资助 本研究是福尔克心胸外科部，斯坦福大学，美国加利福尼亚州斯坦福大学医学院的研究基金的支持。托比亚斯Deuse是由一个德国心脏学会的研究资助。宋佳Schrepfer已收到由德意志研究联合会（DFG）（SCHR992/2-1）的研究补助金。

主动脉支架部署<ol><li>哈伦（印第安纳波利斯，美国）均购自雄性SD大鼠，体重550-600克。众议院大鼠常规条件下，饲喂标准鼠议员和水自由采食。 </li><li>麻醉大鼠异氟醚（2％）和氯胺酮（25毫克/公斤）。在微观下，执行上层的中位数迷你剖腹手术以暴露下腹主动脉。 </li><li>从周围组织解剖腹主动脉，肾动脉水平下降到分叉。注意：无需解剖主动脉，下腔静脉。 </li><li>使用microclamps停止主动脉的血流量。将近端钳，远端钳。 </li><li>打开一个小的横切口主动脉和主动脉与肝素（200单位）冲洗。 </li><li> 2法国福格蒂动脉取栓导管（百特医疗用品，迪尔菲尔德，IL，USA）通过主动脉内皮是光秃秃的。 </li><li>使用任何人类8mm和长度12毫米之间的支架大小，直径2.5毫米- 3MM。注意：支架的直径应不超过10％以上的血管直径，以避免前，后支架狭窄。注意：请勿在同一研究开关支架长度。 </li><li>部署支架，使用合适的球囊压力，以达到所需的直径。 </li><li>关闭小主动脉切口用9-0普理灵缝线（爱惜康，日本东京，德国）。 </li><li>关闭腹部切口层4-0薇乔缝线（爱惜康，日本东京，德国）。 拆除皮肤缝线，仍然应该在7-14天，甚至是用来当Viacryl。 </li><li> 7天在饮水中的动物卡洛芬（6mg/KG），术中和安乃近。 </li></ol>光学相干断层扫描（OCT）成像 OCT图像与M2 OCT成像系统（LightLab成像公司，韦斯特福德，马萨诸塞州，美国）获得。 ImageWire是提供光成像探测的组织和收集的信号。 ImageWire 0.006“（0.15毫米）光纤核心，护套内的一个最大外径为0.019”（0.48毫米）。 <ol><li>麻醉大鼠异氟醚（2％）和氯胺酮（25毫克/公斤）。在微观下，执行中位数重做剖腹探查术，暴露下腹主动脉。 </li><li>夹紧近端的主动脉和两个髂动脉。 </li><li>执行在远端的主动脉横动脉切开术，冲洗1毫升PBS使用28G的导管插入华侨城导管进入主动脉和前瞻性。 </li><li>电动回落OCT成像是在每秒1.0毫米回落率。 </li><li>收购每秒15帧图像，显示图像的颜色查找表和数字存档。 </li><li>关闭动脉切开术。 </li><li>关闭4-0薇乔缝线（爱惜康，日本东京，德国）的腹部及切口。 拆除皮肤缝线，仍然应该在7-14天，甚至是用来当Viacryl。 </li><li>将开发6个星期内支架植入后的最大内膜形成。 </li></ol> OCT图像分析华侨城的测量是使用老鼠基于接口的LightLab OCT成像的专有软件。 <ol><li>反映OCT成像线，这是该系统的标准校准技术校准系统。 </li><li>跟踪管腔支架的横截面区（CSA），每1.0 mm间隔手动。 </li><li>计算斑块跨截面积减去管腔CSA支架CSA。计算％，斑块面积除以支架CSA（％）斑块修正案。计算平均百分之斑块地区。 </li></ol> OCT的技术验证华侨城结果相关的以及与病理组织学（图1）。如上所述，组织学斑块修正案计算。与高密度的纺锤状细胞，只有少数的单核炎症细胞，组织学显示内膜增生。 6周后，支架完全覆盖肉芽组织新生内膜斑块CSA措施1.3 ± 0.4平方毫米 ，在一个2.5mm支架。 <img src="/files/ftp_upload/1346/1346fig1tmb.jpg" border="0" /> 图1：华侨城（A）和组织学（B，放大倍率16倍）图像支架后6个星期的部署。 OCT图像的菌斑修正案获得的结果与病理组织学相关。请<a href="https://www.jove.com/files/ftp_upload/1346/1346fig1.jpg">点击这里</a>看到图1的放大版本。

