We thank the patients who gave their consent and contributed to the publication.

Here, we technically describe the method used for the treatment of sialorrhea in patients with neurological dysphagia. We will not focus on efficacy results, which have been published previously7. In this paper, in the results section, we present data from 5 consecutive patients with long-term follow-up (3 treatment sessions) treated from 2014 to 2016. These patients are considered representative of the entire population, which consists of the botulinum toxin service of San Luigi Gonzaga Hospital. The protocol was approved and follows the guidelines of the local ethical standards committee on human experimentation.
1. Patients&#39; Recruitment
NOTE: Patients eligible for this approach are adults suffering from moderate to severe sialorrhea secondary to neurological dysphagia with a history of failure or contraindication to pharmacological treatments such as anticholinergic drugs.
<ol>
	<li>Ensure that the patient is evaluated by an experienced neurologist before BoNT injection and that the severity and frequency of sialorrhea is assessed with the Drooling Frequency and Severity Scale (DFSS)14. 
	NOTE: The DFSS score is the sum of the frequency (1 = never, 2 = occasionally/not every day, 3 = frequently/part of the day, 4 = constantly) and severity (1 = never drools, 2 = mild or only lips wet, 3 = moderate or wet on lips and chin, 4 = severe or drool extends to clothes wetting, 5 = profuse or clothing, hands, tray, and objects wet) sub-scores14. A score &#62;2 in both of the sub-scores is used as the eligibility cut-off. 
	NOTE: The neurologist scores the scale on the basis of objective and anamnestic data provided by the patient and/or caregiver. In addition, the Visual Analogue Scale (VAS) must be administered for the evaluation of the patients&#39; subjective sufferance related to the sialorrhea. The VAS is a self-reported scale represented by a 10 centimeter horizontal line with a statement at each end representing one extreme of the patient&#39;s discomfort (from 100 = &#34;no discomfort&#34; to 0 = &#34;extreme discomfort&#34;).</li>
	<li>Collect the number of daily saliva aspirations through a saliva aspirator (when applicable) before BoNT injection, as a useful tool for the evaluation of the therapy efficacy (Figure 1).</li>
	<li>Repeat a neurological evaluation along with the DFSS and a daily saliva aspiration assessment during follow-up, 1, 3 and 6 months after the treatment, or according to the sialorrhea re-occurrence. In particular, retreatment must be taken into account when the number of daily aspirations increases over 50% compared to the 1-month follow-up evaluation.</li>
	<li>Collect procedure adverse events (local pain, local bleeding, and facial palsy) immediately after the injection. Collect therapy adverse events (local pain, facial palsy, chewing weakness, viscous saliva, and dry mouth) during follow-up assessments.</li>
</ol>
2. Procedure
<ol>
	<li>BoNT-A preparation 
	NOTE: The abobotulinumtoxinA is a botulinum toxin type A of the Clostridium botulinum complex-hemagglutinin. It is a powder for solution for intramuscular or subcutaneous injection.
	<ol>
		<li>For the treatment of sialorrhea in adults, reconstitute 500 IU abobotulinumtoxinA with 2 mL&#160;of 0.9% sodium chloride solution for injection in order to obtain a solution containing 250 IU/mL&#160;of abobotulinumtoxinA.</li>
		<li>Administer a total of 250 IU of abobotulinumtoxinA per patient as follows: 50 IU in each submandibular gland and 75 IU in each parotid gland. In this way treat two patients with a single vial to avoid drug wastage.</li>
	</ol>
	</li>
	<li>Patients&#39; preparation
	<ol>
		<li>After an adequate explanation of the procedure and of the potential risks and benefits of the treatment, obtain written informed consent from the patient.</li>
		<li>Place the patient on the ultrasound table in the supine position and possibly with the neck extended. 
		NOTE: Consider that not all neurological conditions leading to sialorrhea allow for the complete extension of the neck.</li>
		<li>Disinfect the skin in the area over the parotid and the submandibular glands with an antiseptic. Anesthetize each skin access site with ethylene chloride spray.</li>
	</ol>
	</li>
	<li>Ultrasound guidance 
