这项工作是由韩国医疗技术的R＆D项目，卫生部和福利（HI12C1847，HI12C0066）的支持，生物与医药科技发展计划（2010-0019862）和全球研究实验室（GRL）方案（NRF-2015K1A1A2028228）国家研究基金会，由韩国政府资助。

在这个协议证明了所有的动物程序已经审查并东国大学一山医院动物护理和使用委员会批准，并按照NIH的指南中列出的照顾和动物使用的原则和程序进行的。 1.外源性形成凝块的制备荧光标记标记（图1） <ol><li>在用3％异氟烷，用30％的氧气（1.5L /分钟100％的氧气）混合感应腔麻醉的小鼠。通过观察肌张力，并确认不存在脚趾捏反射确保麻醉深度足够。 </li><li>放置在俯卧位的无菌披盖的动物，并使用吸入面罩和2％异氟醚，用30％的氧气混合，保持麻醉下。通过执行无菌技术和无菌衣/口罩/手套/仪器下面的步骤。保持通过清洗和前一消毒用70％酒精实验区在无菌条件第二手续后。 </li><li>心脏穿刺后6收集约300〜1000微升动脉血。混合70微升的全血用30微升C15探针5（20微摩尔/ L的浓度），一个经Cy5.5荧光探针来活化因子XIII（FXIIIA）凝血酶的血纤维蛋白交联的活性敏感，要荧光标记凝块（ 图1A ）。使用3毫升注射器（23号针）到一个20cm长的聚乙烯管（PE-50，ID0.58毫米）注入该混合血液。聚乙烯管道必须被消毒（或认证的由制造商无菌）和血块必须在组织培养罩无菌制备。 </li><li>通过观察呼吸不足和心脏脉搏的检查动物的死亡。 </li><li>留在室温下将血液装管2小时，然后在4℃下22小时，并执行下列步骤，如先前报道7。 </li><li>切含血栓管进入1.5厘米长的小段。使用3毫升注射器填充有磷酸盐缓冲盐水（PBS），通过轻轻注入PBS成每片管的驱逐血栓到含PBS-6孔板中。用PBS（ 图1B）洗血栓三次。 </li><li>通过仔细地绘制洗涤血栓，同时避免气泡，用生理盐水填充1ml注射器用30用1.5厘米长的血栓装载有15厘米长的PE-10管（ID0.28毫米）的前端部被插入到该管的近端号针头。 </li><li>血栓加载PE-10管连接与修改，以具有锥形端部（ID 200微米）一个3厘米长的PE-50管（ID0.58毫米），这将被放置在大脑中动脉（MCA） - 大脑前在栓塞性中风的小鼠模型（ 图1C）颈内动脉（ICA）的动脉（ACA）的分叉区域。 </li></ol> 2.造型血栓栓塞性中风的小鼠模型（图2） <ol><li> Anesthetiz如先前在用3％异氟烷，用30％的氧气（1.5升/分钟）混合感应室报告7被抚摸EA不同的鼠标。美洛昔康注射（5毫克/公斤）皮下注射，以减轻术后疼痛。通过观察肌张力，并确认不存在脚趾捏反射确保麻醉深度足够。 </li><li>适用兽医软膏少量每只眼睛，以防止麻醉期间干燥。通过执行无菌技术和无菌衣/口罩/手套/仪器下面的外科手术。保持通过在清洗之前，用70％的酒精消毒手术区，无菌条件下，并在手术后。 </li><li>将鼠标移动到手术台。放置在俯卧位的无菌披盖的动物，并使用吸入面罩和2％异氟烷保持麻醉下。然后，使用具有反馈的恒温毯从直肠温度计夹住体温在36.5℃。 </li><li> surgi后校准制备具有优碘和70％的酒精，通过使用在左耳和眼睛之间的头皮手术刀使1cm的垂直切口。胶光纤激光多普勒流量计到暴露的左颞骨表面的远端（1 Mm左和来自前囟低于4毫米）。然后，开始多普勒血流监测（ 图2A）。 </li><li>放下动物。通过拉动与附连到销的字符串上门牙拉直颈部和剃颈部区域。然后，使3厘米的垂直正中切口，传播它开放，通过解剖周围的血管软组织暴露左侧颈动脉球区。要小心，不要伤害迷走神经。 </li><li>结扎用消毒6-0黑丝线缝合左侧近端颈总动脉（CCA），以及结扎左ICA和使用无菌7-0黑丝线缝合左侧翼腭动脉（PPA）。 </li><li>烧灼左甲状腺动脉，这是左外颈动脉USI的一个分支NG单极电烧灼，结扎和左近端颈外动脉（ECA）松散和紧密用无菌7-0黑丝线缝合更远端站点。 </li><li>用微型剪刀，进行ECA的连接点之间的小孔（直径约0.2〜0.25微米）。 </li><li>将含血栓导管插入在ECA孔同时放松近端结扎ECA。推进导管进入CCA为了以收紧导管到位后，重新拧紧近端结扎ECA。 </li><li>烧灼切割ECA远端部分，也就是远离远端结扎部位，并旋转自由近端ECA顺时针它对齐到ICA的方向同时取出从CCA导管。然后，将导管大约9mm到MCA - 远端ICA的ACA分支区域松开ICA结扎后立即生效。