The authors would like to thank past and current members of the Delaware Tendon Research Group who have assisted in data collection using this protocol. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Numbers R01AR072034, R01AR078898, F31AR081663, R21AR067390, and by Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD007490. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Research reported in this publication was also supported by grants provided by the Swedish Research Council for Sport Science, the Stockholm County Council (ALF project), and the Swedish Research Council. This work was&#160;also supported in part by a Promotion of Doctoral Studies Scholarship from the Foundation for&#160;Physical Therapy Research and by the Rheumatology Research Foundation Medical and Graduate Student Preceptorship.

The following protocol follows the guidelines set forth by and has been approved by the University Institutional Review Board to ensure safe and ethical human subject research. All subjects provided informed consent for participation in the research studies and dissemination of data. The full protocol takes approximately 20 minutes to perform by a trained sonographer. However, individual measurements may be taken per assessment need.
1. Marking of the skin
<ol>
	<li>Ask the participant to be seated at the end of a plinth with their legs hanging off the table.</li>
	<li>Palpate and mark the medial tibial plateau and most distal aspect of the medial malleolus bilaterally. Measure the distance between the ipsilateral markings to determine tibial length.</li>
	<li>Calculate 25% of the tibial length. Measure and mark this distance, distal from the medial tibial plateau, to indicate the location for gastrocnemius measurements (Figure 1).</li>
	<li>Calculate 30% of the tibial length. Measure and mark this distance, proximal from the most distal aspect of the medial malleolus, to indicate the location for soleus measurements (Figure 1).</li>
	<li>Record the 25% and 30% tibial length for longitudinally measuring the same location during future evaluations.</li>
	<li>Ask the participant to move into a prone position on the plinth with the knees fully extended and their feet hanging relaxed off the edge of the plinth with ankles in a resting position (Figure 2).</li>
	<li>Transfer the gastrocnemius and soleus markings to the posterior aspect of the lower leg. Palpate and mark the midpoint of the gastrocnemius (the distal most point between the two muscle bellies). Ask the participant to gently plantarflex against manual resistance to palpate this location.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65798/65798fig01.jpg" /> 
Figure 1: Skin measurement markings. Tibial length is measured as the distance from the medial tibial plateau to the most distal aspect of the medial malleolus. Soleus muscle thickness is measured at 30% of the tibial length proximal to the most distal aspect of the medial malleolus (blue mark). Gastrocnemius muscle CSA is measured at 25% of the tibial length distal to the medial tibial plateau (red mark). <a href="https://www.jove.com/files/ftp_upload/65798/65798fig01large.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/65798/65798fig02.jpg" /> 
Figure 2: Participant positioning for ultrasound imaging. The participant is prone with knees fully extended and feet hanging relaxed in a resting ankle position off the edge of the plinth. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Ultrasound imaging
NOTE: All images will be taken on both legs. All images should be taken along the midline of the Achilles tendon. Use palpation of the medial and lateral borders of the Achilles tendon to determine midline location.
<ol>
	<li>Use B-mode ultrasound with a 5 cm linear array transducer with parameters set for visualization of the triceps surae. Set frequency to 10 MHz and depth of measurement to 3.5 cm for visualization. Adjust the foci between 0.75 cm and 1.75 cm with gain adjusted to 49 for optimal visualization of tissue. 
	NOTE: Depth, foci, and gain may be adjusted as appropriate for visualization.</li>
	<li>Place ultrasound gel on the posterior calcaneus. Holding the probe on the long axis, visualize the proximal calcaneal notch and align the notch with the midpoint of the ultrasound probe. Mark this location on the skin (Figure 3).</li>
	<li>Marking the skin for measurement of Achilles tendon thickness and CSA will vary depending on the participant. Use the following guidelines to determine this position.
	<ol>
		<li>For healthy individuals, measure 2 cm proximal from the proximal calcaneal notch and mark this location on the skin.</li>
		<li>For individuals with Achilles tendinopathy, visualize the thickest portion of the Achilles tendon with the ultrasound probe on the long axis and mark this location on the skin. Record the distance of this marking from the proximal calcaneal notch forlongitudinally measuring the same location during future evaluations.</li>
		<li>For individuals with Achilles tendinopathy without visible fusiform thickening, mark the skin at the location of most tendon pain upon palpation.&#160;Record the distance of this marking from the&#160;proximal calcaneal notch for longitudinally measuring the same location during future evaluations.</li>
		<li>For individuals post Achilles tendon rupture, visualize the location of rupture with the ultrasound probe in the long axis and mark this location on the skin. Record the distance of this marking from the proximal calcaneal notch for longitudinally measuring the same location during future evaluations.</li>
