我们非常感谢蓉赵、洁丽和严营的帮助。

这些研究是按照由机构动物保育和使用委员会 (IACUC) 批准的国家卫生和动物议定书研究所出版的《爱护和使用实验室动物指南》进行的。贝勒医学院根据协议编号 AN-4203. 
 1. 用于超声的孕鼠的制备 <ol> <li> 定时交配 <ol> 在1700年后的一夜之间将大坝与一只被证明是可育的雄性老鼠放在一起。分开的水坝和男性由0700小时 (h), 不管阴道堵塞是否存在, 以确保准确的怀孕日期. 
		注意: 交配后的早晨被认为是0.5 天后 coitum (d.p.c.). </li>
	</ol> </li> <li> 为超声的准备 <ol> 将怀孕的鼠标放置在一个密封的麻醉感应容器中. <li>
		<li> 用异氟醚镇静剂 (2%) 吸入麻醉剂和氧气 (1 和 #160; 升/分钟) 直到动物失去了矫正反射 (大约1-2 分钟取决于老鼠的大小) 和自发运动没有被观察 (除呼吸作用以外). </li>
		<li> 用异氟醚 (2%) 吸入麻醉剂和氧气 (1 和 #160;将眼部药膏涂抹在动物和 #39 的眼睛上. </li>
		<li> 用棉签将脱毛膏涂抹到整个腹部、胸骨至阴道, 并侧向侧面. </li>
		<li> 允许奶油保持最多3分钟, 然后用纸巾除去奶油和头发。确保所有头发都被移除, 因为任何剩余的毛发都会降低图像质量. </li>
	</ol> </li> </ol> 2。超声阶段的制备 <ol> <li> 打开传感器. </li>
	<li> 将3维马达工作台连接到传感器. 
	<ol> <li> 通过将3D 马达电缆连接到后面板上的3D 马达接头, 将其插入3D 马达。将3D 马达系统连接到安装系统上, 使用顶部的快速释放开机自检装置, 并在底部安装快速释放装置以固定传感器夹具. </li>
	</ol> </li> <li> 将探头连接到传感器夹具. </li> </ol>
 3. 开始成像怀孕的老鼠 <ol> <li> 将鼠标仰卧在监视平台上。鼠标持续接受异氟醚麻醉 (在 1.5-2.5%) 和氧气 (1 和 #160; 升/分钟) 通过鼻锥。将所有爪子轻轻地贴在平台上的心率监控垫上. 
	<ol> <li> 在腹部应用1-2 毫升超声波传输凝胶. </li>
	</ol> </li> <li> 使用手动马达工作台, 将超声波探头放置在下腹部。
	<ol> <li> 找到膀胱, 它应该显示为一个充满液体的黑色圆圈, 刚好头到阴道开口. </li>
		<li> 一旦膀胱位于, 移动探针非常缓慢头, 以可视化怀孕的子宫, 这应该出现一个圆柱形的圆形区域在怀孕地点。这可以被描述为在链上看起来像珍珠. </li>
		<li> 一旦确定了怀孕的子宫, 就开始2维成像. </li>
	</ol> </li> </ol> 4。2维超声成像 (图 1) <ol> <li> 一旦确定怀孕的子宫, 就从离膀胱最近的妊娠部位开始, 慢慢地并依次移动头, 以确定怀孕地点的数量和位置. </li>
	<li> 如果肾脏、脾脏或肝脏是可视化的, 则当用户走得太远时, 将探针重新定位到靠近头的尾部 (靠近膀胱). </li>
	<li> 以相同的方式图像对侧子宫角. </li>