然后，拧紧结扎ICA。 </li><li>放置血栓进入分叉区轻轻按压注射器1毫升注射血栓。检查多普勒脑血流量（CBF），这应该由30％或更低的降低与基线相比，如果血栓成功闭塞血管（ 图2B）的下降。 </li><li>取出导管，并立即紧紧地结扎ECA近端部位。此外，unligate共同国家评估和PPA。 </li><li>关闭用6-0丝线缝合切口部位。继续进行多普勒监测过的所需时间跨度（在此，为血栓注射后30分钟）后，停止麻醉。返回鼠标在一个空笼子，并保持温暖，取暖灯。直到它已获得了足够的意识，以保持胸骨斜卧不要离开鼠标无人值守。 </li></ol> 3. 在脑血栓的体内显微成像（图3） <ol><li>重新麻醉作为在预先指定的时间点在步骤2.1中所述，用2％异氟醚的小鼠（在此，1小时）的发作以下栓塞性中风的因血栓，在脑动脉的位置。通过确认不存在脚趾捏反射确保麻醉足够深度。适用兽医软膏少量每只眼睛，以防止麻醉期间干燥。 </li><li>在在10mM PBS中的10毫克的Au / ml的浓度重新悬浮的血纤维蛋白靶向金纳米颗粒（FIB-GC-金纳米粒子4），超声处理的血栓成像剂为30分钟，以确保纳米粒子的溶解和分散。注入300μl的FIB-GC-金纳米粒子（10毫克/毫升）插入阴茎静脉。 </li><li>放置在显微机的床中的动物，并且通过拉动与连接到一个销，以减少运动伪影的字符串上门牙拉直颈部。 </li><li>在FIB-GC-金纳米粒子的注射后5分钟，开始获取脑的显微图像。对于这里所描述的实验，请使用以下的成像参数:65 kVp的，60μA，观点26.7点¯x26.7点¯x27.9毫米3场，0.053点¯x0.053×0.054毫米3像素大小，每帧100毫秒，1平均，360视图，512×512的重建矩阵，600片，64秒的扫描时间。 </li><li>返回鼠标在一个空笼子，并保持温暖，取暖灯。直到它已获得了足够的意识，以保持胸骨斜卧不要离开鼠标无人值守。 </li><li>使用安装在显微扫描仪的一个软件包的" 重建 "面板的" 开始 "命令变换医学（DICOM）格式的原始图像数据转换成数字成像和通信。 </li><li>为图像（在步骤6）的定量分析，通过根据制造商的说明使用市售软件包变换DICOM数据转换成TIFF格式。 </li><li>定性分析和定量在一个更简单和更快速的方式进行分析，使用的软件包和原始0.054毫米厚图像以DICOM格式，用于产生一组新的呈现具有1或2毫米（这里，2mm）的厚度，如下轴向和冠状图像。 <ol><li>选择在" 数据源 "树中的DICOM文件夹，单击鼠标右键，并将该文件夹导入到"MasterDB&#39;或&#39;PrivateDB"。 </li><li>点击"MasterDB&#39;或&#39;PrivateDB"中的"数据源"树，然后选择导入文件夹。点击最左边的面板中的"3D"选项卡后，当"加载选项"窗口弹出时，按下" 确定 "导入图像序列中的文件夹作为一个堆栈英寸</li><li>通过点击在轴向和冠状图像窗口单词&#39; 的MRP&#39;和选择弹出菜单上&#39;MIP&#39;改变图像表示以最大强度投影（MIP）格式。在同一个窗口:然后，点击&#39;0毫米] TH&#39;后的图像厚度更改为2毫米。 </li><li>使用2mm宽的3D娜vigator栏，允许探索的图像堆栈，并在适当的角度和位置，它切片，与威利斯（COW）的圆形，其中港口血栓的全覆盖制备2mm厚的轴向截面图像。点击" 输出 "面板上的拍摄按钮（相机图标）。保存为TIFF格式的图像。 </li></ol></li><li>然后，准备五个双毫米，而且从额叶小脑连续覆盖厚冠状切面图像。 <ol><li> ACA血栓 - 首先，由轴向图像上仔细对齐导航栏，以便日冕图像最能直观马华准备第二片。 </li><li>接着，在连续方式制备其他四片。点击" 输出 "面板上的拍摄按钮（相机图标）。 TIFF格式保存图像。 </li></ol></li></ol> 4.溶栓及在脑血栓的体内显微成像（图3） <ol><li>准备100厘米长的PE-10管与在一端上有30号针头，而在另一端的1ml注射器。填用盐水（600微升）或tPA的（在此，24毫克/千克，600微升），同时避免气泡管。 </li><li>如在步骤2.1中所述重新麻醉小鼠。插入动物的阴茎静脉内针尖。小心地将动物到显微机的床。然后，固定并通过将其编带至床稳定导管系统的静脉内注射的一部分。 </li><li>执行后续成像会议前处理基线。