		<li>For the uninvolved limb, make the skin marking at the same distance from the proximal calcaneal notch as the involved limb.</li>
	</ol>
	</li>
	<li>Gastrocnemius tendon length (Full tendon length): Using an extended field-of-view and holding the probe on the long axis, begin the image at the calcaneus by visualizing the Achilles tendon insertion. Glide the probe proximally along the midline of the Achilles tendon towards the mark at the gastrocnemius midpoint until the myotendinous junction is visualized. This will end the image. Take three images of the Achilles&#39; full tendon length (Figure 4).</li>
	<li>Soleus tendon length (Free tendon length): Using an extended field-of-view and holding the probe on the long axis, begin the image at the calcaneus by visualizing the Achilles tendon insertion. Glide the probe proximally along the midline of the Achilles tendon towards the mark at the soleus until the soleus myotendinous junction is visualized. Take three images of the Achilles&#39; free tendon length.</li>
	<li>Achilles tendon thickness: Using the B-mode view, place the probe on the long axis on the skin marking made for Achilles tendon thickness and CSA (step 2.3). Take three images of Achilles tendon thickness.</li>
	<li>Achilles tendon cross-sectional area: Using the B-mode view, place a gel standoff pad on the skin marking made for Achilles tendon thickness and CSA (step 2.3). Holding the probe on the short axis, take three images of the Achilles tendon cross-section. 
	NOTE: Tendon is of an anisotropic quality; depending on the angle of the sound waves, it will reflect back, making it look bright, or reflect away, making it look dark (Figure 5). Therefore, toggling the probe back and forth will help with visualizing the border of the tendon.</li>
	<li>Soleus thickness: Using the B-mode view, place the probe on the long axis on the skin marking made for the soleus (30% of the tibial length from the distal aspect of the medial malleolus). Visualize the anterior border of the soleus muscle. Take three images for soleus thickness19.
	<ol>
		<li>To distinguish between the soleus and flexor hallucis longus, have the participant actively flex and extend the great toe. This will cause movement of the fibers of the flexor hallucis longus without moving the soleus fibers.</li>
	</ol>
	</li>
	<li>Gastrocnemius cross-sectional area: Using an extended field-of-view, hold the ultrasound probe on the short axis and in line with the mark for the gastrocnemius (25% of the tibial length from the medial tibial plateau). Visualize the medial border of the medial gastrocnemius and begin the image by gliding the probe from medial to lateral until the lateral border of the lateral gastrocnemius is visualized. Take three images for gastrocnemius CSA13.
	<ol>
		<li>To capture the most medial aspect of the medial gastrocnemius it may be needed to&#160;ask the participant to abduct their legs and internally rotate the hip slightly. If this is required, ensure the participant is not plantarflexing their foot, as this will impact measurements.</li>
	</ol>
	</li>
	<li>Using the B-mode view, determine the presence or absence of calcifications within the Achilles tendon (Figure 6D) and/or at the Achilles tendon insertion (enthesis; Figure 6C). These calcifications are unattached from the calcaneus and appear as hyperechoic areas with a shadow below, present in multiple planes of view. Take an image to confirm the findings. Note whether the calcifications occur within the Achilles tendon or at the enthesis.</li>
	<li>Using the B-mode view, determine the presence or absence of bursitis. This is visualized by a hypoechoic area deep to the Achilles tendon at the proximal calcaneus (retrocalcaneal bursitis) and/or superficial to the Achilles tendon insertion (subcutaneous calcaneal bursitis). Take an image to confirm the findings (Figure 6B).</li>
	<li>Using the Doppler setting on the ultrasound, assess for the presence or absence of neovascularization within the Achilles tendon.
	<ol>
		<li>Position the Doppler box (i.e., region of interest) on the tendon, hold the ultrasound probe still, and avoid pressing down or distending the tissue as this may occlude and prevent visualization of blood flow.</li>
		<li>Scan the length of the Achilles free tendon, being sure to assess each portion of the tendon without movement of the probe to prevent artifact. If there is vasculature visible within or in contact with the tendon, take three, 3 s videos in the region with maximal blood flow (Figure 6A).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65798/65798fig03.jpg" /> 
Figure 3: Achilles tendon thickness and CSA measurement locations. Extended field of view ultrasound images of (A) a healthy and (B) tendinopathic Achilles tendon. Dotted lines indicate tendon borders. Red arrows indicate measurement locations. Yellow lines indicate the distance proximal to the proximal calcaneal notch (white arrow). In the case of thickening at the 2 cm mark (B), healthy tendon thickness should be taken at a healthy area in the free tendon (blue arrow). <a href="https://www.jove.com/files/ftp_upload/65798/65798fig03large.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/65798/65798fig04.jpg" /> 
