该出版物中报告的研究得到国家牙科和颅面研究研究所 (NIDCR) 和国立卫生研究院全国癌症研究所 (R56 DE025098、UG3 DE027695 和 F30 CA206296) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。这项工作也得到了 NSF DMR 1206219 和 IADR 口腔护理奖 (2016) 创新的支持。
我们要感谢詹 Gavrity 在 IVIS 实验中的帮助。我们要感谢凯伦. 宾利在执行 EM 方面的投入和协助。我们要感谢裴翁对 IHC 的协助。我们要感谢马修·格尔斯在图准备方面的协助。我们要感谢伊莲 Smolock 博士和艾米莉. 吴对这篇手稿的批判性阅读。

下文概述的所有体内程序均由纽约罗切斯特大学动物资源大学委员会批准。
1. 准备
<ol><li>使用32G 颅内导管与线插入, 削减3厘米的油管形成一个斜角结束, 大约45°到长轴。确认导线的长度至少比油管长1厘米。</li>
	<li>将50µL PSMA 纳米粒子溶液 (图 1) 或其他注射材料加载到哈密尔顿注射器中。为减少注射过程中气压伤的几率, 请将导管管、鞘移除、注射器和排出死容积。</li>
	<li>检查注射液, 以确保纳米微粒完全溶解, 以防止导管梗阻后的管理。</li>
	<li>用0.1 毫克/毫升的方法制备阿托品溶液。 注: 由于阿托品是轻敏感和退化随着时间的推移, 这个解决方案应在注射日, 并保护从光, 直到管理。</li>
</ol>2. 进入和可视化导管入口点
<ol><li>用分析天平称 C57/BL6 小鼠。</li>
	<li>使用0.5 毫升注射器与 29G x ½ "针, 麻醉小鼠与腹腔注射无菌盐水溶液100毫克/千克氯胺酮和10毫克/千克甲苯噻嗪。当鼠标不再响应刺激时, 继续执行以下步骤, 通常在注入后5到10分钟内发生。 注: 此过程也可在异氟醚下执行, 但需要一个自定义的鼻锥, 允许进入口腔。</li>
	<li>为了防止在过程中的干燥, 应用润滑剂的眼睛, 并把鼠标放在一个容易的位置在一个自定义阶段。 注意: 为了保持口腔内程序的适当条件, 应在每次使用前对工具进行消毒或消毒。</li>
	<li>通过将上颌门牙固定在金属束上, 打开口腔, 并使用弹性带在下颌门牙后面施加下行张力 (图 2A)。</li>
	<li>在解剖显微镜下对齐鼠标, 使下颚的底座可视化。</li>
	<li>要扩大口腔, 使用一个定制的, 弯曲的钢牵引器, 以双边方式对颊粘膜施加张力。</li>
	<li>为了可视化颌下腺乳突, 用钝钳抓住并轻轻地从口中的舌上抬起舌头。 注: 乳突将出现在舌下的两个苍白突起 (图 2B)。</li>
	<li>为了缓解口腔内的可视化和进一步的操作, 在舌头和颊粘膜之间放置棉花。</li>
</ol>3. 导管插管和线路放置
<ol><li>采用细、弯钳, 用金属丝插入导管。为在插管过程中进行最佳手动控制, 请将油管与镊子的曲率对齐 (图 3A)。</li>
	<li>使用解剖显微镜, 将钳和导线移动到视野中。 注: 导线应从油管上凸出。</li>
	<li>轻轻地将压力压入一个下颌下腺的底部, 使用金属丝插入, 产生一个小的, 表面的, 粘膜穿刺 (0.076 毫米直径), 将方便以后进入导管管 (0.25 毫米直径)。如果遇到阻力, 用锋利的解剖剪刀在油管和导线的插入上切开新的斜面尖。</li>
	<li>进入后, 取出鞘, 并使用解剖显微镜, 确认穿刺部位是否存在唾液。避免对可能导致出血或损害导管完整性的鞘进行强力或突然的运动 (撤回或插入)。</li>
	<li>收回油管内的鞘 (图 3B)。</li>
	<li>为确保注射油管能适应沃顿商学院的管道开放, 插入含有鞘的油管作为先前穿刺的刚性指南 (图 2C)。 注: 如果不快速执行, 局部肿胀可能会阻止重新插入。</li>
	<li>为防止长期导管梗阻的背部压力, 请取出油管。检查, 以验证在显微镜下可见的开口, 可以看到在颌下腺的乳突。如果出现可见出血, 请将鞘和重试从3.2 步移到对立的颌下腺乳突上。</li>
	<li>不移动鼠标, 管理腹腔注射1毫克/千克阿托品溶液, 减少流涎过程中。等待 5-10 分钟。</li>
	<li>掌握注射器导管的末端, 并使用解剖显微镜 (图 3C) 插入孔内。如果遇到阻力, 切一个新的斜面端到油管和重试。</li>
	<li>一旦导管在颌下腺内, 慢慢地向前推进 3-5 毫米进入导管。从镊子上松开油管。</li>
	<li>为了改善导管与颌下腺之间的密封, 用纱布轻轻涂抹1分钟, 使界面干燥。</li>
	<li>检查以确认油管的位置在干燥过程中没有转移。</li>
</ol>4. 注塑
<ol><li>注射材料的速率为10µL/分. 检查以确认鼠标保持镇静, 并没有显示遇险迹象 (图 2D)。 注:15-50 µL 注射液耐受性好。注入较大的体积会导致气压伤。</li>
	<li>注射后, 保持注射器压力为5分钟, 以改善在沃顿商学院的管道和 SMG 的材料保留 (图 4)。定期检查下颌下腺, 以确保导管不出口导管口。</li>
	<li>使用细钳, 抓住并轻轻地从颌下腺的乳突中取出油管。 注意: 观察一些液体从乳突中出口是正常的。</li>
	<li>在将鼠标从舞台上移开之前, 从口腔中取出牵引器和棉。 注意: 动物不应被留在无人看管, 直到它恢复足够的意识, 以维持胸骨卧床。此外, 确保鼠标没有与其他老鼠住在一起, 直到完全恢复。</li>
</ol>5. 核查和分析
注意:体内成像系统 (IVIS) 可用于评估注射后荧光标记的纳米粒子的保留 (如在图 5中注射后显示1小时和24小时)。
<ol><li>为了更好地在 SMG 内通过皮肤可视化荧光信号, 可以通过剃须或使用化学脱毛去除 SMGs 上的腹毛。 注: 在安乐死后, SMG 组织也可以收获, 固定 (隔夜在4% 多聚甲醛), 并染色使用免疫组化, 以确认荧光的持久性标记 NP 一天后注射(图 6)。</li>
</ol>