然后，通过按压针筒柱塞到导管系统注入或60微升生理盐水或tPA的。在推注后，开始注入在一段时间（在此为30分钟）余下的溶液（540微升）。 </li><li>在预先指定的时间点获得使用相同的参数如在步骤3.4显微图像:在这里，在推注后3和24小时。要执行以下体外血栓NIRF大脑的总线成像，通过颈脱位安乐死麻醉下的动物。 </li></ol> 5. 离体 NIRF血栓影像与三苯四唑氯化物（TTC）的脑组织的染色（图4） <ol><li>断头后，取出头皮并通过与从枕骨大孔达向矢状缝剪刀剪头骨。通过仔细地提升用剪刀，同时避免伤害到下面的大脑颅骨切开的边缘，从而一语道破半球删除颅腔。 </li><li>切出视神经在大脑基底尽可能接近到脑表面，因为它们可以与威利斯动脉的圆，应该在NIRF成像可视化重叠。然后，为了清楚地可视化的Y形"MCA / ACA /远端ICA&#39;为NIRF血栓成像，之后轻轻压缩小脑基，以暴露第切出的远端的ICAs尽可能给脑表面Ë切割点（ 图4A）。 </li><li>执行NIRF成像（激发/发射675 / 690nm下1秒的曝光）及其碱朝上（ 图4B，C），其中可视化的荧光标记的血栓在COW的动脉去除的脑组织（ 图4D） 。然后，用脑朝上，该可视化皮质血栓栓塞的顶点执行附加成像。通过将盐水几滴在组织避免大脑的干燥。 </li><li>使用根据制造商的说明脑矩阵器件，快速地准备冠2mm厚的脑切片:6片2mm厚片（2.3，0.3，-1.7，-3.7，从前囟-5.7毫米）。通过使用盐水液滴避免大脑切片干燥，并执行前和各部分的背面两者的NIRF成像。 </li><li>放入2％三苯基氯化四氮唑（TTC）溶液中的大脑切片20分钟，同时避免暴露到二极管灯吨。然后，将切片在4％甲醛溶液在4℃下，同时避免暴露于光。 </li></ol>采用显微图像和ImageJ的（1.49d）软件6.血栓面积的定量（图5） <ol><li>打开图像（ 图5A）。 <ol><li>打开图像序列文件选择" 文件&gt;导入&gt;图像序列 "或" 文件&gt;打开 "创建堆栈文件。通过使用" 图像&gt;类型&gt; 8位 "将图像转换为8位灰度。 </li></ol></li><li>转换英寸该股毫米（ 图5A）。 <ol><li>当CT图像文件中的一个像素对应于0.053毫米，可以使用"分析&gt;设置缩放&#39;进入&#39;1&#39;，&#39;0.053"，并为" 距离像素 &#39;毫米&#39;，&#39; 已知距离 "，和长度的 &#39; 单元 &#39;， 分别。 </li><li>当（只）比例尺是可用的，使用"直线"工具绘制与它的长度等于标尺条的一条线。然后，使用" 分析&gt;设置规模 "，进入毫米的比例尺的长度。 </li></ol></li><li>背景减法（ 图5B） <ol><li>通过使用" 编辑&gt;选项&gt;指定&#39;的地方对脑实质的领域的兴趣（ROI）圆形或矩形区域无血栓或骨相关的高密度地区。因为指定的投资回报率适用于堆栈的每一个切片检查，以确保如果没有在每个片高密度的区域重叠。 </li><li>选择"插件&gt;投资回报率&gt;从投资回报率BG减法&#39;，为&#39;从平均标准偏差数"进入2.0。 </li></ol></li><li>血栓栓塞相关病变高密度分割（ 图5C） <ol><li>选择" 图像&gt;调整&gt;阈值"，然后输入罗"的价值观WER门限电平"和"上限程度"分别为22和255。选择"上/下"，以显示在下方的蓝色灰度，阈值像素下限阈值像素，并且像素绿色上限阈值以上。 </li><li>使用"写意选择工具"绘制围绕血栓相关的高密度灶，而不包括骨性领域的投资回报。在绘图过程中，按住要么[SHIFT]或[ALT]添加或删除一个区域，分别为。 注意:如果高密度血栓栓塞病灶从骨结构遥远，而不是&#39;手绘选择工具"，"棒工具"可以安全地在不改变它的"容差"电平使用。 </li></ol></li><li>该分段病变的定量分析（ 图5D） <ol><li>使用"分析&gt;设置测量&#39;选择&#39;区&#39;，&#39;平均灰度值"和"集成密度（区X平均值）9 ;.检查下列选项:"限价至阈值"和"显示标签"。使用"分析&gt;测量"来获取量化数据。然后，将结果保存为一个"的.xls"文件。 </li><li> TIFF格式存储堆栈文件。此外，使用" 分析&gt;工具&gt;投资回报率经理保存的ROI。 </li></ol></li></ol>

