我们承认博士。斯蒂芬R.华军莫拉诺，卡米洛里科尔迪，安东内洛Pileggi，Speier和丹尼尔Nyqvist富有成果的讨论。我们也感谢鲁特埃尔南德斯和迭戈·埃斯皮诺萨Heidmann技术援助和麦克·巴尔德斯和玛格丽特福莫苏，具有视频录制的帮助。拜伦马尔多纳多记录，编辑和制作最终的视频。研究提供了支持;美国国立卫生研究院糖尿病研究基金会（ <a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ），美国国立卫生研究院/ NIDDK / NIAID（F32DK083226 MHA RO3DK075487 AC; U01DK089538 PO.B.）。通过资金来自卡罗林斯卡医学院，瑞典研究委员会，瑞典糖尿病基金会，家庭二苓佩尔松基金会，家庭克努特和Alice Wallenberg基金会，瑞泰人寿保险股份有限公司，充满活力的进一步的研究提供了支持PO.B（ FP7-228933-2），卡罗林斯卡研究所战略研究计划在糖尿病itutet，诺和诺德基金会，和泊位冯Kantzow的的基础。

下的立体镜执行下面的过程，2步，第一步涉及胰岛加载到套管和第二步骤是实际的移植到ACE。对动物进行的所有程序批准的机构动物护理和使用委员会（IACUC），迈阿密大学的。 1。载入套管用于移植的胰岛<ol><li>中心的胰岛细胞在培养皿中通过旋转的菜在缩小圈子。 </li><li>断开的“蓄水池”，并将其放置在清洁的表面和连接管的套管套管。水库可以做出来的300微升一次性塑料吸管尖不带过滤器（ 图1a）。 </li><li>冲洗气泡时吸进水库的水库，以确保源源不断的小岛。法拉盛水库是通过推动免提电动注射器驱动程序使用的脚踏（ 图1b，c）。这也将在注射器中的空间，以允许吸入的胰岛入储液器（预加载了，如生理盐水，PBS或培养基的无菌溶液）。 </li><li>轻轻吸出所需数量的胰岛入水库。胰岛往往会旋转，当他们进入水库，将继续向底部。愿望是做的向后驱动电动注射器使用脚踏板驱动程序。 </li><li>插管重新连接到储存器经由所述连接管。 </li><li>将导管尖端在培养皿中，并刷新了小岛的水库管，然后在套管。确保小岛留在一起，因为你回填管/套管轻轻“弹”（攻丝）管（ 图1d）。之前或之后的所有气泡停止提前胰岛被冲出插管。如果不能确定，停止小岛的套管剩余的空气进入回气泡领先的胰岛细胞可以帮助防止反流（逆流）的胰岛细胞的ACE。会在一夜之间消失。 </li><li>在这个阶段，你准备好注入到的ACE（请参阅下面的步骤）的小岛。 </li></ol> 2。胰岛移植到眼睛的前房<ol><li>麻醉在一个温暖的鼠标垫在体视镜上。 </li><li>将口鼻部的鼠标连接到氧气/异氟醚麻醉机麻醉“面具”。该掩模是做出来的1毫升的一次性塑料移液管尖（不带过滤器），通过麻醉油管连接的窄端（ 图2A，B）。 </li><li>轻轻缩回眼睑的眼睛，用另一只手的食指和拇指，“啪”的眼睛了更好的接触和方便地访问（ 图2c）被移植。不妨碍呼吸的鼠标，过大的压力，这将需要一些练习来完善颈部或阻止血液流向头部。 </li><li>使用一次性胰岛素注射器（29 - 31G），手术刀，小心地渗透只是冰山在角膜上，使一个单一的横向切口。使切口在角膜和角膜缘的顶点之间的中点位置处，以尽量减少在注射过程中，满分的ACE（ 图2d）回流的胰岛。 </li><li>小心通过切口插入套管（预装小岛）。 </li><li>慢慢地弹出胰岛的套管和虹膜的顶部将它们存放。为了避免由于过大的压力积聚在ACE的胰岛回流，弹出的小岛在短暂的推力体积小（S）可能在象限相反的切口。这可以进一步确保在加载套管（请参阅第1.6步），通过压缩胰岛管。 </li><li>慢慢地缩回套管的ACE。这是关键的一步，特别是，如果将大量的胰岛细胞注射压力由于胰岛回流确保建立内部的ACE可能是不可避免的。为了消除，/尽量减少胰岛回流，轻轻地旋转套管内的ACE释放多余的压力通过切口周围的套管。检查是否有回流的迹象，如果您尝试收回的套管，如果需要的话，等到压力消退之前完全缩回套管的ACE。 </li><li>用无菌PBS或生理盐水冲洗移植眼。 </li><li>注射丁丙诺啡术后镇痛（0.05-0.1毫克/公斤），皮下的第一个48小时。 </li><li>红霉素眼药抗生素软膏后立即移植到移植的眼睛。 </li><li>将动物在加热的笼子，让从麻醉中恢复。 </li></ol>

