（;亨利Dryerre方案LL）和资金由英国心脏基金会（PWFH，BRW，DJW，这项工作是由爱丁堡大学（NSK）和卡内基信托studentships支持RG / 05/008，PG / 05/007; PG / 08/068/25461）和威康信托基金会（JRS，BRW，DJW; 08314 / Z / 07 / Z）。作者感谢为到中心心血管科学所提供的BHF-资助优秀成果奖中心支持他们的工作。 作者是在建立内膜病变生产的手术模型的意见正隆SATA（德岛大学）和伊戈尔Chersehnev博士（在Ernane李嘉欣博士群在医学西奈山医学院）教授特别感谢。视频制作和萨塔等提供。（http://plaza.umin.ac.jp/~msata/english.htm）是特别有用的。

内膜病变在小鼠股动脉1.手术感应<ol><li>使用实验动物应当根据国家和机构的道德要求来执行。所有手术应采用适当的无菌技术下进行。感应内膜病变的是使用由罗克等人 12和萨塔等 13所描述的技术的改进来实现的。 </li><li>称重雄性C57BL6 / J小鼠（年龄10-12周;体重25-30g的），然后通过提供4-5％的异氟醚在感应腔麻醉。一旦麻醉已诱导的小鼠转移到一个加热垫保持体温在37℃。通过面罩持续异氟醚（2-3％）的管理。 </li><li>一旦麻醉适当水平已致（缺乏应对脚趾捏），由丁丙诺啡管理提供镇痛盖（0.1毫克/千克-1）。然后将鼠标在仰卧位的ð剃左后肢的腹面。 </li><li>做一个切口，以暴露上部后肢的肌肉，并且与腘动脉和腹壁的分叉之间，用钝器解剖分离的股动脉和静脉从股神经。用1％w / v的利多卡因根据需要灌溉的伤口。 </li><li>放置近端（靠近腹壁）和远端（紧接在分支下面腘动脉）临时连字（6/0 Mersilk）的股动脉和静脉来控制血流周围。然后隔离腘动脉（约2-5毫米远端的股动脉分支）和远端结扎。将腘动脉下方的第二，解开结扎。 </li><li>作一小切口（动脉切开）的腘动脉，立即远端与股骨动脉分支，防止出血通过施加压力到近端临时结扎。推进直，如雨后春笋般涌现0.014"导丝1-1.5厘米沿股动脉在腹壁的方向并留在原地，持续30秒（ 图1A）。 </li><li>除去导丝和结扎腘动脉的动脉切开以上，使用结扎置于为此目的，并注意不要堵塞股动脉。 注:对于结扎引起的损伤。无伤腔内重塑新生内膜可通过连接股或腘动脉（ 图1B和1C）诱导。为了实现这一目标按照步骤1.1-1.5。然而，不要使动脉切开术，但（避免步骤1.6）或者（i）结扎腘动脉立即远端在与腘动脉分支点的股动脉或（ii）结扎股总动脉。然后继续步骤1.8。 </li><li>删除临时连字，用一个不连续的外部缝线（5/0 Mersilk）闭合伤口和应用EMLA霜（2.5％利多卡因，2.5％丙胺卡因）。让动物重新<html>获得意识（通常5-10分钟），并确保他们自由走动笼子（轻微跛行可能是显而易见的，在受影响的腿，但这应该解决在手术后的第2-3天），返回房间举行之前。小鼠不必在外科手术后单独放置。 </li><li>允许动物恢复长达3个月。小病灶将开始丝伤后7天〜出现后会达到一个稳定的最大尺寸〜21-28天。 </li></ol> 2.诱导动脉粥样硬化病变的载脂蛋白E - / -小鼠<ol><li> 12周为雄性，6周龄ApoE无效小鼠（育成中心）;管理的西方饮食（饮食的研究，美国0.2％的胆固醇）。 </li><li>动脉粥样硬化病变往往在主动脉弓及其主要分支（ 图2）的总检查可见。 </li></ol> 3.试析动脉病变利用光学投影层析成像（OPT） NT"&gt;注:病变在小鼠股动脉和主动脉弓样品的OPT图像用光学投影断层扫描获得的。 <ol><li>安乐死的小鼠通过终端麻醉（80毫克/公斤戊巴比妥钠）根据transcardiac灌注固定和放血，用肝素（10U / ml）的磷酸缓冲盐水（PBS），接着用10％中性缓冲福尔马林中。 </li><li>隔离股动脉或主动脉弓及其主要分支（左颈动脉，左锁骨下动脉，头臂干），适当，并删除多余的围外膜材料。定位后，在10％缓冲的福尔马林中O / N，直到需要存储在70％乙醇之前。 </li><li>在1.5％低熔点琼脂糖嵌入动脉，通过Whatman 113 V纸预过滤。每个样品附着到磁性OPT安装用氰基丙烯酸酯类粘合剂的容器轴线在与所述安装件的线。修剪多余琼脂糖圆锥形。脱水在100％甲醇为至少12小时。 </li><li> C在苯甲醇和苯甲酸苄酯的混合物利尔船只通过浸渍（12-24小时）（1:2体积/体积）。 </li><li>放置清零样品校准层析影像。设置分辨率1024×1024，和确定的光学倍率，使所关注的整个区域被看见。 OPT体积是各向同性的z轴被重构到相同的分辨率（ 即 ，1024×1024×1024），体素尺寸〜200微米。这可能代表分辨率的过度估计，因为有可能是重建伪像。调整样品的位置，使得它旋转时其自身轴线在视场中的亮场，传输信道的中心。 </li><li>在GFP1过滤排放通道（激发滤光片425纳米，具有40纳米的带通;发射过滤:475nm的长通），聚焦样本并调整曝光时间以最大化所产生的图像的动态范围（避免过饱和）。扫描在仅GFP1发射通道的容器中，用0.9°的旋转工序。&lt;/ LI&gt; <li>在完成后，确认使用DataViewer的软件，数据采集的质量。从扫描仪中删除的标本。 </li><li>以允许后续的组织学分析，代替样品在100％甲醇&gt; 24小时前处理，以石蜡为正常。 </li></ol> 4，图像重建及分析使用NRecon软件或类似的执行断层重新构造通过滤波反投影。重建可以进行无人值守，分批。 <ol><li>通过补偿不对准和调整图像亮度等级提高图像质量。 </li><li>检查使用DataViewer中的软件图像重建的质量。 </li><li>确定样本进行分析的相应部分。保持这个长度船只之间是一致的，如果管腔的尺寸要被记录。 </li><li>通过手动跟踪适当的边界1在每50重新构造的横截面限定的病变的轮廓。 </li><li>检查每个交错的横截面，以确保计算机生成的内插是正确的。手动调整边框必要。 </li><li>设定灰度阈使得只有病灶被选择并导出测量数据。 </li><li>对于每次扫描，定义的包含病变兴趣的垂直区域和跟踪媒体和内膜之间的边界（ 即 ，内弹性层的位置）为每50 条扫描线。插值内膜/中边界在软件交错扫描线，以及核查和纠正在需要的契合。进一步段此定义的三维体积来手动定义的强度阈值以产生一个二进制图像组，其中白色像素代表新内膜和黑色象素代表专利内腔。 </li><li>采取测量包括:总病灶体积（容量对象），管腔容积（总体积 - 物体体积）和病变和流明跨教派的分布有理区沿研究容器的轴向长度。 </li></ol>

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The authors have no competing financial interest.

