感谢Daniel Bukala，Natalie Altmeyer和Funda Cay的卓越技术支持。我们感谢Jonathan Cotton，Greg Bowden和Paul Soubiran编辑手稿。这项工作得到Werner西门子基金会和Eberhard Karls大学Tübingen医学院（"Promotionskolleg"）以及DFG通过CRC 156（C3项目）的支持。

本文中描述的所有程序都遵循实验动物护理和使用的指导方针和国际标准，并得到了德国Tuebingen国家委员会动物福利与道德委员会的批准。 8-12周龄的BALB / c和C57BL / 6小鼠保持12小时12小时光线:黑暗循环，并以22±1℃的标准化环境条件容纳在IVC中，用水和2〜5组，食物随意获取。 材料准备<ol><li>根据注射前直接的数据表稀释OI染料进行近红外荧光成像。可活化的OI染料（680nm激发的商业探针） 在体内测量MMPs可以在1.5mL 1x PBS中以20nmol的浓度使用。使用前请轻轻晃动或旋转溶液。 OI染料可以在2 - 8°C下保存长达6个月，防止光照。 </li&gt; <li>静脉注射 （ iv ）制备静脉导管。使用填充有0.9％盐水溶液（含有10μl注射单位的肝素在50mL中）的20U（0.5mL）胰岛素注射器，以及连接到聚乙烯导管的30号针头。注射OI染料每只小鼠推荐剂量2 nmol。 </li></ol> 2.诱发类风湿关节炎和慢性接触性超敏反应<ol><li> 类风湿关节炎: <ol><li>用1x PBS（200μL）稀释100μL含有针对葡萄糖-6-磷酸异构酶（GPI）1:1的抗体（AB）的血清（从K / BxN小鼠10获得）以诱导RA。将稀释的血清储存在-80℃。诱导RA的详细程序由Monach 等人描述 8 。 </li><li>为了诱导RA，通过尾巴轻轻地抬起每只小鼠，并在t的第0天腹膜内（ ip ）注射200μL稀释的血清他实验注射后将鼠标直接放回笼中。 </li><li>可选地，在AB注射之前测量每个踝关节的踝直径并定义"关节炎评分"（ 图1A ）。使用机械测量装置（千分尺）在GPI血清后继续测量踝部肿胀直到第6天。 </li><li>注射可激活的OI染料（步骤1.1-1.2），以测量RA小鼠尾静脉内的体内 MMP活性 在GPI-血清注射后第5天进入对照小鼠的尾静脉。尾静脉注射24 h后进行光学成像实验。 </li></ol></li><li> 接触超敏反应: <ol><li>为了敏化C57BL / 6小鼠，制备溶解在4:1丙酮/油混合物中的5％TNCB溶液。 </li><li>麻醉C57BL / 6小鼠，使用1.5％异氟醚在100％氧气（1.5升/分钟）内蒸发。一旦将小鼠麻醉，就将其置于自发之中<html> È鼻锥（切口5毫升聚乙烯注射器中，连接到1.5体积％的异氟烷​​管）和维持麻醉。对麻醉动物兽医应用眼膏，以防止眼睛干涩，而麻醉。 </li><li>剃使用小动物毛发修剪器仔细腹部（2×2cm）上。避免伤及皮肤，因为这可能会导致动物体内非特异性荧光信号，并能影响研究结果。 </li><li>使用100微升移液管致敏动物申请80μL的5％TNCB溶液慢慢在剃毛的腹部区域。将鼠标轻轻放回笼子照顾，以确保鼠标的眼睛不来与床上用品的接触，避免角膜损伤，并在恢复阶段避免低体温。 </li><li>致敏后6天，制备1％TNCB溶液（溶解在9:1的丙酮/油的1:1混合物），并使用移液管以引起急性和慢性CHSR在右耳处应用20μL。"&gt; 1 <ol><li>开始与在右耳两侧的1 个挑战与用100μL移液管1％TNCB溶液20μL，并重复挑战每隔一天最多五次（致敏后第15天）以引发CHR（ 图1B） 。每日测量耳朵的厚度，使用测微计测量装置。 </li></ol></li><li>注入可活化OI染料（步骤1.1-1.2），以测量在攻击后12个小时的小鼠CHR 体内 MMP活性（具有在680 nm激发商业探针）。进行光学成像实验24小时后尾静脉注射（IV）。 </li></ol></li></ol> 3.动物的制备用于光学成像<ol><li>开关小鼠食物到低或非荧光食物（ 例如 ，无锰），以避免自发荧光的干扰（约700纳米）与所使用的OI剂的荧光信号（在成像前至少3天）。 </li><li>放置动物到麻醉箱和麻醉使用具有氧（1.5L /分钟）蒸发的1.5体积％的异氟烷​​（氧气可以由空气置换，但需要一个实验中标准化的）。 </li><li>当鼠标被可见麻醉，将其放置在一个自制鼻锥（由切口5毫升聚乙烯注射器中，连接到1.5体积％的异氟烷​​管建立）和维持麻醉。 </li><li>仔细剃使用小动物毛发修剪器的目标部位（2厘米×2cm）上对动物。避免伤及皮肤，因为这可能会导致动物体内非特异性荧光信号，并能影响研究结果。 注:毛发可以吸收OI中的荧光信号（依赖于小鼠品系，更多或观察到较少的吸收），这取决于所关注（ROI）的区域。 </li><li>对于静脉内注射，将小鼠尾中温热水，以诱导血管舒张，温和地清洁注射部位用酒精皮肤，并且通过将导管在开始尾部的远端位置。将针的"切割"边缘以20°的角度放入尾静脉，并通过重新悬挂注射器来测试导管的正确放置。 </li><li>如果导管放置正确，请更换注射器并注入OI探针（2 nmol）。注射后，更换注射器并注入25μL的0.9％盐水溶液以完全清除聚乙烯管的死体积。 注意:用于测量体内 MMP活性的所用OI染料（680nm激发的商业探针）的组织半衰期为72小时。为了确保完全清除，在先前注射后7天内不推荐重新注射染料。 </li></ol>光学成像<ol><li>将黑色塑料或纸张放置在视野（FOV）中心的OI扫描仪的黑盒中。 </li><li>设置测量协议并选择合适的波长（激发:680±10 nm，任务:700±10nm）的成像和参数。 注意:在某些OI系统中，几个成像染料的设置是预先定义的。 </li><li>要选择用于荧光成像的正确的协议，打开成像软件（由制造商提供的）和初始化系统。大多数CCD摄像机需要冷却到其工作温度，而这可能需要10分钟。对于可靠的结果，等到系统已准备就绪。 </li><li>观察体内成像系统获取控制面板弹出，并选择的每个过滤器对将所述序列中代表一个图像。在这种情况下，获得一个图像具有用于分别与所述的商业染料和680±10nm的激发和发射波长和700±10nm，则过滤器对，并且开始测量（按"获取序列"）。有关详细说明，请参阅制造商的说明书。 </li><li>合适的标签图像并保存信息中的"编辑图像标签"窗口，其将在"获取序列"之后弹出。 </li><li>在注射OI染料之前对每只动物进行基线扫描，或使用幼稚的对照动物来区分背景信号。 </li><li>在尾静脉注射OI染料之后，将动物置于FOV的中心，以测量OI系统中最高的信号，并开始测量。 注意:重要:低体温可以显着影响成像剂的分布。确保将舞台加热至37°C以避免动物体温过低。成像可以与1至5只小鼠的动物同时进行。根据同时测量的动物数量选择FOV的大小。您可以拍摄明亮的场景图像来检查每个鼠标是否可见。 </li></ol>数据分析注意:使用以下图像软件执行数据分析制造商的协议。 <ol><li>对于RA的测量，使用光子发射的校准单元， 如图2，而慢性CHSR以百分比（效率）描绘为信号强度。 </li><li>手动绘制的ROI，使用工具板。用于RA图像的分析使用标准化的圆，在每个动物的所有脚踝和爪子周围放置最高信号。为了分析CHSR，将投资回报围绕整个权，并根据明视场像左耳。 </li><li>为了测量光子发射或在所述特定绘制ROI，按"测量"信号强度值。该系统将提供用于描述性统计分析绘制的ROI值。 </li></ol>

