为支持这项工作是由国家卫生授予研究院提供＃108229 R01红莲，美国心脏协会的科学家发展津贴＃0830352N，霍华德休斯医学研究所的职业发展奖，雅涛风险投资公司，欧洲共同体的第七框架计划（下授予FP7/2007-2013协议＃235689），麻省总医院的威廉Schreyer奖学金。

在体内动物模型实验AortoIliac动脉粥样硬化的代 1）基线血管造影和球囊剥脱<ol><li>获得基线血管造影和球囊剥脱之前，新西兰白兔喂食高胆固醇（1％）1周的饮食。这种动物是利用平移的相关性为1）在家兔的主动脉iliacs血管是人类冠状动​​脉（2.5 - 3.5毫米）和2）相同口径的高脂血症，球囊损伤模型，生成类似的炎性细胞（巨噬细胞发炎的动脉粥样硬化的轴承）和分子（蛋白酶）在人类动脉粥样硬化。 </li><li>胆固醇喂养，动物与异丙酚和氯胺酮麻醉。一英寸的腹侧中线颈部切口是使用一个大小为15 -手术刀刀片。使用钝性分离技术，筋膜气管右侧下方的肌肉暴露。沿结缔组织交界处，左边的sternocephalicus肌肉分离暴露右侧颈总动脉。动脉是从迷走神经分离。被放置在动脉的近端和远端缝合循环允许回缩和闭塞。一个1到2mm斜面动脉切开术是通过5法语（外径1.67毫米）血管鞘的插入和肝素（1000μ/mL〜150units/kg）管理通过内动脉鞘。 </li><li>然后注入造影剂（优维显）（1至2ml）超过2秒内获得控制远端的主动脉血管造影和两个髂动脉。 </li><li>髂股动脉和主动脉内皮剥脱，然后受伤。使用标准的透视方法，一个3FR福格蒂取栓导管是放置在远端髂动脉，并用0.3〜0.5毫升的对比（50％contrast/50％生理盐水）或空气膨胀。导管，然后撤回其膨胀的状态近端的最大距离沿右髂及远端主动脉左肾动脉起飞。气球剥蚀之后，血管造影是重复文件血管通畅。造影之后，所有的导管鞘被删除和近端右侧颈总动脉结扎，肌肉和筋膜，用4 / 0可吸收缝合线缝合，并用4 / 0非吸收性缝合皮肤切口封闭。 </li><li>动物然后让其恢复的一剂抗生素（头孢唑啉，0.5克的IM）管理。止痛药物，其中包括0.01毫克/公斤丁丙诺啡IM（两次需要一天）。动物，然后继续1％，为4周后，气球剥蚀胆固醇。在第5周，动物过渡到0.3％胆固醇饮食。 </li></ol>集成的多模态成像兔Atheromata 2）标签的蛋白质积极发炎斑块使用注射用纳米传感器;血管造影术，血管内超声（IVUS）， 并在体内血管NIRF成像兔动脉粥样硬化<ol><li> 8周球囊损伤和成像前24小时后，兔子是通过耳缘静脉注射静脉注射500 nmol / kg的Prosense/VM110（珀金埃尔默）。 </li><li>二十四小时注射后，动物麻醉和动脉访问是通过左颈总动脉（见步骤1.2）获得。管理动脉内肝素（150单位/公斤）。基线血管造影获得以上。 </li><li>装上一个血管内超声导管的临床冠状动脉能力0.014英寸线，插入鞘中。使用透视引导，导线的不透X线提示定位到右髂动脉远端。血管内超声导管，然后到近端髂动脉，用一个标准的临床单轨技术先进。 </li><li>发起一个100毫米的回调和图像记录。获得纵向重建的船只和管腔牌匾标识。 </li><li> NIRF导管11,12加载到0.014英寸线（单轨系统），并小心地将导管插入鞘和成像头，右髂内动脉远端定位。 </li><li>多个自动化回调（1毫米/秒纵向回调，每分钟30发），动脉粥样硬化的区域内的荧光信号指出。录制的影像，并取得进一步的处理是适当的缩放和窗口基于信号范围。 </li></ol> 3）安乐死和离体主动脉-髂组织隔离<ol><li>完成安乐死，安乐死代理与1CC（戊巴比妥钠390mg和50mg苯妥英钠溶液），静脉注射，单次注射。 </li><li>动脉树是0.9％生理盐水灌注，直到下腔静脉血液明确。确定动脉粥样硬化的主动脉和髂动脉及周围组织解剖。此外，小的2 × 2李厘米件版，肾，脾，心脏也获得。 </li><li>在这个阶段可以做体外 NIRF成像与血管NIRF成像导管。船只被拉长，NIRF导管重新插入到近端主动脉，直到成像头定位在右髂动脉或分叉。执行上述多个自动回调（见2.6）。 </li></ol> 4） 体外荧光反射成像（FRI）主动脉和髂动脉的解剖<ol><li>解剖组织是摆在10-20毫升生理盐水和运输周五分析（柯达影像站4000MM临，Carestream Health公司）。 </li><li>主动脉，髂血管被拉长，以近似实时的长度和图像在多个波长[白光，绿色荧光通道（前495纳米，EM 515纳米），Cy5的（前565纳米，EM 670纳米）和Cy7（前获得波长650 nm，EM 760纳米）的渠道。一系列的曝光时间，利用每个波长（0.1 - 30秒），获得的图像导出为DICOM或16位的非标度的TIFF进一步分析文件。作为阳性和阴性对照，器官（肝，脾，肾和心脏）成像类似的渠道和曝光时间。 </li><li>增加的近红外通道（780nm的）信号的地方都注意到在动脉粥样硬化的动脉。 </li></ol> 5）组织包埋切片和免疫组织化学分析<ol><li>地区的正常（非受伤的组织，即左髂内动脉）和斑块地区确定并组织5-10毫米的小环嵌入在华侨城（最佳切削温度）媒体。直到切片块保存在-80彗星。 </li><li>切片及免疫组织化学分析的标准技术进行。苏木精和曙红染色，RAM - 11和组织蛋白酶B染色的执行。 </li></ol>多模态图像的分析和集成（血管造影，血管内超声，NIRF和五） 6）NIRF和周五图像处理<ol><li> （近红外780纳米通道）回调DICOM文件包含NIRF和周五成像数据处理使用MATLAB和Osirix软件，分别为。适当的窗口来显示信号强度的全方位的实现。最后将图像导出为TIFF文件。 </li><li>文件导入成标准的图像处理软件（可用于主旨）。图片是一致的参考点（即椎体血管造影，髂分叉，肾动脉）的基础上。正常血管和斑块的领域确定。 </li><li>利息率（ROI）的地区手动追踪（正常组织和地区的斑块）和平均信号强度，使用Osirix和MATLAB获得，分别为周五，NIRF图像。为了引导适当的跟踪，船只的纵向血管内超声图像的使用和正常血管和斑块的识别很容易识别。 </li><li>目标的背景（TBR）的比率计算斑块区。 </li></ol>代表性的成果： 上述协议完成后，我们可以识别和特征增强蛋白酶蛋白酶活性的领域内主动脉和髂血管炎性斑块。注射可激活纳米传感器（Prosense/VM110），使我们能够确定水解积极斑块。这些显示为亮点或信号成像时使用的近红外通道（750纳米）星期五激烈的区域。 NIRF回调关联周五增加信号强度和路线与血管内超声允许NIRF信号解剖登记。计算斑块TBR的星期五和NIRF获得相似（见图3：平均NIRF载重4.2，平均星期五载重2.9）。明亮的斑块的免疫组织化学分析证实存在激烈的RAM - 11和组织蛋白酶B在活动领域的斑块（数据未显示）。 <img alt="图1" src="/files/ftp_upload/2257/2257fig1.jpg" /> 图1。 2D NIRF导管的原理图 ，延长一维NIRF传感方法 6的临床潜力，我们构造了一种新的2 - D导管血管成像NIRF。11,12定制的导管组成的光纤（直径为125微米的安置聚乙烯管材：2.9F）照亮使用750 nm激光激发源。激光灯发出的纤维轴成90度角。该系统采用两个马达（旋转和平移）自动启用伴随360度成像和纵向回调，以获得真正的二维成像。参考11的许可使用的图片。 <img alt="图2" src="/files/ftp_upload/2257/2257fig2.jpg" /> 图2。示意图展示介导的蛋白酶激活的纳米传感器，Prosense/VM110。参考10的许可使用的图像。 <img alt="表1" srC =“/ files/ftp_upload/2257/2257table1.jpg”/&gt; 图3。 在体内和体外斑块TBRs（目标背景比率）

