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>

<ol>
	<li>Torres, V. E., Harris, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+dominant+polycystic+kidney+disease:+the+last+3+years.">Autosomal dominant polycystic kidney disease: the last 3 years.</a> Kidney Int. 76, 149-168 (2009).</li><li>Chapman, A. B., 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=Kidney+volume+and+functional+outcomes+in+autosomal+dominant+polycystic+kidney+disease.">Kidney volume and functional outcomes in autosomal dominant polycystic kidney disease.</a> Clinical journal of the American Society of Nephrology : CJASN. 7, 479-486 (2012).</li><li>Torres, V. E., Harris, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+kidney+disease:+genes,+proteins,+animal+models,+disease+mechanisms+and+therapeutic+opportunities.">Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities.</a> J Intern Med. 261, 17-31 (2007).</li><li>Hateboer, 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=Comparison+of+phenotypes+of+polycystic+kidney+disease+types+1+and+2+European+PKD1-PKD2+Study+Group.">Comparison of phenotypes of polycystic kidney disease types 1 and 2 European PKD1-PKD2 Study Group.</a> Lancet. 353, 103-107 (1999).</li><li>Rossetti, S., 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=Association+of+mutation+position+in+polycystic+kidney+disease+1+(PKD1)+gene+and+development+of+a+vascular+phenotype.">Association of mutation position in polycystic kidney disease 1 (PKD1) gene and development of a vascular phenotype.</a> Lancet. 361, 2196-2201 (2003).</li><li>Chapman, A. B., 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=Renal+structure+in+early+autosomal-dominant+polycystic+kidney+disease+(ADPKD):+The+Consortium+for+Radiologic+Imaging+Studies+of+Polycystic+Kidney+Disease+(CRISP)+cohort.">Renal structure in early autosomal-dominant polycystic kidney disease (ADPKD): The Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) cohort.</a> Kidney international. 64, 1035-1045 (2003).</li><li>Grantham, J. J., 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=Volume+progression+in+polycystic+kidney+disease.">Volume progression in polycystic kidney disease.</a> N Engl J Med. 354, 2122-2130 (2006).</li><li>Schrier, R. W., 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=Blood+Pressure+in+Early+Autosomal+Dominant+Polycystic+Kidney+Disease.">Blood Pressure in Early Autosomal Dominant Polycystic Kidney Disease.</a> The New England journal of medicine. , (2014).</li><li>Torres, V. E., 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=Angiotensin+Blockade+in+Late+Autosomal+Dominant+Polycystic+Kidney+Disease.">Angiotensin Blockade in Late Autosomal Dominant Polycystic Kidney Disease.</a> The New England journal of medicine. , (2014).</li><li>Wilson, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+polycystic+kidney+disease.">Mouse models of polycystic kidney disease.</a> Curr Top Dev Biol. 84, 311-350 (2008).</li><li>Happe, H., Peters, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+research+in+ADPKD:+lessons+from+animal+models.">Translational research in ADPKD: lessons from animal models.</a> Nature reviews. Nephrology. , (2014).</li><li>Frahm, J., Haase, A., Matthaei, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+NMR+imaging+of+dynamic+processes+using+the+FLASH+technique.">Rapid NMR imaging of dynamic processes using the FLASH technique.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 3, 321-327 (1986).</li><li>Bae, K. T., 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=Magnetic+resonance+imaging+evaluation+of+hepatic+cysts+in+early+autosomal-dominant+polycystic+kidney+disease:+the+Consortium+for+Radiologic+Imaging+Studies+of+Polycystic+Kidney+Disease+cohort.">Magnetic resonance imaging evaluation of hepatic cysts in early autosomal-dominant polycystic kidney disease: the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease cohort.</a> Clin J Am Soc Nephrol. 1, 64-69 (2006).</li><li>Hossack, K. F., Leddy, C. L., Johnson, A. M., Schrier, R. W., Gabow, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+findings+in+autosomal+dominant+polycystic+kidney+disease.">Echocardiographic findings in autosomal dominant polycystic kidney disease.</a> N Engl J Med. 319, 907-912 (1988).</li><li>Lumiaho, A., 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=Mitral+valve+prolapse+and+mitral+regurgitation+are+common+in+patients+with+polycystic+kidney+disease+type+1.">Mitral valve prolapse and mitral regurgitation are common in patients with polycystic kidney disease type 1.</a> American journal of kidney diseases : the official journal of the National Kidney Foundation. 38, 1208-1216 (2001).</li><li>Vallee, J. P., Ivancevic, M. K., Nguyen, D., Morel, D. R., Jaconi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+cardiac+MRI+in+small+animals.">Current status of cardiac MRI in small animals.</a> Magma. 17, 149-156 (2004).</li><li>Epstein, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+in+mouse+models+of+cardiac+disease.">MR in mouse models of cardiac disease.</a> NMR Biomed. 20, 238-255 (2007).</li><li>Bloomgarden, D. C., 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=Global+cardiac+function+using+fast+breath-hold+MRI:+validation+of+new+acquisition+and+analysis+techniques.">Global cardiac function using fast breath-hold MRI: validation of new acquisition and analysis techniques.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 37, 683-692 (1997).</li><li>Larson, A. C., 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=Self-gated+cardiac+cine+MRI.">Self-gated cardiac cine MRI.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 51, 93-102 (2004).</li><li>Smith, J. C., Corbin, T. J., McCabe, J. G., Bolon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+with+morphine+is+a+suitable+anaesthetic+regimen+for+embryo+transfer+in+the+production+of+transgenic+rats.">Isoflurane with morphine is a suitable anaesthetic regimen for embryo transfer in the production of transgenic rats.</a> Laboratory animals. 38, 38-43 (2004).</li><li>Ahrens, E. T., Srinivas, M., Capuano, S., Simhan, H. N., Schatten, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+embryonic+and+fetal+development+in+model+systems.">Magnetic resonance imaging of embryonic and fetal development in model systems.</a> Methods Mol Med. 124, 87-101 (2006).</li><li>Zhou, R., Pickup, S., Glickson, J. D., Scott, C. H., Ferrari, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+global+and+regional+myocardial+function+in+the+mouse+using+cine+and+tagged+MRI.">Assessment of global and regional myocardial function in the mouse using cine and tagged MRI.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 49, 760-764 (2003).</li><li>Stimpfel, T. M., Gershey, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+anesthetic+agents+for+human+safety+and+animal+recovery+surgery.">Selecting anesthetic agents for human safety and animal recovery surgery.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 5, 2099-2104 (1991).</li></ol>

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​​...

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 border="0" cellpadding="0" cellspacing="0">
	<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 in vivo small rodent imaging</td>
		</tr>
		<tr>
			<td>TopSpin2.0PV&#160;</td>
			<td>Bruker</td>
			<td>H9088TA2</td>
			<td>Spectrometer processing software&#160;</td>
		</tr>
		<tr>
			<td>Paravision 5.1&#160;</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?...

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 ...

use-ultra-high-field-mri-small-rodent-models-polycystic-kidney

Research

JoVE Journal

Medicine

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

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

科研

医学

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 ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

使用小动物看更衣室反转恢复（SALLI）的MRI大鼠心脏功能和心肌形态学评估

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

体外肠囊评估黏膜通透性在胃肠道疾病模型

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

在多囊肾小型啮齿动物模型采用超高场磁共振成像

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