This work was supported by the Terry Fox Program of the National Cancer Institute of Canada.

注:在本研究中进行的实验程序批准的动物护理委员会森尼布鲁克研究所（多伦多，安大略省，加拿大）。为人道对待动物的程序必须在任何时候都必须遵守。假定研究者被训练在超声成像系统的基本操作。该协议最适合两个人。 1.动物模型<ol><li>队友CD-1雄性和雌性小家鼠获得野生型胚胎灌注研究。 </li><li>分子影像学研究，采用129 /奥拉起源的胚胎干细胞生成工程+/-小鼠同源重组所描述的Bourdeau 等。14。 <ol><li>回交B6- 工程 +/-小鼠，请从米歇尔Letarte博士收购，到CD-1的背景。使用主机 +/- CD-1回交胚胎，以及它们的主机 + / +同窝共ntrols。交配的小鼠产生上演胚胎。 </li></ol></li></ol> 2.实验准备<ol><li>启动小鼠交配和日常检查插头。胚胎天0.5被定义为阴道栓被观察到的一天的中午。 </li><li>通过加入50ml胎牛血清，加入5ml的1M HEPES和5ml青霉素 - 链霉素的（10,000单位青霉素，万微克链霉素）至500制备胚胎媒体毫升Dulbecco改性Eagle培养基（DMEM）中以高浓度葡萄糖（4500毫克/ L）。包装瓶在铝箔，并保持冷藏在4℃。 </li><li>在140 XG 20分钟在50毫升锥形管离心清晰超声凝胶。绘制凝胶成30毫升注射器。补附加（标）30毫升注射器用磷酸缓冲盐水（PBS），备用。胚胎一窝将超声凝胶2-3注射器和4注射器PBS之间的一般要求。 </li><li>拉约30玻璃针，轻轻地拧紧，以橡皮泥条在100×20mm的细胞培养皿。这确保了针被分离，并且在处理过程中的安全。使用玻璃牵拉，使用1×90毫米的玻璃毛细管长丝以下设置:加热器77，副磁铁55，主磁体70。 </li><li>准备十一时五十夹层板（足以保证一个窝内的每个胚胎将接收其自己的盘），使用1:固化剂，以碱的8体积比。倾40毫升碱到50ml锥形管中。添加5-6毫升的固化剂，搅拌用木棒。 <ol><li>倒入60×15毫米培养皿中，填充每个到大约一半。让我们站在O / N设定。 </li></ol></li><li>附加雌性luers至400毫米长片聚氯乙烯（PVC）管（内径:1.3毫米）。管必须从注射泵到超声阶段轻松到达。 </li></ol> 3.实验装置<ol><li>安排手术显微镜下成像平台与三维运动和线性阵列21 MHž传感器。 <ol><li>东方的平台，使轨道安装在左边，与直接定位在显微镜下的阶段。 </li><li>附加3D马达到平台的延伸臂的球窝接头。螺钉传感器夹具的快速释放交成快速释放安装在马达上，面向前方，并在夹紧位置的换能器头，紧固锁闩。 </li><li>固定在该活动端口的换能器连接器的车的前面板上。打开机器。 </li></ol></li><li>启动21 MHz的超声换能器，选择&#39;心脏病&#39;设置分子成像研究，和&#39;一般&#39;的灌注研究。将所有时间增益补偿滑块的确切中间位置。对于"对比度模式"，设置以下参数:频率:18兆赫，发射功率:4％（0.39兆帕），对比度增益:30 dB时，佛慈:6和10毫米，对比度模式:非线性，突发时间:100％在0.1秒，波束宽度:宽。 </li><li>分装40毫升胚胎媒体每进四五十毫升锥形管。