We acknowledge the support from Ruhr-University Bochum for our research on neuromuscular ultrasound.

All examinations for this work were performed in compliance with institutional guidelines of the Ruhr-University Bochum, Germany.
1. Experimental preparations
<ol>
	<li>Patient preparation
	<ol>
		<li>Check the patient inclusion criteria: examine adult patients diagnosed with polyneuropathy, suspicious of inflammatory origin.</li>
		<li>Check the patient exclusion criteria: do not examine patients with open wounds or infections in the regions to be examined.</li>
	</ol>
	</li>
	<li>Instrumental checkpoints
	<ol>
		<li>Check the integrity of the ultrasound machine and all the materials used (see Table of Materials).</li>
		<li>Enter patient name and details in the ultrasound machine before starting the ultrasound examination (depending on the machine).</li>
		<li>Choose an appropriate ultrasound probe (preferred 14-18 MHz) (see Table of Materials) and preset for the neuromuscular ultrasound.</li>
		<li>During the whole examination, adjust the depth and focus on obtaining optimal image quality.</li>
		<li>Whenever possible, examine the complete course of each nerve in a cross-sectional view. 
		NOTE: The nerves recommended for examination are: median, ulnar, radial nerve, cervical roots, brachial plexus, and vagal nerve, as well as tibial, fibular, and sural nerve (Figure 3). The examination of each of these nerves is shown in the next section and the video. The entire ultrasound examination according to the following protocol will take ~30-45 min.</li>
	</ol>
	</li>
</ol>
2. Ultrasound examination
<ol>
	<li>Start examining the arm nerves with the patient sitting in a neutral position with the arm resting supinated on a surface, e.g., the leg.</li>
	<li>Place some ultrasound gel over the transducer probe, the wrist, the forearm, the elbow, and the upper arm.</li>
	<li>For examination of the median nerve, start by performing a transverse scan at the wrist level.</li>
	<li>Move proximally to follow the anatomical course of the median nerve to the upper arm.</li>
	<li>Measure the CSA of the median nerve at the following sites: at the entrance to the carpal tunnel (retinaculum flexorum); at the forearm (10-15 cm proximal to retinaculum flexorum; at the elbow (crook of the elbow); at the upper arm next to the brachial artery (at the middle of the distance between the medial epicondyle and axillary fossa).</li>
	<li>For examination of the ulnar nerve, start by performing a transverse scan at the level of the wrist ulnar to the median nerve.</li>
	<li>Move proximally to follow the anatomical course of the ulnar nerve along the sulcus to the upper arm. 
	NOTE: Moving toward the upper arm, let the patient raise the arm bent at the elbow to examine the sulcus and upper arm.</li>
	<li>Measure the CSA of the ulnar nerve at the following sites: at the entrance to Guyon&#39;s canal; at the forearm (10-15 cm proximal to Guyon&#39;s canal); at the elbow (between the medial epicondyle and olecranon); at the upper arm (at the middle of the distance between the medial epicondyle and axillary fossa).</li>
	<li>To examine the radial nerve, let the patient hold the arm in front of the stomach bent in the elbow and scan the radial nerve directly next to the humerus.</li>
	<li>Use color duplex mode to avoid confusion with the accompanying artery and vein. 
	NOTE: Color duplex mode shows blood flow in arteria profunda brachii, and might show low flow in the corresponding vein, while no flow occurs in the radial nerve. Additionally, the vein can be compressed by exerting external pressure, and the nerve cannot.</li>
	<li>Measure the CSA of the radial nerve at the following site: radial nerve in the spiral groove.</li>
	<li>Carry on with the examination of the vagal nerve, cervical nerve roots, and the brachial plexus.</li>
	<li>Place ultrasound gel at the middle of the neck.</li>
	<li>To examine the vagal nerve, perform a transverse scan at the middle of the neck and find the carotid artery. 
	NOTE: The vagal nerve can be found directly next to the carotid artery and jugular vein.</li>
	<li>Measure the CSA of the vagal nerve at the following site: at carotid sheath at the level of the carotid bifurcation.</li>
	<li>For the examination of cervical nerve roots, C5, C6, C7 move the probe dorsal and a little up and down. 
	NOTE: The cervical nerve roots appear between the anterior and posterior tubercle of the transverse process. C7 can be recognized by the absence of the anterior tubercle from its transverse process, while both anterior and posterior tubercles are found with the other cervical nerve roots.</li>
	<li>Measure either the CSA or the diameter of the cervical nerve roots at the most proximal location possible, where the nerve root exits over the transverse process: C5; C6; C7.</li>
	<li>To examine the brachial plexus, follow the anatomical course of the cervical nerve roots distally and find them perform trunks and cords.</li>
	<li>Measure the CSA of the plexus at the following sites: Intrascalene space (between anterior and medial scalene muscle); Supraclavicular space (next to A. subclavia).</li>
	<li>Carry on with the examination of the leg nerves.</li>
	<li>Let the patient lie down to one side with the legs slightly bent. Place some ultrasound gel over the transducer probe, the popliteal fossa, the fibula, the malleolus, and the lower leg.</li>
	<li>For examination of the fibular nerve, feel the fibular head, place the transducer directly behind it, and then follow the course of the nerve to the popliteal fossa.</li>
	<li>Measure the CSA of the fibular nerve at the following sites: just proximal to the &#64257;bular head; in the popliteal fossa.</li>
	<li>To examine the tibial nerve in the popliteal fossa, find the fibular nerve and the popliteal artery in the popliteal fossa. 
	NOTE: The tibial nerve can be found just above the popliteal artery in most cases.</li>
	<li>Measure the CSA of the tibial nerve at the following site: in the popliteal fossa.</li>
	<li>For examination of the tibial nerve at the ankle, place the probe directly behind the medial malleolus. 
	NOTE: The tibial nerve can be found just next to the posterior tibial artery in most cases.</li>
	<li>Measure the CSA of the tibial nerve at the following site: at the level of the medial ankle.</li>
	<li>For examination of the sural nerve, place the probe at the lateral ankle. 
	NOTE: The sural nerve can be found next to a superficial vein in most cases.</li>
	<li>Follow the anatomical course of the sural nerve proximally to the lower leg.</li>
	<li>Measure the CSA of the sural nerve at the following site: between the lateral and medial head of the gastrocnemius muscle.</li>
	<li>Perform all measurements on both sides.</li>
	<li>Save the results of all the measurements (depending on the ultrasound machine) and end the examination. 
	NOTE: Figure 3 gives an overview of all measuring sites for CSA.</li>
</ol>