	<li> 当超声帧位于植入/妊娠部位的中心时, 将图像冻结, 以备日后分析和测量时保存. 
	注意: 将图像冻结并保存以供以后分析时, 需要不到一秒钟的时间. </li>
	<li> 使用回声蜕反应作为标记来测量植入距离, 方法是手动单击和 #34; 测量和 #34; 工具首先, 然后单击一个植入站点的位置。接下来, 将光标拖动到下一个植入站点, 然后单击以绘制一条计算机将自动报告其距离的直线。然后, 软件将报告手动测量的内容。此步骤不是计算机化的, 但依赖于用户通过在 decidualized 站点之间绘制一条线来标记 decidualized 站点之间的距离, 该程序将提供一个度量值. </li>
	<li> 测量植入大小、胎囊大小和胎极大小. </li>
</ol> 5。胎儿心率 注意: 在 9.5 d.p.c., 胎儿心脏跳动应该清楚地形象化. 
<ol> <li> 同时保持探头静止, 打开脉冲波多普勒并将其定位在可见的心跳上. </li>
	<li> 记录心脏跳动的搏动. 
	注意: 除了心脏外, 这个过程还可以记录脐带的脉动. </li>
</ol> 6. 3 维超声采集 <ol> <li> 怀孕子宫在2维成像中被形象化后, 将探针定位在所需的3维图像的近似中点的区域中。例如, 如果一个人在3维中成像一个苹果, 探针最初应该放在苹果核被预测的位置 (即对象中间). </li>
	<li> 如果探头位于所需图像的中间位置, 则获得3维捕获。3维电机阶段将沿着这段距离横跨子宫的一系列步骤或框架, 目的是要完全捕获调查者的愿望. </li>
	<li> 确认在完成 real-time 超声部分之前, 预期的结构在3维扫描中完全被捕获。保存此3维信息以便以后进行后期处理。2维和3维超声采集的总时间大约10-20 分钟, 由有经验的用户执行. </li>
</ol> 7. 3 维重建后处理 (图 2) <ol> <li> 为3维图像处理加载所需的数据. </li>
	<li> 选择和 #34;p arallel 和旋转方法和 #34, 将所有3D 图像帧加载到一个3维和 #34; 框和 #34; 用户随后将在框架中跟踪所需的对象。选择步骤大小0.08 毫升。从图像块的一端开始, 然后滚动, 以熟悉在空间内捕获的图像. 
	<ol> <li> 从图像的一端开始, 并手动跟踪对象的轮廓. </li>
		<li> 滚动到下一个2维图像切片或 #34; 框架和 #34; 并手动跟踪对象的轮廓. </li>
		<li> 继续此过程, 直到用户手动对所有帧进行了批注/跟踪. </li>
		<li> 单击和 #34; 完成和 #34; 获取3维图像和总体积计算. </li>
	</ol> </li> <li> 在网格和实心叠加之间选择, 它是左上角的箭头. </li>
	<li> 选择保留或删除周围的超声波图像信息以使其清晰。每个3维重建可能需要10到 20 min. </li>
</ol> 8。术后护理 <ol> <li> 当超声波完成后, 关闭麻醉气体, 从平台上取出鼠标, 然后轻轻擦拭并从动物身上清洗任何超声波凝胶. </li>
	<li> 将鼠标放回位于填充区域中俯卧位置的笼子中。监测, 直到动物是清醒和自发移动. </li>
</ol>