放置在冰上。 </li><li>加热1升水中的热板上的2升烧杯中。保持在45℃。添加超声凝胶和PBS中的一个注射器向烧杯预热。保持该温度由温度计在所有时间监视。 </li><li>预留十置十二1毫升注射器和十一点五十21g的针注射微泡。 </li></ol> 4.手术过程注意:有一个助手准备气泡（第5阶段），而外科医生开始手术过程。这里所描述的协议已被改编自怀特利等人 15 <ol><li>收集怀孕母鼠下颈椎脱位E16.5或E17.5胚胎。 注:麻醉对胚胎的影响尚未详细研究16。额外的方法，包括断头，可适当运系统蒸发散的孕鼠的牺牲。 <ol><li>剖开腹部，露出子宫和胚胎。仔细并迅速通过切割在子宫角的尖端（切断卵巢癌和子宫血管），并切断在阴道和膀胱切除子宫。 </li><li>使用分裂镊子，子宫转移到40毫升的冰冷胚胎尽快介质的管，而不会损坏胚胎。抛弃鼠标胴体。 </li></ol></li><li>搬迁到显微镜和舞台。存子宫成100×20mm的细胞培养皿。转移子宫到一个新的培养皿填充用50ml新鲜胚胎介质。 </li><li>使用消毒（70％乙醇喷雾）细镊子，轻轻地撕和扯远了子宫外膜开始一端。揭露胎盘蜕膜，顶叶卵黄囊和赖克特的膜分离和底层卵黄囊删除。 </li><li>解剖出胚胎逐个，保持内脏ÿ白斑囊完好和处理胎盘尽量轻柔17。注意避免卵黄囊破裂的胚胎立即弹出。 <ol><li>使用穿孔勺子将胚胎到另一个盘保持在冰上，用新鲜胚胎媒体。保持冷冻，直到需要的胚胎。改变媒体在胚胎菜每1-1.5小时。这些条件的胚胎是可行的解剖后最多4小时。 </li></ol></li></ol> 5.微泡准备注:使用有针对性和无针对性的进行微分子成像超声。单个实验可能需要多达3个独立的微泡的小瓶:ⅰ）抗体靶向微泡，ⅱ）对照同种型抗体靶向微泡和iii）非靶向微泡。造影剂来作为干冷冻粉末，并且必须在注射前盐水重新配制。有〜2×10 9个微气泡在每个非靶向为MicroBubble小瓶和〜8.8×10 8的微泡中的目标准备小瓶中。微泡是稳定的重构后达3小时。 <ol><li>目标准备小瓶的重构 注:目标准备准备的例子:内皮糖蛋白的有针对性的微泡（MB E，生物素与大鼠MJ 7/18抗体小鼠内皮糖;在内部杂交瘤细胞设施）;血管内皮生长因子受体2（VEGFR2）靶向微泡（MB 为V，生物素的抗小鼠CD309）;大鼠同种型的IgG 2对照抗体靶向微泡（MB C，生物素大鼠同种型的IgG 2的控制目标鼠IgG2a）。 <ol><li>稀盐水的抗体溶液（20微克）在1ml（最终体积）。保持在冰上。 </li><li>填充注射器1毫升生理盐水，清除所有气泡。附加21g的针，慢慢注入溶液注入微泡小瓶。 </li><li>慢慢的撤出柱塞，消除1毫升的空气，并撤回针。平缓搅动。静置5分钟冰上。 注:超声微泡可在高压环境下被破坏。 </li><li>后的经过时间，填充新的注射器与抗体稀释液，并加入到合适的小瓶中。轻轻地搅动。让该混合物（现在是2毫升）中静置10分钟。保持在冰上。假设整个表面缀合，结合的配体的微泡的平均数目是约18 7600配体/μm2以下。 </li></ol></li><li>无目标样品瓶重建<ol><li>填充注射器1毫升生理盐水，清除所有气泡。附加21g的针，慢慢注入溶液注入微泡小瓶。 </li><li>慢慢的撤出柱塞，消除1毫升的空气，并撤回针。轻轻地搅动。静置5分钟。增加一个额外的1毫升盐水，如上所述。