Name: real-time x-ray imaging of lung fluid volumes in neonatal mouse lung
Uploaded: 2016-07-18T13:00:00.000+00:00
Duration: 11 min 26 s
Description: Watch this Scientific Journal Video about Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung at JoVE.com

This work is supported by a grant awarded to MNH by Children's Healthcare of Atlanta, the APS 2014 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) fellowship awarded to PT, and the University of Notre Dame Integrated Imaging Facility.

所有的实验技术，必须按照制度管理和使用委员会的指导方针进行。 1. X射线图像采集<ol><li> 软件概述（参见图1软件设置的概述）。 <ol><li>选择正下方的文件选项卡捕捉按钮。 </li><li>在设置下拉菜单中，选择当前会话 。 </li><li>选择位于下暴露时间下拉框中标准曝光 。 </li><li>设置曝光时间为2.00分钟和第暴露于1。设置X分级和Y分级为2个像素。 见图1。 </li><li>选择导出选项下拉菜单中最后积累 。 </li><li>在照明设置，塞莱CT X射线来自光源下拉菜单。默认KVP被设定在35。 </li><li>在应用参考文件下拉菜单中选择自动选择申请相应的X射线的参考文件。 （见第2节）。 </li><li>在设置摄像头设置 ，设置光圈为2.51。 </li><li>设置FOV（视场），以125.64为PN10老鼠。 </li><li> 焦平面设置为13，并在焦平面滑块旁边的下拉菜单中选择X射线 。 </li><li>对于X射线，设置激发滤光片 ，并在下拉菜单中发射滤光片为0。 </li><li>通过单击新建顶部旁边设置保存当前会话。新输入的名称获得设置文件，如 "朱庇特单曝光事件作出一个协议。"参见图2。 </li></o升&gt; </li><li> 创建的X射线成像协议 <ol><li>点击创建/编辑协议按钮打开的窗口的右侧。 </li><li>由于出现新的协议弹出窗口，点击右上角的角落新建按钮</li><li>输入该协议将在保存，例如"朱庇特示范协议"，然后单击确定的名称。 （参见图3）。 </li><li>在协议的步骤议定书弹出窗口的底部，确保步骤1中红色文本高亮显示， 捕捉新形象被选中， 什么事都不做在之前图像捕捉下拉菜单中选择。 </li><li>在拍照设置，请从步骤1.1.12最近生成的单次曝光的事件。 </li><li>请从拍摄图像后下拉菜单中等待（秒）。 </li><li>单击编辑按钮，典型值Ë180插入弹出框，然后点击确定每2分钟采集后添加一个3分钟的等待时间。 </li><li>重复通过右键单击步骤1选项卡，选择步骤重复步骤1。创建23重复了2小时的观察期。 （参见图4）。 </li><li>在最后的步骤（步骤24）， 图像捕捉设置攻略从等待（秒） 之后 没有改变。 </li><li>单击保存按钮并退出该议定书编辑器。 </li></ol></li></ol> 2.照明参考文件注意:为了在整个实验中获得的X射线图像的检测器的均匀性的变化自动修正应用X射线照射的参考文件到的X射线图像。下面概述的程序是特定于布鲁克体内动物成像系统; 其它体内成像系统可使用。 <ol><li>产生通过打开分子成像软件，点击捕捉按钮的照明参考文件。 </li><li>使用弹出式菜单，建立照明光源的X射线拍摄设置（见上述第1条建议的设置），然后选择在曝光式照明参考 。这可以根据标准曝光下拉框中找到。 （参见图5）。 </li><li>从图像站删除所有样本。设置的X和Y分箱到4×4像素合并。参照表1:照明参考文件的曝光时间 ，以确定准确的曝光时间。 </li><li>新闻揭露 。 </li><li>从下拉菜单下的应用参考文件下拉选择自动选择应用参考文件。 （参见图 6）。照明参考文件将被自动应用到所有的X射线IM使用相同的相机设置拍摄的年龄。步骤2.1 - 2.4将不需要被重复，如果相同的相机设置在随后的实验中使用。 注意:所述的照明参考文件可以在图像采集之后施加，或者如果参考文件的自动选择后出现一条错误消息。应用照明参考使用以下一系列分子成像软件的导航面板中的命令文件的图像后捕获: 图像&gt;图像数学&gt;类型:任务&gt; ​​计算:光量校正 。选择输入图像（X），你想在照明参考文件（Y）申请。重命名更正的文件（Z）。 （参见图7）。 </li></ol> 3，动物处理<ol><li> 获取动物 <ol><li>从商业育种购买怀孕水坝或者按照机构指导方针在12周龄（或以上）繁殖的房子雌性小鼠秒。 </li><li>房子新生小鼠与哺乳期的母亲高达日龄（PN）10。 </li></ol></li><li> 动物麻醉（PN 10） <ol><li>准备氯胺酮/甲苯​​噻嗪鸡尾酒麻醉PN长时间麻醉作用长达40分钟10只。