We thank Dr. John Chang (Radiation Oncology, LSU Health Science Center) for assistance with radiation dose measurements. The study was supported by the American Cancer Society (Grant number: 116945-RSG-09-038-01-CCE), National Institute of Health (Grant number: R21CA173162) and the Feist-Weiller Cancer Center.

Alle Verfahren wurden von der LSU Health, Shreveport, Tierpflege und Use Committee und waren in Übereinstimmung mit den NIH-Richtlinien für die Pflege und Verwendung von Labortieren genehmigt. 1. Kanülierung der Ratte Submandibular Speicheldrüsen <ol><li> Herstellung von Spritzenschlauch Montage <ol><li> Schneiden Sie ein 10 cm Länge von PE10 Polyethylenschlauch mit einem Skalpell. Halten beide Enden der Rohrleitung zwischen dem Zeigefinger und dem Daumen. Heizen Sie den mittleren Abschnitt des Rohres über eine sanfte Flamme und sanft die weiche Schläuche dehnen ihre Länge zu verdoppeln, indem beide Seiten ziehen. </li><li> Schneiden Sie den Schlauch in der Mitte mit einem Skalpell in einem Winkel von 45 o 2 Kanülen zu erhalten - jeweils mit einem verjüngten Ende. Erweitern des nicht verjüngten Ende der Kanüle, indem er über eine 29 G Nadel von 0,5 cc Insulin-Spritze paßt. </li><li> Entfernen Sie die Kanüle, und 220 &amp; mgr; l Salzlösung in die Spritze zu ziehen. Tippen Sie, um Luftblasen entfernen. Den cannulein über die Nadel und den Spritzenkolben drücken Luft auszutreiben und einen freien Fluss der Lösung durch die Kanüle zu gewährleisten. Stellen Sie die Lautstärke auf 200 &amp; mgr; l. </li></ol></li><li> Intra-Oral Kanülierung von Submandibulardrüse Duct HINWEIS: Autoclave Instrumente vor dem Eingriff, und zwischen Verfahren in einem heißen bead Sterilisator zu sterilisieren. <ol><li> Wiegen Sie das Sprague Dawley Ratte und verzichtet das berechnete Volumen von Ketamin (42 mg / kg) / Xylazin (8 mg / kg) / Acepromazin (1,4 mg / kg) Mischung in einer Injektionsspritze. </li><li> Zurückhalten, das Tier durch die Basis des Schwanzes zwischen dem Zeigefinger und dem Daumen einer Hand greifen und mit der anderen Hand über den Körper gleitenden es zu erfassen. Legen Sie das Zeige- und Mittelfinger an den Seiten des Kopfes, während der Rumpf mit dem Daumen und dem restlichen Finger halten. </li><li> Spritzen Sie das Anästhetikum in den hinteren Gliedmaßen Muskulatur. Bestätigen Tiefe der Anästhesie durch toe Prise und Augenlidreflexe. Bewerben Auge Schmiermittel Trockenheit während zu verhindernTier unter Narkose. </li><li> Legen Sie das Tier auf einem speziell konzipierten Plattform (Abbildung 1), und greifen in die oberen Schneidezähne auf der Querstange. Ziehen Sie den Unterkiefer nach unten durch ein Gummiband um die unteren Schneidezähne Looping und sie auf die Plattform zu verankern. </li><li> Übergeben Sie einen sterilen Faden durch die Zunge und heben Sie sie an den Boden des Mundes zu erhöhen. Klemmen Sie die Nähte mit einem hemostat, und gibt sie über den Querbalken. </li><li> Erweitern Sie die Wangen mit einem speziell angefertigten Wangenspreizer (Abbildung 1), und unter einem Binokular die sublinguale Papillen auf dem Boden des Mundes