heterogeneity of clinical symptoms and the diversity of known causes of facial palsy, it is highly likely that more than one pathophysiological mechanism is at play. Our study demonstrates that excitability recordings are feasible in facial palsy due to different aetiologies and has raised the possibility of different excitability profiles according to aetiology, a hypothesis that will require further investigation. Further work is also required to delineate the utility of the technique in determining prognosis and response to treatment. The improved understanding of underlying axonal characteristics which this technique may offer in facial palsy has the potential to guide treatment strategies to improve residual function and explore the origins of debilitating symptoms such as facial muscle spasm and synkinesis. 89

Chapter 3:

Evidence of sodium channel dysfunction in Bell’s palsy 90

Summary

BP is a highly prevalent debilitating facial neuropathy which presents with sudden onset lower motor neuron facial weakness, posterior auricular pain and dysgeusia. The mechanisms leading to axonal dysfunction in BP remain unclear.

This study adapted axonal excitability techniques to assess facial motor nerve excitability in a group of patients with BP. A total of 10 patients with moderate to severe facial weakness of less than 10 days duration were consecutively recruited. In two patients, the facial nerve was inexcitable. In eight patients, a full set of nerve excitability recordings was obtained. Values were compared to healthy controls and to a disease control group of 10 patients with facial palsy due to other causes. Excitability changes in the BP group demonstrated a right shift in stimulus response properties, decreased strength-duration time constant

(p<0.05), decreased refractoriness (p<0.05), decreased subexcitability (p<0.05), and decreased magnitude of response to early depolarising threshold electrotonus (10-20ms). These changes were confined to BP and were different to those in the disease control group. The pattern of change in excitability in the

BP group is characteristic of reduced inward sodium conductances, as previously demonstrated in the setting of tetrodotoxin poisoning. The results suggest that axonal sodium channel dysfunction may be the initial physiological abnormality i BP. 91

Introduction

The aim of this study was to assess the mechanisms of axonal dysfunction in acute BP using nerve excitability techniques (Burke, Kiernan, and Bostock 2001;

Krishnan et al. 2009). While these techniques have been used extensively in the setting of peripheral nerve injury(Sawai et al. 2008; Krishnan et al. 2008; Krarup et al. 2012), there have been no previous studies of excitability in patients with facial palsy of any cause. In the present study, these techniques were adapted for assessment of facial motor nerve properties (Krishnan, Hayes, and Kiernan

2007) in a cohort of patients with acute BP. Separate studies were undertaken in a cohort of healthy controls and in a disease control group of patients with facial palsy due to other causes.

Methods

A total of 10 patients (seven males, three females) were consecutively recruited.

Following recruitment, facial movements were videotaped using a standardised process for facial palsy grading(Lee et al. 2012). House-Brackmann scores, a measure of facial palsy severity, were assigned by two clinicians who independently graded the videotapes (House and Brackmann 1985). The House-

Brackmann scale is a widely used facial palsy assessment tool with six levels of severity. Grade I (normal) represents normal, symmetrical facial function in all areas. Grade II (mild dysfunction) has slight weakness on close examination, the face is symmetrical at rest. Grade III (moderate dysfunction) shows obvious but not disfiguring asymmetry with or without synkinesis and the face is still symmetrical in repose. Grade IV (moderately-severe dysfunction) has obvious, 92 disfiguring asymmetry on movement and eye closure is incomplete. Grade V

(severe dysfunction) shows only a flicker of movement on effort. Grade VI (total paralysis) shows no movement at all.

Studies of facial nerve excitability were also conducted in 27 healthy control subjects (age range 22-64years; mean age 36.1 years) and in a disease control group of 9 patients (age range 34-71 years; mean age 48.9years) with established non-Bell’s facial palsy (NBP). This group comprised of three patients with parotid tumours who had malignant infiltration of the nerve and six patients with facial palsy that developed following surgical operations for tumours near or involving the facial nerve (one intracranial epidermoid, one acoustic neuroma, four parotid tumours). All of the NBP group had established facial palsy for greater than 1 month duration (duration 2-283 months; mean

80months). 93

Patient Days post House- Facial nerve Age number onset brackmann excitable 1 31 7 III Yes 2 47 6 V Yes 3 36 9 III Yes 4 55 5 V Yes 5 50 5 V Yes 6 60 7 V Yes 7 49 5 V Yes 8 50 2 IV Yes 9 40 8 VI No 10 41 9 V No

Table 3.1. Clinical characteristics.

Patients with acute onset unilateral facial palsy attributed to BP. All patients were on or had completed corticosteroid therapy at the time of testing. House-

Brackmann grading I: normal facial function, II: Mild dysfunction, III: Moderate dysfunction, IV: Moderate-severe dysfunction, V: Severe dysfunction, VI: Total paralysis.

Axonal excitability testing was conducted using an established protocol of paired conditioning and subthreshold polarising currents applied through non- polarising surface electrodes(Kiernan et al. 2000). Skin temperature was confirmed as being 33˚C or higher in all subjects. The technical setup for recording facial nerve excitability measures was based on the stimulation of the zygomatic branch of the facial nerve at Zuker’s point (Dorafshar et al. 2013), 5cm along the line between the tragus and the corner of the mouth with the reference electrode located over the corner of the mandible. The compound muscle action potential (CMAP) was recorded from a surface electrode overlying the nasalis muscle, with the reference electrode located in between the eyes on the forehead

(Eviston, Lin, and Krishnan 2014). Data was acquired using QTRAC-S software

(©Institute of Neurology, London) using the TROND protocol(Kiernan et al.

2000). Excitability measures were performed by generating a stimulus response curve using 1ms duration impulses of increasing intensity under the guidance of the user until maximal CMAP was achieved. Using this curve a ~40% target

“threshold” was calculated. Strength-duration, threshold electrotonus (TE), RC and current-threshold (I/V) parameters were acquired automatically.

Analysis

Excitability parameters were calculated and compared using QTRAC-P software

(©Institute of Neurology, London) and SPSS version 22 (©IBM Corp, Armonk,

NY). Student’s t-test was used for comparisons of normally distributed data and

Mann-Whitney U-test was used for non-normally distributed data. Results are 95 presented as mean ± standard error of the mean (SEM) and significance was based on a p value <0.05.

