GHK由NIH / NHLBI K08-HL098565和研究所在芝加哥大学心血管研究的支持。在芝加哥大学实验动物护理和使用委员会批准的所有描述的实验方法。

1。准备成像的小鼠<ol><li>在此之前的影像学研究，麻醉诱导室大坝（2-3％异氟醚）。将动物的感应室，并立即将口鼻部连接的麻醉系统内的鼻锥。电推剪，删除皮草从中期胸部下肢（参见图1）。取出剩余的体毛，脱毛霜。脱毛霜也可用于无电推剪，应彻底洗掉后皮肤使用，以防止刺激。 </li><li>将麻醉小鼠在仰卧位嵌入式心电图上的一个加热垫，导致以保持身体的温度（ 图1）。应用电极凝胶的四只爪子和磁带的ECG电极。 </li><li>获得一个稳定的状态在整个过程中镇静水平（1.0％至1.5％的异氟醚混合100％O 2）。麻醉的水平可以调整，以米aintain目标心跳率每分钟450±50次（BPM）。应特别注意支付，以减少剂量的镇静异氟醚和持续时间不到一小时。 </li><li>通过加热垫，轻轻插入直肠探头（润滑）后，体温监测。重要的是要在一个狭窄的范围内（保持体温37.0°C±0.5°C）。控制麻醉和恒定的体温对母亲和胎儿的血流动力学稳定是至关重要的。 </li></ol>技术注意事项收购胎儿超声心动图是具有挑战性的。从这些研究中获得的数据可以混淆由于水坝和胎儿的应激反应。理想的情况下，动物的温度应保持使用加热成像平台，循环变暖垫，加热灯，或自我调控加热毯。此外，建议常规使用温热声学凝胶。铝虽然主要的关切体温控制，以避免低温，热疗发展应该是平等的关注。不受监控的加热装置，如一个简单的加热垫或卤素照明接近的存在可以导致在快速和危险的体温升高。由于大量的体温波动在两个方向上的动物处于危险之中，应竭尽全力，以维持正常的体温。 获取的图像的超声医师需要避免将与换能器上的模腔的压力过大，因为单独的换能器的重量可能会导致在心脏功能改变。图像采集的持续时间也必须保持到最低限度（理想情况下小于1小时），以减少长时间镇静造成的生理和血流动力学变化。此外，长度的时间和暴露在异氟醚为每个研究必须保持到minimu的米由于潜在的致畸作用的异氟醚12。 2。鉴定胚胎<ol><li>开始成像使用母亲的膀胱为地标，与胎儿标记为L1，2,3， 等 （左侧）和R 1，2,3， 等 （右侧）上的左，右的子宫角（参见图1C）。胎儿位置的表示法是有用的成像后的标本进行检索。 </li><li>标本位于太深腹部扫描，记录他们的存在，但是从数据分析中排除，因为分辨率低。在的能力扫描相邻胚胎的可能帮助跟踪胎儿（ 图2A）。 </li><li>修改扫描平面通过改变鼠标取向的相对于扫描面。画像中得到的2个正交的平面（ 图2）为每个胎儿。努力是获得近似横向的，正面的，或矢状面但Sometimes仅限于斜面的位置在腹部的子宫。旋转扫描头也将允许修改的方向移动大坝。 </li></ol>技术注意事项虽然手持操作的探头是可行的，在成年小鼠的超声心动图在胎儿成像，手持式操作不建议。鉴定胎儿是复杂的变量的性质子宫位置，迂曲和运动的。为了尽量减少与胎儿定位困难，大坝的水平面以最小的运动超出固定换能器的使用（ 图1）是必不可少的。 3。结构和功能的评价<ol><li>扫描B型图像被用来确定心房，室间隔，心室腔室，和左，右的流出道（ 图2），如基本心脏结构。 </li><li> M模式图像s的获得从短轴视图，并且被用于测量心室壁的厚度和腔室的尺寸（ 图3）。如果不能得到正确对准由于胎儿的谎言，从B超图像，测量可用于量化％短轴缩短率（FS）。用于缩短分数（FS）的计算左心室收缩末期尺寸（LVESD）和左室舒张末期内径（LVEDD）在整个心动周期之间的时间变化，如下所示: ％FS = [（LVEDD - LVESD）/ LVEDD]×100 </li><li>左，右心室，识别通过扫描从头部到尾部。左，右两侧应注明。可见流所产生的回声胎儿的血液有利于二尖瓣口多普勒取样容积内的准确位置。二尖瓣心尖四腔和左室长轴意见（ 图4，C）是从左心室流入速度。主动脉流出的测量可以被用于测量systo酒精射血时间（ 图4，D）。心脏速率可以计算从一个流周期的测量周期（ 图4中，C和D）以下流。应小心对齐血流量和多普勒波束多普勒角最小化。超出60度的角度拍摄的值是不准确的，应加以避免。 </li><li> B型扫描图像来识别常见的先天性畸形，如室间隔缺损（ 图5）结构。可以使用，以确定流过室间隔多普勒取样容积内的心室。很容易被监控的其他参数包括胎儿大小，心脏率，流速，心包积液，胎儿水肿。尸检和组织病理学确诊特定的心脏缺陷，需要额外的评估。 </li></ol>技术注意事项对左和右湛鉴定址是很难在胎儿心脏成像，由于在开发过程中的类似尺寸的心室腔。策略之一就是建立实时胎儿左，右方向移动成像平台在水平面上。吻，肢芽，和脊柱的鉴定将有助于确定让我们/右方向的胎儿。如果可能的话，跟踪拱或可视化的主要分歧肺动脉流出道，将允许识别的左心室流出道或右流出道分别。对于每一个胎儿的研究，重要的是要注意所决定的左，右方向上保存的图像。 成像后动物的监测和护理大坝成像完成后，返回到适当的住房和监控按照标准机构程序协议。镇痛跟随此成像过程不是必需的。可以预见在五分钟之内完全恢复正常活动。 4。胎儿超声心动图的代表性成果商业的发展允许高频探头（高于8 MHz），超声心动图的设备，有一个轴向分辨率大约为0.2毫米与0.3毫米的横向分辨率，当图像被放大并在深度为1厘米获得。大多数最近开发的换能器是线性的，具有的优点是避免了近场工件。高频率（30-50兆赫）机械探针已被最近开发为小鼠的胸部和心脏速率是足够的，允许在5-12毫米的深度为约50μm的轴向分辨率。最近，这些高频率的机械探针增加了彩色多普勒能力，使一个完整的评估心室和瓣膜功能和识别的S狩猎病变在胎儿心脏。此处所述的方法进行上VisualSonics的VEVO 770系统，并可以应用到几乎所有的等效系统。目前市售超高频超声系统可以工作在40赫兹，最大成像深度为7〜14毫米至60毫米的横向和50毫米至100毫米的轴向的决议（Vevo770，VisualSonics公司）。与此相比，60 Hz和20毫米的成像深度，具有50至100毫米的轴向和200至500毫米的横向分辨率的临床美国Acuson的红杉超声系统。 由于小尺寸的胎鼠心脏，胎儿超声心动图在小鼠的研究在技术上具有挑战性。与超声心动图在成年小鼠中，必须使用非常规的超声医师超声成像胎儿的体轴定义的飞机。迂曲子宫也影响的取向的胎儿和必须予以考虑。此外，固有的局限性在渗透深度ULTR一个高频率的超声波可以把它难以大量的胎儿图像所有标本在怀孕，。 超声成像策略可以高通量筛选先天性心血管疾病和心外缺陷7。除了遗传改变的研究，这种技术可以用于屏幕在药理学和毒理学研究中的缺陷。此方法也可以用来作为一个指导工具介入的操作过程，如心室压力13的注射剂或测量。 的胎儿超声检查的无创性是有利的，不仅是因为它允许在生理条件下心血管功能进行评估，但也因为这提供了关键的表型的实时信息。纵向检查胚胎的心，虽然技术上是可行的，有以下几个原因仍然充满挑战。串行检查胎儿和IDE一个明显的结构缺陷的情况下ntification在每场考试的胎儿是具有挑战性的。子宫和胎儿的运动可能完全改变方向的标本，从而使纵向跟踪和后续测量困难14。 虽然超声心动图是一个强大的技术鉴定心脏异常，具体诊断的结构性心脏缺陷，需要进一步详细的尸检和组织病理学分型的15。相关的基因型和特定的胎儿需要收获的胎儿后，最好立即回声研究，而大坝是由子宫切开术，麻醉，以尽量减少变化方向和胚胎的位置。 已报道的小鼠胚胎和用户的这种技术室尺寸和功能的正常值应检讨引用的参考文献，这些值6-10。评定心脏瓣膜形态是有限的，但图像分辨率的环形尺寸测量和速度测量，通过大血管是可行的，即使早在ED 9.5。必须小心，以获得足够的血流量和换能器10，16对准。 应该强调的是，心脏尺寸变化根据对小鼠品系，性别和年龄，并迅速改变在胚胎不同时间点和心脏率。重要的是要验证这些参数的组的小鼠进行匹配。胎儿成像，以及不同的小鼠品系。例如，怀孕的CD-1水坝例行C57/BL6应变，因此可能是更难于形象化所有标本相比，包含更多的胚胎。出于这些原因，使用年龄和应变匹配的对照组，每个实验而应使用的参考值。此外，测量的各个参数如左室舒张末期内径和后壁的厚度可能会有所不同在正常小鼠中，高达25％8。 <img alt="图1" src="/files/ftp_upload/4416/4416fig1.jpg" /> 图1。概述设置使用VisualSonics的VEVO 770系统。（A）VisualSonics综合铁路系统与生理监测单位。（B）鼠标的位置，适当的克制加热板上。四肢录音到ECG电极（C）孕鼠的胚胎和布局的示意图。在每个子宫角的胚胎数目可以变化显着除了胎儿的取向。（D）的成像平台上用箭头指出的操纵的平面（X-轴和Y-轴）移动定位坝大坝成像。 Z-轴是指换能器的运动的向上和向下的（如面板B中的箭头表示）。 B，膀胱，L，左，R，RI的高度。 <img alt="图2" src="/files/ftp_upload/4416/4416fig2.jpg" /> 图2。