This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

The protocol demonstrated a clinical protocol for conduction of SGUS and the demonstration of scanning is performed on a de-identified healthy volunteer. Ethical approval is not required as the study is beyond the common rule and FDA definition of human research subject. Verbal consent from patients was obtained for taking clinical photos and their publication.
1. Transducer and machine settings
<ol>
	<li>Select a high-frequency linear transducer of 10 MHz (see the Table of Materials).</li>
	<li>Set to B mode.</li>
	<li>Use the highest frequency of the probe.</li>
	<li>Adjust the depth (3 cm) and gain (50%-60%). 
	NOTE: The depth is usually 1 cm deeper than the target structure. Auto-gain function is available for some ultrasound consoles.</li>
	<li>For a right-handed examiner, place the ultrasound machine next to the patient&#39;s right shoulder.</li>
</ol>
2. Patient preparation
<ol>
	<li>Position the patient in the supine position with the neck slightly extended. Use a pillow to support the patient&#39;s head.</li>
	<li>Remove any clothing or jewelry around the neck that may obstruct the ultrasound transducer.</li>
</ol>
3. Scanning of the thyroid gland
<ol>
	<li>Orient the probe with the marker pointing towards the patient&#39;s right side for transverse images. For longitudinal scanning, orient the marker at the cephalic side.</li>
	<li>Apply gel to the probe surface.</li>
	<li>Place the probe below the thyroid cartilage for scanning of the thyroid gland. The right and left lobes of the thyroid gland are connected by the isthmus. Save the images and take the echogenicity of the thyroid gland as a reference (Figure 1).</li>
	<li>If there is concomitant thyroid disease, compare the echogenicity to that of the surrounding muscles. Normal salivary glands are usually more echogenic than muscle.</li>
</ol>
4. Scanning of the parotid gland
<ol>
	<li>Turn the patient&#39;s head to the opposite side of the gland being examined.</li>
	<li>Place the transducer longitudinally along the anterior border of the parotid gland, just anterior to the ear and parallel to the tragus, moving from the superior to the inferior pole of the gland (Figure 2).</li>
	<li>Place the probe transversely at the angle of the mandible to obtain the transverse scan. Ideally, the parotid gland is located between the mastoid and mandible ramus in transverse view. Scan the parotid gland transversely from the superior to the inferior pole (Figure 3).</li>
	<li>Slide the probe below the ramus of the mandible to visualize the deep part of the parotid gland, which could be partially obscured by the mandible. Place the probe between the mastoid process and the angle of the mandible.</li>
	<li>Save and label the images in both the longitudinal and transverse views.</li>
	<li>Assess the size of the parotid gland.</li>
	<li>Compare the echogenicity with that of the thyroid gland. The echogenicity of the normal parotid gland is similar to that of the thyroid gland.</li>
	<li>The retromandibular vein and external carotid artery divide the superficial and deep parotid lobes. Turn on the color doppler to differentiate blood vessels from the dilated intraglandular duct.</li>
	<li>Capture the degree of inhomogeneity, the clearness of the gland margin, and the presence of hypo-/an-echoic areas within the glands.</li>
	<li>Note the presence of focal lesions and document the size, location, shape, and echogenicity. Turn on the color doppler to look for vascularity.</li>
	<li>Score the degree of sonographic involvement on a scale of 0-3 as defined: Grade 0: normal, homogenous parenchyma; grade 1: mild inhomogeneity without hypo-/an-echoic areas; grade 2: moderate inhomogeneity with the presence of scattered hypo-/an-echoic areas; grade 3: Diffuse inhomogeneity with the presence of hypo-/an-echoic areas in the entire gland4.</li>
	<li>The presence of lymph nodes within the parotid gland could be normal and common, especially when located in the superficial lobe. Measure the diameter of the lymph nodes. Evaluate the shape, echogenicity, and preservation of fatty hilum.</li>
	<li>Repeat the scanning at the contralateral parotid gland. Compare the size and echotexture between the two parotid glands.</li>
</ol>
5. Scanning of the submandibular gland
<ol>
	<li>Position the patient in the supine position with the head maximally tilted to the back.</li>
	<li>To obtain the transverse view of the submandibular gland, position the transducer just below the angle of the mandible at the submandibular area, which is bounded by the mandible, mylohyoid muscles, and the anterior belly of the digastric muscles. Examine the entire submandibular gland from anterior to posterior (Figure 4).</li>
	<li>Obtain the longitudinal view by placing the probe medial to the horizontal body of the mandible. Visualize the facial artery in this plane (Figure 5).</li>
	<li>Save and label the images in both transverse and longitudinal views of the submandibular gland.</li>
	<li>Assess the size of the submandibular gland.</li>
	<li>Examine the echogenicity of the submandibular gland, which is usually less echogenic than the parotid gland.</li>
	<li>Assess the degree of inhomogeneity, the clearness of the gland margin, and the presence of hypo-/an-echoic areas within the glands. Note the presence of focal lesions.</li>
	<li>Grade the degree of inhomogeneity using the OMERACT score: Grade 0: normal parenchyma; Grade 1: minimal change with mild inhomogeneity without hypo-/an-echoic areas; Grade 2: moderate change with moderate inhomogeneity with focal hypo-/an-echoic areas; Grade 3: severe change with diffuse inhomogeneity with hypo-/an-echoic areas occupying all the surface of the gland. If the evaluation of the gland is not possible due to qualitative parameters, the fatty gland refers to grade 1, and the fibrous gland indistinguishable from adjacent tissue corresponds to grade 34.</li>
	<li>Measure the size of the submandibular lymph nodes. Evaluate the shape, echogenicity, and preservation of fatty hilum.</li>
	<li>Repeat the scanning at the contralateral submandibular gland. Compare the size and echotexture between the two submandibular glands.</li>
</ol>
6. Documentation
<ol>
	<li>Document the sonographic involvement on a scale of 0-3 at all four major salivary glands. A Score of &#8805;2 at any gland is considered compatible with SS.</li>
	<li>Document the presence of lymph nodes if any. Record the diameter, shape, and whether fatty hilum is preserved.</li>
	<li>Document the presence of focal abnormalities if any.</li>
</ol>

