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Medicine

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Published: May 06, 2021
doi:
10.3791/62538
,
1Department of Emergency Medicine,Seoul National University Bundang Hospital, 2Graduate School of Medical Science and Engineering,Korea Advanced Institute of Science and Technology, 3KI for Health Science and Technology,Korea Advanced Institute of Science and Technology

Summary

胰 腺的体内高分辨率成像利用胰腺内成像窗口获得便利。

Abstract

在活体小动物模型中，胰腺的直接 体内 细胞分辨率成像在技术上具有挑战性。最近的一项活体成像研究，带有腹部成像窗口，使 体内腹部器官中的细胞动力学可视化。然而，由于小鼠胰腺的软板状结构很容易受到生理运动（如持久性和呼吸）的影响，因此很难在细胞水平上在几周内进行稳定的 纵向体内 成像，以识别、跟踪和量化小鼠胰腺中的胰岛或癌细胞。在此，我们描述了一种植入新颖支撑基座的方法，一个集成的胰腺内成像窗口，可以在空间上将胰腺与肠道分离，用于胰腺微结构的纵向延时活性成像。带成像窗口 的纵向体内 成像可实现稳定的可视化，从而在 3 周内跟踪小岛，并实现微观结构的高分辨率三维成像，这一点在正位胰腺癌模型中就证明了这一点。通过我们的方法，进一步的活体成像研究可以阐明细胞水平上涉及胰腺的各种疾病的病理生理学。

Introduction

胰腺是一种腹部器官，在消化道中具有外分泌功能，内分泌激素在血液中。胰腺的高分辨率细胞成像可以揭示涉及胰腺的各种疾病的病理生理学，包括胰腺炎、胰腺癌和糖尿病1。传统的诊断成像工具，如计算机断层扫描，磁分辨率成像和超声波学在临床领域广泛提供1，2。然而，这些成像模式仅限于可视化只有结构或解剖变化，而改变在细胞或分子水平无法确定。鉴于人类糖尿病或胰腺癌的分子变化可在诊断前10年以上启动，因此，在潜伏期从分子过渡中发现胰腺疾病有可能提供早期诊断和及时干预。因此，通过提供胰腺癌的早期诊断或在糖尿病5进展期间对胰岛变化的提前识别，能够克服分辨率的局限性并提供对功能的宝贵见解的成像将显著获得关注。

特别是小岛，核成像、生物发光成像和光学相干断层扫描被推荐为非侵入性胰岛成像技术6。然而，这些方法的分辨率非常低，典型值从几十微米到几百微米不等，因此检测胰岛细胞水平变化的能力有限。另一方面，以前对小岛的高分辨率研究是在前体7、8（例如， 胰腺切片或消化）、非生理9（例如胰腺外化）和异位条件10、11、12（例如，植入肾胶囊下、肝脏内和眼睛前室），这限制了其解释和临床影响。如果能建立高分辨率成像的体内、生理学和矫形模型，将成为胰岛研究的重要平台。

活体动物的体内成像揭示了一种微观分辨率的病理生理学，最近受到人们的极大关注。 在体内 成像方法中，腹部成像窗口14的开发，将一个窗口植入老鼠的腹部，使得发现了新的发现（即早期肝转移15 的微观美斯塔西阶段和肠道上皮16的干细胞维持机制）。虽然腹部成像窗口提供了宝贵的结果，但这一窗口对胰腺的应用以及基于涉及胰腺的疾病的活体成像研究尚未得到广泛调查。

与人类胰腺的明确定义的实体器官特征不同，小鼠的胰腺是一种分布分散的软组织状结构17。因此，它不断受到生理运动的影响，包括腹膜和呼吸。先前关于为胰腺应用腹部成像窗口的研究表明，流浪是由于排便引起的运动伪影引起的。在生成的平均图像中观察到严重的模糊，这妨碍了微尺度结构的可视化和识别。

在此，我们描述了使用一种新的支持基础集成胰腺内成像窗口结合活体显微镜19，20调查纵向细胞水平事件涉及胰腺的疾病。除了对上一研究18中的方法进行详细描述外，本文还将探讨胰腺成像窗口对涉及胰腺的各种疾病的扩展应用。在此协议中，定制的视频速率激光扫描共聚焦显微镜系统被用作活体显微镜系统。四个激光模块（波长为 405、488、561 和 640 nm）用作激发源，光磁体管 （PMT） 通过带通滤波器 （BPF1： FF01-442/46） 检测到四个发射信号通道：BPF2： FF02-525/50;BPF3： FF01-600/37;BPF4： FF01-685/40）。激光扫描由旋转多边形镜 （X 轴） 和镀锌仪扫描镜 （Y 轴） 组成，使视频速率扫描（每秒 30 帧）成为可能。关于活体显微镜的详细资料在以前的研究中已经描述了10，18，19，20，21，22，23。

