Name: implantation of fibrin gel on mouse lung to study lung-specific angiogenesis
Uploaded: 2014-12-21T14:00:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis at JoVE.com

这项工作是由美国心脏协会（AM），美国国防部（BC074986）和波士顿儿童医院学院职业发展奖学金（TM，AM）的资金支持。作者感谢阿曼达江江伊丽莎白的技术援助。

注: 在动物体内进行了研究，在严格按照指南中的美国国立卫生研究院的实验动物的护理和使用的建议。该协议是审查和批准由波士顿儿童医院的动物护理和使用委员会（协议编号:13-10-2526R，14-02-2568R）。在这个协议中使用的所有药品都是医药级和这些药物在无菌条件下制备。 1.纤维蛋白胶的制备<ol><li>准备包含VEGF和bFGF纤维蛋白胶。 <ol><li>纤维蛋白原和凝血酶的被储存在-80℃至室温（25℃），该解冻储备溶液。 </li><li>加入凝血酶（最终浓度:2.5单位/毫升）， 氯化钙 （终浓度:45毫摩尔），血管内皮生长因子（终浓度:0-100毫微克/毫升）和bFGF（终浓度:0-100毫微克/毫升）的血纤维蛋白原溶液（最终浓度:12.5毫克/毫升在0.9％sodiu米在1.5ml管中的氯化物溶液43-45）。 </li><li>吹打轻轻混匀。 </li><li>轻轻吸取200微升混合物到在用P200枪头一个逐滴方式无菌塑料培养皿中。 </li><li>孵育的液滴在37℃下进行30-60分钟，直到它们固化。 注:该固化的凝胶可以保持在密封塑料盘中，在室温（25℃）数小时植入（ 图1a）之前。 </li></ol></li><li>修剪的血纤维蛋白凝胶成使用小手术剪植入前约3×3×3mm的立方体。 </li></ol> 2.鼠标准备<ol><li>通过腹膜内麻醉成年小鼠（8-12周）（IP）注射氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克），并确认该小鼠得到充分捏鼠标趾麻醉。 <ol><li>使用上的眼睛鼠标兽医药膏，以防止在实验过程中干燥。 </li></ol></li><li>剃须福R经由鼠标的肋笼的左侧。 </li><li>执行鼠标的气管插管。 </li><li>将鼠标上的插管站在倾斜的70度并按住鼠标到位了位于支架顶部的小橡皮圈钩住它的上门牙。 </li><li>使用钝钳轻轻缩回舌头一边。 </li><li>可视化用光纤鹅颈显微镜照明器的帮助下喉部。 </li><li>插入气管内的弹性导管（21 G）的进气管。 </li><li>确认鼠标自发地比较顺利地呼吸（常规100-150次/分钟，没有矛盾或浅的呼吸）。 </li><li>鼠标放置在俯卧位的解剖显微镜下。 </li><li>机械通风使用啮齿类呼吸器鼠标（150次呼吸/分钟和7毫升/公斤潮气量）。 </li><li>算肋定位第 4 次和第 5 次肋之间的肋间空间。 </li><li>在区域b创建一个无菌区Ÿ彻底擦拭用酒精和碘伏。用无菌手术被单充分覆盖手术区域。 </li></ol> 3.鼠标手术<ol><li>局部注射0.25％布比卡因（200微升）中的皮肤后，使横向皮肤切口（大约1厘米长）比使用解剖剪刀肋间空间。 </li><li>注射0.25％布比卡因（200微升）到肋间肌肉后，应采用精小剪刀第 4和第 5肋骨之间的肌肉切口。 </li><li>插入一个解剖拉钩肋骨之间完全可视化的左肺。 <ol><li>刮使用细镊子左肺的中心的内脏胸膜的小区域（1×1平方毫米）。 </li><li>适用于使用消毒棉签，直到出血和空气泄漏被完全控制的地区温和的压力。 </li><li>把少量的纤维蛋白原/凝血酶（纤维蛋白胶）新鲜混合气（步骤1.1.2。20＆＃181，L）在使用P200枪头的区域。 </li></ol></li><li>轻轻使用小镊子在区域（ 图1b）放置一个纤维蛋白胶（步骤1.2）。 <ol><li>确认该凝胶是在肺的呼吸运动以及固定在该地区。 </li></ol></li><li>确保既没有大量的空气泄漏，也没有从肺出血。 </li><li>靠近切口（肌肉和皮肤层）用可吸收的缝线，其不必须去除。 </li><li>吸用27G的针和1ml注射器，以防止气胸胸腔。 </li><li>终止机械通气。 </li></ol> 4.鼠标恢复<ol><li>确保鼠标自发呼吸平稳的方式（常规100-150次/分钟，没有矛盾或浅的呼吸）。 </li><li> IP注射1毫升预热的0.9％的NaCl，以防止脱水。 </li><li>允许小鼠恢复对循环热水垫。 </li><li>取出endotrache确认鼠标有稳定的呼吸后，人管。 </li><li>注入美洛昔康（5毫克/千克，皮下注射（SC），3天，作为术后镇痛。 </li><li>监控鼠标的动作仔细至少15分钟的间隔，直至其胸骨（可滚动到它的肚子，并保持直立），并有意识的。 </li><li>恢复后，返回鼠标到一个新的笼子里，从小鼠中分离无术。 </li><li>监视手术部位感染的迹象（红，肿，放电），动物的基本生物学功能（食物和水的摄入，排尿，排便，体重增加），以及临床症状的困扰（立毛，减少运动）每日继外科手术。 </li></ol> 5.收获肺<ol><li>在植入后7至30天，通过压缩气体源使用的CO 2安乐死鼠标。 </li><li>使xyphoid过程的前端与胸骨之间的切口缺口（正中）和收获全肺与切断气管和解剖所有连接到心脏，肺，气管组织学和生化分析植入凝胶。 </li><li>修复肺部有4％多聚甲醛溶液注入凝胶一夜之间在4ºC，嵌入OCT复合，并利用30微米厚的串行台阶部分。 </li><li>进行组织学（苏木伊红染色）和免疫组化分析（内皮标记:CD31，上皮标记:水通道蛋白（AQP）5和表面活性蛋白（SP）-B）利用共聚焦显微镜22,37,40。 </li><li>编译叠层光学切片（30μm厚），以形成肺血管内皮的使用3D图像分析软件37的三维图像和上皮细胞。 </li><li>量化利用图像分析软件46新形成的血管的投影面积。 </li></ol>