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	<li>Andersson, J., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+immunity+and+atherosclerosis.">Adaptive immunity and atherosclerosis.</a> Clin Immunol. 134, 33-46 (2010).</li><li>Calfon, M. A., Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+near-infrared+fluorescence+molecular+imaging+of+atherosclerosis:+toward+coronary+arterial+visualization+of+biologically+high-risk+plaques.">Intravascular near-infrared fluorescence molecular imaging of atherosclerosis: toward coronary arterial visualization of biologically high-risk plaques.</a> Journal of Biomedical Optics. 15, 011107-011107 (2010).</li><li>Chen, J., Tung, C. -. H. <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+Proteolytic+Activity+in+Atherosclerosis.">In Vivo Imaging of Proteolytic Activity in Atherosclerosis.</a> Circulation. 105, 2766-2771 (2002).</li><li>Jaffer, F. A., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Cardiovascular+Disease.">Molecular Imaging of Cardiovascular Disease.</a> Circulation. 116, 1052-1061 (2007).</li><li>Jaffer, F. A., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+Multimodality+Molecular+Imaging:+Insights+Into+Atherosclerosis.">Optical and Multimodality Molecular Imaging: Insights Into Atherosclerosis.</a> Arterioscler Thromb Vasc Biol. 29, 1017-1024 (2009).</li><li>Jaffer, F. A., Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Catheter+Molecular+Sensing+of+Inflammation+in+Proteolytically+Active+Atherosclerosis.">Real-Time Catheter Molecular Sensing of Inflammation in Proteolytically Active Atherosclerosis.</a> Circulation. 118, 1802-1809 (2008).</li><li>Kim, D. -. E., Kim, J. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease+Imaging+of+Human+Atheromata+Captures+Molecular+Information+of+Atherosclerosis,+Complementing+Anatomic+Imaging.">Protease Imaging of Human Atheromata Captures Molecular Information of Atherosclerosis, Complementing Anatomic Imaging.</a> Arterioscler Thromb Vasc Biol. 30, 449-456 (2010).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420, 868-874 (2002).</li><li>Naghavi, M., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Vulnerable+Plaque+to+Vulnerable+Patient:+A+Call+for+New+Definitions+and+Risk+Assessment+Strategies:+Part+I.">From Vulnerable Plaque to Vulnerable Patient: A Call for New Definitions and Risk Assessment Strategies: Part I.</a> Circulation. 108, 1664-1672 (2003).</li><li>Weissleder, R., Tung, C. -. H. <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+tumors+with+protease-activated+near-infrared+fluorescent+probes.">In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.</a> Nat Biotech. 17, 375-375 (1999).</li><li>Razansky, R. N., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+catheter+system+for+two-dimensional+intravascular+imaging+in+vivo.">Near-infrared fluorescence catheter system for two-dimensional intravascular imaging in vivo.</a> Optics Express. 18, 11372-11381 (2010).</li><li>Jaffer, F. A., Calfon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Dimensional+Intravascular+Near-Infrared+Fluorescence+Molecular+Imaging+of+Inflammation+in+Atherosclerosis+and+Stent-Induced+Vascular+Injury.">Two-Dimensional Intravascular Near-Infrared Fluorescence Molecular Imaging of Inflammation in Atherosclerosis and Stent-Induced Vascular Injury.</a> Journal of the American College of Cardiology. 57, 2516-2526 (2011).</li></ol>

