Name: in vivo vascular permeability detection in mouse submandibular gland
Uploaded: 2022-08-04T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

这项研究得到了中国国家自然科学基金（31972908，81991500，81991502，81771093和81974151）和北京自然科学基金（资助7202082）的支持。

所有实验程序均经北京大学医学部动物研究伦理委员会批准，并符合《实验动物护理和使用指南》（NIH出版物第85-23号，1996年修订）。8-10周年龄组的雄性野生型（WT）小鼠用于本研究。实验动物经过精心治疗，以尽量减少它们的疼痛和不适。
1. 动物程序
<ol><li>准备和管理麻醉剂和示踪剂。<ol><li>在整个研究过程中保持无菌条件，并通过高压灭菌对器械进行灭菌?...

<ol>
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S., 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=Functional+dissociation+of+paracellular+permeability+and+transepithelial+electrical+resistance+and+disruption+of+the+apical-basolateral+intramembrane+diffusion+barrier+by+expression+of+a+mutant+tight+junction+membrane+protein.">Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.</a> The Journal of Cell Biology. 134 (4), 1031-1049 (1996).</li><li>Enis, D. R., 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=Induction,+differentiation,+and+remodeling+of+blood+vessels+after+transplantation+of+Bcl-2-transduced+endothelial+cells.">Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (2), 425-430 (2005).</li><li>Wang, X., 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=Application+of+digital+subtraction+angiography+in+canine+hindlimb+arteriography.">Application of digital subtraction angiography in canine hindlimb arteriography.</a> Vascular. 30 (3), 474-480 (2022).</li><li>Trani, M., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+in+the+control+of+vascular+permeability:+vascular+endothelial-cadherin+and+other+players.">New insights in the control of vascular permeability: vascular endothelial-cadherin and other players.</a> Current Opinion in Hematology. 22 (3), 267-272 (2015).</li><li>Viazzi, F., 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=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertension Research. 31 (5), 873-879 (2008).</li><li>Scheppke, L., 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=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> The Journal of Clinical Investigation. 118 (6), 2337-2346 (2008).</li><li>Blanchet, M. R., 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=Loss+of+CD34+leads+to+exacerbated+autoimmune+arthritis+through+increased+vascular+permeability.">Loss of CD34 leads to exacerbated autoimmune arthritis through increased vascular permeability.</a> Journal of Immunology. 184 (3), 1292-1299 (2010).</li><li>Egawa, G., Ono, S., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging+of+vascular+permeability+by+two-photon+microscopy.">Intravital Imaging of vascular permeability by two-photon microscopy.</a> Methods in Molecular Biology. 2223, 151-157 (2021).</li><li>Vestweber, D., Wessel, F., Nottebaum, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+the+regulation+of+leukocyte+extravasation+and+vascular+permeability.">Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.</a> Seminars in Immunopathology. 36 (2), 177-192 (2014).</li><li>Schulte, 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=Stabilizing+the+VE-cadherin-catenin+complex+blocks+leukocyte+extravasation+and+vascular+permeability.">Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.</a> The EMBO Journal. 30 (20), 4157-4170 (2011).</li><li>Uhl, B., 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=A+novel+experimental+approach+for+in+vivo+analyses+of+the+salivary+gland+microvasculature.">A novel experimental approach for in vivo analyses of the salivary gland microvasculature.</a> Frontiers in Immunology. 11, 604470 (2020).</li></ol>

维持和调节内皮屏障功能对于血管稳态至关重要。内皮细胞及其细胞间连接在维持和控制血管完整性方面起着关键作用12。血流、生长因子和炎症因子的剪切力可引起血管通透性的变化，从而参与高血压、糖尿病和自身免疫性疾病等全身性疾病的发生和发展13，14，15。唾液腺的血管分布和血流是所有器官和组?...