Results

A full set of motor excitability recordings were obtained from 8 of the 10 BP patients recruited into the study. The clinical characteristic of these patients are presented in Table 1.1. In 2 patients, the facial nerve was inexcitable. For the recorded patients, the mean age was 47.2 (range 31-60) with six males and two females. The average time from onset of facial palsy symptoms to clinical testing was 5.8 days (range 2-9days) and the average severity was House-Brackman 4

(range 3-5) with most patients having severe facial weakness.

Nerve excitability comparison with the normal facial nerve

When compared to the normal control facial nerve group significant differences were found across a number of excitability parameters (Table 2). In BP there was a trend for the stimulus response curve to be shifted to the right with an increase in stimulus intensity needed to produce a CMAP 50% of maximal response (BP

4.83 ± 1.07 mA; control 3.86 ± 1.07 mA, p=0.09). The peak response was decreased (BP 1.44 ± 1.25 mV; control 2.33 ± 1.1 mV, p<0.05), rheobase was increased (BP 3.42 ± 1.09 mA; control 2.44 ± 1.08 mA, p<0.05), and the strength

+ duration time constant (τSD), a marker of persistent Na currents (Mogyoros,

Kiernan, and Burke 1996), was significantly decreased (BP 0.36 ± 0.02; control

0.44 ± 0.02, p<0.05). Compared to controls, RC parameters demonstrated a decrease in refractoriness at 2.5ms (BP 17.0 ± 6.1%; control 34.1 ± 3.9%, 96 p<0.05) and a decrease in subexcitability (BP 6.4 ± 2.0%; control 13.3 ± 0.9%, p<0.05). Superexcitability and the relative refractory period were unchanged.

A number of changes in threshold electrotonus, reflecting nodal and internodal conductances, were also noted in BP compared with normal controls. The response to subthreshold depolarising currents (TEd) demonstrated a reduction in the magnitude of responses for 10-20ms currents (BP 55.3 ± 2.0%; control

59.7 ± 1.0%, p<0.05) and a trend towards reduction in TEd(peak) (BP 54. ±

2.0%; control 58.3 ± 0.9%, p=0.06). TEd(undershoot) (BP -10.9 ± 0.8%; control -

14.6 ± 0.5%, p<0.05), and S2 accommodation (BP 15.4 ± 1.0%; control 18.7 ±

0.7%, p<0.05), were also significantly decreased compared to controls. In contrast to the changes noted in depolarising threshold electrotonus, the response to hyperpolarising threshold electrotonus (TEh) demonstrated a significantly reduced response at 10-20ms (BP -65.3 ± 1.6%; control -72.0 ± 1.2, p<0.05), which then normalised for longer duration hyperpolarising threshold electrotonus currents. TEh overshoot tended to be decreased but this was not significant (BP 7.9 ± 1.2%; control 10.0 ± 0.6%, p=0.10). Current-threshold parameters were not significantly different between BP and controls. 97

BP Normal Control value Number of subjects 8 27 M:F 6:2 11:16 NS Age (years) 47.2 (3.4) 36.1 (2.9) NS

Stimulus response Peak response\(mv) 1.44 (1.25) 2.33 (1.1) *

Strength-duration properties Strength-duration time constant (ms) 0.36 (0.02) 0.44 (0.02) * Rheobase (mA) 3.42 (1.09) 2.44 (1.08) *

Threshold electrotonus (TE) TEd(10-20ms) (%) 55.3 (2.0) 59.7 (1.0) * TEh(10-20ms) 65.3(1.6) 72.0 (1.2) * TEd(undershoot) -10.9 (0.8) -14.6 (0.5) ** S2 accommodation 15.4 (1.0) 18.7 (0.7) * TEd (Accom) 15.4 (0.9) 18.4 (0.5) *

Recovery Cycle Refractoriness at 2.5ms (%) 16.97 (6.14) 34.06 (3.95) * Subexcitability (%) 6.41 (2.05) 13.3 (0.9) **

Table 3.2. Comparison of BP and normal facial nerve excitability parameters. Presented are the parameters found to be significantly different between the two groups. Data is presented as mean values (±SEM), NS not significant, *p<0.05, **p<0.01.

Nerve excitability comparison to non-Bell’s facial palsy (NBP)

Figure 3.1 demonstrates the graphical plots of excitability parameters for BP and the disease control group of non-Bell’s facial palsy (NBP), compared to normal control recordings. A number of excitability parameters discriminated between

BP and those of the disease control group (figure 3.2). The mean peak CMAP response of the disease control group was significantly less than those of the BP group (BP 1.4 ± 1.25 mV; NBP 0.53 ± 1.2mV, p<0.005), but other stimulus response properties such as the stimulus for 50% maximal CMAP and rheobase were similar for both groups. TE differences between BP and NBP were marked.

NBP demonstrated a ‘fanned out’ pattern with increased magnitude responses to both depolarising and hyperpolarising subthreshold currents, possibly reflecting

Wallerian degeneration (Figure 3.1). There were a number of threshold electrotonus parameters that were significantly different between the BP and

NBP groups, including TEd(10-20ms) (BP 55.3 ± 2.0%; NBP 65.1 ± 2.3%, p<0.01), TEd (40-60ms) (BP 42.3 ± 2.3%; NBP 50.6 ± 2.7%, p<0.05), TEd (peak)

(BP 54.2 ± 2.0%; NBP 64.0 ± 1.9, p<0.05), TEd(90-100ms) (BP 38.9 ± 1.8%; NBP

47.3 ± 2.9%, p<0.05), TEh (10-20ms) (BP -65.3 ± 1.6%; NBP -81.2 ± 5.4%, p<0.05) and TEh (20-40ms) (BP -82.3 ± 3.0%; NBP 101.8 ± 9.1%, p<0.05).

Other changes between the BP and NBP groups were a decrease in magnitude of superexcitability (%) (BP -9.3 ± 2.1%; NBP -17.1 ± 3.4%, p=0.07) and a decrease i the resting I/V slope of the NBP group (BP 0.69 ± 0.06; NBP 0.53 ± 0.04, p<0.05) reflecting altered nodal conductance in this group. The constellation of findings in the NBP group of ‘fanned out’ threshold electrotonus, decreased 99 resting I/V and increased superexcitability reflect membrane hyperpolarisation i the NBP group(Kiernan 2000) and are analogous to findings shown in studies of Wallerian degeneration in animal models (Moldovan, Alvarez, and Krarup

2009; Sawai et al. 2008) and human studies of regenerating motor axons (Sawai et al. 2008).