FAJ - 前顾问，VisEn医疗;酬金，波士顿科学公司

发炎的高风险或易损斑块可能大多数心肌梗死的负责。如斑块识别到出现症状之前预测的结果和指导药物治疗的重要的临床意义。如X线血管造影常规冠脉动脉成像方式通常侧重于管腔狭窄的表征，而不是启发性的高风险，通常非狭窄病变的潜在的生物学概况。血管NIRF分子成像提供了一个心脏导管插入术实验室的翻译方法，包括利用纳米传感器，识别活跃的巨噬细胞内斑块斑块炎症的生物学，细?...

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<tr>
<td>Prosense 750</td>
<td>&nbsp;</td>
<td>Visen Medical</td>
<td>VM110</td>
<td>500 nmol/kg IV injection</td>
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<tr>
<td>Heparin Sodium</td>
<td>&nbsp;</td>
<td>APP Pharmaceuticals</td>
<td>401586D</td>
<td>&nbsp;</td>
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<td>Cephazolin</td>
<td>&nbsp;</td>
<td>NovaPlus</td>
<td>46015683</td>
<td>&nbsp;</td>
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<td>Lidocaine HCL 2%</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC 0409-4277-01</td>
<td>&nbsp;</td>
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<td>Buprenorphine</td>
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<td>Bedford Laboratories</td>
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<td>Ketamine</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC 0409-2051-05</td>
<td>&nbsp;</td>
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<td>High Cholesterol Diet 1%</td>
<td>&nbsp;</td>
<td>Research Diets</td>
<td>C30293</td>
<td>&nbsp;</td>
<tr>
<td>HIgh Cholesterol Diet 0.3%</td>
<td>&nbsp;</td>
<td>Research Diets</td>
<td>C30255</td>
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in vivo near infrared fluorescence (nirf) intravascular molecular imaging of inflammatory plaque, a multimodal approach to imaging of atherosclerosis