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作者没有什么可以披露的。

OI是临床前研究中非侵入性体内分子成像的非常有用，快速和便宜的工具。 OI的特殊优势是监测高度动态过程（如炎症反应）的能力。此外，OI允许人们在一段时间内跟踪疾病进程，时间范围从几天到几周。 OI具有优于其他体内成像模式（如正电子发射断层扫描（PET）或磁共振成像（MRI））的优点，因为它是非常时间和成本有效的。每次采集多达5只动物的高通量分析是可能的。此外，可以使用大量针对许多不同生物过程的探针。为了提供进一步的解剖信息，OI可以与其他成像技术如MRI或X射线结合。另一个限制是其组织穿透深度低于例如 functional PET成像。此外大量的探针针对许多不同的生物靶标是可用的。 12 麻醉的影响应该不会在体内成像低估英寸而在OI结果麻醉的具体影响没有得到很好的特点，我们的小组有几项研究显示麻醉对PET成像效果的影响。 13，14，15例如，Fuchs 等。分析血液参数，例如的pCO 2，pH和乳酸PET扫描使用不同的呼吸和麻醉协议之前和之后。结果表明，在2的pCO变化显著和麻醉乳酸水平相比有意识氧气或空气呼吸小鼠导致PET结果显著变化。 14他们认为，这种影响主要是由于氧气的呼吸和subsequen吨到动物呼吸性酸中毒，导致在PET成像中的不同的结果。 麻醉参数标准化，如O 2个 -concentrations和剂量的麻醉药物，以及防止低温有助于避免系统故障。当然，OI由物理性质例如光散射，组织自发荧光和光衰减9限定。几组由OI的宏观结果与其它技术，如在体内的荧光显微镜组合或流式细胞术解决这些问题。 在由特定MMP活化的荧光探针MMP活性的体内成像已经像CHS，7炎症的几种实验模型中使用，但也像脑缺血，16个主动脉瘤，17心肌梗塞的疾病18和动脉粥样硬化斑块19以及各种肿瘤模型。 20 例如，Cortez-Retamozo 等人通过在预先致敏的小鼠中鼻内施用卵清蛋白后24小时注射MMP可激活的探针，研究MMPs在过敏性气道炎症模型中的体内活性。 21为了进一步确定MMP活动的位置，本组联合断层扫描OI，NIR光纤支气管镜检查和活体显微镜检查，以更详细地监测疾病过程和治疗反应。 21 测量体内 MMP活性的其他方法主要使用结合MMPs活性位点的MMP抑制剂。 McIntyre 等描述了使用"聚合物基荧光底物" 的体内肿瘤相关的MMP-7检测，其作为"蛋白水解酶IC信标"用于在小鼠异种移植模型的MMPs。22它们表明，MMP-7，可以有选择地通过光学成像检测的活性。23另外，Olson 等人研究了‘可激活的细胞穿透肽’（ACPPs），其能够许多靶向异种移植肿瘤模型，但是直到接头蛋白水解不能吸附到细胞中。他们研究ACPPs它们是选择性的MMP-2和MMP-9。24，25 MMP抑制剂与用于单光子发射计算机断层摄影（SPECT）或PET成像的放射性同位素进行标记被用来研究在动脉粥样硬化26和癌症27 MMP的表达。在最近的研究荧光标记MMP抑制剂相比，MMP活化的探针。 28荧光标记的MMP抑制剂显示出较低的信号噪声比例与可激活的探针相比，但需要较短的摄取时间。 评估病理生理学关键特征的新生物标志物可被OI利用和验证。例如，对危险相关分子模式（DAMP）的反应是炎症反应期间的早期事件。 Vogl 等人在急性耳皮肤皮炎，胶原诱导的关节炎和利什曼原虫感染的模型中使用针对报警器S100A9的Cy5.5标记的抗体，以开发敏感的生物标志物以用于早期的炎症征象。 29 总之，OI代表了一种快速有效的临床前研究方法，能够揭示体内疾病的新机制，表征新的分子靶点，以及监测治疗效果。然而，可能需要使用更多定量技术（如PET）的后续验证过程。

