著者感谢沙大冢女士提供技术援助和帮助实验。部分支持这项研究是由教育部，科学，体育和文化活动，资助青年科学家（二），2010，22700476，Suzuken 2010年纪念基金会为KT这项研究是由研究和开发的支持援助下一代集成模拟生命物质的发展和使用的文部科学省的下一代超级计算机项目的一部分，在垂询的ERATO Suematsu气体生物学项目的一部分。

1。用异硫氰酸荧光素异构体（FITC标记）标记红细胞我们使用的所有实验协议，由庆应义塾大学动物医学院护理委员会的批准。 <ol><li>捐助国小鼠麻醉，做开腹手术切口后退出下腔静脉全血1毫升含有少量的肝素的注射器。巴比妥钠注射液（100毫克/公斤的IP），鼠标立即被安乐死。 </li><li>洗了两次全血离心5分钟400克摆动转头用PBS（pH值7.4）。 </li><li>溶解在100毫米的Na 2 HPO 4 5毫升10毫克的FITC，有0.22微米孔隙过滤膜。 </li><li>在试管中混合与0.15毫升的葡萄糖3毫米，180毫米的NaH 2 PO 4计 ，100毫米和1.5毫升的Na 2 HPO 4 0.25毫升1毫升的FITC标记的解决方案。加入0.2毫升的RBC球，和挖掘混合管。 </li><li>管保持在4°C 2小时，然后在PBS洗两次染色红细胞。 </li><li>幻灯片上的草把少量的红细胞悬液，如果红细胞看起来很健康（即，它们的形状和大小都正常）和荧光强度是足够的。 </li><li>暂停0.9 PBS在pH 7.4毫升0.1毫升，红细胞，并保持悬浮在4°，直到注射Ç。 </li></ol> 2。氧敏感的染料的制备<ol><li> BSA的500毫克溶于10毫升的PBS在pH 7.4。 </li><li>添加30毫克的Pd（Ⅱ）-meso-四（4 - 羧基苯基）卟啉（PD-TCPP的）BSA溶液，搅拌过夜。 </li><li> （可选）BSA绑定钯TCPP的提取利用重力流层析或自旋列从自由钯卟分开。 </li><li>离心除去不溶钯卟，过滤膜过滤器，具有0.22微米的毛孔上清溶液。 &lt;/ LI&gt; <li>店1毫升分装在管，于-20°C避免反复冻融。 </li></ol> 3。动物的制备<ol><li>微循环显微镜观察，准备了一个洞20毫米直径，和磁带洞超过30平方毫米的方形玻璃盖塑料板。 </li><li>麻醉鼠标，除去皮毛，准备的皮肤。放置在尾部使用地下30针连接到一个10厘米的聚乙烯（PE 10）导管肝素化PBS填充注射毒品的徒劳的导管。 </li><li>后正中切口，延长塑料板的主要肝脏小叶，置于胸骨斜卧在鼠标。 </li><li>厨房保鲜膜（3毫米x 8毫米）和周围边缘的的肝叶抑制肝脏与呼吸运动，并保持干燥瓷砖他们准备的小单。 </li><li>在显微镜下观察肝脏微循环（透射光），并确认了B载量流有没有在视野瘀至少15分钟。 </li><li>慢慢地注入0.2毫升的血流量观察钯TCPP的PO 2测量的解决方案或0.2毫升的荧光标记的红细胞。这FITC标记的红细胞数量将占到1/50的可视化区域中的所有流通中的红细胞。 </li></ol> 4。血流量的可视化<ol><li>汞灯光源，通过一个带通滤波器（450-490纳米）1通过辐照激发FITC标记。 </li><li>与CCD相机记录的荧光图像。 </li></ol> 5。 PO 2测量 PD-TCPP的磷光相对薄弱，应检测的高灵敏度探测器。所有实验必须在一个黑暗的房间中进行。 <ol><li>钯TCPP的吸收峰在410 nm和532 nm，激发二次谐波的波长532纳米的建议。这可以产生波长的Nd：YAG脉冲激光2。 </li><li>喂梁钕：YAG激光，到相应的端口上的倒置显微镜和调整的光斑在焦平面的中心位置。空间分辨率取决于激光光斑大小，这是通过针孔梁，改变。 </li><li>要检测的磷光，重视长期通滤波器（620纳米）和光电倍增管（PMT）显微镜的其他港口。光电倍增管，应该​​是红色的波长敏感，尤其是700纳米左右。 </li><li>品尝的磷光信号（在200 kHz的采样500点），并计算拟合指数函数衰减衰减时间常数。 </li><li>校准准备PO 2 150毫米汞柱，0毫米汞柱两个钯TCPP的解决方案的样品。添加亚硫酸钠钠量的1％来样生产缺乏氧气。保持在7.4样品的pH值和温度在校准过程中在37°C TURE。 </li><li>修正的Stern-Volmer常数k q和无氧铜发光衰减时间τ0在方程（1）3。在我们的情况下，与钯TCPP的解决方案，在37℃，PH值为7.4，K q是374毫米汞柱-1 s -1和τ0为0.74毫秒。这些测量参数取决于设备（如激光器，探测器，其他光学装置）和信号处理方法，所以需要校准在每个测量系统4。 τ0 /τ= 1 + K Q•τ0•PO 2（1） </li><li>设置鼠标板显微镜舞台上，观察肝脏微循环，调整目标微血管的位置，激光光斑点。 </li></ol> 6。肝病模型<ol><li>免费获得隔夜水快小鼠。 </li><li>准备一个20毫克每毫升溶液对乙酰氨基酚（APAP）在DMSO。 </li><li> 1 ml/100，Ğ体重（即200毫克/公斤体重）腹腔注射注射。注射后，鼠标就可以免费获得食物和水5。 </li><li>注射后24小时，麻醉鼠标和做正中切口，暴露肝脏。如果实现了肝炎，坏死在pericentral地区将是很容易以肉眼可见。 </li></ol> 7。代表结果例如肝脏微循环图像如图1所示，其中（a）是透射光图像和（b）是一种荧光图像。个人FITC标记的红细胞是在视频电影，观察和门户静脉，血窦和中央静脉血流方向确认。 正如图2所示，激光光斑照射通过X100物镜的中央小静脉approximately直径为10微米。在雄性C57BL / 6小鼠在肝脏微循环的血管PO 2测量， 图3（a）显示PO 2，在门户网站和中央静脉血管直径之间的关系。从门户到中央静脉PO 2梯度表明，红细胞释放出氧气，有效地通过血窦传递时。在图3（b）所示，PO 2平均为59.8毫米汞柱门户的船只，在血窦48.2毫米汞柱，在中央静脉和38.9毫米汞柱。 当我们生产的注射乙醯胺酚腹腔注射的急性肝炎，血管内埔2显着升高肝脏微循环的每个部分：在门静脉和血窦和中央静脉（ 图3（c））P &lt;0.01 P &lt;0.05。 <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="图1" /> 图1。低倍率我法师（一）透射光及（二）荧光显示肝脏微循环。 <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="图2" /> 图2。激光照射有针对性的中央小静脉PO 2测量点的图像。 <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="图3" /> 图3 PO 2在健康对照组和对乙酰氨基酚引起的肝损伤小鼠肝微循环障碍的梯度。 （一）表明PO 2与血管直径为门户静脉（PV）和中央静脉（CV），（b）显示了通过血窦（）氧梯度从光伏到CV。 PO 2 PV和CV之间的差异，反映了肝细胞或其他实质细胞的耗氧量。 （c）所示，PO 2在APAP管理引起的肝炎的条件，在光伏，S，CV和PO 2显着升高梯度下降，表明肝氧耗被攻破。

