Peripheral nerve ultrasound in ALS phenotypes Nerve ultrasound in ALS 1,2
1,2,3
Stefanie Schreiber M.D.* , Susanne Abdulla M.D.*
1
, Grazyna Debska-Vielhaber Ph.D. ,
2
1
Judith Machts M.Sc. , Verena Dannhardt-Stieger M.D.1, Helmut Feistner M.D. , Andreas 1
1
3
3
Oldag M.D. , Michael Goertler M.D. , Susanne Petri M.D. , Katja Kollewe M.D. , Siegfried 4
1,2
3
Kropf Ph.D. , Frank Schreiber M.Sc.5, Hans-Jochen Heinze M.D. , Reinhard Dengler M.D. , 2
Peter J. Nestor M.D. , Stefan Vielhaber M.D.
1,2
* equally contributed 1
2
Department of Neurology, Otto-von-Guericke-University, Magdeburg, Germany German Center for Neurodegenerative Diseases (DZNE) within the Helmholtz Association, Magdeburg, Germany
3
4
Clinic for Neurology, Hannover Medical School, Hannover, Germany Institute
of
Biometry
and
Medical
Informatics,
Otto-von-Guericke-University,
Magdeburg, Germany 5
Institute of Control Engineering, Technical University Braunschweig, Braunschweig, Germany
Corresponding author Stefanie Schreiber, M.D. Department of Neurology, Otto-von-Guericke University, Leipziger Strasse 44, 39120 Magdeburg [email protected] Telephone
+ 49 391 6713431
Fax
+ 49 391 6715233
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24431
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2 Acknowledgements. We would like to thank Anne-Katrin Baum for excellent technical support. This work was supported by a grant to S.V. from the Foundation of Medical Research, Frankfurt / Main, Germany. Disclosures. None. Key words ALS, PLS, median nerve, ulnar nerve, ultrasound Running title Ultrasound in ALS phenotypes
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3
Peripheral nerve ultrasound in ALS phenotypes Abstract Introduction. We sought to determine the cross sectional area (CSA) of peripheral nerves in patients with distinct subtypes of amyotrophic lateral sclerosis (ALS). Methods. Ulnar and median nerve ultrasound was performed in 78 ALS patients [classic, n=21, upper motor neuron dominant (UMND), n=14, lower motor neuron dominant (LMND), n=20, bulbar, n=15, primary lateral sclerosis (PLS) n=8] and 18 matched healthy controls. Results. Compared to controls ALS patients had significant, distally pronounced reductions of ulnar CSA (forearm/wrist level) across all disease groups except for PLS. Median nerve CSA (forearm/wrist level) did not differ between controls and ALS. Conclusion. Ulnar nerve ultrasound in ALS subgroups revealed significant differences in distal CSA values, which suggests it has value as a marker of LMN involvement. Its potential was particularly evident in UMND and PLS groups, which can be hard to separate clinically, yet their accurate separation has important prognostic implications.
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4 Introduction Amyotrophic lateral sclerosis (ALS) is defined by progressive weakness that results from neurodegeneration of upper (UMN) and lower motor neurons (LMN). The variable mix of UMN and LMN signs produces major phenotypic heterogeneity in ALS patients [1-3]. Thus, clinical manifestations of ALS exist on a continuum, ranging from apparently pure LMN dysfunction to severe pyramidal impairment with minor LMN signs [4]. Recently, there has been increasing acceptance of those distinct groups with upper versus lower motor neuron predominant pathology persisting through the course of the disease [5-7]. Briefly, classic ALS is characterized by the combination of LMN and pyramidal signs, upper motor neuron dominant (UMND) ALS by pyramidal signs (spinobulbar spasticity) associated with slight LMN dysfunction [8], lower motor neuron disease or syndrome (LMND) by absence of UMN signs, and progressive bulbar palsy (PBP) by dysarthria and dysphagia [9]. Exclusive UMN involvement with predominant spinobulbar spasticity is the hallmark of primary lateral sclerosis (PLS) [10-12]. Recognition of these variants can be relevant to counseling on prognosis, drug therapy, and tailoring care to the needs of the individual [13]. Neuromuscular ultrasound abnormalities may aid in the diagnosis of ALS [14;15] and also in distinguishing between ALS phenotypes (e.g. UMND versus PLS patients), which has important prognostic implications [6;8]. We hypothesized that categorizing ALS patients by their respective clinical syndromes [16] would reveal quantitative differences in sonographic parameters of the peripheral nerves. Methods Inclusion criteria of ALS patients and control subjects Seventy-eight consecutive patients with sporadic ALS were recruited from the Departments of Neurology at the Universities of Magdeburg and Hannover, Germany, with clinical follow-
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5 up intervals every 3 months. The study was approved by the local ethics committee (No. 150/09), and all subjects gave informed consent. The diagnosis of ALS was based on the revised El Escorial Criteria [17]. Patients with pure lower or upper motor syndromes or bulbar syndromes (who could not be classified in the revised El Escorial criteria) were classified into an additional category of suspected ALS. Patients with clinically obvious dementia at onset were excluded. If relevant, lumbar puncture and magnetic resonance imaging (MRI) of the brain and spinal cord were used to exclude other diagnoses. ALS phenotypes (diagnostic subgroups) were classified according to the operational definitions specified below as: classic (n=21), UMND (n=14), LMND (n=20), bulbar (n=15), or PLS (n=8). At the time of peripheral nerve ultrasound, a variable combination of UMN signs (spastic tone, clonus, etc.) and LMN signs (wasting, weakness, fasciculations) in the upper and lower limbs were found in those designated as classic ALS who, in turn, fulfilled the El Escorial criteria of definite or probable ALS. UMND ALS patients had either no LMN signs (weakness, wasting, or fasciculations), or, if present, (1) they were restricted to only 1 neuraxis level (bulbar, cervical, or lumbosacral), and (2) electromyography (EMG) abnormalities were limited to sparse fibrillation potentials/positive sharp waves or minor enlargement of motor unit potentials in 1 or at most 2 muscles [6;8] for at least 12 months after symptoms onset. The diagnostic criteria for PLS required a period of at least 4 years in which there were only UMN signs on examination [18]. The following were rigorously excluded from the PLS category: patients with clinical or electromyographic (EMG) signs of LMN involvement; those with a disease that could mimic motor neuron disease; a family history of spastic paraparesis/tetraparesis; mutations of genes related to hereditary spastic paraplegia (SPG3A, SPG4. SPG7, SPG10, and SPG11, [12]); and those with symptom onset before age 40 years. All patients with LMND had clinical and electrophysiological evidence of sporadic progressive pure LMN involvement in 1 or more regions without clinical signs of UMN dysfunction. To John Wiley & Sons, Inc.