轻轻搅拌，静置冰上10分钟。 </li></ol></li><li>库尔特计数<ol><li>轻轻搅动所需的微泡瓶和借鉴〜加入50μl用21g的针的溶液到1ml注射器中。注入微泡到空2ml微量管中。 </li><li>吸取的微泡原液的10微升样品到10ml稀释剂。 </li><li>轻轻混合后，通过使用犁刀计数（见材料清单）评估微泡群体的浓度和粒度分布。算使用30微米孔径至少三个50μl的测量（每小瓶样品）。 </li></ol></li><li>注射微泡的准备 注:"注射微泡成胚胎"时，助理执行此步骤（步骤6）开始。每个胚胎注入一次，以随机的助手，使得所有类型的微泡的均匀分布在整个过程，但在一个随机的顺序给定所选微泡的选择用于每次注射的类型。确保外科医生盲目泡沫的类型注入。 <ol><li>确定日Ë体积的需要，以产生1×10 8兆字节/ ml的终浓度在400微升的体积（使用5.3.3测定的库存浓度计算体积）股泡沫溶液。分装盐的相应数量到一个空的离心管。 </li><li>轻轻搅动所选微泡瓶，并使用21g的针吸取溶液的过量体积（〜小于上面计算更大50微升）到1毫升注射器。注入微泡到一个空的微量离心管中。 </li><li>吸取的微泡原液的必要体积，并加入到盐水的等分试样。通过用吸移管的尖端轻轻搅拌混合。 </li><li>绘制使用21g的针稀释微泡溶液到干净的注射器。卸下针头，消除来自注射器的任何气泡，并附加了路厄和油管。慢慢地解决了管制作的最后推确保不产生任何气泡。 </li><li>将syrinGE到注射器注入泵设置为分配所述微泡在为20微升的总体积的比率为20微升/分钟。附加一个拉玻璃针管的末端。移动泵靠近所述注射阶段。 </li></ol></li></ol> 6.注射微泡成胚胎<ol><li>随机选择从冷冻介质盘删除（有穿孔的勺子）一个胚胎。发生在解剖盘位于超声舞台立体镜之下，删除卵黄囊和羊膜用细镊子，切割/从出现血管最少的侧面（在antimesometrial侧）撕裂。这是最容易被刺穿邻近于头部区域的面积来实现。 <ol><li>切，足以从内部取出胚胎，但没有更多。稳定利用小块橡皮泥的解剖菜。 </li></ol></li><li>轻轻地从周围的胚胎操纵囊。定位胚胎在其一侧，与胎盘和脐血管的前面。用昆虫针，拖住卵黄囊（推荐4针），并在必要时胎盘边缘的地方贴上。避免大血管。 </li><li>预热的45°C PBS洗胚胎直到胚胎复苏。识别脐静脉和其相关联的血管网。定位盘，使得注射可以舒适地进行。 注意:一旦温热，血液在胚胎将开始流动，明显地泵送在脐动脉。当流首先开始，脉将是一个明亮的红色，与血液中的两艘船很快出现相同的。从脐静脉所产生的分支通常覆盖那些从脐动脉在胎盘表面。 </li><li>一旦胚复苏，包括它的（但没有胎盘）与预热美国凝胶，一定要精心从周围的胚胎去除任何气泡（使用细镊子）。顶部与预热PBS的菜。 注:请关注soluti水平上在PBS中凝胶的注射器，并根据需要添加备用注射器向加热烧杯。 </li><li>安装在大球橡皮的玻璃针，在解剖盘的边缘和插入针的一端插入PBS中。选择在胎盘盘的绒毛膜表面的静脉，从主分支尽可能合理，用剪刀修剪笔尖的大小。注射是最简单的，如果尖端被切割以微小的角度。取下玻璃使用弹簧剪刀边缘的任何锯齿状边缘。 </li><li>使用注射泵，缓慢地在20μL/分钟注射的微小气泡溶液放入玻璃针，直到所有的空气从针尖排出，微泡可以看出，以自由地流到PBS中。