我们感谢动物生理学核心, 在加州大学洛杉矶分校分子医学分部提供技术支持和开放进入 Vevo 2100 超声生物显微镜观察 (显微镜) 系统。这项研究得到了 NIH/儿童健康研究中心 (5K12HD034610/K12)、加州大学洛杉矶分校-儿童发现研究所和今天和明天儿童基金会的支持, 以及大卫-医学院研究创新奖给 m. 董诚康。

加州大学洛杉矶分校动物保育和使用委员会批准了本议定书所示的所有程序。这项实验是在美国加利福尼亚州洛杉矶加州大学机构动物护理和使用委员会批准的活动动物协议下进行的一项持续研究的一部分进行的。动物处理和关心跟随指南的标准为实验室动物的关心和用途。
1. 高频超声成像系统的研制
<ol><li>打开超声波成像系统和生理学监测单元。</li>
	<li>连接 30/45 MHz 传感器。</li>
	<li>将相应的扫描头放置在成像平台附近的固定架上。</li>
	<li>选择心脏测量程序选项。</li>
	<li>将超声波凝胶倒置在其预热容器设置为37摄氏度。</li>
	<li>确认适当的麻醉管道系统, 并验证氧和异氟醚的水平。</li>
	<li>对成像平台和工作区域进行消毒。</li>
	<li>设置成像平台的热电平, 以保持大坝的恒定体温和心率。</li>
</ol>2. 孕鼠制剂
<ol><li>将怀孕的老鼠 (C57/BL6) 大坝放置在麻醉诱导室中。</li>
	<li>使用连续提供的吸入异氟醚 (异氟醚 2%-3%) 与100% 氧 (100% O2) 混合, 在感应腔内以200毫升/分钟的流速诱导麻醉。</li>
	<li>将镇静的动物转到仰卧位的成像平台上。</li>
	<li>使用连接到麻醉油管系统的面罩提供稳态镇静--将异氟醚 (1.0%-1.5%) 与 100% O2混合在200毫升/分钟。 注意: 使用装有炭过滤器的通风系统控制麻醉气体的泄漏。</li>
	<li>应用电极凝胶后, 将四肢轻轻地贴在嵌入式心电图电极上, 以实现对产妇心脏和呼吸速率的恒定监测。</li>
	<li>调整异氟醚的水平以维持平均心率 (450 +/-50 节拍/分钟 (bpm))。</li>
	<li>保持体温在37.0 摄氏度 +/-0.5 摄氏度范围内。监测生理控制单元中显示的体温和心率。</li>
	<li>在整个成像过程中每15分钟记录镇静鼠的生命体征。</li>
	<li>评估麻醉的水平, 评估鼠标的姿势, 心率, 和脚趾捏的反应。</li>
	<li>应用眼科药膏 (1 滴入每只眼睛), 以防止眼部干燥和角膜损伤。</li>
	<li>使用脱毛霜, 以减少超声衰减, 将皮毛从中胸部水平移到下肢。1-1. 5 分钟后, 通过交替湿和干纱布湿巾, 以防止损害皮肤。</li>
</ol>3. 胚胎识别
<ol><li>触及腹壁轻轻地找到胎儿并将其传播出去。</li>
	<li>使用标记对大坝腹部的每个胚进行注释, 并定义它们的前后和背侧方向。</li>
