Name: perfusion and inflation of the mouse lung for tumor histology
Uploaded: 2020-08-06T15:30:00.000+00:00
Duration: 4 min 24 s
Description: Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

本出版物中报道的研究得到了国家促进转化科学中心的支持，奖励编号为UL1TR003096（MDE）、国家心脏、肺和血液研究所肺病培养计划5T32HL134640（MLD）的博士前研究金。

这里描述的所有方法都已获得伯明翰阿拉巴马大学机构动物护理和使用委员会 （IACUC） 的批准。
1. 实验协议
<ol><li>使用批准的 IACUC 方法牺牲鼠标。在这里，我们使用了用5%异氟素麻醉的老鼠的宫颈错位。使用适当的鼠标进行研究;在这里，我们使用一个8周大的FVB鼠标</li>
	<li>使用手术剪刀，在下腹部中间做一个3.5-5毫米的水平切口。接下来，将手术剪刀插入切口产生的小孔中，并垂直向上切开中心中线，直切到鼠标颈部以下。</li>
	<li>用手指拉回皮肤，检查辅助淋巴结。</li>
	<li>使用手术剪刀，做一个3.5毫米的横切口打开腹腔，然后切入前方向，直至胸腔底部。检查腹腔内的器官:肝脏、脾脏、肾脏等。</li>
	<li>用扁平的手术剪刀，或使用钳子，移动肝脏，以暴露隔膜。检查隔膜是否肿瘤生长或转移。然后，轻轻剪断操作员右侧的隔膜，使其扩展。轻轻地从右到左切割隔膜，露出胸腔和肺部。小心不要割肺。</li>
	<li>穿过左肋笼的横向极端（在操作员的右边），检查肺的左叶。</li>
	<li>轻轻地将肺的右叶移开，切开右肋骨笼的横向极端，取出肋骨笼。 注意:拆除肋骨笼是可选的，虽然去除可以更清楚地看到以后的肺部膨胀。<ol>
<li>如果需要新鲜或冷冻的肺组织，使用造影钳夹住左叶的支气管，并在灌注前使用手术剪刀切除左肺。</li>
	</ol>
</li>
	<li>使用钳子提起覆盖气管的组织，切掉任何多余的组织。然后轻轻切开气管内的薄组织，露出气道。</li>
	<li>用手术剪刀切开肾动脉。</li>
	<li>要给肺部注入活力，请使用 3 mL 注射器与 22 G 针头将 1 倍 PBS 与 10 U/mL 肝素注射到心脏的右心室中。用PBS/肝素缓慢地以大约300微升/秒的速度使肺部充满。肺经常变白。此步骤中通常使用 2.5 mL 的 PBS。</li>
	<li>对于肺部膨胀，使用 3 mL 注射器与 22 G 针，这次与气管平行。将针头插入气管，注射10%的正品，流速不超过±200微升/s，直到肺部完全膨胀。一旦肺部充气，正式素会从气管中回流。将针头保持到位几秒钟，然后取款。<ol>
<li>（可选）在取出针头和肺部膨胀之前，使用缝合线连接气管。为此，使用 4 英寸的缝合线，用一对小钳子保持螺纹点。将螺纹放在气管的后侧，拉过，绕针转。接下来在针头周围打个过手结。拉结紧，从气管中取出针头，紧结。</li>
	</ol>
</li>
	<li>使用钳子抬起心脏，在肺部后面插入手术剪刀，在抬起心脏以切除肺部时切割结缔组织。</li>
	<li>切开心脏，将其从肺中取出。</li>
	<li>将肺部贴上鼠标 ID 或学习 ID 的盒式磁带。将盒式磁带放在 10% 缓冲的正品中，固定 24-48 小时。如果需要，肺可以固定一年以上。</li>
	<li>将含有肺部的盒式磁带转移到70%的乙醇中，然后进行病理学处理。</li>
</ol>

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <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> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, R., 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=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, 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=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</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.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., 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=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, K., 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=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