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DE.K.，JY.K，通道A和KK是纤维蛋白靶向纳米金的专利持有人（10-1474063-0000，韩国知识产权局）。其余的作者都没有透露。

我们展示了使用两个互补的分子成像技术用于在栓塞性中风的实验模型直接血栓成像:血纤维蛋白靶向金纳米颗粒（FIB-GC-金纳米粒子），用于在体内基于Micro-成像和一个FXIIIA针对前光学成像探针体内荧光成像。 FIB-GC-金纳米粒子的静脉内给药后，血栓成为可见的CT为致密的结构，后浓度的梯度引起的颗粒变得由纤维蛋白 - 靶向肽的作用（该颗粒的表面上）包埋在血栓依赖扩散到多孔结构中， 也就是，血栓。这个过程出现仅在一个方向发生，并迅速导致相对低浓度的纳米颗粒在血液中的凝块变得集中，并充分地改变其CT密度，以允许检测。在24小时的最后体内成像显微与离体脑的验尸NIRF成像很好相关，这表明了与光学探针识别被放置在脑动脉之前FXIIIA的纤维蛋白交联的活性被荧光标记的初始血栓后的tPA渣油。以这种方式，两种分子成像探针，使用操作和不同的成像模态，每个报告和不同的机制交叉验证其他的调查结果。 血栓的演变可以用同样的方法来执行;具体地，tPA的溶栓治疗可以遵循在体内 ，允许经CT进行溶栓的成功的迅速及时读出。这对于临床溶栓治疗产生深远的影响，也有新的抗血栓或溶栓疗法的开发研究应用。 在此之前，我们集团能够检测中风治疗的常见并发症，则RE-3血栓发生往往围绕一个部分处理的血块，随后再闭塞和日益恶化的结果。重新血栓形成在同一动物在不同时间4个不同的位置（左CCA远端，左CCA近端，远端右CCA，然后用鼠标右键近端CCA）将1毫米氯化铁-soaked滤纸（等级42）依次为蓝本。在所有的情况下，循环FIB-GC-金纳米粒子进行了论证在血栓积累和渲染这些可见的CT成像。这种效应在此持续时间似乎仅限于FIB-GC-金纳米粒子，这是目前未知的生物半衰期，以及fib-初次注射后已被用于在延长的时间（长达3周）观察GC-金纳米粒子。这是极大的兴趣的临床的，因为纳米颗粒剂的单次剂量可能证明有效用于观察两者的成功和临床治疗方案的失败。需要进一步研究，以评估药代动力学和biodistriFIB-GC-金纳米粒子的bution。 FIB-GC-金纳米粒子的化学合成进行了详细4在前面描述。在EP-2104R磁共振成像使用简言之，将纤维蛋白靶向肽（酪氨酸-D-谷氨酰胺-半胱氨酸羟-L-3- chlorotyrosine -甘氨酸-亮氨酸-半胱氨酸-酪氨酸-异亮氨酸-谷氨酰胺）（MRI）剂8是通过标准的固相的Fmoc肽化学合成，并且用1-乙基-3-（3-二甲氨基丙基）碳化二亚胺和N-羟基琥珀酰亚胺缀合的GC-涂覆金纳米粒子的表面上。最终产品，纯化的FIB-GC-金纳米粒子通过离心收集。 的C15 NIRF探针预制血栓的光学标记是由FXIIIA识别和标记的赖氨酸残基9,10与经Cy5.5荧光染料的ε氨基的15个氨基酸的肽。光学成像剂是由该法的酶的作用共价连接到血栓的纤维蛋白丝ivated凝血因子，当凝块成熟11,12的过程中，交联纤维蛋白丝。的C15 NIRF血栓标记的化学合成也详细4在前面描述。简言之，FXIIIA亲和性的肽（甘氨酸 - 天冬酰胺 - 丙氨酸 - 谷氨酸 - 谷氨酰胺 - 缬氨酸 - 丝氨酸 - 脯氨酸 - 亮氨酸 - 苏氨酸 - 亮氨酸 - 亮氨酸 - 赖氨酸 - 色氨酸）的基础上ALPH-2抗纤溶酶的N-末端被固合成-phase肽合成，然后标记经Cy5.5经由半胱氨酸侧链的荧光染料。用高效液相色谱法，该产品是纯化的和最终的化合物被收集。 同时NIRF和金纳米颗粒制剂的成功是可能的，因为这些药物的生物强的设计特点。 FXIIIA 图11是从根本上重要的凝血酶活化的四聚体的转谷氨酰胺酶，介导纤溶性凝固酶。这种活性酶被认为是新形成的血栓的一个标志。无论我们的代理商符合这一基本血液凝固酶（由FXIIIA感应C15光剂）或它的底物（由纤维蛋白靶向FIB-GC-CT金纳米粒子剂）发生强烈的相互作用。 共定位研究表明，存在由于相互离体荧光（外源性血栓标记物）和用于与纤维蛋白结合的纳米颗粒，这是期望成像脑血栓体内显微密度（内源性血栓标记）之间的优良通信，有关的生物截留机制两者的代理。 有几件事情采用双模式直接血栓成像技术时需要考虑的。首先，为了防止FIB-GC-金纳米粒子从聚集和实现生物相容性，乙二醇脱乙酰壳多糖用于表面涂层13。然而静脉注射前，血栓成像剂应resuspen在PBS DED，并且超声处理以确保纳米粒子的溶解和分散。此外，纳米颗粒的稳定性可以通过使用紫外 - 可见（UV-VIS）吸收光谱进行比较的FIB-GC-金纳米粒子的新鲜与旧股票，这两者应该是在约519纳米的表面等离子体共振峰进行评估。 如增加600至1200片的" 扫描数 "或增加" 平均值 "1至3个，但是，这将增加以完成所需要的时间:第二，显微血栓图像的分辨率可以通过改变扫描参数来改善扫描。 3 5为血栓的本研究中的荧光标记:第三，血液和C15 NIRF成像剂在7:3的比例混合。根据我们的经验，相对低血容量可能妨碍血栓形成，而相对高的血液量可以减少血栓的可探测由于离体从血栓发射荧光信号的衰减。此外，值得注意的是，多种不同的方法可用于血栓14的各种组件的荧光可视化。 第四，与原位 氯化铁介导的血栓形成模型，其中靶动脉仅部分闭塞相比，脑血栓栓塞的显微可视化效果完全堵塞血管要求比较高剂量FIB-GC-金纳米粒子4。因此，有可能是毒性的担心，尽管胶体金被认为是生物相容的和FIB-GC-金纳米粒子没有在先前的研究4展示明显的全身/神经行为毒性。 第五，临床试验表明，血小板靶向或纤维蛋白靶向钆（Gd）为基础的MRI剂可能增加在心脏腔室，颈动脉血栓的高信号，和主动脉弓<html>在T1加权像15。虽然MRI具有极好的软组织的对比度和高空间分辨率，MRI是一种耗时的测试。 CT来自相对贫困的软组织对比度受到影响，但CT扫描可迅速执行。因此，CT保持在行程16的急性管理最广泛使用的成像技术。此外，对于小动物成像显微系统不需要被安装的建筑辐射屏蔽，并提供高清晰度图像。因此，显微与体外反射荧光成像或体内荧光断层17组合是小动物的研究非常有用的。可替代地，使用金-铁MRI / CT双重峰成像纳米颗粒18-20也可以是用于在动物和人类的血栓成像的强大的平台; MRI和CT可以相互补充，与光学成像，它们不从低组织渗透问题的困扰。 内容"&gt;总之，结合CT和荧光成像直接血栓可视化是缺血性中风的调查一个强有力的研究工具，承诺提高检测血栓，治疗的监测，并重新血栓形成的复杂治疗的检测。此外，存在用于这些技术的临床应用显著潜力。