3维分析具有用于替换或添加到2维的组织学技术，仍然支撑大多数动脉损伤形成的调查的巨大潜力。在这里，OPT显示在小鼠动脉（与鼠股动脉大概代表，可以成功地使用这种技术来分析的最小的血管）。然而，它是，也适用于与动脉（和病变）来自其它物种，包括小到中等大小的人类血管使用;本集团已成功地使用这项技术来分析病变的兔主动脉（Bezuidenhout 等;未发表）。 OPT承诺更快的分析和增加的结构信息与传统的组织学比较，并具有不妨碍同时使用组织学和免疫组织化学技术的样品的随后的分析的优点。 使用OPT产生的图象，得到解剖细节，表示病变的形成部位和病变在这些区域的大小。在这些调查中使用的动脉可能接近的技术和图像质量分辨率的限制，因此由伪影（大概从旋转失准，不完整结算，反射/折射在琼脂糖顶点和聚焦的问题引起的）损害至一定程度。尽管这样，所要求的细节（ 即 ，层血管壁）保持可辨别，因此该技术是对各层的量化非常有用的。事实上，图像可以迅速且可再现量化，以提供病变和管腔体积的容器中的斑块支承部分的测量，以及病变的横截面面积和内腔在样品中选择的站点。大（主动脉），中型（股骨，颈动脉，锁骨下）小鼠动脉 - 那些通常用于对动脉粥样硬化和neotintimal损伤形成在小鼠中分析 - 是成功使用该方法进行分析。事实上，我们现在已经使用OPT证明药物干预和遗传操作对动脉粥样硬化和内膜病灶大小的影响。例如，内皮素受体 ​​阻断改变内膜病变的形成而从血管内皮选择删除的内皮素B受体的没有15。在动脉粥样硬化易感小鼠，基因缺失的酶的11β-HSD1 16或半乳凝素3 17被示出，以减少粥样硬化病变的大小。 病灶体积的定量OPT是一个明显的好处。它给出的总损伤负担在动脉4更翔实的指示比通常用组织学方法获得。整个分析病变减少选择偏差和错误，当船只分立部分被选择的分析将不可避免地发生。生产纵向病变型材是OPT的进一步强度通过诱导不同类型的损伤13,16（ 图5）病变的比较强调。例如，既要完成结扎线插入诱导几乎完全闭塞接近femero腘分叉。线损伤，但是，产生了病变沿扫描部的整个长度上延伸，而引起的动脉结扎病变迅速减小尺寸和消失。这个模式是具有较大程度的伤害所造成的插入血管成形术导丝的相一致。生成使用组织切片类似的结果是昂贵的，耗时的和劳动密集的。 OPT的优点包括它产生的图像的质量和它的相对速度和简单（我们已经常规扫描每天20血管）。图像质量出现优越或至少相当，以其他方法，用于产生三维图像的离体 （如MRI和微CT）中，yOPT等需要（我们的研究是典型的1-2sec /形象整合时间）并且是更便宜更短的扫描时间。样品制备延伸到数天，但只需要很少的劳动力，船舶可以分批编制和数据可以在一个会议上获得的。因此，吞吐量高，并且不需要延长扫描器的使用。重要的是，OPT的非破坏性的性质意味着它可以被用来确定用于免疫组化检查感兴趣的地点;从而减少切削的量和染色必需的。这是可能的高分辨率超声发展将为病变动脉这种大小的体积定量的另一种方法，但作者没有察觉到，证明本申请的任何出版物。 也许并不奇怪，在OPT图像质量劣于显微技术（可，当然，只能在较小的样品进行）。建议细化到重构n个数据的可能解决此限制，允许图像质量19,20的未来改进。另一种方法值得关注的是组织处理改变了样品的特性。例如，清除剂的亲脂性，苄醇/苯甲酸苄酯（BABB），很可能是由动脉粥样硬化病变除去脂质，而现有脱水可能会引起收缩率（当然，虽然，脱水和脂质去除步骤也的特征在石蜡包埋的样品制备）。 BABB用于本调查如，在具有亲水结算剂比较（ 例如甘油21）它会导致在形态只有小的变化。 有几种可能性为OPT的进一步发展和完善，特别是关于跟踪关键细胞的3维排列和信令参与动脉重构的因素。动脉组织的强的自体荧光，这是这样一种Ñ ​​优点在解剖图像的产生，不受漂白22的现有的方法猝灭并且可以限制使用荧光探针来评估RNA和蛋白质分布模式。使用比色探针（ 例如 β半乳糖苷酶）通过透射成像可视化可以克服这种限制。 最后，OPT具有病变的动脉小鼠的内膜的三维成像的巨大潜力。它代表了2维的方法通常是劳动密集型的，不能有效地代表总病灶体积相当大的进步。 OPT是比较快的，方便和非破坏性的。在图像分析的新发展的承诺，以进一步提高该技术的功率和效用。

