Name: in vivo imaging of reactive oxygen species in a murine wound model
Uploaded: 2018-11-17T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model at JoVE.com

我们感谢纽约大学医学院的临床前成像核心, 并特别感谢奥兰多阿里斯蒂萨瓦尔和优素福扎姆·瓦德吉里。核心是由 laura 和 isaac perlmutter 癌症中心支持赠款 nihci nhci 5p30ca016087 和 nibib 生物医学技术资源中心 grant nih p41 eb017183 部分支持的共享资源。这项工作得到了美国糖尿病协会 "阻止糖尿病之路" (原案 1-16-ace-08) 和纽约大学应用研究支助基金对 p. r. 的支持。

这里描述的所有方法都得到了纽约大学医学院动物护理和使用机构委员会的批准。所有老鼠都被安置在屏障后面, 所有人员都佩戴适当的个人防护设备。
1. 第0天: 小鼠模型的制备
<ol><li>对糖尿病小鼠 (leprdb/db) 进行麻醉, 年龄为 8-12周, 吸入2% 异氟醚。确认每只鼠标已使用脚垫夹紧测试进行了适当麻醉。在每只眼睛上涂上无菌的眼部润滑剂, 以防止对干燥的刺激。研究?...

<ol>
	<li>Nishinaka, Y., 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=A+new+sensitive+chemiluminescence+probe,+L-012,+for+measuring+the+production+of+superoxide+anion+by+cells.">A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.</a> Biochemical and Biophysical Research Communications. 193 (2), 554-559 (1993).</li><li>Daiber, A., 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=Measurement+of+NAD(P)H+oxidase-derived+superoxide+with+the+luminol+analogue+L-012.">Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.</a> Free Radical Biology and Medicine. 36 (1), 101-111 (2004).</li><li>Imada, I., 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=Analysis+of+reactive+oxygen+species+generated+by+neutrophils+using+a+chemiluminescence+probe+L-012.">Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012.</a> Analytical Biochemistry. 271 (1), 53-58 (1999).</li><li>Sohn, H. Y., Gloe, T., Keller, M., Schoenafinger, K., Pohl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+superoxide+detection+in+vascular+cells+by+the+new+chemiluminescence+dye+L-012.">Sensitive superoxide detection in vascular cells by the new chemiluminescence dye L-012.</a> Journal of Vascular Research. 36 (6), 456-464 (1999).</li><li>Fuchs, 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=In+vivo+Hypoxia+PET+Imaging+Quantifies+the+Severity+of+Arthritic+Joint+Inflammation+in+Line+with+Overexpression+of+Hypoxia-Inducible+Factor+and+Enhanced+Reactive+Oxygen+Species+Generation.">In vivo Hypoxia PET Imaging Quantifies the Severity of Arthritic Joint Inflammation in Line with Overexpression of Hypoxia-Inducible Factor and Enhanced Reactive Oxygen Species Generation.</a> The Journal of Nuclear Medicine. 58 (5), 853-860 (2017).</li><li>Asghar, M. N., 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=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+murine+colitis.">In vivo imaging of reactive oxygen and nitrogen species in murine colitis.</a> Inflammatory Bowel Diseases. 20 (8), 1435-1447 (2014).</li><li>Galiano, R. D., Michaels, J. t., Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+reproducible+murine+model+of+excisional+wound+healing.">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair and Regeneration. 12 (4), 485-492 (2004).</li><li>Soares, M. A., 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=Restoration+of+Nrf2+Signaling+Normalizes+the+Regenerative+Niche.">Restoration of Nrf2 Signaling Normalizes the Regenerative Niche.</a> Diabetes. 65 (3), 633-646 (2016).</li><li>Wang, X., 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=Imaging+ROS+signaling+in+cells+and+animals.">Imaging ROS signaling in cells and animals.</a> Journal of Molecular Medicine. 91 (8), 917-927 (2013).</li><li>Kielland, A., 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=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radical Biology and Medicine. 47 (6), 760-766 (2009).</li><li>Balke, J., 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=Visualizing+Oxidative+Cellular+Stress+Induced+by+Nanoparticles+in+the+Subcytotoxic+Range+Using+Fluorescence+Lifetime+Imaging.">Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime Imaging.</a> Small. , (2018).</li><li>Zielonka, J., Lambeth, J. D., Kalyanaraman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+L-012,+a+luminol-based+chemiluminescent+probe,+for+detecting+superoxide+and+identifying+inhibitors+of+NADPH+oxidase:+a+reevaluation.">On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation.</a> Free Radical Biology and Medicine. 65, 1310-1314 (2013).</li><li>Dikalov, S. I., Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+detection+of+mitochondrial+and+cellular+reactive+oxygen+species.">Methods for detection of mitochondrial and cellular reactive oxygen species.</a> Antioxidants &amp; Redox Signalling. 20 (2), 372-382 (2014).</li><li>Rabbani, P. S., 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=Targeted+Nrf2+activation+therapy+with+RTA+408+enhances+regenerative+capacity+of+diabetic+wounds.">Targeted Nrf2 activation therapy with RTA 408 enhances regenerative capacity of diabetic wounds.</a> Diabetes Research and Clinical Practice. 139, 11-23 (2018).</li><li>Rabbani, P. S., 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=Novel+lipoproteoplex+delivers+Keap1+siRNA+based+gene+therapy+to+accelerate+diabetic+wound+healing.">Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.</a> Biomaterials. 132, 1-15 (2017).</li></ol>