在一个6人将在其一生某个时候中风。缺血性中风是迄今为止最常见的中风类型，占所有中风病例的80％。因为血栓引起广大这些缺血性中风，存在直接血栓成像的兴趣日益增加。 据估计，约200万脑细胞大脑中动脉闭塞1的每一分钟期间死亡，导致的口号"时间就是大脑"。计算机断层扫描（CT）的研究可以快速完成，并且被广泛使用;因为这个原因，CT仍首选初始诊断和治疗超急性缺血性中风的成像。 CT是用于通知重要的早期决定特别有价值的:溶栓施用组织纤维蛋白溶酶原激活剂（tPA）和/或归类（triaging）到血管内凝块检索2。当前的CT-基于血栓成像，但是，不能连续跟踪大脑体内升血栓栓塞，因为它使用间接方法来证明血栓:由碘化造影血池的混浊后，血栓被表现为在容器填充的缺陷。有与碘造影剂的重复给药相关的剂量限值和风险，排除重复的血栓成像用这种方式。 因此，为直接成像方法在中风患者脑血栓迫切需要，允许更快和更好的治疗决定作出。我们提出通过增强的CT，目前使用的前线成像模态对行程的值，与使用血栓寻求纳米粒分子成像剂来实现这一点。 我们已经利用显微计算机断层摄影术（显微），高分辨率的离体或在体内 （小动物）的CT成像版本，允许快速的数据采集&lt;展示了使用本剂的SUP&gt; 3,4。即使有可供小动物显微（比从人类大小的扫描仪差很多）相对较差的软组织对比度，成像剂是能够寻求和使他们对CT，通过分子加强了"密集的血管征"高密度标记血栓成像。 补充CT技术，我们集团之前开发利用经Cy5.5近红外荧光（NIRF）探针可视化脑血栓负担5光学直接血栓成像技术。这是验尸大脑的体外技术，但高度敏感，并提供在研究环境体内数据确认。 拥有CT和NIRF都基于血栓寻求成像技术使我们能够比较和对比这些技术来实现对血栓和血栓成像在缺血性中风的发展进程中的作用非常翔实的数据。 HERE，我们描述了在体内的显微和体外成像NIRF的组合技术的详细协议直接看到血栓栓塞的栓塞性中风的小鼠模型。这些简单的和鲁棒的方法是通过在治疗期间在体内使在血栓负荷/分布和动态血栓演化的表征体内评估准确迅速和定量方式推进我们血栓性疾病的理解是有用的，其次是提供体外数据作为体内成像结果的确认对照和参考标准。

