Scholarly article on topic 'Assessment of the upper motor neuron in amyotrophic lateral sclerosis'

Assessment of the upper motor neuron in amyotrophic lateral sclerosis Academic research paper on "Clinical medicine"

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{"Amyotrophic lateral sclerosis" / "Motor neuron disease" / "Upper motor neuron" / Imaging / "Transcranial magnetic stimulation"}

Abstract of research paper on Clinical medicine, author of scientific article — William Huynh, Neil G. Simon, Julian Grosskreutz, Martin R. Turner, Steve Vucic, et al.

Abstract Clinical signs of upper motor neuron (UMN) involvement are an important component in supporting the diagnosis of amyotrophic lateral sclerosis (ALS), but are often not easily appreciated in a limb that is concurrently affected by muscle wasting and lower motor neuron degeneration, particularly in the early symptomatic stages of ALS. Whilst recent criteria have been proposed to facilitate improved detection of lower motor neuron impairment through electrophysiological features that have improved diagnostic sensitivity, assessment of upper motor neuron involvement remains essentially clinical. As a result, there is often a significant diagnostic delay that in turn may impact institution of disease-modifying therapy and access to other optimal patient management. Biomarkers of pathological UMN involvement are also required to ensure patients with suspected ALS have timely access to appropriate therapeutic trials. The present review provides an analysis of current and recently developed assessment techniques, including novel imaging and electrophysiological approaches used to study corticomotoneuronal pathology in ALS.

Academic research paper on topic "Assessment of the upper motor neuron in amyotrophic lateral sclerosis"

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Clinical Neurophysiology

journal homepage: www.elsevier.com/locate/clinph

Review

Assessment of the upper motor neuron in amyotrophic lateral sclerosis crossM^

William Huynha,b'*, Neil G. Simonc, Julian Grosskreutz d, Martin R. Turnere, Steve Vucicf, Matthew C. Kiernan a

a Brain and Mind Centre, University of Sydney, NSW, Australia b Prince of Wales Clinical School, University of New South Wales, NSW, Australia c Department of Neurology, St Vincent's Hospital, Darlinghurst, Australia d Hans-Berger Department of Neurology, University Hospital Jena, Jena, Germany e Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK f Western Clinical School, University of Sydney, Sydney, Australia

ARTICLE INFO

HIGHLIGHTS

Article history: Accepted 27 April 2016 Available online 5 May 2016

Keywords:

Amyotrophic lateral sclerosis Motor neuron disease Upper motor neuron Imaging

Transcranial magnetic stimulation

• Clinical signs of UMN involvement are an important component in diagnosis of ALS.

• Novel neuroimaging and electrophysiology may facilitate demonstration of UMN degeneration in ALS.

• Improving early ALS diagnosis can facilitate the development of effective therapies.

ABSTRACT

Clinical signs of upper motor neuron (UMN) involvement are an important component in supporting the diagnosis of amyotrophic lateral sclerosis (ALS), but are often not easily appreciated in a limb that is concurrently affected by muscle wasting and lower motor neuron degeneration, particularly in the early symptomatic stages of ALS. Whilst recent criteria have been proposed to facilitate improved detection of lower motor neuron impairment through electrophysiological features that have improved diagnostic sensitivity, assessment of upper motor neuron involvement remains essentially clinical. As a result, there is often a significant diagnostic delay that in turn may impact institution of disease-modifying therapy and access to other optimal patient management. Biomarkers of pathological UMN involvement are also required to ensure patients with suspected ALS have timely access to appropriate therapeutic trials. The present review provides an analysis of current and recently developed assessment techniques, including novel imaging and electrophysiological approaches used to study corticomotoneuronal pathology in ALS. © 2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction................................................................................................................................................................................................................2644

2. Literature search strategy..........................................................................................................................................................................................2644

3. Clinical neurophysiology............................................................................................................................................................................................2645

3.1. Spinal reflex changes in MND/ALS..................................................................................................................................................................2645

3.1.1. Deep tendon reflexes and the H-reflex............................................................................................................................................2645

3.1.2. Plantar reflexes..................................................................................................................................................................................2645

3.1.3. Other spinal interneuronal networks..............................................................................................................................................2645

3.2. Transcranial magnetic stimulation..................................................................................................................................................................2645

3.2.1. Single-pulse TMS..............................................................................................................................................................................2647

3.2.2. Paired-pulse TMS..............................................................................................................................................................................2647

3.2.3. Triple stimulation technique............................................................................................................................................................2649

* Corresponding author at: Brain and Mind Centre, University of Sydney, and Institute of Neurological Sciences, Prince of Wales Hospital, NSW, Australia E-mail address: w.huynh@neura.edu.au (W. Huynh).

http://dx.doi.org/10.1016/j.clinph.2016.04.025

1388-2457/® 2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

3.3. Peristimulus time histograms..................................................................................... 2649

3.4. Beta-band intermuscular coherence................................................................................ 2650

4. Neuroimaging..............................................................................................................................................................................................................2650

4.1. MRI.......................................................................................................... 2651

4.1.1. Structural MRI....................................................................................................................................................................................2651

4.1.2. Magnetic resonance spectroscopy....................................................................................................................................................2653

4.1.3. Functional MRI..................................................................................................................................................................................2654

4.2. Single-photon emission computed tomography...................................................................... 2654

4.3. Positron emission tomography.................................................................................... 2654

5. Combining multiple electrophysiological and imaging modalities..........................................................................................................................2655

6. Conclusions and future directions..............................................................................................................................................................................2655

Acknowledgements....................................................................................................................................................................................................2657

References..................................................................................................................................................................................................................2657

1. Introduction

The term amyotrophic lateral sclerosis was first coined by Charcot, who postulated the primacy of the upper motor neuron in ALS pathogenesis (Charcot and Joffroy, 1869) with loss of Betz cells in the motor cortex being a well-recognised pathological feature (Kaufmann et al., 2004). The clinical diagnosis of classical amy-otrophic lateral sclerosis (ALS) is determined by identification of progressive dysfunction of both cortical ('upper') and spinal ('lower') motor neurons across multiple body regions (chiefly limb and bulbar), much of which was encapsulated by the El Escorial criteria (Brooks, 1994; Brooks et al., 2000b). The variable mix of upper motor neuron (UMN) and lower motor neuron (LMN) signs contribute to the clinical heterogeneity of ALS (Sabatelli et al., 2011).

The initial clinical presentations constituting 90% of all ALS, may be classified according to region: (1) limb-onset ALS; (2) bulbar-onset ALS; or sub-divided into much rarer extremes of LMN or UMN involvement: (1) progressive muscular atrophy, with pure LMN involvement, and typically limb-onset; or (2) primary lateral sclerosis, characterised by predominant UMN involvement, typically lower limb or bulbar in site of onset, both of which are rare (Kiernan et al., 2011). While ALS or Lou Gehrig's disease is the term used to describe all forms of the disease in the USA, motor neuron disease (MND) is the preferred term in Australia and the UK, with ALS reserved for the classical phenotype that presents with a combination of upper and lower motor neuron involvement. In this review, the term MND/ALS will be used to encompass all clinical phenotypes that include the classic ALS, progressive bulbar palsy, progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS).

In the presence of progressive LMN weakness, features of UMN involvement are an important component supporting the diagnosis of MND/ALS (de Carvalho, 2012), but often clinical signs of UMN dysfunction may not be easily appreciated in a limb that is concurrently affected by LMN degeneration particularly in the early stages of MND/ALS (Swash, 2012; Geevasinga et al., 2014). Clinical UMN signs are found to be initially absent in 7-10% of MND patients (Rocha and Maia Júnior, 2012). UMN dysfunction may be identified by the presence of some or all of hyperreflexia with pathological reflex spread, spasticity, and clonus, preserved reflexes in weak wasted limbs and Babinski sign (Brooks et al., 2000b); as well as in some cases, the paucity or impairment in motor control and clumsiness may often be early features of UMN deficit. However, the various components of the UMN syndrome reflect different physiological abnormalities in the descending motor system that is expressed by the intact LMN system, the latter being invariably affected in MND/ALS (Pierrot-Deseilligny and Burke, 2005; Swash, 2012). Furthermore, simultaneous alpha and gamma spinal motor neuron loss in conjunction with spinal interneuron degeneration has an effect on the expression of UMN signs (de Carvalho, 2012; Swash, 2012).