Technique for Salivary Gland Ultrasound Examination of Sjogren's Syndrome

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+College+of+Rheumatology/European+League+Against+Rheumatism+Classification+Criteria+for+Primary+Sj&#246;gren's+Syndrome:+a+consensus+and+data-driven+methodology+involving+three+international+patient+cohorts.">American College of Rheumatology/European League Against Rheumatism Classification Criteria for Primary Sj&#246;gren's Syndrome: a consensus and data-driven methodology involving three international patient cohorts.</a> Arthritis Rheumatol. 69 (1), 35-45 (2017).</li><li>Varoni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+complications+associated+with+labial+salivary+gland+biopsy+for+diagnosis+of+Sj&#246;gren's+Syndrome:+A+retrospective+cohort+study.">Local complications associated with labial salivary gland biopsy for diagnosis of Sj&#246;gren's Syndrome: A retrospective cohort study.</a> J Clin Exp Dent. , e713-e718 (2020).</li><li>Hoc&#780;evar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonographic+changes+of+major+salivary+glands+in+primary+Sjo&#776;gren's+syndrome.+Diagnostic+value+of+a+novel+scoring+system.">Ultrasonographic changes of major salivary glands in primary Sjo&#776;gren's syndrome. Diagnostic value of a novel scoring system.</a> Rheumatology. 44 (6), 768-772 (2005).</li><li>Jousse-Joulin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+clip+assessment+of+a+salivary+gland+ultrasound+scoring+system+in+Sj&#246;gren's+syndrome+using+consensual+definitions:+an+OMERACT+ultrasound+working+group+reliability+exercise.">Video clip assessment of a salivary gland ultrasound scoring system in Sj&#246;gren's syndrome using consensual definitions: an OMERACT ultrasound working group reliability exercise.</a> Ann Rheum Dis. 78 (7), 967-973 (2019).</li><li>Fana, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+OMERACT+Grey-scale+Ultrasound+Scoring+System+for+salivary+glands+in+a+single-centre+cohort+of+patients+with+suspected+Sj&#246;gren's+syndrome.">Application of the OMERACT Grey-scale Ultrasound Scoring System for salivary glands in a single-centre cohort of patients with suspected Sj&#246;gren's syndrome.</a> RMD Open. 7 (2), e001516 (2021).</li><li>Ahuja, A. T., Ying, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonographic+evaluation+of+cervical+lymph+nodes.">Sonographic evaluation of cervical lymph nodes.</a> Am J Roentgenol. 184 (5), 1691-1699 (2005).</li><li>Jousse-Joulin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+salivary+gland+ultrasonography+a+useful+tool+in+Sj&#246;gren's+syndrome?+A+systematic+review.">Is salivary gland ultrasonography a useful tool in Sj&#246;gren's syndrome? A systematic review.</a> Rheumatology. 55 (5), 789-800 (2016).</li><li>De Vita, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+echography+in+primary+and+secondary+Sj&#246;gren's+syndrome.">Salivary gland echography in primary and secondary Sj&#246;gren's syndrome.</a> Clin Exp Rheumatol. 10 (4), 351-356 (1992).</li><li>Cornec, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+salivary+gland+ultrasonography+to+the+diagnosis+of+Sj&#246;gren's+syndrome:+Toward+new+diagnostic+criteria.">Contribution of salivary gland ultrasonography to the diagnosis of Sj&#246;gren's syndrome: Toward new diagnostic criteria.</a> Arthritis Rheum. 65 (1), 216-225 (2013).</li><li>Milic, V. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+salivary+gland+sonography+in+Sj&#246;gren's+syndrome:+diagnostic+value+of+a+novel+ultrasonography+score+(0-12)+for+parenchymal+inhomogeneity.">Major salivary gland sonography in Sj&#246;gren's syndrome: diagnostic value of a novel ultrasonography score (0-12) for parenchymal inhomogeneity.</a> Scand J Rheumatol. 39 (2), 160-166 (2010).