	NOTE: An ultrasound-guided procedure with a linear transducer (5 - 10 MHz) is used for gland localization in order to obtain real-time visualization of muscles, glands, bigger vessels or salivary ducts, and other surrounding structures, as well as of the correct needle position within the glandular tissue.
	<ol>
		<li>Turn on the ultrasound system and press the &#34;Start-end&#34; button to begin the exam.</li>
		<li>Press the &#34;Probe&#34; button to select the high frequency probe (5 - 10 MHz).</li>
		<li>Press the &#34;Patient&#34; button and insert the patient&#39;s name and surname.</li>
		<li>Press the &#34;Protocol&#34; button to select the appropriate protocol and sub-protocol (i.e., protocol: &#34;small tissues&#34;, sub-protocol: &#34;general&#34;).</li>
		<li>Press the &#34;Depth&#34; button and specify the depth of view to 5 cm.</li>
		<li>Press the &#34;Focus&#34; button to select the maximum detail of 2 cm.</li>
		<li>Place the transducer under the mandible, between the anterior and posterior bellies of the digastric muscle, to visualize the submandibular gland, which appears as a hypoechoic area with homogeneous echotexture compared to the surrounding tissues. 
		NOTE: Consider the submandibular gland ideally divided into two quadrants, one cranial and one caudal. Identify the widest gland diameter for the lateral needle access.</li>
		<li>Place the transducer below the external acoustic meatus to visualize the parotid gland, which appears as a hypoechoic area with homogeneous echotexture compared to the surrounding tissues. 
		NOTE: Consider the parotid gland ideally divided into four quadrants, two cranial and two caudal. Identify the two access sites for injection midway between the external auditory canal and the mandible angle, one in the cranial part and one in the caudal part of the gland (Supplemental Figure).</li>
	</ol>
	</li>
	<li>BoNT-A injection 
	NOTE: Use a 22 G needle for injections, with needle penetration at least 0.5 cm from the transducer. The transducer must be oriented longitudinally to the needle so that the needle is visible as a bright echoic line. In this way, the penetration of the needle into the soft tissues can be monitored during the procedure as the needle tip proceeds towards the target, preventing injuries to neurovascular structures.
	<ol>
		<li>Use a lateral short-access approach at the widest gland diameter to access the submandibular gland.</li>
		<li>Inject 25 IU BoNT-A into the upper submandibular quadrant. After the injection, slightly retract the needle and change the direction of the needle tip towards the lower submandibular quadrant, injecting 25 IU BoNT-A for a total of 50 IU to each submandibular gland. 
		NOTE: In this way, two submandibular areas (cranial and caudal) are injected, providing a widespread diffusion of the drug within the glandular tissue.</li>
		<li>Use two access sites for the parotid gland injection. Identify the two accesses midway between the external auditory canal and the mandible angle, one in the cranial part of the gland and one in the caudal part of the gland.</li>
		<li>Using the upper access (in the cranial part of the gland), inject 18 - 19 IU BoNT-A into the medial-cranial quadrant. After the injection, slightly retract the needle, without leaving the site of the cranial part of the gland, and change the direction of the needle tip towards the lateral-cranial quadrant; inject 18 - 19 IU BoNT-A.</li>
		<li>Using the lower access (in the caudal part of the gland), inject 18 - 19 BoNT-A IU into the medial-caudal quadrant. After the injection, slightly retract the needle, without leaving the site of the caudal part of the gland, and change the direction of the needle tip towards the lateral-caudal quadrant; inject 18 - 19 IU BoNT-A. 
		NOTE: In this way, four parotid areas (medial-cranial, lateral-cranial, medial-caudal and lateral-caudal) are injected with a total of 75 IU BoNT-A, providing a widespread diffusion of the drug within the glandular tissue.</li>
		<li>After each injection, dab any bleeding with sterile gauze for 1 - 2 min. Re-evaluate the patient after one hour for any potential adverse events. Administer a nonsteroidal anti-inflammatory drug in case of persisting pain. Schedule a follow-up examination after 1 month.</li>
	</ol>
	</li>
</ol>

