The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

注:在本协议中描述的所有动物的相关手续已在杜克大学机构动物护理和使用委员会（DUIACUC）事先批准。 1.癌症细胞培养和注射<ol><li>培养荧光标记的转移性肿瘤细胞（ 如人力MDAMB231-GFP乳腺癌细胞，从帕特里夏Steeg的博士，NCI和YFP标记的小鼠肉瘤细胞，从大卫·基尔希博士，美国杜克大学医学中心放射肿瘤学系的礼品赠送）在适当的培养基（ 例如 Dulbecco氏改良的Eagle培养基（DMEM）中，用10％胎牛血清和1％青霉素/链霉），在37℃，直到大约90％汇合。 </li><li> Trypsinize细胞，用PBS冲洗2次，计数，然后将它们注入尾静脉异氟烷麻醉10周龄女性裸体老鼠每动物5000000细胞，使用带有27G的针头的注射器。手术水平anesthesIA是由缺乏反应脚趾捏的验证。 </li></ol> 2.监控转移使用显微的<ol><li>用微型CT /微辐射器，以检测转移灶的直径大于约2mm的直径的存在下，每周检查一次大鼠。在显微CT委托如前所述12。 <ol><li>受试者大鼠成像之前的3％异氟烷麻醉。确认通过脚趾捏深麻醉。 </li><li>发病麻醉后，迅速大鼠在成像腔室转移至成像支架和经由鼻锥在2.5-3％连接到一个异氟醚 - 空气混合物。调整在其胸部被定位在显微扫描仪的光子束的方法的摇篮的大鼠的位置，使用在成像支架的外部位置的控制和激光定界符。确保门成像室是封闭的，以从伽马射线遮蔽研究者。 </li><li>控制动物的再次使用位置彩色摄像机。执行低分辨率CT成像试运行，并使用所产生的图像，以视场调整到胸腔的XYZ尺寸。 </li><li>图像使用2毫米的Al滤波器大鼠胸廓在40 kVp的，2.5毫安，并0.008体素在7 FPS和返回动物笼子。一只动物的成像应不超过15或20分钟。不返回已经历手术给其他动物的公司，直到完全恢复的动物。不要让动物无人看管，直到它恢复足够的意识，保持胸骨斜卧。 </li><li>通过相对不透射线的物体的外观不能由胸内的血管进行说明确认转移（ 图1A） </li></ol></li></ol> 3.窗口商会外科<ol><li>麻醉，生命体征和尾静脉插管<ol><li>转移性疾病的存在选择的动物。注入动物腹膜内ðOSE的50毫克/公斤戊巴比妥。出发前确认所捏脚趾手术级别的麻醉。 注:麻醉的协议应该匹配到相应的实验装置。戊巴比妥这里被选为长效麻醉剂，以诱导麻醉深为冗长的程序，同时提供容易的重新给药的选项。但是，动物的损失用药过量​​是一种常见的问题戊巴比妥麻醉。它保留自主反射到更大的程度比巴比妥另一种选择是氯胺酮结合镇静剂如赛拉嗪或美托咪定，仅在单次再给药周期然而其允许。 </li><li>剃上具有转移性疾病，并且在颈部区域中的主体的侧面的动物，使用一个削波器。擦去从皮肤所有剩余松散的头发。后松散的头发被删除，适用兽医药膏眼睛，以防止它们干燥。 注:无胸腺裸鼠体内可能有残留的头发requi在进行外科手术前，水库搬迁。这是非常重要的，以彻底去除所有的头发的，因为它可以与外科手术和成像干涉。 </li><li>修复的动物在被放置在37℃的水中循环加热垫的金属板仰卧位置。前面和后肢肢体被固定在板上用胶带。 注:它是非常有用的控制，并通过使用一个脉冲血氧计，在整个手术和实验程序记录生命体征，如心脏速率和动脉血液充氧。 </li></ol></li><li>气管插管<ol><li>在以设置一个导管的动物的通风，首先使横向颈部皮肤切口，接着沿纵向肌肉腹侧气管的中位数的分离。 </li><li>使用重复打开到关闭动作用尖锐的镊子通过气管的背侧以创建一个通道的缝合。 </li><li>做一个小切口，插入吨气管他腹侧，不超过半圆形，大致在第二和第三气管环之间。留足够长的暴露在背表面气管部，以使所述气管导管的固定。 </li><li>将2.5〜3.0mm的"Y"气管插管进入气管，并拧紧了4-0单丝缝合。确保套管连接到一个压力循环通风机与连接到期满导管填充有6厘米水的瓶子，以维持正肺动脉压。流入的气体应是100％的氧气，除非实验所需的除外。 </li><li>插入一个导管具有25-27ģ针，并且填充有肝素生理盐水到尾静脉大鼠中的一个，并代替用胶带固定。 注:确保尾静脉导管的通畅整个过程通过重复注入少量肝素的生理盐水到尾静脉。此外，奥罗气管插管，即气管指导经由麻醉动物的嘴和过去的喉部进入气管管，是一种可能的替代方案是这里描述的气管切开术过程。