Figure 4: Achilles tendon length measurements. Extended field of view ultrasound image of the Achilles tendon. Free tendon length (yellow line) is measured from the proximal Achilles tendon insertion on the proximal calcaneal notch to the soleus myotendinous junction (MTJ). Full tendon length (red line) is measured from the proximal Achilles tendon insertion on the proximal calcaneal notch to the gastrocnemius myotendinous junction (MTJ)10. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig04large.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/65798/65798fig05.jpg" /> 
Figure 5: Tendon anisotropy. Achilles tendon anisotropy occurs when the ultrasound waves are not perpendicular to the structure. Tilting the ultrasound probe will cause the tendon structure to appear (A) hyperechoic or (B) hypoechoic depending on the relationship of ultrasound waves to the tendon. Dotted lines indicate tendon borders. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65798/65798fig06.jpg" /> 
Figure 6: Additional ultrasound imaging findings. (A) Neovascularization, (B) retrocalcaneal bursitis, (C) entheseal calcifications, (D) intratendinous calcifications. Areas colored red and yellow indicate blood flow within the tendon region of interest (yellow box). Dotted lines indicate bursa borders. Arrows indicate calcifications20. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Ultrasound measurements
NOTE: Ultrasound measurements can be made on the machine based on the settings and tools for the respective ultrasound machine. Images can also be exported for taking measurements on software such as Osirix DICOM viewer. The average of the three trials for each measure will be used for analysis.
<ol>
	<li>Gastrocnemius tendon length (full tendon length): Open the longer extended field-of-view tendon image (taken in step 2.4). Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to the gastrocnemius myotendinous junction (Figure 4).</li>
	<li>Soleus tendon length (free tendon length): Open the shorter extended field-of-view tendon image (taken in step 2.5). Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to the soleus myotendinous junction (Figure 4).</li>
	<li>Tendon thickness at 2 cm: Open the shoter extended field-of-view tendon image. Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to 2 cm, with the end of the measurement on the deep border of the tendon. Measure from this point on the deep border of the tendon to the direct superficial aspect of the tendon to get the 2 cm tendon thickness. This measure should represent healthy tendon thickness (Figure 7A).
	<ol>
		<li>In the case of Achilles tendinopathy, if the tendon is thickened at this 2 cm distance, locate a healthy area in the free tendon to take this measurement and document the distance from the proximal calcaneal notch (Figure 7B). 
		NOTE: Thickness is used to describe the raw thickness of the tendon from the most superficial to deep.</li>
	</ol>
	</li>
	<li>Tendon cross-sectional area: Open image taken in step 2.7.&#160;Visually identify the borders of the Achilles tendon and outline the circumference of the tendon (Figure 8A).</li>
	<li>Tendon thickness: Open image taken in step 2.6.&#160;At the center of the image, measure the Achilles tendon from the superficial border to the deep border of the tendon (Figure 8B).</li>
	<li>Soleus thickness: Open image taken in step 2.8. At the center of the image, measure from the superficial border to the deep border of the soleus muscle (Figure 9).</li>
	<li>Gastrocnemius cross-sectional area: Open image taken in step 2.9.&#160;Visualize the borders of both the medial and lateral heads of the gastrocnemius and outline the medial gastrocnemius and then the lateral gastrocnemius heads (Figure 10).</li>
	<li>Achilles tendon thickening: Use this calculation to determine the degree of thickening of the tendon in Achilles tendinopathy. Subtract thickness at 2 cm (or adjusted location; Figure 7A) from tendon thickness (Figure 7B) to obtain tendon thickening (measurement in step 3.5 minus measurement in step 3.3).</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65798/65798fig07.jpg" /> 
Figure 7: Achilles tendon thickening. Achilles tendon thickening is calculated by subtracting (A) the thickness of the healthy portion of the tendon from (B) the thickness of the thickest portion of the tendon31. Dotted lines indicate tendon borders. Red lines indicate tendon thickness. Yellow lines indicated a 2 cm distance proximal to the proximal calcaneal notch. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65798/65798fig08.jpg" /> 
Figure 8: Achilles tendon cross-sectional area and thickness. Ultrasound images of the Achilles tendon in (A) cross-section and (B) longitudinal view at the thickest portion. Dotted lines indicate tendon borders. The red line indicates tendon thickness. A cross-section of the tendon is shown with and without a border for clarity. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65798/65798fig09.jpg" /> 
Figure 9: Soleus muscle thickness. Ultrasound image of the soleus muscle. Dotted lines indicate muscle borders. The red line indicates muscle thickness. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65798/65798fig10.jpg" /> 
Figure 10: Gastrocnemius muscle cross-sectional area. Extended field of view ultrasound image of medial and lateral gastrocnemius muscles in cross-section. Dotted lines indicate tendon borders. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Ultrasound Evaluation of Achilles Tendon Morphology