在我们之前的小岛研究18中，我们使用转基因小鼠模型（MIP-GFP）24成功地稳定地对活鼠中的胰岛进行了成像，其中小岛被标记为GFP。该方法使小岛的变化在1周内实现了高分辨率可视化。它还有助于对同一小岛进行长达3周的成像，这表明在糖尿病18的发病机制期间，对胰岛进行长期研究以进行功能跟踪或监测的可行性。此外，我们开发了一个正位胰腺癌模型，其中荧光胰腺癌细胞（PANC-1 NucLightRed）25被直接植入小鼠的胰腺。利用胰腺内成像窗口，该模型可作为研究胰腺癌肿瘤微环境细胞和分子病理生理学以及新型药物候选物的治疗监测的平台。

Protocol

本文描述的所有程序均按照《实验室动物护理和使用指南》（2011年 ）第8版（2011年）26 日进行，并经韩国高等科学技术研究院（KAIST）和首尔国立大学邦当医院（SNUBH）机构动物护理和使用委员会批准。 1. 准备窗户和其他材料 定制设计胰腺内成像窗口，使胰腺与腹腔内的肠道分离18（图1A，B）。<…

Representative Results

活体显微镜结合支持基础集成胰腺内成像窗口，使小鼠胰腺的纵向细胞水平成像。此与胰腺内成像窗口的协议提供了长期组织稳定性，使高分辨率成像的获取能够跟踪单个小岛长达 3 周。因此，可以实现扩展视野的马赛克成像、Z 堆栈成像的三维 （3D） 重建以及同一位置的纵向跟踪。此外，我们的活体显微镜提供四个通道（405、488、561 和 647 nm）的采集，从而能够同时实现多个细胞可视化及其相?…

Discussion

此处描述的协议包括胰腺的活体成像，使用从腹部成像窗口修改的新型支持基础集成胰腺内成像窗口。在上述协议中，第一个关键步骤是将活体胰腺成像窗口植入小鼠体内。对于胶水在窗口中的应用，在窗口边缘和盖玻璃之间涂抹胶水很重要，但不要涂在胰腺组织上，因为它可能会显著中断活体成像。如果胶水直接涂在组织上，不仅玻璃和组织之间的胶水本身，而且辅助的灰尘颗粒也会在成像过?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了SNUBH研究基金第14-2020-002号赠款和韩国政府资助的韩国国家研究基金会（NRF-2020R1F1A1058381，NRF-2020R1A1A2C3005694）的支持。

Materials

Alexa Fluor 647 Succinimidyl Esters (NHS esters) Invitrogen A20006 Fluorescent probe for conjugate with antibody
BALB/C Nude OrientBio BALB/C Nude BALB/C Nude
BD Intramedic polyethylene tubing BD Biosciences 427401 PE10 catheter for connection with needle
C57BL/6N OrientBio C57BL/6N C57BL/6N
Cover glasses circular Marienfeld 0111520 Cover glass for pancreatic imaging window
FITC Dextran 2MDa Merck (Former Sigma Aldrich) FD200S For vessel identification
IMARIS 8.1 Bitplane IMARIS Image processing
Intravital Microscopy IVIM tech IVM-C Intravital Microscopy
IRIS Scissor JEUNGDO BIO & PLANT CO, LTD S-1107-10 This product can be replaced with the product from other company
Loctite 401 Henkel 401 N-butyl cyanoacrylate glue
Micro Needle holder JEUNGDO BIO & PLANT CO, LTD H-1126-10 This product can be replaced with the product from other company
Micro rectractor JEUNGDO BIO & PLANT CO, LTD 17004-03 This product can be replaced with the product from other company
Microforceps JEUNGDO BIO & PLANT CO, LTD F-1034 This product can be replaced with the product from other company
MIP-GFP The Jackson Laboratory 006864 B6.Cg-Tg(Ins1-EGFP)1Hara/J
Nylon 4-0 AILEE NB434 Non-Absorbable Suture
Omnican N 100 30G B BRAUN FT9172220S For Vascular Catheter, Use only Needle part
PANC-1 NucLightRed Custom-made Custom-made Made in laboratory
Pancreatic imaging window Geumto Engineering Custom order Pancreatic imaging window – custom order
Physiosuite Kent Scientific PS-02 Homeothermic temperature controller
Purified NA/LE Rat Anti-Mouse CD31 BD Biosciences 553708 Antibody for in vivo vessel labeling
Ring Forceps JEUNGDO BIO & PLANT CO, LTD F-1090-3 This product can be replaced with the product from other company
Rompun Bayer Rompun Anesthetic agent
TMR Dextran 65-85kDa Merck (Former Sigma Aldrich) T1162 For vessel identification
Window holder Geumto Engineering Custom order Window holder – custom order
Zoletil Virbac Zoletil 100 Anesthetic agent
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Tags

Pancreatic Intravital Imaging WindowIn Vivo ImagingCellular-level VisualizationMurine ModelStabilized Longitudinal ImagingPANC-1 Nuclite RedN-Butyl Cyanoacrylate GlueFITC DextranTMR DextranFluorescent ProbesIntravital Microscopy
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Park, I., Kim, P. Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window. J. Vis. Exp. (171), e62538, doi:10.3791/62538 (2021).
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