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6 differentiate this condition from early limb onset ALS, we specified that LMN involvement must be the predominant finding for at least 12 months after the symptom onset. Other LMN diseases, such as multifocal motor neuropathy, spinal muscular atrophy, monomelic amyotrophy, Kennedy disease, and post-polio syndrome were excluded by extensive clinical and laboratory examinations [19;20]. In the PBP group, onset with lower motor neuron dysarthria followed by progressive speech and swallowing difficulties was the dominant clinical finding. To differentiate this condition from early bulbar onset ALS, e.g. we specified that bulbar involvement must be the predominant finding for at least 12 months after the symptom onset. If LMN signs were present, (1) they were restricted to only 1 neuraxis level (cervical, thoracic, or lumbosacral), and (2) EMG abnormalities were limited to sparse fibrillation potentials/positive sharp waves or minor enlargement of motor unit potentials in 1 or at most 2 muscles. Patients with clinical signs of UMN dysfunction were excluded from this category. Pseudobulbar (spastic) paralysis entails no atrophy or fasciculations of the paralyzed muscles, and patients with this condition were also excluded from the PBS category. Other conditions that mimic PBP, such as myasthenia gravis were excluded by appropriate investigations. All patients were graded using the revised ALS functional rating scale (ALSFRS-R) [21]. Disease duration was defined as time in months between symptom onset and the date of the evaluation. Disease-progression rate was calculated as (48 – ALSFRS-R)/disease duration [22]. Additionally, all subjects underwent standard electrophysiological measurements including the determination of distal motor latency (DML); motor and sensory conduction velocity (CV); compound motor action potential (CMAP) amplitude; sensory amplitude of the ulnar nerves with stimulation at the elbow and wrist and of the median nerves with stimulation in the forearm and wrist at 34°C. Based on electrophysiological features, ulnar nerve entrapment at the cubital tunnel (CuT) was confirmed in 8 ALS patients and 3 controls, and median nerve
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7 entrapment at the carpal tunnel was found in 10 ALS patients and 1 control; ultrasound data of these affected ulnar and median nerves were excluded from statistical analysis. The control group consisted of 18 healthy volunteers recruited from the general population via announcement. All participants were free of uncontrolled diabetes mellitus, arterial hypertension, neuromuscular symptoms (e.g., numbness and tingling or weakness), traumarelated peripheral nerve disease, and alcohol dependence syndrome according to International Classification of Diseases (ICD)-10 diagnostic criteria. All had a normal neurological examination. Specifically, an individual who reported symptoms suggesting carpal tunnel syndrome (e.g. hand pain/paresthesia with nocturnal or use-related exacerbation) or ulnar neuropathy (e.g. hand pain/paresthesia, elbow injury, elbow pain) was excluded. As in former peripheral nerve ultrasound studies [14;23-28] diabetes status and vitamin B deficiencies were not sought explicitly or used as exclusion criteria. The control subjects had normal nutritional status (Table 1) and took part in regular preventive medical check-ups in collaboration with health insurance funds, which have specific objectives for identification and treatment of patients with cardiovascular diseases, metabolic disorders, and nutritional disturbances [29]. Ultrasound Ultrasound examinations were performed using a GE High-End LOGIQ®7 System with a 12MHz linear array probe. All subjects were seated with the entire arm extended anteriorly, supinated, and supported by a pillow on a table at approximately mid-thoracic height [30]. The ulnar and median nerves [29;31;32] were identified by their location and morphology [3335] and visualized over the whole distance from the distal wrist crease to the CuT (ulnar nerve) and from the distal wrist crease to the middle third of the forearm (FA, median nerve). As established [29;31;32] transverse images were obtained bilaterally at the CuT (ulnar nerve), the middle third of the FA (ulnar and median nerves), and at the level of the distal wrist crease (ulnar and median nerves [27;29;35-39]). At all sites, the nerve cross sectional area (CSA) was calculated by continuous manual tracing of nerve circumference, excluding
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8 the hyperechoic epineural rim [24]. The ratio between ulnar nerve CSA at the CuT and wrist (ulnar nerve CuT/wrist ratio) and between the median nerve CSA at the FA and wrist (median nerve FA/wrist ratio) were calculated. Three separate CSA measurements were averaged at each nerve site. Sonography images were acquired by a single investigator (S.S.) who was not blinded to diagnosis but to the disease subtypes [29]. Statistics To assess the quality of group matching for gender we used a chi-square test (comparing controls and all patients) and a generalized linear model with subsequent adjustment for multiple comparisons (comparing all diagnostic subgroups). For age, height, weight, disease duration, ALSFRS-R, and disease-progression rate we used a Mann-Whitney-U test (comparing controls and all patients) and a Kruskal-Wallis test (comparing all diagnostic subgroups). A paired t-test was performed to assess possible CSA differences between the right and left upper limb. Tests of between-subjects effects in ANOVA for repeated measures revealed dependency of sonographic data on height, disease duration, and phenotype (diagnostic subgroup). Subsequent comparisons of each ultrasound measurement between all diagnostic subgroups (controls, PLS, UMND, LMND, classic and bulbar phenotype) and ulnar nerve measurements between the disease-onset subtypes (bulbar, limb) and the affected side [right upper limb, left upper limb, equally affected upper limb(s)] using univariate analysis of covariance (ANCOVA) were performed with sonographic parameters (CSA) as dependent variable, height and disease duration as covariates, and phenotype (subgroups or disease-onset subtypes or affected side) as fixed variable; a Bonferroni adjustment for multiple comparisons was applied. CuT/wrist and FA/wrist ratio comparisons between the diagnostic subgroups and controls were performed using a Kruskal-Wallis test adjusted for multiple comparisons. Pearson correlation coefficients were used to assess the associations between the ulnar ultrasound data, clinical outcome measures (e.g. ALSFRS-R), and between ulnar and median nerve CSA and the abductor pollicis brevis muscle
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9 (APB)/abductor digiti minimi muscle (ADM) CMAP amplitude ratio (see below). P-values ≤ 0.05 were deemed to be statistically significant.