</li><li>Finzel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-based+reliability+of+the+Outcome+Measures+in+Rheumatology+(OMERACT)+ultrasound+scoring+system+for+salivary+gland+assessment+in+patients+with+Sj&#246;gren's+syndrome.">Patient-based reliability of the Outcome Measures in Rheumatology (OMERACT) ultrasound scoring system for salivary gland assessment in patients with Sj&#246;gren's syndrome.</a> Rheumatology. 60 (5), 2169-2176 (2021).</li><li>Barrio-Nogal, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+in+the+diagnosis+of+suspected+primary+Sj&#246;gren's+syndrome+and+concordance+with+salivary+gland+biopsy:+a+Spanish+single-center+study.">Ultrasonography in the diagnosis of suspected primary Sj&#246;gren's syndrome and concordance with salivary gland biopsy: a Spanish single-center study.</a> Clin Rheumatol. 42 (9), 2409-2417 (2023).</li><li>Robin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+performances+of+ultrasound+evaluation+of+major+salivary+glands+according+to+the+2019+Outcome+Measures+in+Rheumatology+ultrasound+scoring+system.">Diagnostic performances of ultrasound evaluation of major salivary glands according to the 2019 Outcome Measures in Rheumatology ultrasound scoring system.</a> Arthritis Care Res. 74 (11), 1924-1932 (2022).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+ultrasonography+in+primary+Sj&#246;gren's+syndrome+from+diagnosis+to+clinical+stratification:+a+multicentre+study.">Salivary gland ultrasonography in primary Sj&#246;gren's syndrome from diagnosis to clinical stratification: a multicentre study.</a> Arthritis Res Ther. 23 (1), 305 (2021).</li><li>Tang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value+of+ultrasound+evaluation+of+major+salivary+glands+for+Sj&#246;gren's+syndrome+based+on+the+novel+OMERACT+scoring+system.">Diagnostic value of ultrasound evaluation of major salivary glands for Sj&#246;gren's syndrome based on the novel OMERACT scoring system.</a> Eur J Radiol. 162, 110765 (2023).</li><li>Jousse-Joulin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weight+of+salivary+gland+ultrasonography+compared+to+other+items+of+the+2016+ACR/EULAR+classification+criteria+for+Primary+Sj&#246;gren's+syndrome.">Weight of salivary gland ultrasonography compared to other items of the 2016 ACR/EULAR classification criteria for Primary Sj&#246;gren's syndrome.</a> J Intern Med. 287 (2), 180-188 (2020).</li><li>van Nimwegen, J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+lymphoma+in+primary+Sj&#246;gren's+syndrome:+A+multicenter,+retrospective,+clinical+study+by+the+European+concerted+action+on+Sj&#246;gren's+syndrome.">Malignant lymphoma in primary Sj&#246;gren's syndrome: A multicenter, retrospective, clinical study by the European concerted action on Sj&#246;gren's syndrome.</a> Arthritis Rheum. 42 (8), 1765-1772 (1999).</li><li>Nocturne, G., Pontarini, E., Bombardieri, M., Mariette, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphomas+complicating+primary+Sj&#246;gren's+syndrome:+from+autoimmunity+to+lymphoma.">Lymphomas complicating primary Sj&#246;gren's syndrome: from autoimmunity to lymphoma.</a> Rheumatology. 60 (8), 3513-3521 (2021).</li><li>Lorenzon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonographic+features+of+lymphoma+of+the+major+salivary+glands+diagnosed+with+ultrasound-guided+core+needle+biopsy+in+Sj&#246;gren's+syndrome.">Sonographic features of lymphoma of the major salivary glands diagnosed with ultrasound-guided core needle biopsy in Sj&#246;gren's syndrome.</a> Clin Exp Rheumatol. 39 Suppl 133 (6), 175-183 (2021).</li><li>&#214;zer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+performance+of+gray-scale+ultrasound+and+shear+wave+elastography+in+assessing+salivary+gland+involvement+in+patients+with+primary+Sj&#246;gren's+syndrome.">Diagnostic performance of gray-scale ultrasound and shear wave elastography in assessing salivary gland involvement in patients with primary Sj&#246;gren's syndrome.</a> J Clin Ultrasound. 51 (1), 187-194 (2023).</li></ol>