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PO.B.是的生物技术公司Biocrine的，这是作为一个商业服务平台，要使用眼的前房的创始人之一。 AC是这项技术的专利保护。

小鼠胰岛分离使用胶原酶消化其次密度梯度纯化，如前所述，33。胰岛移植前培养过夜。虽然这可能不是必需的，它建议允许从隔离过程中恢复的胰岛。这是因为这将确保幸存的/强大的胰岛细胞移植时，移植糖尿病的收件人进行。 移植全身麻醉下进行，与氧/异氟醚混合物（1.5-3％）吸入效果。可以用来替代吸入或注射麻醉剂（ 例如，氯胺酮）。如果注射麻醉?...

在活体显微镜的进展显示，预计不会在体外研究中的生理现象。这突出表明在体外培养的方法，通过常规的活的动物的研究结果，在翻译的挑战。在过去的十年中，大大改善生活的动物的组织中的可视化技术进步的成像方式2，3，4，5，6。这促使需要在体内成像方法与可行的在实验动物模型中的应用，使纵向的非侵入性的靶组织的可视化。 启用非侵入性的成像技术，如磁共振成像和正电子发射断层扫描或生物发光成像的器官/组织深身体内的7-8，9。但是，这些技术无法实现单细胞分辨率高的背景信号和低空间分辨率，但使用of高对比度的材料或组织特异性的发光4。这是解决与双光子荧光共焦显微镜10的到来。双光子显微镜活体成像研究，以前所未有的细节11，12，可视化和量化细胞的活动。这导致了在健康和疾病的13，14，15，16中的关键的生物过程的表征。开拓性的活体成像研究主要是“模仿” 在体内切除的组织（ 如淋巴结）的条件，但其他研究已经使用侵入性的方法，图像暴露目标组织在原位 17日，18日，19日，20日，21。其他研究也使用“窗口室模式”，以规避限制，与侵入性方法和有限的成像分辨率在体内 22日，23日，24日，25。在窗口中的腔模型中，一个腔室与一个透明的窗口是在不同的外科手术植入到皮肤租金位置（背侧或耳部皮肤，乳腺脂肪垫，肝等）上的动物（ 如小鼠，大鼠，兔）。虽然这种做法显然使高分辨率成像在体内 ，它需要一个微创手术植入室，可能无法以适应纵向成像研究在数周或数个月22。 这是最近展示了一种微创手术，即移植到前房的眼睛，结合高分辨率的共聚焦显微镜（ACE）提供了一个“自然的身体”窗口“作为一个强大和灵活的体内成像平台，26日，27。移植到ACE在过去的几十年中被用于研究生物方面的各种组织28日，29日，30和其最近的高分辨率成像结合，使胰岛生理研究单细胞分辨率的非侵入和纵向&lt;sup&gt;的26，27。这种方法被用于研究自身免疫反应，在开发过程中的1型糖尿病的动物模型（未发表资料）。它也被用来研究胰腺发育，以及，在肾功能的研究，通过移植到的ACE胰芽或个别肾小球，分别为（未发表的数据）。最近的一份报告使用这种方法进一步证明其研究中的应用胰岛移植31后的免疫反应。更重要的是，本研究表明，移植到的眼球的前房中，提供了一种自然的身体窗口来执行：（1） 在体内的移植组织的纵向的，非侵入性的成像，（2） 在体内 cytolabeling评估细胞表型和生存能力原位 ;（3）实时跟踪，在靶组织中的免疫细胞浸润;（4）通过局部应用或眼内注射的地方干预。 在这里，我们ðemonstrate如何进入前房的眼睛胰岛进行移植。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 说明/意见 </td></tr><tr><td> IsoTHESIA（异氟醚） </td><td> Buttler动物保健供应</td><td> 11695-6775-2 </td><td> 99.9％的异氟醚/毫升</td></tr><tr><td> Ketaset（氯胺酮HCL） </td><td> Fort Dodge动物保健</td><td> 0856-2013-01 </td><td>替代注射麻醉</td></tr><tr><td> Beprenex（丁丙诺啡HCL） </td><td> Reckitt Benckiser公司健康护理（英国）有限公司</td><td> 12496-075-7-1 </td><td> 0.3毫克/毫升</td></tr><tr><td>红霉素眼膏USP，0.5％ </td><td>阿克伦</td><td> 17478-070-35 </td><td>预防移植眼</td></tr><tr><td> 0.9％氯化钠（生理盐水） </td><td>赫士睿公司</td><td> 0409-7983-03 </td><td>静脉注射。无菌</td></tr><tr><td> PBS </td><td> Gibco公司</td><td> 10010-023 </td><td> 1X。无菌</td></tr><tr><td> CMRL中等1066 </td><td> Cellgro </td><td> 98-304-CV </td><td>补充，CIT修改。为小岛的首选媒体</td></tr></tbody></table>

transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time

活体成像已成为生物学研究的一个不可缺少的工具。的过程中，许多成像技术已经发展到在非侵入性动物研究的不同生物过程。然而，一个重要的技术在现有的活体成像方式的限制是无法以非侵入性的结合，纵向成像与单细胞的分辨能力。我们在这里展示，如何移植进入前房的眼睛，会在显着的限制，提供一个多功能的实验平台，使非侵入性的， 体内细胞高分辨率成像与纵向。我们展示了鼠标和移植过程中使用的模型与临床相关性，即胰岛移植提供有代表性的结果。除了能够直接可视化的各种组织移植到眼的前房，这种方法提供了一个平台，卵石Ň药物进行长期的跟进和监测靶组织中。由于它的多功能性，组织/细胞移植到眼睛的前房不仅有利于移植疗法，它延伸到其它在体内应用研究的生理和病理生理过程，如信号转导和癌症或自身免疫性疾病的发展。

有几个参数来定义一个“好”移植。一个良好的移植是会继续进行，而不会出现出血使切口在视频中可以看出，当一个。出血防止/最小化到ACE仅贯通的手术刀（针）的前端（ 图3a）。这也将有助于防止虹膜的接触和穿刺。它也将确保小切口愈合得很好，而不会造成角膜混浊随着时间的推移（ 图3C，D）。一个成功的移植的另一个重要的方面是可移植的胰岛的总的所需量，?...

Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

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

眼内移植和共聚焦显微镜相结合的一种新的方法移植组织内，使纵向的，非侵入性的单细胞分辨率的实时成像<em在体内</em&gt;。我们演示了如何胰岛细胞移植到老鼠的眼睛前房。

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 ...

transplantation-into-anterior-chamber-eye-for-longitudinal-non

Research

JoVE Journal

Medicine

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

13.3K 次观看.  University of Miami Miller School of Medicine. 眼内移植和共聚焦显微镜相结合的一种新的方法移植组织内，使纵向的，非侵入性的单细胞分辨率的实时成像

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

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

科研

医学

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

大鼠肾移植后急性排斥反应的非侵入性的成像使用<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

基于音频的虚拟游戏环境的发展，以帮助盲人的导航技巧

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

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

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

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

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

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 ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...