Name: use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring
Uploaded: 2015-06-23T14:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

We thank Drs. Xiaofang Wang and Katharina Hopp for their invaluable help with the animal models. This work has been supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK090728, DK058816).

开始用活的动物的任何步骤之前，实验方案应该由机构动物护理和使用委员会（IACUC）的批准。 1.扫描仪配置<ol><li>在开始之前，确保加热器处于OFF位置。 </li><li>选择迷你成像梯度和38毫米射频线圈和迷你成像架。 </li><li>在保持器的中心孔安装在变温装配。 </li></ol> 2.动物的制备<ol><li>对于MRI实验中，使用异氟醚蒸发达到最佳的麻醉。麻醉诱导，将动物感应室衬有吸水的组织。调整异氟烷蒸发器的流量计以2.0-2.5升/分钟，并且异氟烷至3％的氧。 </li><li>删除在这个阶段任何金属标签或其它金属物体。适用于动物的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li><li>一旦动物已达到麻醉的手术平面（ 即撤退反射到脚趾捏的损失），将动物与它的鼻子插入鼻锥的持有者。设置麻醉气流在探头2.0-2.5毫升/分钟，在异氟醚浓度为1.5-2.0％的氧。麻醉将通过鼻锥在手术过程中被递送。定期调整异氟醚的浓度取决于动物的年龄和体重，保持40 BPM的〜一呼吸速率。 </li><li>使用动物持有者，以确保动物到位并防止在MRI实验期间运动。变化要扫描视体区域动物保持器的类型。 注意:从实验室塑料（聚丙烯，聚四氟乙烯，聚苯乙烯，聚碳酸酯）定制持有者可以容纳特定的实验并以适合动物大小（从新生小鼠到160克大鼠）。 </li><li>将直肠温度计在动物以监测动物的体温。在EXPeriment，保持动物在35-37℃下，用暖空气流。调节空气的温度（30-38℃）和基于动物的体温反馈流量（1,200-2,000升/小时）。 </li><li>附加气球呼吸压力传感器动物的腹部来监测呼吸速率。 </li><li>固定动物在RF线圈的中心和小心地将射频线圈与动物进入MRI扫描器。 </li></ol> 3. MRI实验<ol><li>调整和射频线圈开始实验，以尽量减少RF功率使用，并最大限度信噪比之前匹配。要启动匹配/调谐: <ol><li>通过点击工具图标，打开光谱仪控制工具。 </li><li>在光谱仪控制工具单击采集→摆盘。一个ACQ / RECO窗口将打开显示摆动曲线。 </li><li>或者调整调谐和匹配电容器（使用调谐和匹配杆）的小步骤，直到反射射频功率被最小化。的目标是要看到以最小的曲线在位于零，在水平轴上，垂直轴。 </li><li>当线圈的校准已经成功地实现了，打了ACQ / RECO窗口中的停止按钮。 </li></ol></li><li>收购侦察图像在三个正交平面创建轴位，冠状面及矢状面上。使用快速的图像序列，例如门内部快速低角度拍摄（IG-FLASH）来获取图像侦察兵12。使用图像侦察设置适当的几何体实际的成像。 </li><li>根据特定的研究目的，选择合适的图像序列和参数，并开始与一个红绿灯扫描。这将校准RF信道，垫片的共振磁体，设置载波频率为水和调整接收机增益，全部自动。 <ol><li>解剖研究和T2加权像，收购二维多片或3D模式。为了缩短实验时间为一个给定的空间分辨率，保持田间-of-视角（FOV）尽可能小，但足够大以避免回绕工件（2.56-3.2厘米）。 </li></ol></li><li>保持选择的序列比动物的呼吸周期稍短的周期通过适当选择的重复时间（TR）和/或切片的数量。这保证了数据在动物的平静期被收集。 <ol><li>例如，对于腹部图像，保持动物的呼吸频率在30〜BPM;那就是每次呼吸约2000毫秒。使用涡轮增压快速采集弛豫增强（RARE）序列，并获得11-19冠状切片，用TR / TE九分之一千五百毫秒，稀有因子8（矩阵256×256，FOV 2.56 x2.56厘米，层厚0.75mm）组合。 注:通过调整TR到1500毫秒，并保持动物的呼吸频率〜30 BPM（每呼吸2000毫秒），我们确保数据在动物的平静期被收集。 </li></ol></li><li>毕竟图像采集已经完成，将扫描的动物在加热垫和监控，直至卧床。恢复后，返回的动物笼子和至少返回到动物设施之前监测1小时。 </li></ol>

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The authors have nothing to disclose.