测量 ros 的常用技术受到需要组织提取或类似侵入性技术的复杂协议的限制。近年来, 氧化应激的测量是在创新成像方式的基础上报告的, 从而允许对 9、10、11进行时空评估。l-012 作为相对于发光醇、露西根宁和 mcla1,4 的化学发光探针, 具有多种优点。该化合物无毒, 易于吸收, 与发光?...

通过炎症过程产生的氧代谢物有助于各种信号级联以及细胞成分的破坏性改变1。利用敏感和特定的技术来测量 ros 是研究炎症过程和描述氧化应激的影响的关键。体内成像是有价值的, 因为它能够提供动态的空间和时间数据在活组织。l-012 是一种合成化学发光探针, 对超氧化物阴离子高度敏感, 在细胞、组织和全血1、2、3、4. 已成功地用于小鼠模型的体内成像, 以研究几种炎症性疾病, 包括关节炎和结肠炎5,6。它尚未被应用于已建立的皮肤伤口愈合模型。测量产生的 ros 对于评估不同条件下伤口愈合的进展同样重要。这种方法的敏感性和非侵入性使其成为研究小鼠模型伤口愈合的一种有前途的技术。
nrf2 是抗氧化反应的主要驱动因素, 也是一种特殊的抗氧化反应元素 (are) 的转录因子, 这些元素是几种抗氧化酶8的启动子区域所共有的。在没有氧化应激的情况下, nrf2 被 keap1 隔离在细胞质中, 从而导致其普遍化和降解。nrf和解1途径的不平衡与不适当的氧化还原稳态和延迟伤口愈合有关, 在氧化应激增加的背景下 9.我们以前已经证明, 抑制 keap1 刺激增加 nrf2 活性, 并促进挽救治疗病理皮肤伤口愈合在糖尿病伤口9。
在这里, 我们描述了一个协议, 利用 l-012 辅助生物发光成像测量 ros 水平在一个切除皮肤伤口愈合模型, 这是至关重要的突出 ros 和伤口愈合之间的联系。这项技术显示了伤口和周围氧化负荷的实时变化。此外, 这种方法可以对影响氧化还原处理的干预措施和机制进行快速评估。在这里, 我们使用keap1 敲除模型来恢复氧化还原稳态, 以评估我们的策略的适用性。因为我们的技术是无创的, 伤口不受干扰, 同一动物可以用于进一步的确认分析的基础上, 组织学或细胞裂解物。

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			<td height="20" style="height:20px;width:265px;">BKS.Cg-Dock7m+/+ Leprdb/J mice</td>
			<td style="width:164px;">Jackson Laboratories</td>
			<td style="width:111px;">000642</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">13 cm x 18 cm Silicone sheet (0.6 mm)</td>
			<td>Sigma Aldrich&#160;</td>
			<td>665581</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3M Tegaderm Transparent Film Dressings</td>
			<td>3M</td>
			<td>88-1626W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000 Transfection Reagent</td>
			<td>Life Technologies&#160;</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keap1 Stealth siRNA</td>
			<td>Thermofisher Scientific</td>
			<td>1299001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silencer negative control&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>AM4635</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM Reduced Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>ThermoFisher Scientific</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methyl-cellulose&#160;</td>
			<td>Sigma Aldrich</td>
			<td>9004-67-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-012</td>
			<td>Wako Chemicals</td>
			<td>120-04891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IVIS Lumina III XR In Vivo Imaging System&#160;</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
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in vivo imaging of reactive oxygen species in a murine wound model

活性氧 (ros) 的产生是炎症过程的标志, 但过度, 氧化应激广泛涉及各种疾病, 如癌症, 动脉粥样硬化和糖尿病。我们以前已经证明, 核因子 (红血球衍生 2) 类似 2 (Nrf2)/开克样红血球细胞衍生蛋白 1 (keap1) 信号通路导致在糖尿病皮肤伤口愈合过程中的极端 ros 不平衡。由于 ros 水平是伤口愈合进展的重要指标, 具体和准确的定量技术是有价值的。介绍了几种用于测量细胞和组织中 ros 的体外检测方法;但是, 它们只为每个样本提供一个累积测量。最近, 基于蛋白质的指标和成像方式的发展使得能够进行独特的时空分析。l-012 (c13h8 cln4nao2) 是一种发光醇衍生物, 可用于在体内和体外化学发光检测 napdh 氧化酶产生的 ros.l-012 发出的信号比其他荧光探针更强, 并且已被证明对检测 ros 既敏感又可靠。l-012 辅助成像的延时适用性提供了有关炎症过程的宝贵信息, 同时减少了牺牲的需要, 并全面减少了研究动物的数量。在这里, 我们描述了一个协议, 利用 l-012 促进的体内成像量化氧化应激模型的切除伤口愈合使用糖尿病小鼠与局部功能失调 nrf2/keap1。

根据已建立的切除伤口模型 (图 1a) 创建双侧伤口三天后, 糖尿病小鼠被放置在成像室。在注射 l-012 以考虑背景信号之前, 拍摄了初始照片和生物发光量 (图 1 b)。在使用 l-012 溶液进行腹腔注射后, 小鼠被重新定位在腔内, 并在检测到 ros 的伤口区域显示生物发光 (图 1c).选择正确的成像和分析设?...