代表B超图像，这个数字代表的胚胎14.5天的胎儿的B超图像。（A）的两个相邻的胎儿的可视化。方框表示胎儿心脏的位置。（B）在胎儿解剖标志来引导方向。一个四腔视图（C），短轴（D）左，右心室，右心室流出道及肺动脉（PA）（E），和左心室流出机智的心脏在胚胎14.5天（LVOT）和主动脉（F）。 <img alt="图3" src="/files/ftp_upload/4416/4416fig3.jpg" /> 图3。这个数字包含代表评估，评估心室功能。图像的二维超声心动图的胚胎14.5天的心脏长轴视图（A），和一个四腔的图（B）（C）的 M模式线表示左，右心室的内部直径在舒张的跟踪（R / LVIDd比值）和收缩（R / LVIDs）从四腔图像平面。室间隔（IVS）是可视化。 <img alt="图4" src="/files/ftp_upload/4416/4416fig4.jpg" /> 图4。代表多普勒评估，这个数字代表图像的二维超声心动图14.5天的胚胎心脏的心尖四腔心切面（A）。左心房和左心室腔进行了概述。（C）（B）代表位置的脉冲多普勒记录二尖瓣流入量为样本。二尖瓣血流多普勒的模式舒张早期velocit的Y（表示"E"）和心房收缩（"A"表示），可以测量速度。（D）代表动脉多普勒波形。主动脉多普勒射流可用于测量喷射时间（ET）。心脏心率（HR），可以计算出一个流动周期下面的流程周期的测量。 <a href="http://www.jove.com/files/ftp_upload/4416/4416fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/4416/4416fig5.jpg" /> 图5。代表检测室间隔缺损，这个数字代表的B超图像的胚胎14.5天心在心尖四腔心切面（A），（B）的左，右心室腔概述。请注意存在的室间隔。（C）的横截面成像心脏与苏木精和曙红染色。（D）B-模式的胚胎14.5天心脏的心室的萼片缺损（VSD）按箭头所示的图像。（E）左，右心室腔概述叠加放置的脉冲波的多普勒取样容积流过的记录室间隔。（F）的横截面成像的心苏木精和曙红染色后，标本的检索。（G）叠加的流量记录的脉冲波的多普勒取样容积放置在整个室间隔。（H）代表多普勒跟踪（G）展示流向由左到右心室。 <a href="http://www.jove.com/files/ftp_upload/4416/4416fig5large.jpg" target="_blank">点击此处查看大图</a> 。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+FOG-2+expression+in+cardiomyocytes+by+microRNA-130a.">Translational control of FOG-2 expression in cardiomyocytes by microRNA-130a.</a> PLoS One. 4, e6161 (2009).</li><li>Momoi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modest+maternal+caffeine+exposure+affects+developing+embryonic+cardiovascular+function+and+growth.">Modest maternal caffeine exposure affects developing embryonic cardiovascular function and growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H2248 (2008).</li><li>Tobita, K., Liu, X., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+modalities+to+assess+structural+birth+defects+in+mutant+mouse+models.">Imaging modalities to assess structural birth defects in mutant mouse models.</a> Birth Defects Res. C Embryo Today. 90, 176 (2010).</li></ol>