这份手稿显示了使用UHF MRI检查体内的表型特征或啮齿动物模型PKD药物监测工具的可行性。 我们描述的实验，在完成16.4 T搭配大口径的Avance III高分辨率核磁共振谱仪配微型和小型成像配件。该光谱仪主要得益于通过Paravision 5.1图像处理软件TopSpin2.0PV控制的采集和处理软件。由于啮齿类动物大小在纵向研究不同，我们使用了小型成像配件与38毫米RF线圈和小型成像支架​​。对于动物的温度控制我们采用标准的高分辨率垂直良莠不齐牛逼emperatureü尼特（BVT VTU 3000数字）通过上旋球2.0指导。从探头的底部供给的气流越过加热器，然后通过其位于紧接在麻醉的啮齿动物低于热电偶。热电偶控制加热器的功率电平连续地改变它，保持空气温度在所需的设定。 对使用超高频MRI对PKD之啮齿动物模型中的表型特征的主要优点是有可能用于获取体内图像，从而允许在相同的动物进行纵向研究。纵向研究的优点包括与磁共振VS常规组织学不全penetrance.Another益处模型下降牧数据变异的成本表型的进展或消退的，以及分析是MR图像呈现更真实的解剖没有收缩和固有失真在组织学切片。此外，MRI可以在3D重建图像。 除了 ​​提供优异的解剖细节，MRI允许体内 TKV测量。 TKV可用于监测随时间的疾病进展，和驴药物间干预措施之前在肾功能发生变化。此外，新生儿成像啮齿动物的可能性，提供了一个重要的切入点在宫内干预措施进行研究。 尽管它的巨大优势， 在体内成像啮齿动物模型的PKD仍然是具有挑战性的。这对于小鼠和新生大鼠更是如此，由于其小尺寸和相比人类更高呼吸和心脏速率。使用超高频MRI和更强的梯度允许更高的信号 - 噪声比和更好的空间分辨图像，但MRI是对运动高度敏感，并且运动伪影可以显著降低图像分辨率，减少了从技术的好处。这是腹部成像它是PKD的主要兴趣尤为重要。屏气扫描，如在人类中获得，也并非没有气管内管（ET）的插入是可行的。控制的可能性的麻醉动物的气道与ET是心脏或呼吸停止的情况下，有利的;然而，啮齿动物的插管需要很高的技术能力和难以掌握。输送吸入麻醉如异氟醚通过面罩的是容易和是选择用于最磁共振程序23的选项。然而，必须考虑缺氧/窒息的可能性，如果动物的位置不正确而在麻醉状态下，并且没有控制在紧急的情况下的气道。因此，仔细监测动物的呼吸频率和呼吸针对性的序列变得非常重要。此外，实现最佳的麻醉和动物的定位是用于在扫描仪获取高分辨率的图像是必不可少的。至于所有活的动物研究中，尤其是在使用病，新生儿或老年动物时，它在手术过程中监测动物的重要参数，并保持稳定的生理状态，以确保动物健康是非常关键的长期成功。 尽管面临挑战，相当大的进展已经取得了UHF MRI允许在PKD小型啮齿动物模型的详细表型信息，成为一个强大的工具， 在体内的表型和药物监测， 在胚胎发育的子宫内的图像允许的早期表征带有基因突变的表型相关联，并且可以识别非存活的胚胎的情况。 体内 MRI是临界实现从PKD的啮齿动物模型的最大利益（或任何其他啮齿动物模型系统），并应在任何实验设计考虑。