然而，该方法需要特别的训练和经验，以避免损坏气管，并且也排除食道的意外插管。 </li></ol></li><li>肺窗应用<ol><li>从胸部其中转移性疾病位于的一侧去除皮肤，通过创建一个切口，然后用钝器解剖分离皮肤。 注:皮肤被切除，并去除随后支队</li><li>通过解剖重叠肌肉（胸，锯，以及背阔肌）的两个层，但留下肋间肌完好进行。在约1.5厘米胸腔直径创建一个穿孔，通过除去通常两个相邻的肋的某些部分。理想情况下，找到穿孔在第六和第七r的地区肠易激综合征。 </li><li>截骨术: <ol><li>为了最大限度地减少出血和损害肺表面上，紧紧地保持肋在切割期间被切割带齿外科钳子。用手术剪，切内侧肋的第一，在大约45°的角度，余下的肋骨指向外面的尖端侧。 </li><li>随后，切开肋骨的侧面以类似的方式，再次离开肋骨指向外部，以防止损坏的肺表面的尖端侧。 </li><li>重复该过程的相邻肋骨，然后切开肋间肌肉，去除切一块。在此过程中，维持的方式，肺表面和肋笼之间的机械相互作用被最小化肺压力。通过适当地调节呼吸机呼吸压力做到这一点。 </li></ol></li><li>插入窗口的: <ol><li>插入定制肺窗口，包括一个玻璃罩即是连接到一个有机玻璃插座（ 图1B）。通过胶粘，或者通过施加非常少量的真空润滑脂附着窗口套接字。插入窗口的方式，表面的转移都位于靠近窗口的中心。如果需要的话，调整所插入的孔通过稍微扩大孔向各个侧带来的微转移到窗口的中心。 注:在胸膜表面转移性疾病可以被识别为肺表面的粉红色，否则对鲑鱼的彩色健康部分出现主要沿裂隙内清楚地辨认白色点或区域。虽然微可以发生在胸腔外的其他领域，被调查的细胞系几乎总是显示浮现在微穿孔区域，一旦转移性疾病可放射学检测。 </li><li>插入插座插入穿孔，以及创建与所述升的内脏胸膜直接接触后UNG，缝合窗框的边缘向周围肋间肌，使用4.0单丝缝合（ 图1C）。使用呼吸机呼吸压力略有增加，以帮助残留的空气逸出，并创建一个密封。 注意:大鼠的呼吸速率可有很大变化，这取决于麻醉，兴奋或焦虑的状态中，吸入空气中的氧浓度（氧合指数2）等的状态，是值得推荐的，调整70和90次之间的呼吸速率。吸气压力应调节谨慎和不被设置为大于大约8厘米的H 2 O（0.6毫米汞柱），以避免损坏肺表面。 </li></ol></li><li>定位动物中，其被设计来消除Z方向运动（ 图1D）上被定位在恒温（电）加热毯的钢板定制设计的阻 ​​挡器，在荧光显微镜下。控制动物的身体温度用直肠热敏电阻。调节动物限制器的螺丝，和呼吸机上的吸气压力来实现横向移动的最佳控制。 注:自然呼吸的哺乳动物包括三个方向运动肺和胸部延伸:双边，背腹和头尾。为了保存自然呼吸运动尽可能，它以最小化Z方向压缩到必要的程度是重要的。因为在Z维约束具有引入可能影响血液流动等参数工件的电位，最好是串联在同一动物重复测量过程中保持恒定的条件。 </li></ol></li></ol> 4.成像测量血液微循环和<ol><li>通过心脏穿刺收集的红细胞和如先前所描述的DiI（1,1 = -dioctadecyl -3,3,3 =，3 =四甲基吲哚羰花青高氯酸盐）标记它们，&lt;SUP&gt; 10。 </li><li>注入300微升标记的红血细胞到尾静脉的大鼠的窗口腔手术完成之前，为了避免首过效应的粘附到玻璃窗口。消除任何气泡在注射器或导管，如引入空气进入静脉将抑制任何进一步的注射。 </li><li>图像血流用显微镜CCD照相机在-40℃冷却的芯片温度，和大约100X总分辨率（ 即用10X显微镜物镜，和10X预相机目镜）。使用标准的罗丹明/ TRITC滤光片组（激发波长450-490，发射&gt; 515纳米）。记录所得到的图像序列的实际帧速率和像素分辨率。每个堆栈记录至少200（最好约300）的图像，以确保流速的成功分析。 </li><li>通过注入每一个小时约1毫升生理盐水知识产权的补充在动物体液丢失。 注:涉及干预实验设置， 如一种药物，用于修改在肺癌转移的血流速度，需要血流速度的肺转移癌的重复测量。对于这些扩展实验，以补充足够的流体，动物是非常重要的。 </li><li>安乐死的动物通过输注3毫升3N氯化钾到尾静脉</li><li>评估采用发表，公开可用的基于Matlab的计算机算法，将创建流速灰度和彩色编码图所有的血流痕迹10,11图像栈。随后评价所形成的灰度图像通过使用商业或公开可用的图像分析软件，如的Image J，阈值化的折扣值，表明血细胞的无运动， 即没有活性脉管之后。 </li></ol>