The authors declare no conflicts of interest related to this manuscript.

Nerve ultrasound is a helpful additional diagnostic tool in polyneuropathies. It can give information on the possible causes of polyneuropathy depending on the extent and pattern of nerve enlargement. Moreover, CSA alterations in the longitudinal disease course of patients with CIDP were described to correlate to clinical disease course and treatment response33,34,35,36.
Critical steps within the protocol 
For obtaining reproducible results, consistent methodology and standardization of the examinations are essential37,38. Each examiner must consider deviations resulting from different ultrasound devices and local differences of demographics. To ensure the high quality and reproducibility of ultrasound measurements, specific training of the examiner is also necessary13,14,15,21,39.
Modifications and troubleshooting of the technique 
Typical findings in demyelinating inflammatory neuropathies are multifocal nerve swellings with inhomogeneous fascicles33,40. Therefore, the measurement of CSA of specific nerve sites is necessary, but the whole nerve must be scanned. Also, evaluation of fascicular structure and echogenicity can help in inconclusive cases, as not only CSA increase of the entire nerve but also intrafascicular swellings as well as hypoechoic and hyperechoic nerves are found in CIDP. Hypoechoic nerves are considered to result from acute edema, while hyperechoic nerve result from fibrous remodeling40,41.
Limitations of the technique 
There are anatomical limitations for nerve ultrasound, i.e., examination of cervical nerve roots can be difficult to impossible in patients with obesity and short neck. Also, imaging of proximal nerve roots of the lower extremity nerves of the lumbosacral plexus is not possible due to the limited penetration depth of the ultrasonic rays. An alternative method, evaluating these nerves, is possible by MRN42, but ultrasound is the more common method due to its spatial and temporal flexibility and cost-effective use43.
Significance with respect to existing methods 
Nerve ultrasound is recommended as an additional and complementary tool in diagnosing polyneuropathies to evaluate nerve morphology. Standard diagnostic workouts, including nerve conduction studies and other tools such as cerebrospinal fluid analysis, should still be performed.
Future applications of the technique 
For experts in polyneuropathies, nerve ultrasound is of interest for diagnosis in clinical routine and as it can give insights into possible pathophysiologic aspects, i.e., represent inflammation. Therefore, nerve ultrasound is a promising method not only in clinical use but also in neuromuscular research. Also, with increasing progress in ultrasound technology, future ultrasound characteristics such as shear wave elastography or vascularization of peripheral nerves may add further aspects in assessing polyneuropathies.