<ol>
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作者没有什么可透露的。

这项新的使用3维高频成像显示的能力, 成功地检测, 可视化, 并表征胚胎植入点在早期小鼠怀孕的非侵入性的方式。该技术提供了明显的改善比目前的方法, 这依赖于中断妊娠的总组织和病理特征。然而, 应该指出的是, 组织学方法将仍然被认为是更优化, 当表征在一个更放大和更多的细胞水平是需要的, 或当基因和蛋白质分析是必需的。随着越来越多的新的鼠标模型显示植入缺陷和早期怀孕的损失19,20,21,22, 这种先进的超声技术的能力来检测早期妊娠从5.5 迪普 c 起提供明显的优势比早期的超声方法, 仅限于检测在中期12,13。此外, 该方法在心脏开始跳动时立即检测心脏运动, 介于 8.5-9.5 迪普 c23,24允许确认存活胎儿。在同一动物中跟踪早孕事件的能力意味着每项实验需要更少的动物, 更准确的分析可以用这样的纵向实验设计来完成25,26, 27。
应当指出的是, 议定书中最关键的三步骤是: (1) 知道确切的孕日;(2) 操作者在正确的解剖部位定位子宫和定位探头的能力;和 (3) 精确的技术, 以产生3维重建。以下的问题拍摄指南证明是有帮助的: 当它是很难找到子宫的超声, 用户可以开始, 以确保膀胱首先确定。膀胱通常被视为一个黑色的圆圈, 并立即头到阴道开口。即使是在超声检查之前已经排尿的小鼠, 膀胱也从来没有完全空过, 因此 (在我们的经验中) 可以是一个可靠的标记。一旦膀胱被确定, 探针应该慢慢地移动头直到子宫被看见。如果膀胱是小的或无法形象化, 用户也可以尝试想象子宫由开始在卵巢, 这可以发现在侧翼, 不如肾脏。采用这种系统化的方法, 可以观察到每个植入部位和胎儿发育情况, 如图 1所示。有时, 由于子宫在腹部的定位方式, 在超声检查中可能很难获得子宫的所需区域, 如图 2所示, 子宫在体内的不同形状和位置。用户可以倾斜的鼠标平台平面转移或调整子宫内部到一个更有利的位置, 其中所需的子宫区域可以被超声波捕获。最后, 用户可以确认, 所需的子宫图像是通过快速滚动超声图像后不久, 超声波会议完成后获得的超声影像。一个小的限制是, 3 维网状/固体覆盖的视觉重建不能执行的超声会议期间。为了确保所获得的超声图像是准确的, 我们建议, 随着研究人员的熟练程度, 这些动物会在超声后进行安乐死, 以便将超声结果的准确性与大体组织标本相关联。测量和解剖学。
当我们在这里描述正常的小鼠妊娠模型, 这项技术的未来应用将用于量化异常早孕事件。高频和3维重建将允许调查人员检测和监测在生长和/或发育异常的怀孕的数量、大小和地点。例如, 非侵入性表型特征的异常-即有缺陷的子宫内膜蜕, 异常胚胎着床分布, 以及滋养细胞入侵和生长不足-显示的增加利用这种成像方法, 现在可以纵向分析转基因小鼠的数量, 而不会中断妊娠。

复发性早孕损失是一个最常见的并发症后受孕和影响约1% 的夫妇试图受孕1,2。早期妊娠损失的基本机制多种多样: 从内在的胚胎异常和母体并存到子宫内膜容受性的缺陷1,3,4。由于其遗传可, 小鼠模型已被广泛应用于早期胚胎着床和妊娠的研究。此外, 鼠标的短孕龄和执行 large-scale 研究的能力, 确保了在解决人类生殖医学的关键临床问题5中, 老鼠的日益增加的效用。尽管如此, 绝大多数的小鼠实验设计仍然要求在妊娠天数内对大量的大坝进行安乐死, 以量化和分析怀孕期间的植入地点、数量、大小和间隔模式6, 7,8, 从而排除对同一动物的纵向研究。
在临床中, 超声是一个可靠的和无创的工具, 以监测人的胎儿生存能力和发展的非侵入性的方式9,10,11。最近, 高频超声 (高频) 已经开始发现有限的应用在鼠标作为一种方法, 监测胎儿的生存能力和生长在怀孕期间12,13,14。最近的技术进步在超声成像允许应用三维 (3) 数据为视觉重建动物器官和随后监视病症15,16, 17。使用这种先进的成像技术显著提高了检测小体积波动的能力, 减少 inter-animal 的变异性, 并监测病理的进展或治疗干预的效果17。虽然这项技术的主要效用是监测恶性进展的 oncomouse 模型15,16, 3 维高频成像最近才被用于定量和监测胚胎着床的主动增长和胎儿发育在小鼠子宫18。
在这里, 我们演示如何执行高频成像, 以产生2维和3维数据, 以生成早期怀孕的小鼠子宫的重建。我们证明了这种新方法的效用, 以检测这些早期胚胎植入事件不需要终止妊娠, 使研究人员收集数据的非侵入性的方式。

<table>
	<tbody>
		<tr>
			<td>VisualSonics Vevo 2100 Ultrasound Imaging Platform/Machine</td>
			<td>VisualSonics, inc.</td>
			<td>VS-11945</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Imaging Station</td>
			<td>VisualSonics, inc.</td>
			<td>SA-11982</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquasonic 100 Ultrasound Transmission Gel</td>
			<td>Parker</td>
			<td>#SKU PLI 01-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (IsoThesia) 100mL bottle</td>
			<td>Henry Shein</td>
			<td>#29404</td>
			<td></td>
		</tr>
		<tr>
			<td>PuraLubenAnimal Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>#12920060</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse

