直立活体显微术制备小鼠颌下腺涎腺
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 佩戴个人防护用品, 包括实验室外套和手套。 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。 准备乙酰…
Representative Results
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?…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Narketan 10 % (Ketamine) 20ml (100 mg/ml) | Vetoquinol | 3605877535982 | |
| Rompun 2% (Xylazine) 25 ml (20 mg/ml) | Bayer | 680538150144 | |
| Saline NaCl 0.9% | B. Braun | 3535789 | |
| Prequillan 1% (Acepromazine) 10 ml (10 mg/ml) | Fatro | 6805671900029 | |
| Electric shaver | Wahl | 9818L | or similar |
| Hair removal cream | Veet | 4002448090656 | |
| Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m) | Durapore (3M) | 1538-1 | |
| Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m) | Durapore (3M) | 1538-0 | |
| Super glue Ultra gel, instantaneous glue | Pattex, Henkel | 4015000415040 | |
| Microscope cover glass slides 20 mm and 22 mm | Menzel-Gläser | 631-1343/ 631-1344 | |
| Grease for laboratories 60 g glisseal N | Borer (VWR supplier) | DECO514215.00-CA15 | |
| Surgical scissors | Fine Science Tools (F.S.T ) | 14090-09 | or similar |
| Fine Forceps | Fine Science Tools (F.S.T ) | 11252-20 | or similar |
| Cotton swab | Migros | 617027988254 | or similar |
| Gauze Gazin 5 x 5 cm | Lohmann and Rauscher | 18500 | or similar |
| Stereomicroscope | Leica | MZ16 | or similar |
| Texas Red dextran 70kDa | Molecular Probes | D1864 | |
| Cascade Blue dextran 10kDa | invitrogen | D1976 | |
| Two-photon system | LaVision Biotec | TrimScope I and II | or similar |
| XLUMPLANFL 20x/0.95 W objective | Olympus | n/a | or other water immersion objective |
| Digital thermometer | Fluke | 95969077651 |
Riferimenti
- Pakurar, A. S., Bigbee, J. W. Digestive System. Digital Histology. , 101-121 (2005).
- Carpenter, G. Role of Saliva in the Oral Processing of Food. Food Oral Processing. , 45-60 (2012).
- Masedunskas, A., Weigert, R. Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents. Traffic. 9 (10), 1801-1810 (2008).
- Amano, O., Mizobe, K., Bando, Y., Sakiyama, K. Anatomy and histology of rodent and human major salivary glands. Acta Histochem Cytochem. 45 (5), 241-250 (2012).
- Sequeira, S. J., Larsen, M., DeVine, T. Extracellular matrix and growth factors in salivary gland development. Front Oral Biol. 14, 48-77 (2010).
- Takeyama, A., Yoshikawa, Y., Ikeo, T., Morita, S., Hieda, Y. Expression patterns of CD66a and CD117 in the mouse submandibular gland. Acta Histochem. 117 (1), 76-82 (2015).
- Hata, M., Ueki, T., Sato, A., Kojima, H., Sawa, Y. Expression of podoplanin in the mouse salivary glands. Arch Oral Biol. 53 (9), 835-841 (2008).
- Proctor, G. B., Carpenter, G. H. Regulation of salivary gland function by autonomic nerves. Auton Neurosci. 133 (1), 3-18 (2007).
- Le, A., Saverin, M., Hand, A. R. Distribution of Dendritic Cells in Normal Human Salivary Glands. Acta Histochem Cytochem. 44 (4), 165-173 (2011).
- Hofmann, M., Pircher, H. E-cadherin promotes accumulation of a unique memory CD8 T-cell population in murine salivary glands. Proc Natl Acad Sci. 108 (40), 16741-16746 (2011).
- Bombardieri, M., Barone, F., Lucchesi, D., et al. Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice. J Immunol. 189 (7), 3767-3776 (2012).
- Szwarc, M. M., Kommagani, R., Jacob, A. P., Dougall, W. C., Ittmann, M. M., Lydon, J. P. Aberrant activation of the RANK signaling receptor induces murine salivary gland tumors. PLoS One. 10 (6), e0128467 (2015).
- Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A. Intravital microscopy: A novel tool to study cell biology in living animals. Histochem Cell Biol. 133 (5), 481-491 (2010).
- Masedunskas, A., Milberg, O., Porat-Shliom, N., et al. Intravital microscopy. Bioarchitecture. 2 (5), 143-157 (2012).
- Zipfel, W. R., Williams, R. M., Webb, W. W. Nonlinear magic: Multiphoton microscopy in the biosciences. Nat Biotechnol. 21 (11), 1369-1377 (2003).
- Theer, P., Hasan, M. T., Denk, W. Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al_2O_3 regenerative amplifier. Opt Lett. 28 (12), 1022 (2003).