The authors have no conflicts of interest to declare.

Ultrasonography of the major salivary glands is a non-invasive and accessible imaging modality with high spatial resolution. The procedure of performing SGUS at the major salivary glands for patients with suspected SS is described in this article to facilitate reproducible and standardized examination. Parotid and submandibular glands are superficially located, which permits detailed examination by high-frequency ultrasound. SGUS is useful in evaluating various clinical conditions, including SS, sialadenitis, sialolithia...

Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration in exocrine glands, including the salivary and lacrimal glands. Xerostomia, keratoconjunctivitis sicca, and extraglandular manifestations, including arthralgia, cytopenia, and lymphoma, are described in patients with SS. SS can be divided into primary and secondary types, in which the latter occurs in association with other connective tissue diseases. In the 2016 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for primary SS, anti-SSA/Ro positivity and lymphocytic infiltration in labial salivary gland biopsy contributed to the two major criteria, whereas Schirmer's test, ocular staining score, and unstimulated salivary flow represented the rest of the minor criteria1. The criteria are summarized in Table 1. Labial biopsy is an invasive procedure with potential complications, including sampling error, sensory loss, and hematoma formation2. Ocular staining tests and salivary flow assessment require specialized settings and might not be widely available. 
Salivary gland ultrasonography (SGUS) is a non-invasive imaging modality that provides a detailed examination of the superficial salivary gland structure, which is commonly affected in patients with SS. Lymphocytic infiltration and inflammation in the salivary glands often lead to fatty infiltration, fibrosis, and loss of parenchyma, resulting in parenchymal inhomogeneity detected in SGUS3. The Outcome Measures in Rheumatology Clinical Trials (OMERACT) working group derived an ultrasound scoring system from grades 0 to 3 to semi-quantify inhomogeneity and the presence of hypoechoic areas within the parotid or submandibular glands4. A cutoff of grade 2 or above in at least one gland was found to correlate with positive labial biopsies, sialometry, and positive autoantibodies5. It demonstrated excellent specificity and good sensitivity in fulfilling the 2016 ACR/EULAR classification criteria for primary SS when given similar weight as the minor criteria. This protocol presents a standardized and practical approach for diagnosing SGUS in SS in clinical settings.

<table><tbody><tr><td>Examination couch</td><td></td><td>/</td><td>Quantity: 1</td></tr><tr><td>High frequency linear transducer of at least 10 MHz</td><td></td><td>SL2325 18-6 MHz linear transducer</td><td>Quantity:&amp;nbsp;1</td></tr><tr><td>Ultrasound console</td><td></td><td>Esaote my lab seven</td><td>Quantity:&amp;nbsp;1</td></tr><tr><td>Ultrasound gel</td><td></td><td>/</td><td>Quantity:&amp;nbsp;1</td></tr></tbody></table>

ultrasonographic evaluation of salivary glands for sjogren's syndrome: diagnostic and monitoring insights

