<ol style="padding:5px;"><li>设备设置<ol style="padding:5px;"><li>选择面积在实验室中，室温在20 ° C或以上</li><li>避免在空调通风口和/或大声喧哗附近的位置。 </li><li>避免暴露动物的气味，可能会引起刺激和/或压力，如香水，古龙水，化学品，和清洁工，。 </li><li>打开尾声控制器。 </li><li>打开变暖的平台，并设置为3级。如果室温高于25 ° C时，启动1级或2级。 </li><li>控制器诊断测试<ol style="padding:5px;"><li>打开尾声软件。 </li><li>尾声设备上按一下选择。 </li><li>点击“选择测试设备”。 </li><li>选择袖口和渠道进行测试，然后单击“测试”。 </li><li>测试完成后，关闭“设备测试”窗口。 </li></ol></li></ol></li><li>软件设置<ol style="padding:5px;"><li>管理人员和动物<ol style="padding:5px;"><li>管理人员<ol style="padding:5px;"><li>选择“工具”&gt;“管理人员”。 </li><li> “研究员”，点击更改名称。 </li><li>在随后的行，点击添加额外的研究人员。 </li><li> “技术员标签”，点击修改或增加的技术人员。 </li><li>点击“标本”选项卡，以易于识别的名称的动物和选择动物类型;鼠或鼠标。 </li><li> “动物”选项卡上点击改变的动物种类和灵敏度。 </li></ol></li></ol></li><li>新实验<ol style="padding:5px;"><li>选择File&gt; New&gt;实验</li><li>开始一个新的实验<ol style="padding:5px;"><li>输入实验的名称。 </li><li>选择的主要研究人员。 </li><li>选择开始日期，然后单击“下一步”。 </li></ol></li><li>基本会话信息<ol style="padding:5px;"><li>输入会议名称</li><li>设置的“驯化周期”至5。如果你不喜欢，包括驯化周期设置为零。为了协助acclimating持有人及袖口的动物，你的实验中添加额外的周期和运行连续5天的实验。 </li><li> “套数”设置为1。 </li><li> “集之间的时间”设置为30秒多套</li><li>设置“循环”，每套20个周期。 </li><li> “周期之间的时间”设置为5秒，点击“下一步”。 </li></ol></li></ol></li><li>样本的选择<ol style="padding:5px;"><li style="list-style-type:none;"><ol style="padding:5px;"><li>选择从标本池的标本，将其分配给第一个可用的通道，并点击“下一步”。 </li></ol></li><li>会话参数<ol style="padding:5px;"><li>设置250mmHg“最大闭塞压”。 </li><li>设置“通缩时代”小鼠，大鼠15秒至20秒。 </li><li>设置“最低交易量”到15ul。 </li><li>设置“显示样式”，每图的一个通道。 </li><li>添加额外的研究人员和技术，并点击“下一步”。 </li></ol></li><li>会话脚本<ol style="padding:5px;"><li>审查会话脚本，点击“下一步”。 </li><li>不要选择完成，直到您完成动物准备部分。 </li></ol></li></ol></li></ol></li><li>动物的制备<ol style="padding:5px;"><li>动物设置的<ol style="padding:5px;"><li>动物持有人<ol style="padding:5px;"><li>培养和提高动物的驯化，添加额外的周期，您的实验和运行连续五天的实验。您还可以协助配售持有人的动物，15分钟，连续3天前的实际研究与驯化。 </li><li>持有人拿起动物的尾巴，轻轻放入动物的持有人面临的鼻锥开口端的后方放置在每个动物。注：动物在选定的持有人的大小尺寸。动物应该能自由进入的持有人。 </li><li>小心地保护持有人的后舱门打开后舱门红色的螺丝帽。应注意避免捏尾巴或身体其他部位，同时确保后方舱口。 </li><li>滑向后方舱口的鼻锥，限制动物的运动。鼻锥，应在一个位置，限制从动物转向左右，而里面的持有人。 </li><li>将变暖平台上的持有人在指定的位置。如果测试通过一段时间的同一组的动物，可以肯定的旋转变暖的平台上持有人的地位，提高你的血压测量的一致性和准确性。允许的动物，至少5分钟，以适应环境的持有人。 </li><li>请勿触摸或处理的动物，而里面的持有人。增加接触可以刺激动物。 </li><li>切勿持有人留在无人看管的动物。 </li></ol></li><li>袖口安置<ol style =“填充：5px;”&gt; <li>穿过尾巴“闭塞的袖口。”广场袖口尽可能靠近基地的尾巴，没有力量。 </li><li>安全的“闭塞袖口油管”持有人的后上方的缺口。 </li><li>穿过“VPR袖口”的尾巴，它放置在2mm的“闭塞袖口”。 </li><li>保护持有人的后上方的缺口“VPR袖口油管”。 </li><li>将袖口尾声控制器。 </li></ol></li><li>动物温度<ol style="padding:5px;"><li>允许的动物，至少5分钟，以thermoregulate。 </li><li>经常用红外测温仪记录动物的温度。动物的温度应在32至35 ° C。 </li></ol></li></ol></li></ol></li><li>实验<ol style="padding:5px;"><li>开始实验<ol style="padding:5px;"><li>点击“完成”，并实验将立即开始。 </li></ol></li><li>结束实验<ol style="padding:5px;"><li>动物<ol style="padding:5px;"><li>立即从袖口和持有人的动物。 </li><li>返回后的笼子里的动物的“简单的会话总结报告”显示。 </li></ol></li><li>数据收集<ol style="padding:5px;"><li>接受周期尾声应用程序的试算表内格式将被自动显示最后一个周期完成后，点击“导出到Excel”按钮。 </li><li>选择放置文件的文件夹，命名该文件，并点击“保存”。 </li><li>打开Excel应用程序，然后单击“打开”。 </li><li>查看所有文件类型，以显示“。csv文件保存”，在文件上单击。 </li></ol></li><li>数据处理<ol style="padding:5px;"><li>有几种方法来处理Excel文件内的数据。通常的做法是获得的平均值和标准偏差。 </li><li>删除测量，如果标准偏差大于30。 </li><li>此外，拒绝周期可能被视为整个数据库可以很容易地导出到Excel。咨询用户的其他信息手册。 </li></ol></li></ol></li></ol></li></ol>