<table border="0" cellpadding="0" cellspacing="0" width="588">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Machines</td>
			<td style="width:219px;"></td>
			<td style="width:119px;"></td>
			<td style="width:96px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microCT</td>
			<td>NanoFocusRay, JeonJu, Korea</td>
			<td>NFR Polaris-G90</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIRF imaging system</td>
			<td>Roper-scientific,Tucson, AZ</td>
			<td>coolsnap-Ez</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Doppler flowmeter</td>
			<td>Perimed, Stockholm, Sweden</td>
			<td>PeriFlux System 5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical microscope</td>
			<td>Leica Microsystems, Seoul, Korea</td>
			<td>EZ4HD</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Inhalation anesthesia machine</td>
			<td>PerkinElmer, Massachusetts, USA</td>
			<td>XGI-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">NFR control</td>
			<td>NanoFocusRay, JeonJu, Korea</td>
			<td>NFR Polaris-G90</td>
			<td style="width:96px;">microCT control software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Lucion</td>
			<td>Infinitt, Seoul, Korea</td>
			<td>Lucion</td>
			<td style="width:96px;">3D render imaging software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab chart 7</td>
			<td>ADInstruments, Colorado, USA</td>
			<td>Lab chart 7</td>
			<td>rCBF</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Image J software</td>
			<td>Wanye Rasband, NIH, USA</td>
			<td>1.49d</td>
			<td>imaging analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Devices/Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Infusion pump</td>
			<td>Harvard, Massachusetts, USA</td>
			<td>pump 22(55-2226)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homeothermic blanket</td>
			<td>Panlab, Barcelona, Spain</td>
			<td>HB101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pocket cautery</td>
			<td>Daejong, Seoul, Korea</td>
			<td>DJE-39</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain matrice</td>
			<td>Ted pella, CA, USA</td>
			<td>15003</td>
			<td style="width:96px;">coronal section</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-50 tubing</td>
			<td>Natsume, Tokyo, Japan</td>
			<td>SP-45(PE-50)</td>
			<td style="width:96px;">I.D. 0.58 mm O.D. 0.96 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-10 tubing</td>
			<td>Natsume, Tokyo, Japan</td>
			<td>SP-10(PE-10)</td>
			<td style="width:96px;">I.D. 0.28mm O.D. 0.61 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">30 gauge needle</td>
			<td>sungshim-medical, Seoul, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>CPL-medical, Ansan, Korea</td>
			<td></td>
			<td>1 &#38; 3 cc</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gauze</td>
			<td>Panamedic, Cheonan, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tape</td>
			<td>Scotch, Seoul, Korea</td>
			<td>3M-810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro forceps</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td>&#160;11253-27</td>
			<td>Dumont #L5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro scissor</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td style="width:119px;">15000-03</td>
			<td>Vannas spring</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissor</td>
			<td>Fine Science Tools, Vancouver, Canada</td>
			<td style="width:119px;">14084-08</td>
			<td>8.5 cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black silk suture</td>
			<td>Ailee, Busan, Korea</td>
			<td>SK6071, SK728</td>
			<td>6-0 and 7-0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">meloxicam</td>
			<td>Yuhan, Seoul, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vet ointment</td>
			<td>Novartis, Basel, Swiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% Povidone-iodine (betadine)</td>
			<td>Firson, Cheon-an, Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FeCl3</td>
			<td>Sigma, Missouri, United States</td>
			<td>157740-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TTC</td>
			<td>Amresco, Ohio, USA</td>
			<td>0765-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Hana-Pham, Gyeonggi, Korea</td>
			<td>Ifran</td>
			<td>100 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Welgene, Daegu, Korea</td>
			<td style="width:119px;">LB001-02</td>
			<td>500 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold nanoparticles</td>
			<td></td>
			<td></td>
			<td>Synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C15 optical agent</td>
			<td></td>
			<td></td>
			<td>Synthesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue plasminogen activator</td>
			<td style="width:219px;">Boehringer Ingelheim, Biberach, Germany</td>
			<td style="width:119px;">rtPA(actilyse)</td>
			<td>20 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Daihan Pham, Seoul, Korea</td>
			<td>48N3AF3</td>
			<td>20 mL</td>
		</tr>
	</tbody>
</table>