Next to clinical examination, evaluating any large-fiber polyneuropathy includes an electrophysiological examination to characterize the motor or sensory system&#39;s involvement and differentiate axonal from demyelinating damage1. In axonal polyneuropathy, toxic and diabetic neuropathy are the leading causes, while in demyelinating polyneuropathies, hereditary or inflammatory neuropathies such as CIDP should be considered2,3,4. Commonly used diagnostic criteria for CIDP are the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) criteria established in 2005 and revised in 2010 and 20215. These define clinical and electrophysiological criteria to diagnose CIDP and describe additional criteria such as nerve biopsy to detect demyelination or inflammation. However, in some cases, despite a thorough diagnostic workup, the cause of neuropathy remains ambiguous. In these cases, nerve ultrasound offers a complementary method to examine the nerves not functionally but morphologically6. Several studies proved the use of nerve ultrasound as an additional tool in diagnosing CIDP, so that the 2021 revised EFNS/PNS criteria implemented nerve ultrasound in the guideline5. The advantage of nerve ultrasound compared to other imaging methods such as magnet resonance neurography (MRN) is that it can be used directly by the treating neurologists as a bedside tool; it is relatively cost-efficient. It can be used repeatedly, as it is noninvasive and not painful.
Typical characteristics of CIDP observed in nerve ultrasound are cross-sectional-area (CSA) increase7,8, also found in hereditary polyneuropathies. In CIDP, this affects individual nerve segments heterogeneously7,9.
A variety of examination protocols have been published10,11,12,13,14,15&#160;trying to clarify normal CSA values and determine the adequate anatomical positions of ultrasound examination. Some of these positions are similar in most examination protocols. However, a widely accepted protocol to standardize the examination process and simplify the interpretation of the measurements does not exist.
This article demonstrates the nerve ultrasound examination using a standardized protocol for polyneuropathies, presents various reference values for the CSA, and shows typical pathological findings in patients with inflammatory neuropathies.
Technical requirements for nerve ultrasound 
The neuromuscular ultrasound is performed in B-mode (Brightness mode, two-dimensional image with gray levels) using the compound imaging of the corresponding sonographic device6,16. Compound imaging enables electronic control of the piezoelectric elements in the sonic probe (transducer) to illuminate the target structure from different angles17. The ultrasound waves are reflected in several directions due to the histological structure of the peripheral nerves. As a result of the sound coming from different angles, a more significant part of the otherwise lost reflections gets back to the sound probe (receiver) and can generate images. For neuromuscular ultrasound, a high-resolution ultrasound probe with 18 MHz linear array transducer, for deeper nerves, an additional 12 MHz linear array probe (e.g., to display tibial and fibular nerve in the popliteal fossa) is used6,16. Transducers with lower frequencies result in reduced spatial and lateral resolution so that the differentiation of the nerve boundaries from the surrounding structures is less precise. The optimal settings can be kept constant using a preset for neuromuscular imaging provided by the manufacturer. During the examination, the image depth and the focus position must be adjusted to the structure to be examined and constantly adapted to the position of the nerve. The B-image gain and the depth-dependent gain can be adjusted for image optimization with uniform brightness. Blood vessels are often close to neural structures and are often used as landmarks to make the measurements at the same position. To depict their anatomical interaction and distinguish between nerves and vessels, it is also necessary to display the flow velocity and direction using pulsed Doppler and color-coded duplex sonography16,18. The pulse repetition frequency must be adapted to the expected low flow velocities in the&#160;blood vessels of the extremities, or the power Doppler must be selected for color-coding16.
Nerves reflect the ultrasound waves differently from different angles of incidence so that the sonographic image varies in echogenicity (anisotropy)16,19. The best image is achieved from an orthograde angle since the ultrasonic waves are reflected most strongly by the nerves in this angle. For avoiding artificial anisotropy or nerve deformity, the probe must therefore be held in a neutral position during the examination without applying additional pressure perpendicular to the nerves (Figure 1). The cross-sectional area (CSA) is measured within the thin, hyperechoic epineurium (Figure 2) to avoid alterations of the epinerval tissue in the measurement19. More details on technical ultrasound can be found in References6,16,17,18,19,20,21.