多囊肾（PKD），包括一组特征是肾囊肿发展单基因疾病。其中包括常染色体显性多囊肾病（ADPKD）和常染色体隐性多囊肾病（ARPKD），它代表了最常见的类型1,2。多囊肾，遗传性肾囊肿性疾病的最常见形式，起源由PKD1或PKD2基因突变。它的特点是迟发性，多发性双侧肾囊肿，伴随可变外肾囊肿，以及心血管和肌肉骨骼异常。 ARPKD，最常见的影响新生儿和幼儿，是通过突变PKHD1引起，其特征在于放大回声肾脏和先天性肝纤维化3。 重要的是，多囊肾的特征在于异质性，无论是在基因（基因）和突变（等位基因）的水平，这将导致大量的phenotypic变异。在PKD1基因突变与严重临床表现（无数囊肿，早期诊断，高血压和血尿），以及快速进展到终末期肾病（20岁之前的患者PKD2突变）相关联4。严重多囊肝（PLD）和血管异常可与在两个PKD1与PKD2 5突变相关联。多数ADPKD的肾脏并发症的出现主要是由于囊肿扩张连同相关的炎症和纤维化的结果。囊肿发展开始在子宫内 ，并通过患者生存期仍在继续。肾脏通常保持自己的肾形的形状，即使他们可能达到20倍以上的正常肾脏体积。大多数肾囊肿的患者当前两国的分布，但在某些特殊情况下，可以囊肿发展单边或不对称的格局。 一个主要的查林GE为以下肾病ADPKD患者或实施治疗的是疾病的自然史。在它的大部分，当然​​，肾功能保持正常和肾功能开始下降的时间，大部分的肾脏已替换囊肿。当疗法在稍后阶段实现，这是不太可能成功，因为患者可能已经达到了一个点的慢性肾脏疾病没有返回。相反，当治疗在早期阶段被启动，它是难以确定仅仅基于肾小球滤过率的响应。其结果是，肾脏体积作为疾病进展的标志物的概念受到关注。 该联合体多囊肾病（CRISP）研究的放射影像学研究表明，在ADPKD患者的增加肾和囊肿的卷直接与肾功能恶化相关，强调总肾体积（TKV），为作为潜在urrogate标志为疾病进展6,7。因此，TKV目前用作在用于ADPKD 2,8,9多个临床试验初级或次级端点。 多个小鼠模型包括自发突变和基因工程已经阐明了PKD 10,11的发病轻。 PKD1 PKD2或模型（无论是在Pkd1基因或突变PKD2）已经成为最流行 ​​的，因为他们完全模拟人类疾病。另外，啮齿动物模型中，在基因比Pkd1基因或PKD2基因其它突变已被用来作为实验平台来阐明信令相关的疾病的途径。此外，几个这些模型已经用于测试潜在疗法。然而，对于PKD的限制因素，在许多啮齿类动物的研究通常是缺乏有效的非侵入性的方法，以顺序地分析在肾脏解剖和功能的变化。 磁 - [Resonance成像（MRI）是当前的金标准的成像技术来遵循ADPKD患者，提供优良的软组织对比度和解剖细节，并允许TKV测量。尽管MRI是公认的解剖成像在较大的动物和人类，成像小老鼠体内需要额外的技术挑战，其中获得高分辨率图像的能力可能会限制其实用性。在引入超高场（UHF）核磁共振（7-16.4 T）和强梯度的发展，现在有可能实现更高的信号 - 噪声比，并与一个诊断质量的空间MRI图像的分辨率相似的在人类获得。因此，采用UHF MRI对体内成像的小啮齿动物模型为PKD已经成为一个强大的工具，为研究人员。

<table><tbody><tr><td>AVANCEIII-700 (16.4 T)</td><td>Bruker</td><td>BH067206</td><td>Wide-bore two channel multinuclear spectrometer equipped with mini and micro-imaging accessories for &lt;em&gt;in vivo&lt;/em&gt; small rodent imaging</td></tr><tr><td>TopSpin2.0PV</td><td>Bruker</td><td>H9088TA2</td><td>Spectrometer processing software</td></tr><tr><td>Paravision 5.1</td><td>Bruker</td><td>T10314L5</td><td>Imaging sofware</td></tr><tr><td>VTU BVT 3000 digital</td><td>Bruker</td><td>W1101095</td><td>Temperature controller</td></tr></tbody></table>