	<li>使用子宫颈的镇静剂水坝作为一个地标。将左侧和右侧子宫角上的胎儿标记为 L1、L2,L 3、等(左侧) 和 R1、R2、R3、等(右侧), 分别 (图 1A)。 注意: 避免强行传播胎儿。1-2 个胎儿在每个垃圾可能重叠与其他, 使他们的定位和成像不可靠。从分析中排除这些胎儿。</li>
</ol>4. 胎儿心脏的可视化和注释
<ol><li>将预预热的超声凝胶敷在腹部, 并仔细涂抹以避免气泡形成。在扫描成像区域添加额外的凝胶量。</li>
	<li>将超声波探头放在其机械支架上, 并在使用扫描 B 模式 (图 1) 的同时, 在寻找跳动的心脏时, 逐步向皮肤进行接触。</li>
	<li>单击扫描B 模式按钮以获取2维图像。使用膀胱作为标志, 以确定第一个胎儿定位在右侧或左子宫角, 并标记为 R1 或 L1, 分别。</li>
	<li>通过在水平平面上移动成像平台, 实时确认单个胎儿的左右方向。从头部到尾部进行扫描, 以注释鼻、四肢和脊柱为地标 (图 1B, 视频 1)。</li>
	<li>可视化跳动的心脏和注释左心室 (LV) 和右心室 (RV)。使用彩色多普勒模式优化心脏可视化 (图 1 C G, 视频 1-2)。</li>
	<li>单击扫描B 模式按钮以获得胸骨旁短轴视图, 使 LV 和 RV 显示在数据获取帧中心的最大直径。启动实时映像 (图 1B-c)。</li>
	<li>更改鼠标相对于扫描平面的方向, 以获得纵向四室视图 (图 1D)。首先, 确定心脏的其余结构, 如心房, 室隔膜, 左, 右流出大片。其次, 将心室和心房腔显示在其最大直径。然后开始图像采集。</li>
	<li>从最终分析中排除非最佳的斜图像。单击Cini按钮可获得连续录制的 "Cineloops", 最低为十年代, 然后保存录制的图像。</li>
</ol>5. 评估胎儿心率和心室功能
<ol><li>单击扫描M 模式按钮可从四腔平面 (视频 3) 获取心脏图像。</li>
	<li>在所有胚胎的图像完成后, 查看记录的列表以进行分析。</li>
	<li>从最终分析中排除非最佳的斜图像。</li>
	<li>单击分析按钮以测量舒张壁厚度和左/右心室内径 (LVID, d;RVID, d) 和收缩 (LVID, s;RVID), 如图 2所示。</li>
	<li>通过播放每个记录的 M 模式跟踪并计算一个流循环到以周期 ( 相邻峰值之间的间距 ) 来确定平均胎心率。</li>
	<li>执行多个测量 (每次跟踪至少5个) 以获得平均心率 (图 2)。</li>
	<li>在整个心脏循环中测量左心室内舒张直径 (LVID、d) 和左心室内径在终收缩 (LVID) 之间的时间变化。然后计算分数缩短百分比 (FS%), 如下所示: FS% = [(LVID, d-LVID, s)/LVID, d] x100。</li>