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S., White, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submandibular+duct+relocation+for+drooling:+a+10-year+experience+with+194+patients.">Submandibular duct relocation for drooling: a 10-year experience with 194 patients.</a> Otolaryngol Head Neck. 101 (1), 87-92 (1989).</li><li>Squires, N., Wills, A., Rowson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+management+of+drooling+in+adults+with+neurological+conditions.">The management of drooling in adults with neurological conditions.</a> Curr Opin Otolaryngol Head Neck Surg. 20 (3), 171-176 (2012).</li><li>Pickett, A., Perrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+composition+of+botulinum+toxins+in+clinical+use.">Formulation composition of botulinum toxins in clinical use.</a> J Drugs Dermatol. 9 (9), 1085-1091 (2010).</li><li>Barbero, P., 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=Long-term+follow-up+of+ultrasound-guided+botulinum+toxin-A+injections+for+sialorrhea+in+neurological+dysphagia.">Long-term follow-up of ultrasound-guided botulinum toxin-A injections for sialorrhea in neurological dysphagia.</a> J Neurol. 262 (12), 2662-2667 (2015).</li><li>Guidubaldi, 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=Botulinum+toxin+A+versus+B+in+sialorrhea:+a+prospective,+randomized,+double-blind,+crossover+pilot+study+in+patients+with+amyotrophic+lateral+sclerosis+or+Parkinson's+disease.">Botulinum toxin A versus B in sialorrhea: a prospective, randomized, double-blind, crossover pilot study in patients with amyotrophic lateral sclerosis or Parkinson's disease.</a> Mov Disord. 26, 313-319 (2011).</li><li>Standring, S., Gray, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gray's anatomy: The anatomical basis of clinical practice. 41st ed. , 527 (2016).</li><li>Standring, S., Gray, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gray's anatomy: The anatomical basis of clinical practice. 41st ed. , 504 (2016).</li><li>Standring, S., Gray, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gray's anatomy: The anatomical basis of clinical practice. 41st ed. , 528 (2016).</li><li>Gomez-Caravaca, M. T., 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=The+use+of+botulinum+toxin+in+the+treatment+of+sialorrhea+in+parkinsonian+disorders.">The use of botulinum toxin in the treatment of sialorrhea in parkinsonian disorders.</a> Neurol Sci. 36 (2), 275-279 (2015).</li><li>Walter, U., Dressler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+botulinum+toxininjections+in+neurology:+technique,+indications+and+future+perspectives.">Ultrasound-guided botulinum toxininjections in neurology: technique, indications and future perspectives.</a> Expert Rev Neurother. 14 (8), 923-936 (2014).</li><li>Thomas-Stonell, N., Greenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+treatment+approaches+and+clinical+factors+in+the+reduction+of+drooling.">Three treatment approaches and clinical factors in the reduction of drooling.</a> Dysphagia. 3 (2), 73-78 (1988).</li><li>Sriskandan, N., Moody, A., Howlett, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+submandibular+gland+injection+of+botulinum+toxin+for+hypersalivation+in+cerebral+palsy.">Ultrasound-guided submandibular gland injection of botulinum toxin for hypersalivation in cerebral palsy.</a> Br J Oral Maxillofac Surg. 48 (1), 58-60 (2010).</li><li>Contarino, M. 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=Botulinum+toxin+B+ultrasound-guided+injections+for+sialorrhea+in+amyotrophic+lateral+sclerosis+and+Parkinson's+disease.">Botulinum toxin B ultrasound-guided injections for sialorrhea in amyotrophic lateral sclerosis and Parkinson's disease.</a> Parkinsonism Relat Disord. 13 (5), 299-303 (2007).</li><li>Chinnapongse, R., 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=Safety+and+efficacy+of+botulinum+toxin+type+B+for+treatment+of+sialorrhea+in+Parkinson's+disease:+a+prospective+double-blind+trial.+Mov+Disord.">Safety and efficacy of botulinum toxin type B for treatment of sialorrhea in Parkinson's disease: a prospective double-blind trial. Mov Disord.</a> Mov Disord. 27 (2), 219-226 (2012).</li><li>Reid, S. 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=Secondary+effects+of+botulinum+toxin+injections+into+salivary+glands+for+the+management+of+pediatric+drooling.">Secondary effects of botulinum toxin injections into salivary glands for the management of pediatric drooling.</a> J Craniofac Surg. 24 (1), 28-33 (2013).</li><li>Chan, K. 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=Long-term+safety+and+efficacy+data+on+botulinum+toxin+type+A:+an+injection+for+sialorrhea.">Long-term safety and efficacy data on botulinum toxin type A: an injection for sialorrhea.</a> JAMA Otolaryngol Head Neck Surg. 139 (2), 134-138 (2013).</li><li>Petracca, 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=Botulinum+Toxin+A+and+B+in+sialorrhea:+Long-term+data+and+literature+overview.">Botulinum Toxin A and B in sialorrhea: Long-term data and literature overview.</a> Toxicon. 1 (107), 129-140 (2015).</li></ol>

