作者感谢技术咨询安娜Cotrim博士和博士Monzur设备Murshed。这项工作是由加拿大卫生研究院资助部分。

1。程序说明<ol><li>皮下注射每只小鼠注入1μL/ g体重0.5毫克/毫升硫酸阿托品水合物（＃11330猫，Fluka公司，美国），以防止破坏输注的运动唾液腺分泌物。 </li><li>麻醉注射1μL/ g的体重为100毫克/毫升氯胺酮和20毫克/毫升甲苯噻嗪（凤凰科学，AR，美国）解决方案的每鼠腹腔（知识产权） </li><li>在腹侧的位置上放置定做的塑料平台的鼠标被锁定在一个金属丝，上颌切牙和下颌切牙迷上弹性字符串，以保持口开放。 </li><li>使用胰岛素注射器内放置一个直径为0.58毫米的聚乙烯管（＃427410，猫，医学博士， 美国碧迪图2）（猫＃600145，泰科医疗，马萨诸塞州，美国）一个29号针头。聚乙烯管插入超过了29号针头的针尖5毫米。针是用来作为一个刚性的导轨移动到沃顿商学院的管聚乙烯管。聚乙烯管插入导管内3-5毫米。请注意，只有聚乙烯管进入唾液腺管，而针外管仍然。通过过滤0.4％台盼蓝染色解决方案（猫＃15250，GIBCO，美国）的沃顿商学院的管，这是位于下方的舌头50ul注入颌下腺。输液交付稳步正压注射器活塞，活塞上的手指仍然即使所有的输液后已交付的腺体，以防止回流输液，口服内。台盼蓝溶液是用于视频展示技术作为一种染料。 </li><li>后注射台盼蓝溶液，鼠标安乐死之前剥离。唾液腺手术暴露，以确定有多少的注入实际上仍然在腺体。 </li></ol> 2。代表性的成果: 后分别注射输液，鼠标解剖手术暴露后空心唾液腺。图像2所示，沃顿商学院的管暗着色提供的证据表明，台盼蓝溶液注入成功。 <img src="/files/ftp_upload/3074/3074fig1.jpg" alt="图1" /> 图1小鼠颌下腺的管子。 <img src="/files/ftp_upload/3074/3074fig2.jpg" alt="图2" /> 图2。聚乙烯管了29号针头的针尖周围包裹。作为一个刚性的导轨移动到沃顿商学院的管聚乙烯管注射器针头。聚乙烯管插入管道内（空心）3-5毫米。请注意，只有聚乙烯管进入唾液腺管，而针外管仍然。

<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>

所有的实验已经大学动物保健委员会批准，并按照加拿大动物保健委员会的指引。

使用此插管方法是一个困难的注入解决方案，以防止回流。台盼蓝溶液，即使暂时成功地迁移到唾液腺，唾液分泌可能会干扰与输液，造成回流的台盼蓝溶液腺体。作为一项预防措施，除使用阿托品抑制唾液的分泌，聚乙烯套管应保持在沃顿商学院的管道和60秒后举行的注入已存入的注射器活塞。这将使大部分的注入的解决方案保持腺体内。 按照惯例，全身应用，即静脉注射...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>阿托品</td><td>药物</td><td> Fluka公司</td><td> 11330 </td><td>为了抑制唾液分泌</td></tr><tr><td>氯胺酮</td><td>药物</td><td>凤凰科学</td><td></td><td>镇静</td></tr><tr><td>甲苯噻嗪</td><td>药物</td><td>凤凰科学</td><td></td><td></td></tr><tr><td>聚乙烯管材（直径0.58毫米） </td><td>管</td><td> Intramedic PE - 10，碧迪诊断系统</td><td> 427410 </td><td>要插入比针尖插入到沃顿商学院的管道，以避免在组织损伤。 </td></tr><tr><td>超舒适胰岛素注射器（带29G针） </td><td>注射器</td><td>泰科医疗</td><td> 600145 </td><td></td></tr><tr><td> 4％台盼蓝</td><td>污点</td><td> Gibco公司</td><td> 15250 </td><td></td></tr></tbody></table>