use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

在这个手稿中，我们的目标是展示UHF MRI的效用并在体内的表型特征或药物监测的啮齿动物模型为PKD和其他肾脏疾病的工具。所有的实验都是批准的IA​​CUC实验方案的一部分。 在小型啮齿动物模型使用UHF MRI PKD 体内表型: 所有成像研究是异氟烷麻醉下对活的动物进行的，用Bruker AVANCEIII-700（16.4 T）的垂直孔两种通道多核光谱仪，配备有小型和微型成像配件用于体内和体外 NMR光谱和显微镜。 腹部: 在PKD的主要结构的变化是囊肿发展和成长，负责大多数肾并发症。最常见的肾外表现的多囊肾病是肝囊肿和c的存在一个被发现在高达90％的受感染的成人13。使用超高频的MRI，能够获得高清晰度，小PKD啮齿动物，其允许在体内评估囊性表型和肾体积测量的解剖腹部图像图1A - D显示多个2D的T2加权解剖腹部图像不同啮齿动物模型PKD的。使用38 mm容积射频线圈被收购腹部图像。一种MRI相容支架是用来放置动物垂直沿磁场。气囊传感器，用于监测呼吸。进行呼吸门控。然后，冠状，矢状和轴，侦察图像，以找到肾脏，并规定其几何形状被收购。一种涡轮快速采集弛豫增强（RARE）序列，11-19冠状切片与TR / TE 1500/9毫秒，稀有因子8，（矩阵256×256，FOV 2.56 x2.56厘米，层厚0.75mm）组合来收集解剖图像。 <p类="jove_step"&gt;心: 心血管并发症保持ADPKD患者的一个重要问题，具有增加的发病率和死亡率14,15相关联。实验和临床MRI允许心脏结构和功能16-18精确和可重复的评估。 MRI具有高时空分辨率，允许最佳的可视化和小型，快速跳动的心脏灭鼠分析。出于这个原因，使用UHF MRI获得心脏图像计算舒张末期容积（EDV）是可行的，收缩末期容积（ESV），以及在几个啮齿动物模型从覆盖整个心脏顺序短轴cines心肌质量的PKD。 图2显示了小鼠心肌的MRI图像。心脏电影图像使用内门，快速低角度拍摄（IG-FLASH）序列19（11短轴切片，TR / TE 3.5 / 1.45毫秒，重复100，矩阵256×256，FOV 2.56 x2.56厘米，SL后天冰厚1毫米）。 大脑: 许多ciliopathies已与其他缺陷中脑畸形有关。在过去几年中，MRI已经成为黄金标准为大脑的非侵入性成像。与组织学研究，MRI检测提供的解剖变化无准备的文物，与正常考试造成干扰。我们使用超高频的MRI评估的多个小鼠模型PKD，或与之相关的疾病的其他修饰基因的脑表型。 图3显示了小鼠大脑的解剖图像。使用涡轮RARE序列获得图像，11-13轴位片和25-29冠状切片与TR / TE 1500/9毫秒，稀有因子8，（矩阵256×256，FOV 2.56 x2.56厘米，层厚0.75mm）组合。 在母体子宫内胚胎小鼠体内 MRI: 聚集在体内有关胚胎数，viabil的可能性性，发育阶段，以及评估胚的表型差异，是很重要的探索灭活特定信号传导途径与Pkd1基因或PKD2特异性突变结合的效果时尤其如此。通过执行怀孕的女性体内 MRI，它能够检测胚胎致死并评估表型异常，并且当存在时，确定在它们发生的初期阶段， 图4示出了详细的胚胎的信息可以从使用怀孕女性获得的一个例子体内 UHF MRI检查。被收购腹部图像作为先前用于非妊娠动物说明。异氟烷可以安全地使用在怀孕啮齿类和麻醉实现如在非妊娠动物20。由胚胎13天（E13），它能够识别许多解剖特征，如肢芽，中脑，端脑和心脏。从E14-15后肾可以指出，出现<html>出的用作卵形结构（1-1.5毫米，长度）与髓质和皮层部件21。 在体内的疾病进展或小型啮齿动物模型使用UHF MRI PKD治疗监测: 除了提供优异的解剖细节，超高频的MRI允许在啮齿动物模型为PKD TKV测量。如患者，TKV可用于监测疾病进展或评估药物干预肾功能的改变可以感知之前。此外，新生儿成像啮齿动物的可能性，提供了一个重要的切入点在宫内干预进行MRI检查。 图5显示了多个二维T2加权解剖腹部图像所用TKV作为终点一PCK大鼠的研究。被收购腹部图像如前所述。 res.jpg"WIDTH ="467"/&gt; 图1:解剖冠状腹部MRI图像为PKD（A）24月龄的Pkd1 RC / RC鼠标表示肾组织由囊肿（箭头），以此模仿多囊肾典型变化几乎完全更换不同啮齿动物模型 ， （B）4.月龄PKD2 - / WS25鼠标显示双侧肾囊肿（箭头）和残存的肾实质和肝囊肿（箭头）， （c）及 4个月大的PKHD1 LSL / LSL鼠标显示肝纤维化和肾脏无囊肿，和 （ 四）21日龄大鼠PCK显示多个双侧肾囊肿（箭头）主要在皮髓质区和外髓质和温和的胆管扩张。图片显示出良好的解剖细节的表型特征，在平面分辨率100微米/像素，切片厚度750微米。注意相比正常出现肾脏（C）的多囊性肾脏（A，B和D）之间的差异。标尺:10毫米<a href="https://www.jove.com/files/ftp_upload/52757/52757fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52757/52757fig2highres.jpg" width="700" /> 图2:心脏形态和功能。 （ 一 ）从短轴，IG-FLASH序列的小鼠心脏的解剖图像。 （ 二）心内膜边界的舒张末期（红色）大纲允许计算舒张末期容积（EDV）为每个切片。同样的程序可以收缩末期容积（ESV）来完成。此外，心肌体积（红-蓝）可被计算为每个切片从短轴，电影图像22。 （C </stro纳克&gt;）长轴图像显示，整体的EDV和ESV可以通过添加每个切片的体积来获得，并进一步计算每搏输出量和射血分数。标尺:10毫米<a href="https://www.jove.com/files/ftp_upload/52757/52757fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52757/52757fig3highres.jpg" width="467" /> 图3:老鼠大脑MRI UHF解剖成像（A）和 （B）的轴向，（C）和（D）的大脑中多囊肾小鼠模型中，在二维的T2加权涡轮RARE序列获得的冠状图像。该图像平面分辨率为100微米/像素，切片厚度750微米使得毛利率分析大脑解剖。在（B）和（D）的白色箭头指向蛛网膜半胱氨酸TS第四脑室的地区。标尺:10毫米<a href="https://www.jove.com/files/ftp_upload/52757/52757fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52757/52757fig4highres.jpg" width="700" /> 图4: 在 E14小鼠胚胎子宫内影像在母体冠状面（A）和（B）产妇轴面T2收购加权涡轮罕见影像的显示4种不同的胚胎1-4和B胚1和4，和额外的胚胎没有见过的 。 上镶石，放大的图像从胚胎1，显示胎儿的矢状面A，定位与头部朝上，右，其背部左侧，而且胎儿的冠状面<html> b。将图像平面分辨率100微米/像素的可以识别的许多解剖学特征，如肢芽，中脑，端脑，心脏和肝脏。图片比例尺:10毫米<a href="https://www.jove.com/files/ftp_upload/52757/52757fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52757/52757fig5highres.jpg" width="700" /> 图5:解剖冠状腹部MRI图像的PCK大鼠研究（A - C）表示对照组（生理盐水）。从相同的动物在P3，P10和p21获得图像。 （D - F）的代表治疗组（1-脱氨基-8-D-精氨酸加压素处理的）。从相同的动物被收购在同一年龄作为控制图像。比例尺:10mm以下。p_upload / 52757 / 52757fig5highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