100

100 100 A B

0 (%) (%) (%) Threshold change Threshold

Threshold reduction Threshold -100 0

-200 0 100 200 10 100 Delay (ms) Interstimulus interval (ms)

100 100 C D

0 (%) (%) (%) -100 Threshold change Threshold

Threshold reduction Threshold 0

-200

0 100 200 10 100 Delay (ms) Interstimulus interval (ms)

Figure 3.1. Comparison of excitability measures.

BP recordings (filled circle) compared to normal control (hollow circles) in panels A and B, and with NBP disease controls (hollow triangles) in panels C and

D. Panels A and C are threshold electrotonus recordings representing the percentage change in threshold following ±40% subthreshold polarising currents of a duration up to 100ms. By convention a reduction in threshold is represented as being the positive direction of change. Panels B and D represent

RC recordings which track the change at different timepoints following a conditioning supramaximal stimulus. 101

1 100 A B

NS *

* TEd(10-20ms) TEd(10-20ms) Strength-duration time constant (ms) (ms) constant time *

NC BP NBP NC BP NBP

-50 C D

-50

NS * *

-100 ** TEh(10-20ms) TEh(10-20ms) TEh(20-40ms)

-100 -150

NC BP NBP NC BP NBP

Figure 3.2. Variables which discriminate between BP and NBP facial palsy.

Panels A-D demonstrate excitability variables which highlight the different patterns of change in strength-duration time constant (A) and early threshold electrotonus values (B-D) between normal controls (NC), acute BP and NBP.

Significance based on comparison to normal controls. NS: not significant, * p<0.05, **p<0.005. 102

Discussion

This is the first study to apply axonal excitability techniques to a BP cohort.

Prominent changes in excitability were observed including a reduced τSD, reduced refractoriness, reduced late subexcitability, and reduced magnitude responses in depolarising threshold electrotonus and early hyperpolarising threshold electrotonus. The pattern of changes is broadly consistent with excitability changes that have previously been reported in the setting of sodium channel dysfunction due to tetrodotoxin (TTX) ingestion (Kiernan, Isbister, et al.

2005), mexiletine treatment (Kuwabara et al. 2005), and genetic mutations in the sodium channel subunit, SCN1B (Kiernan, Krishnan, et al. 2005). The changes are most similar to those observed in tetrodotoxin as shown in Figure

3.3. The differences between our recordings in BP and those due to TTX were in late hyperpolarising electrotonus at 90-100ms, hyperpolarising I/V and superexcitability. However, it should be noted that even in TTX ingestion, these changes were not present at baseline but were demonstrated following the addition of hyperpolarisation to reproduce sodium channel dysfunction in a mathematical model of the human motor axon(Kiernan, Isbister, et al. 2005). In our study, the concordant changes in refractoriness, subexcitability, depolarising threshold electotonus, rheobase and τSD are identical to those noted in

TTX(Kiernan, Isbister, et al. 2005).

The excitability changes in BP are unlikely to reflect membrane hyperpolarisation on to basis of Wallerian degeneration which would be the expected finding in the setting of focal compression or ischaemia. This would be expected to cause increased magnitude (‘fanning out’) in depolarising threshold 103 electrotonus and increased magnitude of superexcitability. Indeed, the changes in the NBP group are characteristic of membrane hyperpolarisation and correspond with those of Wallerian degeneration(Moldovan and Krarup 2004a;

Moldovan, Alvarez, and Krarup 2009). Moreover, all BP subjects were tested within 10 days of symptom onset, which would argue against a role for structural alterations as a contributing factor to the neurophysiological abnormalities. The difference in duration of facial palsy between the BP and NBP may be a contributing factor to the differences observed between the two groups and future longitudinal studies of the course of BP may help clarify how the physiological profile of the nerve evolves over time.

The implication of acute sodium channel dysfunction in BP is an important finding for the pathophysiology of the disease, particularly in relation to translating established basic science mechanisms of virus-mediated nerve injury.

Although some controversy has existed as to the role of HSV in the past

(Kennedy 2010) , most clinicians now acknowledge its involvement based on its presence in endoneural fluid sampled during facial nerve decompression(Murakami et al. 1996), its ability to induce BP in animal models

(Sugita et al. 1995; Takahashi, Hitsumoto, and Honda 2001; Wakisaka et al.

2002), and the clinical similarities between BP and varicella zoster induced facial palsy (Hato, Murakami, and Gyo 2008).

Focal facial nerve compression due to swelling in the facial canal of the temporal bone has also been postulated to contribute to the pathogenesis of BP. Evidence against this has been the presence of human histopathological (1976; Adour et 104 al. 1978; Liston and Kleid 1989) and animal data(Wakisaka et al. 2002) demonstrating diffuse pathological changes along the length of the facial nerve in

BP, and the ability to induce BP in settings where bony constriction is not present(Sugita et al. 1995). Clinically, BP has been observed to involve other cranial nerves(Adour et al. 1978) which is also suggestive of a more diffuse process. The diffuse nature of nerve involvement in BP may explain the occurrence of excitability changes in our study, which were demonstrated downstream from the geniculate ganglion. 105

100 10 (mV) (mV) 1 (%) Peak response Peak

Threshold change Threshold 0

.1 10 100 .1 1 10 Interstimulus interval (ms) Stimulus current (mA) 100

0 0 (%) (%) Current

(% threshold) threshold) (% -100 Threshold reduction Threshold

-100 -200 -500 0 0 100 200 Threshold reduction (%) Delay (ms)

Figure 3.3. A side-by-side comparison of excitability measures for acute BP

and TTX poisoning in humans (inset). Normal control facial nerve recordings

(grey), BP (bold, black), TTX poising (solid line, inset) and median nerve controls

(inset dotted grey). Inset panels are reprinted with permission (Kiernan, Isbister,

et al. 2005) ©Wiley, with the black line demonstrating changes noted in TTX

ingestion and the dotted line representing normal control recordings from the

median nerve. 106

HSV is a double stranded DNA alpha herpes virus which commonly causes latent infection in peripheral nerve ganglia(Koyuncu, Hogue, and Enquist 2013). HSV shares genetic and structural similarities with other alpha herpes viruses including varicella zoster (VZV) (Steiner, Kennedy, and Pachner 2007; Kennedy

2002) and asymptomatic carriage in the geniculate ganglion and other cranial nerve ganglia is highly prevalent(Kennedy 2010). In a pivotal study, in-vitro exposure of peripheral nerve to HSV led to acute changes in sodium conductances secondary to selective internalisation of sodium channels via a process which was blocked by chloroquine (Storey 2002). Further evidence of direct axonal interactions by alpha herpes viruses has been demonstrated in a compartmentalised model(Koyuncu, Perlman, and Enquist 2013) where viral exposure induced direct, localised axonal pro-inflammatory mRNA transcription.