Sjogren's syndrome (SS) is a chronic autoimmune condition commonly affecting the exocrine glands, causing oral or ocular dryness and extraglandular manifestations including arthralgia, cytopenia, and lymphoma. The presence of autoantibodies against SSA/Ro, labial salivary gland biopsy, ocular staining, Schirmer's test, or salivary flow assessment are included in the current classification criteria of SS. However, the availability and invasiveness of these procedures limit their widespread use in clinical settings. Salivary gland ultrasonography (SGUS) is a non-invasive imaging modality for the evaluation of the salivary gland parenchyma and is increasingly utilized to aid diagnosis and monitoring in SS. 
This article presents the protocol of SGUS for image acquisition at the parotid and submandibular glands. The objective is to present a standardized, reproducible, and practical approach to diagnostic SGUS for SS in daily clinical settings. Major salivary glands are scanned in a stepwise approach, beginning at the angle of the mandible for the superficial lobe of the parotid gland, followed by the deep lobe below the ramus of the mandible. Submandibular areas are then scanned for the submandibular glands. The steps in obtaining salivary gland images at each anatomical site are explained in the accompanying video. The echogenicity and echotexture at the thyroid gland are taken as a reference. The homogeneity, the presence and distribution of hypoechoic areas within the glands, and the border of the salivary glands are examined. The sizes and features of intra-/peri-glandular lymph nodes are recorded. The most distinctive sonographic feature in SS is glandular heterogeneity with the presence of hypoechoic/hyperechoic areas within the glands. 
In summary, while SGUS cannot diagnose SS on its own, it can supplement the current classification criteria of SS and guide the clinical decision for salivary gland biopsy to support the diagnosis of SS in patients with sicca syndrome or suspicious systemic features, combined with autoantibody testing.

Here we described the interpretation of salivary gland ultrasound images to aid diagnosis of SS. The anatomical annotation at the scanning sites is summarized in Figure 1, Figure 2, and Figure 3. The echotexture of the thyroid gland is taken as the reference. Normal parotid glands should appear homogeneous with clear demarcation with overlying tissues and muscles. The echotexture of normal parotid glands is comparable to that of the...

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Author Spotlight: A Focus on Standardized Salivary Gland Ultrasound Protocol in Connective Tissue Disease Research

Salivary gland ultrasonography is a promising tool for the diagnosis and evaluation of disease activity and complications in Sjogren's syndrome (SS). The scanning technique for salivary gland ultrasound examination relevant to SS is described in the article.

Ultrasonographic Evaluation of Salivary Glands for Sjogren's Syndrome: Diagnostic and Monitoring Insights

Sjogren's syndrome (SS) is a chronic autoimmune condition commonly affecting the exocrine glands, causing oral or ocular dryness and extraglandular ...

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1.9K Views.  The University of Hong Kong. Ons onderzoek richt zich op bindweefselziekten, waaronder het syndroom van Sjögren, en we zullen een gestandaardiseerde benadering van speeksel en echografie beschrijven bij het diagnosticeren van het syndroom van Sjögren. We hopen dat het protocol nuttig zal zijn voor clinici die niet bekend zijn met echografie om de toepassing ervan in de klinische praktijk te verbeteren. Echografie is een krachtig hulpmiddel bij het evalueren van verschillende reumatologische aandoeningen, waaronder artr...

Video: Auteur in de schijnwerpers: een focus op het gestandaardiseerde echografieprotocol voor speekselklieren in onderzoek naar bindweefselziekten

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Immunology and Infection

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Ultrasound-guided Botulinum Toxin-A Injections: A Method of Treating Sialorrhea

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an impaired quality of life, often worsened by correlated complications such as aspiration pneumonia, oral infections, dental caries, and maceration of the skin. Diverse therapeutic approaches have been proposed for the treatment of sialorrhea, including surgery and the use of anticholinergic agents, with limited results and the possible occurrence of serious adverse events. Recently, botulinum toxin (BoNT) injection within the major salivary glands has been proposed in patients refractory to anticholinergic therapy, with the aim of inhibiting local acetylcholine release and gland activity. 
In order to obtain a better outcome in terms of reduction of saliva production, efficacy, duration, and avoidance of major adverse events, we developed an ultrasound-guided BoNT-type A injection technique accurately described in the text. Here we present a method of treating sialorrhea with bilateral parotid and submandibular gland BoNT-type A injections under ultrasound guidance. Four quadrants of the parotid gland and two quadrants of the submandibular gland are visualized and injected using two accesses and one access, respectively. 
The ultrasound-guided procedure provides a simple, non-invasive, real-time visualization of the muscular and glandular tissues and their surrounding structures, optimizing treatment efficacy and safety.