加入500微升氯胺酮（100毫克/毫升）的对甲苯噻嗪的75微升（100毫克/毫升）。在0.9％的盐溶液，以使一个氯胺酮（100毫克/千克）/甲苯噻嗪（10毫克/千克）麻醉剂鸡尾酒稀释1:10。 </li><li>称量新生小鼠。 </li><li>使用3/10注射器用31克5/16英寸（8 MM）针，辖10微升/ g体重麻醉用腹膜内注射。 </li><li>动物保持干燥，隔热，以防止体内热量过度流失。 </li></ol></li></ol> 4.气管滴注<ol><li>制备由140 mM氯化钠，5mM的氯化钾，1mM的氯化钙和10mM的HEPES的气管内盐水溶液; pH值= 7.4。此溶液的渗透压应319 mosmol /公斤H 2 O. </li><li>安装了麻醉动物腹侧到用外科胶带倾斜的手术板。 Ensurethat动物的头是在倾斜的顶部。 </li><li>执行脚趾捏，以确保动物麻醉，准备手术。消毒所有外科领域，仪器，和胸部的动物的区域。 </li><li>做一个小（3毫米），颈部（喉区）的前侧 - 内侧腹方面的采用外科手术刀切开，大小11推开颈阔肌，并以可视化和访问气管前壁用钝钳气管肌肉。 </li><li>灌输3微升/ g的重量（约10 - 30微升最终体积）的生理盐水通过使用31克5/16英寸（8 MM）针暴露气管。该小切口留给动物成像期间开放，通常能自动恢复的很好。与动物资源的本地司协商，以确定是否该切口也同样可以保持打开。否则，可能缝线是必需的。 </li></ol> 5.动物成像<ol><li>仔兔腹侧一个明确的底部，可拆卸，动物成像盘位置。居中的动物，以使X射线束会直接在胸区。 </li><li>为奇数动物队列，直接放置在第一小狗在托盘的中间，和为偶数队列放置第小狗刚刚离开中心的，这样，当其它动物被放置在托盘中的所有动物都集中在。 </li><li>返回成像盘的X射线成像柜和关闭柜体的门。 </li><li>打开动物的热控制单元，以保持麻醉动物的体温。使用高设置，以实现约35室温度 - 37℃。转动物麻醉单元（通过氧输送汽化异氟醚），以确保动物麻醉并固定在整个2小时的成像过程的持续时间。 </li><li>执行X射线成像协议<ol><li>点击捕获并选择适当的协议， 例如，"朱庇特示范协议"，从协议下拉菜单。 （参见图8）。 </li><li>点击分子成像软件执行选择的 协议按钮。 注意:一个弹出式窗口将出现跟踪图像采集的状态，该协议完成后，窗口会消失的时候。 X射线的剂量低，&lt;0.3 MREM或大于牙科用X射线小约10倍。与其它的X射线的程序，没有残留辐射。 </li><li>当2小时采集进程完成后，从成像托盘上取出的动物，并将其退回到自己的笼子里。回到机架之前监视动物完全恢复。 </li></ol></li></ol> 6.数据分析注:分子成像软件可以量化和XR翻译唉像素强度到肺液清除率。以下步骤概述正常化的X射线图像和在感兴趣（ROI）定义的区域量化强度所需要的程序。 <ol><li> 设计一个模板，投资回报率 注意:兴趣模板的一个区域必须特定于捕获的X射线图像的2小时的研究过程中创建的，并应以比较的X射线强度之间的实验组使用。由于生理盐水的挑战少量肺4-6的左上叶通常积累，投资回报率（S）应侧重于肺的这一部分。 <ol><li>打开在2小时集中的第一个和最后的X射线图像。选择第一个x射线图像的窗口。 </li><li>在导航工具栏中，选择手动的投资回报 &gt; 新的投资回报率设置 。 </li><li>点击ROI椭圆 ，并创建一个投资回报率，足可涵盖鼠标的左lung.An投资回报率直到红色没有定义，概述了投资回报率是博士agged到不同的位置。已定义的投资回报率会以蓝色列出了一些。 </li><li>如果多个幼仔成像，一下就红了，勾画ROI并拖动到其他老鼠的左肺在同一组，以创造更多的个人投资回报率。红，概述了投资回报率拖到一个区域有明显的背景下，以创建一个背景的投资回报率。 <ol><li>选择鼠标选择来定位的ROI趴在每只小鼠的左肺，直属第二肋。 </li></ol></li><li>在顶部的工具栏上，单击图像显示 。 </li><li>检查覆盖在图像显示对话框覆盖的最后一个X射线图像，同时保持设定的ROI的位置。如果有必要，导航工具栏上选择鼠标选择调整的ROI的位置，并确保在两个图像足够的肺覆盖。 </li><li>在手动ROI对话框中，选择模板 &gt; 保 ​​存为模板 。名T及他模板，然后单击确定。 </li><li>关闭这两个图像。提示保存更改时，选择否 。 </li><li>应用在ROI模板捕获通过打开在研究期间拍摄的所有图像来分析液体清除各X射线图像。选择一个打开的文件开始，然后单击手册投资回报率 &gt; 模板 。 </li><li>从下拉菜单以前创建的ROI模板，然后单击应用到所有打开的文档。 </li></ol></li><li> 从图片到电子表格导出数值数据投资回报率 <ol><li>在左上角，点击File&gt; 导出数据 &gt; 投资回报率 。 </li><li> 支票在弹出的对话框中显示 ，并自动在Excel中打开 。 </li><li>选择导出所有打开的文档 。 </li><li>命名该文件，然后单击保存 。 </li><li>关闭分子成像软件。 </li></ol></li></OL&gt;

