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?...

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）。

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

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

Universidad de Cartagena UDEC

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

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

科研

生物工程

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 ...

免疫与感染

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

医学

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

非侵入性 ACL 损伤和手术对小鼠蛋白酶活性的影响

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

作者聚焦：研究 ACL 损伤对骨关节炎进展的早期事件和长期影响

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

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与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

在体内成像方法区分急性和慢性炎症

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

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摘要

探索更多视频

此视频中的章节

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

在体内成像方法区分急性和慢性炎症

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像