	<li>执行多个测量 (每次跟踪至少5个) 以获得平均 FS% 值。</li>
</ol>6. 评价心肺流动参数
<ol><li>在小于 60o的购置角度调整扇区。单击多普勒按钮, 通过使用45兆赫传感器, 从2维四室成像平面执行脉冲波多普勒测量。<ol>
<li>首先, 可视化肺动脉的分岔, 以确定右流出道。接下来, 单击脉冲波多普勒按钮以获得通过肺和主动脉瓣 (图 3A, 视频 4) 的流模式。</li>
	</ol>
</li>
	<li>从脉冲波多普勒追踪获得肺流量测量, 包括峰值收缩速度 (PkV)、加速度时间 (AT) 和弹射时间 (ET)。</li>
	<li>执行多个测量 (每次跟踪至少5个) 以获得平均值测量, 如图 3A (右) 所示。</li>
	<li>计算每个流出阀的正/ET 比, 作为流出道通畅和血流的指示器。</li>
	<li>采用脉冲波多普勒方法, 从2维顶端四腔的角度进行二尖瓣和主动脉血流模式的提取。首先, 确定左心房和左心室室。其次, 将脉冲波多普勒样本量用于记录二尖瓣流入多普勒模式, 测量早期舒张速度 (E) 和心房收缩速度 (A) (图 3B)24,25。</li>
	<li>调整多普勒样本量, 获得主动脉多普勒射流模式。使用主动脉多普勒射流跟踪测量加速度时间 (AT) 和弹射时间 (ET), 如图 3B (右) (视频 5)</li>
</ol>7. 评估胎儿-胎盘轴
<ol><li>使用 45 MHz 传感器 (图 4A), 利用彩色多普勒扫描可视化子宫动脉和胎儿胎盘血管树。</li>
	<li>当脐带从胎儿腹中流出后, 在脐带的羊膜腔内发现脐血管 (两条动脉和一条静脉)。</li>
	<li>将脉冲波多普勒样本量用于获得脐动脉流模式 (图 4A)。</li>
	<li>利用脉冲波多普勒扫描记录 (图 4B) 测量血管峰值流量参数, 包括加速度时间 (AT)、弹射时间 (ET) 和末端收缩 (PkV) 的峰值流速。</li>
	<li>在没有胎儿运动和产妇呼吸运动的情况下, 在每艘船上连续获得5个波形, 以测量每条血管的平均峰值速度。</li>
	<li>进入下一个胚胎。</li>
</ol>8. 成像后动物监测
<ol><li>在成像过程完成后关闭异氟醚容器。</li>
	<li>在恢复阶段继续监测体温、呼吸率和心率。</li>
	<li>一旦大坝开始自发运动, 面罩和连接的油管系统就会被拆除。</li>
	<li>将大坝返回适当的住房, 并根据标准的机构后程序协议继续进行观察。</li>
	<li>记录正常活动完全恢复的时间。</li>
</ol>9. 性能要求和技术考虑事项
<ol><li>将8胎儿的处理时间限制在大约1小时左右, 以避免长期麻醉对生命体征和生理参数的不良影响。</li>
	<li>对8-10 只怀孕小鼠进行完整训练, 优化图像采集和短时间内的流型跟踪技术。</li>
</ol>