Objective UMN markers are critical to the diagnosis, as purely LMN syndromes may be caused not only by MND/ALS (Turner et al., 2013; Simon et al., 2014), but mimics including progressive muscular atrophy, various motor neuropathies, Kennedy's disease and adult-onset spinal muscular atrophy (SMA). Importantly, autopsy reports have identified UMN degeneration in 50-75% patients without apparent clinical signs affecting the corticospinal tract (Lawyer and Netsky, 1953; Ince et al., 2003; Kaufmann et al., 2004). Failure to recognise UMN features in patients presenting with suspected MND/ALS consequently results in diagnostic uncertainty and thereby delay which according to population studies is more than a year from symptom onset to diagnosis. This will inevitably delay the commencement of potentially disease modifying or neuroprotective therapy, most effective when started early in the disease course, in addition to adversely affecting enrolment into therapeutic trials (Turner et al., 2009; Hardiman et al., 2011; Vucic et al., 2013a).

The more recent Awaji criteria, developed to increase diagnostic sensitivity for MND/ALS, incorporated objective neurophysiologi-cal biomarkers of LMN dysfunction which included chronic neuro-genic changes and features of active denervation that also incorporated the presence of MND fasciculations (de Carvalho et al., 2008; Costa et al., 2012). Assessment of UMN involvement however, has remained clinically-based despite the improved diagnostic sensitivity using the Awaji criteria. For this reason, there remains a critical need to develop better in vivo UMN markers to improve diagnostic certainty that would in turn facilitate enrolment of patients with suspected MND/ALS into appropriate treatment and clinical trials. As such, the current review will provide an overview of recently developed techniques, including functional and structural imaging as well as novel electrophysiological approaches to study the integrity of the corticomotoneuronal (that part of the corticospinal tract with monosynaptic connections to spinal cord motor neurons) system in MND/ALS. There is no formal definition for the ''early stages" of MND with some investigators suggesting that this may encompass those with minimal disability as defined by the ALS-FRS score, within a year of symptom onset, or based on the revised El-Escorial subgroups. In this current review, ''early stages" of MND will be referred to those patients in either the clinically ''suspected" or ''possible" El-Escorial groups and especially those without clinically evident UMN signs. A detailed description of the actual methodology of the techniques discussed are beyond the scope of this review and readers are encouraged to refer to referenced papers for such discussion.

2. Literature search strategy

A systematic literature review was performed using PubMed (National Library of Medicine) during the period between 1970 and 2016. The search strategy used the following key words or

statements in various combinations: amyotrophic lateral sclerosis or motor neuron disease, primary lateral sclerosis, upper motor neuron, corticospinal tract, neuroimaging, imaging, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), single photon emission computed spectroscopy (SPECT), positron emission tomography (PET), diffusion tensor imaging (DTI), elec-trophysiology, neurophysiology, F waves, H reflexes, spinal reflexes, transcranial magnetic stimulation (TMS), cortical excitability, sensitivity and specificity. Further relevant references within publications were hand-searched.

3. Clinical neurophysiology

3.1. Spinal reflex changes in MND/ALS

3.1.1. Deep tendon reflexes and the H-reflex

Hyperactive deep tendon reflexes may be masked when there are severe concomitant LMN changes (Swash, 2012), although the presence of any deep tendon reflex in a muscle with significant wasting may be consider as a sign of UMN dysfunction (Simon et al., 2015). Besides LMN loss, spinal interneurons degenerate alongside anterior horn cells in MND/ALS (Stephens et al., 2006), which may contribute to variable segmental excitability of remaining alpha-motoneurons, and in turn prevent the expected hyperactive deep tendon reflex secondary to corticomotoneuronal injury (Swash, 2012).

The H-reflex represents the neurophysiological correlate of the deep tendon reflex and is a monosynaptic reflex evoked by stimulation of Ia sensory afferents that project on homonymous motoneurons. At the most simple level, presence of an H-reflex in a clinically wasted and weak muscle, is clear evidence of upper motor neuronal dysfunction, a finding that will facilitate an early diagnosis of MND/ALS. The H-reflex has been used to assess segmental motoneuronal excitability in a variety of neurological diseases usually through measurement of Hmax/Mmax ratio, or the ratio of the amplitude of the maximal H-reflex relative to the maximal compound muscle action potential (CMAP, M-wave) amplitude (Pierrot-Deseilligny and Burke, 2012). Typically Hmax/Mmax is increased in disorders characterised by spasticity and hyper-reflexia (Misiaszek, 2003). Of interest, MNd/alS patients do not demonstrate significantly higher Hmax/Mmax relative to normal controls, despite the presence of clinical UMN signs (Raynor and Shefner, 1994; Mazzini et al., 1997), although increased Hmax/Mmax did significantly correlate with hyperreflexia and the Babinski sign (Mazzini et al., 1997).

This unexpected finding was further explored in recent studies of the H-reflex in MND/ALS patients (Simon et al., 2015). Hmax/Mmax was similar between MND/ALS patients and healthy subjects. Like previous studies, Hmax/Mmax did correlate with a composite clinical score of clinical UMN signs. However, Hmax/Mmax was affected by the position of the H reflex recruitment curve relative to the M wave recruitment curve. It was inferred that preferential loss of fast-conducting motoneurons in MND/ALS alters the dynamics of H-reflex recruitment and attenuates the measurable Hmax, hence reducing Hmax/Mmax (Simon et al., 2015).

An alternative measure, Hh/Mh, is independent of the effects of collision of antidromic and orthodromic impulses generated by direct nerve stimulation and the descending H reflex respectively. Hff and Mh are calculated from the slope angle of the earliest rising phase of the H- and M-wave recruitment curves. It is generally accepted that recruitment of the H-reflex follows the size principle, such that the initial phase of the H-reflex recruitment curve is formed from activation of smaller calibre motor neurons (Buchthal and Schmalbruch, 1970). Conversely, the largest calibre motor neurons have the lowest threshold to electrical stimulation (Feiereisen et al., 1997), and as such the earliest portion of the

M-wave recruitment curve is comprised of potentials produced by the largest calibre motor axons. As such, Hh and Mh are independent of the artifactual influence of collision where Hmax and Mmax are not.

The ratio of the recruitment slope of the H reflex and M wave (equivalent to He/Me) is increased in spastic limbs (Funase et al., 1994, 1996; Higashi et al., 2001). Similarly, in MND/ALS patients, Hg/Mg was increased relative to healthy subjects and was closely correlated with clinical UMN signs (Simon et al., 2015). As such, Hg/Mg may be a more suitable measure of segmental motoneuronal hyperexcitability in MND/ALS than the more traditional Hmax/Mmax

(Fig. 1).

3.1.2. Plantar reflexes

It has been estimated that only 30% of MND/ALS patients demonstrate an extensor plantar response (Baek and Desai, 2007). However, electrophysiological assessment of the plantar reflex in MND/ALS patients shows a typical 'extensor reflex' pattern with activation of extensor hallucis longus, extensor digito-rum longus and tibialis anterior superimposed on tonic activation of flexor hallucis brevis irrespective of whether the clinical response was extensor or flexor (Landau and Clare, 1959). In addition, studies of patients with unexpectedly absent extensor plantar responses despite the presence of other UMN signs indicate that an extensor plantar response may only be evident if the intraspinal pathways of the flexion reflex is intact (Van Gijn, 1978). Thus the absence of extensor plantar responses in MND/ALS may reflect injury to the spinal segmental connections contributing to the extensor plantar response (Swash, 2012).

3.1.3. Other spinal interneuronal networks

Neurophysiological studies of spinal interneuronal function

other than the H reflex are less extensive in MND/ALS. However, analysis of spinal interneuronal dysfunction may provide further insights into mechanism of LMN loss.

Excitability of the F-wave is influenced by UMN dysfunction (Drory et al., 1993). In MND/ALS, F-wave excitability has been measured using persistence and the ratio of the F-wave and corresponding compound muscle action potential amplitude (F/M ratio) (Drory et al., 2001). While F-wave persistence diminishes with advancing disease (de Carvalho et al., 2003), the F/M ratio increases in MND/ALS relative to controls, although these changes are not influenced by the degree of clinical UMN involvement (Drory et al., 2001). However, increase in the F/M ratio is not restricted to UMN lesions, as it is also seen in polyneuropathy (Fisher, 1988), limiting its value as a measure of UMN dysfunction.

