这项工作是支持的补助伊娃Sevick：NIH R01 CA128919和美国国立卫生研究院R01 HL092923。

所有动物的研究进行了大学，得克萨斯大学健康科学中心（休斯顿，德克萨斯州），比较医学系，分子成像后，审查和批准该协议由各自的机构动物保健中心的标准和使用委员会（IACUC）或动物福利委员会（AWC）。 1。制备动物24小时之前，成像 必须完成下面的步骤中（如需要）的前一天，淋巴造影发生。 <ol><li>将动物的感应箱与异氟醚和稳重。 </li><li>一旦动物是状态的深刻麻醉（用脚趾夹机动监测），在尿片/的绒毛垫和位置鼻的鼻锥连接到异氟醚气体的地方镇静动物。 </li><li>所有头发夹/皮草（如有）周围的区域进行成像。 </li><li>脱毛剂（NAIR）应用的裁剪区域，并把它Øn的皮肤3分钟。 </li><li>与温暖，潮湿的纱布或纸巾轻轻擦去所有的脱毛剂。 </li><li>皮肤用温水轻轻冲洗，并用纱布或纸巾轻轻地擦干地区。 </li><li>让动物恢复加热垫或加热灯，并返回到他们的笼子。 </li></ol> 2。一天的影像<ol><li>复溶显像剂，然后用无菌水稀释使用无菌的，正常的（0.85％）生理盐水CABD-IRDye800 ICG或200μM（680μg/10微升）的达到645μM（5μg/10微升）， 保持在黑暗中的解决方案重建后的6小时内的条件和使用。 </li><li>将动物的感应箱与异氟醚和稳重。 </li><li>一旦动物是在深刻麻醉（用脚趾夹机动监测），在其一侧连接到气异氟醚在鼻锥上的尿片/的绒毛垫和位置的鼻子的地方镇静动物的状态。 </li><li>关灯（所以房间暗）。如果需要的话，一张小桌子卤素光源，可用于光看注射少量的。 </li><li>用31号针头用胰岛素注射器，注入ID为5微升50微升的ICG或CABD IRDye800在每个后爪和/或背侧基地的尾巴上的左侧和右侧，这取决于该地区的利益（见讨论）。每个注射剂量的范围可以从0.083到1.25毫克/公斤（ICG），或0.113至1.7毫克/公斤（CABD-IRDye800）。注射体积将随动物应变和注射部位。对于无胸腺小鼠中，注射的体积可以是5微升（后肢）或10μl（基尾）， 如果动物是没有的成像系统下的喷射（），将动物的成像系统下后立即注射（）。 </li><li>如果没有染料的吸收是在淋巴管可见，需要重复步骤2.5将作为每只动物协议需要。 </li><li>一旦被视为淋巴管，包括注射部位用黑色电工胶带或黑色PA元。 </li><li>长达1小时使用获取淋巴图像V +软件和小动物，NIRF成像系统。 （动物镇静异氟醚和呼吸时监视图像获取）。虽然小动物，NIRF成像市售的，我们利用一个定制的，小动物NIRF成像系统组成的785纳米激光二极管（1005-9mm的78503的，强烈的，位于North Brunswick，NJ）配备（-B C24TME​​，Thorlabs公司，牛顿，新泽西州）的非球面透镜，扩散器（ED1-C20，Thorlabs公司），和过滤器（LD01-785/10-25的，Semrock，纽约州罗切斯特市），以产生均匀的照射动物的激励磁场的入射积分通量率小于1.4毫瓦每平方厘米10。电子倍增电荷耦合器件（EMCCD，PhotonMax512，普林斯顿仪器，特伦顿，NJ）系统有两个830纳米的的滤波器（AND11333，Andover的公司，在新罕布什尔州Salem）和28毫米的尼克尔镜头（1992年，尼康相机，纽约州Melville）是用来捕捉到淋巴的图像融合添ES 200毫秒的动态影像和800毫秒的静态影像5。请参阅图1系统配置，表中的每个组件的更多细节，并讨论了简短的讨论关键的成像性能。 </li><li>让动物恢复加热垫或加热灯和返回到他们的笼子，或安乐死。 </li><li>使用ImageJ或MATLAB软件分析图像。参见图6。 </li></ol>

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作者有没有透露，但有些作者列出的专利。

我们使用一个自定义的，小动物NIRF成像系统捕捉到的图像标记的小鼠淋巴管。为了构建电影淋巴结运动，300或更多个图像被收集。有关从电影淋巴管的功能分析，两个或两个以上的ROI手工画出的沿淋巴容器。的感兴趣区的尺寸中为每个容器中，并保持恒定的直径约为该船只。虽然整个动物的空间分辨率可以描绘荧光淋巴管的100微米或更小，可以采用更精细的分辨率的图像的macrolens 10。为?...