自身免疫疾病如类风湿关节炎（RA）或牛皮癣被分级为延迟型超敏反应（DTHR）。 1 RA是以侵蚀性滑膜炎和关节破坏为特征的常见自身免疫性疾病。 2炎性关节炎关节表现出炎症细胞的浸润和增殖，增加炎症细胞的表达，导致血管us形成，软骨和骨破坏。 3，4细胞外基质分子如基质金属蛋白酶（MMPs）的胶原蛋白的切割对组织转化和血管生成至关重要，并导致组织破坏。 5，6接触超敏反应（CHR）的特征是中性粒细胞的聚集，导致氧化爆发。 7与RA相似，CHR中的MMPs是不同的粘弹性阻尼器在组织转化，细胞迁移和血管生成，以便建立慢性炎症。 为了研究RA，使用了葡萄糖-6-磷酸异构酶（GPI） - 血清注射小鼠模型。从含有抗GPI抗体的转基因K / BxN小鼠8血清，注入幼稚BALB / c小鼠后，其风湿性炎症开始与GPI-血清注射后第6天溶胀最大脚踝的24个小时（参见1.1）中培养。为了分析慢性CHR，C57BL / 6小鼠用在腹部trinitrochlorobenzene（TNCB）致敏。右耳被质疑到开始致敏1周后5次（也参见1.1和1.2）。 非侵入性的小动物OI是基于荧光基团，chemiluminescent-和生物发光-信号，这些信号主要用于在临床前研究的体内研究的技术。所获得的半定量的数据给出了分析上市公司molec在机关ular机制和健康组织以及患病实验动物模型，并且使得纵向后续测量（ 例如 ，以评估在体内治疗响应简档）。纵向研究的一大优势是动物数量的减少，因为相同的动物可以在后续的研究在几个时间点，而不是每个时间点使用不同的小鼠中测得的。 OI的分辨率允许器官的详细的功能成像和在实验动物中甚至更小的组织结构。 的特定激发和发射滤光片具有窄透射光谱，由遮光对散射光保护"暗盒"和一个敏感的电荷耦合器件（CCD）相机，其在许多设备冷却到-70℃，使用允许高度特异性和荧光信号的敏感测量。 通过使用荧光剂与excitation-和近红外荧光窗（650 - 950 nm）的发射光谱可以显着提高信噪比。近红外荧光窗的特征在于通过血红蛋白和水对信号的相对较低的吸收以及低的背景自动荧光。 9允许小动物组织中的穿透深度达2厘米。 OI探针可以直接（ 例如通过荧光标记的抗体）靶向靶标，或者可以在靶组织中（ 例如通过蛋白酶）活化。由于Förster共振能量转移（FRET）到淬灭部分，活化的OI探针以其失活形式光学沉默，其将分子内的激发能转移到另一个结构域。如果染料被切割（例如通过蛋白酶），则能量不再在分子内转移，并且可以通过OI检测荧光信号。这允许设计具有高特异性的OI探针y用于不同的生物过程和优异的信噪比。 以下协议详细说明了动物的制备，使用可活化OI探针在体内成像MMP-2，-3，-9和-13活性的OI测量和两个炎症实验模型（RA，CHR）。

