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 border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Name of Material/ Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fibrinogen from human placenta</td>
			<td>Sigma</td>
			<td>F4883</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T9549</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse VEGF 164</td>
			<td>R&#38;D</td>
			<td>493-MV</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse bFGF</td>
			<td>R&#38;D</td>
			<td>3139-FB</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Rodent Intubation Stand</td>
			<td style="width:177px;">Braintree Scientific INC</td>
			<td style="width:147px;">RIS 100</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fiber-Optic Light Source</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-565-35</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">20G Elastic catheter</td>
			<td>B.Braun</td>
			<td>4251652-02</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">MiniVent Ventilator</td>
			<td style="width:177px;">Harvard Apparatus</td>
			<td style="width:147px;">CGS-8009</td>
			<td>For ventilation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Stemi DV4 Steromicroscope</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-070-515</td>
			<td>For surgey</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Absobable suture</td>
			<td>Ethicon</td>
			<td>PDP304</td>
			<td>Surgical suture</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against CD31</td>
			<td>BD Biosciences</td>
			<td align="right">553370</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against AQP5</td>
			<td style="width:177px;">Abcam</td>
			<td>AB78486</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against SP-B</td>
			<td style="width:177px;">Millipore</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

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

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

科研

医学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

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 Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

无创性气管内插管，以研究小鼠肺的病理生理

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

插管介导气管内（IMIT）灌输：一种无创，龙专用输送系统

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

根尖切除小鼠模型来研究早期哺乳动物心脏再生

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...