<table border="1"><tbody><tr><td> 解决方案，试剂和设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>吲哚菁绿（ICG） </td><td> Patheon Italia SpA的</td><td> NDC 25431-424-02 </td><td>重组至645μM（5μg/10μL） </td></tr><tr><td>循环白蛋白结合结构域（CABD） </td><td>巴赫姆</td><td>习惯</td><td>复溶至200μM，（6.8μg/10μL） </td></tr><tr><td> IRDye800 </td><td> LI-COR </td><td> IRDye 800CW </td><td>共轭CABD equilmolar浓度重组根据制造商的指示; </td></tr><tr><td>无菌水</td><td> Hospira公司，公司的Lake Forest，IL </td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td> NAIR </td><td>教堂和德怀特有限公司，公司</td><td>当地的商店</td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td>成像系统（以下组件） </td><td>分子影像学研究中心</td><td> N / A </td><td>自定义建立在我们的实验室。 </td></tr><tr><td>电子倍增电荷耦合器件（EMCCD）相机</td><td>普林斯顿仪器公司，美国新泽西州特伦顿， </td><td>光子最多512 </td><td></td></tr><tr><td>尼康相机镜头</td><td>尼康公司，纽约梅尔维尔， </td><td> 1992年，型号尼克尔28mm </td><td></td></tr><tr><td>光学过滤器</td><td> Andover的公司，在新罕布什尔州Salem </td><td> ANDV11333 </td><td>两个830.0/10.0 nm的带通滤波器中使用的镜头前</td></tr><tr><td> 785纳米激光二极管</td><td>激烈有限公司，北不伦瑞克，新泽西州</td><td> 1005-9MM-78503 </td><td> 500 mW的光输出</td></tr&gt; <tr><td>准直光学器件</td><td> Thorlabs公司，美国新泽西州牛顿， </td><td> C240TME-B </td><td>准直的激光输出之前清理过滤器</td></tr><tr><td>清洁过滤器</td><td> Semrock公司，在纽约州罗切斯特</td><td> LD01-785/10-25 </td><td>移除激光发射的荧光带</td></tr><tr><td>光学扩散器</td><td> Thorlabs公司，美国新泽西州牛顿， </td><td> ED1-C20 </td><td>在动物扩散的激光</td></tr><tr><td> V + </td><td>数码光学，布朗湾，奥克兰，新西兰</td><td> 5.0版</td><td>软件用于控制摄像系统和图像保存到计算机。 <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td>以下软件包分析软件，可用于图像分析</td></tr><tr><td> ImageJ的</td><td>国家Institutes的健康，马里兰州贝塞斯达</td><td>目前大多数版本</td><td>免费提供<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> MathWorks公司，Natic​​k郡，MA </td><td> 2008A或更高版本</td><td> <a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a> </td></tr></tbody></table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

淋巴血管系统的循环系统，保持体液平衡，提供了免疫监视，并介导对脂肪的吸收，在肠道中的重要组成部分。然而，尽管其关键的功能，有相对较少的了解淋巴系统如何适应为这些功能在健康和疾病的1。最近，我们表现出的能力，动态图像淋巴管架构和淋巴结在正常的人类受试者以及患有淋巴功能障碍的近红外荧光（NIRF）染料的使用跟踪管理和自定义的“抽水”行动，第三代强化成像系统2-4。 NIRF成像显示出了巨大的变化，在淋巴管的结构和功能与人类疾病。目前还不清楚这些变化是如何发生的和正在开发的新的动物模型，阐明它们的遗传和分子基础。在这个协议中，我们提出了NIRF淋巴管，S使用吲哚菁绿（ICG），已被用于在人类750年的染料，和一个NIRF染料标记的环状白蛋白结合结构域（CABD-IRDye800）肽，优先结合鼠标和人白蛋白8商场动物成像5,6 。约5.5倍的亮度比ICG，CABD IRDye800有一个类似的淋巴间隙轮廓比，ICG以实现足够的的NIRF信号为成像8，可以在较小的剂量注入。由于组织间隙8白蛋白的的CABD-IRDye800和ICG绑定，它们都可以描绘出活性蛋白运输和淋巴管内。皮内（ID）注射液（5-50微升）的ICG（645μM）或CABD-IRDye800（200μM），在盐水给药每个后爪和/或左，右侧的基极的背侧尾的异氟醚麻醉的小鼠。在动物所得的染料浓度是83-1,250微克/千克的ICG或113-1,700微克/千克CABD，IRDye800。紧随注射剂，功能性淋巴成像长达1小时，使用一个定制的，小动物NIRF成像系统进行。可以描绘整个动物的空间分辨率为100微米或更小的荧光淋巴管，和图像的结构至3厘米的深度可以获取9。图像采集采用V + +软件，并使用ImageJ或MATLAB软件进行分析。在分析过程中，连续绘制地区的利益（投资回报率），覆盖整个容器直径沿淋巴管。每个ROI测量定量评估通过血管淋巴移动的“数据包”，对于一个给定的容器和NIRF强度，每个ROI的尺寸保持不变。

NIRF在小鼠淋巴造影的例子当ICG注入或CABD IRDye800 ID在基地正常小鼠的尾部，在尾巴根部之间的注射部位和腹股沟淋巴结（LN）的淋巴血管应立即可视化。注射后不久（几秒钟到几分钟），淋巴管之间的腹股沟LN和腋下淋巴结应可视化，如在图2中看出。由于小鼠的淋巴管的变化在动物，因为他们在人类，动物之间的体系结构的变化，如在图3中所示，可以?...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

最近开发的成像技术，利用近红外荧光（NIRF）可能有助于阐明淋巴系统在肿瘤转移中发挥的作用，免疫反应，创面修复，以及其他相关的淋巴管疾病。

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

non-invasive-optical-imaging-of-the-lymphatic-vasculature-of-a-mouse

Research

JoVE Journal

Immunology and Infection

16.0K 次观看.  University of Texas Health Science Center-Houston. 最近开发的成像技术，利用近红外荧光（NIRF）可能有助于阐明淋巴系统在肿瘤转移中发挥的作用，免疫反应，创面修复，以及其他相关的淋巴管疾病。

视频: Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...