<table><tbody><tr><td>Affiniti 70</td><td>Philips GmbH</td><td>n/a</td><td>with preset for neuromuscular ultrasound</td></tr><tr><td>L18-5 linear array transducer</td><td>Philips GmbH</td><td>n/a</td><td></td></tr><tr><td>Ultrasound gel</td><td>C + V Pharma Depot GmbH</td><td>n/a</td><td></td></tr></tbody></table>

nerve ultrasound protocol to detect dysimmune neuropathies

Nerve ultrasound is increasingly used in the differential diagnosis of polyneuropathy as a complementary tool to nerve conduction studies. Morphological alterations of the peripheral nerves, such as increasing the cross-sectional area (CSA), have been described in various immune-mediated polyneuropathies. The most prominent morphological changes in nerve ultrasound have been described for the chronic inflammatory demyelinating polyneuropathy (CIDP)-spectrum disease. CIDP may be distinguished from hereditary and other polyneuropathies by measuring the extent and pattern of nerve swellings (CSA increase). Typical findings in demyelinating inflammatory neuropathies are multifocal nerve swellings with inhomogeneous fascicular structure, while CSA increase in demyelinating hereditary neuropathies occurs in a more generalized and homogenous manner. In other non-inflammatory axonal neuropathies, nerves can appear with normal or slight CSA increases, especially in typical entrapment sites. This article presents technical requirements for nerve ultrasound, an examination procedure using a standardized examination protocol, current reference values for the CSA, and typical sonographic pathological findings in patients with inflammatory neuropathies.