The authors have nothing to disclose.

This protocol describes US-guided BoNT-A injection in the parotid and submandibular glands for the treatment of moderate to severe sialorrhea. Sialorrhea secondary to dysphagia is a frequent complication of the advanced stages of different neurological diseases, and when pharmacological therapy with anticholinergic agents demonstrates scarce efficacy or tolerability, salivary gland BoNT injection represents a useful therapeutic option2,7. However, salivary gland BoNT injection guided by anatomical landmarks may be a therapy of low efficacy and possibly burdened with serious adverse events, since the correct injection of the glands is not guaranteed13. On the basis of literature data, BoNT salivary injection for the treatment of sialorrhea showed a wide range in success rates, ranging from 30% to 70%. This difference should be attributed to the differences in the published studies in terms of study designs (prospective17,18 or retrospective19,20), sample sizes, injection sites, and techniques. On the other hand, Lakray and collegues2 recently described a very low complication rate with ultrasound-guided injections of BoNT for the management of sialorrhea, demonstrating a better outcome for the US-guided injection technique compared to the landmarks method. Furthermore, the same review did not evidence the superiority of one therapy brand over another.
As we recently reported7, ultrasound-guided injections of both the parotid and submandibular glands proved to be an effective technique to obtain real-time and precise needle visualization for correct BoNT administration, providing a reliable sialorrhea treatment with prolonged efficacy and a low rate of serious adverse events. Indeed, the visualization of the glands and of the needle penetration provided by the ultrasound-guided approach allows for the widespread administration of BoNT-A within the glands, significantly reducing the severity and frequency of drooling and improving patient quality of life7,13,15. Moreover, our protocol produces long-lasting efficacy in the reduction of sialorrhea, with a treatment-free interval of about 6 months and with a low rate of side effects7. The choice of the dose and type of BoNT is based on the clinical practice of our center and is supported by literature data7,8,16, suggesting that the visual target identification and the ultrasound-guided injection allows for a reproducible and reliable effect with the administration of an identical BoNT-A dose for every patient. Therefore, this technique aims to avoid repeated treatments for the identification of each patient's effective dose, avoiding patient discomfort and reducing costs in terms of time and drug wastage. 
The first step for a good outcome is correct patient selection; scales assessing the severity of sialorrhea, such as the DFSS14, are useful tools for the identification of patients eligible for this treatment. In addition, the evaluation of the number of daily aspirations is of further help in the assessment of subjects with severe sialorrhea; this assessment also represents an objective outcome of the treatment. After patient selection, the critical step of the procedure is the US identification of the targets, specifically the parotid and submandibular glands. The correct visualization of the targets and of needle penetration is crucial to obtain optimal results, since the technique described here allows safe needle tip introduction and widespread BoNT-A distribution within the glandular tissue using multiple injection sites. 
Limitations of this procedure include the need for experience in the use of ecographs and for knowledge of the maxillofacial anatomical structures, or for the assistance of a radiologist expert in US procedures. Moreover, the use of this technique in patients with coagulation abnormalities or in those receiving anticoagulant therapy is not recommended due to the high risk of bleeding. 
As neurologists, we developed this technique for the treatment of sialorrhea in adult neurological patients, demonstrating its efficacy in subjects with different neurological diseases7. However, this approach can potentially be used for the treatment of every subject suffering from moderate to severe sialorrhea, independent of its etiology. 
In conclusion, this technique produces a demonstrable and long-lasting improvement of sialorrhea in neurological dysphagia, encouraging the use of BoNT-A injection in the salivary glands and highlighting the role of ultrasound guidance to obtain the best outcomes in terms of efficacy and safety.