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我们什么都没有透露。

组织提供氧气，是微循环的重要作用，和很多文件描述微血管解剖和流变6有关的疾病。肝血流的动脉和静脉血液的组合。肝脏的血流量约30％是提供由门静脉，肝动脉和其他的70％提供的含氧血，脾和消化道7不佳的含氧的血液从静脉流出。肝脏疾病，如脂肪肝，休克，或肿瘤在肝脏微循环的血流量变化的。为了确定组织氧合，需要测量的微循环血流量和氧浓度。市售仪器的缺乏，...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>钯（II）meso-四（4 - 羧基苯基）卟啉</td><td>科学前沿，公司</td><td> PdT790 </td></tr><tr><td>异硫氰酸荧光素异构体的我</td><td> Aldrich公司</td><td> F7250 </td></tr><tr><td>对乙酰氨基酚</td><td> Aldrich公司</td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>光电倍增管</td><td>滨松光子学株式会社</td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

因为肝脏耗氧量是比较高的，但约有70％的肝脏血液供应不足含氧从胃肠道，脾的门静脉血液，在肝脏中的氧气供应和需求之间有一个相当大的差异。氧气从门静脉的终末支流入通过血窦中央小静脉血液运送到肝，这使得在肝小叶的氧气梯度。氧梯度是一个重要的物理参数，包括酶在肝脏微循环的上游和下游的表达，但在肝脏微循环的氧气消耗测量技术的缺乏，已经推迟了与氧气在肝脏代谢机制的澄清。因此，我们用FITC标记的红细胞，肝脏微循环的可视化和使用激光辅助磷光测量氧气在微血管氧分压。 noncontaCT和可以量化的连续光学测量血流速度，血管直径，有关肝脏的氧气消耗的氧气梯度。在急性肝炎模型中，我们通过管理对乙酰氨基酚对小鼠，我们发现增加了门户网站和中央静脉氧气压力，但在血窦减少氧梯度，表明肝细胞坏死在pericentral区域转移氧气压力和影响酶的表达在汇管区。总之，我们的光学测量肝血流动力学和氧消耗的方法可以揭示有关肝病的机制。

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

光学系统的开发，可视化与FITC标记的红细胞肝脏微循环和氧气在微血管局部压力测量激光辅助磷光。这种方法可以用来研究生理和病理机制，通过分析微血管结构，直径，血流速度和氧张力。

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

visualization-analysis-blood-flow-oxygen-consumption-hepatic

Research

JoVE Journal

Medicine

12.5K 次观看.  Keio University. 光学系统的开发，可视化与FITC标记的红细胞肝脏微循环和氧气在微血管局部压力测量激光辅助磷光。这种方法可以用来研究生理和病理机制，通过分析微血管结构，直径，血流速度和氧张力。

视频: Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

发光法小鼠肝肿瘤模型

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

科研

医学

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

在急性肝衰竭异位辅助性大鼠肝移植流量调节门静脉动脉

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

蛛网膜下腔出血急性和后遗症的研究兔血分流型号：技术问题

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

超声造影成像脊髓血流量的评估实验脊髓损伤

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.