This ability of an axon to modify protein transcription at the site of exposure or injury has emerged as a key mechanism in peripheral nerve biology(Jung, Yoon, and Holt 2012) and provides a mechanism which may underlie the sodium channel changes we have demonstrated in this clinical cohort.

From a clinical perspective, conduction block induced by acute sodium channel changes secondary to a direct viral-axonal interaction or a maladaptive axonal response to the viral pathogen would provide a possible explanation for the findings. Acute sodium channel changes in conjunction with inflammation are an important driving mechanism of intra-axonal degeneration pathways(Persson et al. 2013; Conforti, Gilley, and Coleman 2014) and may explain the high prevalence of nerve degeneration and persistent disability seen in a portion of 107 patients who suffer BP. Future studies are needed to examine whether there is a connection between the degree of acute change in sodium channel parameters and long-term axonal degeneration and physical disability.

In conclusion, the data presented in this study demonstrate evidence for acute sodium channel dysfunction in the setting of acute BP in a pattern similar to TTX ingestion. The studies suggest that reduced inward sodium conductances may underlie the development of acute weakness in BP. 108

Chapter 4:

Axonal dysfunction in facial synkinesis

109

Introduction

Synkinesis is the involuntary contraction of a muscle group triggered by the voluntary contraction of another muscle group. For patients who have suffered facial palsy it is a common and debilitating complication which results in facial asymmetry, disfigurement and disruption of normal emotional expression(Beurskens, Oosterhof, and Nijhuis-van der Sanden 2010). Synkinesis is usually attributed to aberrant regeneration(Moran and Neely 1996), as has been demonstrated in animal models(D. Choi and Raisman 2002; Yamada et al.

2010) (Baker et al. 1994). It is commonly accompanied by involuntary and myokymic style discharges(Valls-Sole, Tolosa, and Pujol 2004; Bacsi and Kiernan

2008) which share common neurophysiological features with hemifacial spasm

(HFS), including lateral spread on blink reflex studies(Eekhof et al. 2000) and enhanced F-wave responses(Oge et al. 2005). These findings have been interpreted as enhanced facial nucleus excitability.

As many as 40-55%(Salles et al. 2015) of patients with long term facial palsy will develop synkinesis. Patterns include oro-ocular, oculo-oral, mentalis and platysma synkinesis. Current treatment approaches to facial synkinesis necessitate a multidisciplinary approach encompassing facial biofeedback and neuromuscular retraining (Lee et al. 2014), selective chemodenervation with botulinum toxin injection(Mehta and Hadlock 2008; Eviston et al. 2015) and surgical myectomy or neuroctomy (Hohman, Lee, and Hadlock 2013; Yoshioka

2015).

110

While previous studies have explored possible structural mechanisms that may lead to synkinesis, there have been no previous studies exploring functional changes in the peripheral nerve trunk itself which may contribute to the development of the ectopic motor features that characterise facial synkinesis.

In the present study, facial nerve function was assessed using axonal excitability techniques, an established neurophysiological method for determining the biophysical properties of peripheral nerve axons in situ(Krishnan et al. 2009;

Nodera and Kaji 2006; Kiernan and Kaji 2013). Experience with facial nerve excitability techniques to date have been limited to normal controls(Eviston, Lin, and Krishnan 2014) and hemifacial spasm(Krishnan, Hayes, and Kiernan 2007).

This is the first application of the technique to the study of synkinesis.

Methods

18 patients with chronic facial palsy were recruited. Inclusion criteria for the study were patients with established facial palsy with and without synkinesis.

Patients were clinically assessed and the severity of facial palsy and synkinesis.

The severity of synkinesis was determined based on the severity of midface

(oculo-oral or oro-ocular) synkinesis using the synkinesis domains of the

Sunnybrook grading scale. This applies 0-3 grading of severity to each region of the face up to a score of 15. Patients were determined for inclusion in the synkinesis group if they scored 2 or 3 in the midface components. This was based on the justification that axonal excitability recordings would be sampling from the zygomatic branch to nasalis and the involvement of these muscles 111 should be a requirement. Studies were also undertaken in patients who scored 0 or 1 and the data obtained was analyzed as a non-synkinesis facial palsy group.

Statistical analysis

Analysis of the recorded axonal excitability measures was performed using

QTRAC-P (©Institute of Neurology, London) and SPSS version 22. Mean data was compared using student’s t-test if it was normally distributed or Mann-Whitney

U if the data was not normally distributed. A normal control group of 27 healthy, age-matched recordings was also used for comparison.

Results

Clinical characteristics

Eight patients (4 females, 4 males; mean age 42.5years; range 28-65years) with severe midface (oculo-oral or oro-ocular) synkinesis were assessed. The severity of facial palsy, based on the House-Brackmann facial nerve grading scale, was 3.75 (range 3-5). The mean Sunnybrook synkinesis score was 5.5

(range 4-7). The disease control (DC) group of non-synkinesis facial palsy comprised 10 patients (7 females, 3 males; mean age 43; range 29-66). The mean

House-Brackmann score was 3.1 (range 2-4) and the mean Sunnybrook

Synkinesis score was 1 (range 0-2).

Nerve excitability findings

Synkinesis patients demonstrated marked alterations in a number of excitability parameters compared to normal controls. Stimulus response parameters demonstrated a significant decrease in peak response (synkinesis 0.70 ± 1.37mV; 112 controls 2.33 ± 1.1mV, p<0.0001), a right shift in the stimulus for 50% maximal

CMAP (synkinesis 7.08 ± 1.09 mA; controls 3.86 ± 1.07 mA, p<0.0001), an increase in rheobase (synkinesis 5.14 ± 1.11 mA; controls 2.44 ± 1.08 mA, p<0.0001) and a decrease in latency (synkinesis 5.69 ± 0.42 ms; controls 7.07 ±

0.15 ms, p<0.0001).