Sialorrhea is an excessive flow of saliva representing a common and disabling complication of several neurological disorders, including stroke, cerebral palsy, multiple sclerosis, and neurodegenerative diseases1. 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 skin2. Various therapeutic options are available for the treatment of sialorrhea, ranging from surgery to pharmacological therapy, with the aim of reducing saliva production.
Surgical options include denervation, excision of the salivary glands, and transposition or ligation of the salivary ducts3,4. However, despite its efficacy, surgery is by definition an invasive procedure with potentially serious adverse events, such as salivary fistula and cyst formation, infections, loss of taste, loss of hearing, dysarthria, and irreversible effects on salivary gland production3. In addition, it has been observed that surgical approaches may have only temporary efficacy due to reinnervation of the salivary glands two or more years after the procedure2,5. A more common and less invasive treatment for severe sialorrhea is the use of systemic drugs with anticholinergic properties. However, the chronic use of anticholinergic agents is often associated with systemic side effects such as confusion, memory problems, drowsiness, urinary retention, and paralytic ileus2,5.
Botulinum toxin (BoNT) salivary injection is an emerging treatment option for sialorrhea. BoNT are proteases (seven, from A to G), produced by the bacterium Clostridium botulinum, that are able to halt acetylcholine release from the presynaptic nerve terminals of the neuromuscular junction, blocking muscle contraction and gland activity at the orthosympathetic and parasympathetic postganglionic synapses. BoNT type A (BoNT-A) and type B (BoNT-B) are commonly used for therapeutic management of dystonia and spasticity6. Recently, BoNT injection within the major salivary glands proved to be a useful therapeutic option in moderate to severe sialorrhea with suboptimal response to other medical therapies due to a lack of efficacy or the presence of significant side effects2,7,8.
Human salivary glands are classified into two groups: major and minor. Major salivary glands include a pair of parotid glands, a pair of submandibular glands, and a pair of sublingual glands; the minor salivary glands consist of about 1,000 small glands spread within the sub-mucosa of the oral cavity9. Approximately 0.5 liters of saliva are secreted per day. The three pairs of major salivary glands are responsible for more than 90% of the saliva production, approximately divided as follows: 20% for the parotid glands, 65% for the submandibular glands and 5%, for the sublingual glands9. The parotid gland is the largest one and is located below the external acoustic meatus, between the mandible and the sternocleidomastoid muscle; it projects forward on the surface of the masseter muscle10. Important structures pass through the gland, such as the facial nerve, external carotid artery, and retromandibular vein, and several other important neurovascular structures are close to the gland. The submandibular gland is a seromucous gland situated behind and below the ramus of the mandible, in the region of the submandibular triangle, between the anterior and posterior bellies of the digastric muscle and around the posterior border of the mylohyoid muscle11.
BoNT salivary gland injection is a local treatment with the potential to avoid discomfort and/or systemic side effects correlated with the other therapies, such as oral, transdermal, and surgical options. Here, we show a reliable and easily reproducible method to treat sialorrhea with BoNT-A injections in the parotid and submandibular glands under ultrasound (US) guidance in order to obtain a satisfying and long-lasting reduction of drooling while strongly limiting the occurrence of serious adverse events. Data on BoNT-A injection efficacy in sialorrhea in the absence of US guidance are reported in the literature12; however, the US-guided identification of glands and their surrounding tissues, along with real-time visualization of the needle position, allows for a reduction in adverse events and a more accurate injection, leading to increased efficacy and outcome reproducibility7,13.

<table><tbody><tr><td>abobotulinumtoxinA (Dysport)</td><td>IpsenBiopharm, Wrexham, UK</td><td></td><td>Delivered drug</td></tr><tr><td>MyLab Twice</td><td>Esaote</td><td></td><td>Ultraonograph</td></tr><tr><td>LA523 5&amp;ndash;10 MHz</td><td>Esaote</td><td></td><td>Linear transducer</td></tr></tbody></table>

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.