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I., Lemke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+determination+of+the+optimized+atrioventricular+delay+in+patients+with+implanted+biventricular+pacing+devices.">Non-invasive determination of the optimized atrioventricular delay in patients with implanted biventricular pacing devices.</a> Indian Pacing Electrophysiol J. 10 (2), 73-85 (2010).</li><li>Kono, M., Kisanuki, A., Ueya, N., Kubota, K., Kuwahara, E., Takasaki, K., Yuasa, T., Mizukami, N., Miyata, M., Tei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+global+systolic+dysfunction+has+a+significant+role+in+the+development+of+diastolic+heart+failure+in+patients+with+systemic+hypertension.">Left ventricular global systolic dysfunction has a significant role in the development of diastolic heart failure in patients with systemic hypertension.</a> Hypertens Res. 33 (11), 1167-1173 (2010).</li></ol>

没有宣布利益冲突。

心血管畸形和疾病在很大程度上受遗传因素和环境因素的影响19。我们以前已经表明, 孕产妇的热量限制, 开始在第二个月, 对胎儿胎盘循环流和胎儿心脏功能9的重大影响。
产前缺氧是胎儿发育过程中的另一个常见的压力因子, 可能会极大地影响胎儿-胎盘生理和循环系统。在冠心病导致围产儿适应产后生活的情况下, 产前缺氧暴露的影响可能更为深远。本研究发现的心率异常和心脏指数确实是心脏应激和改变胎盘循环生理学的重要指标, 是检测发育缺陷的主要因素,由于产前缺氧应激导致早期心力衰竭, 血流动力学改变可能会进一步明显。与预期相反, 缺氧暴露的胎儿的心率较低。这一现象可能反映了胎儿小鼠在 GD18.5 缺氧时的未成熟心脏自动调节功能机制。然而, 确切的发病机制仍不得而知。
尽管其他先进的成像方法, 如胎儿心脏 MRI, 允许心脏结构的活成像在发展期间20, 血液动力学状态经常丢失由于静态图像和冗长的过程。另一方面, 无创超声技术允许执行维持基线生理学的体内动态成像。此外, 随着高频传感器的可用性提高分辨率, 在不同发育阶段的胎儿心脏的可视化, 可以变得更可行的转基因小鼠通过优化胎儿注释方法。最后, 使用这种方法, 每个实验的成本都要少得多。
在 Kim GH et的前一份报告中, 作者提供了有关使用前代高频超声成像系统21进行数据采集的成像计划优化的重要和新颖的见解。周 YQ et的另一份报告, 采用高频超声, 配备彩色多普勒系统22, 建立了在生理水平上胎儿循环的标准化基线测量。因此, 此处提出的协议补充了以前建立的协议, 并扩展了一个在实验环境中实时可行和切实可行的综合方法。本研究采用一种先进的高灵敏度高频超声系统, 以扫描胎儿-胎盘回路为单位。概述的协议是简单和规范化的使用这个强大的系统有效地通过实现量化测量缺氧对胎儿循环的影响在 GD18.5 的小鼠。
然而, 我们应该承认致命心脏成像的重要局限性和挑战: 首先, 包括异氟醚在内的麻醉药物可能会影响胎儿的生理参数。长时间的麻醉、脱发和超声凝胶会导致体温过低, 这会影响到大坝的心率和血流动力学指标以及胎儿。目前尚无有效的方法评价麻醉药的水平及其对胎儿的影响。为了规避这一限制, 我们滴定吸入异氟醚的水平, 以达到适当的镇静的水坝, 同时保持其基本心率和生命体征。第二, 在腹部深处可视化的胎儿是困难和不理想的, 导致这些胎儿被排除在最终的数据分析中。彩色多普勒可以提高成像剖面的优化和传感器与血流之间的适当对准。三是对所有胎儿进行同步分析, 需要快速、准确的可视化和图像采集操作者的效率, 这就意味着实践训练的重要性。
最后, 该方法的关键步骤需要强调, 包括 1) 适当的准备系统。2) 保持对怀孕小鼠体温和心率的稳定。3) 优化异氟醚的流速, 保持胚胎的基线生理状态, 获得可靠的数据。4) 在最短的时间内实现一致、高效的图像采集。5) 孕龄、性别和动物应变是重要的变量, 可能会显著影响结果。因此, 应该仔细设计实验协议, 在数据分析和解释中包括相同动物应变的匹配控制来解释这些变量。
总之, 高频超声系统是一种有效的方法来实现胎儿心血管系统的表型特征在子宫内具有重要的实验和科学价值和潜在的未来应用, 可能包括 1) 了解心脏发育过程中的生理动态。2) 对 CHDs 3 的遗传模型进行综合表型分析, 阐明胎儿胎盘循环对心脏室发育、成熟和适应应激的影响。4) 进行超声引导胎儿注射, 以研究毒素, 致畸因子, 或治疗剂的未来。6) 实施散斑追踪和应变分析能力, 获得心肌发育的详细区域心肌功能, 可为今后的研究提供依据。