TE properties showed changes in parameters affected by paranodal and internodal K+ conductance including hyperpolarising electrotonus TEh (10-

20ms) (synkinesis 5.14 ± 1.11 mA; controls 2.44 ± 1.08 mA) and TEh (slope 101-

140ms) (synkinesis 5.14 ± 1.11 mA; controls 2.44 ± 1.08 mA). S2 accomodation and TEd (undershoot).

RC parameters were unchanged between normal control and Synkinesis patients. Current-threshold (I/V) demonstrated alterations to minimum I/V slope (synkinesis 5.14 ± 1.11 mA; controls 2.44 ± 1.08 mA) and hyperpolarising

I/V slope (synkinesis 5.14 ± 1.11 mA; controls 2.44 ± 1.08 mA). This is a reflection of increased rectification properties to prolonged hyperpolarising

(200ms) currents and is seen in membrane depolarisation.

Comparison of the synkinesis group with the non-synkinesis facial palsy group

(figure 4.1-4.3) demonstrated marked differences in properties dependent on resting membrane potential. In particular, the ‘fanning in’ of threshold electrotonus, decrease in magnitude of superexcitability and increase in slope of hyperpolarising I/V are consistent with membrane depolarisation. Parameters 113 found to be significantly changed included TEd (10-20ms) (synkinesis 58.3 ±

1.9%; non synkinesis 64.3 ± 1.7%, p<0.05), TEd(40-60ms)(synkinesis 43.4 ±

2.2%; non synkinesis 50.0 ± 1.8%, p<0.05) , TEd (peak)(synkinesis 55.9 ± 1.5%; non synkinesis 63.4 ± 1.3%, p<0.001), TEd(undershoot)(synkinesis -12.0 ± 1.1%; non synkinesi -18.1 ± 2.1%, p<0.05), TEh (10-20ms) (synkinesis -64.8 ± 5.1%; non synkinesi -86.5 ± 6.3%, p<0.05), TEh(20-40ms) (synkinesis -84.0 ± 3.7%; non synkinesis -114.2 ± 8.1%, p<0.01), TEh (90-100ms) (synkinesis -104.8 ±

4.8%; non synkinesis -152.7 ± 11.4%, p<0.01), TEh (slope 101-140ms)

(synkinesis 1.54 ± 1.08; non synkinesis 2.62 ± 1.12, p<0.01). The magnitude of superexcitability (%) (synkinesis -10.3 ± 2.9%; non synkinesis -18.0 ± 2.0%, p<0.05) was significantly decreased (figure 3) and superexcitability at 7ms was approaching significance (synkinesis -8.6 ± 2.9%; non synkinesis -15.5 ± 2.2%, p=0.07). Resting I/V slope (synkinesis 0.68 ± 0.05%; non synkinesis 0.54 ±

0.04%, p<0.05) and hyperpolarising I/V slope were approaching significance

(synkinesis 0.48 ± 0.09%; non synkinesis 0.29 ± 0.02%, p=0.07). 114

100 100 A B C

0 0 (%) (%) (%) Current

(% threshold) threshold) (% -100 0 Threshold change Threshold Threshold reduction Threshold

-100 -200 -500 0 0 100 200 10 100 Threshold reduction (%) Delay (ms) Interstimulus interval (ms)

100 100 D E F

0 0 (%) (%) (%)

Current -100

(% threshold) threshold) (% 0 Threshold change Threshold Threshold reduction Threshold

-200

-100 -500 0 0 100 200 10 100 Threshold reduction (%) Delay (ms) Interstimulus interval (ms)

Figure 4.1. Excitability properties in the synkinesis group.

Axonal excitability properties of normal control (grey triangle) compared with

synkinesis (black circle) (panels A-C) and non-synkinesis facial palsy (hollow

circle) and synkinesis (panels D-F) are demonstrated. A, D current-threshold

relationship; B,E threshold electotonus; C, F, recovery cycle. 115

10 0 A B

-10 5 * *

** -20

* TEd(undershoot) TEh(slope 101-140ms) 101-140ms) TEh(slope -30

-40

synkinesis DC NC synkinesis DC NC

100 1 C D

TEd(peak) TEd(peak) NS

** slope I/V Hyperpol. **

50 0

synkinesis DC NC synkinesis DC NC

Figure 4.2. Discriminating values between synkinesis and non-synkinesis.

TE and I/V properties show distinct alterations in synkinesis compared to non- synkinesis facial palsy (DC) and normal controls (NC).

116

100 A B 100

10 (mv) (mv) TEd(peak) TEd(peak) Peak response Peak

synkinesis DC NC synkinesis DC NC

-50 10 C D

Latency (ms) (ms) Latency

0 Superexcitability at 7 ms (%) (%) ms 7 at Superexcitability

synkinesis DC NC synkinesis DC NC

Figure 4.3. Comparison between selected variables.

Scatter plots demonstrating synkinesis, non-synkinesis facial palsy (DC), and normal control results for peak response (A), peak threshold change to depolarising subthreshold currents in threshold electotonus, TEd (peak) (B), superexcitability following supramaximal stimulation in RC at 7ms (C), and latency (D).

117

Discussion

The present study assessed the excitability of facial nerve motor axons using axonal excitability techniques. The study has demonstrated that there are differences in facial nerve excitability in patients who develop synkinesis when compared to those who do not. Excitability studies reflect changes in voltage- gated ion channel function and membrane potential of nerve fibres within the peripheral nerve trunk and the study therefore suggests that there are changes i ion channel expression in the facial nerve trunk itself. The prominent changes i threshold electrotonus recordings in particular occurred in a pattern that is consistent with altered nerve membrane potential in the synkinesis group.

Specifically, the results suggest a depolarised resting membrane potential in patients with synkinesis(Kiernan 2000). As membrane depolarisation refers to a reduction in the charge separation across the axonal membrane, this may predispose to axonal hyperexcitability. In contrast to the results in the synkinesis group, excitability findings in the non-synkinesis group suggested axonal hyperpolarisation, which may be explained by Wallerian degeneration in facial nerve fibres(Moldovan, Alvarez, and Krarup 2009). Na+ channel kinetics in the nodal membrane demonstrate a regenerative(Krishnan et al. 2009) kinetic profile which reinforces increased inward current towards threshold as the resting membrane potential becomes more depolarised. Clinically, this is an important findings which provides an explanation for the movement abnormalities seen in post facial palsy synkinesis and explains the myokymic discharges(Valls-Sole, Tolosa, and Pujol 2004; Bacsi and Kiernan 2008) reported in previous studies. The finding is also distinct to the changes seen in hemifacial spasm in a previous study which did not demonstrate any axonal excitability 118 changes between the HFS group and normal controls(Krishnan, Hayes, and

Kiernan 2007).