Data from all patients (n=78) and controls (n=18) were used to calculate a Cronbach α in order to assess the reliability of repeated CSA measurements. The respective reliabilities of ulnar and median nerve CSA measurements were as follows: patients, 0.984/0.991 (wrist), 0.952/0.954 (FA), 0.991 (CuT), and controls, 0.996/0.981 (wrist), 0.965/0.965 (FA), 0.995 (CuT).
Results Subjects Clinical and demographic data are summarized in Table 1. There were no significant group differences for gender, height and weight between the controls and the whole patient cohort (Table 1) and between the controls and the different diagnostic subgroups (data not shown in detail). The age distribution was comparable between the controls and the whole patient cohort (Table 1) and between the controls and all diagnostic subgroups. The ALSFRS-R score did not differ between diagnostic subgroups (Table 1). As expected, disease duration and disease-progression rate differed significantly between the subgroups in that PLS patients had the longest disease duration and the slowest disease-progression rate (Table 1). Ultrasound data Compared to controls, ulnar nerve CSA at the wrist and FA was reduced significantly in all ALS diagnostic subgroups except for PLS (Figures 1 & 2, Table 2). The FA and wrist ulnar nerve mean differences ranged from 1.66 (FA left, LMND) to 3.02 (FA right, UMND) between the controls and the different ALS subgroups (UMND, LMND, classic and bulbar phenotype) John Wiley & Sons, Inc.
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10 and from 0.43 (FA left) to 1.20 (, FA right) between controls and the PLS subgroup. Ulnar nerve CSA at the level of CuT and median nerve CSA at wrist and FA did not differ between any of the diagnostic subgroups (Table 2). Consequently, in the ALS subgroups (UMND, LMND, classic, and bulbar phenotype) the CuT/wrist ratio was increased significantly compared to the controls, while it did not differ between the PLS subgroup and the control subjects (Table 3). Median nerve FA/wrist ratios did not differ between any of the diagnostic subgroups (PLS, UMND, LMND, classic and bulbar phenotype) and the control subjects (data not shown). No consistent correlations were found in either the total ALS cohort or in the subgroups between any ulnar nerve CSA measurement and clinical outcome measures, e.g. ALSFRSR, disease-progression rate, or electrophysiological measures [e.g. compound muscle action potential (CMAP) of the thenar (for the median nerve) and hypothenar (for the ulnar nerve)].
Additional statistical analysis with respect to further clinical classifications [disease-onset subtype (bulbar, limb), side of most clinically affected upper limb] including all subgroups (PLS, UMND, LMND, classic and bulbar phenotype) revealed no consistent effects on the ulnar nerve sonographic data. Interestingly, in ALS patients (non-PLS variants) who had asymmetric upper limb involvement (present in n = 31), ulnar nerve CSA did not differ between the right and left upper limb and ulnar nerve CSA of the clinically less affected (n = 24) or unaffected upper limb (n = 7) was likewise significantly smaller compared to the control values (data not shown).
Discussion Stratifying ALS patients into clinical subgroups revealed small but significant reductions in ulnar nerve CSA at the wrist and FA in all groups with the exception of PLS. Although the PLS group was the smallest, the lack of statistically significant effect is unlikely to reflect lack of power, because the mean differences in PLS were also considerably less than those seen
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11 for the other groups. No significant changes were found in ulnar nerve CSA at the CuT level and median nerve CSA for any group.
Control CSA values of the ulnar and median nerves were in line with those of previous studies [29;31;32]. Reliability of CSA measurements was high, which was in keeping with previous reports applying the same method [25;29]. As in former studies, CSA values are influenced by height [28;31;36], but not by weight [36].