combined near-infrared fluorescent imaging and micro-computed tomography for directly visualizing cerebral thromboemboli

直接血栓成像可视化血栓梗塞的根本原因。如果能够像血栓允许直接行程不是依靠间接测量的更好的调查，将是一个强有力的和强大的血管的研究工具。我们使用标签与分子成像血栓血栓标记的光学成像方法 - 该凝块的成熟过程中被共价连接至由活化的凝血因子的ⅩⅢa的血纤维蛋白交联的酶作用的血栓的纤维蛋白丝一经Cy5.5近红外线荧光（NIRF）探针。的微型计算机断层扫描（显微）基的方法是使用官能化以靶向凝块的主要成分血栓求金粒子（AuNPs）:纤维蛋白。本文描述了组合的体内显微和栓塞性中风的小鼠模型血栓栓塞的体外 NIRF成像的详细协议。我们表明， 在体内&lt;/ em&gt;的显微和纤维蛋白靶向乙二醇壳聚糖纳米金（FIB-GC-金纳米粒子）可同时用于原位血栓和脑栓塞血栓可视化。我们还描述了使用在体内基于Micro-直接血栓成像以串行监控组织纤溶酶原激活介导的溶栓治疗效果。最后成像会议之后，我们证明通过体外 NIRF成像的程度和残留血栓栓塞的在脑中的分布。最后，我们描述了显微及成像NIRF数据定量分析图像。直接血栓成像的组合技术允许血栓可视的两个独立的方法来进行比较:血栓有关的荧光信号对区域体外 NIRF成像与高密度显微血栓体内的体积。