先天性畸形的心脏是在早期心脏发育期间发生的异质结构缺陷。目前, 操作程序的技术进展使 CHDs1、2的婴儿存活率显著提高。然而, 生活质量经常被损害次要到长期住院和需要为分期的外科修理规程1,2,3,4,5。产前检测 CHDs 的基本分子机制是至关重要的, 以便计划早期干预, 实施新的预防战略, 并改善终生结局6,7。
虽然多重遗传和环境因素已牵连 CHDs 发病机制, 建立因果关系仍然是未满足的需要, 以改善诊断, 治疗和预防策略1,8,9 ,10,11,12。此外, 检查在子宫内应激因子和表观遗传修饰符的作用, 为将来的调查打开新的场所11,12。过去的十年确实见证了下一代测序技术的飞速发展, 包括单核苷酸多态基因芯片、全 exome 测序和全基因组甲基化研究, 它们在基因研究中的应用复杂的人类疾病的原因, 包括 CHDs1,8,9,10,11铺平了道路, 以确定新的突变和遗传变种尚未被在合适的动物模型中测试其致病性。
在不同的疾病模型系统中, 鼠标是选择的动物模型, 不仅用于 CHDs 早期 cardiogenesis 的调查机制13,14,15,16, 还要阐明其对产前和围产期应激因素对心室成熟和晚期妊娠功能的影响。因此, 在发育的早期和后期, 对突变胎鼠心脏的体内表型特征进行分析, 对于了解这些遗传变异和环境因素对心脏发育的作用至关重要, 并未来对小鼠室特异性成熟过程的潜在影响。
在开发过程中早期发现和准确诊断心脏缺损是干预计划的关键17,18。胎儿超声是安全、简单、便携和可重复的, 实际上已经成为临床心脏评估的标准影像技术。多普勒超声对胎儿循环的评价在临床实践中得到了广泛应用, 不仅用于检测心脏缺损, 而且能检测血管畸形、胎盘功能不全和宫内生长受限, 并评估胎儿的福祉, 以回应在子宫内侮辱包括低氧血症, 产妇疾病和药物毒性17,18。在评估人类缺陷和疾病的价值的同时, 超声对胎儿小鼠的评估在实验设置中得到了越来越大的效用19,20,21,22, 23。特别是, 胎儿心脏超声 (超声心动图) 允许连续在体内可视化的发展心脏。许多实验研究都采用超声成像技术观察转基因胎鼠的胎儿心血管发育。多普勒超声对于阐明病理生理学参数, 如在生理挑战或疾病条件下的胎儿循环中的流动模式特别有用10,19。在人类和动物中, 异常的血流或胎儿的氧气供应可能会因各种情况而导致胎儿环境在子宫内和影响 fetoplacental 轴, 包括胎盘异常, 产妇缺氧,妊娠期糖尿病和药学诱导的血管收缩15,22。因此, 建立标准化的方法来执行多普勒超声波对胎儿小鼠将极大地增强未来的研究 CHDs 通过促进监测流型和关键血流动力学指标的心血管回路在基因小鼠模型心脏发育的不同阶段.
高频超声已成为测量小鼠模型和人类疾病中心血管系统发育和生理参数的有力工具18。这项技术近年来得到了进一步的改进。我们和其他研究人员已经证明了该系统在胎儿小鼠心脏进行超高频超声研究的可行性15,19,20,21,22 ,23。该系统配备多普勒彩色流映射和线性阵列传感器, 产生二维, 动态图像在高频 (30 至50兆赫) 帧率。这些优势, 相比于低频超声系统和前一代高频超声21,22, 提供必要的灵敏度和分辨率, 以深入评估胎儿循环系统, 包括心脏结构的综合表征, 室功能, 以及胎儿小鼠在实验环境中的流动指标。在此, 我们概述了使用高频系统在胚胎日 E18.5体内进行体外循环和胎儿胎盘循环快速评估的方法。我们选择了一个30/45 兆赫传感器, 提供了大约60µm 的轴向分辨率和150µm 的横向分辨率。然而, 可以选择一种更高频率的传感器 (40/50 兆赫) 来分析早期的发育阶段, 方法是采用类似的方法。所选的 M 模式允许在高时间分辨率水平 (1000 帧/秒) 的运动中组织的可视化。最后, 我们证明了高超声对胎儿心血管血流动力学状态和功能的详细的综合表型描述的可行性在小鼠基线和应对产前缺氧应激。

<table border="0" cellpadding="0" cellspacing="0" width="1606">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:189px;">Vevo 2100&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:291px;">VisualSonics, Toronto, Ontario, Canada</td>
			<td style="width:301px;">N/A</td>
			<td style="width:827px;">High Freequency Ultrasound Biomicroscopy. The set up is available in animal physiology core facility, division of molecular medicine, UCLA. USA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">inbred mice (c57/BL6)&#160;&#160;</td>
			<td>Charles River Laboratories</td>
			<td>N/A</td>
			<td>Inbread wild type mouse strain</td>
		</tr>
	</tbody>
</table>

fetal mouse cardiovascular imaging using a high-frequency ultrasound (30/45mhz) system