动脉损伤的形成是中央的高发病率和死亡率与心血管疾病相关联。损伤形成被认为是由一不受约束的炎症反应引起的动脉损伤2。动脉粥样硬化病变慢慢形成响应于慢性损伤动脉壁而再狭窄病变中迅速以下急性机械损坏（例如，支架植入后）。有助于动脉病变的发展的机制已经相当澄清通过使用适当的动物模型，通常是与相关的基因操作1结合。 病变大小和组成的分析已经经典很大程度上取决于体外 ，二维组织学（虽然这是改变与改进的方法的开发用于体内和离体检测和病变的小动物的分析&lt;SUP&gt; 3）。动脉损伤的组织学分析是劳动密集，耗时且提供3维结构的有限信息。例如，病变负担通常通过测量病变的横截面面积进行评估（无论是在随机选择的地点或在最大闭塞部位）。这提供了整体的病变负担一个不完整的分析。整个安装三维成像技术提供了可能解决这一问题，但令人惊讶的几个合适的方法已被描述。这可能是由于以主要的小鼠动脉这对单光子共聚焦显微镜太大而是用于磁共振成像（MRI）4和X射线计算机断层扫描（CT）5太小的尺寸。 体外 MRI微型CT应用和动脉粥样硬化病变的小鼠研究表明，他们提供有限的分辨率，即使在比较大的动脉。加入到这一点，需要相对较长的采集时间限制吞吐量（和提高扫描费用）4,6。 新的光学成像方式（如光学相干断层扫描3,7和光声断层8）发展提供了巨大的潜力，改善病灶成像在小鼠动脉。类似电位示由光学投影层析（OPT），其被开发以允许小鼠胚胎的分析。 OPT被设计图像标本范围从〜0.3-10毫米，直径9。透射成像记录一个半透明样品多色可见光的不透明度，并且可用于识别的解剖结构。光激发之后在来自内特定的波长（ 例如 ，胶原，弹性蛋白）和样品中的外源性荧光团发射的成像记录发射。这也可以提供解剖信息（因为不同的组织成分所用的类型和自发荧光物种的密度不同当下）。此外，免疫反应性或基因表达的分布可以通过使用适当的荧光探针10来确定。对于任一成像模式（传输或发射），光被聚焦到一个电荷耦合器件，以允许迭代图像捕获作为样品旋转（通常为400张图像，0.9°增量）。这些可用于体积由标准层析重建方法（例如滤波反投影（使用锥算法）或迭代重建）的计算。 该视频展示了我们新的应用OPT进行快速，可量化的和具有成本效益的三维和动脉粥样硬化病变内膜的分析，如以前在柯比等 11描述。该技术被证明是适合于在三个常用模型定量损伤大小:（ⅰ）股动脉线损伤; （二）股动脉结扎，和（iii）膳食诱导的动脉粥样硬化中apolipoprotei否E缺陷（ApoE基因- / - ）小鼠。

