Name: stabilized longitudinal in vivo cellular-level visualization of the pancreas in a murine model with a pancreatic intravital imaging window
Uploaded: 2021-05-06T14:30:00
Description: Watch this Scientific Journal Video about Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window at JoVE.com

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。

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
<ol><li>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）。<...

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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 Succinimidyl Esters (NHS esters)</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>Fluorescent probe for conjugate with antibody</td>
		</tr>
		<tr>
			<td>BALB/C Nude</td>
			<td>OrientBio</td>
			<td>BALB/C Nude</td>
			<td>BALB/C Nude</td>
		</tr>
		<tr>
			<td>BD Intramedic polyethylene tubing</td>
			<td>BD Biosciences</td>
			<td>427401</td>
			<td>PE10 catheter for connection with needle</td>
		</tr>
		<tr>
			<td>C57BL/6N</td>
			<td>OrientBio</td>
			<td>C57BL/6N</td>
			<td>C57BL/6N</td>
		</tr>
		<tr>
			<td>Cover glasses circular</td>
			<td>Marienfeld</td>
			<td>0111520</td>
			<td>Cover glass for pancreatic imaging window</td>
		</tr>
		<tr>
			<td>FITC Dextran 2MDa</td>
			<td>Merck (Former Sigma Aldrich)</td>
			<td>FD200S</td>
			<td>For vessel identification</td>
		</tr>
		<tr>
			<td>IMARIS 8.1</td>
			<td>Bitplane</td>
			<td>IMARIS</td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>Intravital Microscopy</td>
			<td>IVIM tech</td>
			<td>IVM-C</td>
			<td>Intravital Microscopy</td>
		</tr>
		<tr>
			<td>IRIS Scissor</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>S-1107-10</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Loctite 401</td>
			<td>Henkel</td>
			<td>401</td>
			<td>N-butyl cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>Micro Needle holder</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>H-1126-10</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Micro rectractor</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>17004-03</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Microforceps</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>F-1034</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>MIP-GFP</td>
			<td>The Jackson Laboratory</td>
			<td>006864</td>
			<td>B6.Cg-Tg(Ins1-EGFP)1Hara/J</td>
		</tr>
		<tr>
			<td>Nylon 4-0</td>
			<td>AILEE</td>
			<td>NB434</td>
			<td>Non-Absorbable Suture</td>
		</tr>
		<tr>
			<td>Omnican N 100 30G</td>
			<td>B BRAUN</td>
			<td>FT9172220S</td>
			<td>For Vascular Catheter, Use only Needle part</td>
		</tr>
		<tr>
			<td>PANC-1 NucLightRed</td>
			<td>Custom-made</td>
			<td>Custom-made</td>
			<td>Made in laboratory</td>
		</tr>
		<tr>
			<td>Pancreatic imaging window</td>
			<td>Geumto Engineering</td>
			<td>Custom order</td>
			<td>Pancreatic imaging window - custom order</td>
		</tr>
		<tr>
			<td>Physiosuite</td>
			<td>Kent Scientific</td>
			<td>PS-02</td>
			<td>Homeothermic temperature controller</td>
		</tr>
		<tr>
			<td>Purified NA/LE Rat Anti-Mouse CD31</td>
			<td>BD Biosciences</td>
			<td>553708</td>
			<td>Antibody for in vivo vessel labeling</td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>F-1090-3</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td>Rompun</td>
			<td>Anesthetic agent</td>
		</tr>
		<tr>
			<td>TMR Dextran 65-85kDa</td>
			<td>Merck (Former Sigma Aldrich)</td>
			<td>T1162</td>
			<td>For vessel identification</td>
		</tr>
		<tr>
			<td>Window holder</td>
			<td>Geumto Engineering</td>
			<td>Custom order</td>
			<td>Window holder - custom order</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td>Zoletil 100</td>
			<td>Anesthetic agent</td>
		</tr>
	</tbody>
</table>

stabilized longitudinal in vivo cellular-level visualization of the pancreas in a murine model with a pancreatic intravital imaging window

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。

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?...

