作者感谢她的技术援助和执行组织学克里斯蒂安Pahrmann。

<html>年龄在8至12周的最低重量为20g的BALB / C小鼠均购自查尔斯河（Sandhofer WEG 7，D - 97633 Sulzfeld）。 小鼠被安置在常规条件下，美联储的标准鼠标颗粒和水自由采食 。麻醉与异氟醚使用感应室（2％）的鼠标。 <ol><li>剃除颈部，左侧的肋骨和消毒用80％乙醇。 </li><li>在它的后面放置鼠标放在它的鼻子和嘴的口罩，以保持麻醉。 </li><li>检查的反射捏尾巴和后腿的脚，以确保足够anesthesized鼠标。 </li><li>在微观执行颈中线切口，分离皮肤，肌肉和组织覆盖气管。 </li><li>当暴露气管切开一个小孔，插入气管导管（图1）进入组织之间的声门以下两个墨盒环。插入气管导管，持有使用微型外科forecepts的气管颅。检查胸的运动，以确保通风良好，两肺。 </li><li>呼吸频率（RR）应约每分钟110，与17日至18厘米H 2 O的吸气压力小心打开鼠标，躺在其右侧，面对它的左边。执行第3和第 4肋骨之间的leftsided开胸，并仔细解剖组织和肌肉，使用考特，以防止出血。 </li><li>小心打开胸廓，一旦打开胸腔，发现心脏，没有任何尖锐的物体接触肺。现在，删除的部分，是覆盖在心脏的心包囊。 </li><li>位于法援署之间的肺动脉和左耳廓。使用8-0普理灵缝合（爱惜康，日本东京，德国）结扎的LAD近端与一个单一的缝合（图2）。 </li><li>放置胸管（28G，腐败的导管）， 第 4和第 5肋骨之间。 </li><li>关闭层的胸切口，用6-0普理灵运行缝线（爱惜康，日本东京，德国），以适应肋骨和4-0普理灵运行缝线（爱惜康，日本东京，德国），以接近皮肤。 </li><li>漏了一个2ml注射器小心（图3）的帮助下胸部。将鼠标在它的后面。立即采取气管插管和气管墨盒戒指适应单一使用7-0普理灵缝合针（爱惜康，日本东京，德国）。 </li><li>放在鼠标上的口罩，并关闭皮肤用4-0普理灵运行缝线（爱惜康，日本东京，德国）。 </li></ol>验证有不同的可能性，确认成功的LAD结扎。 肌钙蛋白测试可以进行手术后6至18小时，使用，而不需要安乐死的动物中，只有150升血液。血是适用于一个定制的肌钙蛋白检测试剂盒（热带T敏感，罗氏，曼海姆）（图4）。肌钙蛋白是一种调节蛋白在肌肉细胞的肌动蛋白丝，有肌肉的三种不同的亚型。 cTnI和cTnT的亚型被发现在心里，并释放组织损伤。两次心脏特异性单克隆抗体，一个陷阱，在血液样本肌钙蛋白肌钙蛋白T（cTnT）的标准化测试的基础上，第二个是一个标记。 梗死区可确定宏观后第3天（图5）。 TTC（2,3,5 - 三苯基氯化）染色措施，组织活力用于评估心肌梗死面积。伊文思蓝染料（1,5％，1.0mL），磷酸盐缓冲液（PBS）进入左心室腔注入测量心肌缺血区。鼠标是安乐死的心是收获和分段成片。组织切片在37℃孵育时间为20分钟在1％TTC PBS液，pH值7.4。组织固定在10％PBS缓冲福尔马林一夜之间在2-8 ° C。 TTC是管理的体外染料伊文思蓝阴性区（图6）。 对于组织学的鼠标已经被安乐死的心已被嵌入作进一步处理。执行石蜡切片后，与H＆E（苏木精和曙红）染色幻灯片或三色可视化纤维组织（图7）。 <img src="/files/ftp_upload/1438/1438fig1.jpg" alt="图1" /> 图1：气管套管。 1A：暴露气管，颈椎后腹切口。 1B：气管开了两个尾鳍喉软骨环。 1C：气管导管插入进气管。 <img src="/files/ftp_upload/1438/1438fig2.jpg" alt="图2" /> 图2：通过左侧开胸暴露心脏，在LAD结扎。 T“&gt; <img src="/files/ftp_upload/1438/1438fig3.jpg" alt="图3" /> 图3：后关闭胸腔，胸膜漏。鼠标仍然在麻醉状态下。 <img src="/files/ftp_upload/1438/1438fig4.jpg" alt="图4" /> 图4：两条线路，几分钟后出现时肌钙蛋白测试为阳性。 <img src="/files/ftp_upload/1438/1438fig5.jpg" alt="图5" /> 图5：显示explanted的心 。 5A：宏观的角度与心脏缺血区的一个褪了色发白的颜色。 5B：横截面梗死心脏缺血区显示在右上角。