The US-guided injection is an effective technique to obtain real-time and precise needle visualization for BoNT administration in the salivary glands. This approach provides a reliable sialorrhea treatment, with prolonged efficacy and the absence of serious adverse events. Indeed, the visualization of the glands and of the needle penetration provided by the US-guided approach proposed here allows a widespread administration of BoNT-A within the major salivary glands with significant and long-lasting outcomes. Sialorrhea reduction can be easily observed 1 w after the treatment through the administration of the DFSS and VAS and through the collection of the number of daily aspirations through a saliva aspirator (if appropriate). These data allow a quick follow-up assessment and are useful to evaluate the need for retreatment. We recommend patients for another BoNT-A injection when the number of daily aspirations increases to over 50% of the best efficacy obtained after the previous injection (obtained about 1 month after the treatment). This technique usually allows a treatment interval of 6 months and leads to a significant reduction in severity and frequency of sialorrhea, with a decrease of about 50% in the DFSS. In detail, the DFSS severity score showed a significant reduction from the pretreatment average score of 4.06 ± 0.81 to the average 1-month post-treatment score of 1.73 ± 1.65, and the DFSS frequency score reduced from the pretreatment average of 3.46 ± 0.75 to the average 1-month post-treatment of 1.82 ± 0.56. The VAS score changed from the pretreatment average of 3.01 ± 1.21 to the average 1-month post-treatment of 8.33 ± 1.10. Finally, the need of daily saliva aspirations lowered from the pretreatment number of 11.90 ± .88 to the 1-month post-treatment number of 4.44 ± 1.43. On average, sialorrhea improvement can be observed as soon as 1 w after injection (Figure 3a, b, c, d).
The US identification of the different tissues and of the needle route also provides an important reduction in adverse events, avoiding BoNT injection into incorrect targets and needle penetration through neurovascular structures. The most common adverse events observed with this technique are mild and consist local pain, dry mouth, viscous saliva, and local bleeding7,13,15.
<img alt="Decision tree for sialorrhea management; flowchart; BoNT injection; anticholinergic treatment." src="/files/ftp_upload/54606/54606fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Flow-chart of Patient Selection. Patient evaluation and selection for BoNT-A treatment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54606/54606fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Ultrasound image showing tissue layers; arrow indicates region of interest; medical imaging analysis." src="/files/ftp_upload/54606/54606fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Ultrasound Injection of Parotid Gland. The arrow indicates the needle penetration, visible as a bright echoic line, within the parotid glandular tissue. Unit scale is expressed in cm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54606/54606fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Daily aspirations and DFSS scores bar charts; visualizes treatment effects over time intervals." src="/files/ftp_upload/54606/54606fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Clinical Outcome of BoNT-A Treatment. Pre = evaluation before therapy; post = evaluation 1 month after therapy; DA = number of Daily Aspirations; VAS = Visual Analogue Scale; FS = Drooling Frequency and Severity Scale - frequency subscore; SS = Drooling Frequency and Severity Scale - severity subscore. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54606/54606fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Facial nerve pathways diagram; labeled regions A and B; anatomical study; 1 cm scale included." src="/files/ftp_upload/54606/54606fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Supplemental Figure: Representation of the Ideal Injection Sites. A) Schematic division of the parotid gland into four quadrants, two cranial and two caudal, for correct injection into four different glandular sites. B) Schematic division of the submandibular gland into one cranial and one caudal quadrant for correct injection into two different glandular sites.

Watch this Scientific Journal Video about Ultrasound-guided Botulinum Toxin-A Injections: A Method of Treating Sialorrhea at JoVE.com

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

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 ...

ultrasound-guided-botulinum-toxin-injections-method-treating

Nanyang Technological University

Research

JoVE Journal

Medicine

22.8K Views.  University of Torino, San Luigi Gonzaga Hospital. 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.

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

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Neuroscience

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if any, therapies exist that target age-related laryngeal muscle dysfunction. Neurotrophins are involved in muscle innervation and differentiation of neuromuscular junctions (NMJs). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. The neuromuscular junctions (NMJs) become smaller and less abundant in aging rat laryngeal muscles, with evidence of functional denervation. We explored the effects of NTF4 for future clinical use as a therapeutic to improve function in aging human laryngeal muscles. Here, we provide the detailed protocol for systemic application and direct injection of NTF4 to investigate the ability of aging rat laryngeal muscle to remodel in response to NTF4 application. In this method, rats either received NTF4 either systemically via osmotic pump or by direct injection through the vocal folds. Laryngeal muscles were then dissected and used for histological examination of morphology and age-related denervation.

Here, we present a protocol to describe the use of neurotrophin 4 (NTF4) systemically and directly to remodel rat aging laryngeal muscles.

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

Developmental Biology

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Bioengineering

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. ...

Subcutaneous Trigeminal Nerve Field Stimulation for Refractory Facial Pain

Chronic or neuropathic trigeminal facial pain can be challenging to treat. Neurosurgical procedures should be applied when conservative treatment fails. Neuromodulation techniques for chronic facial pain include deep brain stimulation and motor cortex stimulation, which are complex to perform. Subcutaneous nerve field stimulation is certified for chronic back pain and is the least invasive form of neuromodulation. We applied this technique to treat chronic and neuropathic trigeminal pain as an individual therapy concept. First, trial stimulation is performed. Subcutaneous leads are placed in the painful trigeminal dermatome under local anesthesia. The leads are connected to an external neurostimulator that applies constant stimulation. Patients undergo a 12 day outpatient trial to assess the effect of the stimulation. Electrodes are removed after the trial. If the patient reports pain reduction of at least 50% in intensity and/or attack frequency, a reduction in medication or increase in quality of life, permanent implantation is scheduled. New electrodes are implanted under general anesthesia and are subcutaneously tunneled to an infraclavicular internal pulse generator. Patients are able to turn stimulation on and off and to increase or decrease the stimulation amplitude as needed.
This technique represents a minimal invasive alternative to other more invasive means of neuromodulation for trigeminal pain such as motor cortex stimulation or deep brain stimulation.

