Peripheral Nerve Ultrasonography in Chronic Inflammatory Demyelinating Polyradiculoneuropathy and Multifocal Motor Neuropathy: Correlations with Clinical and Neurophysiological Data

Objective. This cross-sectional study analyzes the pattern of ultrasound peripheral nerve alterations in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN) at different stages of functional disability. Material and Methods. 22 CIDP and 10 MMN patients and a group of 70 healthy controls were evaluated with an ultrasound scan of the median, ulnar, peroneal, tibial, and sural nerves. Results were correlated with clinical disability scales and nerve conduction studies. Results. Patients with intermediate functional impairment showed relatively larger cross-sectional areas than subjects with either a milder (p < 0.05) or more severe impairment (p < 0.05), both in CIDP and in MMN. In addition, MMN was associated with greater side-to-side intranerve variability (p < 0.05), while higher cross-sectional areas were observed in CIDP (p < 0.05) and in nerve segments with predominantly demyelinating features (p < 0.05). Higher CSA values were observed in nerves with demyelinating features versus axonal damage (p < 0.05 for CIDP; p < 0.05 for MMN). Discussion and Conclusions. Greater extent of quantitative and qualitative US alterations was observed in patients at intermediate versus higher functional disability and in nerves with demyelinating versus axonal damage. CIDP and MMN showed differential US aspects, with greater side-to-side intranerve variability in MMN and higher cross-sectional areas in CIDP.


Introduction
Multifocal motor neuropathy (MMN) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are acquired immune-mediated peripheral neuropathies (PN). MMN is a pure motor neuropathy syndrome usually beginning in one or both hands and principally affecting the upper extremities, characterized by a chronic or stepwise progressive asymmetrical limb weakness and muscle atrophy [1]. CIDP is an immune-mediated peripheral neuropathy that may cause weakness, paralysis, and/or impairment in both motor and sensory functions, usually affecting both sides of the body (symmetrical) [2].
The neurophysiological hallmark of MMN is conduction blocks (CB) outside the usual sites of nerve compression [1,3], while CIDP typical features are CB, slowed motor, and sensory nerves conduction velocities and prolonged distal latencies [4]. In addition, both diseases may present a variable extent of axonal loss [5,6], which has been attributed both to recurrent demyelinating insults and intrinsic pathogenic features, especially in the case of MMN [1].
Neuromuscular ultrasound (US) is a noninvasive, painless, and radiation-free complementary imaging technique for the diagnostic work-up of PN [7,8]. Focal nerve enlargements can be observed in the majority of MMN patients and generalized nerve enlargements can be observed in CIDP patients, interestingly showing alterations also in limbs without signs of neurophysiological dysfunction [9][10][11]. However, the correlation between US, neurophysiological findings, and functional disability is still partially controversial [12][13][14]. Some authors have found an association between disease duration and the extent of nerve enlargement [15], while 2 Neurology Research International others have suggested a specific US pattern in relation to different mechanisms of injury [13]: demyelinating insults might result in swollen, enlarged, and hypoechoic nerves, while axonal damage may be characterized by hyperechoic atrophic bundles of fascicles. In addition, a variable combination of axonal and demyelinating insults may also coexist, resulting in hyperechoic and hypertrophic nerves. Most of literature data have been collected on CIDP patients, while a few sonographic-clinical-electrophysiological studies have been currently reported in MMN [9,11,[16][17][18].
The aim of this study is to analyze US findings in patients with CIDP and MMN at different functional disability, in order to correlate US qualitative and quantitative measures with clinical and neurophysiological features.  [3,19] and, at the time of US examination, were receiving a monthly treatment with intravenous immunoglobulin (IVIg) (1-2 g/kg/month) for at least 6 months. Written informed consent and local ethical committee (AOU San Giovanni Battista di Torino) approval were obtained. The authors acted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

Clinical Assessment.
A complete neurological examination was performed by means of the inflammatory neuropathy cause and treatment (INCAT) disability scale [20], the Medical Research Council (MRC) score in 8 muscle groups bilaterally (shoulder abduction, elbow flexion, wrist flexion, first dorsal interosseous, hip flexion, knee extension, and ankle flexion/extension), and the Overall Neuropathy Limitation Scale (ONLS).
According to the INCAT disability scale, upper limb activities were scored as "no/minimal impairment" (not affected), "moderate impairment" (affected but not prevented), or "severe impairment" (prevented), while the evaluation of walking difficulties was based on the use of aids: "no/minimal impairment" (no/minimal gait impairment); "moderate impairment" (moderate gait impairment, independent or with unilateral support); "severe impairment" (severe gait impairment, bilateral supports or wheelchair).