没有利益冲突的声明。

进行串行测量和检测突变的胎儿心脏缺陷的能力，突出的实用性超声心动图研究正常和不正常的心血管疾病的发展。 体内的心脏结构和功能的分析已成为胎儿的正常发育遗传和非遗传修饰的描述中不可或缺的一部分。 2D制导多普勒的可用性使得它可以监测心脏率和血流模式的同时，获得实时图像。发展可能存在心脏畸形，如室间隔缺损和检测。尽管当前的成像平台的高分辨率能力，收购高...

<table border="1"><tbody><tr><td> VEVO 770成像系统 </td><td> VisualSonics </td><td></td><td> （加拿大，多伦多） </td></tr><tr><td> RMV707B.15-45 MHz传感器</td><td></td><td></td><td></td></tr><tr><td> Tec的异氟醚蒸发器</td><td></td><td></td><td></td></tr><tr><td>异氟醚（2 - 氯-2 - （二氟甲氧基）-1,1,1 - 三氟乙烷） </td><td></td><td></td><td></td></tr></tbody></table>

murine fetal echocardiography

转基因小鼠显示心脏的发育和功能的异常，正常心血管功能和人体心血管疾病的病理生理基础的分子机制的理解一个强大的工具。胎儿和围产期死亡是一个共同的特点，在研究基因的改变影响心脏发育1-3。为了研究遗传或药物的变化，在发展的初期心功能的作用，早期识别异常和纵向后续的活胎儿的超声成像已经成为一个重要的工具。无创性的超声显像是一种理想的方法，用于检测和研究先天畸形和心功能的影响之前，死亡4。它允许在客厅胎儿的异常和疾病的进展可以跟在子宫内的纵向研究5,6的早期识别。直到最近，成像胎鼠心脏经常涉及侵入性的方法。胎儿必须进行磁共振显微镜和电子显微镜或透射镜手术交付的牺牲。高频探头与传统2-D和脉冲多普勒成像中的应用已被证明在胚胎发育过程中提供测量心脏收缩和心脏率与数据库的正常发育变化，现已6-10。 M模式成像进一步提供重要的功能的数据，虽然，往往很难获得适当的成像平面。高频超声成像对胎儿已提高了2-D分辨率，心脏结构11的早期发展，并能提供优秀的信息。

Watch this Scientific Journal Video about Murine Fetal Echocardiography at JoVE.com

Murine Fetal Echocardiography

学习时，胎儿和围产期死亡的共同特点是影响心脏发育的基因的改变。高频超声成像已经提高了2-D分辨率，可以提供优秀的信息对早期心脏发育是一个理想的方法来检测心脏结构和功能的影响，在死亡前。

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 ...

Research

JoVE Journal

Medicine

17.1K 次观看.  University of Chicago. 学习时，胎儿和围产期死亡的共同特点是影响心脏发育的基因的改变。高频超声成像已经提高了2-D分辨率，可以提供优秀的信息对早期心脏发育是一个理想的方法来检测心脏结构和功能的影响，在死亡前。

视频: Murine Fetal Echocardiography

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

经胸超声心动图在小鼠

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

科研

医学

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

高频超声心动图：非侵入性的反复测量小鼠心脏缺血再灌注心肌劳损，收缩力，二尖瓣关闭不全的第一个证据

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

小鼠超声心动图和超声成像

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

左心房，主动脉和肺动脉的超声心动图小鼠

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

axolotl 中的二维和三维超声心动图

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Acquiring and maintaining the necessary surgical skills require consistent practice, a critical caseload, and time. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. Here we describe realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. We describe the skills that are especially important in conducting placental laser coagulation that the surgeon can practice on the model and apply in a clinical case. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. Such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome.

模型外科训练: 用逼真模拟器 Fetoscopic 激光光凝单绒毛膜 Diamniotic 双胎胎盘的技能习得

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

经食管超声心动图在心脏手术中无创地测定涡旋形成时间

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...