The absence of median nerve CSA reductions differs from the only previous ALS nerve ultrasound study, which revealed a significant reduction of median nerve CSA [14]. In that study, however, the median nerve CSA was measured at a more proximal site between the medial epicondyle and the axilla [14]. At first glance, our data seem to be also incompatible with a recent EMG study describing a preferential involvement of median nerve innervated muscles (part of C8/Th1 innervation pattern) in ALS [40]. We speculate that these findings can be reconciled in a model that considers the relative proportion of motor and sensory fibers along the nerve course and between different nerves. The internal grouping and proportion of motor and sensory branches of the median nerve differs from that of the ulnar nerve [41;42]. Results should, therefore, be influenced by both varying proportions of sensory axons in the 2 nerves and by the anatomic location studied along the course of each nerve. Indeed, compared to proximal sections, the distal ulnar nerve predominantly consists of motor branches, whereas the median nerve is a mixed nerve, especially in its more distal sections. This could explain both the circumscribed distal ulnar CSA reduction and also the preserved ulnar nerve CSA at the more proximal site of the CuT. Furthermore, this model would predict that median nerve CSA reductions would be more evident in the upper arm, where the nerve still contains the motor axons that will supply the forearm muscles. Finally, the model is compatible with past EMG findings of severe distal median denervation [43-45] if the proportion of motor axons in the distal median nerve are relatively fewer than in the
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12 ulnar nerve but these same median motor fibers are severely affected, one would expect little impact on CSA but profound changes on EMG. The absence of correlations with clinical measures such as the ALSFRS-R might be due to the fact that the sonographic data set was not distributed adequately in and between the subgroups to result in rigorous correlations. It is not surprising, though, that no clear relationship is found when one considers that: (1) the ALSFRS-R is a composite score from 3 different domains (bulbar, motor, and respiratory function) and therefore unlikely to reflect changes sensitively in a specific peripheral nerve; and (2) that clinical heterogeneity across the different sub-syndromes of ALS means that similar ALSFRS-R scores can be generated in spite of major between-subject differences in the degree of UMN and LMN involvement. A more direct issue is reconciling these ultrasound findings to the dissociated atrophy of the intrinsic hand muscles, the ’split hand’. In ALS, the APB usually shows a greater reduction of motor unit number estimates compared to the ADM which is reflected in the APB/ADM CMAP amplitude ratio (< 0.6, [40;46;47]). No significant correlations could be detected between the APB/ADM ratio and the corresponding ulnar and median nerve CSA. A split hand according to the above mentioned electrophysiological criteria was found in 40% of our ALS patients, which is in line with findings of other groups [47]. From this, and the fact that the degree of atrophy of muscles is usually not symmetric in ALS, one would also expect more pronounced axonal loss and subsequent nerve caliber reduction in the more severely atrophic upper limb. We could not, however, find any significant ulnar nerve CSA asymmetries in those ALS subjects (n = 31) that exhibited clinically asymmetric upper limb involvement. When EMG changes were considered by region, there was a relatively uniform detection rate of neurogenic abnormalities including limbs without apparent clinical weakness [48], which is consistent with our sonographic findings. In any case, the model proposed above can accommodate the split hand phenomenon by considering that ultrasound could be insensitive to significant distal median nerve motor fiber loss.
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13 Our data cannot answer the question of whether ALS and PLS are distinct disorders [10;49;50], but they at least allow us to state that the sonographic appearance of the ulnar nerve in our PLS group was similar to that of the controls. The distinction between ALS and PLS relates primarily to the degree of lower motor neuron involvement. Indeed, the fact that the UMND variant was indistinguishable from the other non-PLS variants in this sonographic study. This indicates probable motor axonal loss as a pathological substrate for ulnar nerve CSA reduction and therefore common LMN involvement, at least in the chosen ALS subgroup classification. Morphometric analysis of peripheral nerves in ALS showed progressive reduction of large myelinated fibers [51] but also a transient increase of small myelinated fibers suggestive of reinnervation [52]. Overall, peripheral nerves in ALS become progressively thinner [53]. This is an area that should be explored in more detail in future ultrasound studies with additional measurement of individual nerve fascicles to permit more definitive conclusions.
In conclusion, the findings of this study suggest that ultrasound as a quantitative measure in conjunction with other systematic investigations such as electrophysiological studies may offer promise in distinguishing patients with pure UMN involvement from those in whom UMN is the predominant but not exclusive manifestation. Prospective studies will be needed to confirm this finding as well as to determine whether other disease subgroup classifications (e.g. according to genotype, myotomes affected, rate of progression) would result in a different distribution of sonographic data. Nevertheless, we believe that even without splitting the patients into further subcategories such as flail arm and flail leg variants or typical ALS with arm-onset and leg-onset, our findings serve as a good working model to advance this field and to lead to further investigations using larger sample sizes, including such subcategories and perhaps genetically defined ALS variants. Further studies could also focus on the relationship between peripheral biomarkers such as nerve CSA and central biomarkers such as diffusion tensor imaging of the corticospinal tract.
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14
Gender
Controls
PLS
UMND
LMND
Classic
Bulbar
P
14 m, 4w
5 m, 3 w
11 m, 3 w
14 m, 6 w
12 m, 9 w
6 m, 9 w
0.217
Age
63.8
(9.5)
63.5 (16.8) 54.1
(8.3)
59.2 (14.5) 58.5 (12.1) 66.1 (11.4)
0.093
Height
174.0 (8.7) 172.7 (13.2) 171.0 (8.3) 173.5 (9.5) 171.1 (8.5) 169.4 (10.2)
0.734
Weight
81.0 (12.7) 75.0 (17.2) 79.4 (16.5) 79.4 (16.7) 70.9 (10.1) 70.3 (14.9)
0.119
Disease duration
n.a.