finden. </li><li> Fassen Sie das verjüngte Ende des vorgeformten PE10 Schlauch, eine empfindliche Pinzette. manipulieren Sie vorsichtig die Spitze der Kanüle in die Gangöffnung auf der sublingualen Papillen. Bestätigen Sie die Platzierung der Kanüle durch Einfädeln von 3 bis 5 mm in den Kanal; sicherzustellen, daß sie ohne Behinderung hindurchgeht. </li></ol></li><li> Submandibulardrüse Instillation <ol><li> Injizieren Atropin (0,5 mg / kg) subkutan in das Genick des Halses, und warten Sie 10 Minuten für eine Verringerung der Speichelsekret. </li><li> Sichern Sie die Kanüle in die Kanalöffnung mit einem Tropfen Cyanacrylat (Kleber) und trocknen lassen. Vermitteln die Lösung in der Drüse (200 ul / gland) langsam den Spritzenkolben mit einer Rate von ~ 50 &amp; mgr; l / min drücken. </li><li> Crush den Schlauch mit einem hemostat und vorsichtig die Spritze entfernen. Bewahren Sie den Schlauch in den Kanal für 30 - 60 min, bis das Tier das Bewusstsein wiedererlangt. Entfernen Sie die Naht, die die Zunge hält. </li><li> Übertragen Sie das Tier in einen separaten Käfig und einer Wärmelampe verwenden Sie es während der Wiederherstellung warm zu halten. Nicht das Tier unbeaufsichtigt lassen, bis er das Bewusstsein zu halten sternal recumbence wiedergewonnen hat. </li><li> Nachdem das Tier vollständig ambulante, Haus es im Vivarium mit uneingeschränkten Zugang zu Nahrung und Wasser. </li></ol></li></ol> 2. Lokalisierte fraktionierten Bestrahlungvon Submandibulardrüse <ol><li> Zurückhalten, das Tier, wie zuvor beschrieben, und betäuben mit intramuskulären Verabreichung von Ketamin (33 mg / kg) / Xylazin (6 mg / kg) / Acepromazin (1 mg / kg) Mix in der hinteren Gliedmaßen. Bestätigen Tiefe der Anästhesie und Schmiermittel auf die Augen. </li><li> Legen Sie das Tier in Rückenlage auf dem Linearbeschleuniger Tischplatte, und verlängern den Hals durch den Kopf kippen. Kollimieren das Strahlungsfeld (3 cm Spaltbreite), um den Bereich von der unteren Grenze des Unterkiefers an die Spitze des Brustbeins zu umfassen. </li><li> Legen Sie eine 1 cm gewebeäquivalenten Bolus über die Region, und stellen Sie den Abstand zwischen der Strahlungsquelle und der Oberseite des Bolus zu 100 cm. </li><li> Bestrahlen, um das Tier (2,5 Gy), um eine 6 MV Photonenstrahl von einem Linearbeschleuniger verwendet wird. Dosisleistung, Feldgröße und Abstand von der Strahlungsquelle Oberfläche Bolus wird die Belichtungszeit diktieren. In der aktuellen eingerichtet wurden die Tiere mit einer Dosisrate von ~ 1 Gy / min bestrahlt. </li><li> Wiederholen Sie Belichtung; 2,5 Gy / Tag für insgesamt 8Tage; 4 Tage / Woche mit einem 2-Tage-Intervall dazwischen. Halten Sie das Tier warm und während der Genesung. Übertragen Sie das Tier auf das Vivarium nachdem sie vollständig ambulante ist. </li><li> Sammeln Sie stimulierte Submandibulardrüse Speichel 8 Wochen nach der Strahlung 14 Drüsenfunktion zu messen. Euthanize Tiere unter Narkose durch Herzperfusion von kaltem 4% Paraformaldehyd / Phosphat-gepufferte Kochsalzlösung pH 7,2. Ausrotten Submandibulardrüse zur histologischen und immunhistochemischen 14 analysiert. </li></ol>