<ol>
	<li>Langeveld, B., Roks, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commentary:+rat+restenosis+models:+means+for+thorough+restenosis+research.">Commentary: rat restenosis models: means for thorough restenosis research.</a> Journal of endovascular therapy. 12 (3), 343-345 (2005).</li><li>Roguin, A., Grenadier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stent-based+percutaneous+coronary+interventions+in+small+coronary+arteries.">Stent-based percutaneous coronary interventions in small coronary arteries.</a> Acute Card Care. 8, 70-74 (2006).</li><li>Post, M. J., Waltenberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Is+Beautiful:+A+Miniature+Stent+Model.">Small Is Beautiful: A Miniature Stent Model.</a> Arterioscler. Thromb. Vasc. Biol. 27, 701-702 (2007).</li><li>Langeveld, B., Roks, A. J. M., Tio, R. A., Boven, A. J. v. a. n., Want, J. J. L. v. a. n. d. e. r., Henning, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+abdominal+aorta+stenting:+a+new+and+reliable+small+animal+model+for+in-stent+restenosis.">Rat abdominal aorta stenting: a new and reliable small animal model for in-stent restenosis.</a> Journal of vascular research. 41 (5), 377-386 (2004).</li><li>Lowe, H. C., Chesterman, C. N., Khachigian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+aortic+stenting:+toward+a+simple+model+of+in-stent+restenosis.">Rat aortic stenting: toward a simple model of in-stent restenosis.</a> The American journal of cardiology. 88 (6), 720-721 (2001).</li><li>Groenewegen, R., Harst, R. v. a. n. d. e. r., Roks, R., Buikema, R., Zijlstra, R., Gilst, R. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+angiotensin+II+and+angiotensin+II+type+1+receptor+blockade+on+neointimal+formation+after+stent+implantation.">Effects of angiotensin II and angiotensin II type 1 receptor blockade on neointimal formation after stent implantation.</a> International Journal of Cardiology. , (2007).</li><li>de Smet, B. J. G. L., Zijlstra, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+look+at+drug+eluting+stents+with+optical+coherence+tomography.">A look at drug eluting stents with optical coherence tomography.</a> European Heart Journal. 28 (8), 918-919 (2007).</li><li>Pinto, T. L., Waksman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+optical+coherence+tomography.">Clinical applications of optical coherence tomography.</a> Journal of interventional cardiology. 19 (6), 566-573 (2006).</li><li>Matsumoto, D., Shite, J., Shinke, T., Otake, H., Tanino, Y., Ogasawara, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neointimal+coverage+of+sirolimus-eluting+stents+at+6-month+follow-up:+evaluated+by+optical+coherence+tomography.">Neointimal coverage of sirolimus-eluting stents at 6-month follow-up: evaluated by optical coherence tomography.</a> European Heart Journal. 28 (8), 961-967 (2007).</li><li>Kawase, Y., Suzuki, Y., Ikeno, F., Yoneyama, R., Hoshino, K., Ly, H. Q. <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+nonuniform+rotational+distortion+between+mechanical+IVUS+and+OCT+using+a+phantom+model.+Ultrasound+in+medicine+&amp;amp;+biology.">Comparison of nonuniform rotational distortion between mechanical IVUS and OCT using a phantom model. Ultrasound in medicine &amp;amp; biology.</a> 33 (1), 67-73 (2007).</li></ol>

在斯坦福大学医学中心（斯坦福大学，加利福尼亚州），标准温度，湿度和照明条件下，所有大鼠均安置在动物保健设施，并提供了鼠议员和水自由采食。调查符合“ 指南 ”由美国国立卫生研究院（NIH出版物，编号85-23，1996年修订）出版的“实验动物的关心和使用。研究方案，斯坦福大学的实验动物管理委员会批准。