There is reduced presynaptic inhibition in MND/ALS (Bour et al., 1991; Lee et al., 2014), consistent with findings in patients with brain or spinal cord lesions producing spasticity (Hultborn et al., 1987). However, reciprocal inhibition, a marker of Renshaw cell interneuronal function in the spinal cord, was significantly reduced in MND/ALS patients relative to normal controls, opposite to what was identified in patients with spinal cord injury (Raynor and Shefner, 1994). These findings suggest that interneuronal influences in MND/ALS are more complex than those seen in more focal lesions (Turner et al., 2012).

3.2. Transcranial magnetic stimulation

Traditional approaches such as nerve conduction studies (NCS) and EMG provide a quantitative measure of LMN loss such as CMAP amplitude and motor unit recruitment although quantifying UMN loss remains more difficult. An important focus in the development of a neurophysiological biomarker of MND/ALS has been the identification of cortical hyperexcitability and the quantification of UMN dysfunction (Turner et al., 2009). Transcranial

Fig. 1. Changes in the H-reflex pathway in ALS patients. Comparison of the slope angle of the H-reflex recruitment curve (Hh), the slope angle of the M-wave recruitment curve (M„), their ratio He/Me (slope ratio), and Hmax/Mmax between ALS (black filled bar), healthy control (open bar) and UMN disease control groups (grey filled bar). He/Me was significantly increased in the ALS and disease control groups (A), and Hmax/Mmax was increased in disease controls but similar between healthy control and ALS groups. Mh was reduced in the ALS group but Hh did not differ significantly between groups (B). The UMN Score was associated with changes in both He/Me (C) and Hmax/Mmax (D) in ALS patients with this relationship strongest for He/Me. Illustrative M-recruitment curves in ALS patients are depicted (E) to illustrate the relationship between Mh, Mthresh and clinical UMN signs. Patient 22 (lower limb onset, filled circles) displayed prominent UMN signs in the displayed lower limb (UMNS 5 out of 6), and both Mh and Mthresh were relatively low. Patient 15 (upper limb onset, open triangles) did not display clinical UMN signs on examination (UMNS 0 out of 6) and Mh and Mthresh were relatively high. A healthy control subject is superimposed (open circles; Mthresh = 6.3 mA, Mh = 47.6°). Mh was strongly correlated with M threshold (F) in ALS patients, which in turn was correlated with clinical UMN signs. Reproduced with permission (Simon et al., 2015).

magnetic stimulation (TMS) techniques have gained credibility as a clinical tool to investigate the integrity of the corticomotoneuronal system in MND/ALS. Single, paired, and triple-pulse TMS techniques have all been utilised, with such parameters as motor threshold (MT), central motor conduction time (CMCT), cortical silent period (CSP), intracortical inhibition and facilitation, taken to reflect corticomotoneuronal function (Vucic and Kiernan, 2013). Features of cortical hyperexcitability were characterised

by functional abnormalities, including reduced short-interval intracortical inhibition and CSP duration, as well as an increase in intracortical facilitation, along with inexcitability in the motor cortex (Grieve et al., 2015).

Moreover there were significant bilateral TMS abnormalities evident in the MND/ALS cohort at an early stage of the disease process, in keeping with previous studies reporting that functional abnormalities of the motor cortex are an early and specific feature

of MND/ALS, preceding the development of LMN dysfunction (Vucic and Kiernan, 2006; Vucic et al., 2008, 2013c; Geevasinga et al., 2014; Grieve et al., 2015; Menon et al., 2015b).

3.2.1. Single-pulse TMS

The time interval between stimulation of the motor cortex and onset of the resultant motor evoked potential (MEP) response from the arrival of corticospinal volley at the spinal motor neuron, is termed the central motor conduction time (CMCT) (Vucic and Kiernan, 2013). Multiple factors contribute to the CMCT including time to activate the corticospinal cells, conduction time of the descending volley down the corticospinal tract, synaptic transmission and activation of spinal motor neurons (Vucic et al., 2013c). CMCT is reported to be prolonged in a variable proportion of MND/ALS patients (16-100%) (Mills and Nithi, 1998; Miscio et al., 1999; Schulte-Mattler et al., 1999; Triggs et al., 1999; Pouget et al., 2000; Kaufmann et al., 2004; Vucic and Kiernan, 2013), with a specificity of 38% (Kaufmann et al., 2004). Additionally, CMCT abnormalities in patients without clinically overt UMN involvement had a sensitivity of 50-71% (Miscio et al., 1999; Schulte-Mattler et al., 1999). Although the mechanisms underlying prolongation of the CMCT in MND/ALS remain to be fully elucidated, an increased desynchronization of corticomotoneuronal volleys, secondary to axonal loss, as well as axonal degeneration of the fastest conducting corticomotoneuronal fibres have been proposed as likely mechanisms (Eisen et al., 1996; Vucic and Kiernan, 2013; Vucic and Kiernan, 2013b). It should also be noted that CMCT calculations are associated with technique-dependent variations that likely account for the large discrepancies in sensitivity reported by previous studies.

Corticomotor threshold or the resting motor threshold (RMT), reflects the ease with which corticomotoneurons are excited and the density of UMN projections onto motor neurons (Rossini et al., 1999) with the highest projections for intrinsic hand muscles being reflected in the lowest motor thresholds for these muscles (Vucic and Kiernan, 2013). RMTs are influenced by the glutamater-gic neurotransmitter system, through a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors, whereby excessive glutamate activity reduces MTs as well as being modulated by sodium channel blockers (Vucic et al., 2013b). RMT in MND/ALS varied between studies but in general appeared to be lower early on in the disease course and higher or even inexcitable as disease progressed (Eisen et al., 1993; Mills and Nithi, 1997; Triggs et al., 1999; Grieve et al., 2015). Reduced RMT may be modulated by increased glutamate excitation, reduced gamma-Aminobutyric acid (GABA) inhibition or a combination of both (Vucic et al., 2013b). Given that motor threshold may be modulated by glutamate activity, the finding of reduced motor threshold early in the disease course of MND/ALS may support a dying-forward process, whereby cortical hyperexcitability underlies the development of progressive neurodegeneration via glutamate tox-icity (Vucic and Kiernan, 2013). The motor cortex is inexcitable in about 20% of patients and appeared to be a finding late in the disease course (Grieve et al., 2015), as well as being a relatively frequent early finding in patients with PLS (Geevasinga et al., 2015b).

The interruption of voluntary electromyography (EMG) activity in the target muscle following stimulation of the motor cortex is referred to as the cortical silent period (CSP) (Cantello et al., 1992). Mechanisms underlying the generation of the CSP are complex but primarily mediated by activation of cortical inhibitory neurons, through long-lasting inhibitory postsynaptic potentials, acting via GABA-B receptors (Ziemann et al., 1993; Vucic and Kiernan, 2013). However, a contribution from spinal mechanisms has also been suggested for early cortical silent period segments (Cantello et al., 1992; Vucic and Kiernan, 2013). The CSP duration has been reported to show a reduction or absence in patients with

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0 5 10 15 20 25 JO

Intersliniulus interval (ills)

Fig. 2. Paired-pulse subthreshold conditioning TMS (a) An illustrative example disclosing short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) in a healthy control. SICI was absent whilst ICF was increased in an ALS patient. (b) Group data revealed a significant reduction of SICI and increase of ICF in ALS. Reproduced with permission (Geevasinga et al., 2014).

MND/ALS across all phenotypes (Uozumi et al., 1991; Desiato and Caramia, 1997; Pouget et al., 2000; Vucic and Kiernan, 2006, 2013; Grieve et al., 2015) and is reported in both sporadic and familial MND/ALS, with the degree of duration reduction being most prominent early in the disease course (Prout and Eisen, 1994; Vucic and Kiernan, 2006, 2013; Vucic et al., 2008). Of relevance, this reduction in CSP duration appeared specific for MND/ALS, being normal in mimic disorders such as Kennedy's disease, acquired neuromyotonia, and distal hereditary motor neuronopa-thy with pyramidal features (Vucic and Kiernan, 2008; Vucic et al., 2010a,b, 2011; Menon et al., 2015a). The CSP duration reduction in MND/ALS is likely to represent a combination of decreased corticomotoneuronal drive and reduced GABAergic inhibition, due to either degeneration of inhibitory interneurons or dysfunction of GABAB-mediated receptor inhibition (Vucic and Kiernan, 2013). Absent or prolonged ipsilateral CSP duration has also been identified in the early stage of MND/ALS, potentially representing dysfunction of inhibitory transcallosal neurons, a notion supported by recent MRI studies (Turner et al., 2009; Vucic et al., 2013).