停泵并重置为20微升的注射体积。不允许空气喷射期间进入胚胎的血管系统。 </li><li>将玻璃针尖轻轻插入静脉的一个胎盘，并确保它是不动的。旋开立方米的Oscope头和位置胚胎上述换能器。启动成像。 </li></ol> 7.超声分子成像注意:使用预先设定的对比度非线性成像条件下，定位传感器，以使胚胎被均匀地位于所述焦点之间在6和10毫米。一旦定位，开始推注的微泡。当注射完成时，启动定时器。 <ol><li>灌注成像<ol><li>确保选中按" 学习管理 "按钮，然后点击" 偏好设置 "选项卡，在研究浏览器顶部的非线性对比度模式帧的最大数量。检查在" 首选项 "窗口中的相应的框。 </li><li>通过启动非线性成像调节成像设置。按控制面板上的" 对比度模式 "按钮。通过调整" 增益 ＆＃细化2D增益8217;拨至30dB，用&#39; 威力 &#39;拨号降低功率〜4％，使用" 频率 "拨号并设置波束宽度（左下角）使用滚球/小鼠宽降低频率为1赫兹。 注:所有控制位于超声系统的控制面板上。 </li><li>通过记录20分钟电影回放以1赫兹的帧频捕获的微小气泡丸药的整个洗入和耗散。按下扫描/冻结，开始数据采集。 </li><li>一旦图像的全序列已被抓获，按" 电影商店 "按钮位于控制面板上的保存图像序列中的系统缓冲区。系统创建取得的非线性模型的对比数据的日期戳电影圈。 </li></ol></li><li>有针对性的微泡成像<ol><li>在" 偏好设置 "，设置突发时间0.1秒。设置使用控制DES适当的成像参数cribed上述（7.1.2。）。 </li><li>按控制面板上的" 对比度模式 "按钮，启动非线性造影。确保胚胎正确坐落在换能器和该微泡造影剂的通过胚胎的流动是在非线性对比度模式图像可见。 </li><li>为了评估靶向微泡在胚胎分子成像研究结合，使微泡循环静置3分钟，完成注射后40秒。这个时间是，允许保留的循环微泡。按"扫描/冻结"在此期间停止成像。 </li><li>在3分钟和40秒，恢复影像，以确认该胚胎仍然可见，被充分地覆盖，用PBS，而微泡继续流通。按"扫描/冻结"启动成像。 </li><li>通过记录一个"预destruc获得3分，50秒后注射破坏/补货序列在29赫兹化"声学响应序列。按"扫描/冻结"，开始数据采集。在4分钟后注射18,19，启动0.1秒的高频声爆裂。按"爆发"按钮来实现了一阵。 </li><li>继续记录帧的序列的其余部分（直到缓冲区行已满），按"电影店"拯救电影圈。 </li><li>收集微循环的附加电影圈。这以后的"后销毁"成像序列被认为包含有补充光束只循环泡沫的信号。按"扫描/冻结"启动成像和"电影店"收集图像。 </li><li>成像每个胚胎后标注电影循环。按"学习管理"，采用滚珠突出文件和"选择"按钮，适当的，按"图像标签"和名称。 </li></ol></li&gt; </ol> 8.处理胚胎注射后<ol><li>后成像，移动传感器，取消固定胚胎和安乐死（通过斩首，颈椎脱位，二氧化碳窒息，或麻醉后快速冷冻或固定），根据需要。 </li><li>对于每个后续的胚胎注入新鲜的解剖盘，针头，注射器和管段必须使用，用解剖和复兴在准备下一批微气泡注射液正在发生。 </li><li>保存组织样品（ 例如 ，尾，脑），用于附加的分析（ 例如 ，基因分型通过孤立尾DNA，lacZ启动染色，或生物标志物表达的使用蛋白质印迹21的半定量测量PCR分析测定）。 </li></ol>