先天性心脏缺损 (CHDs) 是儿童发病率和早期死亡的最常见病因。产前检测 CHDs 的潜在分子机制对于发明新的预防和治疗策略至关重要。突变小鼠模型是发现新机制和环境应激修饰剂的有力工具, 推动心脏发育及其在 CHDs 的潜在改变。然而, 建立这些假定的贡献者因果关系的努力仅限于非生存动物实验的组织学和分子研究, 其中监测关键的生理和血流动力学参数往往是缺席的。实时影像技术已成为建立 CHDs 病因的重要工具。特别是, 超声成像可以使用产前没有手术暴露胎儿, 允许保持他们的基线生理学, 同时监测环境压力对心脏室的血流动力学和结构方面的影响发展。在此, 我们使用高频超声 (30/45) 系统来检查胎儿在 E18.5子宫内在基线和应对产前缺氧暴露的小鼠心血管系统。实验表明, 该系统能测量心脏室大小、形态学、心室功能、胎心率和脐动脉血流指数, 并对其在子宫内暴露于系统性慢性缺氧的胎儿小鼠中的变化进行了分析, 并在实际中进行了研究。时间。

对心脏和血流动力学指标的统计分析离线进行。计算了3种最佳图像中连续5次测量的方法。这些数据被表示为 "平均" 电子扫描电镜. 学生的t测试用于推断团体比较。≤0.05 的P值被认为具有统计学意义。
根据上述协议, 我们的特点是长期暴露于产前缺氧对胎鼠晚期胎儿心血管状况的影响, 通过获得 C57/BL6 定时怀孕小鼠的实时高频超声记录妊娠日 (GD) 18.5。
育种组建立后, 成功的交配得到证实。定时怀孕水坝在12小时黑暗的政权下保持在笼子里与食物和水ad 随意直到。在 GD14.5, 怀孕的小鼠被分配到 normoxia 组 (在环境空气中维持) 或缺氧组 (放置在缺氧室在10% 不负担装卸2, 以诱发系统性缺氧)。出生后, 水坝和幼崽仍然被分配到他们的实验条件, 直到产后 7 (P7)。
在这些实验中, 总共研究了6座大坝, 并在 GD18.5 成功地拍摄了42只胎儿。其中, 从36个胎儿获得的数据用于后续分析 (表 1)。对 GD18.5 胎儿心率的分析表明, 缺氧胎儿的胎儿心动过缓 (心率降低), 胎儿心功能指数显著下降 (EF% 和 FS%)(表 1)。显著地, 缺氧暴露的胎儿的脐动脉 PkVs 的峰值流速 (PkVs) 减少 (图 4B和表 1)。此外, 脐动脉加速时间/射出时间 (ET) 比常氧胎儿显著降低缺氧值, 提示脐血管阻力增加。协议中, 在2维/M 模式图像上测量的缺氧胎儿右心室壁厚度增加 (图 5)。由于 rv 在胎儿发育过程中具有主导的泵功能, 而胎盘作为氧合的主要血管床, 这些数据统称为胎儿-胎盘血管回路中升高的流动阻力导致 RV肥厚。重要的是, 缺氧暴露的新生儿面临产后早期致死。RV 衰竭和增加的血管阻力引起的慢性暴露于产前缺氧是潜在的贡献原因。其他因素, 如再氧损伤造成的氧化还原毒性, 不良喂养和产妇疾病不能排除。然而, 在今后的研究中仍有待确定产前缺氧诱发心脏发病机制和胎儿早期致死的确切机理。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57210/57210fig1.jpg"> 图 1: 使用扫描 B 模式和彩色多普勒审讯的胎儿小鼠注释和心脏可视化在子宫内。() 胎儿小鼠识别和注释的示意图表示 (左, R: 右).(B) 在胎儿解剖地标的代表性形象, 以指导孕日18.5 胎心的方向从左心室 (LV), 右心室 (RV) 和室间隔 (IVS) 的胸骨旁短轴视图。(C) 对 LV 和 RV 的胸骨旁短轴视图的代表图像进行颜色审问, 以方便心室可视化。(D) LV 和 RV、左心房 (LA) 和右心房 (RA) 彩色多普勒的纵向四室视图。(E) LV 和 RV 的纵向四室视图, 用彩色多普勒审讯方便了流出通道的可视化: 右心室流出道 (RVOT)、左心室流出道 (LVOT)、主动脉 (AO) 和右心室流出道 (RVOT)。(F) 代表彩色多普勒 RVOT 和 PA 的审讯。(G) 代表彩色多普勒 LVOT 和 AO 的审讯。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/57210fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57210/57210fig2.jpg"> 图 2: 对胎儿心率和心室功能的评估.(A) 代表性的 M 模式跟踪, 从 GD 18.5 的长轴4室视图中获得。(LV: 左心室;右心室;左心房;RA: 右心房)。(B) 代表量化 (万箭穿心线) 方法的心室尺寸, 包括左和右心室内径在舒张 (LVID, d;RVID, d) 和收缩 (LVID, s;RVID, 左、右心室壁厚度舒张 (LVAW, d;RVW、d)、室间隔 (IVS) 和节拍对 HR 的测量均显示在四室成像平面上。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/57210fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57210/57210fig3.jpg"> 图 3: 脉冲波多普勒追踪胎儿肺、主动脉和二尖瓣流量指数.