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Name of Reagent/Material</td>
			<td style="width:180px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:180px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Operating Microscope</td>
			<td style="width:180px;">Zeiss, Germany</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;">OPMI Pico i</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Anaesthetic Machine</td>
			<td style="width:180px;">Vet Tech, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Fluovac</td>
			<td style="width:180px;">Harvard Apparatus UK</td>
			<td style="width:137px;">340387</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Fluosorber</td>
			<td style="width:180px;">Harvard Apparatus UK</td>
			<td style="width:137px;">340415</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Bead Steriliser</td>
			<td style="width:180px;">Fine Science Tools, UK</td>
			<td style="width:137px;">1800-45</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Heated Mat</td>
			<td style="width:180px;">Fine Sceince Tools, UK</td>
			<td style="width:137px;">21061-10</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Balance</td>
			<td style="width:180px;">Mettler Toledo</td>
			<td style="width:137px;">MS1602S</td>
			<td style="width:180px;">PB1502 or equivalent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Sutures</td>
			<td style="width:180px;">Ethicon, UK</td>
			<td style="width:137px;"></td>
			<td style="width:180px;">5/0 Mersilk</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Guidewire</td>
			<td style="width:180px;">Cook Inc, USA</td>
			<td style="width:137px;">C-PMS-251</td>
			<td style="width:180px;">0.014&#8221;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Suture Silk</td>
			<td style="width:180px;">Fine Science Tools, UK</td>
			<td style="width:137px;">18020-60</td>
			<td style="width:180px;">6/0 Mersilk</td>
		</tr>
		<tr height="21">
			<td height="169" rowspan="6" style="height:169px;width:195px;">Surgical Tools</td>
			<td rowspan="6" style="width:180px;">Fine Science Tools, UK</td>
			<td style="width:137px;">14058-09</td>
			<td style="width:180px;">Toughcut Iris scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:137px;">15000-01</td>
			<td style="width:180px;">Cohan-Vannas Spring Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:137px;">11251-35</td>
			<td style="width:180px;">Dumont #5/45 Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:137px;">11370-31</td>
			<td style="width:180px;">Moria Iris Forceps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:137px;">13008-12</td>
			<td style="width:180px;">Halsted-Mosquito Haemostat</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:137px;">18050-35</td>
			<td style="width:180px;">Bulldog clips</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Bioptonics 3001 Tomograph&#160;</td>
			<td style="width:180px;">Bioptonics, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Magnetic OPT Mount</td>
			<td style="width:180px;">Bioptonics, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Computer</td>
			<td style="width:180px;">Dell Inc, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Peristaltic pump</td>
			<td style="width:180px;">Gilson</td>
			<td style="width:137px;">F117606</td>
			<td style="width:180px;">Minipuls 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">DataViewer software&#160;</td>
			<td style="width:180px;">Skyscan, Belgium</td>
			<td style="width:137px;">v.1.4.4</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">NRecon software&#160;</td>
			<td style="width:180px;">Skyscan, Belgium</td>
			<td style="width:137px;">v.1.6.8</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">CTan software</td>
			<td style="width:180px;">Skyscan, Belgium</td>
			<td style="width:137px;">v.1.12</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:180px;"></td>
			<td style="width:137px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;"></td>
			<td style="width:180px;"></td>
			<td style="width:137px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Isoflurane</td>
			<td style="width:180px;">Merial Animal Health Ltd, UK</td>
			<td style="width:137px;">AP/Drugs/220/96</td>
			<td style="width:180px;">100% Inhalation vapour, liquid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Medical Oxygen</td>
			<td style="width:180px;">BOC Medical, UK</td>
			<td style="width:137px;">UN1072</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Vetergesic</td>
			<td style="width:180px;">Alstoe Animal Health Ltd, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;">0.3mg/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">1% Lignocaine</td>
			<td style="width:180px;">Hamlen Pharmaceuticals, UK</td>
			<td style="width:137px;">LD1010</td>
			<td style="width:180px;">10ml ampoule</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">EMLA Cream</td>
			<td style="width:180px;">Astra Zeneca, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium Pentobarbital</td>
			<td style="width:180px;">Ceva Animal Health Ltd, UK</td>
			<td style="width:137px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Western Diet</td>
			<td style="width:180px;">Research Diets, USA</td>
			<td style="width:137px;">D12079B</td>
			<td style="width:180px;">0.2% cholesterol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Phosphate Buffered Saline</td>
			<td style="width:180px;">Sigma UK</td>
			<td style="width:137px;">P4417</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Heparin (Mucous)</td>
			<td style="width:180px;">Leo Laboratories, UK</td>
			<td style="width:137px;">PL0043/003GR</td>
			<td style="width:180px;">25, 0000 Units</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Neutral Buffered Formalin</td>
			<td style="width:180px;">Sigma, UK</td>
			<td style="width:137px;">HT501128</td>
			<td style="width:180px;">10%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Ethanol</td>
			<td style="width:180px;">VWR BDH Prolabo, UK</td>
			<td style="width:137px;">20821.33</td>
			<td style="width:180px;">Absolute AnalaR&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Agarose</td>
			<td style="width:180px;">Invitrogen, UK</td>
			<td style="width:137px;">16020050</td>
			<td style="width:180px;">Low melting point</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Filter Paper</td>
			<td style="width:180px;">GE Healthcare, UK</td>
			<td style="width:137px;">113v</td>
			<td style="width:180px;">Whatman</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Cyanoacrylate adhesive</td>
			<td style="width:180px;">Henkel, UK</td>
			<td style="width:137px;">4304</td>
			<td style="width:180px;">Loctite</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Benzyl alcohol</td>
			<td style="width:180px;">Sigma, UK</td>
			<td style="width:137px;">B6630</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Benzyl benzoate</td>
			<td style="width:180px;">Sigma, UK</td>
			<td style="width:137px;">402834</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Methanol</td>
			<td style="width:180px;">VWR BDH Prolabo, UK</td>
			<td style="width:137px;">20856.296</td>
			<td style="width:180px;">100%</td>
		</tr>
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generation and 3-dimensional quantitation of arterial lesions in mice using optical projection tomography