Botulinum toxin is injected into the salivary glands for the treatment of sialorrhea. The ultrasound-guided botulinum toxin injection in both the parotid and submandibular glands demonstrates a sustained efficacy and the absence of serious side effects.

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

Diagnosis and Strain Identification of pSS-Related Oral Candida

Examination of Oral Candida Infection in Primary Sj&#246;gren&#39;s Syndrome Patients

Primary Sj&#246;gren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due to the hyposalivation experienced by pSS patients, oral dysbacteriosis often occurs. A common complication of pSS is the oral Candida infection. In this article, the authors describe systematic methods that can effectively diagnose oral Candida infection and identify the Candida strains using saliva, oral mucosal swabs, or mouthwash from pSS patients. The Sabouraud's Dextrose Agar (SDA), hyphal formation assay, potassium hydroxide (KOH) smear test, and calcofluor white (CFW) staining assay are used for the diagnosis of oral Candida infection. A Candida diagnostic agar is used for the identification of Candida strains. Finally, antifungal susceptibility testing is used to determine appropriate antifungal drug treatment. This standardized method can enhance the diagnosis, treatment, and future research of pSS-related oral Candida infections. Early diagnosis, using this method, can also prevent any complications arising due to delay in receiving appropriate treatment.

Author Spotlight: Oral Candida Diagnosis to Advance Clinical Treatment Regimen for pSS Patients

Here, we present a protocol for the examination of oral Candida infection in patients with primary Sj&#246;gren's syndrome, which can be used for timely treatment and, thereafter, avoiding related complications.

Primary Sj&#246;gren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due ...

Key Molecular Targets Linking Primary Sjogren's Syndrome and Lung Adenocarcinoma

Primary Sjogren's Syndrome Associated with Lung Adenocarcinoma: Probing the Potential Common Pathogenic Mechanisms and Experimental Verification

This study aimed to probe the potential common pathogenic mechanisms linking primary Sjogren's syndrome (pSS) and lung adenocarcinoma (LUAD) through bioinformatics analysis and experimental verification. The relevant genes associated with pSS and LUAD were retrieved from the Gene Expression Omnibus (GEO) database and Genecard database. Subsequently, differentially expressed genes (DEGs) associated with pSS and LUAD were screened as pSS-LUAD-DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed to elucidate the significant biological functions of pSS-LUAD-DEGs. Core targets were identified by constructing the protein-protein interaction (PPI) network, further assessing hub gene diagnostic accuracy through Receiver Operating Characteristic (ROC) curve analyses. In this study, NOD/Ltj mice served as pSS animal models and were stimulated with particulate matter 2.5 (PM2.5) to generate an inflammatory reaction. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting were employed for relevant molecular biology experiment verification. The results revealed through KEGG and GO enrichment analyses indicate that inflammation plays a critical role in linking pSS and LUAD. IL6, CCNA2, JAK2, IL1B, ASPM, CCNB2, NUSAP1, and CEP55 were determined as key targets of pSS-LUAD. BALB/c mice and NOD/Ltj mice exhibited enhanced expression of inflammatory cytokines IL-6 and IL-1&#946; in lung tissues following 21 days of stimulation with PM2.5, activating the JAK2/STAT3 signaling pathway and up-regulating the expression of tumor-associated genes CCNA2, CCNB2, and CEP55, with NOD/Ltj mice exhibiting more pronounced changes than BALB/c mice. This protocol demonstrates that carcinogenesis induced by the pulmonary inflammatory microenvironment may be a key reason for the high incidence of LUAD in pSS patients. Additionally, blocking-related mechanisms may help prevent the occurrence of LUAD in pSS patients.

Author Spotlight: Exploring the Role of Inflammation in the Co-occurrence of Primary Sjogren's Syndrome and Lung Adenocarcinoma

This manuscript describes the operational procedures and precautions to probe the potential common pathogenic mechanisms linking primary Sjogren's syndrome and lung adenocarcinoma through bioinformatics analysis and experimental verification.

This study aimed to probe the potential common pathogenic mechanisms linking primary Sjogren's syndrome (pSS) and lung adenocarcinoma (LUAD) through ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Ultrasonographic Evaluation of Salivary Glands for Sjogren's Syndrome: Diagnostic and Monitoring Insights

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