Name: retroductal nanoparticle injection to the murine submandibular gland
Uploaded: 2018-05-03T14:15:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research (NIDCR) and the National Cancer Institute (NCI) of the National Institutes of Health under Award Number R56 DE025098, UG3 DE027695, and F30 CA206296. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the NSF DMR 1206219 and the IADR Innovation in Oral Care Award (2016). 
We would like to thank Jayne Gavrity for her assistance in performing IVIS experiments. We would like to thank Karen Bentley for her input and assistance in performing EM. We would like to thank Pei-Lun Weng for his assistance with IHC. We would like to thank Matthew Ingalls for his assistance in figure preparation. We would like to thank Dr. Elaine Smolock and Emily Wu for critical reading of this manuscript.

All in vivo procedures outlined below were approved by the University Committee on Animal Resources at the University of Rochester, Rochester,&#160;NY.
1. Preparation
<ol>
	<li>Using 32G intracranial catheter tubing with wire inset, cut 3 cm of the tubing to form a beveled end, approximately 45&#176; to the long axis. Confirm that the wire is at least 1 cm longer than the tubing.</li>
	<li>Load 50 &#181;L of PSMA nanoparticle solution (Figure 1), or other injection material, into a Hamilton syringe. To reduce the probability of barotrauma during injection, attach the catheter tubing, with the stylet removed, to the syringe and expel dead volume.</li>
	<li>Inspect the injection solution to ensure the nanoparticle is fully solvated to prevent ductal obstruction following the administration.</li>
	<li>Prepare atropine solution at 0.1 mg/mL. 
	NOTE: Because atropine is light sensitive and degrades over time, this solution should be made the day of injection, and protected from light until administered.</li>
</ol>
2. Accessing and Visualizing Ductal Entry Point
<ol>
	<li>Weigh C57/BL6 mice using an analytical balance.</li>
	<li>Using a 0.5 mL syringe with 29G x &#189;&#34; needle, anesthetize mice with an intraperitoneally injected sterile saline solution of 100 mg/kg ketamine and 10 mg/kg xylazine. Proceed to the following step when the mouse no longer responds to stimuli, which generally occurs within 5 to 10 min following the injection. 
	NOTE: This procedure may also be performed under isoflurane, but will require a custom nose cone that permits access to the oral cavity.</li>
	<li>To prevent dryness during the procedure, apply lubricant to eyes and place the mouse in a prone position on a custom stage. 
	NOTE: To maintain appropriate conditions for intra-oral procedure, tools should be disinfected or sterilized prior to each use.</li>
	<li>Open the oral cavity by securing the maxillary incisors over a metal beam, and use an elastic band to apply downward tension behind the mandibular incisors (Figure 2A).</li>
	<li>Align the mouse beneath the dissecting microscope such that the base of the jaw is visualized.</li>
	<li>To widen the mouth, use a custom, curved steel retractor to apply tension bilaterally to the buccal mucosa.</li>
	<li>To visualize the submandibular papillae, grasp and gently lift the tongue from the floor of the mouth using blunt forceps. 
	NOTE: The papillae will appear as two pale protrusions beneath the tongue (Figure 2B).</li>
	<li>To ease the visualization and further manipulation within the oral cavity, place cotton between the tongue and the buccal mucosa.</li>
</ol>
3. Ductal Cannulation and Line Placement
<ol>
	<li>Using fine, curved forceps, grasp catheter tubing with the wire inset. For optimum manual control during cannulation, align the tubing with the curvature of the forceps (Figure 3A).</li>
	<li>Using the dissecting microscope, move the forceps and wire into the field of view. 
	NOTE: The wire should be protruding from the tubing.</li>
	<li>Gently apply pressure into the base of one submandibular papilla using the wire inset to produce a small, superficial, mucosal puncture (0.076 mm diameter) that will facilitate later entry of the catheter tubing (0.25 mm diameter). If resistance is encountered, cut fresh beveled tips on both the tubing and wire inset with sharp dissecting scissors.</li>
	<li>Following the entry, withdraw the stylet and, using the dissecting microscope, confirm the presence of saliva at the puncture site. Avoid forceful or sudden movement (either withdrawal or insertion) of the stylet that may cause bleeding or compromise ductal integrity.</li>
	<li>Retract the stylet within the tubing (Figure 3B).</li>
	<li>To ensure that injection tubing will fit into Wharton&#39;s duct opening, insert tubing containing the stylet as a rigid guide into the previously made puncture (Figure 2C). 
	NOTE: If not performed quickly, local swelling may prevent re-insertion.</li>
	<li>To prevent back pressure from prolonged ductal obstruction, withdraw the tubing. Inspect to verify that an opening, visible under microscopy, can be seen in the submandibular papilla. If visible bleeding occurs, remove the stylet and reattempt from step 3.2 on the opposing submandibular papillae.</li>
	<li>Without moving the mouse, administer intraperitoneal injection of 1 mg/kg atropine solution, to reduce salivation during the procedure. Wait 5 - 10 min.</li>
	<li>Grasp the end of the syringe tubing, and insert into the orifice using the dissecting microscope (Figure 3C). If resistance is encountered, cut a fresh beveled end to the tubing and reattempt.</li>
	<li>Once the tubing is in place within the submandibular papilla, slowly advance 3 - 5 mm into the duct. Release the tubing from the forceps.</li>
	<li>To improve the seal between the tubing and the submandibular papilla, dry the interface by gently blotting with gauze for 1 min.</li>
	<li>Inspect to confirm that the position of the tubing has not shifted during drying.</li>
</ol>
4. Injection
<ol>
	<li>Inject material at a rate of 10 &#181;L/min. Inspect to confirm that the mouse remains sedated and is not showing signs of distress (Figure 2D). 
	NOTE: Injections of 15 - 50 &#181;L are well tolerated. Injection of larger volumes can result in barotrauma.</li>
	<li>Following the injection, maintain syringe pressure for 5 min to improve the retention of material within Wharton&#39;s duct and SMG (Figure 4). Inspect the submandibular papilla periodically to ensure that tubing does not exit the ductal orifice.</li>
	<li>Using fine forceps, grasp and gently withdraw the tubing from the submandibular papillae. 
	NOTE: It is normal to observe some fluid egress from the papillae.</li>
	<li>Remove the retractor and cotton from the oral cavity before moving the mouse from the stage. 
	NOTE: The animal should not be left unattended until it has regained sufficient consciousness to maintain sternal recumbency. Furthermore, ensure that the mouse is not housed with other mice until fully recovered.</li>
</ol>
5. Verification and Analysis
Note: An in vivo Imaging System (IVIS) can be used to assess retention of fluorescently labeled nanoparticles following injection (as shown 1 h and 24 h after injection in Figure 5).
<ol>
	<li>To better visualize fluorescent signal within the SMG through the skin, remove the ventral fur overlying the SMGs either by shaving or using a chemical depilatory. 
	Note: Following euthanasia, SMG tissue can also be harvested, fixed (overnight in 4% paraformaldehyde), and stained using immunohistochemistry to confirm persistence of fluorescently labeled NP one day following injection (Figure 6).</li>
</ol>

