Name: induction of mouse lung injury by endotracheal injection of bleomycin
Uploaded: 2019-04-30T12:30:00
Description: Watch this Scientific Journal Video about Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin at JoVE.com

这项工作得到了意大利部长德拉·萨洛(阿曼多·加布里埃利)的RF-2011-02352331赠款的支持。

所有动物护理和实验程序均获得意大利卫生部的批准(授权书456/2016-PR),并根据《赫尔辛基公约宣言》执行。
1. 老鼠
<ol><li>购买后,让小鼠在注射前至少7天适应。 注:小鼠在无病原体条件下被安置在动物设施中,在12小时光/暗循环的恒定温度和湿度下保持,并免费获得水和标准颗粒食物。</li>
	<li>使用雌性C57BL/6小鼠,在12至16周大时注射。</li>
</ol>2. 内切治疗注射?...

<ol>
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内切管管理是向肺部输送外源性药物的优先途径。几年来,将肌霉素直接注射到气管中已被广泛用于诱发肺纤维化13,最近,已经开发出更先进的非侵入性技术,以实现这14,15 ,16.
此处描述的方法提供了一些有意义的好处,以超过一些潜在的限制。气管的注射需要手术干预,携带手术本身引起的并?...

肺纤维化是一种渐进的病理过程,其特点是细胞外基质成分(主要是I型胶原蛋白)在肺间质内沉积过多,导致肺功能受损。它是几种具有不同病因的人类肺部疾病的标志,是临床预后不良因素。由于目前的疗法相当有限1,小鼠模型仍然是一个重要工具,以进一步调查影响疾病发病和进展的致病机制,以及开发新的抗纤维化药物策略2,3.
迄今为止,博洛霉素的施用一直是实验诱发肺纤维化最常见的应用模型。除了多种分娩方法(包括静脉注射、腹管内、皮下和吸入外,宫内注射的博洛霉素已成为最常用的途径4、5。本文所描述的方法已经开发出来,以避免霉素对气管粘管的烫伤作用。事实上,通过将气管外在并通过操作显微镜进行可视化,可以实现将整个体积的博洛霉素溶液直接注入下气道,而不会在上气道中溢出。当有所需的外科专业知识和仪器可用时,此方法允许安全、可靠和可重复的诱导肺部炎症和纤维化,如下所述。

<table border="0" cellpadding="0" cellspacing="0" style="width:1024px;" width="1023">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">C57BL/6 mice</td>
			<td style="width:236px;">Charles River</td>
			<td style="width:239px;">Jax Mice Stock n. 000664</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">2,2,2-Tribromoethanol (Avertin)&#160;</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:239px;">T48402</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Barraquer Micro Needle Holder</td>
			<td style="width:236px;">Lawton</td>
			<td style="width:239px;">62-3755</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Bleomycin sulfate</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>B1141000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">B&#252;rker chamber</td>
			<td style="width:236px;">Brand&#160;</td>
			<td style="width:239px;">718905</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture Flasks&#160;</td>
			<td style="width:236px;">EuroClone</td>
			<td style="width:239px;">ET7076</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Disposable razors</td>
			<td style="width:236px;">Unigloves</td>
			<td style="width:239px;">4080</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Dissecting Forceps</td>
			<td style="width:236px;">Aesculap Surgical Instruments</td>
			<td style="width:239px;">BD311R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">DPBS</td>
			<td style="width:236px;">Gibco</td>
			<td style="width:239px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Heating pad</td>
			<td style="width:236px;">2Biological Instruments</td>
			<td style="width:239px;">557023</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Isoflurane Vet</td>
			<td style="width:236px;">Merial Italia</td>
			<td>N01AB06</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Operating Microscope</td>
			<td style="width:236px;">Carl Zeiss</td>
			<td style="width:239px;">Model OPM 16</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">TrypLE Select Enzyme</td>
			<td style="width:236px;">Gibco</td>
			<td style="width:239px;">12563-029</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Vannas Micro Scissors</td>
			<td style="width:236px;">Aesculap Surgical Instruments</td>
			<td style="width:239px;">OC498R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:369px;">Vicryl Plus 4/0 Absorbable Suture, FS-2 needle 19 mm</td>
			<td style="width:236px;">Ethicon</td>
			<td style="width:239px;">VCP392ZH</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of mouse lung injury by endotracheal injection of bleomycin

肺纤维化是几种人类肺病的标志,病因不同。由于目前的疗法相当有限,小鼠模型仍然是开发新的抗纤维化策略的重要工具。在这里,我们提供了一种有效的方法,以研究从全脐带(hUC-MSC)获得的人类中位血质基质细胞在体内抗纤维化活性,以减轻博霉素引起的肺损伤。C57BL/6小鼠接受单次内切注射布洛霉素(1.5 U/kg体重),然后两次向尾静脉注入hUC-MSC(2.5 x 10 5),在布洛霉素给予后24小时和7天。在第8天、第14天或第21天牺牲时,将分析炎症和纤维化变化、胶原蛋白含量和hUC-MSC在外植肺组织中的存在。将小霉素注射到小鼠气管中,可直接瞄准肺部,导致广泛的肺部炎症和纤维化。双剂量hUC-MSC的系统施用导致博霉素引起的肺损伤的早期钝化。静脉注射hUC-MSC被暂时移植到小鼠肺部,在那里他们发挥抗炎和抗纤维化活性。最后,该方案已成功应用于人类肺纤维化实验小鼠模型hUC-MSC的临床前测试。然而,这种技术可以很容易地扩展,既研究不同内切施用的物质对肺部病理生理学的影响,也验证新的抗炎和抗纤维化全身疗法。

