Name: preparation of murine submandibular salivary gland for upright intravital microscopy
Uploaded: 2018-05-07T12:30:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

这项工作由瑞士国家基金会 (SNF) 项目赠款31003A_135649、31003A_153457 和 31003A_172994 (JVS) 提供资金, Leopoldina 2011-16 (至 BS)。这项工作得益于在伯尔尼大学的 "显微成像中心" (MIC) 的光学设置。

所有动物的工作都已得到州动物试验委员会的批准, 并根据联邦准则进行。
1. 麻醉鼠标
<ol><li>佩戴个人防护用品, 包括实验室外套和手套。</li>
	<li>将氯胺酮、甲苯噻嗪和生理盐水混合在一起, 其工作浓度分别为20毫克/毫升和1毫克/毫升。将工作库存腹腔 (ip) 注入8-10 µL/克鼠标。把鼠标放回笼子里。 注: 本协议已测试6至40周的男性和女性小鼠的 C57BL/6 背景。</li>
	<li>准备乙酰丙嗪溶液在2.5 毫克/毫升, 并注入30µL, 15 分钟后, 氯胺酮-甲苯噻嗪注射 (75 µg/小鼠)。</li>
	<li>5-15 分钟后, 检查是否有足够的麻醉和镇痛通过控制反射,例如, 撤回反射的爪子或角膜反射。</li>
	<li>控制的反射大约每30分钟在整个实验期间。皮下注射氯胺酮/甲苯噻嗪在1.5 µL/克鼠, 每60分钟或更早, 如果反射是阳性的。 注意: 由于老鼠的固定, 吸入性麻醉 (如异氟醚) 不能与此协议一起使用。</li>
</ol>2. 从操作区取出毛皮
<ol><li>使用电动剃刀刮胡子的区域, 大致从5毫米的嘴尾向前腿, 伸展在颌骨对两个耳朵。</li>
	<li>用棉签将脱毛膏涂抹在剃光区。经过3分钟的仔细和彻底清除,例如, 用湿纸巾擦拭。</li>
</ol>3. 将鼠标固定在舞台上
注: 本协议使用自定义阶段 (图 1), 配备两个可自由调节的盖子滑动架 (一个附在舞台上的螺钉, 另一个固定在它上);可调节的立体定向耳棒;可以用螺钉拧紧的绳子;可自由调节的金属加热环;和一个凸起的区域, 指定放置鼠标的躯干。这一阶段是从腘淋巴结阶段修改, 增加了立体定向支架和支持下盖滑动持有形象化 SMG。本阶段的详细计划或报价可根据要求提供。
<ol><li>通过拆卸加热环和可拆卸盖滑动架的支架来准备舞台。拧松耳棒支架和固定护盖滑动架的螺钉。把一块纱布贴到舞台的中心区域, 充当老鼠的床上用品。</li>
	<li>使用胶水 (请参阅材料表) 将20毫米直径的盖玻璃连接到圆柱形金属支架的顶部 (这将是下盖玻璃)。将25毫米的盖子玻璃粘附在扁金属架的底部 (这将是上盖玻璃)。确保盖眼镜与金属持有者重叠约2-3 毫米, 其余的玻璃仍然没有油脂和胶水。</li>
	<li>将鼠标放回纱布上, 垂直于伸展的绳子, 头部居中在耳棒之间。</li>
	<li>用手术胶带将鼠标的上肢固定在舞台上。</li>
	<li>接下来, 把绳子钩到前门牙上, 收紧, 直到下巴达到水平位置。使用一个有角的弯曲钳稍微拉出舌头从嘴, 这有助于呼吸。</li>
	<li>紧紧的, 但不是有力的, 拉尾巴和磁带到舞台上。</li>
	<li>将耳条对齐, 以在条形和舞台边缘之间达到近似的30°角。为了确保这个角度, 使用三角形标尺在一张纸上绘制一个右和左的30°角, 并将透明的舞台放在纸张上以相应地对齐。</li>
	<li>通过同时将左右杆向内移动, 直到下巴 immotile, 拧紧螺钉, 固定耳条之间的颚部。在固定下颚之前、期间和之后观察胸部运动。呼吸减少或缺席表明, 酒吧是固定在颈部, 而不是颚骨;在这种情况下, 请立即重复安装过程。</li>
	<li>给鼠标加热,例如, 在舞台下面放置一盏暖气灯和/或加热垫。</li>
</ol>4. 用显微镜手术暴露涎腺
注意: 以下步骤需要使用显微镜、细钳和手术剪刀。由于过程是终端, 没有必要保持无菌的操作条件。乙醇清洗仪器足够了。
<ol><li>找到右侧的 SMG 叶的近似位置, 寻找一个轻微的突起在皮肤大约15毫米尾骶部和5毫米内侧的颚线。抬起一个小的皮肤褶皱的中心隆起和削减它创造和开放约5毫米长度。</li>
	<li>立即在伤口周围的皮肤上涂上4毫米高的真空油脂环。(使用真空油脂填充注射器与钝25克皮下针)。</li>
	<li>用盐水填充油脂环, 以确保组织在手术过程中保持湿润。</li>