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			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
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		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
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			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
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		<tr>
			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
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			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
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		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
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		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
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			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
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			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
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		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
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			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
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			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
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			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
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			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
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			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
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		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
		</tr>
		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
		</tr>
		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
		</tr>
	</tbody>
</table>

non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

本文介绍了一种非侵入性的方法，用于成像基质金属蛋白酶（MMP）由活化的荧光探针-activity， 通过 体内荧光光学成像（OI），在炎症的两个不同的小鼠模型:类风湿关节炎（RA）和接触超敏反应（CHR）模型。在近红外（NIR）窗口的波长的光（650 - 950 nm）的允许更深的组织穿透和最小的信号的吸收相比，低于650纳米的波长。使用荧光OI的主要优点是，它是便宜，快速，易于在不同的动物模型来实现。 活化的荧光探针在它们的失活状态的光学沉默，但通过蛋白酶激活时变得高度荧光的。活化蛋白酶引起组织破坏并播放在迟发型超敏反应（DTHRs）如RA和CHR疾病进展中起重要作用。此外，MMP是对于软骨和骨降解和键的蛋白酶是由巨噬细胞，成纤维细胞和软骨细胞响应于促炎细胞因子引起的。在这里，我们使用由密钥的MMP活化等MMP-2，-3探针，-9和-13和描述的成像协议用于RA MMP活性的近红外荧光的OI和对照小鼠6天疾病诱导后以及如急性（1X挑战）和慢性（5次挑战）在右耳CHR比健康耳朵的小鼠。