各种唾液腺产生唾液，唾液主要作为抵御感染的第一道防线并帮助消化，从而在口腔和整体健康中发挥重要作用1。血液供应对唾液腺分泌至关重要，因为它不断提供形成主要唾液的水、电解质和分子。由紧密连接（TJ）复合物调节的内皮屏障功能严格而微妙地限制了毛细血管的渗透，毛细血管对水，溶质，蛋白质甚至从循环血管移动到唾液腺组织的细胞都具有高度渗透性2，3。我们之前发现，响应胆碱能刺激而打开内皮TJs有助于唾液分泌，而内皮屏障功能的损害与干燥综合征4中下颌下腺（SMG）的分泌不足和淋巴细胞浸润相互关联。这些数据表明，对于多种唾液腺疾病，内皮屏障功能的贡献需要引起足够的重视。
双光子激光扫描显微镜是观察体内完整组织中细胞动力学的有力工具。该技术的优点之一是，当标本被近红外激发时，近红外光（NIR）比可见光或紫外光具有更深的组织穿透力，并且在适当的条件下不会对组织造成明显的光损伤5，6。事实上，唾液腺是一种非常均匀和浅表的组织，其中表面腺泡细胞距离腺体表面仅约 30 μm7，8。已经表明，活体共聚焦显微镜可以在亚细胞分辨率8下研究活小鼠唾液腺中的外分泌和肌动蛋白细胞骨架。然而，双光子激光扫描显微镜不仅具有传统共聚焦显微镜的优势，而且还可用于更清晰地检测更深的组织和图像。在这里，荧光标记的葡聚糖经常用作细胞旁通透性示踪剂，并且具有不同尺寸的优点，可用于测试TJ孔9的大小。本研究建立了一种活体实时双光子激光扫描显微镜技术，用于小鼠SMGs内皮屏障功能的原位评价。小鼠SMG中体内血管通透性检测的每个工作步骤在当前协议中描述。这是在小鼠SMG导管结扎模型中检测内皮屏障功能的示例。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-photon microscope (TCS-SP8 DIVE)</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 kDa FITC-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>70 kDa rhodamine B-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>R9379</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt tissue separation nickel</td>
			<td>Bejinghuabo Company</td>
			<td>NZW28</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile syringe</td>
			<td>Zhiyu Company</td>
			<td></td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>0253316</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Leica Application Suite X software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 1x</td>
			<td>Servicebio</td>
			<td>G4207-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Bejinghuabo Company</td>
			<td>M286-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>JITIAN Bio</td>
			<td>JT0781</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo vascular permeability detection in mouse submandibular gland

唾液在口腔和整体健康中起着重要作用。血管完整的内皮屏障功能使唾液分泌，而内皮屏障功能障碍与许多唾液腺分泌疾病有关。本协议描述了一种 体内 细胞旁通透性检测方法，以评估小鼠下颌下腺（SMG）中内皮紧密连接（TJ）的功能。首先，将具有不同分子量（4 kDa，40 kDa或70 kDa）的荧光标记葡聚糖注射到小鼠的角静脉中。之后，在双光子激光扫描显微镜下解剖单侧SMG并将其固定在定制的支架中，然后捕获血管，腺泡和导管的图像。利用该方法，监测不同大小示踪剂从血管到腺泡基底侧甚至穿过腺泡上皮进入导管的实时动态泄漏，以评估生理或病理生理条件下内皮屏障功能的改变。

按照协议，将单侧SMG连接到定制的支架上，并且腺体尽可能远离小鼠身体，以防止呼吸引起运动伪影。在显微镜下观察到血管中红细胞（黑点）的快速流动。在发现人工晶状体下的组织区域后，必须切换到操作显微镜软件。在对照组中，两种示踪剂都存在于小鼠SMG的血管中。特别是，由于其分子量小，FD4能够从血管泄漏到腺泡和导管的基底侧，从而清楚地描绘出腺泡和导管的形状（如<strong class="x...

Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

在本协议中，通过在双光子激光扫描显微镜下将不同分子加权荧光示踪剂注射到 体内 测试动物模型的角静脉中来评估下颌下腺（SMG）的内皮屏障功能。

体内 小鼠下颌下腺血管通透性检测

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

本协议描述了一种体内细胞旁通透性检测措施，以评估小鼠下颌下腺中紧密内皮连接的功能。然而，双光子激光扫描显微镜不仅具有传统共聚焦显微镜的优势，而且还可用于更清晰地检测更深的组织和图像。本实验为评估不同组织特别是动物表面组织和器官的血管通透性提供了很好的方法测量方法。首先选择合适的示踪剂，例如荧光素、异硫氰酸酯（标记的葡聚糖）和罗丹明B（标记的右旋糖酐）具有不同的激发发射光谱，以最大程度地减少渗透性测定中荧光信号之间的干扰。将痕量稀释到无菌磷酸盐缓冲盐水中，制成每毫升100毫克的储备液，并将等分试样储存在零下20摄氏度的避光处。麻醉8至10周龄雄性野生型小鼠后，轻轻握住小鼠的头部和颈部，使眼球的一侧略微突出。沿眼角以直角插入含有100微升荧光示踪剂溶液混合物的胰岛素注射器。注射后，快速将鼠标以仰卧姿势放在纸板上，四肢和头部贴上胶带。对于下颌下腺或SMG分离，在体视显微镜下，用一般组织剪刀切开颈部表皮，以暴露SMG的两侧。接下来，使用钝的组织分离针轻轻地将胶囊与腺体表面分开，而不会损坏腺体组织、血管和腺体结构。将定制的支架连接到负压装置上，并提前检查负压效果是否正确实现。将暴露的SMG轻轻放在定制的支架上，并通过负压吸力吸起组织，尽可能远离小鼠身体，以减少呼吸和其他条件引起的运动伪影。选择腺体部位后不久开始成像。沿Z轴获取连续图像，从腺体表面相距2微米，进入组织70至140微米，以生成三维结构。接下来，获取血管的延时成像以测量SMG中的血管通透性。在“资源管理器”对话框中，单击“添加新文件夹”并更改文件名。然后，返回到“流量获取”列。在 XY 中，格式为 512 x 512，速度为 400 赫兹。打开双向 X“，将缩放系数设置为 0.75 和 1.5 以进行缩放。接下来，在“采集模式”下选择XYT“获取延时成像。根据实验需要将持续时间设置为20秒，30分钟或更长。设置条件后，单击Live“以观察照片形成框中两个通道和重叠通道的血管成像并选择感兴趣的区域。点击 开始“开始成像。体内血管通透性测定，以及小鼠血管的三维图像。此处显示了下颌下腺。在对照组中，两种示踪剂都存在于SMG的血管中。由于其分子量小，FD4能够从血管泄漏到腺泡和导管的基底侧，从而清楚地描绘出腺泡和导管的形状。RD70分布在大血管和微型血管中。在导管结扎组中，FD4和RD70均外渗至腺泡基底侧，表明导管结扎可破坏内皮屏障功能，增加对大分子的通透性。此外，半定量FD4和RD70荧光强度结果证实了这些观察结果。与对照组相比，三维图像显示结扎组血管周围FD4和RD70的荧光更加模糊。仔细的SMG隔离和适当的放置以及显微镜是成功检测的必要先决条件。按照这些程序，可以通过结合红细胞的运动来计算血流速率，并且可以追踪荧光标记的血细胞的浸润。

Research

JoVE Journal

Biology

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Single-cell Suction Recordings from Mouse Cone Photoreceptors

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Evaluation of Mammary Gland Development and Function in Mouse Models

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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 ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

The Mouse Isolated Perfused Kidney Technique

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

在体内人细胞 Rb 蛋白 sumo 的检测与分析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

体内 小鼠下颌下腺血管通透性检测

体内小鼠下颌下腺血管通透性检测