	<li>在显微镜下, 使用以下程序扰乱结缔组织: 用一个细钳牢牢地握住直接附着在 SMG 上的结缔组织, 然后用第二个钳把远端结缔组织拉到破裂。确保某些结缔组织仍然附着在 SMG 上;这是可取的, 因为它允许处理。不要用镊子触摸腺体本身, 因为这会导致出血。</li>
	<li>重复这种扰乱结缔组织的运动首先在腺体的顶端, 然后在内侧 (在 SMG 的右和左叶之间)。请勿将舌下与颌下腺分开。</li>
	<li>继续逆时针移动外侧结缔组织 (内侧至尾部, 到远端, 延髓)。</li>
	<li>确保血液供应, 传出淋巴管和涎腺导管, 这些都位于腺体的颅骨部位的捆绑, 没有破裂。</li>
	<li>一旦外侧结缔组织被破坏, 使用镊子把连接到 smg 尾端的结缔组织向上提起, 以解除 smg 的尾端。从尾部开始, 并向延髓端移动, 扰乱了 SMG 叶下方的结缔组织。</li>
	<li>继续, 直到所有的连接组织的所有地点的 SMG 被删除 (不包括延髓网站与主要喂养/排泄血管和 WD)。</li>
</ol>5. 固定腺体
<ol><li>确认含生理盐水的润滑脂环至少有4毫米高且未泄漏 (见步骤 4.2)。</li>
	<li>安装下盖玻璃 (见步骤 3.2), 通过附加金属棒到其高度可调的持有人。确保盖板滑动架调整到最高位置, 并在连接到舞台时不接触润滑脂环。将持有者从尾部方向定位到小鼠的角度, 其盖子滑动覆盖了露出的裂片的大约两个 (尾部和远端部分)。把盖子玻璃放低, 直到它与腺体接触。</li>
	<li>完成盖玻璃顶部的润滑脂环, 形成新的盐水库。</li>
	<li>小心地拉上的 SMG 瓣顶部的盖子滑与镊子, 只接触连接到它的结缔组织 (器官可以翻转与底部的网站面对向上)。</li>
	<li>确保润滑脂环的均匀高度至少为4毫米, 不泄漏。如有必要, 用30克填充生理盐水的注射器重新填充盐水水库。</li>
	<li>确保上盖玻璃的盖玻璃架处于其最高位置。使用螺钉将金属支架与 25 mm 上盖玻璃连接在支架上。使用可调节螺丝的持有人定位的封面玻璃, 使 SMG 在中间居中。</li>
	<li>使用可调支架螺钉, 将顶盖玻璃降低至直接与 SMG 接触。在此步骤中观察腺体的形状和着色。如果腺体表面积较大, 或者着色改变 (例如从粉红色到白色), 立即将盖玻璃置于更高的位置, 以减轻 SMG 的压力。</li>
	<li>在水库外寻找气泡和盐水, 检查盐水水库是否漏水。如果检测到泄漏或气泡, 请卸下盖板和所有油脂, 然后从步骤2重新启动。</li>
	<li>螺丝关闭所有固定螺钉。</li>
</ol>6. 固定加热环和温度探头
<ol><li>将温度探头插入储层并将其放置在靠近腺体的位置, 然后将探头导线贴到舞台上。</li>
	<li>将润滑脂环应用到顶盖滑动上, 与在中心的 SMG (润滑脂环的直径应对应于加热环的直径)。将加热环放在盖子滑动顶部, 用油脂密封。将加热环的油管胶带放到舞台上。</li>
</ol>7. 成像
注: 我们使用直立双光子显微镜系统, 配备可调谐 Ti: Sa 激光器和荧光显微镜与水浸泡20X 或25X 目标。通过使用自动阶段和 VivoFollow19, 可以实现在采集过程中对组织漂移进行实时校正。这提高了图像采集的稳定性, 但不是必需的。
<ol><li>使用显微镜制造商提供的适当软件打开直立显微镜系统。将 Ti: Sa 激光器调至 780-900 nm 的激发波长。</li>
	<li>将加热管连接至加热环。蠕动式水泵循环热水 (通过在80°c 水浴中浸没部分油管加热) 通过环来温暖 SMG。</li>
	<li>将温度探头连接到数字温度计。如果温度不介于摄氏35-39 摄氏度之间, 则调整蠕动泵的速度。泵的工作速度越快, 对象得到的温度越热, 反之亦然。</li>
	<li>在开始图像采集之前, 对血液流速进行视觉控制。仔细检查白细胞通过薄毛细血管或荧光葡聚糖注射液。 注: 静脉注射的荧光葡聚糖在血液流动是生理的时候, 会立即填满整个 SMG 的血管。这在这个过程中几乎总是如此。</li>
	<li>为了遵循免疫细胞, 成像深度通常介于20-150 µm 的 2nd谐波生成信号所识别的 SMG 表面之下。记录图像栈150-300 µm 的视野, 像素分辨率约 512 x 512 或更高, z 深度20-60 µm 与间距2-4 µm 之间的图像。每20-30 秒重复一次堆栈捕获, 总工期为20-60 分钟。</li>
	<li>使用数字温度计控制在获得一个图像序列后的最低和最高温度水平。如果最小或最大值不在37到2°c 的范围内, 则丢弃图像序列。在图像序列之间重新填充水以实现目标的浸入。</li>
	<li>在成像会话结束时, 弄死在麻醉下通过 CO2窒息老鼠。 注: 这是根据瑞士联邦动物实验指南;其他有关安乐死的准则可能适用于其他地方。</li>
</ol>