An explanation for the altered resting membrane potential may be altered ion channel transcription distribution following nerve injury (Waxman 2001).

Altered sodium channel transcription has been demonstrated experimentally in nerves as an injury response (Akopian, Sivilotti, and Wood 1996) and in response to alpha herpes viruses infection(Kennedy et al. 2013). This may be the link between the viral causality in BP and HZO and the subsequent abnormal function commonly noted following recovery. The altered kinetics associated with changes in channel isoform expression may lead to secondary changes in resting membrane potential(Krishnan et al. 2009).

The finding of membrane depolarisation in the post-facial palsy setting is contrary to expected findings following Wallerian degeneration. A number of animal and human studies(Moldovan, Alvarez, and Krarup 2009; Sawai et al.

2008; Moldovan and Krarup 2004a) using axonal excitability techniques have all demonstrated hyperpolarisation in various stages of degeneration and regeneration. These changes usually persist(Moldovan and Krarup 2004b) and are thought to reflect structural changes(Moldovan and Krarup 2007) in the axon. This difference between findings in facial palsy without synkinesis and the synkinesis group (figure 4.1 and figure 4.2) underscores the significance of this finding as a cause for the clinical symptoms that patients experience.

119

Currently, botulinum toxin and physiotherapy are the core components of treatment for post facial palsy synkinesis(Salles et al. 2015; Eviston et al. 2015; J.

M. Lee et al. 2014). Due to underlying weakness, chemodenervation therapy needs to be used with caution to avoid exacerbating the underlying facial palsy(Eviston et al. 2015). This treatment balance between facial weakness and synkinesis symptoms is a delicate and ongoing problem for patients and clinicians. The recurrent need for injections is also inconvenient.

The finding of a depolarised resting membrane potential associated with hyperkinesis and myokymia in post-facial palsy synkinesis is o clinical significance. It provides justification for the use of pharmacotherapies which stabilise resting membrane potential as being a feasible adjunctive treatment in the setting of facial synkinesis. Medications which exert a membrane stabilising effect through peripheral nerve sodium channel blockade such as phenytoin, carbamazepine(Jensen 2002) or mexiletine(Kuwabara et al. 2005) seem reasonable given their mode of action. Axonal excitability measures may be useful in patient selection for such a study as evidenced by the findings of this study. 120

Chapter 5:

Axonal abnormalities in the contralateral face in the setting of facial palsy 121

Introduction

It is frequently noted in clinical practice that the unaffected side of the face becomes hyperkinetic following the onset of unilateral facial palsy(Valls-Sole

2002). This results in an exaggeration of the cosmetic deformity(de Maio and

Bento 2007) and in some cases excessive blinking or blepharospasm (Baker,

Miwa, Pastor 1998). The use of botulinum toxin on the contralateral “healthy” side to treat this hyperkinesis and reduce the asymmetry is well documented (de

Maio and Bento 2007; Choi et al. 2013; Kim 2013). Neurophysiological studies of blink reflexes in the setting of unilateral facial palsy have demonstrated altered blink reflex responses on the contralateral side suggestive of hyperexcitability

(Oge 2005; Valls-Sole et al. 2004). This implies that these hyperkinetic changes which occur on the contralateral face have a neurophysiological basis and are not just perceived differences in muscle tone. In this study we apply axonal excitability techniques to examine the biophysical properties of the facial nerve on the contralateral face in patients with facial palsy to evaluate for underlying axonal function changes in this clinical setting. Patients with facial palsy due to a broad cross section of aetiologies were included.

Methods

Institutional ethics approval was attained for the study. 22 patients with established facial palsy of greater than 3 months duration were recruited for the study from participating clinical sites. These included a specialist facial nerve clinic (Sydney Facial Nerve Service), a head and neck cancer clinic (Liverpool 122 hospital) and a neurology service (Prince of Wales hospital). Following informed consent patients underwent clinical assessment that screened for conditions which may alter excitability studies.

Inclusion criteria for the study were persons greater than 18 years of age without pre-existing other neurological disease who had suffered unilateral facial palsy for greater than 3 months duration. Lower motor neuron facial paralysis due to all aetiologies was included. Exclusion criteria were previous botulinum toxin use, pregnancy, pre-existing neurological disease, diabetes, active malignancy receiving chemotherapy, chronic kidney disease requiring renal replacement therapy. Patients with facial palsy of less than 3 months duration were not included in this study on the basis that structural of functional changes in axonal function may take time to develop.

123

Table 5.1. Clinical characteristics

DURATION OF PATIENT AGE AETIOLOGY SEVERITY CLASSIFICATION FACIAL PALSY (months) SURGICAL 1 35 3 moderate 25 INJURY 2 39 BELL'S 4 moderate 49 3 51 HZO 3 moderate 100 4 32 BELL'S 3 moderate 39 5 42 BELL'S 3 moderate 204 6 29 BELL'S 2 mild 160 7 34 HZO 2 mild 9 8 65 BELL'S 2 mild 65 9 34 BELL'S 3 moderate 25 10 52 HZO 4 moderate 35 11 32 BELL'S 2 mild 3 12 28 HZO 2 mild 9 13 37 BELL'S 4 moderate 6 14 40 BELL'S 6 severe 3 SURGICAL 15 39 5/6 severe 21 INJURY SKULL 16 32 6 severe 62 FRACTURE 17 53 BELL'S 5* severe 117 SURGICAL 18 45 5* severe 283 INJURY SURGICAL 19 61 4/5* severe 240 INJURY 20 65 BELL'S 4/5* severe 358 SURGICAL 21 39 5 severe 5 INJURY 22 44 BELL'S 6 severe 2 124

Results

22 patients (7 males, 15 females, mean age 42.2 years; range 28-65) were included in the study. The average severity of facial palsy was HB 4 (range 2-6).