肺损伤是由在100μL无菌盐水中单次注射1.5 U/kg的硫酸洋霉素体重引起的。对照动物接受相同量盐水的内切注射。两针hUC-MSC(2.5 x 105在200 μL无菌盐水)注入小鼠尾静脉,24小时和7天后,博洛霉素。对照动物接受同等体积的无菌盐水的静脉注射。小鼠在博霉素给给后的第8天、第14天和第21天被牺牲用于肺外植和组织处理(图1)。
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Watch this Scientific Journal Video about Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin at JoVE.com

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

在这里,我们提出了一种有效的方法,以研究静脉注射人类中发性血质细胞的抗纤维化活性,从整个脐带获得后,通过在C57BL的内切治疗注射注射肺损伤/6 小鼠。该协议可以很容易地扩展到其他疗法的临床前测试。

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 ...

该协议可以通过血管注射Bleomycin，在肺部诱导重要的炎症和纤维反应，并验证新的抗炎和抗纤维化全身疗法的尾静脉输液。该技术的主要优点是，将Bleomycin直接注射到小鼠气管中，可确保快速瞄准肺部，而上气道没有任何溢出物，从而安全、可靠且可重复地诱导肺炎和纤维化。演示这个程序的将是来自我们合作实验室的兽医菲奥伦扎·奥兰多博士。首先，将麻醉小鼠放在手术平台上。巧妙地用手术胶带固定它的腿，以固定到位。要过度伸展小鼠的脖子，请在其颈椎区域下放置一个支撑，如牙棉卷。然后用剃刀轻轻剃喉咙。使用一对解剖钳来捏皮。使用一对环处理弯曲钝剪刀，使短切口约半厘米长，以对应小鼠胸肌。使用棉布棒止血。然后通过钝解剖将气管外化，然后轻轻地从脂肪和其他组织中清洁。旋转手术平台，使鼠标头部朝向操作者，使操作员在注射过程中正确角度对注射器进行角。要可视化气管，请将鼠标置于手术显微镜下。调整照明并设置 1 和 1.2 之间的放大倍率，然后调整焦点和锐度。气管可以很容易地区分为白色半透明管与清晰可见的气管环。轻轻移液器，混合 Bleomycin 溶液，用 25 量针将 100 微升吸气吸入 0.5 毫升注射器，避免气泡形成。一旦气管被清晰地可视化，小心地用针尖以30度角刺穿气管。慢慢地将100微升的Bleomycin直接注射到气管的流明中。等待几秒钟后，整个体积向下针移动，将其从气管中取出。当针头正确插入气管时，观察几秒发生的呼吸暂停，表明整个液体量已被吸入。安全丢弃注射器和针头。用5-0可吸收的缝合线关闭皮下筋膜和皮肤伤口，让动物恢复。再次麻醉小鼠后，一旦失去知觉得到确认，将鼠标放在无菌罩下。在整个实验中，使用连续流量为1.5%异氟的面膜保持温和的麻醉。为了促进血管扩张和更容易注射，将尾巴浸泡在温水中两分钟。通过轻轻移液混合人体脐带中质质的悬浮细胞，以避免细胞团形成。用26规格的针头将200微升吸入一毫升注射器，避免气泡形成。抓住尖端的尾巴，轻轻拉直它。然后找到尾巴的横向静脉。用手术刀轻轻刮取尾巴，然后用70%乙醇擦拭。从尾部的远角开始，以 15 度角将针头插入静脉。慢慢地注入200微升的人类脐带中质质质细胞。要监测成功的静脉输液，观察进入静脉的液体无阻力和缺乏外注射。等待几秒钟后，直到整个体积沿着针头向下移动，将其从静脉中去除。然后用无菌纱布短暂地对入口伤口施加压力，以防止出血，让动物恢复。输液后安全丢弃注射器和针头。Bleomycin注射后，与盐水注射后的正常肺结构相比，由血氧林和Eosin和Picrosirius红染色评估肺组织病理变化，确认广泛炎症渗透和纤维化。由血红素引起的肺部炎症和纤维化在很大程度上被人类脐带间质细胞输液减轻，但不受盐水影响。胶原蛋白沉积通过羟基丙酸测定进行测量。在布洛霉素注射后，羟基丙酸水平从第8天逐渐增加至第14天和21日。而人类脐带中质细胞的加入显著降低了血红素的影响。全肺mRNA中Col1A1型一型胶原蛋白基因的表达水平在第8天、第14天和21日也出现逐渐增加，由于人类脐带中质细胞治疗部分降低。含有特异性抗体的免疫功能表明，注入人类脐带的细胞细胞迅速有效地到达小鼠肺部，尽管只检测到少数细胞。这些数据表明，尽管具有长期的保护作用，但细胞从现场损伤中迅速脱位。使用操作显微镜可确保在安装前准确监控针头正确放入小鼠气管中，从而最大限度地降低损坏针头的风险，从而确保成功的信心。

Research

JoVE Journal

Immunology and Infection

Transurethral Induction of Mouse Urinary Tract Infection

经尿道诱导小鼠尿路感染

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

科研

免疫与感染

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Isolation of Mouse Lung Dendritic Cells

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Pseudomonas aeruginosa Induced Lung Injury Model

Pseudomonas aeruginosa Induced Lung Injury Model

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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

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 ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

单方支气管内酸灌输自我限制急性肺损伤模型

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...