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(51), e3074 (2011).</li><li>Chen, G. Y., Nu&#241;ez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation:+Sensing+and+reacting+to+damage.">Sterile inflammation: Sensing and reacting to damage.</a> Nat Rev Immunol. 10 (12), 826-837 (2010).</li><li>McLaren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+causes+of+variation+of+body+temperature+in+mice.">Some causes of variation of body temperature in mice.</a> Q J Exp Physiol Cogn Med Sci. 46 (1), 38-45 (1961).</li><li>Baumgart, K., Wagner, F., Gr&#246;ger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+metabolic+effects+of+hypothermia+and+inhaled+hydrogen+sulfide+in+anesthetized+and+ventilated+mice.">Cardiac and metabolic effects of hypothermia and inhaled hydrogen sulfide in anesthetized and ventilated mice.</a> Crit Care Med. 38 (2), 588-595 (2010).</li><li>Crouch, A. 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L., Shakhar, G., Dudziak, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+dendritic+cell+networks+in+vivo.">Visualizing dendritic cell networks in vivo.</a> Nat Immunol. 5 (12), 1243-1250 (2004).</li><li>Riedl, J., Flynn, K. C., Raducanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact+mice+for+studying+F-actin+dynamics.">Lifeact mice for studying F-actin dynamics.</a> Nat Methods. 7 (3), 168-169 (2010).</li><li>Chtanova, T., Hampton, H. R., Waterhouse, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+interactive+two-photon+photoconversion+of+recirculating+lymphocytes+for+discontinuous+cell+tracking+in+live+adult+mice.">Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice.</a> J Biophotonics. 7 (6), 425-433 (2014).</li><li>Kyratsous, N. I., Bauer, I. J., Zhang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+context-dependent+calcium+signaling+in+encephalitogenic+T+cells+in+vivo+by+two-photon+microscopy.">Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.</a> Proc Natl Acad Sci U S A. 114 (31), E6381-E6389 (2017).</li><li>Mank, M., Reiff, D. F., Heim, N., Friedrich, M. W., Borst, A., Griesbeck, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys J. 90 (5), 1790-1796 (2006).</li><li>Tsyboulski, D., Orlova, N., Saggau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplitude+modulation+of+femtosecond+laser+pulses+in+the+megahertz+range+for+frequency-multiplexed+two-photon+imaging.">Amplitude modulation of femtosecond laser pulses in the megahertz range for frequency-multiplexed two-photon imaging.</a> Opt Express. 25 (8), 9435 (2017).</li><li>Potma, E. O., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single+lipid+bilayers+with+coherent+anti-Stokes+Raman+scattering+(CARS)+microscopy.">Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy.</a> J Raman Spectrosc. 34 (9), 642-650 (2003).</li></ol>