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本文是关于多式联运临床前成像的特殊问题，布鲁克BIOSPIN赞助的一部分。

使用X射线成像，新生儿肺部清晰的图像可以被分析为肺液的卷。我们7,3,11和10人，已成功地利用X射线成像，以确定自由呼吸麻醉动物模型肺液量动态变化，以及这种技术带来了巨大希望推进新生儿肺损伤的研究。在使用我们的方法来评估肺流体体积（相对于例如X射线相位constrast 10）的一个主要优点是，最多五个PN10小鼠幼仔可以使用成像系统，该系统是在研究设施和芯共?...

在出生，新生儿肺部必须从流体分泌过渡到一个流体再吸收器官建立足够的通风和主体的氧合。有利于在出生时肺液有效地清除的机制（或妨碍）仍不清楚。在建模C57BL / 6新生小鼠的幼崽肺泡液体清除率将导致更好地理解监管因素，可以提高或降低液体的吸收率。它也可以适用于急性肺损伤或感染的其他新生模型，并可能导致对呼吸窘迫新生儿新的治疗策略。 由于新生儿的肺相比，成人肺是微不足道的，依赖于灌洗或gravimetrical测量肺泡液体清除常规措施可能不适合准确地研究新生儿肺部模型肺液清关。在这个协议中，我们证明的测定法，允许肺泡液体清除率的准确测定日龄10 C57BL / 6小鼠的幼崽用小动物成像仪。使用荧光方法的一个主要的好处是，将动物体内成像。他们在自由地呼吸，可能会为未来的观测和研究这种微创法恢复。该方法的总的目标是肺水肿在新生儿肺部建模，并评估在新生儿肺部肺泡液体清除的速率。这种技术被开发，部分作为减少策略，以减少所需的动物数量，然而最大化试验输出。这种技术还允许肺流体体积的优越检测用X射线成像，并需要在基本动物克制熟练度和处理1;小动物手术和气管滴入2，小动物成像仪，和基本的图像分析软件。谁愿意在体内评价肺流体体积研究者（BRE自如athing麻醉动物模型）可能会发现适合自己应用此过程。最后，该协议可能会增加在支气管肺发育不良，包括高氧性肺损伤，机械通气和肺部炎症3车型的机理研究中使用的新生儿肺损伤等现有车型。