Watch this Scientific Journal Video about Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window at JoVE.com

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

胰 腺的体内高分辨率成像利用胰腺内成像窗口获得便利。

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

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

此协议使用新的胰腺内成像窗口描述胰腺的稳定和重复的细胞水平在体内成像。这项技术的主要优点是，在活老鼠的胰腺中，在三周内可以进行一动不动的三维成像，分辨率达到细胞水平。首先准备手术平台，用70%乙醇对表面进行消毒。麻醉后，使用带有自热控制加热垫的直肠探头来监测体温。从鼠标的左侧取下头发，然后用酒精和碘替代，从切口部位旋转到外表面，永不回到切口部位，以碘结束。然后，在老鼠的左侧做一个1.5厘米的切口，解剖皮肤和肌肉。使用黑色或尼龙 4-0 缝合线在切口边缘执行钱包串缝合。用环钳小心地拉动脾脏，识别胰腺。将窗口放在鼠标的侧翼，通过窗户的空地穿过脾脏和胰腺。然后，轻轻地将胰腺放在成像窗的板上，同时将脾脏放在窗口的空地上。将PANC-1核素红色直接注射到胰腺中。使用 31 口径导管针将一滴 n-丁基氰酸丙烯酸酯胶水涂抹到成像窗口边缘，确保使用少量胶水。轻轻地将 12 毫米圆形盖玻璃涂抹到成像窗的边缘。然后，握住缝合圈，使其适合窗口的横向凹槽，并将其绑住三次。最后，为了防止这些紧缝的中断，当小鼠清醒时，切开领带的最大近端部位。首先打开活性显微镜，包括激光电源。要插入血管导管，请用食指和第三指对尾部近端施加压力。如有必要，用灯加热尾部。用 70% 乙醇喷雾消毒尾静脉，然后将 30 度导管插入横向尾静脉，并可视化 PE-10 管中的血液回流。在导管上涂抹丝胶带以稳定导管。根据荧光探头的组合，酌情注射FITC德克斯特兰和TMR德克斯特兰或其他荧光探针。插入直肠探头，通过家用热加热垫系统自动控制体温。然后，将在活体显微镜设置期间准备的胰腺成像窗口插入窗口支架。将鼠标从手术平台转移到成像阶段。要执行活体成像，请从低放大倍率（如四次）对胰腺进行成像开始，以扫描胰腺成像窗口中胰腺的整个视图。确定感兴趣区域后，切换到更高的放大倍率目标透镜（如 20 倍或 40 倍），分别使用约 0.5 微米和 3 微米的横向和轴向分辨率进行细胞级成像。执行 Z 堆栈或延时成像，以观察 3D 结构或细胞级动态（如细胞迁移）。活性显微镜与胰腺内成像窗口相结合，可提供长期组织稳定性，使高分辨率成像的采集能够跟踪单个小岛长达三周。窗户植入C57黑色6N小鼠与静脉注射抗CD31抗体，与Alexa 647荧光素结合，促进大面积成像和放大胰腺的3D成像。在相邻血管的胰腺组织平均图像中识别出阴性细胞，分别使用自发光和抗CD31抗体进行可视化。使用马赛克成像方法，将广域视图与高分辨率成像相结合，在 MIP-GFP 鼠标中可视化大约 40 到 50 个小岛与相邻的血管。先前的研究表明，使用这种稳定的成像方法，可以跟踪小岛长达三周。为了可视化癌细胞，PANC-1核酸红细胞在手术过程中被直接植入小鼠胰腺，附近的血管被与Alexa 647结合的抗CD31染色。使用此协议，生成胰腺癌的广域图像。这有助于划定肿瘤的边缘，以及实现高分辨率的3D图像在单细胞水平。这种方法最关键的一步是巧妙地将胰腺成像窗口植入小鼠体内。利用荧光小鼠细胞和抗体探针的其他组合，可以识别附近细胞与小岛或取消细胞的动态相互作用。这种方法可以广泛应用于那些在胰腺癌的各种病理生理条件和微观环境中探索胰岛变化的人。

Research

JoVE Journal

Medicine

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

成像神经胶质瘤启动<em在体内</em&gt;通过抛光和增强的薄膜头骨的颅窗

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

科研

医学

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

纵向，非侵入性的移植到眼睛的前房<em在体内</em&gt;单细胞分辨率的实时成像

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;：单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像：相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

慢性胰腺炎的机器人侧胰腺肌切除术

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

阿霉素诱导的体内扩张型心肌病小鼠模型

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

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

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