104.1 (77.7) 32.8 (22.4) 37.0 (26.0) 20.9 (12.8) 15.7
(5.7)
1,2,3
Stefanie Schreiber M.D.* , Susanne Abdulla M.D.*
1
, Grazyna Debska-Vielhaber Ph.D. ,
2
1
Judith Machts M.Sc. , Verena Dannhardt-Stieger M.D.1, Helmut Feistner M.D. , Andreas 1
1
3
3
Oldag M.D. , Michael Goertler M.D. , Susanne Petri M.D. , Katja Kollewe M.D. , Siegfried 4
1,2
3
Kropf Ph.D. , Frank Schreiber M.Sc.5, Hans-Jochen Heinze M.D. , Reinhard Dengler M.D. , 2
Peter J. Nestor M.D. , Stefan Vielhaber M.D.
1,2
* equally contributed 1
2
Department of Neurology, Otto-von-Guericke-University, Magdeburg, Germany German Center for Neurodegenerative Diseases (DZNE) within the Helmholtz Association, Magdeburg, Germany
3
4
Clinic for Neurology, Hannover Medical School, Hannover, Germany Institute
of
Biometry
and
Medical
Informatics,
Otto-von-Guericke-University,
Magdeburg, Germany 5
Institute of Control Engineering, Technical University Braunschweig, Braunschweig, Germany
Corresponding author Stefanie Schreiber, M.D. Department of Neurology, Otto-von-Guericke University, Leipziger Strasse 44, 39120 Magdeburg [email protected] Telephone
+ 49 391 6713431
Fax
+ 49 391 6715233
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24431
Muscle & Nerve
Page 2 of 27
2 Acknowledgements. We would like to thank Anne-Katrin Baum for excellent technical support. This work was supported by a grant to S.V. from the Foundation of Medical Research, Frankfurt / Main, Germany. Disclosures. None. Key words ALS, PLS, median nerve, ulnar nerve, ultrasound Running title Ultrasound in ALS phenotypes
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3
Peripheral nerve ultrasound in ALS phenotypes Abstract Introduction. We sought to determine the cross sectional area (CSA) of peripheral nerves in patients with distinct subtypes of amyotrophic lateral sclerosis (ALS). Methods. Ulnar and median nerve ultrasound was performed in 78 ALS patients [classic, n=21, upper motor neuron dominant (UMND), n=14, lower motor neuron dominant (LMND), n=20, bulbar, n=15, primary lateral sclerosis (PLS) n=8] and 18 matched healthy controls. Results. Compared to controls ALS patients had significant, distally pronounced reductions of ulnar CSA (forearm/wrist level) across all disease groups except for PLS. Median nerve CSA (forearm/wrist level) did not differ between controls and ALS. Conclusion. Ulnar nerve ultrasound in ALS subgroups revealed significant differences in distal CSA values, which suggests it has value as a marker of LMN involvement. Its potential was particularly evident in UMND and PLS groups, which can be hard to separate clinically, yet their accurate separation has important prognostic implications.
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4 Introduction Amyotrophic lateral sclerosis (ALS) is defined by progressive weakness that results from neurodegeneration of upper (UMN) and lower motor neurons (LMN). The variable mix of UMN and LMN signs produces major phenotypic heterogeneity in ALS patients [1-3]. Thus, clinical manifestations of ALS exist on a continuum, ranging from apparently pure LMN dysfunction to severe pyramidal impairment with minor LMN signs [4]. Recently, there has been increasing acceptance of those distinct groups with upper versus lower motor neuron predominant pathology persisting through the course of the disease [5-7]. Briefly, classic ALS is characterized by the combination of LMN and pyramidal signs, upper motor neuron dominant (UMND) ALS by pyramidal signs (spinobulbar spasticity) associated with slight LMN dysfunction [8], lower motor neuron disease or syndrome (LMND) by absence of UMN signs, and progressive bulbar palsy (PBP) by dysarthria and dysphagia [9]. Exclusive UMN involvement with predominant spinobulbar spasticity is the hallmark of primary lateral sclerosis (PLS) [10-12]. Recognition of these variants can be relevant to counseling on prognosis, drug therapy, and tailoring care to the needs of the individual [13]. Neuromuscular ultrasound abnormalities may aid in the diagnosis of ALS [14;15] and also in distinguishing between ALS phenotypes (e.g. UMND versus PLS patients), which has important prognostic implications [6;8]. We hypothesized that categorizing ALS patients by their respective clinical syndromes [16] would reveal quantitative differences in sonographic parameters of the peripheral nerves. Methods Inclusion criteria of ALS patients and control subjects Seventy-eight consecutive patients with sporadic ALS were recruited from the Departments of Neurology at the Universities of Magdeburg and Hannover, Germany, with clinical follow-
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5 up intervals every 3 months. The study was approved by the local ethics committee (No. 150/09), and all subjects gave informed consent. The diagnosis of ALS was based on the revised El Escorial Criteria [17]. Patients with pure lower or upper motor syndromes or bulbar syndromes (who could not be classified in the revised El Escorial criteria) were classified into an additional category of suspected ALS. Patients with clinically obvious dementia at onset were excluded. If relevant, lumbar puncture and magnetic resonance imaging (MRI) of the brain and spinal cord were used to exclude other diagnoses. ALS phenotypes (diagnostic subgroups) were classified according to the operational definitions specified below as: classic (n=21), UMND (n=14), LMND (n=20), bulbar (n=15), or PLS (n=8). At the time of peripheral nerve ultrasound, a variable combination of UMN signs (spastic tone, clonus, etc.) and LMN signs (wasting, weakness, fasciculations) in the upper and lower limbs were found in those designated as classic ALS who, in turn, fulfilled the El Escorial criteria of definite or probable ALS. UMND ALS patients had either no LMN signs (weakness, wasting, or fasciculations), or, if present, (1) they were restricted to only 1 neuraxis level (bulbar, cervical, or lumbosacral), and (2) electromyography (EMG) abnormalities were limited to sparse fibrillation potentials/positive sharp waves or minor enlargement of motor unit potentials in 1 or at most 2 muscles [6;8] for at least 12 months after symptoms onset. The diagnostic criteria for PLS required a period of at least 4 years in which there were only UMN signs on examination [18]. The following were rigorously excluded from the PLS category: patients with clinical or electromyographic (EMG) signs of LMN involvement; those with a disease that could mimic motor neuron disease; a family history of spastic paraparesis/tetraparesis; mutations of genes related to hereditary spastic paraplegia (SPG3A, SPG4. SPG7, SPG10, and SPG11, [12]); and those with symptom onset before age 40 years. All patients with LMND had clinical and electrophysiological evidence of sporadic progressive pure LMN involvement in 1 or more regions without clinical signs of UMN dysfunction. To John Wiley & Sons, Inc.