(A) 有代表性的肺动脉脉冲波多普勒追踪 (左) 图像。定量方法 (线) 肺流量指数 PkV (峰值速度), 在 (加速时间), ET (弹射时间) 显示 (右) 从纵向四室的看法。(B) 有代表性的二尖瓣和主动脉脉冲多普勒血流模式 (左) 和定量的二尖瓣流量指数 E (早期舒张速度) 和 A (心房收缩) 和主动脉流量指数在, ET 和 PkV (右) 显示从四室成像平面。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/57210fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57210/57210fig4v2.jpg"> 图 4: 评估胎儿-胎盘循环.(A)使用彩色多普勒审讯 (上部) 和产妇心电图记录 (下) 的胎儿胎盘血管回路的代表图像. (B)有代表性的脉冲波多普勒记录和量化测量 (线) 在缺氧 (上) 和 normoxia 控制暴露的胎儿小鼠 (较低) 的数字检测 (行). PkV (峰值速度), 在 (加速度时间), ET (弹射时间)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/57210fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57210/57210fig5.jpg"> 图 5: 低氧治疗胎鼠右心室壁厚度的评估.(A, B)在 GD 18.5 normoxia 和缺氧条件下从长轴四室视图获得的典型 m-模式跟踪。左心室, RV: 右心室, 右心室壁。线表示收缩 (s) 和舒张 (d) 中 RVW 厚度的定量测量。(C) RVW 的量化显示, 与 normoxia 相比, 缺氧处理的胎鼠 RVW 厚度增加。误差条: 平均值的标准误差。(D) 在 GD 18.5 中具有代表性的横断面图像, 描述了低氧治疗和 normoxia 治疗组中 RV 壁厚度的增加。原始放大倍数10X。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/57210fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>参数、单位</td>
			<td>Normoxia</td>
			<td>产前缺氧</td>
		</tr>
<tr>
<td>成功成像胎儿数</td>
			<td>20</td>
			<td>16</td>
		</tr>
<tr>
<td>产后死亡率</td>
			<td>5%</td>
			<td>68.75%</td>
		</tr>
<tr>
<td>血流动力学参数</td>
			<td>(指电子扫描电镜)</td>
			<td>(指电子扫描电镜)</td>
		</tr>
<tr>
<td>胎儿心率, bpm</td>
			<td>138 @ 4</td>
			<td>89 8 **</td>
		</tr>
<tr>
<td>左心室 EF,%</td>
			<td>71.2 @ 3</td>
			<td>55 ~ 2 **</td>
		</tr>
<tr>
<td>左心室 FS,%</td>
			<td>43 @ 2</td>
			<td>29 ~ 4 **</td>
		</tr>
<tr>
<td>肺动脉 PkV, 毫米/秒</td>
			<td>102 @ 10</td>
			<td>129 ~ 8 **</td>
		</tr>
<tr>
<td>肺动脉到 ET 比值</td>
			<td>0.42 @ 0.05</td>
			<td>0.35 @ 0.03 *</td>
		</tr>
<tr>
<td>脐动脉 PkV, 毫米/秒</td>
			<td>58 @ 4</td>
			<td>40 1.5 **</td>
		</tr>
<tr>
<td>脐动脉到 ET 比值</td>
			<td>0.5 @ 0.03</td>
			<td>0.42 @ 0.025 *</td>
		</tr>
<tr>
<td>脐静脉 PkV, 毫米/秒</td>
			<td>13 @ 1。2</td>
			<td>19.6 ~ 3 **</td>
		</tr>
<tr>
<td>脐动脉-静脉延迟, ms</td>
			<td>122 @ 4</td>
			<td>238 @ 20 *</td>
		</tr>
<tr>
<td colspan="3">EF, 弹射分数;FS, 分数缩短;NA, 不可用;NS, 不显著;PkV, 峰值速度;PkV, d, 舒张期峰值速度;收缩期 PkV、s、峰值速度;学生的 t 检验被用来推断团体的差异。P < 0.005。** < 0.01。* P < 0.05 代表了群体间比较的显著差异。学生的t 测试。不重要的是留空白。</td>