血管损伤反应是一个精心策划的炎症反应的巨噬细胞内的血管壁，导致积累，管腔内充满脂质斑块，平滑肌细胞增殖和血管腔逐渐缩小的积累引发。这种易损斑块容易破裂，多数情况下急性心肌梗死的基础的形成。在复杂的分子和细胞炎症级联反应是精心策划的招聘T淋巴细胞和巨噬细胞和内皮细胞和平滑肌细胞的旁分泌的影响。 在动脉粥样硬化的分子成像已经演变成一个重要的临床和研究的工具，允许在体内炎症和其他生物过程的可视化。最近的几个例子证明能够及时发现高风险患者的斑块，并评估在动脉粥样硬化pharmacotherapeutics的影响。虽然数量（特别是MRI和PET）的分子成像方法可以形象大血管，如颈生物方面动脉，冠状动脉成像中存在缺乏选项。2高分辨率光学成像战略的问世，尤其是近红外荧光（NIRF），再加上与激活的荧光探针，提高灵敏度，并导致新的血管的战略发展以改善人类冠状动​​脉粥样硬化的生物成像。 分子成像利用近红外荧光（NIRF）定义的频带宽度的光子源（650-900纳米），的光造影剂或荧光探针交付时，发出近红外窗口，可以检测到的荧光激发光使用适当的排放过滤器和一个高灵敏度的电荷耦合相机。相对于可见光，近红外光深深地渗透到组织，如血红蛋白，血脂和水的内源性光子吸收剂明显减弱，并允许由于在近红外窗口，以减少自体荧光的高目标背景比率。成像在近红外“窗口”可以大幅度提高体内成像的潜力。 2,5 已经很好的研究炎症半胱氨酸蛋白酶激活NIRF探头10，在动脉粥样硬化形成中发挥重要作用。通过降解细胞外基质，半胱氨酸蛋白酶作出重要贡献8动脉粥样硬化的进展和并发症。在斑块组织蛋白酶B活性，特别是半胱氨酸蛋白酶，组织蛋白酶B，高表达和colocalizes与巨噬细胞，在实验小鼠，兔，和人类 atheromata此外3,6,7，可以利用先前描述1体内检测- ð血管内的近红外荧光技术6，结合在一个多聚赖氨酸的聚合物骨架多个近红外荧光（VM110/Prosense750，EX / EM 750/780nm，VisEn医疗，沃，MA）的衍生物组成的纳米传感器与注射剂在基线强分子内淬火，结果10以下有针对性的组织蛋白酶B（已知colocalize斑块巨噬细胞），单独的荧光，如半胱氨酸蛋白酶的酶裂解，导致大幅扩增NIRF信号。利用新颖的二维血管NIRF导管血管内的近红外荧光信号检测，使高清晰度，几何体内发炎斑块组织蛋白酶B活性检测的准确。 在体内分子成像使用导管为基础的2D NIRF成像，而不是以前的1 - D光谱方法的动脉粥样硬化，是一种新型的和有前途的工具，利用增强巨噬细胞丰富的斑块蛋白酶活性检测血管炎症 。11,12以下的研究协议介绍，血管内的2维NIRF导管的使用形象和斑块结构特征，利用斑块生物学的关键环节。这是一个翻译的平台，整合现有的临床成像技术，包括血管造影和血管内超声（IVUS），提供了一个独特新颖的综合多式联运分子成像技术，区分炎症atheromata，并允许在人类大小的冠状动脉血管NIRF信号检测。

Watch this Scientific Journal Video about In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis at JoVE.co

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

我们详细介绍了一个新的近红外荧光（NIRF）导管二维血管内斑块的生物学分子成像<em&gt;在体内</em&gt;。 NIRF导管可以想像报告斑块狂热的激活和有针对性的近红外荧光存在的关键生物过程，如炎症。导管采用临床工程和电力的需求和目标是应用在人体冠状动脉。以下的研究描述了一个利用了一种新型的多模态成像战略<em&gt;在体内</em&gt;血管NIRF导管的形象和量化水解积极发炎兔atheromata炎性斑块。

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

in-vivo-near-infrared-fluorescence-nirf-intravascular-molecular

Research

JoVE Journal

Medicine

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

17.9K 次观看.  Harvard Medical School. 我们详细介绍了一个新的近红外荧光（NIRF）导管二维血管内斑块的生物学分子成像在体内。 NIRF导管可以想像报告斑块狂热的激活和有针对性的近红外荧光存在的关键生物过程，如炎症。导管采用临床工程和电力的需求和目标是应用在人体冠状动脉。以下的研究描述了一个利用了一种新型的多模态成像战略在体内血管NIRF导管的形象和量?...

视频: In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

科研

医学

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

一个人力<em&gt;前体内</em&gt;动脉粥样硬化斑块模型研究病变的生物学

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

组织鬼怪模拟，以评估乳癌手术潜在的近红外荧光成像中的应用

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...