生成和血管病变的分析合适的动物模型是研究心血管疾病的基石，在产生病变形成的发病机制和新疗法的行动的重要信息。使用动脉硬化多发小鼠，病变感应的手术方法，和饮食改变的极大地提高，有助于疾病的发展和新的治疗药物的潜在机制的理解。 经典地，使用2-维组织学技术进行病变分析离体进行 。本文介绍的应用光学投影层析成像（OPT）动脉病变的三维定量。由于该技术是非破坏性的，它可以用来作为一种辅助标准的组织学和免疫组织化学分析。 内膜病变引起的线插入鼠标股骨艺术或结扎ERY而动脉粥样硬化病变由动脉粥样硬化的饮食ApoE基因缺陷小鼠产生的管理。 自体荧光使用发射OPT成像其次是互补的组织学和免疫组化分析病灶进行了检查。 OPT从底层血管壁明确区分病变。病变大小用面积法计算出在2维节，使损伤体积和最大截面积的计算中。使用OPT生成的数据是使用组织学获得的测量结果一致，证实了该技术的精确度和其作为一种补充（而不是替代），以传统的分析方法的潜力。 这项工作表明成像动脉粥样硬化和新生内膜病变OPT的潜力。它提供了一种快速，非常需要体外技术血管重塑的常规三维定量。

