下面的协议说明，在20-35克鼠效果很好的系统。该方法可以很容易地适应于更大或更小的小鼠通过简单地改变导管的大小。所有动物方案都经约翰霍普金斯大学动物护理和使用委员会。 1.肺灌注<ol><li>选择市售一英寸长的20个克静脉插管以用于插管。 </li><li>修改导管尖端手动弯曲它产生如图1在前端具有微小曲率。 </li><li>麻醉小鼠用氯胺酮（100毫克/千克）和赛拉嗪（15毫克/千克）的混合物中IP注射，并且由于没有反射运动的确认麻醉。麻醉后立即应用兽医软膏的眼睛。麻醉适用兽医药膏对眼睛紧随，并给予卡洛芬（5-10毫克/公斤SQ），用于手术后和滴注镇痛。 </li><li>将谅解备忘录SE仰卧在倾斜的平台。 如图2，一间大办公室粘合剂缝合环上的作品贴得很清楚。 </li><li>剃颈部和清洁的腹侧部分和消毒用70％酒精的颈部区域。用新的latex-和无粉手套，使用消毒用70％酒精手术器械。 </li><li>使用锋利的剪刀做一个小的手术切口在约12毫米以下的下颌切牙颈部。 </li><li>用镊子轻轻拉扯皮肤的脖子直到尾端气管的腹壁可见。 </li><li>轻轻拉回舌和与弯曲尖端的倾斜朝向鼠标的腹面插入插管。正如在1.4，拉轻轻在脖子上的皮肤，然后将导管插入气管。 注:稍加练习，导管将可见向下移动气管。如果它在食道，就会有导管的运动没有视觉瞄准。无切口在气管制成。 </li><li>一旦导管被认为是在颈部气管，其推进约5mm至可靠地传递声带但仍明显高于隆突。 </li><li>准备灌输高达50微升的液体通过与凝胶装载枪头导管注入。放置在鲁尔毂的前端，但注入外表仔细观察在前端同步用鼠标的呼吸的流体的移动之前。然后注入滴注。 </li><li>用1ml注射器，立即做为0.6ml空气进入肺部相对快速充气穿过导管，以帮助分配液体深在肺部。拆下套管。 </li><li>拆下套管。 </li><li>用少量氰基丙烯酸酯类粘合剂的以关闭小外科伤口中为每个VetBond包装插页指示。将小鼠单独的笼子，并直观地监视他们，直到他们醒来，通常表现得没有任何迹象显示discomf的ORT。 </li></ol> 2.肺固定注意:一旦所有的实验过程中的小鼠进行，肺可以通过固定用甲醛（或任何其它期望的固定剂）被准备好进行组织学处理。 <ol><li>牺牲鼠标的IACUC接受的过程。用于在视频显示代表鼠标，麻醉小鼠的颈脱位被使用。 </li><li>通过手术暴露气管腹侧，做一个小切口，并插入18G的存根针头插入气管，用线扎它进行气管造口术（如果尚未完成）。 </li><li>小心打开了中线切开胸腔，切开膈肌，并卸下侧面胸壁揭露肺部。 </li><li>针的接头末端连接到一个环形支架含甲醛水库。 参见图3。 </li><li>将甲醛的顶面25厘米以上的谅解备忘录的水平东南。 参见图3。下一步要确保没有在固定管没有空气通过运行流体出活塞的结束。连接气管套管到储存管的路厄氏端。打开活塞膨胀肺与甲醛。离开肺部压力下至少20分钟。 </li><li>打开活塞膨胀肺与甲醛。离开肺部压力下至少20分钟。 </li><li>接着，打结气管超出存根针的末端。它可以帮助拉回来慢慢针揭露更多uncannulated气管。当捆绑牢固，取出活塞。 </li><li>小心解剖出肺。 </li><li>将甲醛肺部过夜。较长的时间都很好，有的污渍或程序可以指定特定的时间。也任何其它液体固定剂，例如z修复可用于滴注和浸泡。 </li><li>在进一步的组织学处理，测量如下所述固定肺容积。 </li></ol> 3.测量固定肺容积<ol><li>测量使用如图4阿基米德原理的肺容积。从甲醛卸下固定肺和解剖心脏和任何其他非肺组织。 </li><li>使用用于保持肺部完全在水下以前构造简单的自制线支撑装置。 注:此装置需要进行与取平衡正在使用兼容。 图 5中所示的典型装置从塑料移液器和薄（20 G）的金属丝制成。这个系统可以很好地在视频中使用的平衡，但可以很容易地适应大多数实验室天平。 </li><li>放置一个烧杯≈200毫升水对水的平衡和皮与支撑笼到位。 参见图6中取出的金属笼子。将肺在水面上和下水按同笼。 </li><li>记录天平上的权重。这个数字反映了水置换的体积，因此是一个直接测量肺体积。要小心，以确保肺或缝合或部分铁丝笼不接触的侧面或烧杯底部。 </li><li>对于精度，重复这种测量。除去从水肺，并在组织干。再次重重的抽带保持架到位的烧杯中，重复肺容积测量。两个体积测量然后应平均。 注:如果肺部留在福尔马林超过约一周，空气在肺部将溶解在液体中。当这种情况发生时，肺会下沉，所以不再需要使用任何设备作为图 5中，以保持浸没肺。在这种情况下，体积可以通过简单地进行测量通过保持肺由缝线串之一，直到它被完全淹没，如图4。 </li></ol>