作者没有什么可透露的。

上述用于小鼠肺的灌注、膨胀和固定的程序是快速高效地准备小鼠肺进行肺肿瘤病理学和病理分析的理想方法。该程序不需要任何特殊设备，每只鼠标可在 6 分钟内完成。该程序不需要固定的通货膨胀量，也不要求恒定的流动压力。由于此程序不标准化，因此不建议希望对肺部进行固态或形态学分析。需要这种标准化的程序已更好地描述1，7。
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从复杂的 GEMM 到致癌素诱导模型，再到合成和异种移植模型，存在各种小鼠模型，其中癌细胞通过内脏、胸内、尾静脉或其他方法在肺内注射。所有这些模型都有着对肺病理学和病理学进行病理学评估的共同需要。因此，有必要有一个强大而快速的方法，在给肺注入时对小鼠进行尸检，以去除多余的血液，并充气和固定肺部，以清晰地可视化肺部结构。速度是这个过程的关键组成部分，因为可能需要在一个时间点从几十只小鼠身上收集肺部。此程序可以在每个鼠标不到 6 分钟内执行。
虽然这个程序是足以评估肿瘤组织学，它不建议那些谁想要执行立体学或肺部的形态测量。这种测量要求肺膨胀标准化，肺的绝对表面积、绝对体积、肺活体大小和1号的计算也是如此。这种方法对于某些成像方法也不理想。例如，通过μCT对肺部进行成像，以便进行肺活体测量分析，要求肺部保持充满空气2。当空气空间和尺寸的保存是主要关注，建议修复肺部的灌注脱水技术3，4。这种模型最大的担忧之一是肺壁破裂的可能性，减少其在肺气肿研究中的使用:然而，建议的肺部固定程序，以研究肺气肿仍然相当相似，因为它建议修复肺部，无论是通过在恒定的液体压力下10%的体温（类似于这里描述的协议）或原位固定5。
这里描述的程序的优点是，它不需要恒定的液体压力，而是充气肺，直到他们完全扩大，从而减少了手术所需的时间。这里的程序与毒理病理学学会军械库建议的方法非常相似，该协会成立了一个小组委员会，为毒理学研究推荐最佳的肺固定方法。这个小组委员会中的大多数科学家建议用注射器在体内灌输来固定肺部，尽管关于肺留在固定6中的时间有不同的建议。因此，在存在多种肺部膨胀和固定方法的同时，本文所述的方法被认为是快速膨胀和固定肺部以进行下游肿瘤病理学评估的最佳方法。

<table><tbody><tr><td>10% buffered formalin</td><td>Fisher</td><td>23-245685</td><td></td></tr><tr><td>22 G Needle</td><td>BD</td><td>305155</td><td></td></tr><tr><td>3 mL syringe</td><td>BD</td><td>309656</td><td></td></tr><tr><td>70% Ethanol</td><td>Decon</td><td>2405</td><td></td></tr><tr><td>Forceps</td><td>Harvard Apparatus</td><td>72-8595</td><td></td></tr><tr><td>Heparin</td><td>Fisher</td><td>H19</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Surgical scissors</td><td>Harvard Apparatus</td><td>72-8428</td><td></td></tr></tbody></table>

perfusion and inflation of the mouse lung for tumor histology

评估肺组织学的能力对肺癌研究和癌症转移领域至关重要。同样重要的是，在不牺牲所采购组织质量的情况下，从研究中快速有效地进行尸检。本协议的目标是提出一种方法，快速灌注，充气，并固定小鼠肺下游的病理学分析。这种方法不能规范肺部膨胀:因此，它不需要任何特殊的程序或设备，而是简单地通过气管直接灌输固定后，通过心脏灌注。这允许充分估计肿瘤大小，病理学和评分。这也允许在肺组织固定之前收集冷冻组织。这种方法是有限的，因为它不允许以后的肺的形态定量:然而，它足以从基因工程小鼠模型（GEMM）、合成模型以及异种移植肿瘤和转移研究进行肺肿瘤分析。

上述协议允许快速灌注、通货膨胀和固定小鼠肺。下图表示每一步的重要性。 图1 描绘了H&E染色的肺部，这些肺部与PBS和肺部一起渗透，其中灌注步骤已被跳过或肺部未能正确灌注。如前所示，灌注不良的肺部的过量血液导致组织学不理想，因此很难完全观察肺部结构。 图2 既显示了通货膨胀的重要性，也显示了过度通货膨胀的危险。由于肺泡的压缩?...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

该方法旨在为小鼠肺的灌注、膨胀和固定提供一种简单高效的方法，用于检查肺肿瘤病理学和对肺转移的评价。

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 ...

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

18.2K 次观看.  University of Alabama at Birmingham. 该方法旨在为小鼠肺的灌注、膨胀和固定提供一种简单高效的方法，用于检查肺肿瘤病理学和对肺转移的评价。

视频: Perfusion and Inflation of the Mouse Lung for Tumor Histology

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

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

利用18f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

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 ...

医学

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

通过小鼠肺切除术和假体植入测量内部肺表面积的标准方法

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

发育生物学

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

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 research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

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 ...

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 ...

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

血管灌注固定的穆林肺的空气膨胀

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, ...

神经科学

<meta charset="utf8"></head><body>小鼠肺移植中细胞动力学的实时监测

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>作者聚焦：通过活体成像推进肺移植免疫学

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植小鼠肺的活体双光子显微镜检查

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

肿瘤病理学鼠肺的灌注和膨胀

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长期高分辨率活体显微镜在肺与真空稳定成像窗口

利用¹⁸f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

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

通过小鼠肺切除术和假体植入测量内部肺表面积的标准方法

灌输和固定方法有用的小鼠肺癌研究

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

血管灌注固定的穆林肺的空气膨胀

移植小鼠肺的活体双光子显微镜检查

移植小鼠肺的活体双光子显微镜检查

移植小鼠肺的活体双光子显微镜检查