3.2.2. Paired-pulse TMS

Cortical excitability may be assessed effectively using a paired-pulse technique, in which a conditioning stimulus is utilised to modulate the effect of a second test stimulus. A potential limitation of the original ''constant stimulus" paired-pulse TMS method was the significant variability in the MEP amplitude with consecutive stimuli (Kiers et al., 1993), resulting in fluctuations in the resting threshold of cortical neurons. For this reason, to ensure validity

Fig. 3. (a) Averaged short-interval intracortical inhibition (SICI), between interstimulus interval (ISI) 1-7 ms, was significantly reduced in amyotrophic lateral sclerosis (ALS). (b) The reduction of averaged SICI was comparable in Awaji subgroups. Peak SICI at ISI (c) 1 ms and (d) 3 ms was significantly reduced in Awaji subgroups. ""P < 0.0001. Reproduced with permission (Geevasinga et al., 2014).

of measurement, multiple stimuli were required to be delivered at each conditioning-test stimulus interval. In order to overcome this limitation, a novel threshold tracking technique was recently developed, in which a constant target MEP response is tracked by a test stimulus, utilising a range of different conditioning-test paradigms (Fisher et al., 2002; Vucic et al., 2006). Using this technique, applying a subthreshold conditioning stimulus (set to 70%RMT) at predefined time intervals prior to a suprathreshold test stimulus has been used to investigate short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) (Kujirai et al., 1993; Vucic et al., 2006).

SICI, a biomarker of inhibitory GABAergic cortical interneuronal function, was significantly reduced or absent in MND/ALS patients compared to controls and there was no significant difference in the degree of reduction between the right and left sides, between patients with less severe versus more prominent LMN dysfunction, or between differing sites of onset (Turner et al., 2009; Vucic and Kiernan, 2013; Geevasinga et al., 2014; Grieve et al., 2015) (Fig. 2). The reduction in SICI has been widely documented in both sporadic and familial forms of MND/ALS and appears to be an early feature in sporadic MND/ALS (Hanajima et al., 1996; Yokota et al., 1996; Ziemann et al., 1997; Zanette et al., 2002b; Vucic and Kiernan, 2006, 2013; Vucic et al., 2008, 2010b,c; Geevasinga et al., 2015a). With either abnormal SICI or inexcitable motor cortex, the threshold-tracking TMS technique was shown to have a sensitivity of 73.21% and a specificity of 80.88% (Menon et al., 2015a) and there were no differences in the sensitivity of the technique between patients with or without UMN signs (Geevasinga et al., 2014; Menon et al., 2015a). Furthermore, an absent SICI demonstrated a sensitivity of 97% (Vucic et al., 2013b). When the established cut-off value of SICI (<5.5%) (Vucic et al., 2011) was combined with parameters such as prolonged CMCT or inexcitable motor cortex, TMS abnormalities were evident in 77% of MND/ALS patients with frequency of abnormalities similar across Awaji diagnostic groups (Geevasinga et al., 2014). And the presence of such TMS abnormalities resulted in 88% of Awaji possible patients being reclassified as Awaji probably/definite

Fig. 4. (a) Short-interval intracortical inhibition (SICI) was significantly reduced in amyotrophic lateral sclerosis (ALS) when compared to non-ALS syndrome (NALS) patients and controls. (b) Averaged SICI, between interstimulus intervals (ISI) 1-7 ms, was significantly reduced in ALS patients compared to NALS patients and controls. However, SICI was comparable between NALS patients and controls. ""P< 0.001. Reproduced with permission (Vucic et al., 2011).

(Geevasinga et al., 2014). Specifically, 56% of Awaji possible patients had abnormally reduced SICI (Geevasinga et al., 2014) (Fig. 3). Using the threshold-tracking technique, SICI abnormalities seem to be the most robust diagnostic variable suggestive of UMN

dysfunction in MND/ALS patients (Geevasinga et al., 2014; Menon et al., 2015a,b).

Abnormalities derived from the use of this technique may potentially be limited by the end-point result obtained from the target muscle studied which in most cases, have been an intrinsic hand muscle. However, previous studies have also identified similar abnormalities in SICI from abductor pollicis brevis (APB) muscles in patients with the flail-leg variant of MND/ALS where the disease is confined to the lower limbs only (Menon et al., 2016). In addition, combining TMS with novel imaging studies have also revealed multiple areas of cortical abnormalities beyond those of the motor cortex (Huynh et al., 2013a,b,2016; Grieve et al., 2015). Reduction of SICI as occurs in MND/ALS is mediated by a loss of GABA-secreting inhibitory cortical interneurons (Vucic and Kiernan, 2013), a finding supported by neuropathological studies disclosing loss of parvalbumin positive inhibitory interneurons in MND/ALS (Nihei et al., 1993). These results suggest that the abnormalities in cortical excitability obtained from a target hand muscle reflect a more widespread abnormality not only in the motor cortex itself but also in other regions of the brain. In addition, a contributory role for a glutamate-mediated excitotoxic mechanism was also suggested by partial restoration of SICI in patients with MND/ALS who were treated with the glutamate antagonist, riluzole (Stefan et al., 2001; Vucic et al., 2009, 2013a).

Intracortical facilitation, a potential biomarker of cortical glu-taminergic function, has been reported to be significantly increased in MND/ALS patients (Vucic and Kiernan, 2013; Grieve et al., 2015) (Fig. 1) although not in other studies (Zanette et al., 2002a,b). The use of cortical excitability testing using this technique may be able to unveil upper motor neuron involvement in MND/ALS patients particularly those phenotypes without clinically evident upper motor neuron signs on examination such as the flail arm variant of MND/ALS (Vucic and Kiernan, 2007). When compared to the Awaji criteria alone, an extra 34% of ALS patients could be diagnosed with ALS at initial assessment when using the threshold tracking TMS parameters combined with clinical and conventional electrophysiological assessments (Menon et al., 2015a). Of further relevance, the technique enabled reliable distinction between MND/ALS and other neurological conditions mimicking the disorder such as in Kennedy's disease, spinal muscular atrophy, peripheral nerve hyperexcitability disorders, multifocal motor neuropathy, Hirayama disease, lead neuropathy, CIDP, hereditary motor neuropathy with pyramidal features, and hereditary spastic paraparesis (Vucic and Kiernan, 2008, 2010a,b; 2011; Farrar et al., 2012) (Fig. 4).

3.2.3. Triple stimulation technique

The triple stimulation technique (TST) was developed as a means of increasing the sensitivity of TMS techniques in detecting corticomotoneuronal dysfunction. The TST is a collision technique, whereby the degree of MEP desynchronization (which normally occurs with single cortical stimulus) may be suppressed (Magistris et al., 1998, 1999; Vucic et al., 2013b). Using TST, measurement of the evoked response over a hand muscle is performed following stimulation of motor cortex, wrist and Erb's point in succession. This complex technique is performed by first delivering a high-intensity magnetic stimulus to motor cortex followed by supramaximal electrical stimulation of the peripheral nerve supplying the target muscle at the wrist such that the descending cor-ticomotoneuronal volley is 'collided' out by the antidromic action potentials. Collision takes place along the proximal segment of the peripheral nerve at the upper arm. A third stimulus is subsequently delivered to Erb's point (axilla) after an appropriate delay, eliciting a highly synchronised motor response in those fibres in which the collision had occurred. The amplitude and area of this test CMAP response are compared with the response induced by

the conditioned TST paradigm (Erb's point-wrist-Erb's point stimulation) yielding an amplitude ratio of >93% and area ratio of >92% in healthy controls (Magistris et al., 1998, 1999; Vucic et al., 2013b). The derived TST amplitude ratio allows the estimate of a proportion of surviving corticospinal motor neurons. In MND/ ALS, the TST has been suggested to be sensitive at detecting subclinical UMN dysfunction with an apparent 100% sensitivity in those with suspected or possible MND categories (Komissarow et al., 2004), thereby enabling an earlier diagnosis of MND/ALS (Kleine et al., 2010). However, an overall sensitivity was found to be of around only 54% (Furtula et al., 2013), as there were about 48% of patients with clinically apparent UMN and normal ratios. Furthermore, the test was limited in patients with significant LMN loss (Rosler et al., 2000), suggesting that the test may be useful primarily in the early stages of the disease when there are sub-clinical UMN signs. The TST amplitude ratio was smaller in MND/ ALS patients with pure UMN or mixed UMN and LMN involvement compared to those without clinical UMN signs (Rosler et al., 2000). It was also able to distinguish between certain mimic disorders such as inclusion body myositis and other peripheral nerve disorders (Kleine et al., 2010). The triple stimulation technique offers promise to increase the sensitivity to UMN lesions but it is not yet being used routinely as it is difficult to apply.