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作者没有什么可透露的。

Retroductal 注射液对涎腺的局部药物传递至关重要。该技术在筛选治疗剂的条件, 包括干燥综合征和 RIH9,10,28的应用。通过 retroductal 注射液直接向 SMG 提供药物, 在降低靶向效应 (包括免疫激活11) 的潜在效果方面比系统性管理具有重要优势。在不积累周围组织的情况下, 最大限度地提高当地药物的输送能力, 也能使治疗试验在更广泛的剂量范围内进行, 而不是系统地实现。
我们提出这个协议, 与故障排除和质量检查步骤, 作为一个详细的和更新的方法, 通过沃顿商学院的管道提供聚合物纳米材料的小鼠 SMG20。例如, 正确使用导线指南有助于套管放置。 此外, 通过使用干印迹代替氰胶水在注射过程中保持套管到位, 减少了粘膜损伤的风险。此方法可用于处理具有一系列化合物的小鼠, 并且可以用相同的鼠标在多天内执行, 以评估重复管理11的 timecourse。
正常腺体分泌物将提供一个简单而直接的清除机制, 为过剩的有效载荷, 但这一策略应该通过仔细选择注射物质和阿托品剂量滴定来优化不同的应用。在这种情况下, NPs 在 SMG 中坚持至少24小时。通过使用能够进行药物加载的 NPs 或类似的纳米材料, 今后这项工作的应用包括克服溶解度限制, 否则将防止使用 retroductal 注入20,21测试疏水性剂。