Treating chronic or neuropathic facial pain can be challenging when medical or standard treatment fails. Subcutaneous nerve field stimulation is the least invasive form of neuromodulation and is used for chronic back pain. We applied this technology to treat chronic and neuropathic trigeminal facial pain.

Chronic or neuropathic trigeminal facial pain can be challenging to treat. Neurosurgical procedures should be applied when conservative treatment fails. ...

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen&#39;s Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (&#60;0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

Immunology and Infection

Technique for Salivary Gland Ultrasound Examination of Sjogren's Syndrome

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.

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.

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

Innovative Approach to Minimize Serious Complications in Cosmetic Dermal Filling

Cosmetic dermal filling represents a significant advance in aesthetic medicine, providing non-invasive treatments with immediate and durable outcomes. Despite the generally safe nature of dermal fillers and avoidable complications with proper technique and planning, they are still risky, with potential complications varying from mild and transient to severe and enduring. This study introduces innovative strategies designed to decrease the risk of serious complications, such as vascular occlusion. These include the use of ultrasound guidance, the application of ice, auxiliary gestures, effective withdrawal, etc. Throughout our research, we observed no severe complications like embolism, skin necrosis, or ocular issues. However, minor complications such as erythema, edema, and bruising were noted. It is important to recognize that while most severe complications can be avoided with meticulous technique and planning, the inherent risk of unexpected outcomes persists in any medical procedure. Our findings suggest that with these innovative strategies, the safety of cosmetic dermal filling can be significantly enhanced, thereby minimizing the risk of severe adverse effects.

The protocol proposes an innovative approach to cosmetic dermal filling focusing on ultrasound, icing, pressing, auxiliary gestures, and effective withdrawal to reduce the risk of serious complications like vascular occlusion. Despite the general safety of dermal fillers, accidents can still occur because of the inherent risks.

Cosmetic dermal filling represents a significant advance in aesthetic medicine, providing non-invasive treatments with immediate and durable outcomes. ...

Nasolacrimal Lavage as a Treatment for Ocular Surface Toxic Soup Syndrome

Ocular surface toxic soup syndrome (OSTSS) is characterized by inadequate tear drainage through the nasolacrimal duct system, leading to the accumulation of inflammatory mediators in the tear film. This condition can result in toxic keratoconjunctivitis, with symptoms such as conjunctival hyperemia, itchiness, discomfort, and epiphora. Dilation and irrigation are common diagnostic procedures in both optometry and ophthalmology, used to assess nasolacrimal duct obstruction in cases of epiphora. This technique involves the injection of saline into the nasolacrimal duct system through the puncta, followed by the evaluation of reflux, which indicates obstruction. Although intended to be diagnostic, many patients report significant improvements in epiphora and ocular comfort after the procedure. In this paper, we present a case study in which a patient achieved complete resolution of epiphora and itchiness following therapeutic nasolacrimal lavage performed every 2 months. Subjective improvements in symptoms in 3 additional patients are also reported. We propose nasolacrimal lavage not only as a diagnostic tool but also as an effective therapeutic intervention for managing OSTSS.

OSTSS leads to pooled inflammatory mediators in the tear film, causing symptoms like epiphora and discomfort. Here, we present a case where therapeutic nasolacrimal lavage every 2 months resolved epiphora and itchiness, suggesting its efficacy as a therapeutic intervention for OSTSS. Additionally, we report subjective improvements in symptoms in 3 additional patients.

Ocular surface toxic soup syndrome (OSTSS) is characterized by inadequate tear drainage through the nasolacrimal duct system, leading to the accumulation ...

An Ultrasound-Guided Injection of Therapeutic Drugs into Human Salivary Glands

Source: Barbero, P., et al,&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54606">Ultrasound-guided Botulinum Toxin-A Injections: A Method of Treating Sialorrhea.</a> J. Vis. Exp. (2016)
The video demonstrates an ultrasound-guided injection technique for administering therapeutic drugs into the salivary glands of a human patient.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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

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