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The authors have nothing to disclose.

Retroductal injection is critical for localized drug delivery to the salivary gland. This technique has applications in screening therapeutic agents for conditions including Sjogren's syndrome and RIH9,10,28. Direct drug delivery into the SMG via retroductal injection provides a key advantage over systemic administration in its potential to reduce off-target effects, including immune activation11. The ability to maximize local drug delivery, without accumulation in surrounding tissues can also enable therapeutic testing in a wider dose range than could be achieved systemically.
We present this protocol, with troubleshooting and quality check steps, as a detailed and updated method to deliver polymeric nanomaterials through Wharton's duct to the murine SMG20. For example, proper use of a wire guide facilitates cannula placement. Furthermore, by using dry blotting instead of cyanoacrylate glues to hold the cannula in place during the injection, the risk of mucosal trauma is minimized. This method can be used to treat mice with a range of compounds, and can be performed over multiple days with the same mouse to evaluate a timecourse of repeat administration11. 
Normal gland secretion will provide a simple and straightforward clearance mechanism for excess payload, though this strategy should be optimized for different applications by careful selection of injected substance and titration of atropine dosage. In this case, NPs persist in the SMG for at least 24 h. By using NPs capable of drug loading, or similar nanomaterials, future applications of this work include overcoming the solubility limit that would otherwise prevent testing hydrophobic agents with retroductal injection20,21.