- Gomez-Conde, I., Caetano, S. S., Tadokoro, C. E., Olivieri, D. N. Stabilizing 3D in vivo intravital microscopy images with an iteratively refined soft-tissue model for immunology experiments. Comput Biol Med. 64, 246-260 (2015).
- Parslow, A., Cardona, A., Bryson-Richardson, R. J. Sample drift correction following 4D confocal time-lapse imaging. J Vis Exp. (86), (2014).
- Vladymyrov, M., Abe, J., Moalli, F., Stein, J. V., Ariga, A. Real-time tissue offset correction system for intravital multiphoton microscopy. J Immunol Methods. 438, 35-41 (2016).
- Haghayegh Jahromi, N., Tardent, H., Enzmann, G., et al. A novel cervical spinal cord window preparation allows for two-photon imaging of T-Cell interactions with the cervical spinal cord microvasculature during experimental autoimmune encephalomyelitis. Front Immunol. 8, 406 (2017).
- Heymann, F., Niemietz, P. M., Peusquens, J., et al. Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice. J Vis Exp. (97), e52607 (2015).
- Gaylo, A., Overstreet, M. G., Fowell, D. J. Imaging CD4 T cell interstitial migration in the inflamed dermis. J Vis Exp. (109), e53585 (2016).
- Looney, M. R., Thornton, E. E., Sen, D., Lamm, W. J., Glenny, R. W., Krummel, M. F. Stabilized imaging of immune surveillance in the mouse lung. Nat Methods. 8 (8), 91-96 (2011).
- Liou, H. L. R., Myers, J. T., Barkauskas, D. S., Huang, A. Y. Intravital imaging of the mouse popliteal lymph node. J Vis Exp. (60), e3720 (2012).
- Sramkova, M., Masedunskas, A., Parente, L., Molinolo, A., Weigert, R. Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals. Am J Physiol Cell Physiol. 297 (6), C1347-C1357 (2009).
- Masedunskas, A., Porat-shliom, N., Tora, M., Milberg, O., Weigert, R. Intravital microscopy for imaging subcellular structures in live mice expressing fluorescent proteins. J Vis Exp. (79), e50558 (2013).
- Masedunskas, A., Sramkova, M., Parente, L., et al. Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy. Proc Natl Acad Sci. 108 (33), 13552-13557 (2011).
- Milberg, O., Shitara, A., Ebrahim, S., et al. Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals. J Cell Biol. 216 (7), 1925-1936 (2017).
- Kuriki, Y., Liu, Y., Xia, D., et al. Cannulation of the mouse submandibular salivary gland via the Wharton’s duct. J Vis Exp. (51), e3074 (2011).
- Chen, G. Y., Nuñez, G. Sterile inflammation: Sensing and reacting to damage. Nat Rev Immunol. 10 (12), 826-837 (2010).
- McLaren, A. Some causes of variation of body temperature in mice. Q J Exp Physiol Cogn Med Sci. 46 (1), 38-45 (1961).
- Baumgart, K., Wagner, F., Gröger, M., et al. Cardiac and metabolic effects of hypothermia and inhaled hydrogen sulfide in anesthetized and ventilated mice. Crit Care Med. 38 (2), 588-595 (2010).
- Crouch, A. C., Manders, A. B., Cao, A. A., Scheven, U. M., Greve, J. M. Cross-sectional area of the murine aorta linearly increases with increasing core body temperature. Int J Hyperth. , 1-13 (2017).
- Smith, C. J., Caldeira-Dantas, S., Turula, H., Snyder, C. M. Murine CMV infection induces the continuous production of mucosal resident T cells. Cell Rep. 13 (6), 1137-1148 (2015).
- Lindquist, R. L., Shakhar, G., Dudziak, D., et al. Visualizing dendritic cell networks in vivo. Nat Immunol. 5 (12), 1243-1250 (2004).
- Riedl, J., Flynn, K. C., Raducanu, A., et al. Lifeact mice for studying F-actin dynamics. Nat Methods. 7 (3), 168-169 (2010).
- Chtanova, T., Hampton, H. R., Waterhouse, L. A., et al. Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice. J Biophotonics. 7 (6), 425-433 (2014).
- Kyratsous, N. I., Bauer, I. J., Zhang, G., et al. Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy. Proc Natl Acad Sci U S A. 114 (31), E6381-E6389 (2017).
- Mank, M., Reiff, D. F., Heim, N., Friedrich, M. W., Borst, A., Griesbeck, O. A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change. Biophys J. 90 (5), 1790-1796 (2006).
- Tsyboulski, D., Orlova, N., Saggau, P. Amplitude modulation of femtosecond laser pulses in the megahertz range for frequency-multiplexed two-photon imaging. Opt Express. 25 (8), 9435 (2017).
- Potma, E. O., Xie, X. S. Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy. J Raman Spectrosc. 34 (9), 642-650 (2003).
Citazione di questo articolo
Ficht, X., Thelen, F., Stolp, B., Stein, J. V. Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy. J. Vis. Exp. (135), e57283, doi:10.3791/57283 (2018).