<ol>
	<li>Atkins, B. Z., Hueman, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+cardiomyoplasty+improves+diastolic+properties+of+injured+Heart.">Cellular cardiomyoplasty improves diastolic properties of injured Heart.</a> J Surg Res. 85 (2), 234-242 (1999).</li><li>Jaquet, K., KT, K. r. a. u. s. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+myocardial+scar+size+after+implantation+of+mesenchymal+stem+cells+in+rats:+what+is+the+mechanism.">Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism.</a> Stem Cells Dev. 14 (3), 299-309 (2005).</li><li>Joung, B., Kim, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mononuclear+stem+cells+transplanted+in+rat+infarct+myocardium+improved+the+electrical+conduction+without+evidence+of+proarrhythmic+effects.">Bone marrow mononuclear stem cells transplanted in rat infarct myocardium improved the electrical conduction without evidence of proarrhythmic effects.</a> Yonsei Med J. 48 (5), 754-764 (2007).</li></ol>

资助本研究是由汉堡大学医院（MK，女适应训练格兰特），东风集团（SS，SCHR992/3-1），ISHLT（TD，2009年研究奖）的支持。 声明所有的动物都收到符合实验动物保健的原则“的人文关怀，对动物的实验是在由国家医学研究和指南学会出版的”实验动物的关心和使用规定的准则和法规美国国立卫生研究院（美国国立卫生出版物85-23研究院，1985年修订）。 所有动物均来自查尔斯河实验室（德国Sulzfeld，），并保持在汉堡大学医院的Eppendorf的动物保健设施。动物标准的议员和水自由采食。

综上所述，本视频一步一步的LAD结扎模型可视化和标识在手术过程中很容易可行的小鼠法援署义务。

lad-ligation: a murine model of myocardial infarction

梗死和心肌缺血的研究模型是必不可少的调查在心肌缺血的急性和慢性的病理生物学和病理生理过程，并开发和优化未来的治疗。 两种不同的方法建立心肌缺血是在啮齿类实验动物。第一种方法是创建一个快速但不精确的技术，冷冻梗死，其中一个冷冻笔的心（1-3）表面上应用。科学家使用这种方法不能保证冷冻疤痕导致缺血，也显示病理生理方是不相关的心肌梗死的效果，创建了广阔的心肌损伤。第二种方法是永久性结扎左冠状动脉前降支（LAD）。这里法援署是一个单针结扎，形成了一个几乎立即可以看到的缺血。关闭法援署，没有进一步的血流量是允许在这一领域，而周围的心肌组织中几乎没有受到影响。这种外科手术模拟发生在梗死心肌缺血的病理生物学和病理生理方面。 在这个视频中介绍的方法演示鼠标梗死模型的外科手​​术结扎法援署。这种模式是心肌梗死的病理生物学和病理生理以及免疫生物学研究的方便。显示技术提供高精确度和关联以及与组织学切片。

Watch this Scientific Journal Video about LAD-Ligation: A Murine Model of Myocardial Infarction at JoVE.com

LAD-Ligation: A Murine Model of Myocardial Infarction

本视频演示了如何使用快捷及可靠的模型来研究心肌缺血的病理生物学和病理生理过程。

法援署结扎：有心肌梗死的小鼠模型

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

lad-ligation-a-murine-model-of-myocardial-infarction

Research

JoVE Journal

Biology

44.4K 次观看.  University Heart Center Hamburg. 本视频演示了如何使用快捷及可靠的模型来研究心肌缺血的病理生物学和病理生理过程。

视频: LAD-Ligation: A Murine Model of Myocardial Infarction

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

小鼠垂直套胃切除术的动物模型

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

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.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...