This group included 12 patients with facial palsy due to unrecovered BP, four due to unresolved facial palsy following HZO (Ramsay Hunt syndrome), five due to facial nerve injury during surgical procedures for tumour removal (two accoustic neuromas, one intracranial epidermoid, two parotid gland tumours), and one patient with facial palsy following a base of skull fracture. Four patients had received nerve grafts for facial reanimation procedures (three cross-facial nerve grafts and one VII-XII transfer). The clinical characteristics of the group are summarised in table 5.1.

Comparison of the contralateral facial recordings for the whole group with normal control recordings did not demonstrate systemic changes. However, subgroup analysis was also performed by dividing patients into two groups based on the severity of their facial palsy, a mild-moderate disease (FP) group encompassing HB 2-4 facial palsy, and a severe (SFP) group including those with

HB 5-6 facial palsy. Six of the seven patients with SFP had inexcitable facial nerves on the affected side. The patient with an excitable facial nerve had a

CMAP of 0.28mV. The mean CMAP for the mild/moderate group was 0.79 ± 1.3 mV. Figure 5.1 demonstrates the emergence of these patterns of change when the contralateral facial palsy group is analysed based on severity. 125

FP vs normal controls

Increase Resting I/V slope ** TEh (10-20ms) *** TEh (20-40ms) *** TEh (90-100ms) **

Decrease TEd (10-20ms) ** TEd (40-60ms) ** TEd (90-100ms) ** TEd (peak) ** TEh (slope 101-140ms) **

SFP vs normal controls

Increase TEd (peak) * TE (40-60ms) * TEd (90-100ms) *

Decrease Resting I/V slope ** Superexcitability (%) * Refractoriness at 2.5ms *

Table 5.2. Changes in axonal properties in the contralateral facial nerve.

Values which demonstrated significant change on statistical analysis. All recordings are from the “unaffected” contralateral facial nerve. FP was defined as

HB 1-4 facial palsy. Severe was HB 5-6; * p<0.05; ** p<0.01; ***p<0.001. 126

100 100 A B C

0 0 (%) (%) (%) Current

(% threshold) threshold) (% -100 0 Threshold change Threshold Threshold reduction Threshold

-100 -200 -500 0 0 100 200 10 100 Threshold reduction (%) Delay (ms) Interstimulus interval (ms)

100 100 D E F

0 0 (%) (%) (%) Current

(% threshold) threshold) (% -100 0 Threshold change Threshold Threshold reduction Threshold

-100 -200 -500 0 0 100 200 10 100 Threshold reduction (%) Delay (ms) Interstimulus interval (ms)

Figure 5.1. Axonal excitability properties in the contralateral face.

Panels A-C demonstrate mean normal control (plain line) compared with

contralateral facial nerve recordings (hollow circles). Panels D-F demonstrate

subgroup plots of contralateral face recording from patients with mild-moderate

(gray diamond) and severe (black triangle) facial palsy. A, D current-threshold.

B,D threshold electrotonus. C, F recovery cycle. 127

Mild-moderate facial palsy (FP)

Contralateral facial nerve recordings from this subgroup of 13 patients with HB scores of 2-4 demonstrated significant changes compared to normal controls. A summary of changes is given in table 2. Stimulus response properties and peak

CMAP were not different between the disease group and normal controls. TE demonstrated a ‘fanned in’ pattern (figure 2) with a decrease in the magnitude of threshold change for both depolarising and hyperpolarising conditioning currents. Variables which demonstrated significant change included TEd (10-

20ms) (FP 54.9 ± 1.4%; control 59.7 ± 1.0%, p<0.01), TEd (40-60ms) (FP 39.6 ±

1.1%; control 43.2 ± 0.7%, p<0.01), TEd (90-100ms)(FP 36.7 ± 1.0%; control

40.2 ± 0.7%, p<0.01), and TEd (peak) (FP 53. ± 1.4%; control 58.3 ± 0.9%, p<0.01). TEh(10-20ms) (FP -64.3 ± 1.5%; control -72.0 ± 1.2%, p<0.001), TEh

(20-40ms) (FP -76.8 ± 2.1%; control 88.8 ± 2.0%, p<0.001), TEh (90-100ms)(FP

-89.9 ± 3.6%; control -113.2 ± -4.2%, p<0.01) and TEh (slope 101-140ms)(FP

1.453 ± 0.087; control 1.964 ± 0.091, p<0.01) also demonstrated significant changes. This pattern of change is typical of membrane depolarisation.

Significant changes were also noted in resting I/V slope (FP 0.803 ± 0.031%; control 0.68 ± 0.021, p<0.01). Superexcitability (%) values were reduced but did not achieve significance (FP -6.5 ± 1.3%; control 10.3 ± 1.3%, p=0.06). Also the reduction in τSD did not achieve significance (FP 0.419 ± 0.018; control 0.436

± 0.018, p=0.57).

128

100

0 (%) (%)

-100 Threshold reduction Threshold

-200 0 100 200 Delay (ms)

Figure 5.2. Threshold electrotonus in FP vs control. plot of normal control facial nerve (line) and contralateral recordings from patients with FP unilateral facial palsy (gray diamond). Curves are generated using hyperpolarising and depolarising subthreshold currents of ±40% of between 10ms and 100ms duration and examining the stimulus required to achieve a target threshold. Arrows represent direction of change in electrotonus values demonstrating a ‘fanning in’.

129

Severe facial palsy (SFP)

9 patients were classified as having severe facial palsy (HB 5-6). Significant changes were noted between the contralateral face recordings from this group and normal controls and the direction of change was noted to be opposite to those of the FP group. Resting I/V slope was decreased (SFP 0.573 ± 0.033%; control 0.689 ± 0.021, p<0.01). In RC recordings, the magnitude of superexcitability was increased (SFP -16.8 ± 1.7%; control -10.3 ± 1.3%, p<0.01), and refractoriness was decreased (SFP 15.9 ± 6.8%; control 34.1 ± 3.9%, p<0.05). In TE recordings, the SFP group demonstrated a ‘fanned out’ pattern with increases in magnitude of both hyperpolarising and depolarising threshold electrotonus (figure 5.3). Specific values which achieved significance were TEd

(peak) (SFP 62.5 ± 1.9%; control 58.3 ± 0.9%, p<0.05), TEd (40-60ms) (SFP 47.1

± 2.3%; control 43.2 ± 0.7%, p<0.05), TEd (90-100ms)(SFP 43.7 ± 1.8%; control

40.2 ± 0.7%, p<0.05), and TEh (90-100ms) was approaching significance (SFP -

127.3 ± 3.9%; control -113.2 ± 4.2%, p=0.07). τSD was increased in the SFP group compared to controls but this did not achieve significance (SFP 0.483 ±

0.072; control 0.436 ± 0.018, p=0.33). The combination of ‘fanning out’ of threshold electrotonus, increased magnitude of superexcitability and the decrease in resting I/V slope are consistent with hyperpolarisation of the resting membrane potential. 130

100

0 (%) (%)

-100 Threshold reduction Threshold

-200 0 100 200 Delay (ms)

Figure 5.3. Threshold electrotonus, control Vs SFP.