US Assessment.
The US assessment was performed by means of a SonoSite M-Turbo Ultrasound Machine equipped with a broadband linear transducer (frequency band 6-15 MHz). The US scan was performed the same day of the neurological assessment by an evaluator (Michela Rosso), who was blinded to the clinical and neurophysiological data.
The following quantitative and qualitative US parameters were collected for the median, ulnar, peroneal, tibial, and sural nerves bilaterally.
Nerve Cross-Sectional Area (CSA). CSA was measured by tracing the nerve just inside the hyperechoic rims with the "ellipse method" when applicable (when the nerve in the transverse scan had an elliptical or roundish shape) or alternatively tracing the nerve shape (when the nerve had an irregular shape) [13]. CSA was evaluated for each nerve at predetermined sites: median nerve was evaluated at wrist (entrance of carpal tunnel), middle third of the forearm, elbow (before penetrating the pronator teres muscle next to the brachial artery), and middle third of the arm (middle of the distance between medial epicondyle and axillary fossa); ulnar nerve at wrist (Guyon's canal), middle third of the forearm, elbow (between medial epicondyle and olecranon), and middle third of the arm (middle of the distance between medial epicondyle and axillary fossa); peroneal nerve at the fibular head and popliteal fossa; tibial nerve at popliteal fossa and at medial malleolus before its division into plantar nerves (ankle); sural nerve at the ankle. Maximal CSA (CSA max ) enlargement was recorded for each nerve; median and ulnar nerves were scanned along the entire viewable tract, from the wrist to the middle third of the arm. Abnormal CSA values at entrapment sites (wrist, elbow, and fibular head) were excluded to avoid confounding local neuropathies.
Intranerve CSA Variability. Intranerve CSA variability was calculated as the ratio between CSA max /CSA min , for each nerve (available for median and ulnar nerves) [21].
Side-to-Side Intranerve Variability. Side-to-side intranerve variability was calculated as the "side-to-side" ratio of the intranerve CSA variability [12].
Qualitative Analysis of Nerve Fascicles. Nerves were classified as abnormal if ≥ 3 fascicles showed a cross-sectional area ≥ 2 mm 2 , regardless of the CSA value [22].
Normative US reference values were obtained by the assessment of healthy controls (Table 1), considering the upper threshold of the normality range (UT) to be the average value + 2 standard deviations. Then, in order to compare the CSA of different nerves taking into account their relative normative values, a normalized CSA (CSA NORM ) was calculated by dividing the CSA max of each nerve to the corresponding UT value (CSA NORM = CSA max /CSA UT ).

Nerve Conduction Studies.
Nerve conduction studies were performed by means of a KeyPoint (Natus Medical Incorporated, San Carlos, CA, USA) electromyography (EMG) machine by evaluators blinded to the US study, assessing the bilateral peroneal, tibial, ulnar, and median motor nerves and the bilateral sural, median, and ulnar sensory nerves. Nerve conduction velocities (CV), compound muscle action potentials (CMAP), and sensory action potentials (SNAP) were collected and compared to the normality cutoff values of our laboratory; all patients were checked for skin temperature with a probe on the EMG machine. If needed, the body temperature was maintained above +34 ∘ C by means of an infrared lamp. CB was defined in accordance with the EFNS/PNS criteria [3,19], excluding possible sites of entrapment (wrist, elbow, and fibular head) to avoid confounding focal neuropathies. Moreover, neurophysiological alterations of nerve segments were stratified in predominantly "myelin damage" or "axonal damage" in accordance with the classification already proposed by Di Pasquale et al. [22].

Statistical Analysis.
Descriptive statistics (mean, standard deviation, and range) were used for continuous variables. Mann-Whitney and Cramer's tests were used for comparison between patients with different disease severity and neurophysiological alterations and between CIDP and MMN patients. Kruskal-Wallis test was applied for comparison among groups. Spearman's rho, Kendall's tau-b, and Pearson's correlations were used to estimate correlations between clinical, US, and electrophysiological characteristics, while a linear regression model was applied to estimate the influence of age, disease and treatment duration, and IVIg doses on CSA values. Bonferroni's correction for multiple comparisons was applied to avoid statistical biases of repeated testing effects. The average CSA values in bilaterally measured nerves were obtained pooling together data of the two sides. However, in order to take into account the asymmetrical involvement typical of MMN, we also considered the side-toside intranerve variability, calculated by dividing the intranerve variability of the most affected side with the intranerve variability of the less affected side. All values reported are two-tailed, considering 0.05 as statistical threshold. Analyses were performed with SPSS Statistics 21.0 for Mac.