		</tr>
</tbody></table>表 1: 常氧和缺氧胎鼠在妊娠日的血流动力学参数 18.5 。EF, 弹射分数;FS, 分数缩短;PkV, 峰值速度;在, 加速度时间;等等, 弹射时间。学生的t测试用于推断团体差异。** P < 0.005。**p < 0.01 和 * P < 0.05 代表了组间比较的显著差异。
<img alt="Video 1" src="/files/ftp_upload/57210/57210vid1v2.jpg"> 视频 1: B 模式短轴视图.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/Video_1-B_mode-Short_axis_View.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Video 2" src="/files/ftp_upload/57210/57210vid2v2.jpg"> 视频 2: 彩色多普勒-顶端纵向视图.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/Video_2-Color_Doppler-Apical_Longtudinal_View.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Video 3" src="/files/ftp_upload/57210/57210vid3v2.jpg"> 视频 3: M 模式.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/Video_3-M-Mode.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Video 4" src="/files/ftp_upload/57210/57210vid4v2.jpg"> 视频 4: 肺动脉-脉冲波多普勒.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57210/Video_4-Pulmonary_Artery_Pulsed_wave_Doppler.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>

Watch this Scientific Journal Video about Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System at JoVE.com

使用高频超声 30/45MHZ 系统进行胎儿小鼠心血管成像 |发育生物学|视频

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

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

高频超声成像的胎儿小鼠有改进的影像分辨率, 可以提供精确的非侵入性的心脏发育和结构缺陷的特点。此处概述的协议旨在执行实时的胎儿小鼠超声心动图在体内。

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

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

Human Biology

Circulatory and Pulmonary Systems

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11.5K 次观看.  University of California, Los Angeles. 高频超声成像的胎儿小鼠有改进的影像分辨率, 可以提供精确的非侵入性的心脏发育和结构缺陷的特点。此处概述的协议旨在执行实时的胎儿小鼠超声心动图在体内。

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

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.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

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 ...

科研

发育生物学

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

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 ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...