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F.S. Foster acknowledges his role as consultant to VisualSonics Inc.

超声造影剂注入晚期妊娠小鼠胚胎和非线性对比度的图像被收购来衡量灌注参数和有针对性的微泡结合。内胚胎脉管系统的微泡的成功的成像依赖于许多因素，第一个是胚胎的生存能力。所有设备和装置，以减少所需的胚胎的分离从子宫到注射开始时预先准备的。由于单次或重复暴露于麻醉对胚胎的小鼠的影响还没有被详细研究，在母亲的使用麻醉被避免前牺牲16。期间胚胎隔离，这是重...

超声造影成像是利用微泡造影剂的可视化和表征血管环境。这些药物可使微循环，血管和心血管功能的无创性评估。此外，气泡的表面的改性可以导致靶向微泡结合到内皮的生物标志物，其表现在血管生成，动脉粥样硬化和炎症1,2-使得可能血管事件的分子超声成像临床前应用中。超声造影，因此可以用来识别影响健康和患病的血管状态3-5的复杂和多样的环境。 在多年以前数，微泡成像工具的兴趣已经扩展到通用的小鼠胚胎模型。哺乳动物的发展模式，引入微泡成胚胎血管增强生理显影循环系统（ 例如 ，血流量，心输出量），并在转基因箱子和心脏疾病6,7的靶向突变体小鼠模型，研究可能会产生见解因素如何遗传改变心血管功能。事实上，定量和定性的2D胚胎脑血管分析已经实现8。另外，小鼠胚胎呈现为用于检查靶向微泡的血管标记物的结合在体内的优良模型。 Bartelle 等 9，例如，引入了抗生物素微泡成胚胎心室评估目标锁定在Biotag，生物素化酶BirA转基因胚胎具有约束力，并检查血管解剖。的杂合和纯合小鼠模型的产生也可被用作替代物肿瘤模型研究，目的来定义分子超声波的定量性质 - 在把这种技术应用到临床的重要基准。 8-10暴露引入到胚胎循环。 在子宫内注射，但是，面临着许多挑战。这些包括喷射引导，有必要以对抗在母亲和形象化胚胎运动，维护血流动力学生存能力在母亲和形象化胚胎，寻址麻醉和并发症引起的出血11的长期影响。因此，调查的目标是开发用于注射微泡成隔离的生活晚期胚胎12的技术。这个选项提供了更多的自由，在喷射控制和定位，所述成像平面的再现无阻碍，并简化图像分析和定量方面。 在本研究中，我们概述了一种新的方法用于注射微泡到活鼠胚胎FOř研究微泡动力学行为和的目的​​，正在学靶向微泡结合的内源性内皮表面标记。非线性对比度特定超声成像是用于测量的一些基本灌注参数包括峰增强（PE），洗入速率和达峰时间（TTP）中分离的E17.5胚胎。我们还表明，胚胎模型用于评估的功能的转基因小鼠模型，其中内皮糖蛋白是一种临床上相关的目标胚胎内皮糖损失分子超声波的定量性质的有效性，由于其高表达在血管内皮细胞在血管生成活性的13位点。的内皮糖蛋白靶向（MB E），大鼠同种型的IgG 2控制（MB C）和不相关的（MB U）的微泡粘附在杂内皮糖蛋白（英文+/-）和纯合子内皮糖蛋白（英文+ / +）表达的胚胎进行了评价。目标宾迪分析吴透露，超声分子能够内皮糖蛋白的基因型之间的区别，并与受体密度，以量化的超分子水平。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt; (biotinylated, eBioscience) &amp;#8212; Antibody choice depends on the experiment</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;rat isotype IgG&lt;sub&gt;2&lt;/sub&gt; control&lt;/li&gt;&lt;/ul&gt;</td><td>eBioscience</td><td>13-4321-85</td><td>This antibody/microbubble combination is often required as experimental control&amp;nbsp;</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;biotin anti-mouse CD309&lt;/li&gt;&lt;/ul&gt;</td><td>eBioscience</td><td>13-5821-85</td><td></td></tr><tr><td>Biotinylated rat MJ 7/18 antibody to mouse endoglin</td><td>In house hybridoma</td><td></td><td>Outside antibodies may also be appropriate: we&amp;nbsp; have used eBioscience (13-1051-85 ) in the past</td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Embryo media&lt;/strong&gt;</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;500 ml Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium with high glucose&lt;/li&gt;&lt;/ul&gt;</td><td>Sigma</td><td>D5796</td><td></td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;50 ml Fetal Bovine Serum&lt;/li&gt;&lt;/ul&gt;</td><td>ATCC</td><td>30-2020</td><td>lot # 7592456</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;Hepes&lt;/li&gt;&lt;/ul&gt;</td><td>Gibco</td><td>15630</td><td>5 ml, 1 M</td></tr><tr><td>&lt;ul style=&quot;padding:0px;margin:0px&quot;&gt;&lt;li&gt;Penicillin-Streptomycin&lt;/li&gt;&lt;/ul&gt;</td><td>Gibco</td><td>15140-122</td><td>5 ml, 10,000 units Pen., 10,000 &amp;#956;g Strep</td></tr><tr><td>Ethanol, 70%</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>76240</td><td>4%</td></tr><tr><td>Phosphate Buffered Saline [1x]&amp;nbsp;</td><td>Sigma</td><td>D8537</td><td>1x, w/o calcium chloride &amp;amp; magnesium chloride</td></tr><tr><td>Pregnant mouse, CD-1</td><td>Charles River Laboratories Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>0.9% sodium chloride (saline)</td><td>Hospira</td><td>0409-7984-11</td><td></td></tr><tr><td>Ultrasound contrast agent, target ready and untargeted</td><td>MicroMarker; VisualSonics Inc.