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<li>Zellers, J. A., Cortes, D. H., Pohlig, R. T., Silbernagel, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tendon+morphology+and+mechanical+properties+assessed+by+ultrasound+show+change+early+in+recovery+and+potential+prognostic+ability+for+6-month+outcomes%5BTitle%5D)+AND+%22Knee+Surg+Sports+Traumatol+Arthrosc%22%5BJournal%5D)">Tendon morphology and mechanical properties assessed by ultrasound show change early in recovery and potential prognostic ability for 6-month outcomes.</a> Knee Surg Sports Traumatol Arthrosc. 27 (9), 2831-2839 (2019).</li>
<li>Zellers, J. A., Bley, B. C., Pohlig, R. T., Alghamdi, N. H., Silbernagel, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Frequency+of+pathology+on+diagnostic+ultrasound+and+relationship+to+patient+demographics+in+individuals+with+insertional+Achilles+tendinopathy%5BTitle%5D)+AND+%22Int+J+Sports+Phys+Ther%22%5BJournal%5D)">Frequency of pathology on diagnostic ultrasound and relationship to patient demographics in individuals with insertional Achilles tendinopathy.</a> Int J Sports Phys Ther. 14 (5), 761-769 (2019).</li>
<li>de Jonge, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incidence+of+midportion+Achilles+tendinopathy+in+the+general+population%5BTitle%5D)+AND+%22Br+J+Sports+Med%22%5BJournal%5D)">Incidence of midportion Achilles tendinopathy in the general population.</a> Br J Sports Med. 45 (13), 1026-1028 (2011).</li>
<li>Lemme, N. J., Li, N. Y., DeFroda, S. F., Kleiner, J., Owens, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+of+Achilles+tendon+ruptures+in+the+United+States%3A+athletic+and+nonathletic+injuries+from+2012+to+2016%5BTitle%5D)+AND+%22Orthop+J+Sports+Med%22%5BJournal%5D)">Epidemiology of Achilles tendon ruptures in the United States: athletic and nonathletic injuries from 2012 to 2016.</a> Orthop J Sports Med. 6 (11), 2325967118808238(2018).</li>
<li>Jackson, J. B. 3rd, Chu, C. H., Williams, K. A., Bornemann, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+ultrasonographic+parameters+of+the+posterior+tibial%2C+peroneal%2C+and+Achilles+tendons%5BTitle%5D)+AND+%22Foot+Ankle+Spec%22%5BJournal%5D)">Normal ultrasonographic parameters of the posterior tibial, peroneal, and Achilles tendons.</a> Foot Ankle Spec. 12 (5), 480-485 (2019).</li>
<li>Schmidt, W. A., Schmidt, H., Schicke, B., Gromnica-Ihle, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standard+reference+values+for+musculoskeletal+ultrasonography%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">Standard reference values for musculoskeletal ultrasonography.</a> Ann Rheum Dis. 63 (8), 988-994 (2004).</li>
<li>Ackermann, P. W., Hart, D. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAckermann%2C+PW+%22Metabolic+Influences+on+Risk+for+Tendon+Disorders%22">Metabolic Influences on Risk for Tendon Disorders</a>. , Springer International Publishing, Switzerland. (2016).</li>
<li>Westh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+habitual+exercise+on+the+structural+and+mechanical+properties+of+human+tendon%2C+in+vivo%2C+in+men+and+women%5BTitle%5D)+AND+%22Scand+J+Med+Sci+Sports%22%5BJournal%5D)">Effect of habitual exercise on the structural and mechanical properties of human tendon, in vivo, in men and women.</a> Scand J Med Sci Sports. 18 (1), 23-30 (2008).</li>