<ol>
	<li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J Appl Physiol. 87, 2362-2365 (1999).</li><li>Walters, D. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+endotracheal+intubation+of+mice.">A novel and simple method for endotracheal intubation of mice.</a> Laboratory. 41, 128-135 (2007).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+retrograde+intubation+in+mice.">A technique for retrograde intubation in mice.</a> Lab animal. 35, 39-42 (2006).</li><li>Rivera, B., Miller, S., Brown, E., Price, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+endotracheal+intubation+of+mice+and+rats+used+in+imaging+studies.">A novel method for endotracheal intubation of mice and rats used in imaging studies.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 44, 52-55 (2005).</li><li>Vergari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+orotracheal+intubation+in+mice.">A new method of orotracheal intubation in mice.</a> European review for medical and pharmacological sciences. 8, 103-106 (2004).</li><li>Vergari, A., Polito, A., Musumeci, M., Palazzesi, S., Marano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-assisted+orotracheal+intubation+in+mice.">Video-assisted orotracheal intubation in mice.</a> Laboratory animals. 37, 204-206 (2003).</li><li>Hastings, R. H., Summers-Torres, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Laryngoscopy+in+Mice.">Direct Laryngoscopy in Mice.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 38, 33-35 (1999).</li><li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> Am J Respir Crit Care Med. 181, 394-418 (2010).</li><li>Foster, W. M., Walters, D. M., Longphre, M., Macri, K., Miller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+the+measurement+of+mucociliary+function+in+the+mouse+by+scintigraphy.">Methodology for the measurement of mucociliary function in the mouse by scintigraphy.</a> J Appl Physiol. 90, 1111-1117 (2001).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> Journal of visualized experiments : JoVE. , e50601 (2013).</li><li>Das, S., Macdonald, K., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> Journal of visualized experiments : JoVE. , (2013).</li><li>MacDonald, K. D., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+simple+method+of+mouse+lung+intubation.">An improved simple method of mouse lung intubation.</a> J Appl Physiol. 106, 984-987 (2009).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage.</a> American Reveiw of Respiratory Diseases. 132, 1078-1083 (1985).</li><li>Bishai, J., Fields, M. J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Mouse+Lung+Volumes+Inflated+with+Air+and+Instilled+Fixatives.">Comparison of Mouse Lung Volumes Inflated with Air and Instilled Fixatives.</a> Proc Am Thorac Soc. 3, A549 (2006).</li><li>Mitzner, W., Brown, R., Lee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+lung+volumes+in+mice.">In vivo measurement of lung volumes in mice.</a> Physiol Genomics. 4, 215-221 (2001).</li></ol>

此处描述的方法有若干优点。第一所需设备是简单且廉价的。第二，该插管可迅速用很少的错误进行。第三，要修复肺以恒定压力，然后测量所述固定肺容积的能力允许结构或细胞在肺10正确定量。 一个可能的缺点插管是小手术。这可能会限制重复该过程如果需要2次滴注的能力。然而，随着粘接剂的精心手术和申请中，我们已经能够每周定期做第二次滴注或?...

由于一些原因，肺癌还没有被广泛研究，在小鼠。其中一个原因是，获得的肺是非常困难的体内 ，并固定肺部定量分析操作并不常见。本文介绍的方法旨在改变这种状况。这里的目标是描述简单的方法，将在设置和小鼠肺分析肺癌或其它病理极大地帮助。而所有这些方法是全新的，它们没有被一起呈现如在这里所描述的简化方式的独立的方法。 已经有许多已描述的方法进行小鼠肺插管主要做重复肺功能或支气管肺泡灌洗在纵向研究单个老鼠的目的手稿。由于该原始文件，但已在该所描述的不同的方法来谅解备忘录几个其他文件Ë插管1-9。虽然所有这些方法都可以成功地被使用，它们通常需要相当大的训练，并且通常不是没有一个非平凡的故障率。另外，在为了进行肺功能测量中，套管需要适应气管得足够紧以便没有空气泄漏。然而，另一个实际使用气管插管是直接提供特定的药物（如癌细胞或其他侮辱），或治疗药物到肺。这样的程序并不需要紧密配合插管，也没有任何复杂的肺功能设备。此处示出了该方法的新颖特征包括一个小的外科手术，其允许插管而不进入食道插管的任何可能性。这个简单的方法使插管成功相对较少的培训或经验。多达30只小鼠/小时可以使用此方法具有故障率接近零对待。 一旦吨他老鼠是准备来牺牲，受伤或癌肺然后可以进行组织学和病理分析中删除。然而，为了正确地量化任何组织学变量与其它的肺相比，有必要标准化的固定程序和适当地量化固定肺容积10。本文详细介绍了简单的程序，使标准化的固定程序，以及一个方法来衡量固定的肺容积。该卷在组织学的量化的重要指标，因为如果没有这样的容积测定，只有相对密度可以测量10。一旦肺容积是已知的，然而，细胞和肺中的其他结构的测量绝对测量然后可以定量。