涎腺功能障碍有许多病因, 包括 Sjögren 综合征, 自身免疫介导的功能性分泌组织丧失, 以及辐射诱发的 hyposalivation (RIH), 共同 sequella 头颈肿瘤放疗1。由于任一条件而导致的唾液功能丧失会诱发个人口腔和全身感染、蛀牙、消化和吞咽功能障碍、言语障碍和主要抑郁症1,2,3。因此, 生活质量显著受损, 干预措施仅限于缓解症状, 而不是治疗4。为了研究新的治疗方法在体内, 直接对涎腺进行生物活性化合物是有好处的。
Retroductal 注射液是一种很有价值的方法, 可以直接向唾液腺提供生物活性化合物, 并检测疾病、损伤或正常组织稳态下的疗效。三个主要唾液腺是腮腺 (PG), 颌下腺 (SMG) 和舌下 (SLG), 所有这些都空入口腔通过排泄导管。该小鼠的解剖允许通过插管的沃顿商学院的管道直接进入, 位于舌下的口底部5。插管后, 溶解药物可以直接管理到 SMG。在 retroductal 分娩后, 附加腺的扩散受周围组织胶囊的限制, 该囊调节与周围结构的材料交换6。smg 和它的导管是相似的结构在人, 并且经常被访问在 SMG 手术期间和 sialoendoscopy7。在人类和小鼠, PG 同样可以通过 Stensen 的导管在颊粘膜8。
在 RIH 的小鼠模型中, SMG retroductal 注射液用于提供治疗方法, 包括生长因子、原代细胞、腺病毒载体、细胞因子和抗氧化化合物, 以调节细胞对损伤的反应, 并减少由此产生的组织损伤5,9,10,11,12,13,14,15,16。retroductal 注射液的临床成功最显著的是对腺病毒载体的管理, 以直接表达的水道 (水通道蛋白 1;AQP1) 治疗头颈癌放疗后的患者17。
以前, 我们已经开发并显示了 retroductally 注射聚合物纳米 siRNA 系统的功效, 以保护唾液腺功能从 RIH11,18,19,20。作为我们过去工作的延伸, 在这里, 我们展示了我们的协议 retroductal SMG 注射使用一个荧光标记纳米粒子 (NP) 能够加载和交付否则不太可溶性药物21,22,23。
我们已经合成了 NP 从两嵌段共聚物组成的聚 (苯乙烯-alt-马来酸酐)-b 聚 (苯乙烯) (PSMA) 通过可逆添加链碎片 (筏) 聚合, 如前所述21。通过溶剂交换, 这些聚合物自发地自组装成胶束 NP 结构, 具有疏水性内部和亲水性外部的21。NPs 被标记与得克萨斯红色荧光允许验证 NP 交付入腺体, 不用牺牲动物。活体动物成像和 SMG 免疫组化显示在注射后1小时和1天。
此更新和可重现的插管协议应使其他人能够实现 retroductal 注射。我们预计, 这种精制技术将成为体内研究和治疗发展的关键24,25。

<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

涎腺治疗的两个共同目标是在自身免疫或辐射损伤后组织功能障碍的预防和治疗。通过局部向涎腺提供生物活性化合物, 可以安全地实现更大的组织浓度, 而不是系统性的管理。此外, 离体超腺堆积物的靶组织效应可以显著降低。在这方面, retroductal 注射液是一种广泛应用的方法, 以调查涎腺生物学和病理生理学。Retroductal 对生长因子、原代细胞、腺病毒载体和小分子药物的管理在损伤的设置中支持腺体功能。我们以前已经证明了 retroductally 注射纳米粒子-siRNA 策略在辐照后维持腺体功能的有效性。在这里, 一个高效和可重复的方法管理纳米材料的小鼠颌下腺通过沃顿的管道是详细 (图 1)。我们描述了进入口腔, 并概述了必要的步骤 cannulate 沃顿的管道, 并进一步的意见, 作为质量检查整个过程。