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Nanyang Technological University

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Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

11.4K 次观看.  Mayo Clinic. The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

视频: Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

科研

癌症研究

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

神经科学

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

医学

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

使用3D机器人超 声进行小鼠 肾脏体内分析

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

使用2光子显微镜量化慢性单侧输尿管梗阻对肾小球过程的影响

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

小动物高分辨率心脏正电子发射断层扫描/计算机断层扫描

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

<meta charset="utf8"></head><body>用于小鼠肝脏微血管系统分析的超快多普勒超声

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

<meta charset="utf8"></head><body>使用超快多普勒超声对小鼠肝脉管系统进行成像和定量

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

使用超快多普勒超声对小鼠肝脉管系统进行成像和定量

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

在多囊肾小型啮齿动物模型采用超高场磁共振成像

摘要

探索更多视频

此视频中的章节

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

多鼠神经解剖学磁共振成像

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

在鼠害自觉肾功能的评估皮

小鼠肾小球疾病模型肾脏功能的评价

使用3D机器人超声进行小鼠肾脏体内分析

使用2光子显微镜量化慢性单侧输尿管梗阻对肾小球过程的影响

小动物高分辨率心脏正电子发射断层扫描/计算机断层扫描

使用超快多普勒超声对小鼠肝脉管系统进行成像和定量

在多囊肾小型啮齿动物模型采用超高场磁共振成像

摘要

探索更多视频

此视频中的章节

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

多鼠神经解剖学磁共振成像

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

在鼠害自觉肾功能的评估皮

小鼠肾小球疾病模型肾脏功能的评价

使用3D机器人超 声进行小鼠 肾脏体内分析

使用2光子显微镜量化慢性单侧输尿管梗阻对肾小球过程的影响

小动物高分辨率心脏正电子发射断层扫描/计算机断层扫描

使用超快多普勒超声对小鼠肝脉管系统进行成像和定量

使用3D机器人超声进行小鼠肾脏体内分析