Each ultrasound laboratory should establish its CSA reference values by collecting data from the healthy local population, as specific ultrasound machines and examiner or population-dependent variables can lead to slightly different results in each laboratory. However, to indicate which CSA values can be considered normal, data from two leading German nerve ultrasound groups and a recent meta-analysis of all published reference values so far13,14,15,22,23 are summarized in Table 1. Reference values for patients studied under this protocol in our department are those by Kerasnoudis et al.22 (Table 1).
Typical findings in demyelinating inflammatory neuropathies are multifocal nerve swellings with inhomogeneous fascicles, while nerve swellings in demyelinating hereditary neuropathies occur more generalized and homogenous12,24. The histologic correlate of increased CSA is assumed to be acute inflammation and repeated de- and remyelination; however, this remains to be investigated7. In other non-inflammatory axonal neuropathies, nerves can appear normal or slightly increased in size, especially in typical entrapment sites25,26,27,28.
To simplify the interpretation of the results, the adjusted Bochum Ultrasound Score is suggested as a scoring system, which helps to distinguish chronic inflammatory neuropathies such as CIDP from non-inflammatory neuropathies.
The adjusted Bochum Ultrasound Score is calculated from the number of sites with significantly enlarged CSA of six of the above-described measurement sites: median nerve at the forearm, the median nerve at the upper arm, ulnar nerve at the forearm, ulnar nerve at the upper arm, the radial nerve at the upper arm and sural nerve at the calf. Examination of only these six sites will take ~15 min. Each of these six sites is scored with 1 point if the nerve shows pathological CSA enlargement on one or both sides of the body. Thus, the minimum score is 0 points, and the maximum score is 6 points. With this scoring system, if &#8805;2 points are assigned, the diagnosis of CIDP is possible with a sensitivity of ~53% and a specificity of ~83%, even if additional axonal damage in nerve conduction studies results in difficult detection by electrophysiological criteria.
Different groups have proposed other scoring systems to differentiate between neuropathies10,11,18,29,30. None of these scores is widely used. The adjusted Bochum Ultrasound Score is based on earlier publications which describe the Bochum Ultrasound Score10 derived from four measurement sites to distinguish CIDP from Guillain-Barr&#233; Syndrome and the Nerve ultrasound protocol30 derived from nine measurement sites to differentiate CIDP from MMN, MADSAM and vasculitic or paraproteinemic neuropathy. These different scores should be used according to the exact question. The adjusted Bochum Ultrasound Score was developed to diagnose CIDP if nerve conduction studies show possible CIDP defined by electrophysiological EFNS/PNS criteria5.
However, even if the adjusted Bochum Ultrasound Score only uses six nerve sites for calculation, still all other described nerve sites and the whole course of each nerve should be examined to detect focal lesions31 or exclude homogenous enlargement. In the case of homogenous nerve enlargement, hereditary neuropathy should be considered24. Scoring systems for homogeneity and alterations of the fascicular structure were described before and may aid in evaluating homogeneity8,24,32.
For ultrasound images of a healthy person, see Figure 4; for example, images from a CIDP patient, see Figure 5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62900/62900fig01.jpg" /> 
Figure 1: Examination of the median nerve at the wrist. To avoid artificial anisotropy or nerve deformity, the probe must be held in a neutral position during the examination without applying additional pressure perpendicular to the nerves. <a href="https://www.jove.com/files/ftp_upload/62900/62900fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62900/62900fig02.jpg" /> 
Figure 2:&#160;Measurement of the cross-sectional area (CSA). The cross-sectional area is measured within the thin, hyperechoic epineurium. <a href="https://www.jove.com/files/ftp_upload/62900/62900fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62900/62900fig03.jpg" /> 
Figure 3: Overview of measuring sites for CSA. Blue stars - median nerve, green stars - ulnar nerve, red star - radial nerve, pink star - vagal nerve, yellow stars - cervical roots and brachial plexus, white stars - fibular nerve, purple stars - tibial nerve, brown star - sural nerve. <a href="https://www.jove.com/files/ftp_upload/62900/62900fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62900/62900fig04.jpg" /> 