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The authors have nothing to disclose.

Speicheldrüsen erhalten oft Strahlendosen über die Schwelle von Gewebewiederherstellung bei Patienten Strahlentherapie bei Kopf-Hals-Krebs, elektiven Ablation von Hals Knoten oder regionalen malignen hämatologischen Erkrankungen unterziehen. Obwohl die Flüssigkeit absondernden, Azinuszellen der Drüse ausdifferenzierten sind, sind sie gegenüber Strahlung paradoxerweise empfindlich. Die sekretorischen Funktion fällt innerhalb der ersten Wochen der Strahlung und irreversible Drüse Schädigung führt zu einer chroni...

Sicherheiten Zerstörung von gesundem Gewebe entfallen eine Reihe von nachteiligen Nebenwirkungen von Krebsbehandlungen. Ein Teil oder die Gesamtheit der großen Speicheldrüsen, die mit den Strahlungsfeldern liegen, werden unweigerlich zerstört. Daher sind die meisten Patienten Strahlentherapie bei Kopf- und Halskrebs, Gebärmutterhalskrebs - Lymphom unterziehen, oder Ganzkörperbestrahlung vor der Transplantation des Knochenmarks leiden eine der häufigsten und persistent negativen Auswirkungen der Strahlung, Speicheldrüsenunterfunktion 2-6. Die Fluid produzierenden Azinuszellen der Speicheldrüsen sind äußerst empfindlich gegenüber Strahlung. Eine Schädigung der Speicheldrüsen bewirkt eine drastische Verminderung der Speichelfluss, eine Bedingung als Speichelunterfunktion bezeichnet. Die chronische Verringerung der Speichelfluss beeinträchtigt Schlüssel mündliche Aktivitäten wie Kauen, Schlucken, Sprechen und Geschmack, aber die krankhafte Folgen von starken Schmerzen, Schleimhaut Tränen, Dysphagie, opportunistischen Infektionen und Karies verschlechtert sich einPatienten Wohlbefinden und die Funktion 2,3. Da Strahlentherapie-assoziierte Verlust Speichel Zelle irreversibel ist, gibt es keine korrigierende Behandlung von Xerostomie. Aktuelle Behandlung , die auf beschwichtigende Symptome , die mit künstlichen Speichelersatzmittel und prosecretory Medikamente unwirksam zur Linderung langfristigen Fokus 6. Obwohl verbesserte Strahlungsabgabetechniken , um die Schwere der Erkrankung zu verringern haben dazu beigetragen, normalen Gewebetoxizität und ihre Komplikationen bleiben ein limitierender Faktor bei der Krebsbehandlung 6,7. Präventive Maßnahmen Strahlentherapie-assoziierten Komplikationen zu vermeiden sind daher immer die Norm. Radio - Schutzmittel , die durch freie Radikale Sauerstoffspezies, Pflege Zelle Neubesiedlung scavenge oder verbessern die DNA - Reparatur werden erforscht 11.08 Speichelunterfunktion zu verhindern. Sekrete von exokrinen Speicheldrüsen münden in den Mund durch die Hauptausführungsgänge. Intraorale Kanülierung the Ausführungsgänge zur Injektion von Kontrastmitteln wird routinemäßig als ein ambulantes Verfahren durchgeführt. Unter Verwendung eines ähnlichen Ansatzes können Speicheldrüsen direkt für lokalisierte 12 Behandlung ausgerichtet sein. Neben der Verringerung des Risikos von systemischen Nebenwirkungen hat retroductal gland Instillation Vorteile gegeben. Die einschichtigen Anordnung von Speichelzellen um den duktalen Baum aller Speichel Epithelzellen ermöglicht Targeting und die faserige Verkapselung der Drüse wirkt als Barriere unerwünschte therapeutische Ausbreitung zu reduzieren. Im Wesentlichen sind Speicheldrüsen für die gezielte Behandlung von Drüsen Beschwerden optimal geeignet wie strahlungsinduzierte Speichelunterfunktion. (- 2,5 Gy / Fraktion / Tag, fünf Tage die Woche 1.8) für einen Zeitraum von Wochen konventionellen Strahlentherapie zur Behandlung von Krebs wird in kleinen Dosen geliefert. Daher ist ein Radio-Schutz therapeutischen, die Wirksamkeit gegen ein langwieriger Strahlung Schema in experimentellen Modellen zeigt eine größere klinische Lager. compromiert Speichelfunktion nach fraktioniert Strahlung hat bei kleinen Tieren aufgezeichnet worden, aber Strahlungsquelle, Dosisfraktion und verwendeten Protokolle sind vielfältig 9,10,13. Dieser Bericht stellt Methoden für retroductal Lieferung und lokalisierte Strahlung von Submandibulardrüse Ratte unter Verwendung von patientenrelevanten Strahlungsquelle und Dosisanteil.

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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.

Anpassung eines minimal-invasiven Sialographie Technik ist die lokale Behandlung der großen Speicheldrüsen möglich. Retroductal Verabreichung in submandibular Speicheldrüsen der Ratte wurde von intraoralen Kanülierung der Kanäle Wharton versucht (Abbildung 2). Die Speichelgänge von Wharton offen auf dem sublingualen Papillen auf dem Boden des Mundes, aber die Öffnungen sind nicht ohne weiteres sichtbar. Einführen der Kanüle wurde daher durchgeführt durch vorsi...

Watch this Scientific Journal Video about Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction at JoVE.com

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

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.

Retroductal Submandibulardrüse Einträufeln und Lokalisierte fraktionierten Bestrahlung in einem Rattenmodell von Speichel- Unterfunktion

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

retroductal-submandibular-gland-instillation-localized-fractionated

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11.3K Aufrufe.  Louisiana State University Health Sciences Center. 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.

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

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Kanülierung der Maus Submandibular Speicheldrüsen über die Wharton-Gang

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

Forschung

Medizin

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolierung von Maus Speicheldrüsen Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biologie

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.

Retroductal Nanopartikel Injektion an der murinen mandibulären Drüse

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

Biotechnik

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.

Eine Methode zur Messung der Speicheldrüse Funktion bei Mäusen

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

Immunologie und Infektion

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.

Vorbereitung der murinen mandibulären Speicheldrüse aufrecht Intravitalen Mikroskopie

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.

Ultraschall-gesteuerte Botulinumtoxin-A-Injektionen: ein Verfahren zur Behandlung Sialorrhö

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.

Murine Speichel funktionale Bewertung über Pilocarpin Stimulation folgenden fraktioniert Bestrahlung

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

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.

Retrograde Ohrspeicheldrüseninfusion durch Stensen-Kanal in einem nicht-menschlichen Primaten für vektorierte Genabgabe

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

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Strahlenbehandlung von organischen Kulturen aus Submandibulären und Parotid-Salivarienmodels Schlüssel in Vivo-Leiten

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

Echtzeit-Live-Bildgebung der Regeneration der submandibulären Drüse der Maus

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.

Autor im Rampenlicht: Untersuchung von Makrophagen-Epithelzell-Interaktionen bei der Regeneration von Speicheldrüsen nach Verletzungen 

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.

Untersuchung von Zell-Zell-Interaktionen in der Speicheldrüse mittels ex vivo Lebendzell-Bildgebung

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