Retroductal 注射液可用于管理 NPs 到小鼠 SMG (图 1)。在这里, 我们提供了50µg PSMA NPs 标记与德州红荧光。
正确放置鼠标可以方便地访问和可视化口底 (图 2A-b)。颌下腺的乳突被确定为舌下的两个肉质突起。在插管 (图 2C) 和阿托品注射液之后, 注射器导管可以放入颌下腺乳突 (图 2D)。
为便于插管, 在下颌下腺的一小口穿刺首先使用导管管内的导线鞘 (图 3A)。完成此操作后, 鞘应在油管内缩回, 以作为刚性导轨, 同时进行较大的开口 (图 3B)。鞘的直径为0.076 毫米, 而导管管的外径为0.25 毫米。在这个更大的开口的创建之后, 附加到注射注射器上的预加载导管油管可以引导进入导管孔 (图 3C)。
注射后, 建议将注射器固定, 并保持注射压力。如果压力不被应用, 交付将是成功的, 虽然效率和重现性较低。这表明, 通过注射50µL 1% 甲苯胺蓝染料双边和观察微弱的染色在腺体没有保持压力后, 注射 (图 4)。
为了验证 NP 传递, IVIS 可以用于检测鼠标中的荧光信号, 这是一侧到注入区域 1 h post 管理 (图 5)。此方法可在不 euthanizing 鼠标的情况下进行确认, 并且可以纵向持续, 直到信号不再被检测到26、27。
为了确认在 24 h 以下注射后的 NP 持久性, 腺体可以切片, 并通过荧光成像查看。Aqp5 和 Krt5 IHC 分别标记了 SMG 的分泌和导管细胞, 并在两个车厢中显示 NPs (图 6)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57521/57521fig1.jpg"> 图 1.逆行注射示意图.导管插管和注射器放置后, 50 µL 1 毫克/毫升聚合 NP 溶液注入到 SMG。具有代表性的透射电子显微图像 (TEM) 显示单分散 (多分散性指数 = 0.2) NP 人口。<a href="/files/ftp_upload/57521/57521fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57521/57521fig2.jpg"> 图 2.逆行注射步骤.(A)通过将上颌和下颌门牙分开来访问口腔。(B)可视化口腔底部舌下的乳突 (盒装), 这标志着沃顿商学院管道的位置。(C)使用带插入线的导管, 轻轻地 cannulate 下颌下腺的基底。(D)插管后, 导管管可与注射器油管交换<a href="/files/ftp_upload/57521/57521fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57521/57521fig3.jpg"> 图 3.鞘导管插管的有效定位.(A)将油管与镊子的曲率对齐, 并在油管和导线上切割一个斜面末端, 最初刺穿舌下的乳突。(B)收回油管内的鞘, 使其在舌下的乳突内插入导管的刚性指南。(C)在先前制作的孔内插入导管导管 (鞘取出), 加入注射注射器。<a href="/files/ftp_upload/57521/57521fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57521/57521fig4.jpg"> 图 4.注射后保持注射器压力可以提高材料的保留率.在注射50µL 甲苯胺蓝后, 注射器压力要么维持5分钟 (右 retroductal 第一次注射), 要么注射器被立即撤回 (左 smg 第二次注射)。<a href="/files/ftp_upload/57521/57521fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57521/57521fig5.jpg"> 图 5.retroductal NP 后注入的确认.(A)体内成像系统 (IVIS) 显示了红色荧光信号的侧向性 (左) 侧的小鼠 1 h 后注射。(B) NP IVIS 信号在24小时内显著降低。<a href="/files/ftp_upload/57521/57521fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57521/57521fig6.jpg"> 图 6.确认 retroductal NP 持久性 24 h 后注入.A、C。Uninjected 控制 Aqp5 和 Krt5 染色, 分别标记分泌腺泡和导管细胞。B、D。在 retroductal NP 注入的 SMG, Aqp5 和 Krt5 污渍显示正常腺体形态和 NPs 占去了腺泡细胞和管道 (鳞片酒吧:75 µm)。<a href="/files/ftp_upload/57521/57521fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</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

局部药物向颌下腺的传递对了解涎腺生物学和新疗法的发展有很浓厚的兴趣。我们提出了一个更新和详细的 retroductal 注射协议, 旨在提高交付准确性和实验再现性。本文提出的应用是高分子纳米粒子的传递。

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.0K 次观看.  University of Rochester. 局部药物向颌下腺的传递对了解涎腺生物学和新疗法的发展有很浓厚的兴趣。我们提出了一个更新和详细的 retroductal 注射协议, 旨在提高交付准确性和实验再现性。本文提出的应用是高分子纳米粒子的传递。

视频: 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.

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 ...

科研

医学

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.

可生物降解聚合物颗粒的淋巴内结注射

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

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. ...

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

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

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.

导管内注射局部给药的小鼠乳腺

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.

逆行帕罗蒂德格兰德输液通过斯滕森的管道在非人类灵长类病原体矢量基因交付

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

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 ...

Retroductal 纳米微粒注射液对小鼠颌下腺的治疗

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此视频中的章节

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

可生物降解聚合物颗粒的淋巴内结注射

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

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

导管内注射局部给药的小鼠乳腺

逆行帕罗蒂德格兰德输液通过斯滕森的管道在非人类灵长类病原体矢量基因交付

通过离体活细胞成像询问唾液腺中的细胞间相互作用

通过离体活细胞成像询问唾液腺中的细胞间相互作用

通过离体活细胞成像询问唾液腺中的细胞间相互作用