Watch this Scientific Journal Video about In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model at JoVE.com

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

我们描述了一种非侵入性的体内成像协议, 该协议具有简化且具有成本效益的特性, 它利用 l-012 (一种化学发光发光-模拟) 来可视化和量化小鼠切除伤口模型中产生的活性氧物种 (ros)。

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

这种方法可以帮助回答组织修复和再生领域有关细胞保护通路、氧化应激和伤口愈合反应之间的关联性方面的关键问题。这种强大的技术的主要优点是，您可以实时测量伤口中的活性氧种类，以确定它们对组织再生的影响。与我展示这个程序的将是詹妮弗·光，一个实验室的研究助理。使用血糖仪测量每只麻醉的8至12周大糖尿病小鼠的血糖。并使用头发修剪剂和脱毛霜去除第一动物的后毛。使用酒精擦拭两次清洁裸露的皮肤，并覆盖动物，使只有手术区暴露。当酒精干燥时，使用无菌的10毫米活检冲孔，根据成熟的切除伤口愈合技术，产生两个10毫米全厚度伤口，通过潘尼库鲁斯肌龙体。使用 0.5 毫米厚的硅胶片，带 10 毫米圆形切口，将伤口夹开，用中断的 4/0 丝线缝合固定伤口。然后将小鼠放在热垫上，进行监测，直到完全恢复，然后返回其单独的笼子，提供纸巾作为额外的筑巢材料两周。第二天，将控制无稽之谈小干扰RNA凝胶或实验性小干扰Keap1凝胶应用到每个动物的伤口顶部，用透明薄膜敷料包裹每个躯干，以保持凝胶就位，同时保持四肢自由。在实验的第三天，将一毫米注射器装上一个装有27规格的针头，并装上新准备的L-012溶液，并包裹注射器，以保护溶液免受光线的光。要成像伤口，请轻轻从每只小鼠身上取下透明膜敷料，而不干扰伤口，将小鼠放入生物发光成像系统的成像室。将成像系统流入和感应室氧含量设置为每分钟一升，并在基线上通过生物发光和明亮场对小鼠进行成像。接下来，用酒精湿巾擦拭腹部，然后每200克体重注射5毫克L-012溶液，IP，一旦酒精干燥，注入每只小鼠。注射L-012溶液而不干扰后皮的伤口至关重要，因为任何变形都会影响伤口愈合轨迹和数据解释。确保动物在注射前安全地在背背下。注射后，立即将小鼠放回成像室中各自的位置，并在60分钟的成像周期内每四分钟对小鼠进行一次成像，将10毫米伤口定义为确定活性氧物种水平的感兴趣区域。然后将小鼠放在热垫上，进行监测，直到完全恢复，然后再将动物返回到各自的笼子。根据既定的切除伤口模型创建双边伤口三天后，在L-012注射之前未观察到生物发光。在内酮内L-012溶液注射后，在检测到活性氧物种的伤口区域观察到生物发光。在 60 分钟内每 4 到 5 分钟记录一分钟的生物发光记录，可显示感兴趣的区域随着时间的推移，最佳成像时间达到完整的 L-012 饱和度，约 50 分钟。然后，可以通过将光强的总计数除以该区域来计算每个伤口区域中对L-012注射到无稽之谈小干扰RNA治疗或Keap1小干扰RNA治疗小鼠的生物发光。分析第10天伤口组织部分，以确认通过H&E染色进行活性氧物种水平测量的准确性，发现与小型干扰性无意义处理组织相比，细胞渗透到小干扰Keap1治疗的伤口中，表明实验性凝胶处理动物的炎症形态减少。F4/80的免疫反应，一种在伤口组织部分的蛋白质巨噬细胞标记，表明与小干扰性无意义治疗的伤口相比，小干扰Keap1治疗伤口的巨噬细胞数量减少，进一步突出了这种方法的正确性。尝试此过程时，请确保确认 L-012 未过期，并在受光线保护的位置搅拌溶液。按照这个程序，靶向探针和基于免疫测定的方法可用于研究活性氧物种及其相关物的亚细胞定位，从而能够快速评估影响伤口重用统计的干预措施和机制。

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Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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 ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

慢性假单位病伤口感染的延迟接种模型

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

在伤口反应期间斑马鱼中性粒细胞中趋化因子受体的实时成像

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...