The authors declare that they have no competing financial interests.

本文介绍了一种新的方法来植入活的成年小鼠的肺表面的生物材料。有了这个系统，主机肺源性血管被成功概括材料内部。这个系统使研究人员能够内皮细胞之间探索串扰，其他细胞（ 例如 ，上皮细胞，间质细胞，免疫细胞）和各种细胞外基质成分所需要的地方的血管生成50-53和肺泡再生24,54。尽管传统的体内皮下植入的水凝胶已被广泛地用于研究血管发生37-39?...

该协议的总体目标是引进一种方法来对植入成年小鼠，这使研究人员能够表征肺血管和肺泡发育的分子机制，肺表面纤维蛋白胶，并为了发展仿生材料能够利用这些知识的扼要生理肺血管和肺泡的形成来治疗各种肺部疾病。 超过3500万美国人患有慢性肺部疾病，包括慢性阻塞性肺疾病，肺纤维化。这些患者有持久的慢性呼吸道症状，如呼吸急促，胸闷，咳嗽唠叨，和疲倦，其中显著影响他们的日常生活1-3。尽管努力开发有效的治疗这些肺病量很大，目前还没有治愈;因此，生活质量对这些患者差，经济和人力成本是喜GH 4-7。目前，肺移植是挽救患者的终末期慢性肺部疾病的唯一途径。由于移植的供体，成本高，严重的并发症，成活率低8-11短缺然而，移植不是一个最佳的方法。在组织工程技术的最新进展迅速使研究人员通过重新填充脱细胞的全肺与各种类型的祖细胞或诱导的多能干细胞（iPS细胞）12,13，以生物工程植入肺。然而，这些生物工程肺部功能的宿主动物只植入12,14,15后的几个小时。利用生物材料再生肺的复杂结构和功能也已经相当成功。这可能是因为支配成人肺再生键生物过程没有得到很好的研究。在肺，形成血管系统是国内最早，最重要的事件之一杜里吴发育和再生16-21。在肺中，不仅输送氧气，营养物质和所需的器官形成的各种细胞成分，而且还提供了有益的调节信号到周围的细胞22-25新形成的脉管。因此，血管生成在成年肺部24,26,27再生alveolarization关键作用。此外，失调的血管生成有助于慢性肺部疾病，如慢性阻塞性肺疾病（COPD）28，支气管肺发育不良（BPD）21-23，和肺纤维化29。因此，开发用于工程肺或治疗慢性肺疾病更有效的策略，有必要了解肺特异性血管生成的基本机制。 每个器官显示独特的机械和化学性质，其可生理和病理条件30-33之间不同。这些器官特异性microenvironments调节内皮细胞行为和协调在器官特异性方式24,34-36血管网形成。因此，开发用于肺再生更有效的策略，需要底层肺特异性血管生成的机制，来理解。而常规的体内血管发生测定法，如皮下植入的水凝胶已被广泛地用于研究血管发生37-39，这些方法不重述器官特异性血管生成。最近，一种新的方法，以在植入的小鼠的肺的弹性模基底膜已经开发并示出成功地招募血管和肺上皮细胞进入凝胶22。这种独特的方法将允许研究人员探索生理和病理条件下肺特异性血管生成的血管和非血管肺细胞的机制，以及相互作用。自1）的Matrigel不适合临床应用; 2）电子商务用于铸造的凝胶可能影响水凝胶和宿主肺组织和3）对肺的弹性模呼吸期间潜在地导致肺功能和疼痛的障碍之间的相互作用，作为更临床相关的方式LASTIC模​​具，三维纤维蛋白基质含有血管生成因子（血管内皮生长因子（VEGF）/碱性成纤维细胞生长因子（bFGF））已被植入在小鼠肺而不浇铸在弹性模，并已成功地概括宿主肺源性血管生成。纤维蛋白胶，从凝血酶裂解纤维蛋白原产生聚合物纤维，是已知的陷阱多种血管生成因子，如bFGF和VEGF的加速血管生成在体内 40,41。因为它的再生能力和可生物降解的性质的42，纤维蛋白胶被广泛应用于组织工程领域。 本文介绍了一种新颖而独特的方式植入活adul肺表面纤维蛋白胶吨小鼠并表明宿主肺源性血管生成被概括在体内的凝胶内。该方法中，这使得研究人员研究肺特异性血管生成，将有可能导致新的治疗方法用于各种类型的肺部疾病的发展和显著前进努力成功再生成人肺。