Figure 4: Example images of a healthy person of the six nerve sites used in adjusted Bochum ultrasound score. A - median nerve at the forearm, B - median nerve at the upper arm, C - radial nerve at the upper arm, D - ulnar nerve at the forearm, E - ulnar nerve at the upper arm, F - sural nerve at the calf. <a href="https://www.jove.com/files/ftp_upload/62900/62900fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62900/62900fig05.jpg" /> 
Figure 5: Example images of a patient with CIDP of the six nerve sites used in adjusted Bochum ultrasound score. A - median nerve at the forearm, B - median nerve at the upper arm, C - radial nerve at the upper arm, D - ulnar nerve at the forearm, E - ulnar nerve at the upper arm, F - sural nerve at the calf. <a href="https://www.jove.com/files/ftp_upload/62900/62900fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2" rowspan="2"></td>
			<td colspan="2" rowspan="2">Kerasnoudis et al.22</td>
			<td rowspan="2">Grimm et al.23</td>
			<td colspan="2">Meta-Analysis by</td>
		</tr>
		<tr>
			<td colspan="2">Fisse et al.13-15</td>
		</tr>
		<tr>
			<td rowspan="2">Nerve</td>
			<td rowspan="2">Site</td>
			<td>mean CSA</td>
			<td rowspan="2">standard deviation</td>
			<td>mean CSA</td>
			<td>mean CSA</td>
			<td>95% CI</td>
		</tr>
		<tr>
			<td>(mm2)</td>
			<td>(mm2)</td>
			<td>(mm2)</td>
			<td>(mm2)</td>
		</tr>
		<tr>
			<td>Median nerve</td>
			<td>Wrist</td>
			<td>8.43</td>
			<td>2.07</td>
			<td>10.6</td>
			<td>8.3</td>
			<td>7.9 - 8.7</td>
		</tr>
		<tr>
			<td></td>
			<td>Forearm</td>
			<td>6.6</td>
			<td>1.6</td>
			<td>7.2</td>
			<td>6.4</td>
			<td>5.9 - 6.9</td>
		</tr>
		<tr>
			<td></td>
			<td>Elbow</td>
			<td>-</td>
			<td>-</td>
			<td>9.2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td>Upper arm</td>
			<td>8.4</td>
			<td>2.87</td>
			<td>9.1</td>
			<td>8.3</td>
			<td>7.5 - 9.0</td>
		</tr>
		<tr>
			<td>Ulnar nerve</td>
			<td>Guyon loge</td>
			<td>5.16</td>
			<td>1.03</td>
			<td>-</td>
			<td>4.1</td>
			<td>3.6 - 4.6</td>
		</tr>
		<tr>
			<td></td>
			<td>Forearm</td>
			<td>5.46</td>
			<td>1.26</td>
			<td>5.9</td>
			<td>5.2</td>
			<td>4.8 - 5.7</td>
		</tr>
		<tr>
			<td></td>
			<td>Elbow</td>
			<td>5.33</td>
			<td>1.4</td>
			<td>8.7</td>
			<td>5.9</td>
			<td>5.4 - 6.5</td>
		</tr>
		<tr>
			<td></td>
			<td>Upper arm</td>
			<td>6.53</td>
			<td>1.82</td>
			<td>7</td>
			<td>6.6</td>
			<td>5.1 - 6.1</td>
		</tr>
		<tr>
			<td>Radial nerve</td>
			<td>Upper arm</td>
			<td>3.26</td>
			<td>1.52</td>
			<td>-</td>
			<td>5.1</td>
			<td>4.0 - 6.2</td>
		</tr>
		<tr>
			<td>Vagal nerve</td>
			<td>Carotid sheath</td>
			<td>-</td>
			<td>-</td>
			<td>2.2</td>
			<td>2.2</td>
			<td>1.5 &#8211; 2.9</td>
		</tr>
		<tr>
			<td>C5</td>
			<td></td>
			<td>-</td>
			<td>-</td>
			<td>2.4*</td>
			<td>5.6</td>
			<td>4.6 &#8211; 6.7</td>
		</tr>
		<tr>
			<td>C6</td>
			<td></td>
			<td>-</td>
			<td>-</td>
			<td>3.4*</td>
			<td>8.8</td>
			<td>7.4 &#8211; 10.3</td>
		</tr>
		<tr>
			<td>C7</td>
			<td></td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>9.5</td>
			<td>8.0 &#8211; 10.9</td>
		</tr>
		<tr>
			<td>Brachial plexus</td>
			<td>Intrascalene space</td>
			<td>30.93</td>
			<td>10.82</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td>Supraclavicular space</td>
			<td>46.13</td>
			<td>18.27</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fibular nerve</td>
			<td>Fibula Head</td>
			<td>7.1</td>
			<td>2.3</td>
			<td>-</td>
			<td>8.4</td>
			<td>6.8 &#8211; 9.9</td>
		</tr>
		<tr>
			<td></td>
			<td>Popliteal fossa</td>
			<td>8.60</td>
			<td>1.77</td>
			<td>8.4</td>
			<td>7.9</td>
			<td>6.6 &#8211; 9.2</td>
		</tr>
		<tr>
			<td>Tibial nerve</td>
			<td>Popliteal fossa</td>
			<td>8.43</td>
			<td>2.68</td>
			<td>23.2</td>
			<td>25.9</td>
			<td>17.5 &#8211; 34.4</td>
		</tr>
		<tr>
			<td></td>
			<td>Malleolus</td>
			<td>6.36</td>
			<td>1.45</td>
			<td>10.2</td>
			<td>10</td>
			<td>7.7 &#8211; 12.4</td>
		</tr>
		<tr>
			<td>Sural nerve</td>
			<td>Heads of gastrocnemius muscle</td>
			<td>1.82</td>
			<td>0.64</td>
			<td>2.2**</td>
			<td>2.4</td>
			<td>1.7 &#8211; 3.1</td>
		</tr>
		<tr>
			<td colspan="7">* Grimm et al.23 measured diameter, not the CSA for C5 and C6 root.</td>
		</tr>
		<tr>
			<td colspan="7">** Grimm et al.23 measured sural nerve at the lateral ankle.</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Reference CSA values for patients. Proposed reference values are based on the publication of Kerasnoudis et al.22, Grimm et al.23 and a recent meta-analysis by Fisse et al.13,14,15. Reference values for patients studied in our department&#160;under the protocol presented here are those by Kerasnoudis et al.22.