Over the years, paired-pulse TMS and in particular assessment of SICI has emerged to be the more favoured and accurate diagnostic technique over that of the traditional single-pulsed techniques. Triple-stimulation techniques offer a promising novel approach to enhance the diagnostic sensitivity for detecting subclinical UMN involvement but the patient numbers studied thus far are very small and further larger-scale studies are required to establish its potential clinical utility in MND/ALS. Until then, the use of abnormalities in SICI and an inexcitable motor cortex would offer the most valuable approach in disclosing UMN dysfunction in MND/ ALS patients (Burrell et al., 2011; Vucic and Kiernan, 2013; Geevasinga et al., 2014; Menon et al., 2014, 2015a).

3.3. Peristimulus time histograms

Peristimulus time histograms (PSTH) can be used to assess the integrity of the corticomotoneuronal system (Weber and Eisen, 2000) whereby the modulation in firing of a single motor unit in response to a stimulus can be assessed (Eisen and Weber, 2001; Vucic and Kiernan, 2013). The primary peak (PP) measure changes in the firing probability of a voluntarily activated motor unit subjected to a series of transcranial magnetic stimuli (Eisen and Weber, 2000; Weber and Eisen, 2000). In healthy controls, a well synchronised primary peak was evident at a latency of approximately 20-30 ms (Vucic et al., 2013b). PSTHs have shown a variety of abnormalities in MND/ALS that suggest there is a supraspinal defect, which is pre-synaptic to the spinal motor neuron. The major abnormality observed in MND/ALS patients was a desynchronised, complex, primary peak that had an increased duration and delayed. This result was best explained by an increase in the repetitive firing of the corticomotoneuron associated with greater temporal dispersion of the descending cortical volley (Awiszus and Feistner, 1993, 1995; Mills, 1995; Eisen et al., 1996; Nakajima et al., 1997; Eisen and Weber, 2000, 2001; Weber and Eisen, 2000; Weber et al., 2000). In addition, the amplitude of the primary peak was increased and additional subcomponents were evident, all of which are suggestive of corticomotoneuronal hyperexcitabil-ity (Eisen et al., 1999). An argument in favour of a supraspinal origin for abnormal PSTH results in MND/ALS was suggested by the lack of abnormalities demonstrated in Kennedy's disease, which affect only the LMNs (Eisen and Weber, 2000) (Fig. 5).

With increased disease duration, progressive desynchronization occurs in PPs, characterised by an increased number of excess bins

Fig. 5. Peristimulus time histograms in normal controls (a) and patients with Kennedy's disease (b) compared to that in ALS (c). Reproduced with permissions (Eisen and Weber, 2000).

and prolonged duration (Weber and Eisen, 2000). Changes over time have been explained by the activation of slow-conducting pathways and progressive loss of fast monosynaptic pathways from pyramidal Betz cells (Weber and Eisen, 2000). The desynchro-nization is thought to reflect repetitive firing of the cortical colony, which in part may be due to glutamate excitotoxicity (Eisen and Weber, 2000).

The sensitivity of PSTHs in MND/ALS has not been established but there is good correlation between PP abnormalities and UMN signs without correlation with LMN signs (Weber and Eisen, 2000). However, most PP abnormalities are not specific to MND/ ALS and can be observed in other central nervous system disorders such as stroke and MS although the double PP appears typical to MND/ALS reflecting activation of the slow-conducting pathways (Weber and Eisen, 2000).

3.4. Beta-band intermuscular coherence

Rhythmic activity can be recorded from the motor cortex in the alpha (15-30 Hz) and beta (8-12 Hz) frequency bands. Intermuscular coherence during a sustained muscle contraction is dependent on supraspinal structures including the corticospinal tract. A

recent study using this technique demonstrated that the betaband coherence was observed in all healthy controls as well as patients with PMA where there is an intact UMN integrity and not substantially altered by loss of LMNs. Furthermore, the results were abnormal in all patients with PLS and hence suggested that assessment using this technique could provide information regarding upper motor neuron involvement and facilitate a more definitive diagnosis of MND/ALS in patients with otherwise subclinical UMN signs (Fisher et al., 2012).

4. Neuroimaging

Previously, the World Federation of Neurology stated that there was no role for neuroimaging in confirming the diagnosis of MND/ ALS (Brooks, 1994), and conventional imaging methods have been used primarily to exclude other potential diagnoses that may be mimicking the disorder, particular in those with clinically probable or possible disease. In more recent times however, advanced neu-roimaging techniques have facilitated investigation of the central nervous system for atrophy and alterations in microstructure, biochemistry, neural networks, metabolism and neuronal receptors that may occur in patients with MND/ALS as means to identify

Fig. 6. Cerebral MRI of a patient with ALS demonstrating (a, b) signal hyperintensities along the corticospinal tract (arrows). Reproduced with permission (Rocha and Maia Júnior, 2012).

Fig. 7. Voxel-based intensitometry reflects disease severity in the corticospinal tract, transcallosal fibres and brainstem. Whole brain group-mean of T1 images projected on generic brain (a, b) and Maximum Intensity Projection of significantly different regions (d, e). The extent of MRI intensity change in 'low' disability group (a, d) is significantly less widespread than in the 'high' disability group (b, e). Inference was done using Threshold Free Cluster Enhancement (10,000 permutations) und Family-wise error rate correction for multiple comparisons. Colour spectrum giving p-value indication (c). Reproduced with permission (Hartung et al., 2014). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

an objective marker of UMN dysfunction especially at the earlier stages of disease (Foerster et al., 2013b).

4.1. MRI

4.1.1. Structural MRI

Conventional MRI have identified hypointensities over the motor cortices in patients with MND/ALS whilst hyperintense signalling was observed on T2-weighted images involving the corticospinal tracts (CST) (Peretti-Viton et al., 1999; Agosta et al.,

2010a; Huynh et al., 2010; Rocha and Maia Júnior, 2012), but neither were specific to the disease itself being also present in other neurodegenerative disorders as well as older age controls. More specifically, abnormal signal intensity in the posterior third of the posterior limb of the internal capsule correlated with degeneration of the corticospinal tract on histopathology (Yagishita et al., 1994). Hyperintensities involving the CST was associated with low sensitivity (640%) and specificity (670%) (Fig. 6), and the sensitivity may be lower in patients with less severe motor symptoms or in the early stages of the disease (Chan et al., 2003). This is

despite degeneration of the entire CST reportedly occurring in approximately 47% of autopsy specimens from patients with MND (Brownell et al., 1970). In addition, the CST signal changes did not appear to correlate with clinical scores (Agosta et al., 2010a). The combination of these two findings however may be more specific for MND/ALS (Goodin et al., 1988; Luis et al., 1990; Oba et al., 1993; Shiozawa et al., 2000).

More advanced techniques based on high resolution T1 images such as volume-based morphometry (VBM) measures relative grey and white matter volumes in specific brain regions whilst surfaced-based morphometry (SBM) measures cortical thickness. White matter changes are more reliably detected using the recently developed VBM derivative voxel-based intensitometry (Hartung et al., 2014) (Fig. 7). Most VBM analyses have demonstrated widespread grey matter atrophy involving the motor cortex extending into the frontal and parietal regions in patients with MND/ALS whilst SBM show consistent reductions in cortical thickness in MND/ALS motor cortices with progressive thinning over time (Grosskreutz et al., 2006; Foerster et al., 2013b; Zhu et al., 2015). Some studies have proposed such cortical thinning as a potential early biomarker of UMN dysfunction (Verstraete et al., 2012; Mezzapesa et al., 2013; Thorns et al., 2013; Walhout et al., 2015). In other studies, bitemporal cortical thinning were evident in MND/ALS patients and independent of functional abnormalities with 56% of motor cortices analysed revealing significant thinning below the previously established diagnostic cut-off values (<2.48 mm) (Grieve et al., 2015; Walhout et al., 2015). Correlation with disease severity is low in grey matter analyses, possibly due to the high physiological inter-individual anatomical variability of gyri (Verstraete and Foerster, 2015). White matter signal increase in VBI, however, correlates linearly with ALSFRS-R reduction in large portions of the CST from subcortical regions through the internal capsule into the brainstem (Hartung et al., 2014).