<table><tbody><tr><td>Fibrinogen from human placenta</td><td>Sigma</td><td>F4883</td><td>For fabrication of fibrin gel</td></tr><tr><td>Thrombin from bovine plasma</td><td>Sigma</td><td>T9549</td><td>For fabrication of fibrin gel</td></tr><tr><td>Recombinant mouse VEGF 164</td><td>R&amp;amp;D</td><td>493-MV</td><td>For supplementation to fibrin gel</td></tr><tr><td>Recombinant mouse bFGF</td><td>R&amp;amp;D</td><td>3139-FB</td><td>For supplementation to fibrin gel</td></tr><tr><td>Rodent Intubation Stand</td><td>Braintree Scientific INC</td><td>RIS 100</td><td>For intubation</td></tr><tr><td>Fiber-Optic Light Source</td><td>Fisher Scientific</td><td>12-565-35</td><td>For intubation</td></tr><tr><td>21 G Elastic catheter</td><td>B.Braun</td><td>4251652-02</td><td>For intubation</td></tr><tr><td>MiniVent Ventilator</td><td>Harvard Apparatus</td><td>CGS-8009</td><td>For ventilation</td></tr><tr><td>Stemi DV4 Steromicroscope</td><td>Fisher Scientific</td><td>12-070-515</td><td>For surgey</td></tr><tr><td>Absobable suture</td><td>Ethicon</td><td>PDP304</td><td>Surgical suture</td></tr><tr><td>Antibody against CD31</td><td>BD Biosciences</td><td>553370</td><td>Immunohistochemistry</td></tr><tr><td>Antibody against AQP5</td><td>Abcam</td><td>AB78486</td><td>Immunohistochemistry</td></tr><tr><td>Antibody against SP-B</td><td>Abcam</td><td>AB40876</td><td>Immunohistochemistry</td></tr></tbody></table>

implantation of fibrin gel on mouse lung to study lung-specific angiogenesis

在干细胞研究，生物工程技术的最新显著的进步在利用生物材料的再生和修复，在骨科和牙周领域简单的组织损伤取得了很大进展。然而，试图再生的结构和更复杂的三维（3D）的器官的功能，如肺部并不是非常成功的，因为器官再生的生物过程还没有被很好的探讨。这是越来越明显，血管生成，新血管的形成，起着器官再生的关键角色。新形成的脉管不仅输送氧气，营养物质和所需要的器官再生的各种细胞成分，但也提供了有益的信号，以再生局部组织。因此，为了成功地再生成人在肺部，有必要概括的肺特异性的微环境，其中血管发生的局部肺组织的驱动器再生。虽然合作nventional 体内血管发生测定法，诸如细胞外基质（ECM）的富水凝胶（ 例如 ，纤维蛋白或胶原凝胶或基质胶-细胞外基质蛋白混合物通过Engelbreth-Holm的-群小鼠肉瘤细胞分泌）的皮下植入，被广泛用于探索血管生成的一般机制，肺特异性血管发生还没有得到很好的特点，因为在肺癌生物材料的原位移植方法尚未确立。该协议的目的是介绍一种独特的方法来植入活成年小鼠的肺表面纤维蛋白胶，允许主机肺源性血管生成的成功概括的凝胶内。这种方法使研究人员能够探索由肺特定的微环境控制血管形成和牙槽骨的再生在正常和病理条件下的机制。由于植入的生物材料释放并提供物理和化学信号到相邻的升UNG组织中，对患病的肺这些生物材料的植入可以潜在正常化相邻患病组织，使研究人员开发新的治疗方法用于各种类型的肺部疾病。

检查宿主肺源性血管形成是否被概括植入肺中的生物材料的内部，纤维蛋白凝胶补充有大血管生成因子VEGF和bFGF（0,10和100ng / ml的各自）植入的活小鼠肺部的表面上使用基底膜22日报道。纤维蛋白凝胶47包含这些血管生成生长因子被制造， 如图1a所示 。开胸后，左肺表面的一个小区域，使用镊子刮下和所制造的纤维蛋白凝胶植入在成年小鼠的使用量小的纤维蛋白胶的肺?...

Watch this Scientific Journal Video about Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis at JoVE.com

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

implantation-fibrin-gel-on-mouse-lung-to-study-lung-specific

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.0K 次观看.  Boston Children's Hospital and Harvard Medical School. Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse. 

视频: Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

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

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

生物工程

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

在缺血大鼠肺的血管生成

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

研究癌症转移至肺部的永久窗口

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

癌症研究

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

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 ...

免疫与感染

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

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 ...

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 ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

使用脱细胞小鼠肺和原代人内皮细胞移植生物工程肺

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...