没有宣布利益冲突。

该协议提供了一个简单的方法,在体内成像的小鼠颌下腺和舌下涎腺使用直立非线性显微镜经常使用的免疫学领域。该方法可用于头颈区其他外分泌腺体的成像。例如, 我们的实验室以类似的方式对泪腺进行了成像 (不显示)。
除去在 SMG 周围的结缔组织是这个协议最关键的步骤, 因为意外的组织损伤可能发生。由于 smg 的组织会变得缺氧, 因此对 smg 的主要血液供应造成的?...

唾液由外分泌腺体分泌来润滑食物, 保护口腔粘膜表面, 并提供消化酶和抗菌物质1,2。除了在口腔黏膜下层散置的小涎腺外, 根据其位置1、2, 还有三个双侧的主要腺体被确定为腮腺、舌下和颌下腺。金字塔状上皮细胞, 组织成烧瓶状囊 (腺泡细胞) 或 demilunes 包围的上皮细胞和基底膜, 分泌的浆液和粘液成分的唾液1。腺泡细胞狭窄的腔内空间排水成夹层导管, 直到它们最终加入一个排泄管道1。SMG 的主要排泄管称为沃顿商学院的管道 (WD), 并打开到舌下阜3,4。因此, SMG 上皮隔间是一个高度 arborized 的结构, 具有流形终端端点, 类似于一束葡萄1,5,6。SMG 间质由血液和淋巴管组成, 内含于结缔组织7中, 内含副交感神经8和细胞外基质 5. 正常的人和啮齿动物的涎腺也含有 T 细胞, 巨噬细胞和树突状干细胞9, 以及分泌免疫球蛋白 A (IgA) 到唾液9,10的血浆细胞。由于其在健康和疾病方面的多方面作用, SMG 在许多生物研究领域都有兴趣, 包括牙科4、免疫学11、肿瘤学12、生理学8和细胞生物学3。
动态细胞过程和交互的成像是生物学研究中的一个强有力的工具13,14。基于非线性光学 (NLO) 的深部组织成像和创新 inmicroscopes 的发展, 它依赖于通过样本对多个光子的散射或吸收, 从而可以直接检查复杂组织中的细胞过程13 ,15。多光子的吸收涉及用低能光子传递总励磁能量, 这限制了荧光对焦平面的激发, 从而允许更深层的组织穿透, 减少了光损伤和噪声。励磁13,15。这一原则采用双光子显微镜 (2PM), 并允许在高达1毫米15,16深度的荧光标本上进行成像。虽然商用2PM 设置已变得方便和可靠, 活体成像的主要挑战是仔细暴露和稳定麻醉小鼠的靶器官, 特别是对时间失效序列的成像。数据获取后数字漂移校正的几种方法已发布17,18和我们最近开发了 "VivoFollow", 一个自动校正系统, 它抵消缓慢的组织漂移实时使用计算机化阶段19。然而, 对于高质量的成像, 要尽量减少组织运动, 尤其是呼吸或心跳导致的快速运动, 这仍然是至关重要的19。为多个器官 (包括脊髓20、肝脏21、皮肤22、肺23和淋巴结24) 发布了准备和稳定程序。此外, 大鼠涎腺成像模型已经开发了3,25和进一步完善的高分辨率活体成像的小鼠 SMG 量身定做的倒置显微镜设置26,27,28。
在这里, 我们提出了一个实用的和适应性的协议, 活体成像的小鼠的直立非线性显微镜, 这是普遍用于活体成像在免疫学领域。为此, 我们改良了一个广泛使用的固定化阶段用于腘淋巴结准备。