<table><tbody><tr><td>Laboratory Balance</td><td>Ohaus</td><td>Adventurer Pro Model AV 313&amp;nbsp;</td><td>Other balances can be used if they have a range of 1-300 g</td></tr><tr><td>20 g Luer intravenous catheter</td><td>Insylte</td><td></td><td>Several other possible vendors, &lt;em&gt;e.g.&lt;/em&gt;, Jelco Optiva</td></tr><tr><td>500 ml laboratory bottle</td><td>Various</td><td></td><td>Several other possible vendors</td></tr></tbody></table>

instillation and fixation methods useful in mouse lung cancer research

灌输活剂，细胞或化学物质直接进入肺而不伤害或杀死小鼠的能力是在肺癌的研究的一个重要工具。虽然有许多方法，这些方法已经发表，显示如何插管老鼠肺功能的测量，没有一个是不为小鼠大同伙快速气管滴注的潜在问题。在本文中，一个简单快捷的方法进行说明，使调查员进行这种滴注以有效的方式。该方法不需要任何特殊的工具或照明，并且可以用很少的实施而得知。它涉及麻醉小鼠，使一个小切口，在颈部以可视化的气管，然后直接插入静脉导管。小切口被快速关闭用组织粘合剂，并且将小鼠允许恢复。一个熟练的学生或技术人员可以在2分钟/小鼠的平均速率做滴注。一旦吨他癌症成立时，经常有必要在肺部的定量组织学分析。传统病理学家平时也懒得固定在规范肺胀，并分析往往是基于一个计分制度，可以说是相当主观的。虽然这有时可能是足够足够的肺肿瘤的大小，总的估计，肺结构或细胞的任何适当的体视学定量分析需要一个可重复的固定程序和后续的肺容积测量。在这里，我们描述了两个固定在压力下的肺部，然后精确测量固定肺容积简单可靠的方法。唯一的要求是一个实验室的平衡，是准确的范围内为1mg-300克。这里提出这样的程序可以大大提高创建，处理和分析肺癌小鼠的能力。

该过程描述在第一协议本身不导致任何广义的结果。这里只描述一个非常可靠的手段来灌输物质直接进入气管。 图7示出了其中锥虫蓝滴注用这里描述的方法的肺的一个例子。有染料的广泛分布，类似于已经看到的直接给出进入气管或小鼠11,12其他染料或示踪剂。我们也用这种方法来提供或者博莱霉素或弹性蛋白酶对肺，这导致广泛纤维化或肺气肿，分别。 <p class="jove_conten...

Watch this Scientific Journal Video about Instillation and Fixation Methods Useful in Mouse Lung Cancer Research at JoVE.com

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

本文的目的是描述简单的方法，将在设置和小鼠肺分析肺癌或其它病理极大地帮助。我们提出3个协议能够简单，可靠地进行肺滴注，固定，和肺容积的测量。

灌输和固定方法有用的小鼠肺癌研究

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

instillation-and-fixation-methods-useful-in-mouse-lung-cancer-research

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

28.2K 次观看.  Johns Hopkins University. 本文的目的是描述简单的方法，将在设置和小鼠肺分析肺癌或其它病理极大地帮助。我们提出3个协议能够简单，可靠地进行肺滴注，固定，和肺容积的测量。 

视频: Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

科研

医学

Perfusion and Inflation of the Mouse Lung for Tumor Histology

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

肿瘤病理学鼠肺的灌注和膨胀

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

癌症研究

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

小鼠肺气肿评价持续压力下的肺固定

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

直接注射内管理,改善小鼠肺内的选择性代理沉积

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

<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 ...

免疫与感染

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...

灌输和固定方法有用的小鼠肺癌研究

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此视频中的章节

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

肿瘤病理学鼠肺的灌注和膨胀

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

小鼠肺气肿评价持续压力下的肺固定

直接注射内管理,改善小鼠肺内的选择性代理沉积

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

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

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化