Name: in vivo imaging method to distinguish acute and chronic inflammation
Uploaded: 2013-08-16T12:00:00
Description: Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

这项工作是支持的的南希·劳瑞商标基金会。我们感谢南希E.科尔博士批判性阅读的手稿。我们感谢黄健K.他协助在准备这个视频报告。

注：所有认可机构协议和动物保健指引下进行的动物研究。 1。试剂和解决方案<ol><li> PMA溶液sc的接种：准备股票的佛波醇12 - 肉豆蔻酸酯-13 - 乙酸酯（PMA）在5毫克/毫升在DMSO溶液。原液储存在-20℃下接种前，解冻原液和稀释到1毫克/毫升PMA在PBS。 </li><li> SC接种LPS溶液：溶解在无菌PBS脂多糖（LPS的沙门氏菌血清型肠炎）在1毫克/毫升皮下注射接种前。 </li...

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在这份报告中，我们展示了生物发光法在生活动物炎症的非侵入性的成像。利用两种发光底物，鲁米诺，光泽精，该方法可以区分炎症的不同阶段。鲁米诺生物发光是与嗜中性粒细胞在炎症的急性期，而光泽精的生物发光是介导的巨噬细胞在慢性期。相对较小（分子量=177.16克/摩尔）和电不带电荷的，，鲁米诺可以很容易地穿透血浆和吞噬体膜25-29。此外，鲁米诺发光特定的胞内9?...

炎症是一个高度管制的生物反应中所涉及的各种人类疾病，包括微生物感染1，伤口愈合2，糖尿病，癌症，心血管5，6神经变性，和自身免疫疾病7。组织炎症，需要妥善协调各种免疫细胞，以达到病原体清除，组织修复和疾病的分辨率。嗜中性粒细胞和巨噬细胞的免疫组织炎症介质是关键。嗜中性粒细胞在炎症的急性期，各种有害刺激和组织损伤8的第一反应。中性粒细胞迅速从循环损伤的部位外渗，细胞释放抗微生物颗粒和吞噬入侵微生物的灭活。吞噬过程中，中性粒细胞吞噬微生物入侵到吞噬小体，其内细胞产生高水平的superoxIDE（O 2· - ）。吞噬体超氧化物歧化酶是许多下游的活性氧物种（ROS）的主要来源。例如，超氧化物歧可以dismutated过氧化氢 ​​（H 2 O 2）自发歧化或超氧化物歧化酶（SOD）9，10。在嗜中性粒细胞，髓过氧化物酶（MPO）进一步将过氧化氢 ​​抗微生物次氯酸（HOCl分子）11。由于炎症反应继续下去，循环单核细胞逐渐迁移到损伤部位，并分化成成熟的巨噬细胞的吞噬功能有助于去除灭活的病原体和细胞碎片。此外，作为一个关键的调节炎症的后期阶段中，巨噬细胞促进组织修复产生抗炎性细胞因子12和产生细胞外的活性氧（ROS）在一个较低的水平9。在稍后阶段产生的ROS调节组织重塑，新血管的形成，并reepithelia显逢13。 吞噬细胞的NADPH氧化酶（phox）的表达是在嗜中性粒细胞和巨噬细胞9超生产的主要来源。 PHOX是一个多亚基复合物的装配是被严格调控9。全酶包含一些胞浆调节亚基（PHOX P67，P47 PHOX，p40的PHOX，和RAC）和一种膜结合的异源二聚体细胞色素 b 558（包括的的亚基CYBA和CYBB）。 细胞色素b 558内的反应芯CYBB亚基（也被称为P91 PHOX和NOX2）进行的主要的氧化还原链反应9。有趣的是，它的组装基地，是中性粒细胞和巨噬细胞的差异。在休息的嗜中性粒细胞，细胞色素 b 558主要是存在于胞内储存颗粒的膜14。吞噬过程中，中性粒细胞聚集的holoenzymes吞噬体，其中也存在高水平的MPO活性。中性粒细胞PHOX迅速消耗氧气，并发挥其杀菌能力的活性氧的产生，这种现象称为呼吸爆发11。与此相反，巨噬细胞具有较低的MPO的表达水平和细胞色素b 558主要存在于质膜15，16。因此，嗜中性粒细胞产生高水平的抗微生物活性，超氧化物歧，而巨噬细胞产生较少的超监管功能15。 由于炎症体内过程是一个复杂的，非侵入性的成像方法，具体有炎症的不同阶段将允许纵向和定量评估疾病模型。使用机械的研究，我们先前已经表明使用两种化学发光剂，鲁米诺（5 - 氨基-2,3 - 二氢-1,4 - 二氮杂萘二酮），光泽精（双-N-甲基吖啶硝酸盐），急性和后期（慢性）的炎症阶段，分别为17的非侵入性的成像。鲁米诺使可视化的嗜中性粒细胞的髓过氧化物酶（MPO）活性在炎症的急性期18-20，而光泽精的生物发光，可用于评估相关联的巨噬细胞活性的晚期或慢性炎症17。在这篇稿件中，我们使用了两个实验性炎症模型（SC PMA和SC LPS），以证明这些成像技术。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>P8139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype enteritidis</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>L2012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>A4685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucigenin (bis-N-methylacridinium nitrate)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>M8010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum imaging system with Living Imaging 4.2 software package</td>
			<td>Caliper LS/Perkin Elmer, Hopkinton, MA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vivo imaging method to distinguish acute and chronic inflammation