</td><td></td><td></td></tr><tr><td>Ultrasound gel (Aquasonic 100, colourless)</td><td>CSP Medical</td><td>133-1009</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Cell culture plates (4) :&amp;nbsp; 100&amp;nbsp;x&amp;nbsp;20&amp;nbsp;mm</td><td>Fisher Scientific</td><td>08-772-22</td><td></td></tr><tr><td>Cell culture plates (12) : 60&amp;nbsp;x&amp;nbsp;15&amp;nbsp;mm</td><td>Sigma</td><td>D8054</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall Legend RT centrifuge&amp;nbsp;</td><td></td><td></td></tr><tr><td>Conical tubes, 50 ml BD Falcon</td><td>VWR</td><td>21008-938</td><td></td></tr><tr><td>Diluent</td><td>Beckman Coulter</td><td>Isoton II Diluent, 8448011</td><td></td></tr><tr><td>Dissection scissors (Wagner)</td><td>Fine Science Tools</td><td>Wagner 14068-12</td><td></td></tr><tr><td>Forceps (2), Dumont SS (0.10 x 0.06 mm)</td><td>Fine Science Tools</td><td>11200-33</td><td></td></tr><tr><td>Forceps, splinter</td><td>VWR</td><td>25601-134</td><td></td></tr><tr><td>Glass beaker, 2 L (Griffin Beaker)</td><td>VWR</td><td>89000-216</td><td></td></tr><tr><td>Glass capillaries, 1 x 90&amp;nbsp;mm GD-1 with filament</td><td>Narishige</td><td>GD-1</td><td></td></tr><tr><td>Glass needle puller</td><td>Narishige</td><td>PN-30</td><td></td></tr><tr><td>Gloves</td><td>Ansell</td><td>4002</td><td></td></tr><tr><td>Gross anatomy probe</td><td>Fine Science Tools</td><td>10088-15</td><td></td></tr><tr><td>Hot plate</td><td>VWR</td><td>89090-994</td><td></td></tr><tr><td>Ice bucket</td><td>Cole Parmer</td><td>RK 06274-01</td><td></td></tr><tr><td>Imaging Platform</td><td>VisualSonics Inc.</td><td>Integrated Rail System</td><td></td></tr><tr><td>Light source, fiber-optic</td><td>Fisher Scientific</td><td>12-562-36</td><td>Ideally has adjustable arms</td></tr><tr><td>Luers (12), polypropylene barbed female &amp;frac14;-28 UNF thread</td><td>Cole Parmer</td><td>45500-30</td><td></td></tr><tr><td>Micro-ultrasound system, high-frequency</td><td>VisualSonics Inc.</td><td>Vevo2100</td><td></td></tr><tr><td>Needles, 21 gauge&amp;nbsp; (1&amp;rdquo;)</td><td>VWR</td><td>305165</td><td></td></tr><tr><td>Particle size analyzer</td><td>Beckman Coulter</td><td>Multisizer 3 Coulter Counter</td><td></td></tr><tr><td>Perforated spoon (Moria)</td><td>Fine Science Tools</td><td>MC 17 10373-17</td><td></td></tr><tr><td>Pins (6), black anodized minutien 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Pipettors [2-20 &amp;#956;l, 20-200 &amp;#956;l, 100-1,000 &amp;#956;l]</td><td>Eppendorf</td><td>Research Plus&amp;nbsp; adjustable 3120000038;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 3120000054;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 3120000062</td><td></td></tr><tr><td>Pipettor tips [2-200&amp;nbsp;&amp;#956;l, 50-1,000&amp;nbsp;&amp;#956;l]</td><td>Eppendorf</td><td>epT.I.P.S.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 22491334;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 022491351</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Tubing, Tygon laboratory 1/32&amp;rdquo; x 3/32&amp;rdquo;</td><td>VWR</td><td>63010-007</td><td></td></tr><tr><td>Wooden applicator stick (swab, cotton head)</td><td>VWR</td><td>CA89031-270</td><td></td></tr><tr><td>Surgical microscope 5-8X magnification</td><td>Fisher Scientific</td><td>Steromaster</td><td></td></tr><tr><td>Syringes, 1 ml Normject</td><td>Fisher</td><td>14-817-25</td><td></td></tr><tr><td>Syringes (10), 30 ml</td><td>VWR</td><td>CA64000-041</td><td></td></tr><tr><td>Syringe infusion pump&amp;nbsp;</td><td>Bio-lynx&amp;nbsp;</td><td>NE-1000</td><td></td></tr><tr><td>Thermometer, -20-110&amp;nbsp;&amp;#176;C</td><td>VWR</td><td>89095-598</td><td></td></tr><tr><td>Timer</td><td>VWR</td><td>33501-418</td><td></td></tr><tr><td>Tubes, Eppendorf</td><td>VWR</td><td>20170-577</td><td></td></tr><tr><td>Tube racks (3)</td><td>VWR</td><td>82024-462</td><td></td></tr><tr><td>Ultrasound transducer, 20 MHz</td><td>VisualSonics Inc.</td><td>MS250</td><td></td></tr><tr><td>Vannas-Tubingen, angled up</td><td>Fine Science Tools</td><td>15005-08</td><td></td></tr></tbody></table>

