体内 小鼠下颌下腺血管通透性检测
1Department of Physiology and Pathophysiology, Peking University School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences,Ministry of Education, and Beijing Key Laboratory of Cardiovascular Receptors Research, 2Department of Oral and Maxillofacial Surgery,Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Reseah Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, 3State Key Laboratory of Natural and Biomimetic Drugs,Peking University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 准备和管理麻醉剂和示踪剂。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?…
Representative Results
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…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2-photon microscope (TCS-SP8 DIVE) | Leica, Germany | ||
| 4 kDa FITC-labeled dextran | Sigma Aldrich | 46944 | |
| 70 kDa rhodamine B-labeled dextran | Sigma Aldrich | R9379 | |
| Blunt tissue separation nickel | Bejinghuabo Company | NZW28 | |
| Depilatory cream | Veet | ||
| Disposable sterile syringe | Zhiyu Company | 1 mL | |
| Image J software | National Institutes of Health | ||
| Insulin syringe | Becton, Dickinson and Company | 0253316 | 1 mL |
| Leica Application Suite X software | Leica Microsystems | ||
| Microtubes | Axygen | MCT-150-C | 1.5 mL |
| Phosphate buffered saline 1x | Servicebio | G4207-500 | |
| Tissue scissors | Bejinghuabo Company | M286-05 | |
| Tribromoethanol | JITIAN Bio | JT0781 |
Riferimenti
- Carpenter, G. H. The secretion, components, and properties of saliva. Annual Review of Food Science and Technology. 4, 267-276 (2013).
- Garrett, J. R. The proper role of nerves in salivary secretion: A review. Journal of Dental Research. 66 (2), 387-397 (1987).
- Berndt, P., et al. Tight junction proteins at the blood-brain barrier: Far more than claudin-5. Cellular and Molecular Life Sciences. 76 (10), 1987-2002 (2019).
- Cong, X., et al. Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sjögren’s syndrome. Biochimica et Biophysica Acta – Molecular Basis of Disease. 1864 (10), 3154-3163 (2018).
- Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nature Methods. 2 (12), 932-940 (2005).
- Zipfel, W. R., Williams, R. M., Webb, W. W. Nonlinear magic: Multiphoton microscopy in the biosciences. Nature Biotechnology. 21 (11), 1369-1377 (2003).
- Masedunskas, A., Sramkova, M., Weigert, R. Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents. Bioarchitecture. 1 (5), 225-229 (2011).
- Masedunskas, A., et al. Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy. Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13552-13557 (2011).
- Balda, M. S., et al. 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. The Journal of Cell Biology. 134 (4), 1031-1049 (1996).
- Enis, D. R., et al. Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells. Proceedings of the National Academy of Sciences of the United States of America. 102 (2), 425-430 (2005).
- Wang, X., et al. Application of digital subtraction angiography in canine hindlimb arteriography. Vascular. 30 (3), 474-480 (2022).
- Trani, M., Dejana, E. New insights in the control of vascular permeability: vascular endothelial-cadherin and other players. Current Opinion in Hematology. 22 (3), 267-272 (2015).
- Viazzi, F., et al. Vascular permeability, blood pressure, and organ damage in primary hypertension. Hypertension Research. 31 (5), 873-879 (2008).
- Scheppke, L., et al. Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits. The Journal of Clinical Investigation. 118 (6), 2337-2346 (2008).
- Blanchet, M. R., et al. Loss of CD34 leads to exacerbated autoimmune arthritis through increased vascular permeability. Journal of Immunology. 184 (3), 1292-1299 (2010).
- Egawa, G., Ono, S., Kabashima, K. Intravital Imaging of vascular permeability by two-photon microscopy. Methods in Molecular Biology. 2223, 151-157 (2021).
- Vestweber, D., Wessel, F., Nottebaum, A. F. Similarities and differences in the regulation of leukocyte extravasation and vascular permeability. Seminars in Immunopathology. 36 (2), 177-192 (2014).
- Schulte, D., et al. Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability. The EMBO Journal. 30 (20), 4157-4170 (2011).
- Uhl, B., et al. A novel experimental approach for in vivo analyses of the salivary gland microvasculature. Frontiers in Immunology. 11, 604470 (2020).
Citazione di questo articolo
Mao, X., Min, S., He, Q., Cong, X. In Vivo Vascular Permeability Detection in Mouse Submandibular Gland. J. Vis. Exp. (186), e64167, doi:10.3791/64167 (2022).