The authors are grateful to Dr. Ana Cotrim for technical consultation and to Dr. Monzur Murshed for equipment. This work was in part funded by the Canadian Institutes for Health Research.

1. Procedural Description
<ol>
	<li>Subcutaneously inject 1 &mu;l / g body weight of 0.5 mg / ml atropine sulfate monohydrate (Cat# 11330, Fluka, USA) into each mouse to prevent the salivary
	secretions from disrupting the movement of the infusions.</li>
	<li>Anesthetize each mouse with 1 &mu;l / g body weight of a 100 mg / ml ketamine and 20 mg / ml xylazine (Phoenix Scientific, AR, USA) solution 
	injected intra-peritoneal (I.P.)</li>
	<li>Place the mouse on a custom-made plastic platform in the ventral position: the maxillary incisors are locked on a metal wire, and the mandibular incisors are hooked on an elastic string in order to hold the mouth open.</li>
	<li>Use an Insulin syringe with a 29-gauge needle (Cat# 600145, Tyco Healthcare, MA, USA) placed inside a 0.58 mm diameter polyethylene tube (Cat# 427410, Becton 
	Dickinson, MD, USA; Fig 2). The polyethylene tube is inserted 5mm over the tip of a 29-gauge needle. The needle is used as a rigid guide to move the 
	polyethylene tube into the Wharton's duct. The polyethylene tube is inserted 3-5 mm inside the duct. Note that only the polyethylene tube enters the salivary gland 
	duct while the needle remains outside the duct. Inject 50ul of filtered 0.4% trypan blue stain solution (Cat# 15250, Gibco, USA) into the submandibular gland via the 
	Wharton's duct, which is located right beneath the tongue. The infusion is delivered steadily with positive pressure on the syringe piston; the finger remains on the 
	piston even after all of the infusion has been delivered to the gland, in order to prevent a backflow of the infusion intra-orally. The trypan blue solution is used in 
	the video as a dye to demonstrate the technique.</li>
	<li>fter the injection of the trypan blue solution, the mouse was euthanized prior to dissection. The salivary gland is surgically exposed in order to determine 
	how much of the infusions have actually remained in the gland.</li>
</ol>
2. Representative Results:
After the infusions were injected, the mouse was dissected to surgically expose the post-cannulated salivary gland. As shown on Image-2, the dark coloration of the Wharton's duct provides evidence that the trypan blue solution was infused successfully. 
<img src="/files/ftp_upload/3074/3074fig1.jpg" alt="Figure 1" /> 
Figure 1. Cannulation of mouse submandibular gland.
<img src="/files/ftp_upload/3074/3074fig2.jpg" alt="Figure 2" /> 
Figure 2. Polyethylene tube is encased around the tip of a 29-gauge needle. The syringe needle is used as a rigid guide to move the polyethylene tube into the Wharton's duct. The polyethylene tube is inserted (cannulated) 3-5 mm inside the duct. Note that only the polyethylene tube enters the salivary gland duct while the needle remains outside the duct.

<ol>
	<li>Fox, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+salivary+dysfunction.+Drugs+and+radiation.">Acquired salivary dysfunction. Drugs and radiation.</a> Ann N Y Acad Sci. 842, 132-137 (1998).</li><li>Bhide, S. A., Miah, A. B., Harrington, K. J., Newbold, K. L., Nutting, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+xerostomia:+pathophysiology,+prevention+and+treatment.">Radiation-induced xerostomia: pathophysiology, prevention and treatment.</a> Clin Oncol (R Coll Radiol). 21, 737-744 (2009).</li><li>Redman, R. S., Ball, W. D., Mezey, E., Key, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+donor+salivary+gland+cells+are+widely+distributed+in+the+recipient+gland+when+infused+up+the+ductal+tree.">Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.</a> Biotech Histochem. 84, 253-260 (2009).</li></ol>

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.

A difficulty using this cannulation method is to prevent the backflow of the injected solution. Even if the trypan blue solution has temporarily migrated into the salivary gland successfully, the saliva secretion might interfere with the infusions and cause backflow of the trypan blue solution out of the gland. As a precaution, in addition to using atropine to inhibit secretion of saliva, the polyethylene cannula should remain in the Wharton's duct and the piston of the syringe held for 60 seconds after the infusions hav...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Atropine</td>
 <td>Drug</td>
 <td>Fluka</td>
 <td>11330</td>
 <td>To inhibit saliva secretion</td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Drug</td>
 <td>Phoenix Scientific</td>
 <td>&nbsp;</td>
 <td>Sedation</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Drug</td>
 <td>Phoenix Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethylene tubing (0.58 mm diameter) </td>
 <td>Tube</td>
 <td>Intramedic PE-10, Becton Dickinson Diagnostic Systems</td>
 <td>427410</td>
 <td>To insert over tip of needle to avoid tissue injury during insertion into Wharton&#x2019;s duct.</td>
 </tr>
 <tr>
 <td>Ultra Comfort Insulin syringe (with a 29G needle) </td>
 <td>Syringe</td>
 <td>Tyco Healthcare</td>
 <td>600145</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4% trypan blue </td>
 <td>Stain </td>
 <td>Gibco</td>
 <td>15250 </td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct at JoVE.com

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton&#039;s Duct

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.

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 ...

cannulation-mouse-submandibular-salivary-gland-via-wharton-s

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25.9K Views.  McGill University. Un protocollo per l'incannulazione della ghiandola salivare sottomandibolare del mouse tramite il dotto di Wharton è descritto. Per questo esperimento, la soluzione trypan blu è usato come un tintore per dimostrare quanto questa tecnica offre efficacemente infusi nella ghiandola mirati, e di suggerire l'affidabilità di questo nuovo approccio clinico come un potenziale farmaco / terapia cellulare per la rigenerazione delle ghiandole salivari.

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

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

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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.

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. ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

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

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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.

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Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

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Laparoscopic Common Bile Duct Exploration in Patients with a Previous History of Biliary Tract Surgery

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Programmed surgery based on surgical methods and anatomical markers helps shorten the operation time, reduce complications, and improve the safety of the surgery. This study investigated and summarized the surgical methods and anatomic markers of laparoscopic common bile duct exploration in patients with a history of biliary tract surgery.

For recurrent choledocholithiasis, abdominal adhesions in previous surgeries lead to changes in anatomical structures, and a secondary injury occurs ...