<table><tbody><tr><td>Preclinical Imaging System (In- Vivo MS FX PRO)</td><td>Bruker; Billerica, MA</td><td></td><td></td></tr><tr><td>Ketamine</td><td>Ketaset; Fort Dodge Animal Health, IA</td><td>26637-411-01</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories; Shenandoah, IA</td><td>4821</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (with Calcium and Magnesium)&amp;nbsp;</td><td>Lonza; Walkersville, MD</td><td>17-513F</td><td></td></tr><tr><td>Sodium chloride</td><td>Amresco; Solon, OH</td><td>241</td><td></td></tr><tr><td>Potassuim chloride</td><td>Fisher Scientific; Fair Lawn, NJ</td><td>P217-3</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich; St. Loius, MO</td><td>C5080</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich; St. Loius, MO</td><td>H3375</td><td></td></tr><tr><td>0.3 ml insulin syringe with 31 G x 5/16&quot; (8 mm) needle</td><td>BD Insulin Syringe; Franklin Lakes, NJ</td><td>328438</td><td></td></tr></tbody></table>

real-time x-ray imaging of lung fluid volumes in neonatal mouse lung

出生时，肺经历从分泌吸收了深刻的表型开关，它允许以适应自主呼吸。促进和维持这种表型是整个生命的正常肺泡生长和气体交换至关重要。几个 体外研究其特征关键调节蛋白，信号分子，和类固醇激素，可以影响肺液清除率的作用。然而， 在体内的检查，必须执行以评估这些调控因子是否在调节围产期肺液体吸收中起重要的生理作用。因此，实时X射线成像的使用情况，确定围产期肺液清，或肺水肿，代表在该领域的技术进步。在此，我们解释和说明的方法来评估在10后产后我们每天C57BL / 6小鼠肺部肺泡液体清除和肺泡洪水率ING X射线成像和分析。成功地执行本协议要求的体制动物护理和使用委员会（IACUC）， 体内小动物X射线成像系统，以及兼容的分子成像软件事先批准。

图9左侧面板- 10是在基线成像PN 10小鼠肺的（预注入）。这些图像显示的盐水挑战成功滴注到新生儿肺部的左叶。在图9中 ，小鼠肺中气管与上述定义的盐溶液滴入（参见第2.1节）。 图9的中间和右侧板是从得到的5分钟和2小时后滴注相同的鼠标的X射线图像;这种动物已成功清除了生理盐水的挑战。具体地说，该动物ROI的X射...

Watch this Scientific Journal Video about Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung at JoVE.com

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

在新生小鼠肺肺流体体积的实时X射线成像

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

real-time-x-ray-imaging-of-lung-fluid-volumes-in-neonatal-mouse-lung

Research

JoVE Journal

Medicine

8.6K 次观看.  Emory University. We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology. 

视频: Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

科研

癌症研究

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

免疫与感染

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

发育生物学

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

革兰阴性细菌败血症的新生儿成像模型

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

生物工程

<meta charset="utf8"></head><body>保留肺部真菌孢子定位以进行荧光显微镜分析

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

<meta charset="utf8"></head><body>保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

在新生小鼠肺肺流体体积的实时X射线成像

摘要

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此视频中的章节

长期高分辨率活体显微镜在肺与真空稳定成像窗口

超声成像对小鼠肺肿瘤进展的检测

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

用于微CT成像的成人和早期产后小鼠肺的血管铸造

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

革兰阴性细菌败血症的新生儿成像模型

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

Hyperpolarized ¹²⁹Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法

保留气道中真菌孢子空间动力学的小鼠肺成像的有效方法