Discussion
This study reports the peripheral nerve US findings of 32 CIDP and MMN patients at different functional disabilities. Lower CSA values were associated with more severe clinical alterations and/or axonal damage, while higher CSA values were associated with intermediate functional disability (clinical alterations without loss of functionality) and/or demyelinating damage.
These data are in accordance with the findings reported by Di Pasquale et al. [22], who observed that nerve segments characterized by predominantly myelin damage had greater CSA than nerves with predominantly axonal damage and normal nerves, which virtually overlapped.
In addition, we found some differential aspects between MMN and CIDP: greater side-to-side intranerve variability was observed in MMN, in line with the pattern of heterogeneous and multifocal involvement characteristic of the disease; patients with CIDP showed higher CSA values, potentially indicating more prominent demyelinating processes; qualitative US analyses revealed a different distribution  of abnormal fascicles in the upper and lower limbs, with more prominent US alterations in district affected by predominantly demyelinating damage (frequently associated with a less marked functional impairment) compared to district affected by secondary axonal degeneration. The majority of literature data reported increased CSA values in CIDP, with a possible association between intranerve variability and functional scores [23,24]. Less data are available for MMN, where asymmetric and focal CSA enlargements have also been reported in nerves without neurophysiological alterations, suggesting disease processes that could extend beyond the sensitivity of standard diagnostic techniques [9,11,[16][17][18]. Several complex phenomena, such as segmental demyelination, proliferation of Schwann cells in response to repeated inflammatory insults, onion bulbs formation, and a variable degree of axonal loss might underlie these US findings [22,25,26]. However, their correlation with the mechanisms of nerve damage and repair still remains to be clarified.
Our data support the complementary role of US in the assessment of CIDP and MMN, suggesting a different pattern in nerves with demyelinating versus axonal damage and in CIDP versus MMN patients, in possible relationship with the different pathological processes involved.
Previous studies reported a correlation between disease duration and CSA values [14,22,27], while in our heterogeneous sample of patients we observed a "U-shaped" relationship between CSA values and functional impairment. We speculate that different disease phases might be associated with different US patterns, with an initial/intermediate phase of inflammation and myelin damage, characterized by increased CSA and enlarged swollen fascicles and a 8 Neurology Research International late phase of severe axonal degeneration, characterized by small atrophic fascicles, reduced CSA and greater functional impairment.
Other factors, such as IVIg pharmacological treatment and/or individual inflammatory response, might also be implicated in the morphological modifications of peripheral nerves [14]. However, the similar therapeutic regimen (IVIg) administered to our patients did not allow a post hoc analysis of treatment effects on CSA values. Finally, patients with CIDP, characterized by more prominent inflammatory and demyelinating features, might display greater nerve enlargement compared to MMN or to peripheral neuropathies characterized by primary axonal degeneration (i.e., chronic idiopathic axonal polyneuropathy).
Taken together, these findings suggest variable applications for US in the field of immune-mediated peripheral neuropathies, ranging from the more accurate clinicopathophysiologic phenotyping to the early detection of morphological changes associated with critical disease milestones. In addition innovative US score, such as the Bochum Ultrasound Score [10], will likely allow a more accurate differentiation between CIDP and other acquired or inherited peripheral neuropathies. However, US examinations require adequate training and experience to obtain reliable results.

Conclusions
Our findings suggest that CIDP and MMN patients with an intermediate functional disability may present more pronounced quantitative and qualitative US alterations than patients with higher disability. Moreover, some differential aspects can be recognized in CIDP versus MMN and greater US alterations might be observed in nerve segments with demyelinating versus axonal damage.
The strength of our findings is partially limited by the relatively small sample size and the lack of serial prospective follow-up assessments. In addition, two aspects should be considered in the interpretation of data: (a) the "U-shaped" relationship between US findings and functional impairment, which might result in a similar US pattern in patients with either minimal or severe disability; (b) the association of CSA reduction with two different factors (axonal damage and functional impairment), indicating the need of further prospective studies to analyze which of the two features primarily correlates with nerve size reduction.