<li>Cassel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+Achilles+and+patellar+tendinopathy+and+their+association+to+intratendinous+changes+in+adolescent+athletes%5BTitle%5D)+AND+%22Scand+J+Med+Sci+Sports%22%5BJournal%5D)">Prevalence of Achilles and patellar tendinopathy and their association to intratendinous changes in adolescent athletes.</a> Scand J Med Sci Sports. 25 (3), e310-e318 (2015).</li>
<li>Hirschmüller, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Achilles+tendon+power+Doppler+sonography+in+953+long+distance+runners+-+a+cross+sectional+study%5BTitle%5D)+AND+%22Ultraschall+Med%22%5BJournal%5D)">Achilles tendon power Doppler sonography in 953 long distance runners - a cross sectional study.</a> Ultraschall Med. 31 (4), 387-393 (2010).</li>
<li>Radovanović, G., Bohm, S., Arampatzis, A., Legerlotz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Achilles+tendinopathy+the+symptomatic+tendon+differs+from+the+asymptomatic+tendon+while+exercise+therapy+has+little+effect+on+asymmetries-an+ancillary+analysis+of+data+from+a+controlled+clinical+trial%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">In Achilles tendinopathy the symptomatic tendon differs from the asymptomatic tendon while exercise therapy has little effect on asymmetries-an ancillary analysis of data from a controlled clinical trial.</a> J Clin Med. 12 (3), 1102(2023).</li>
<li>Corrigan, P., Cortes, D. H., Pontiggia, L., Silbernagel, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+degree+of+tendinosis+is+related+to+symptom+severity+and+physical+activity+levels+in+patients+with+midportion+Achilles+tendinopathy%5BTitle%5D)+AND+%22Int+J+Sports+Phys+Ther%22%5BJournal%5D)">The degree of tendinosis is related to symptom severity and physical activity levels in patients with midportion Achilles tendinopathy.</a> Int J Sports Phys Ther. 13 (2), 196-207 (2018).</li>
<li>Enriquez, J. L., Wu, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+introduction+to+ultrasound+equipment+and+knobology%5BTitle%5D)+AND+%22Crit+Care+Clin%22%5BJournal%5D)">An introduction to ultrasound equipment and knobology.</a> Crit Care Clin. 30 (1), 25-45 (2014).</li>
<li>Jacobson, J. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJacobson%2C+JA+%22Fundamentals+of+Musculoskeletal+Ultrasound+3rd+Edition%22">Fundamentals of Musculoskeletal Ultrasound 3rd Edition</a>. , Elsevier, Philadelphia. (2018).</li>
<li>Scanlan, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonographic+artifacts+and+their+origins%5BTitle%5D)+AND+%22AJR+Am+J+Roentgenol%22%5BJournal%5D)">Sonographic artifacts and their origins.</a> AJR Am J Roentgenol. 156 (6), 1267-1272 (1991).</li>
<li>Gimber, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artifacts+at+musculoskeletal+US%3A+resident+and+fellow+education+feature%5BTitle%5D)+AND+%22Radiographics%22%5BJournal%5D)">Artifacts at musculoskeletal US: resident and fellow education feature.</a> Radiographics. 36 (2), 479-480 (2016).</li>
</ol>