Salivary gland dysfunction has many etiologies, including Sj&#246;gren&#39;s syndrome, an autoimmune mediated loss of functional secretory tissue, and radiation induced hyposalivation (RIH), a common sequella of head and neck cancer radiotherapy1. Loss of salivary function due to either condition predisposes individuals to oral and systemic infection, tooth decay, digestive and swallowing dysfunction, speech impairment, and major depression1,2,3. As a result, quality of life significantly suffers, with interventions limited to palliation of symptoms rather than cure4. To investigate novel therapies in vivo, it is of interest to administer bioactive compounds directly to the salivary gland.
Retroductal injection is a valuable method to deliver bioactive compounds directly to the salivary glands and test the efficacy in disease, injury, or under normal tissue homeostasis. The three major salivary glands are the parotid (PG), the submandibular (SMG), and the sublingual (SLG), all of which empty into the oral cavity through excretory ducts. The anatomy of the murine SMG permits direct access through cannulation of Wharton&#39;s duct, located in the floor of the mouth beneath the tongue5. Following the cannulation, solvated drugs can be administered directly to the SMG. Following retroductal delivery, extra-glandular diffusion is restricted by the surrounding tissue capsule which regulates the exchange of material with surrounding structures6. The SMG and its duct are similarly structured in humans, and are routinely accessed during SMG surgery and sialoendoscopy7. In humans and mice, the PG is likewise accessible via Stensen&#39;s duct in the buccal mucosa8.
In murine models of RIH, SMG retroductal injection has been used to deliver therapeutics including growth factors, primary cells, adenoviral vectors, cytokines, and antioxidant compounds to modulate the cellular response to injury, and reduce the resulting tissue damage5,9,10,11,12,13,14,15,16. The most notable clinical success of retroductal injection is the administration of adenoviral vector to direct expression of a water channel (Aquaporin 1; AQP1) in patients following the radiation for head and neck cancer17.
Previously, we have developed and shown the efficacy of a retroductally injected polymeric nanoparticle-siRNA system to protect salivary gland function from RIH11,18,19,20. As an extension of our past work, here, we demonstrate our protocol for retroductal SMG injection using a fluorescently labeled nanoparticle (NP) capable of loading and delivering otherwise poorly soluble drugs21,22,23.
We have synthesized the NP from a diblock copolymer comprised of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA) through reversible addition chain fragmentation (RAFT) polymerization, as described previously21. Through solvent exchange, these polymers spontaneously self-assemble into micelle NP structures with a hydrophobic interior and hydrophilic exterior21. The NPs are labeled with Texas-Red fluorophore to permit the verification of NP delivery into the glands without sacrificing the animal. Live animal imaging and SMG immunohistochemistry is shown at 1 h and 1 day following the injection.
This updated and reproducible cannulation protocol should enable others to achieve retroductal injection. We expect that this refined technique will become critical for in vivo studies and therapeutic development24,25.

<table><tbody><tr><td>Pilocarpine hydrochloride</td><td>Sigma Aldrich</td><td>P6503</td><td>Pilocarpine</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91500-9</td><td>Spring Scissors for Tracheostomy</td></tr><tr><td>Sterile Saline Solution</td><td>Medline</td><td>RDI30296H</td><td>Saline</td></tr><tr><td>Dumont #7 Forceps</td><td>Fine Science Tools</td><td>11274-20</td><td>Curved Forceps</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>Straight Forceps</td></tr><tr><td>Standard Pattern Forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Blunt Forceps</td></tr><tr><td>Fine Scissors- Tungsten Carbide</td><td>Fine Science Tools</td><td>14568-09</td><td>Dissection Scissors</td></tr><tr><td>Microhematocrit Heparinized Capillary Tubes</td><td>Fisher Scientific</td><td>22362566</td><td>Capillary tubes</td></tr><tr><td>Lubricant Eye Ointment</td><td>Refresh</td><td>N/A</td><td>Refresh Lacri-Lube</td></tr><tr><td>Goat polyclonal anti-Nkcc1</td><td>Santa Cruz Biotech</td><td>SC-21545</td><td>Nkcc1 Antibody</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>DAPI</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>ver6.0</td><td>Statistical Software</td></tr><tr><td>Cotton tipped applicator</td><td>Medline</td><td>MDS202000</td><td>Applicator for eye ointment</td></tr><tr><td>0.5cc Insulin Syringe, 29G x 1/2&quot;</td><td>BD</td><td>7629</td><td>Syringe for intraperitoneal injection</td></tr></tbody></table>

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.