虽然兔髂动脉和猪冠状动脉模型是最常见的为1支架置入术，放射和手术设备需要结合使用，动物房能力是有限的，购买成本高。支架大鼠模型的局限性是老鼠必要使用专门设计的支架，金属动脉的比例， 从而导致更多的血管损伤2，人为的高发病率和血栓形成的3。 大鼠支架置入模型是一个简单，价格低廉，快速，准确的临床前模型 4 。 Lowe等的大鼠主动脉直接支架术的初步报告后5，这个支架内再狭窄的病理生理的评价模型的可行性和适用性已被彻底显示5,6。大鼠主动脉的直径是足够的，允许在不破坏生理血管结构市售支架扩张。它已被证明的病理生理机制，如血栓形成，炎症，SMC增殖，，在这些大鼠模型开发，因为他们在兔子和猪。因此，这些模型的再狭窄的实际过程中良​​好的交涉。 华侨城高分辨率成像技术是有用的，以评估内膜增生。穿透深度只有1.5-2毫米，但其分辨率是一个幅度大于血管内超声（IVUS）7,8。华侨城与血管内超声比较多的研究得出结论，华侨城是目前首选的技术评价支架植入后8-10内膜增生。特别是在小血管直径的小动物，华侨城的高分辨率，使再狭窄的评价最好的成像方式。 综上所述，本视频显示：（1）大鼠主动脉支架置入术是可行的方便，（2）大鼠腹主动脉支架置入术是适合商业生产的用于测试支架（3）OCT成像是一个准确和优雅的技术，在支架的纵向监测再狭窄。

imaging in-stent restenosis: an inexpensive, reliable, and rapid preclinical model

必须解开的病理生理过程，导致支架内再狭窄和优化现有和未来的药物洗脱支架再狭窄的临床前模型。 在大鼠体内的各​​种抗体和转基因和基因敲除株。因此，支架内再狭窄大鼠模型，将病理生物学和病理生理研究的方便。 在这段视频中，我们目前的全部程序和坑瀑布老鼠支架模型，适用于高吞吐量的支架研究。我们将展示的外科手术，支架部署和使用华侨城最优雅的技术（光学相干断层扫描）的支架再狭窄的评估。这种技术提供了高精确度，在评估斑块修正案（截面领域）和关联，这就需要特殊和费时的嵌入和切片技术与组织学切片。 OCT成像还允许在同一动物的纵向监测支架内再狭窄的发展相比，一次性快照，用组织学。

Watch this Scientific Journal Video about Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model at JoVE.com

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

本视频演示了如何使用廉价和可靠的临床前模型来研究在支架内再狭窄发展的病理生物学和病理生理过程。纵向使用OCT（光学相干断层成像）和OCT图像分析在体内监测也表明。

支架内再狭窄的影像：一种廉价的，可靠，快速的临床前模型

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

imaging-stent-restenosis-an-inexpensive-reliable-rapid-preclinical

Research

JoVE Journal

Biology

13.7K 次观看.  Stanford University School of Medicine. 本视频演示了如何使用廉价和可靠的临床前模型来研究在支架内再狭窄发展的病理生物学和病理生理过程。纵向使用OCT（光学相干断层成像）和OCT图像分析在体内监测也表明。

视频: Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

PDMS微流体装置的廉价制造的非等离子键合

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

基础X射线计算机断层数据的三维打印设置

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac resynchronization therapy (CRT), and LBBB has become one of the important predictors for CRT response. The conventional tachypacing-induced HF model has several major limitations, including absence of stable LBBB and rapid reversal of left ventricular (LV) dysfunction after cessation of pacing. Hence, it is essential to establish an optimal model of chronic HF with isolated LBBB for studying CRT benefits. In the present study, a canine model of asynchronous HF induced by left bundle branch (LBB) ablation and 4 weeks of rapid right ventricular (RV) pacing is established. The RV and right atrial (RA) pacing electrodes via the jugular vein approach, together with an epicardial LV pacing electrode, were implanted for CRT performance. Presented here are the detailed protocols of radiofrequency (RF) catheter ablation, pacing leads implantation, and rapid pacing strategy. Intracardiac and surface electrograms during operation were also provided for a better understanding of LBB ablation. Two-dimensional speckle tracking imaging and aortic velocity time integral (aVTI) were acquired to validate the chronic stable HF model with LV asynchrony and CRT benefits. By coordinating ventricular activation and contraction, CRT uniformed the LV mechanical work and restored LV pump function, which was followed by reversal of LV dilation. Moreover, the histopathological study revealed a significant restoration of cardiomyocyte diameter and collagen volume fraction (CVF) after CRT performance, indicating a histologic and cellular reverse remodeling elicited by CRT. In this report, we described a feasible and valid method to develop a chronic asynchronous HF model, which was suitable for studying structural and biologic reverse remodeling following CRT.

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

心脏再同步治疗在左束分支消融和快速起搏诱发的异步心力衰竭模型中的益处

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

利用斑马鱼对肠道转运时间进行高通量测量

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.