VBM and SBM studies have also demonstrated reduction in frontotemporal cortical regions that correlated with cognitive impairment in MND/ALS patients, in that those with cognitive impairment and patients with the ALS-FTD overlap syndrome showing the greatest change (Abrahams et al., 2005; Chang et al., 2005; Grosskreutz et al., 2006; Murphy et al., 2007; Agosta et al., 2012).

Despite evidence of regional atrophy identified on VBM in the abovementioned studies, other investigators have not observed such differences (Kiernan and Hudson, 1994; Ellis et al., 2001; Abrahams et al., 2005; Mezzapesa et al., 2007), and combined with its limited sensitivity, render the utility of this MRI technique for detecting UMN dysfunction in MND patients limited at present. Part of the reason for this may be the small and heterogeneous patient groups together with methodological differences or limitations. Moreover, longitudinal studies in MND patients are

challenging as progressive bulbar and respiratory weakness pose a challenge to serial scanning.

The use of diffusion tensor imaging (DTI) measure water movement within intact neuronal pathways and is highly anisotropic (i.e., non-random) with parameters such as reduced fractional ani-sotropy (FA) and, less commonly, increased mean diffusivity being potential surrogate markers for loss of neuronal integrity within white matter tracts (Turner et al., 2009; Simon and Kliot, 2014). MRI studies using DTI in MND/ALS patients have demonstrated reduced FA in the CST, with the lowest values observed in the PLS phenotype and more modest reductions in ALS and least with PMA subgroups (Abe et al., 2004; Sach et al., 2004; Thivard et al., 2007; Iwata et al., 2008, 2011; Sage et al., 2009; Agosta et al., 2010b; Cosottini et al., 2010; Filippini et al., 2010; Chapman et al., 2014). More specifically, FA values in the CST appear to be correlated with UMN scores (Wong et al., 2007; Sage et al., 2009) and FA was also reduced in several frontal white matter regions that correlated with cognitive scores and executive function (Abe et al., 2004; Sach et al., 2004; Sage et al., 2009; Keil et al., 2012). Others however, have observed FA values in the posterior limb of the internal capsule of PMA patients to be similar to those of ALS patients with obvious UMN signs on initial assessment (Graham et al., 2004; Sach et al., 2004), and the eventual development of clinical UMN features (Sach et al., 2004). This suggests a potential clinical utility for its use to detect subclinical UMN features in suspected patients, although conflicting data exists from other studies (Cosottini et al., 2005). Bulbar-onset patients also appear to have the more marked reductions in FA (Turner and Kiernan, 2012). Pooled sensitivity and specificity for DTI in the assessment of changes observed in MND patients was found to be 68% and 73% respectively (Foerster et al., 2013a). The spreading of FA reduction into frontal regions and the cerebellum (Keil et al., 2012) in longitudinal studies also correlated with ALSFRS-R, disease duration and disease progression (Jacob et al., 2003; Agosta et al., 2009; Zhang et al., 2011; Keil et al., 2012). In addition, FA was also found to be reduced in the corpus callosum of patients with MND/ALS and particular, the middle posterior body of the corpus that connects the motor and motor association cortices thus supporting the hypothesis that disease propagates along structural connections (Sach et al., 2004; Filippini et al., 2010; Bak and Chandran, 2012; Foerster et al., 2013b; Chapman et al., 2014) (Fig. 8).

Magnetic transfer imaging (MTI) is based on the exchange of magnetisation between spins in two proton pools: bound immobile protons associated with macromolecules (such as myelin) and free mobile protons associated with free water. The technique can demonstrate presence of structural changes in tissue associated with disease, even in the absence of changes observed in other sequences (Rocha and Maia Júnior, 2012c). The use of MTI in detecting structural changes involving the CST in MND patients

Fig. 8. Diffusion Tensor Imaging. TBSS P value maps fully corrected for multiple comparisons of voxel-wise differences between patients and healthy controls for fractional anisotropy (FA). Results displayed at significant levels (p < 0.05, fully corrected for multiple comparisons) in Montreal Neurological Institute (MNI) standard space overlaid on the mean FA image derived from all participants. Major fibre tracts as determined by TBSS are displayed in green. Red-yellow clusters indicate locations of significant corrected differences between patients and controls (p < 0.05). Locations of images in standard space are X = -1, Y = -17, Z = 19, MNI mm. Reproduced with permission (Chapman et al., 2014). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

have demonstrated CST hyperintensities on Tl-weighted spin-echo magnetisation transfer contrast (T1 SE MTC), with impressive sensitivity (80%) and specificity (100%) (da Rocha et al., 2004; Rocha and Maia Júnior, 2012). In addition, the sequence is fast and simple to acquire, and shown to be particularly useful in the early course of the disease to detect signal hyperintensity in the CST. The changes however were overrepresented in the cohort of MND patients with overt clinical UMN features and seldom in those with LMN predominant phenotypes such as PMA rendering the technique of limited utility in detecting subclinical UMN dysfunction (Carrara et al., 2012).

4.1.2. Magnetic resonance spectroscopy

Proton magnetic resonance spectroscopy (MRS) measure regional biochemistry in vivo and can detect proton-containing metabolites including N-acetyl aspartate (NAA), choline, and creatine (Cr) (Kaufmann and Mitsumoto, 2002; Kaufmann et al., 2004). NAA occur in neurons but not in glia (Kaufmann and Mitsumoto, 2002) and is marker of neuronal integrity or neuronal loss or damage depending on whether expressed as ratio to creatine or choline (respectively) (Turner et al., 2009).

Studies using MRS in MND/ALS patients have described a significant decrease in the ratio of NAA to creatine or choline in the motor cortex and other regions of the CST (Pioro et al., 1994; Giroud et al., 1996; Block et al., 1998; Rooney et al., 1998; Pohl et al., 2001a; Kaufmann et al., 2004) suggesting reduced neuronal integrity in the motor cortices (Foerster et al., 2013b), although at what stage of the disease process these changes become evidence remains conflicting (Figs. 9 and 10). Less dramatic changes were also observed in the bilateral frontal and parietal regions (Rule et al., 2004). Such levels of NAA were found to decrease longitudinally as disease progresses (Rule et al., 2004; Unrath et al., 2007; van der Graaff et al., 2010) but improved following institution of Riluzole (Kaufmann et al., 2004). The changes were also observed to correlate with disease severity, extent of UMN signs and clinical scores although these were based on cross-sectional studies (Agosta et al., 2010a). The mean decrease in NAA varied between studies and reportedly between 8% and 56% (Kaufmann and Mitsumoto, 2002) with one study suggesting a NAA/Cr ratio cut-off of 2.5 for normal values (Chan et al., 1999). This abnormality observed in MRS has been reported across all MND/ALS pheno-types with similar reductions observed in ALS and PLS and less

pronounced changes with PMA (Gredal et al., 1997; Mitsumoto et al., 2007). Furthermore, the reductions appeared more dramatic in MND/ALS patients with bulbar onset compared to limb onset (Ellis et al., 1998). Of particular relevance, abnormal MRS was demonstrated in a small number of autopsy cases without significant loss of Betz cells, suggesting that MRS may be able to detect UMN changes before Betz cell loss is visible using conventional stains or that sampling of cortex may miss affected areas (Kaufmann et al., 2004). Taken together, this suggests that the use of NAA reduction observed in MRS may be able to uncover sub-clinical UMN involvement in patients with MND/ALS although the results are more likely to be abnormal later on in the disease course rather than in the early stages. Moreover, MRS was able to distinguish MND/ALS from mimics such as SMA without UMN signs which showed no changes in the NAA ratio (Pioro et al., 1994). MRS sensitivity in detecting UMN involvement in MND/ ALS is reported to be around 86% with a specificity of about 37%, with the sensitivity somewhat lower at 63% in patients with PMA and no clear UMN signs clinically (Kaufmann et al., 2004). For this reason, although the method can be used to monitor disease progression, the diagnostic value of MRS remains poor because of considerable overlap in parameters found in MND patients with those of healthy controls (Rocha and Maia Júnior, 2012).