The authors have no conflicts of interest to disclose.

Critical steps in the protocol to ensure the validity and reliability of the methods include the use of skin markings on participants to guide the ultrasound exam and the required training of individuals performing the measurements used in research studies. Skin markings at specific documented locations assist in the ability to assess variables of interest consistently and accurately at the same locations in the same individual over time. Furthermore, applying skin markings to different individuals in the same systematic...

The Achilles tendon originates at the myotendinous junctions of the gastrocnemius and soleus muscles and inserts on the posterior calcaneus. The Achilles tendon consists primarily of densely packed, organized collagen tissue arranged in a hierarchical fashion to maximize tensile strength1. Despite its ability to withstand heavy forces, the Achilles tendon is susceptible to several types of injuries throughout one&#39;s lifespan. These injuries, such as Achilles tendinopathy and Achilles tendon ruptures, are often accompanied by changes in the structure of the triceps surae and surrounding tissues. In Achilles tendinopathy, patients often exhibit fusiform tendon thickening, tendinosis, collagen disorganization, and neovascularization, a process of proliferation of vascular and neural tissue into the tendon2. Additionally, pathological changes associated with Achilles tendinopathy include paratendinitis, intratendinous, and/or entheseal calcifications, and bursitis2,3. After Achilles tendon ruptures, structural changes are common occurrences, irrespective of treatment, and include Achilles tendon thickening and increased tendon length4,5. Furthermore, muscular changes, such as triceps surae muscular atrophy, are also commonly associated with Achilles tendon injuries5,6.
The ability to assess triceps surae and surrounding tissue structures provides valuable insight into structural integrity, tissue quality, and size, which are known to relate to symptoms, function, and prognosis4,7,8,9. Ultrasound imaging is a reliable and valid assessment tool of these structures, including, but not limited to, Achilles tendon length10, thickness10,11, cross-sectional area (CSA)12, gastrocnemius anatomical CSA13, and neovascularization14,15. Assessment of these measures provides valuable insight into the understanding of healthy triceps surae tissue, as well as quantification of structural alterations to evaluate injury risk, severity, and recovery, as well as understanding healthy tissue qualities16.
Despite the clinical and research utility of ultrasound imaging in assessing triceps surae structure, there are often differences in imaging techniques and measurement parameters among clinical and research studies17,18. As a result, comparisons between studies are difficult. Therefore, the purpose of this methods paper is to describe a valid and reliable protocol for efficiently evaluating triceps surae muscle and tendon structure using musculoskeletal ultrasound imaging. This protocol aims to demonstrate the feasibility of incorporating this tool into research and clinical settings through its entirety or as specific parts in healthy and injured individuals. In addition, representative values for healthy and injured triceps surae are provided.