cannulation of the mouse submandibular salivary gland via the wharton's duct

在干燥综合征，并接受他们的头部和颈部区域，用于治疗癌症的治疗照射患者经常发现严重唾液腺功能减退症。两组患者出现症状，如口干（口干），吞咽困难（咀嚼和吞咽障），严重龋齿，味觉改变，口咽感染（念珠菌），口腔粘膜炎，疼痛和不适。 在RS雷德曼，2009年发送Mezey等再生医学的一个创新的方法治疗的唾液腺低功能推测:中，干细胞可以直接存入腺体由插管在恢复受损的功能的有力方法器官。据推测，移居国外的干细胞会分化成腺细胞作为主机唾液腺的一部分功能。此外，这种插管技术是一种快捷和有效地用于临床的基因转移中的应用的传递方法。 在这里，我们说明了沃顿商学院的管（ 图1）通过对小鼠颌下腺唾液腺在执行插管过程中所涉及的步骤。 C3H鼠（查尔斯河，蒙特利尔，QC，加拿大）用于本实验中，已清洁常规条件下保持在麦吉尔大学动物资源中心。所有的实验已经大学动物保健委员会批准，并按照加拿大动物保健委员会的指引。 在这个实验中，台盼蓝溶液注入腺体内，通过使用胰岛素注射器与一个29号针头包裹聚乙烯管内一个开放的沃顿商学院的管。随后，鼠标解剖显示，输注成功迁移进入腺体。

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's Duct

鼠标颌下唾液腺通过沃顿商学院的导管插管协议描述。在这个实验中，台盼蓝溶液作为一个代尔，证明这种技术如何有效地注入到有针对性的腺，并建议作为一个潜在的临床药物/唾液腺的再生细胞治疗这种新方法的可靠性。

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

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

Research

JoVE Journal

Medicine

26.0K 次观看.  McGill University. 鼠标颌下唾液腺通过沃顿商学院的导管插管协议描述。在这个实验中，台盼蓝溶液作为一个代尔，证明这种技术如何有效地注入到有针对性的腺，并建议作为一个潜在的临床药物/唾液腺的再生细胞治疗这种新方法的可靠性。

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

<meta charset="utf8"></head><body>小鼠模型中注射后的颈总动脉修复

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

<meta charset="utf8"></head><body>作者聚焦:通过溶瘤病毒和 MicroRNA 的动脉内递送策略推进胶质母细胞瘤治疗

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

科研

癌症研究

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 纳米微粒注射液对小鼠颌下腺的治疗

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

生物工程

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.

Retroductal颌下腺灌输和本地化分割照射在涎腺功能减退症大鼠模型

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

医学

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.

直立活体显微术制备小鼠颌下腺涎腺

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

免疫与感染

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

将套管植入啮齿动物的麦格纳池中

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

神经科学

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.

小鼠涎腺照射后通过匹属刺激进行唾液功能评估

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

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.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

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, ...

生物学

小鼠下颌下腺再生的实时实时成像

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.

作者聚焦:研究损伤后唾液腺再生中巨噬细胞-上皮细胞的相互作用 

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.

通过离体活细胞成像询问唾液腺中的细胞间相互作用

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

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

摘要

探索更多视频

此视频中的章节

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

Retroductal 纳米微粒注射液对小鼠颌下腺的治疗

Retroductal颌下腺灌输和本地化分割照射在涎腺功能减退症大鼠模型

直立活体显微术制备小鼠颌下腺涎腺

将套管植入啮齿动物的麦格纳池中

小鼠涎腺照射后通过匹属刺激进行唾液功能评估

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

通过离体活细胞成像询问唾液腺中的细胞间相互作用