A potential issue that may affect the interpretation of MRS results arise from use of metabolite ratios rather than absolute NAA quantification. The use of ratios such as NAA/Cr or NAA/Cho, which are relatively easier to achieve with little processing time required after scanning, rests on the assumption that creatine and choline concentrations remain constant in the presence of disease (Chan et al., 2003). A study however, revealed decreases in values of NAA, creatine and choline in the brainstem of MND/ALS patients and thus cautioned against the use of concentration ratios (Hanstock et al., 2002). Furthermore, other studies have demonstrated that the levels of NAA, choline and creatine all decline with MND/ALS disease progression resulting in a relatively stable NAA metabolite ration, thus rendering the use of metabolite ratio for UMN assessment in longitudinal studies potentially unreliable. In view of this, absolute NAA quantification may be the preferred method as technology and software improves that will significantly cut back on the scanning and post-processing times that currently limits its feasibility. In addition, the use of single-voxel

Fig. 10. Proton magnetic resonance spectroscopy (MRS) in ALS compared to control groups in the left (a) and right (b) precentral gyrus demonstrating a reduction in the NAA/creatine ratio in patients with ALS. Line within the box represents the median whilst the 25-75% quartiles are shown by the box. Maximal and minimal values are represented by the short horizontal lines. Reproduced with permission (Sivak et al., 2010).

technique in most MRS studies likely imposes limits to the sensitivity because of only a relatively small brain being assessed, and will be improved by the future development of multivoxel MRS that has greater spatial resolution and allow several different axial sections to be sampled simultaneously, but may be limited by its more complex scanning procedure and longer scan time and larger data sets to process afterwards (Chan et al., 2003; Kaufmann et al., 2004). This is especially relevant given that reductions in NAA have also been observed in multiple regions of the CST (Govind et al., 2012; Stagg et al., 2013) including the brainstem with most changes seen in the pons and upper medulla in patients with prominent UMN or bulbar signs (Cwik et al., 1998).

4.1.3. Functional MRI

Functional MRI (fMRl) is able to assess physiological function of the upper motor neurons. It provides high-resolution measures of neural activity that is reflected by changes in blood flow to local vasculature in response to activity. During index-thumb opposition in patients with MND/ALS of lower motor neuron onset, results were similar to normal controls revealing activation over Ml and some activation over the sensorimotor cortex. In

MND/ALS patients with upper motor neuron signs the activation appear more widespread, involving the supplementary motor, premotor, and sensory cortex (Brooks et al., 2000a; Eisen and Weber, 2001; Agosta et al., 2010a), which was also observed on the contralateral hemisphere (Schoenfeld et al., 2005). Much of these changes were thought to be secondary to task-difficulty associated with the motor impairment and/or a compensatory role of these recruited extra-motor related brain regions (Agosta et al., 2010a).

ln other studies, decreases in regional patterns of activation were observed in MND/ALS patients during a motor task, with activation in other regions that positively correlated with UMN burden. Furthermore, activation increased with physical impairment during follow-up (Tessitore et al., 2006; Lule et al., 2007). Of relevance however, there is difficulty in controlling task performance in MND patients that may be result in variability of results using fMRl.

Resting state functional connectivity studies have demonstrated motor network changes in patients with MND/ALS (Foerster et al., 2013b). Connectivity appeared to be decreased in the motor network early in the disease whilst increased connectivity was observed with loss of interhemispheric inhibition as disease progressed and disease burden more pronounced (Mohammadi et al., 2009; Jelsone-Swain et al., 2010; Verstraete et al., 2010; Agosta et al., 2011; Douaud et al., 2011; Tedeschi et al., 2012).

With a view to establishing consensus regarding various applications of novel MRl techniques to the study of MND, and to explore the possibility of multicentre collaboration, the first Neu-roimaging Symposium in ALS (NISALS) was held Oxford University, UK, in November of 2010. There was a recognised need to balance a multiparametric approach to increase the potential biomarker yield, with simplicity, reproducibility, and tolerability (Turner et al., 2011). Consensus was reached about essential and desirable protocols for MRl as well as the inclusion of critical clinical information for MND studies, in order to achieve standardised approaches to develop robust biomarkers through international multicentre and longitudinal studies.

4.2. Single-photon emission computed tomography

Using single-photon emission computed tomography (SPECT), areas of widespread cortical hypoperfusion have been demonstrated in motor areas as well as frontoparietal regions in patients with MND/ALS (Anzai et al., 1990; Abe et al., 1993), that were more pronounced in those with longer disease duration (Ludolph et al., 1989). The more characteristic reduced uptake confined to the motor cortex was found only in 29-45% of patients with MND/ ALS (Chan et al., 2003).

4.3. Positron emission tomography

Positron emission tomography (PET) imaging allows qualitative and quantitative studies of brain metabolism in vivo (Kaufmann and Mitsumoto, 2002). ln MND/ALS patients, frontal hypometabo-lism has been uniformly demonstrated using 18FDG-PET in all patients. The most commonly reported regions with hypometabo-lism involved the perirolandic and frontal brain regions and appeared to be a sensitive marker of MND/ALS (Dalakas et al., 1987; Shiozawa et al., 2000; Foerster et al., 2013b; Pagani et al., 2014; Van Laere et al., 2014) (Fig. 11), with a diagnostic accuracy of greater than 90% for differentiating MND patients from health controls (Chio and Traynor, 2015) and sensitivity and specificity greater than 90% and 80%, respectively (Pagani et al., 2014; Van Laere et al., 2014). Of relevance, patients clinically diagnosed with PMA also demonstrated a similar pattern of hypometabolism (Van Laere et al., 2014) that underscores the presence of subclinical

Fig. 11. 18F-fluorodeoxyglucose PET analysis in ALS patients demonstrating hypometabolism. The images show three-dimensional rendering of the brain cortical surface of the clusters of voxels in which patients with ALS show hypometabolism compared with healthy controls. Uptake is substantially impaired mainly in the frontal and anterior cingulate cortex. Reproduced with permission (Chio et al., 2014).

corticomotoneuronal involvement as well as PMA being part of the MND/ALS clinical spectrum. In addition, patients with MND/ALS also showed clusters of relative hypermetabolism within the cerebellum, occipital cortex, upper brain stem, and medial temporal cortex (Van Laere et al., 2014). Patients with PLS demonstrated a more widespread abnormality with symmetrically reduced metabolism bilaterally in the prefrontal cortex, anterior cingulate, pericentral cortex, and thalamus (Van Laere et al., 2014). One study proposed that the most discriminating regions (between pheno-types of MND: ALS, PMA and PLS) were the prefrontal cortex, thalamus, posterior cingulate, and anterior cingulate (Van Laere et al., 2014). Reductions in metabolism in frontal lobe regions were prominent in MND/ALS patients with cognitive impairment especially those with executive dysfunction such as verbal fluency (Abrahams et al., 1996; Cistaro et al., 2012), and extensive fron-totemporal hypometabolism was predictive for a lower survival (Burrell et al., 2016; Van Weehaeghe et al., 2016). Further studies however are needed to compare patients with MND to patients with mimic disorders to better define the sensitivity and specificity of 18FDG-PET in the diagnosis of MND. Longitudinal studies are also required to investigate both the neuroradiological course of the disease using 18FDG-PET but its change in relation to potentially disease-modifying agents.

15O2 and H215O were originally used to study regional cerebral blood flow (rCBF), permitting the introduction of activation studies (Kew et al., 1993). Attention shifted to the utilisation of tracer ligands such as 18F-6-fluorodopa and 11C-Flumazenil to identify changes in specific neurochemical and cellular systems in MND/ ALS (Turner and Leigh, 2000). Studies using Flumazenil as a GABA-A receptor ligand demonstrated similar regions of decreased tracer uptake in the motor and extramotor regions of the brain (Cistaro et al., 2014), and profound serotonergic receptor binding reductions were observed in frontotemporal regions of non-depressed, non-demented MND/ALS patients (Turner et al., 2005b).