Retroductal injection can be used to administer NPs to the murine SMG (Figure 1). Here, we deliver 50 &#181;g PSMA NPs labeled with Texas Red fluorophore.
Proper placement of the mouse allows facile access and visualization of the floor of the mouth (Figure 2A-B). The submandibular papillae are identified as two fleshy protrusions beneath the tongue. Following the cannulation (Figure 2C) and atropine injection, syringe tubing can be placed into the submandibular papillae (Figure 2D).
To facilitate the cannulation, a small puncture in the submandibular papilla is first made using the wire stylet inside of the catheter tubing (Figure 3A). Once this is done, the stylet should be retracted within the tubing to serve as a rigid guide while a larger opening is made (Figure 3B). The stylet has a diameter of 0.076 mm, while the catheter tubing has an outer diameter of 0.25 mm. Following the creation of this larger opening, the pre-loaded catheter tubing, attached to the injection syringe, can then be guided into the ductal orifice (Figure 3C).
Following the injection, it is recommended that the syringe be immobilized and injection pressure maintained. If the pressure is not applied, delivery will be successful, albeit with less efficiency and reproducibility. This is demonstrated by injecting 50 &#181;L of 1% toluidine blue dye bilaterally and observing fainter staining in the gland without maintained pressure following the injection (Figure 4).
To verify NP delivery, the IVIS can be used to detect fluorescent signal within the mouse, which is lateralized to the injected region 1 h post administration (Figure 5). This approach enables the confirmation without euthanizing the mouse and can be continued longitudinally until signal is no longer detectable26,27.
To confirm NP persistence in the SMG 24 h following injection, glands can be sectioned, and viewed by fluorescent imaging. Aqp5 and Krt5 IHC mark secretory and ductal cells of the SMG, respectively, and show NPs in both compartments (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57521/57521fig1.jpg" /> 
Figure 1. Retrograde injection schematic. Following ductal cannulation and syringe placement, 50 &#181;L of 1 mg/mL polymeric NP solution is injected into the SMG. Representative transmission electron micrograph (TEM) shows monodisperse (polydispersity index = 0.2) NP population. <a href="/files/ftp_upload/57521/57521fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57521/57521fig2.jpg" /> 
Figure 2. Retrograde injection steps. (A)&#160;Access the oral cavity by separating the maxillary and mandibular incisors. (B)&#160;Visualize the papillae (boxed) below the tongue at the floor of the mouth, which mark the location of Wharton&#39;s duct. (C)&#160;Using a catheter with wire inset, gently cannulate the base of the submandibular papilla. (D)&#160;Following cannulation, the catheter tubing can be exchanged with syringe tubing <a href="/files/ftp_upload/57521/57521fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57521/57521fig3.jpg" /> 
Figure 3. Effective positioning of catheter and stylet for Wharton&#39;s duct cannulation. (A)&#160;Align the tubing with the curvature of the forceps, and cut a beveled end on the tubing and wire to initially puncture the sublingual papilla. (B)&#160;Retract the stylet within the tubing to make a rigid guide to insert the tubing within the sublingual papilla. (C)&#160;Insert catheter tubing (stylet removed), joined to injection syringe, within the previously made orifice. <a href="/files/ftp_upload/57521/57521fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57521/57521fig4.jpg" /> 
Figure 4. Maintaining syringe pressure following injection improves material retention. Following retroductal injections of 50 &#181;L of 1% toluidine blue, syringe pressure was either maintained for 5 min (Right SMG - first injection) or the syringe was withdrawn immediately after the injection (Left SMG - second injection). <a href="/files/ftp_upload/57521/57521fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57521/57521fig5.jpg" /> 
Figure 5. Confirmation of retroductal NP delivery post injection. (A)&#160;In vivo Imaging System (IVIS) shows the lateralization of red fluorescent signal to the treated (left) side of the mouse 1 h post injection. (B)&#160;NP IVIS signal at 24 h has decreased significantly. <a href="/files/ftp_upload/57521/57521fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57521/57521fig6.jpg" /> 
Figure 6. Confirmation of retroductal NP persistence 24 h post injection. A, C. Uninjected control SMG stained for Aqp5 and Krt5, marking secretory acinar and ductal cells, respectively. B, D. In retroductal NP injected SMG, Aqp5 and Krt5 stains show normal gland morphology and NPs taken up in both acini and ducts (scale bars: 75 &#181;m). <a href="/files/ftp_upload/57521/57521fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

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 ...

retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.9K Views.  University of Rochester. 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.

Video: Retroductal Nanoparticle Injection to the Murine Submandibular Gland

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.

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

Medicine

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

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. ...

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, ...

Immunology and Infection

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Biology

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

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 ...

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 ...

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 ...

Intraductal Injection: Delivering Injection Mix into the Ducts of the Mouse Mammary Gland

Source: Xiang, D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/59502/modeling-breast-cancer-via-an-intraductal-injection-cre-expressing">Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland.</a> J. Vis. Exp (2019).
This video describes an intraductal injection technique where we deliver injection mix into the ducts of mouse mammary glands.

Source: Xiang, D., et al. Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland. J. Vis. Exp ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Summary

Explore More Videos

Chapters in this video

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

Intra-lymph Node Injection of Biodegradable Polymer Particles

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

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

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

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Intraductal Injection: Delivering Injection Mix into the Ducts of the Mouse Mammary Gland