<table><tbody><tr><td>Narketan 10 %&amp;nbsp; (Ketamine) 20ml (100 mg/ml)</td><td>Vetoquinol</td><td>3605877535982</td><td></td></tr><tr><td>Rompun 2% (Xylazine) 25 ml (20 mg/ml)</td><td>Bayer</td><td>680538150144</td><td></td></tr><tr><td>Saline NaCl 0.9%</td><td>B. Braun</td><td>3535789</td><td></td></tr><tr><td>Prequillan 1% (Acepromazine) 10 ml (10 mg/ml)</td><td>Fatro</td><td>6805671900029</td><td></td></tr><tr><td>Electric shaver</td><td>Wahl</td><td>9818L</td><td>or similar</td></tr><tr><td>Hair removal cream</td><td>Veet</td><td>4002448090656</td><td></td></tr><tr><td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td><td>Durapore (3M)</td><td>1538-1</td><td></td></tr><tr><td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td><td>Durapore (3M)</td><td>1538-0</td><td></td></tr><tr><td>Super glue Ultra gel, instantaneous glue</td><td>Pattex, Henkel</td><td>4015000415040</td><td></td></tr><tr><td>Microscope cover glass slides 20 mm and 22 mm</td><td>Menzel-Gl&amp;auml;ser</td><td>631-1343/ 631-1344</td><td></td></tr><tr><td>Grease for laboratories 60 g glisseal N</td><td>Borer (VWR supplier)</td><td>DECO514215.00-CA15</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools (F.S.T )</td><td>14090-09</td><td>or similar</td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools (F.S.T )</td><td>11252-20</td><td>or similar</td></tr><tr><td>Cotton swab</td><td>Migros</td><td>617027988254</td><td>or similar</td></tr><tr><td>Gauze Gazin 5 x 5 cm</td><td>Lohmann and Rauscher</td><td>18500</td><td>or similar</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ16</td><td>or similar</td></tr><tr><td>Texas Red dextran 70kDa</td><td>&amp;nbsp;Molecular Probes</td><td>D1864</td><td></td></tr><tr><td>Cascade Blue dextran 10kDa</td><td>invitrogen</td><td>D1976</td><td></td></tr><tr><td>Two-photon system</td><td>LaVision Biotec</td><td>TrimScope I and II</td><td>or similar</td></tr><tr><td>XLUMPLANFL 20x/0.95 W objective</td><td>Olympus</td><td>n/a</td><td>or other water immersion objective&amp;nbsp;</td></tr><tr><td>Digital thermometer</td><td>Fluke</td><td>95969077651</td><td></td></tr></tbody></table>

preparation of murine submandibular salivary gland for upright intravital microscopy

颌下腺 (SMG) 是三大涎腺之一, 对许多不同领域的生物研究, 包括细胞生物学, 肿瘤学, 牙科和免疫学感兴趣。SMG 是由分泌上皮细胞、肌、内皮细胞、神经和细胞外基质组成的分泌腺。大鼠和小鼠的动态细胞过程以前已经被成像, 主要是使用倒置的多光子显微镜系统。在这里, 我们描述了一个简单的协议, 以手术准备和稳定麻醉小鼠在体内的在活体成像与直立多光子显微镜系统。我们提出了具有代表性的活体图像集内源性和过继转输转移荧光细胞, 包括标签的血管或唾液导管和第二次谐波生成, 以可视化纤维胶原。总之, 我们的协议允许手术制备小鼠涎腺在直立显微镜系统, 这是通常用于活体成像在免疫学领域。

该协议允许成像几乎所有的背部或腹侧的 SMG。视野通常也包括舌下涎腺, 这与在细胞成分中的 SMG 略有不同4。两个腺体由纤维胶原蛋白封装, 并细分成裂片。大多数2PM 系统可以通过测量 2nd谐波信号来产生纤维胶原的无标签图像, 但通常需要荧光分子来可视化细胞和亚型结构。例如, 绿色荧光蛋白 (GFP) 的无处不在的转基因表达与 2nd谐波信?...

Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

直立活体显微术制备小鼠颌下腺涎腺

我们描述了一个协议, 以手术揭露和稳定的小鼠颌下腺腺活体成像使用直立活体显微镜。该协议很容易适应小鼠和其他小啮齿目动物头部和颈部区域外分泌腺体。

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

preparation-murine-submandibular-salivary-gland-for-upright

Research

JoVE Journal

Immunology and Infection

8.1K 次观看.  University of Bern. 我们描述了一个协议, 以手术揭露和稳定的小鼠颌下腺腺活体成像使用直立活体显微镜。该协议很容易适应小鼠和其他小啮齿目动物头部和颈部区域外分泌腺体。

视频: Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

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

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. ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

生物学

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, ...

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

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

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

下颌骨和腮腺表型模型对组织型培养的放射治疗是机体活力特征的关键

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

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 ...

直立活体显微术制备小鼠颌下腺涎腺

摘要

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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

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

基因改造和重组唾液的腺体器官培养

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

下颌骨和腮腺表型模型对组织型培养的放射治疗是机体活力特征的关键

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