为了在初始的BALB / c小鼠中诱导类风湿性关节炎（RA），在第0天将动物与GPI自动抗体（1×1稀释，1x PBS）腹腔注射。该GPI-血清诱导的最大炎症（踝肿胀） RA模型是在第6天后注射11 。因此，制备2nmol的可激活的OI染料，并在注射后第24天（第6天）将iv注射到关节炎小鼠和健康对照动物的尾静脉中，小鼠被麻醉并按照第3节（ 图1A ）。 为了诱导CHR，C57BL / 6小鼠在剃须腹部的第0天用5％TNCB致敏。在感染后第7天，使用1％TNCB的右耳第一次挑战开始。进行五次挑战（第15天）以诱发慢性炎症。 7之后12点最后一次挑战，注射20nmol的OI染料iv 。在注射后第15天，24小时，如上所述进行OI（ 图1B ）。 与对照踝关节相比， 图2显示疾病诱导后第6天RA小鼠的后爪，脚踝和前爪中的明显增强的荧光信号（ 图2A ）。在RA小鼠和健康小鼠的器官中的MMP活性的生物分布分析在RA小鼠的关节中仅在后爪，脚踝和前爪中显示强烈增强的信号。有趣的是，当与健康对照小鼠的肾脏相比时，我们确定了所有测量的RA小鼠的两个肾脏中的增强的MMP信号（ 图2A ;右）。此外，我们测量了肝脏和肠道中增强的MMP信号，无论小鼠是否患有RA。在t中没有发现RA与健康小鼠之间的差异他的胰腺，肺，脾和心脏。 在慢性CHR（右耳五个TNCB挑战）期间，我们测量了与健康左对照耳相比，右发炎耳朵的MMP活性高度增加（ 图2B ）。 因此，我们的数据表明在炎性疾病模型中疾病进展期间MMP活性的主要作用。 用于生物发光和荧光的工具包允许定量例如pmol的荧光染料或表达荧光的细胞数。数据可以显示在1.计数:显示未照相的相机光子的测量值。2.辐射（光子）:表示在"光子/秒/ cm 2 /球面"中校准的光子发射测量，推荐用于发光成像或3adiant效率（荧光）:显示每次入射激发功率的荧光发射辐射，推荐用于荧光测量。 在分析OI图像时使用辐射单位的一个主要优点是可以在一个实验过程中改变相机设置，并且对图像本身或测量的ROI数据没有影响，这进一步允许比较来自不同IVIS系统的图像。 感兴趣区域（ROI）表示光学图像中用户感兴趣的特定区域。工具栏可以绘制3个不同的投资回报率:测量（测量图像的ROI中的信号强度），平均背景（测量用户定义的背景区域的平均信号强度）或对象ROI（识别主题动物一个图像）。 如果主题dis播放高度非特异性的背景信号本身，通过从目标ROI中减去背景信号（ROI）来获得背景校正的ROI测量。将图像最小值设置为零，以确定图像中的自动荧光或自然发生的背景发光。将图像链接到自定义（用户特定）背景图像（ROI），这主要是测量的特定图像。 通过使用Living Image软件在关节炎和对照踝关节以及发炎和健康的耳朵中绘制标准化的感兴趣区域（ROI）来进行图像的半定量分析。由于动物数量不足，本手稿中仅对体内数据进行了描述性统计学分析。与对照踝关节相比，关节炎和健康踝关节或爪的信号强度分析显示关节炎中MMP信号强度增加7倍。<html> A 3倍的关节炎小鼠的前爪检测MMP更高的信号强度相比于健康动物（ 图3A）的。 即使第1 个挑战， 第三后进一步增加，并保持在相同的水平，直到第 5 次攻击后的急性和慢性CHR相比左侧未攻击控制耳朵的耳朵的分析显示显著增加MMP信号（ 图3B） 。与由于小鼠的搔抓的接触变应原在左耳污染的结果，我们确定在左侧稍微增加MMP信号（未TNCB激发的）控制的耳朵。结果表明MMP的重要作用，GPI-关节炎和慢性炎症过程中由一个高度增加的荧光信号明证，如通过OI测量。 / 55180 / 55180fig1.jpg"/&gt; 图1:RA和CHR模型，疾病诱导的时间过程和OI的时间点。 （A） BALB / c小鼠（B）中RA诱导的时间过程和C57BL / 6小鼠的慢性CHR诱导。在两种模型中用于测量MMP活性的OI染料的相应体内成像时间点和相关联的注射时间点的概述。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55180/55180fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55180/55180fig2.jpg" /> 图2:RA，CHR和对照动物中的MMP OI。 （A）在GPI-关节炎诱导后6天和健康对照鼠（左侧）的关节炎小鼠中测量的MMP活性的代表性结果。明显较高的信号（Radiance（p / s / cm 2 / sr））int与健康小鼠相比，在关节炎中观察到一致。右侧的图像显示后爪中的MMP活性，包括OI染料注射后处死的GPI-关节炎小鼠的脚踝，前爪，胰腺，肺，脾，心，肾，肝和肠。无论小鼠注射GPI血清（RA小鼠）还是对照血清（健康对照小鼠），OI在RA小鼠和肝脏和肠道的前爪，脚踝和后爪中都发现了高信号。 （B）具有慢性CHR（左）和对照动物（右）的耳朵中的MMP荧光信号。与对照耳朵相比，发炎的右耳（挑战5次）显示出高度增强的信号。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55180/55180fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55180/55180fig3.jpg" /> &lt;图3:使用OI的炎性和健康的踝关节和耳朵组织的半定量分析。 （A）前爪和踝关节RA和健康动物的荧光信号的半定量分析。与健康的踝关节和前爪相比，关节炎前爪和踝关节显示出显着（** p &lt;0.05）（n = 3）的MMP信号。数据表示为平均值±标准差。 （B）第1 次 ，第3 次 ， 第 5 次攻击后左右耳（受挑战）耳朵的半定量分析。 MMP信号从第一次到第五次攻击显着增加，并且在第三次攻击后（** p &lt;0.05）（n = 3），右侧挑战的耳朵已经显示出最大的信号强度（对照耳部被TNCB部分污染由于老鼠的伤害，这产生了一个增强的信号，这并不重要）。数据以平均值表示±SEM。双尾学生t检验用于分析炎症（GPI关节炎脚踝，右治疗耳朵）和对照（对照踝，未治疗的耳朵）两种模型的差异。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55180/55180fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

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

本文阐述了荧光成像的使用可活化的光学成像探针可视化键基质金属蛋白酶的体内活性在炎症的两个不同的实验模型中的应用。

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 ...

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

8.7K 次观看.  Eberhard Karls University of Tübingen. 本文阐述了荧光成像的使用可活化的光学成像探针可视化键基质金属蛋白酶的体内活性在炎症的两个不同的实验模型中的应用。 

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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 provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Bioluminescent Bacterial Imaging In Vivo

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 bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

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 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

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 ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

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

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (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 ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...