健康（未病变）小鼠股动脉初步扫描（N = 5）表明，透射成像并没有提供有用的图像。这是清除动脉变得太透明（而不是过于不透明）用于传输imaging.However的结果，这是有利的发射成像，因为这里所发射的信号的没有吸收/散射。与此相反，股动脉强烈autofluoresce在发射信道，具有以下的激发在405-445纳米（用410nm的激发峰为弹性蛋白14一致）的最大信号。从这些图像重构的二维切片显然从管腔和外膜和管腔区分介质。 在小鼠股动脉无线收获后28天（N = 6）或ligation-（N = 5）性损伤血管内膜增厚是无断层排放量预测明显（ 图3A）。在重建的2-三维地球有理切片，同心内膜病变可以从媒体通过弱发射（ 图3B和图S1）不同。 主动脉弓整装样本的动脉粥样硬化的小鼠OPT发射图像及其主要分支（N = 8），确定了与预期解剖分布病变（ 即 ，在主动脉弓，头臂动脉，起源小弯左侧颈动脉和左锁骨下动脉（ 图4A）。的截面图像表明，这些是典型的偏心病变和均容易从媒体和管腔分辨（ 图4B，图S2和S3）。 用于组织学分析以下OPT处理动脉证实OPT的非破坏性的性质，使用的组织学（美国三色，PICR成功染色切片osirius红色）和免疫组织化学（α-SMA，苹果-2）的技术（ 图3C和4C）。 使用OPT病灶大小的测量已经证明是使用从相同动脉11采取组织切片图像分析获得的测量结果是一致的。 病变区域的由OPT和组织学通过线性回归密切相关为无线（R 2 = 0.92），并结扎诱导的（R 2 = 0.89）新内膜病变和动脉粥样硬化斑块（R 2 = 0.85）得到的地上物的测量。 OPT的一个重要的好处是它能够实现3维分析。通过开发病灶体积定量使用该技术，我们能够在无线记录 ​​病灶体积（0.1100±0.0091毫米3; N = 6），并结扎损伤股动脉（0.0200±0.0089毫米3; N = 5），并且还在动脉粥样硬化头臂动脉（0。180±0.018毫米3; N = 8）。测量是高度可再现的（变异5.4％分别系数，11.4％和4.8％，中，n = 4）对所有类型病变。新内膜病变丝损伤器皿都较大（P &lt;0.0001）比由结扎产生，具有较大程度的破坏由前造成的一致。 所产生的数据也可以被表示为病灶剖面（ 图5）和呈现为动态的，定性评价（ 见图S1 - S3）。这种方法明显地证明了损伤形成的程度响应于不同损伤程序和突出的损伤形成在损伤血管分布不均。 <img alt="图1" src="/files/ftp_upload/50627/50627fig1.jpg" /> 图1:方法开始在小鼠股动脉病变的形成＆＃。160;导丝进入股动脉，通过在腘动脉动脉切口的装置的血管成形术（A） 的逆行插入刺激损伤形成响应于伸展损伤和除去内皮。血流重新建立在容器的受伤部分。在没有腔内伸展，剥蚀或中断血流（B）的新内膜增殖可通过连接两种股动脉或腘动脉立即远端股骨动脉分叉处诱导。 （C）的一个更严重的非剥脱损伤/增殖反应可通过在普通股动脉的分支点结扎两个股骨和腘动脉诱导。这种技术也将阻止在股动脉的远端部分的血流量。 <img alt="图2" src="/files/ftp_upload/50627/50627fig2.jpg" /> 图2:动脉粥样硬化的小鼠主动脉弓的特点沉积容易发生动脉粥样硬化（Apolipopotein E基因小鼠）喂食高胆固醇的西方饮食12周，病变发展沉积在主动脉弓及其主要分支的特征模式。这表现，病变是可见（箭头），由肉眼检查在解剖显微镜下，在主动脉弓，头臂动脉，并在左颈动脉和左锁骨下动脉的窦口。 <img alt="图3" src="/files/ftp_upload/50627/50627fig3.jpg" /> 图3:病变形成以下左侧股动脉结扎 （A） 的非断层荧光发射的图像（反转以增加清晰度-暗区对应于更强的发射）允许鉴定内膜增厚（红色arrowhe广告）。 （B）的鲜明的血管区域和内腔可以在断层重建被区分。 （三）组织学分析（美国三色）强调清晰的相似之处使用OPT获得的图像。比例尺在交流（AC）200毫米。从柯比朗斯等人在（AC）11比例尺适于是200微米。 <img alt="图4" src="/files/ftp_upload/50627/50627fig4.jpg" /> 图4:动脉粥样硬化的动脉粥样硬化易感小鼠的主动脉弓成像（A）的粥样硬化（红色箭头）是在非断层图像显而易见（反转，使较暗区域表示强排放，从而提高了清晰度）主动脉弓，在网站预测为动脉粥样硬化轴承通过在光学显微镜下检查（见图2）。 （ 二）分布的这种模式被证实在断层CROSS-部分。 （ 三）组织学（美国三色）染色展示了使用几种不同的抗体强调OPT的互补性与传统方法损伤分析断层段密切的相似性，和免疫组化。比例尺的（A-B）为1毫米; （C）中比例尺为250微米。 RSA，右锁骨下动脉; RCA，右颈总动脉; LCA，左侧颈总动脉; LSA，左锁骨下动脉; BCA，头臂动脉; AAO，升主动脉;道，降主动脉。从柯比等改编。11 <img alt="图5" src="/files/ftp_upload/50627/50627fig5.jpg" /> 图5:病变和管腔轮廓分析表明不同的内膜增生程度响应于动脉损伤的方法不同，光学投影层析允许病变和管腔横断面measuremen。TS被作图沿股动脉的距离。这清楚地表明，与未受伤的动脉（A），部分结扎（B）的比较产生小的，相对独立的病变，而总结扎（C）的产生完全闭塞在结扎的网站，但病变不沿动脉远延伸。腔内丝损伤（D）中产生的损伤，几乎完全封闭了样品的远端部分和沿着动脉的扫描部分的整个长度延伸。从柯比等改编。11 图S1。动画重建以下结扎损伤来自小鼠股动脉得到截面图像的，这种类型的动画形象的是用于定性和定量分析非常有用。作为动画从近侧移动到动脉的远侧部分的闭塞新内膜的逐渐发展，光盘从管腔和媒体ernible，是显而易见的。侧分支可以容易地识别并且有明显的管腔闭塞和动脉作为病灶的大小增加的向外重塑。一旦结扎的部位达到血管完全闭塞的发生。从柯比等改编。11 。图S2动画重建从动脉粥样硬化倾向小鼠的主动脉弓的横截面图像的动画开始于横截面的上升（左-这似乎第一）和降序（右）主动脉。小病灶出现在升主动脉作为扫描移动时在主动脉弓的方向。这些图像然后将穿过拱门，显示头臂（左），左侧颈（中）和左锁骨下（右）动脉损伤严重奥斯蒂亚。由于扫描移动远端沿着这些分支病变逐渐减少和消失，先在SUBCLavian的台词动脉，然后在颈动脉，最后在头臂动脉。有趣的是，病变在头臂动脉移动到分流作为该容器分为右颈动脉和右锁骨下动脉。从柯比等改编。11 图S3。动画，从动脉粥样硬化倾向小鼠的主动脉弓的体绘制图像。光学投影层析允许产生的三维图像，在这种情况下，显示出在一个载脂蛋白E缺陷小鼠的主动脉弓病灶分布。 （A） 的粥样硬化是存在于预期位点（整个头臂动脉，在左侧颈动脉和锁骨下动脉的窦口和在主动脉弓的小弯）。 （B）的分割与病变（显示为红色）的渲染轴承横截面强调斑块的分布在原始图像上叠加时。从柯比等改编。11