5. Combining multiple electrophysiological and imaging modalities

As the abovementioned novel electrophysiological and imaging become more readily accessible in clinical practice, it seems likely that combining structural and functional diagnostic biomarkers

more likely increases the likelihood of objectively demonstrating UMN dysfunction in MND thereby facilitating earlier diagnosis and institution of potentially disease-modifying agents (Pohl et al., 2001b; Kaufmann et al., 2004; Turner et al., 2005a; Furtula et al., 2013; Grieve et al., 2015; Bae et al., 2016). In a study that combined structural imaging and electrophysiological approaches revealed that the presence of precentral gyrus cortical thinning or paired-pulse TMS abnormalities was evident in 88% of MND patients, while temporal region cortical thinning or TMS abnormalities were evident in 96% (Grieve et al., 2015). Further, TMS abnormalities did not correlate with cortical thinning, suggesting that functional and structural cortical abnormalities may act as complementary diagnostic biomarkers of UMN dysfunction in MND/ALS (Grieve et al., 2015). Prior to this study, another group of investigators also combined the use of MRS with single-pulsed TMS techniques to examine the presence of UMN abnormalities in patients with MND/ALS (Pohl et al., 2001b). The authors found that abnormal MRS findings were present in 53% whilst abnormal TMS in 63%. Abnormalities in either study was demonstrated in 78% whilst abnormalities in both in 39%, again suggesting that the use of a combined imaging and electrophysiological approach may complement each other and increase the yield of detecting UMN abnormalities. Of particular relevance, the combined approach detected UMN abnormalities in 60% of those MND patients without clinical UMN signs (that included patients in the suspected and possible EEC categories). Furthermore, more than 60% of these patients upon follow-up later developed clinical UMN signs (Pohl et al., 2001b).

6. Conclusions and future directions

There remains considerable challenges in the development of a diagnostic tool that fulfils all of the requirements necessary to constitute a biomarker of UMN dysfunction in MND/ALS patients (Table 1). It requires replicable data in a large number of patients with different clinical phenotypes and at various stages of the disease. Currently, neurophysiological and neuroimaging techniques utilised in the assessment of UMN function are either not readily accessible outside a research setting or exhibit poor sensitivity or specificity in early stage of the disease process. For this reason, formulating a diagnostic algorithm based on the use of these novel

Table 1

Comparison between the techniques used to assess upper motor neuron degeneration in MND.

Technique Sensitivity Specificity Utility in subclinical Utility in Prognostic Limitations/Problems Longitudinal

UMN phenotypes mimics value studies

TMS (threshold-tacking) 73.2-97%] 88.9%1 Yes Yes Yes Limited availability Yes

TST 54%2 Maybe higher in those with earlier stage3 Yes No Not studied • Limited in those with significant LMN loss • Limited number of patients and studies to date No

PSTH - Low No Yes Not studied Lack specificity and abnormalities seen in Yes

other CNS disorders

T2 MRI 640%4 670%4 No No Not studied Low sensitivity No

VBM/SBM MRI 25%5 No No Not studied • Conflicting data • Late stage disease mainly • Small patient numbers No

DTI MRI 25-86%2,6,7 P 70%6,7 Yes No Yes • Non-standardised data • Limited patient numbers • Late stage disease mainly Yes

MTI-MRI 80%8 100%8 No No No • Very small numbers to date • Needs further studies No

MRS 71-86%9,10 37-75%9,10 Yes Yes No • Considerable overlap between healthy controls and patients • Limited patient numbers • Late stage disease mainly Yes

fMRI - - No No No Diagnostic value uncertain No

SPECT 29-45%4 - No No No Diagnostic value uncertain No

PET 89-95%1u2 P80%1112 Yes No No Need to compare with MND mimics No

TMS, transcranial magnetic stimulation; TST, triple stimulation technique; PSTH, peristimulus time histograms; MRI, magnetic resonance imaging; VBM, volume-based morphometry; SBM, surfaced-based morphometry; DTI, diffusion-tensor imaging; MTI, magnetisation transfer imaging; MRS, magnetic resonance spectroscopy; MTI, magnetic transfer imaging; fMRI, functional MRI; SPECT, single-photon emission computed tomography; PET, positron emission tomography. Table References:

1 Menon et al. (2015a).

2 Furtula et al. (2013).

3 Komissarow et al. (2004).

4 Chan et al. (2003).

5 Chen and Ma (2010).

6 Foerster et al. (2013a).

7 Foerster et al. (2014).

8 da Rocha et al. (2004).

9 Cervo et al. (2015).

10 Pohl et al. (2001b).

11 Pagani et al. (2014).

12 Van Laere et al. (2014).

techniques will be difficult in a clinical setting and will depend largely on the regional availability and accessibility of these techniques.

Of relevance, most neuroimaging studies to date have involved relatively small numbers of patients with long disease duration that may result in skewed sampling, and may explain some of the discrepant neuroimaging results in the literature, and potentially limiting the generalizability of the findings (Foerster et al., 2013b). International efforts have produced a consensus statement paper from the Neuroimaging Society in ALS to facilitate neu-roimaging biomarker development that will be standardised across centres (Filippi et al., 2015). More recently, a multicentre DTI study showed that by applying correction procedures to regress and account for centre-specific variability, it is possible to increase the sensitivity and specificity of DTI findings, thereby suggesting the feasibility to pool scans across sites, despite significant differences across protocols, to facilitate multisite collaboration (Hornberger and Kiernan, 2016; Muller et al., 2016).

In addition, UMN abnormalities demonstrated in many neu-roimaging studies have mainly been observed in those with clinically apparent UMN features (majority of patients in the clinically probably or definite categories) with the changes correlating to the degree of UMN scores, casting doubt on their utility in uncovering reliable changes in patient's in the early stages where LMN features predominate. The recruitment pool also differed in those patients selected for neuroimaging studies that were

predominantly from retrospective cohorts of clinical patients referred to imaging centres, thereby explaining why majority were in the later stages of disease. This is in comparison to many electro-physiological studies that were prospective assessment of a large number of patients that were referred to specialised MND/ALS academic and research centres with a substantial proportion in their early stages. Furthermore, the ability to distinguish MND from mimics in neuroimaging studies have not been sufficiently explored (Chio and Traynor, 2015). Further limitations of current neuroimaging studies are attributable to respiratory involvement in MND patients that result in their inability to take part in longitudinal studies as their disease progresses to a later stage when lying flat for the study poses significant issues. Moreover, there are issue relating to the availability and cost of installing these advanced imaging modalities outside of research or academic facilities.

Without doubt, it is likely that in searching for an ideal diagnostic biomarker of UMN degeneration in MND, there will be a need for collaboration amongst neurologists, neuroimaging specialists and neurophysiologists, and that a combination of electrophysio-logical and neuroimaging modalities will prove to be the most attractive approach as they become more widespread and accessible to the clinical setting in the not too distant future. A multi-modality structural and functional approach may also prove useful to further our understanding of the pathophysiological mechanisms and natural history of MND in a way that may

complement neuropathological theories of primary induction of disease-causing pTDP-43 protein aggregates in the corticomo-toneurons and their subsequent dissemination via axonal transport to more distant sites such as the spinal cord or other brain regions (Braak et al., 2013). Such multimodal approaches may also facilitate comprehensive longitudinal studies thereby paving the way for the development of therapeutic biomarkers to investigate nervous system changes in response to potential novel therapeutics. Until then, neurologists will need to utilise whichever modality that is currently accessible, and being able to appropriately apply the research results when addressing individual patients but keeping in mind their current limitations in clinical practice. Based on the authors experience and the current review, a combination of threshold tracking TMS and FDG-PET imaging represent an attractive approach given their high sensitivity and specificity based on large numbers of patients studied, their relative availability (particularly with PET imaging) and cost, as well as the ability for the tests to complement each other. In particular, PET studies are able to identify a larger area of abnormality (motor and extramotor) that is not captured by a single TMS parameter from a specific cortical region examined, thereby allowing for a more comprehensive approach to studying disease pathophysiology, natural history and the effects of disease-modifying agents. After decades of seemingly incremental progress, it now seems tangible that objective biomarkers of UMN dysfunction will be available at the clinic, that in turn will facilitate an earlier diagnosis of MND/ALS, more timely commencement of neuroprotective therapy and in turn, enrolment into future trials and novel treatment strategies.

Acknowledgements

WH was supported by the University of Sydney post-doctoral research fellowship. This work was supported by funding to Forefront, a collaborative research group dedicated to the study of motor neuron disease, from the National Health and Medical Research Council of Australia program grant (#1037746). MRT is funded by the Medical Research Council and Motor Neuron Disease Association Lady Edith Wolfson Fellowship (MR/K01014X/1).

Conflict of interest: None of the authors have potential conflicts of interest to be disclosed.

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