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作者什么都没有透露。

一个模型提出即是可行的，使用活体显微镜和计算血流分析在血液微循环和大鼠的肺转移其它动态过程，图像的变化。而其他方法存在于啮齿动物的开放ribcages暴露肺部进行显微镜，这种模​​式也是通过胸壁穿孔封闭胸设置的第一个图像肺转移。使用这种方法的可行性被示为测量药理学诱导的变化的微循环血液肺转移的流动。 两种基本方法存在图像的生活啮齿类是由自发血...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

在实体瘤的脉管系统是已知的，从正常供血显著不同，表示更大程度的扭曲的，并且更高intervascular距离13。因此，当相比于正常肺微循环（ 图2A，上图）中的实验性肺乳腺癌和肉瘤转移血流磁道具有不规则的形状和大intervascular间隙（ 图2A，下两个面板）。在先前的研究中，肺窗口方法的能力证明，可以执行正常的肺10的变化中的血流速度的自动测量。为?...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

微循环血流速度在大鼠肺转移自动测量

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K 次观看.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

视频: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

科研

医学

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

增加肺动脉脉动流改善缺氧性肺动脉高压的仔猪

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

经皮微循环成像在早产儿

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

肱血流介导的扩张和大鼠股浅动脉超声评估

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

心脏重构通过大鼠腹主动脉缩窄的模型

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

锁骨下静脉穿刺作为大鼠采血的一种替代方法

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

左肺切除术联合单氯联氨酸或苏根为大鼠肺动脉高压的模型

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

大鼠同时心电图记录及侵入性血压测量

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...