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Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies

This article presents a protocol for nerve ultrasound in polyneuropathies to aid the diagnosis of inflammatory neuropathies.

Nerve ultrasound is increasingly used in the differential diagnosis of polyneuropathy as a complementary tool to nerve conduction studies. Morphological ...

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2.6K Views.  Ruhr-University Bochum. This article presents a protocol for nerve ultrasound in polyneuropathies to aid the diagnosis of inflammatory neuropathies.

Video: Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

Neuroscience

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Bioengineering

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

High-frequency Ultrasonography of the Digital and Palmar Nerve Branches of the Hand in Peripheral Nerve Diseases

Peripheral nerve ultrasound is a well-established imaging technique to evaluate certain peripheral nerve pathologies. However, there is a poor correlation between ultrasound abnormalities of peripheral nerves and electrodiagnostic or clinical evidence of axonal loss. This is a significant limitation of peripheral nerve ultrasound, as many peripheral nerve diseases encountered in clinical settings are related to axonal loss. Furthermore, clinical and electrodiagnostic evidence of axonal loss directly correlates with disability in all peripheral nerve diseases. However, due to the floor effects often encountered in electrodiagnostic studies, these correlations, as well as definitive diagnoses, are often challenging. Thus, imaging techniques that correlate with axonal loss are essential for expanding the utility of peripheral nerve ultrasound as a potential biomarker for peripheral nerve diseases. With new technological advancements and the ever-increasing imaging capabilities of high-frequency ultrasound, the palmar and digital nerve branches of the hand can be imaged with exceptionally high resolution even using point-of-care ultrasound devices. Their superficial and distal-most anatomic locations are ideal for evaluating polyneuropathies, as these branches degenerate earliest during axonal loss. However, no studies have systematically evaluated these nerve branches to determine if they can be reproducibly measured with ultrasound. The current protocol was adapted for the systematic assessment of cross-sectional areas of the median and ulnar nerves in the palmar surface and digits of the hand. This protocol provides reference data for a subset of nerves that demonstrate high intraclass correlation coefficients between three separate ultrasonographers. Finally, as a proof of concept and to demonstrate the clinical applications of this protocol, representative data from individuals with genetically confirmed inherited polyneuropathies are compared with established normative data to examine cross-sectional area differences.

The present protocol describes high-frequency neuromuscular ultrasonography of the digital and palmar branches of the median and ulnar nerve, which can aid in localizing peripheral nerve diseases and be adapted to evaluate digital nerve injuries.

Peripheral nerve ultrasound is a well-established imaging technique to evaluate certain peripheral nerve pathologies. However, there is a poor correlation ...

Ultrasound-Guided Needle Release Combined with Corticosteroid Injection

Ultrasound-Guided Needle Release Combined with Corticosteroid Injection for the Treatment of Supinator Syndrome

The two main branches of the radial nerve (RN) are the deep branch (DBRN) and the superficial branch (SBRN). The RN splits into two main branches at the elbow. The DBRN runs between the deep and shallow layers of the supinator. The DBRN can be easily compressed at the arcade of Frohse (AF) due to its anatomical features. This work focuses on a 42-year-old male patient who had injured his left forearm 1 month prior. Multiple muscles of the forearm (extensor digitorum, extensor digiti minimi, and extensor carpi ulnaris) were sutured in another hospital. After that, he had dorsiflexion limitations in his left ring and little fingers. The patient was reluctant to undergo another operation because he had undergone suture surgeries for multiple muscles 1 month prior. Ultrasound revealed that the deep branch of the radial nerve (DBRN) had edema and was thickened. The exit point of the DBRN had deeply adhered to the surrounding tissue. To relieve this, ultrasound-guided needle release plus a corticosteroid injection were performed on the DBRN. Nearly 3 months later, the dorsal extension in the patient's ring and little fingers was significantly improved (ring finger: &#8722;10&#176;, little finger: &#8722;15&#176;). Then, the same treatment was done for the second time. Nearly 1 month after that, the dorsal extension of the ring and the little finger was normal when the joints of the fingers were fully straightened. Ultrasound could evaluate the condition of the DBRN and its relationship with the surrounding tissues. Ultrasound-guided needle release combined with corticosteroid injection is an effective and safe treatment for DBRN adhesion.