Curve demonstrates normal control facial nerve recordings (line) with

contralateral facial nerve recordings from patients with SFP (black triangle).

Arrows indicate the ‘fanning out’ of values in the disease group representing a

magnitude increase in response to both depolarising and hyperpolarising

conditioning currents.

131

60 A B

50 -100

* NS

TEd(90-100ms) TEd(90-100ms) 40 TEh(90-100ms) **

30 -200

Control severe mild/mod Control severe mild/mod

-50 100 C D

* * Superexcitability (%) (%) Superexcitability Refractoriness at 2.5ms (%) (%) 2.5ms at Refractoriness NS 0

Control severe mild/mod Control severe mild/mod

Figure 5.4. Discriminating values between FP and SFP

The following select variables demonstrate the distinct contradictory patterns of change between the contralateral facial nerve recordings from patients with SFP

(black) and FP (grey).

132

Discussion

This study demonstrates that there are distinct alterations in axonal function in the contralateral facial nerve in the setting of facial palsy. These changes are affected by the severity of facial palsy and are different for those with severe paralysis compared to those who have residual movement (mild or moderate facial palsy).

The changes in the SFP group are typical of those seen in membrane hyperpolarisation. There was a ‘fanning out’ of threshold electrotonus demonstrating increased magnitude responses to conditioning polarising currents, an increase in superexcitability, shortening in the refractory period and a decrease in resting I/V slope. All of these changes are consistent with changes due to axonal hyperpolarisation(Kiernan 2000). It is possible that this represents activity-dependent changes secondary to increased impulse conduction on the uninvolved side.

The changes in the mild-moderate facial palsy were different to those in the severe group. The decrease in magnitude to both hyperpolarising and depolarising electrotonus currents demonstrates a ‘fanning in’ response suggestive of membrane depolarisation. The exact reasons for this remain unclear but it is possible that the results in this cohort were confounded by other treatments that had been undertaken including physiotherapy, neuromuscular retraining and biofeedback instructions.

133

A point of difference which may be of relevance between the two subgroups is that all patients in the FP group had a facial nerve which was physically intact and all had recovered a degree of facial movement. In contrast, patients in the

SFP group had nerve transection or resection resulting in physical disruption of the axonal path and others had not recovered any facial movement. It is possible that these distinct patterns may alter synaptic and cell body responses at the level of the facial nerve nuclei and have a direct or indirect impact on contralateral facial nerve function. Future studies exploring functional imaging and central reorganisation may assist in exploring these changes.

It has been observed in some studies that in the setting of denervation the contralateral facial nerve will innervate muscles on the affected side adjacent to the midline(Casanova-Molla et al. 2011). This chemotactic stimulus driving the contralateral facial nerve to sprout toward the denervated tissue may be another explanation for altered channel and membrane properties in the contralateral nerve in patients with severe dysfunction. The mechanisms for regionally altered axonal mRNA translation and axonal structural change in response to stimuli have emerged recently and provide a scientific basis for axonal structural and functional changes in response to peripheral stimuli (Gomes et al. 2013; Jung,

Yoon, and Holt 2012).

In conclusion, this study has provided physiological evidence demonstrating that the observation of hyperkinesis in the unaffected side of facial palsy is associated with altered axonal function. These findings reinforce that a holistic approach to facial assessment and treatment is required in the setting of facial palsy and that 134 the contralateral face does not represent normal facial function. This discovery of abnormal contralateral nerve function in the setting of a unilateral nerve injury may be a stereotyped process with functional implications in other nerve injury settings. 135

Summary an Conclusions 136

This thesis lays important groundwork for the future study of facial nerve disorders. Axonal excitability measures provide valuable insight into the biophysical properties of peripheral nerves in health and disease and the development of this technique for application into the study of facial nerve disorders is an important step towards understanding the pathophysiology of common causes of facial neuropathy.

In Chapter 1, the techniques and technical setup for determining facial nerve excitability properties were developed and refined. This work established that the technique was feasible across a broad range of ages, face shapes and skin types. The normative data from this study lay the foundation for comparison with disease states in subsequent chapters.

Chapter 2: This study applied the techniques developed in chapter one to a disease cohort. The study demonstrated that excitability recordings are feasible i facial palsy due to different aetiologies and raised the possibility that different excitability profiles occur in the setting of different aetiologies, a hypothesis that will require further investigation. Further work is also required to delineate the utility of the technique in determining prognosis and response to treatment. The improved understanding of underlying axonal characteristics which this technique may offer in facial palsy has the potential to guide treatment strategies to improve residual function and explore the origins of debilitating symptoms such as facial muscle spasm and synkinesis.

137

Chapter 3: Bell’s palsy excitability measures were compared with normal control and non-Bell’s Palsy groups. The data presented in this study demonstrate evidence for acute sodium channel dysfunction in the setting of acute BP in a pattern similar to TTX ingestion. The studies suggest that reduced inward sodium conductances may underlie the development of acute weakness in BP.

Chapter 4: Patients with synkinesis were compared with non-synkinesis facial palsy to investigate if there were axonal changes associated with synkinesis. The findings of the study demonstrated that post-facial palsy synkinesis is associated with membrane depolarisation. This may provide justification for the use of pharmacotherapies which stabilise resting membrane potential as being a feasible adjunctive treatment modality in the setting of post facial palsy synkinesis.

Chapter 5: Axonal excitability studies were used to explore the basis for hyperkinesis seen on the contralateral side during facial palsy. The study demonstrated that axonal excitability properties change on the contralateral side i two distinct ways depending on the severity of facial palsy. These findings reinforce that in the setting of facial palsy the contralateral face does not represent normal facial function. This discovery of abnormal contralateral nerve function in the setting of a unilateral nerve injury may be a stereotyped process with functional implications in other nerve injury settings. 138

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