Watch this Scientific Journal Video about Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography at JoVE.com

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

generation-3-dimensional-quantitation-arterial-lesions-mice-using

Research

JoVE Journal

Medicine

9.7K 次观看.  The Queen's Medical Research Institute. Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

视频: Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

多普勒光学相干断层扫描视网膜循环

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

科研

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

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 &amp;#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 ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Ablation of Ischemic Ventricular Tachycardia Using a Multipolar Catheter and 3-dimensional Mapping System for High-density Electro-anatomical Reconstruction

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars represent zones of slow conduction that allow the occurrence and maintenance of reentrant circuits. Catheter ablation enables substrate modification of those low voltage areas and thus can help to alter the scar tissue in such a way that arrhythmias cannot appear anymore. Hospitalizations of concerned patients decrease, quality of life and outcome rise. Consequently, VT ablation represents a growing field in electrophysiology, especially for patients with endocardial scars in ischemic heart disease after myocardial infarction. However, ablation of ventricular tachycardia remains one of the most challenging procedures in the electrophysiology lab. Precise scar definition and localization of abnormal potentials are critical for ablation success. The following manuscript describes the use of a multipolar mapping catheter and 3-dimensional (3D) mapping system to create a high density electro-anatomical map of the left ventricle including a precise scar representation as well as mapping of fractionated and late potentials in order to allow a highly accurate substrate modification.

With the following protocol, we provide an approach to Ventricular Tachycardia (VT) ablation using high density mapping with a multipolar catheter and 3D mapping system enhancing the success of the procedure.

采用多极导管三维测图系统进行缺血性室性心动过速的高密度电解剖重建

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

使用电子地图的光映射数据的高通量分析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...