炎症是许多人类疾病的一个基本方面。在这个视频报告中，我们展示了非侵入性的生物发光成像技术，区分急性和慢性炎症的小鼠模型。 ，中性粒细胞与组织损伤或病原体入侵的第一道防线，调解急性炎症反应中发挥了重要作用。由于炎症反应过程中，循环单核细胞逐渐迁移到损伤部位，并分化成成熟的巨噬细胞，去除组织碎片和生产抗炎细胞因子介导的慢性炎症，促进组织修复。腹腔注射鲁米诺（5 - 氨基-2,3 - 二氢-1,4 - 二氮杂萘二酮，钠盐）能够检测大部分组织中浸润的嗜中性粒细胞介导的​​急性炎症。鲁米诺特异性反应与中性粒细胞的吞噬体，因为从髓过氧化物酶的生物发光的结果（M内产生的超氧化物歧PO）介导的反应。光泽精（双-N-甲基吖啶硝酸盐）也与超氧化物反应，以便产生生物发光。然而，光泽精生物发光是独立MPO和它完全依赖于巨噬细胞的吞噬细胞NADPH氧化酶（了PhoX）在慢性炎症过程中。在一起，鲁米诺，光泽精允许非侵入性的可视化和纵向评估不同吞噬细胞种群的跨急性和慢性炎症相。由于在多种人类疾病的重要作用，炎症，我们相信这种非侵入性的成像方法可以帮助在各种病理条件下的差的嗜中性粒细胞和巨噬细胞的作用。

我们进行了纵向生物发光成像评估急性和慢性炎症的实验性炎症模型。佛波醇12 -肉豆蔻酸酯-13 -乙酸酯（PMA）是一种强效的蛋白激酶C（PKC）激动剂激活PHOX产生超氧阴离子，触发激烈的急性炎症反应的21。皮下注射50毫克的PMA在NCR裸鼠引起的快速皮肤刺激性和急性炎症注射部位18，22，23。我们每天进行4天成像PMA注射早3小时后获得的第一个图像， 图1展示了使用鲁米诺和...

Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

我们描述了一种非侵入性的成像方法，用于区分炎症阶段。系统交付鲁米诺揭示中性粒细胞MPO活性依赖于急性炎症。与此相反，注射光泽精允许可视化PHOX在巨噬细胞的活性依赖于慢性炎症。

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Research

JoVE Journal

Immunology and Infection

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Bioluminescent Bacterial Imaging In Vivo

Bioluminescent Bacterial Imaging In Vivo

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...