The authors have nothing to disclose.

measuring blood pressure in mice using volume pressure recording, a tail-cuff method

尾声，8通道高通量非侵入性血压系统测量血压，同时8只小鼠或大鼠。尾声尾袖系统使用卷压力记录（VPR）来衡量确定的尾巴血容量血压。一个特别设计的差动压力传感器和一个闭塞的尾巴袖口措施在尾部的总血量，而不需要获得个人的脉冲信号。特别注意的是给予闭塞袖口的长度，为了获得最准确的血压读数。 VPR可以很容易地获得对皮肤黝黑的啮齿类动物，如C57BL6小鼠，的读数和核磁共振兼容。尾声系统提供的六（6）不同的血压参数的测量，收缩压和舒张压，心率，平均血压，尾巴的血流量，和尾血容量。测量，可无论是清醒或麻醉的小鼠或大鼠。尾声系统包括一个控制器，笔记本电脑，软件，袖口，动物持有人，红外变暖垫，红外测温仪。有七个小大900克8克大鼠，小鼠的不同规模的持有人。

Watch this Scientific Journal Video about Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method at JoVE.com

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

尾声，8通道高通量非侵入性血压系统测量血压，同时8只小鼠或大鼠。这尾袖系统采用体积压力记录（VPR）来衡量确定的尾巴血容量血压。

使用卷压力记录，一条尾巴袖套法小鼠的血压测量

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

measuring-blood-pressure-mice-using-volume-pressure-recording-tail

Research

JoVE Journal

Biology

51.3K 次观看.  University of Kentucky. 尾声，8通道高通量非侵入性血压系统测量血压，同时8只小鼠或大鼠。这尾袖系统采用体积压力记录（VPR）来衡量确定的尾巴血容量血压。

视频: Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

科研

生物学

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

初压雌核发育二倍体的斑马鱼

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

用奔放的整体体积描记测量呼吸功能的影响

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

心脏压力容量环分析小鼠使用电导导管

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

测量压力容量环中的鼠标

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...