contrast imaging in mouse embryos using high-frequency ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

超声造影剂到子宫外小鼠胚胎的注射依赖于活，晚期妊娠阶段从子宫和维持生存力在注射和相关的超声成像的过程中胚胎的成功分离。一旦胚胎已形象化和定位， 如图1中 ，小心注射造影剂到胚胎脉管系统是可能的。一个E17.5小鼠胚胎的一个典型的B模式超声图像示于图2A中 。洗入微泡和相应的增强中，下腔静脉，心脏，大脑，并且在整个动物可以利用非线性对比度...

Watch this Scientific Journal Video about Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound at JoVE.com

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

在这里，我们提出了一个协议注入超声微泡造影剂进入活的，孤立的晚期妊娠阶段小鼠胚胎。此方法使灌注参数和使用对比增强高频超声成像的胚胎内血管分子标志物的研究。

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

contrast-imaging-in-mouse-embryos-using-high-frequency-ultrasound

Research

JoVE Journal

Developmental Biology

15.3K 次观看.  University of Toronto. 在这里，我们提出了一个协议注入超声微泡造影剂进入活的，孤立的晚期妊娠阶段小鼠胚胎。此方法使灌注参数和使用对比增强高频超声成像的胚胎内血管分子标志物的研究。 

视频: Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

科研

神经科学

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

小鼠胎儿超声心动图

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

医学

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

发育生物学

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

高频超声在胎儿和胎盘发育分析中的影响

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

癌症研究

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

鸡卵外胚胎和脉络膜尿囊血管的制备作为体内模型，用于对比增强超声成像和微泡介导的药物递送研究

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

生物工程

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://www.jove.com/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
High-frequency ultrasound systems are used to acquire high resolution images. Here, the use of a state-of-the-art system will be demonstrated to image the morphology and hemodynamics of small pulsatile arteries and veins found in mice and rats. Ultrasound is a relatively inexpensive, portable, and versatile method for the noninvasive assessment of vessels in humans as well as large and small animals. These are several key advantages that ultraound offers compared to other techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence tomography (NIRF). CT requires ionizing radiation and MRI can be prohibitively expensive and even impractical in some scenarios. NIRF, on the other hand, is limited by the penetration depth of light required to excite the fluorescent contrast agents.
Ultrasound has limitations in terms of imaging depth; however, this may be overcome by sacrificing resolution and using a lower frequency transducer. Abdominal gas and excess body weight can severely diminish image quality. In the first case, the propagation of sound waves is limited, while in the latter case, they are attenuated by overlying tissues, such as fat and connective tissue. As a result, no contrast or faint contrast may be observed. Finally, ultrasound is a highly user-dependent technique, requiring the sonographer to be familiar with anatomy and to be able to work around issues, such as the appearance of imaging artifacts or acoustic interference.

High-frequency Ultrasound Imaging and Image Acquisition

腹大塔的高频超声成像

Source: Amelia R. Adelsperger, Evan H. Phillips, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 
...

教育

JoVE Science Education

工程学

生物医学工程

Injection of Microbubbles into Isolated Mouse Embryos: A Technique to Deliver Microbubble Contrast Agents into the Vasculature of Living Murine Embryos

Source: Denbeigh, J. M. et al. <a href="https://www.jove.com/video/52520" target="_blank">Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound</a>. J. Vis. Exp. (2015)
This video demonstrates a procedure to inject microbubble contrast agents into an isolated late gestation stage mouse embryo. The microbubble contrast agent-based ultrasound imaging helps visualize and characterize the vascular environment of the embryo.

将微气泡注射到离体小鼠胚胎中:一种将微泡造影剂输送到活小鼠胚胎的脉管系统的技术

Source: Denbeigh, J. M. et al. Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound. J. Vis. Exp. (2015)
This video demonstrates a procedure ...