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6 differentiate this condition from early limb onset ALS, we specified that LMN involvement must be the predominant finding for at least 12 months after the symptom onset. Other LMN diseases, such as multifocal motor neuropathy, spinal muscular atrophy, monomelic amyotrophy, Kennedy disease, and post-polio syndrome were excluded by extensive clinical and laboratory examinations [19;20]. In the PBP group, onset with lower motor neuron dysarthria followed by progressive speech and swallowing difficulties was the dominant clinical finding. To differentiate this condition from early bulbar onset ALS, e.g. we specified that bulbar involvement must be the predominant finding for at least 12 months after the symptom onset. If LMN signs were present, (1) they were restricted to only 1 neuraxis level (cervical, thoracic, or lumbosacral), and (2) EMG abnormalities were limited to sparse fibrillation potentials/positive sharp waves or minor enlargement of motor unit potentials in 1 or at most 2 muscles. Patients with clinical signs of UMN dysfunction were excluded from this category. Pseudobulbar (spastic) paralysis entails no atrophy or fasciculations of the paralyzed muscles, and patients with this condition were also excluded from the PBS category. Other conditions that mimic PBP, such as myasthenia gravis were excluded by appropriate investigations. All patients were graded using the revised ALS functional rating scale (ALSFRS-R) [21]. Disease duration was defined as time in months between symptom onset and the date of the evaluation. Disease-progression rate was calculated as (48 – ALSFRS-R)/disease duration [22]. Additionally, all subjects underwent standard electrophysiological measurements including the determination of distal motor latency (DML); motor and sensory conduction velocity (CV); compound motor action potential (CMAP) amplitude; sensory amplitude of the ulnar nerves with stimulation at the elbow and wrist and of the median nerves with stimulation in the forearm and wrist at 34°C. Based on electrophysiological features, ulnar nerve entrapment at the cubital tunnel (CuT) was confirmed in 8 ALS patients and 3 controls, and median nerve
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7 entrapment at the carpal tunnel was found in 10 ALS patients and 1 control; ultrasound data of these affected ulnar and median nerves were excluded from statistical analysis. The control group consisted of 18 healthy volunteers recruited from the general population via announcement. All participants were free of uncontrolled diabetes mellitus, arterial hypertension, neuromuscular symptoms (e.g., numbness and tingling or weakness), traumarelated peripheral nerve disease, and alcohol dependence syndrome according to International Classification of Diseases (ICD)-10 diagnostic criteria. All had a normal neurological examination. Specifically, an individual who reported symptoms suggesting carpal tunnel syndrome (e.g. hand pain/paresthesia with nocturnal or use-related exacerbation) or ulnar neuropathy (e.g. hand pain/paresthesia, elbow injury, elbow pain) was excluded. As in former peripheral nerve ultrasound studies [14;23-28] diabetes status and vitamin B deficiencies were not sought explicitly or used as exclusion criteria. The control subjects had normal nutritional status (Table 1) and took part in regular preventive medical check-ups in collaboration with health insurance funds, which have specific objectives for identification and treatment of patients with cardiovascular diseases, metabolic disorders, and nutritional disturbances [29]. Ultrasound Ultrasound examinations were performed using a GE High-End LOGIQ®7 System with a 12MHz linear array probe. All subjects were seated with the entire arm extended anteriorly, supinated, and supported by a pillow on a table at approximately mid-thoracic height [30]. The ulnar and median nerves [29;31;32] were identified by their location and morphology [3335] and visualized over the whole distance from the distal wrist crease to the CuT (ulnar nerve) and from the distal wrist crease to the middle third of the forearm (FA, median nerve). As established [29;31;32] transverse images were obtained bilaterally at the CuT (ulnar nerve), the middle third of the FA (ulnar and median nerves), and at the level of the distal wrist crease (ulnar and median nerves [27;29;35-39]). At all sites, the nerve cross sectional area (CSA) was calculated by continuous manual tracing of nerve circumference, excluding
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8 the hyperechoic epineural rim [24]. The ratio between ulnar nerve CSA at the CuT and wrist (ulnar nerve CuT/wrist ratio) and between the median nerve CSA at the FA and wrist (median nerve FA/wrist ratio) were calculated. Three separate CSA measurements were averaged at each nerve site. Sonography images were acquired by a single investigator (S.S.) who was not blinded to diagnosis but to the disease subtypes [29]. Statistics To assess the quality of group matching for gender we used a chi-square test (comparing controls and all patients) and a generalized linear model with subsequent adjustment for multiple comparisons (comparing all diagnostic subgroups). For age, height, weight, disease duration, ALSFRS-R, and disease-progression rate we used a Mann-Whitney-U test (comparing controls and all patients) and a Kruskal-Wallis test (comparing all diagnostic subgroups). A paired t-test was performed to assess possible CSA differences between the right and left upper limb. Tests of between-subjects effects in ANOVA for repeated measures revealed dependency of sonographic data on height, disease duration, and phenotype (diagnostic subgroup). Subsequent comparisons of each ultrasound measurement between all diagnostic subgroups (controls, PLS, UMND, LMND, classic and bulbar phenotype) and ulnar nerve measurements between the disease-onset subtypes (bulbar, limb) and the affected side [right upper limb, left upper limb, equally affected upper limb(s)] using univariate analysis of covariance (ANCOVA) were performed with sonographic parameters (CSA) as dependent variable, height and disease duration as covariates, and phenotype (subgroups or disease-onset subtypes or affected side) as fixed variable; a Bonferroni adjustment for multiple comparisons was applied. CuT/wrist and FA/wrist ratio comparisons between the diagnostic subgroups and controls were performed using a Kruskal-Wallis test adjusted for multiple comparisons. Pearson correlation coefficients were used to assess the associations between the ulnar ultrasound data, clinical outcome measures (e.g. ALSFRS-R), and between ulnar and median nerve CSA and the abductor pollicis brevis muscle
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9 (APB)/abductor digiti minimi muscle (ADM) CMAP amplitude ratio (see below). P-values ≤ 0.05 were deemed to be statistically significant.