基线显微图像， 在体内得到施用FIB-GC-金纳米粒子（10毫克/毫升，300微升）以栓塞性中风后1小时，明显地显现在MCA脑血栓后-远端颈内动脉ACA分叉区（ 图6 ）。后续显微成像显示，用生理盐水治疗血栓COW没有变化。然而，tPA的治疗显示COW血栓的逐渐溶解（ 图6中的蓝色箭头）。这一发现表明，显微血栓成像可以允许溶栓治疗监测。有一个在24小时在体内 CT密度和体外 NIRF之间一个很好的对应残留血栓。请注意，由于内源性血栓标志着由纤维蛋白靶向纳米金很好地共定位与因NIRF信号区的高密度显微领域外源性血栓标记，这是EMBO前进行LIC冲程通过使用经Cy5.5荧光探针到凝固XIIIa因子介导的纤维蛋白的交联的预形成的凝块的成熟过程的活性（ 图6）很敏感。由TTC染色法直接血栓成像演示的tPA介导的溶栓效果的综合体现技术组织中风结果。 TTC染色表明在缺血性脑损伤的量为动物接受tPA的溶栓明显减少（ 图6中发白区域）。 <img alt="图1" src="/files/ftp_upload/54294/54294fig1.jpg" /> 图1:荧光标记的血液凝块的制备概述 （A）和经Cy5.5近红外线荧光的新鲜全血混合（NIRF）探针（C15），该共价由血纤维蛋白连接于血栓的纤维蛋白丝激活混凝的-crosslinking酶的作用凝块的成熟过程中的相关特征研XIIIa因子。该混合血液注入到聚乙烯（PE）-50管，放置24小时，以允许凝块形成和成熟。 （B）的NIRF成像确认与C15探针血栓的光学标记。注意洗涤说明，从PE管取出血栓后，持续的荧光。 （C）的血栓加载PE-10管，在PE-50管修饰以具有锥形端部（直径为200微米），用于在大脑中动脉栓塞-小鼠颈内动脉的前脑动脉分叉区。比例尺:2毫米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54294/54294fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54294/54294fig2.jpg" /> 图2:救援人员到场建模的小鼠模型概述mboembolic脑梗塞。（A）的前和大脑中动脉（MCA）闭塞后激光多普勒监测脑血流量（CBF）的，与光纤的激光多普勒流量计粘到左颞骨表面的前端（1 Mm左和来自前囟低于4毫米）。注意多普勒CBF的闭塞后下降，这应该由30％或更低的降低相比基线。 （B）的含血栓导管插入（ 例如，向远侧逐渐变细的聚乙烯-50管中图1C）插入到颈外动脉（ECA），孔接着（最终）通过推进导管大约9mm到MCA - 远端颈内动脉（ICA）的大脑前动脉（ACA）的分叉区域。 CCA，颈总动脉; PPA，翼腭动脉。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54294/54294fig2large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="图3" src="/files/ftp_upload/54294/54294fig3.jpg" /> 图3:概述在体内显微型纤维蛋白采用针对性的金纳米粒子可视化脑血栓和监控组织型纤溶酶原激活剂（tPA）介导的溶栓作用血栓直接成像 （A）纤维蛋白靶向乙二醇壳聚糖纳米金的静脉注射。 （FIB-GC-金纳米粒子）与血栓栓塞性中风鼠标。 （B）中的小鼠被放置在显微成像器的床。 （C）FIB-GC-金纳米粒子注射后5分钟获取显微图像。 （D）图像重建使用安装在显微扫描器软件包变换在医学（DICOM）格式的原始图像数据转换成数字成像和通信。 （E）的图像分析和准备轴向/ C的oronal图像渲染采用最大密度投影模式有2个毫米的厚度。 （F）静脉的tPA介导的溶栓治疗，这可以通过使用基于Micro-直接血栓成像方法进行监测和分析。（C - E） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54294/54294fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54294/54294fig4.jpg" /> 图4: 前体内近红外荧光（NIRF）血栓栓塞脑影像学 （A和B）代表的脑组织从血栓栓塞性中风鼠标删除概述 。采取预防措施，不要在大脑中动脉血栓栓塞输（红色箭头）（MCA） - 远端颈内动脉大脑前动脉（ACA）分支区域（ICA，蓝色箭头）去除脑部的时候。 （C和D） 体外血栓NIRF成像。脑组织被放置在NIRF成像仪（C）的血栓（D中红色箭头）预先标记的与MCA的闭塞栓塞前经Cy5.5荧光标记C15的可视化的透光的室内- ACA分叉区。比较图5的CT表现。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54294/54294fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54294/54294fig5.jpg" /> 图5:采用显微图像和量化血栓卷ImageJ的概述（1.49d）（A）将图像导入到一个新的堆栈文件，文件格式转换成8位灰度，并改变单位。毫米。 （B）背景减法。 （ 三 ）通过围绕高密度区域绘制感兴趣区域（ROI）的区域，同时排除骨性结构的血栓栓塞病灶分割。 （D）通过使用菜单&#39;分析 &gt; 办法"为获取投资回报的量化数据。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54294/54294fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54294/54294fig6.jpg" /> 图6:代表直接脑血栓成像数据获得之前和治疗小鼠抚摸咸水与组织型纤溶酶原激活剂（tPA）（A）显微（MCT）血栓成像，以便组织型纤溶酶原激活物治疗监测后剂（tPA）介导的溶栓。 （B和<str翁&gt; C） 在体内 CT密度（A）和体外 （B之间的一个很好的对应关系）的近红外荧光（NIRF; c）对于后tPA的残留血栓栓塞在24小时左右。对大脑（D）和较小的白色梗死上拆下的大脑中的TPA处理的动物的相应部分（E）的TTC染色的冠状体内显微计算机断层摄影图像（D和E）减去残余的血栓（黄色箭头头）比在盐水治疗的动物。请参阅详细的解释正文（ 代表结果 ）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54294/54294fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli at JoVE.com

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

本协议描述相结合近红外荧光（NIRF）成像和微计算机断层扫描（显微）的可视化脑血栓中的应用。这种技术允许的血栓负担和演化的量化。该NIRF成像技术可视化荧光标记在切除脑血栓，而显微技术，可视化使用金的纳米粒子的生活里面的动物血栓。

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 ...

combined-near-infrared-fluorescent-imaging-micro-computed-tomography

Nanyang Technological University

Research

JoVE Journal

Medicine

9.8K 次观看.  Dongguk University College of Medicine. 本协议描述相结合近红外荧光（NIRF）成像和微计算机断层扫描（显微）的可视化脑血栓中的应用。这种技术允许的血栓负担和演化的量化。该NIRF成像技术可视化荧光标记在切除脑血栓，而显微技术，可视化使用金的纳米粒子的生活里面的动物血栓。 

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

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

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

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

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

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 ...

科研

医学

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

无创成像和脑缺血分析生活中的大鼠使用正电子发射断层扫描<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

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 ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

如何管理危重新生儿、婴儿和儿童近红外光谱

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...