<table><tbody><tr><td>Aquaflex Stand Off Pad</td><td>Parker Laboratories&amp;nbsp;</td><td>E8317C</td><td></td></tr><tr><td>Aquasonic ultrasound Gel</td><td>Parker Laboratories&amp;nbsp;</td><td>E8365AF</td><td></td></tr><tr><td>Linear Array Ultrasound Probe L4-12t-RS</td><td>GE Healthcare</td><td>5495987</td><td></td></tr><tr><td>LOGIC &lt;em&gt;e&lt;/em&gt; Ultrasound</td><td>GE Healthcare</td><td>E8349PA</td><td></td></tr><tr><td>Osirix Dicom Viewer</td><td>Pixmeo SARL</td><td></td><td>Software for measurements</td></tr></tbody></table>

measurement of healthy and injured triceps surae morphology

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally classified as an overuse injury associated with fusiform tendon thickening, neovascularization, and interstitial tendon degeneration. Current literature suggests these structural changes are associated with&#160;symptoms and lower physical activity levels, as well as&#160;symptoms and lower extremity function in the long term. Surgically and non-surgically managed Achilles tendon ruptures result in increased tendon cross-sectional area (CSA) and a lengthened Achilles tendon. Both structural outcomes have clinical implications, as larger CSA positively predicts function, whereas increased tendon lengthening predicts reduced function after Achilles tendon rupture. Given the relationship between structural changes associated with Achilles tendon injuries for both injury severity and injury recovery, it is critical to be able to quantify Achilles tendon structure reliably and accurately. Silbernagel&#39;s group has established a valid and reliable method for efficiently evaluating triceps surae muscle and tendon structure. In this protocol, B-mode musculoskeletal ultrasound imaging is used to measure triceps surae structure, including Achilles tendon thickness and CSA, soleus thickness, and the presence of additional findings (calcifications and bursitis). B-mode extended field-of-view is used to measure Achilles tendon length and gastrocnemius anatomical CSA. Finally, power Doppler is used to identify intratendinous neovascularization. Quantification of triceps surae structure allows for comparison between limbs as well as longitudinal changes in response to exercise and treatment for healthy individuals and those with Achilles tendon injuries. This protocol has been used in many research studies to date and proves valuable in understanding the relationship between tendon structure and injury development, severity, and recovery. As ultrasound devices are becoming more affordable and portable, this protocol proves promising as a clinical tool, given its quick and efficient methods.

The measures in this protocol have been demonstrated to be reliable and valid10,20. Excellent reliability has been reported for extended field-of-view Achilles tendon length to gastrocnemius (interclass correlation coefficient (ICC): 0.944) and Achilles tendon length to soleus (ICC: 0.898)10. The minimal detectable change at the group level (MDCgroup) for Achilles tendon length measures is 0.43 cm for length to gastrocnemius and...

Watch this Scientific Journal Video about Measurement of Healthy and Injured Triceps Surae Morphology at JoVE.com

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &#8212; Research and Future Directions

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

Measurement of Healthy and Injured Triceps Surae Morphology

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally ...

measurement-of-healthy-and-injured-triceps-surae-morphology

Research

JoVE Journal

Medicine

640 Views.  University of Delaware. Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons. 

Video: Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries — Research and Future Directions

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Cancer Research

Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue remodeling. Based on this interest, several analytical techniques have been developed to measure local tissue strains within tendon explants during loading. However, in several cases, the accuracy and sensitivity of these techniques have not been reported, and none of the algorithms are publicly available. This has made it difficult for the more widespread measurement of local tissue strains in tendon explants. Therefore, the objective of this paper was to create a validated analysis tool for measuring local tissue strains in tendon explants that is readily available and easy to use. Specifically, a publicly available augmented-Lagrangian digital image correlation (ALDIC) algorithm was adapted for measuring 2D strains by tracking the displacements of cell nuclei within mouse Achilles tendons under uniaxial tension. Additionally, the accuracy of the calculated strains was validated by analyzing digitally transformed images, as well as by comparing the strains with values determined from an independent technique (i.e., photobleached lines). Finally, a technique was incorporated into the algorithm to reconstruct the reference image using the calculated displacement field, which can be used to assess the accuracy of the algorithm in the absence of known strain values or a secondary measurement technique. The algorithm is capable of measuring strains up to 0.1 with an accuracy of 0.00015. The technique for comparing a reconstructed reference image with the actual reference image successfully identified samples that had erroneous data and indicated that, in samples with good data, approximately 85% of the displacement field was accurate. Finally, the strains measured in mouse Achilles tendons were consistent with the prior literature. Therefore, this algorithm is a highly useful and adaptable tool for accurately measuring local tissue strains in tendons.

Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation

This paper describes an open-source digital image correlation algorithm for measuring local 2D tissue strains within tendon explants. The accuracy of the technique has been validated using multiple techniques, and it is available for public use.

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue ...

Engineering

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Bioengineering

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Evaluating the Function of the Foot Core System in the Elderly

As a complex structure to link the body and the ground, the foot contributes to postural control in human static and dynamic activities. The foot core is rooted in the functional interdependence of the passive, active, and neural subsystems, which combine into the foot core system controlling foot motion and stability. The foot arch (passive subsystem), responsible for load, is considered the functional core of the foot, and its stability is necessary for normal foot functions. The functional abnormalities of the foot have been widely reported in the elderly, such as weakness of toe flexor muscles, abnormal foot postures, and decreased plantar sensory sensitivity. In this paper, a comprehensive approach is introduced for evaluating the foot function based on foot core subsystems. The strength and morphology of the foot intrinsic and extrinsic muscles were used to evaluate the foot muscle (active subsystem) function. The doming strength test was applied to determine the function of foot intrinsic muscles, while the toe flexion strength test focused more on the function of extrinsic muscles. The navicular drop test and foot posture index were applied to evaluate foot arch (passive subsystem) function. For the neural subsystem, the plantar light touch threshold test and two-point discrimination test were used to assess plantar tactile sensitivity at nine regions of the foot. This study provides new insights into the foot core function in the elderly and other populations.

The functional core stability of the foot contributes to the human static posture and dynamic activities. This paper proposes a comprehensive evaluation for the function of the foot core system, which combines three subsystems. It may provide increased awareness and multifaceted protocol to explore the foot function among different populations.

As a complex structure to link the body and the ground, the foot contributes to postural control in human static and dynamic activities. The foot core is ...

Neuroscience

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

Ultrasound Protocol for Perioperative Frailty Assessment

Identifying Frailty Using Point-of-Care Ultrasonography: Image Acquisition and Assessment

Frailty is a significant predictor of a range of adverse outcomes in surgical patients, including increased mechanical ventilation time, longer hospital stays, unplanned readmissions, stroke, delirium, and death. However, accessible tools for screening in clinical settings are limited. Computed tomography of the psoas muscle is the current standard imaging device for measuring frailty, but it is expensive, time-consuming, and exposes the patient to ionizing radiation. Recently, the use of point-of-care ultrasound (POCUS) has emerged as a potential tool to determine the presence of frailty and has been shown to accurately predict frailty and postoperative outcomes. In this article, we will describe the image acquisition of the quadriceps muscles and explain how they can be used to determine frailty and predict postoperative adverse events. We will present information on probe selection, patient positioning, and troubleshooting. Images from a demonstration will be used to present the POCUS technique and example results. The article will culminate in a discussion of the use of these images in medical decision-making and potential limitations.

Author Spotlight: Assessing Surgical Frailty with Point-of-Care Ultrasound of Quadriceps Muscles

Here, we present a protocol to assess frailty in the perioperative setting using point-of-care ultrasound to measure quadriceps thickness. This method offers a practical, non-invasive alternative to traditional assessment methods, potentially enhancing perioperative care by quickly identifying frail patients.

Frailty is a significant predictor of a range of adverse outcomes in surgical patients, including increased mechanical ventilation time, longer hospital ...

Measurement of Healthy and Injured Triceps Surae Morphology

Summary

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3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

Evaluating the Function of the Foot Core System in the Elderly

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Identifying Frailty Using Point-of-Care Ultrasonography: Image Acquisition and Assessment