Data from all patients (n=78) and controls (n=18) were used to calculate a Cronbach α in order to assess the reliability of repeated CSA measurements. The respective reliabilities of ulnar and median nerve CSA measurements were as follows: patients, 0.984/0.991 (wrist), 0.952/0.954 (FA), 0.991 (CuT), and controls, 0.996/0.981 (wrist), 0.965/0.965 (FA), 0.995 (CuT).
Results Subjects Clinical and demographic data are summarized in Table 1. There were no significant group differences for gender, height and weight between the controls and the whole patient cohort (Table 1) and between the controls and the different diagnostic subgroups (data not shown in detail). The age distribution was comparable between the controls and the whole patient cohort (Table 1) and between the controls and all diagnostic subgroups. The ALSFRS-R score did not differ between diagnostic subgroups (Table 1). As expected, disease duration and disease-progression rate differed significantly between the subgroups in that PLS patients had the longest disease duration and the slowest disease-progression rate (Table 1). Ultrasound data Compared to controls, ulnar nerve CSA at the wrist and FA was reduced significantly in all ALS diagnostic subgroups except for PLS (Figures 1 & 2, Table 2). The FA and wrist ulnar nerve mean differences ranged from 1.66 (FA left, LMND) to 3.02 (FA right, UMND) between the controls and the different ALS subgroups (UMND, LMND, classic and bulbar phenotype) John Wiley & Sons, Inc.
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10 and from 0.43 (FA left) to 1.20 (, FA right) between controls and the PLS subgroup. Ulnar nerve CSA at the level of CuT and median nerve CSA at wrist and FA did not differ between any of the diagnostic subgroups (Table 2). Consequently, in the ALS subgroups (UMND, LMND, classic, and bulbar phenotype) the CuT/wrist ratio was increased significantly compared to the controls, while it did not differ between the PLS subgroup and the control subjects (Table 3). Median nerve FA/wrist ratios did not differ between any of the diagnostic subgroups (PLS, UMND, LMND, classic and bulbar phenotype) and the control subjects (data not shown). No consistent correlations were found in either the total ALS cohort or in the subgroups between any ulnar nerve CSA measurement and clinical outcome measures, e.g. ALSFRSR, disease-progression rate, or electrophysiological measures [e.g. compound muscle action potential (CMAP) of the thenar (for the median nerve) and hypothenar (for the ulnar nerve)].
Additional statistical analysis with respect to further clinical classifications [disease-onset subtype (bulbar, limb), side of most clinically affected upper limb] including all subgroups (PLS, UMND, LMND, classic and bulbar phenotype) revealed no consistent effects on the ulnar nerve sonographic data. Interestingly, in ALS patients (non-PLS variants) who had asymmetric upper limb involvement (present in n = 31), ulnar nerve CSA did not differ between the right and left upper limb and ulnar nerve CSA of the clinically less affected (n = 24) or unaffected upper limb (n = 7) was likewise significantly smaller compared to the control values (data not shown).
Discussion Stratifying ALS patients into clinical subgroups revealed small but significant reductions in ulnar nerve CSA at the wrist and FA in all groups with the exception of PLS. Although the PLS group was the smallest, the lack of statistically significant effect is unlikely to reflect lack of power, because the mean differences in PLS were also considerably less than those seen
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11 for the other groups. No significant changes were found in ulnar nerve CSA at the CuT level and median nerve CSA for any group.
Control CSA values of the ulnar and median nerves were in line with those of previous studies [29;31;32]. Reliability of CSA measurements was high, which was in keeping with previous reports applying the same method [25;29]. As in former studies, CSA values are influenced by height [28;31;36], but not by weight [36].