Author Spotlight: Ultrasound-Guided Needle Release Combined with Corticosteroid Injection for the Treatment of Supinator Syndrome  

The deep branch of the radial nerve can easily be compressed at the arcade of Frohse due to its anatomical features. Ultrasound-guided needle release combined with corticosteroid injection is an effective and safe treatment for deep branch radial nerve adhesion.

The two main branches of the radial nerve (RN) are the deep branch (DBRN) and the superficial branch (SBRN). The RN splits into two main branches at the ...

Neuromuscular Ultrasound of the Median Nerve at the Carpal Tunnel

The use of neuromuscular ultrasound greatly enhances the evaluation of carpal tunnel syndrome as an adjunct diagnostic tool as it provides dynamic and structural information about the median nerve and its surrounding anatomy.&#8239;Neuromuscular ultrasound aids in diagnostic accuracy (when used with electrodiagnostic testing) and offers etiologic information as a non-invasive, painless, cost-effective, and radiation-free imaging technology that can be easily carried out at the bedside for immediate interpretation. Neuromuscular ultrasound has the limitation of subjectivity, and the need for training and experience will affect the interpretation of results. This article describes a basic practical guide to visualizing the median nerve using neuromuscular ultrasound in a step-by-step manner to aid in the evaluation of carpal tunnel syndrome. Even though the use of ultrasound in the assessment of median nerve entrapment has been long established, there has been no recognized standard protocol. The present protocol aims to provide clear and concise instructions to describe a standard technique to visualize the median nerve through diagnostic ultrasound.

The present protocol describes a technique for the standardization of median nerve ultrasound in cases of suspected carpal tunnel syndrome.

The use of neuromuscular ultrasound greatly enhances the evaluation of carpal tunnel syndrome as an adjunct diagnostic tool as it provides dynamic and ...

Ultrasound Scanning Protocol in the Assessment of Ulnar Neuropathy at the Elbow

Ulnar neuropathy at the elbow is commonly encountered in clinical practice and is the second most common entrapment neuropathy. Left untreated, ulnar neuropathy at the elbow can result in significant disability due to loss of dexterity and grip strength secondary to the weakness of intrinsic hand muscles. Precisely localizing a lesion in ulnar neuropathy can be challenging with electrodiagnostic testing alone. Ultrasound is a relatively quick and useful adjunctive diagnostic modality in overcoming this limitation, as an increase in the cross-sectional area (CSA) of the nerve is a common and validated finding in ulnar neuropathies at the elbow. Sonographic assessment of the nerve's echotexture and vascularity can provide additional diagnostic clues. Ultrasound also offers the unique benefit of detecting ulnar nerve subluxation or dislocation out of the retroepicondylar groove during dynamic assessment, although the clinical significance of this is controversial. Finally, ultrasound can also identify structural abnormalities leading to nerve compressions, such as the presence of bony abnormalities, scar tissue, and space-occupying lesions. These findings may influence management strategies and surgical planning. This protocol aims to illustrate the technique of static and dynamic sonographic imaging of the ulnar nerve around the elbow as a complement to electrodiagnostic testing in the assessment of ulnar neuropathy at the elbow.

This is a standardized protocol for evaluating the ulnar nerve at the elbow using ultrasound.

Ulnar neuropathy at the elbow is commonly encountered in clinical practice and is the second most common entrapment neuropathy. Left untreated, ulnar ...

Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies

Summary

Explore More Videos

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

High-frequency Ultrasonography of the Digital and Palmar Nerve Branches of the Hand in Peripheral Nerve Diseases

Ultrasound-Guided Needle Release Combined with Corticosteroid Injection for the Treatment of Supinator Syndrome

Neuromuscular Ultrasound of the Median Nerve at the Carpal Tunnel

Ultrasound Scanning Protocol in the Assessment of Ulnar Neuropathy at the Elbow