高频超声 (高频) 是性监测胎儿子宫内real-time 发育的常用方法。小鼠通常被用作一个体内模型来研究胚胎着床和妊娠进展。不幸的是, 这种小鼠的研究需要怀孕中断, 以使随访表型分析。为了解决这个问题, 我们使用了三维 (3-D) 重建高频成像数据, 早期发现和表征小鼠胚胎着床地点和各自的发展进程在子宫内。结合高频成像与3维重建和建模, 我们能够准确地量化胚胎植入点的数量, 并监测发育进展的怀孕 C57BL6J/129S 小鼠从5.5 天后交媾 (d.p.c.) 到 9.5 d.p.c。使用变频器。测量包括: 数量, 位置和体积的植入点, 以及 inter-implantation 的场地间距;通过心脏活动监测评估胚胎的生存能力。在直接 post-implantation 期间 (5.5 到 8.5 d.p.c.), 妊娠子宫的3维重建在网格和坚实叠加格式使发展的怀孕的视觉表示法在每个子宫垫铁之内成为了。由于基因工程小鼠继续被用来表征从子宫功能障碍中获得的女性生殖表型, 这种方法提供了一种新的方法来检测、量化和表征早期植入事件在体内。这项新的使用3维高频成像显示的能力, 成功地检测, 可视化, 并表征胚胎植入点在早期小鼠怀孕的非侵入性的方式。该技术提供了明显的改善比目前的方法, 这依赖于中断妊娠的总组织和病理特征。在这里, 我们使用视频和文本格式来描述如何成功地执行超声波早期小鼠怀孕, 以产生可靠和重现性的数据与重建的子宫形式的网状和固体3维图像。

如图 1所示, 高频超声可以从5.5 迪普 c 的时间点开始检测植入站点的开发。使用打火机回声 decidualized 子宫内膜作为一个标记的植入点在6.5 迪普 c 允许数量的植入地点和间距这些网站的量化。随着怀孕的进展到 7.5 d.p.c., 一个较暗的回声妊娠囊和胎极也容易辨认。
如图 2所示, 完成后的小鼠子宫三维重建的后处理组合能够被可视化。在6.5 迪普 c, 3 维高频超声组成的网状和固体覆盖格式可以用来提供一个视觉描绘。另一个例子, 这一次在7.5 迪普 c, 演示了最后的复合图像的另一子宫的网状和固体覆盖。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56207/56207fig1.jpg"> 
图 1: 高频超声检测植入部位发育和监测整个孕期胎儿生长情况.如图 1A所示, 在 5.5 d.p.c. 上, 打火机回声 decidualized 子宫内膜 (d) 允许数量的植入点和间距的量化。如图 1B所示, 在另一个子宫角的蜕点 (D) 由子宫角 (U) 连接。由 7.5 d.p.c., 较暗的回声囊 (GS) 和胎儿极 (F) 易于识别图 1C。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56207/56207fig1large.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/56207/56207fig2.jpg"> 
图 2: 早期妊娠时小鼠子宫的三维重建.在6.5 迪普 c, 如图 2A所示, 显示了网格和实体叠加格式的3维高频超声。在7.5 迪普 c, 如图 2B所示, 显示了带有网格和实心叠加格式的3维高频超声图像。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56207/56207fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

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

三维高频超声对小鼠早期妊娠特征的新应用

老鼠被广泛用于研究妊娠生物学。但是, 这类研究需要终止妊娠, 因为这一问题排除了纵向调查, 必须使用大量动物。因此, 我们描述了一种非侵入性技术的高频超声早期检测和监测的 post-implantation 事件在怀孕的鼠标。

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 ...

a-novel-use-three-dimensional-high-frequency-ultrasonography-for

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  Baylor College of Medicine. 老鼠被广泛用于研究妊娠生物学。但是, 这类研究需要终止妊娠, 因为这一问题排除了纵向调查, 必须使用大量动物。因此, 我们描述了一种非侵入性技术的高频超声早期检测和监测的 post-implantation 事件在怀孕的鼠标。

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

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

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

牙龈三维炎症人体组织当量

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

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 ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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 ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...