The absence of median nerve CSA reductions differs from the only previous ALS nerve ultrasound study, which revealed a significant reduction of median nerve CSA [14]. In that study, however, the median nerve CSA was measured at a more proximal site between the medial epicondyle and the axilla [14]. At first glance, our data seem to be also incompatible with a recent EMG study describing a preferential involvement of median nerve innervated muscles (part of C8/Th1 innervation pattern) in ALS [40]. We speculate that these findings can be reconciled in a model that considers the relative proportion of motor and sensory fibers along the nerve course and between different nerves. The internal grouping and proportion of motor and sensory branches of the median nerve differs from that of the ulnar nerve [41;42]. Results should, therefore, be influenced by both varying proportions of sensory axons in the 2 nerves and by the anatomic location studied along the course of each nerve. Indeed, compared to proximal sections, the distal ulnar nerve predominantly consists of motor branches, whereas the median nerve is a mixed nerve, especially in its more distal sections. This could explain both the circumscribed distal ulnar CSA reduction and also the preserved ulnar nerve CSA at the more proximal site of the CuT. Furthermore, this model would predict that median nerve CSA reductions would be more evident in the upper arm, where the nerve still contains the motor axons that will supply the forearm muscles. Finally, the model is compatible with past EMG findings of severe distal median denervation [43-45] if the proportion of motor axons in the distal median nerve are relatively fewer than in the
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12 ulnar nerve but these same median motor fibers are severely affected, one would expect little impact on CSA but profound changes on EMG. The absence of correlations with clinical measures such as the ALSFRS-R might be due to the fact that the sonographic data set was not distributed adequately in and between the subgroups to result in rigorous correlations. It is not surprising, though, that no clear relationship is found when one considers that: (1) the ALSFRS-R is a composite score from 3 different domains (bulbar, motor, and respiratory function) and therefore unlikely to reflect changes sensitively in a specific peripheral nerve; and (2) that clinical heterogeneity across the different sub-syndromes of ALS means that similar ALSFRS-R scores can be generated in spite of major between-subject differences in the degree of UMN and LMN involvement. A more direct issue is reconciling these ultrasound findings to the dissociated atrophy of the intrinsic hand muscles, the ’split hand’. In ALS, the APB usually shows a greater reduction of motor unit number estimates compared to the ADM which is reflected in the APB/ADM CMAP amplitude ratio (< 0.6, [40;46;47]). No significant correlations could be detected between the APB/ADM ratio and the corresponding ulnar and median nerve CSA. A split hand according to the above mentioned electrophysiological criteria was found in 40% of our ALS patients, which is in line with findings of other groups [47]. From this, and the fact that the degree of atrophy of muscles is usually not symmetric in ALS, one would also expect more pronounced axonal loss and subsequent nerve caliber reduction in the more severely atrophic upper limb. We could not, however, find any significant ulnar nerve CSA asymmetries in those ALS subjects (n = 31) that exhibited clinically asymmetric upper limb involvement. When EMG changes were considered by region, there was a relatively uniform detection rate of neurogenic abnormalities including limbs without apparent clinical weakness [48], which is consistent with our sonographic findings. In any case, the model proposed above can accommodate the split hand phenomenon by considering that ultrasound could be insensitive to significant distal median nerve motor fiber loss.
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13 Our data cannot answer the question of whether ALS and PLS are distinct disorders [10;49;50], but they at least allow us to state that the sonographic appearance of the ulnar nerve in our PLS group was similar to that of the controls. The distinction between ALS and PLS relates primarily to the degree of lower motor neuron involvement. Indeed, the fact that the UMND variant was indistinguishable from the other non-PLS variants in this sonographic study. This indicates probable motor axonal loss as a pathological substrate for ulnar nerve CSA reduction and therefore common LMN involvement, at least in the chosen ALS subgroup classification. Morphometric analysis of peripheral nerves in ALS showed progressive reduction of large myelinated fibers [51] but also a transient increase of small myelinated fibers suggestive of reinnervation [52]. Overall, peripheral nerves in ALS become progressively thinner [53]. This is an area that should be explored in more detail in future ultrasound studies with additional measurement of individual nerve fascicles to permit more definitive conclusions.
In conclusion, the findings of this study suggest that ultrasound as a quantitative measure in conjunction with other systematic investigations such as electrophysiological studies may offer promise in distinguishing patients with pure UMN involvement from those in whom UMN is the predominant but not exclusive manifestation. Prospective studies will be needed to confirm this finding as well as to determine whether other disease subgroup classifications (e.g. according to genotype, myotomes affected, rate of progression) would result in a different distribution of sonographic data. Nevertheless, we believe that even without splitting the patients into further subcategories such as flail arm and flail leg variants or typical ALS with arm-onset and leg-onset, our findings serve as a good working model to advance this field and to lead to further investigations using larger sample sizes, including such subcategories and perhaps genetically defined ALS variants. Further studies could also focus on the relationship between peripheral biomarkers such as nerve CSA and central biomarkers such as diffusion tensor imaging of the corticospinal tract.
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14
Gender
Controls
PLS
UMND
LMND
Classic
Bulbar
P
14 m, 4w
5 m, 3 w
11 m, 3 w
14 m, 6 w
12 m, 9 w
6 m, 9 w
0.217
Age
63.8
(9.5)
63.5 (16.8) 54.1
(8.3)
59.2 (14.5) 58.5 (12.1) 66.1 (11.4)
0.093
Height
174.0 (8.7) 172.7 (13.2) 171.0 (8.3) 173.5 (9.5) 171.1 (8.5) 169.4 (10.2)
0.734
Weight
81.0 (12.7) 75.0 (17.2) 79.4 (16.5) 79.4 (16.7) 70.9 (10.1) 70.3 (14.9)
0.119
Disease duration
n.a.
104.1 (77.7) 32.8 (22.4) 37.0 (26.0) 20.9 (12.8) 15.7
(5.7)
