作者感谢安德烈阿克斯特尔，伊藤聪，MD，杰夫Velotta，医学博士，格兰特霍伊特，罗伯特C ·罗宾斯博士，金钰，医学博士，添多伊尔，博士和斯坦福大学的小动物成像核心技术援助。作者还感谢支持兽医设备AM比克福德公司。支持这项研究是由美国国立卫生研究院的研究补助金（R01 HL - 75774，R01，R21 HL085743，CA098303 1K12 HL087746），加利福尼亚州烟草相关疾病的研究计划的加州大学（15IT - 0257和1514RT - 0169） ，并为加州再生医学（RS1 - 00183）研究所。新罕布什尔州是支持由美国心脏协会的奖学金。

1。单侧肢体缺血诱导<ol><li>此操作所需的手术工具包括：细尖头镊子，尖头镊子，弹簧剪刀，手术剪，持针器，并牵开。我们做我们自己的拉钩使用回形针，因为它是比市售拉钩小。术前高压灭菌器或热珠灭菌消毒这些工具。一个烧灼的工具和消毒罚款指出棉签将也需要这种手术。建议在操作过程中所需的技巧重新消毒的工具。 </li><li>当工具都准备好，放入含有1-3％，在100％的氧气，在流速为1L/min时异氟醚麻醉诱导室的鼠标。 </li><li>留在鼠标感应室，直到它是对外界刺激的反应迟钝。然后从动物感应室。建议从包装盒中冲洗麻醉前，打开盖子，以减少操作人员暴露，以异氟醚。 </li><li>然后仰卧位放置在上手术台前，动物和它连接到一个异氟醚的连续流。使用电动剃须刀，头发从后肢删除。应用脱毛膏彻底清除头发。 </li><li>鼠标放置在褶上手术台的加热垫仰卧位，并把它连接到一个异氟醚的连续流。一块磁带扩展和安全的后肢。一旦后肢是安全的，请用三个交替优碘和酒精擦洗皮肤。对于手术过程的其余部分，使用10X或20X放大倍率一个解剖镜下获得的后肢地区扩大。 </li><li>用细镊子和手术剪，使皮肤切口，约1厘米长，从膝盖向大腿内侧。 </li><li>使用磷酸盐缓冲液（PBS）的湿细尖的棉签，轻轻地刷了皮下脂肪组织周围的大腿肌肉。 </li><li>接下来，应用烧灼横向切割，并通过皮下脂肪组织的解剖，揭示了潜在的股动脉。 </li><li>用牵开器打开伤口，并获得更好的下肢血管。 </li><li>透过膜股鞘罚款钳和罚款指出棉签，轻轻捅破，暴露的神经血管束。 </li><li>然后，使用一个精细镊子和棉签清洁，解剖和分离股动脉，股静脉和神经在腹股沟附近的近端位置。剥离后，通过7-0丝线缝合下方的股动脉近端钢绞线。近端闭塞的股动脉，使用双海里。近端血管放置在伤口尽可能的配合，以假为第二领带的长度和干预段将被切断。 </li><li>从股静脉在远端位置靠近膝盖的分离股动脉。传递一个7-0下方的股动脉，腘动脉近端远端缝合链。闭塞的使用双海里的船只。 </li><li>闭塞远端与近端节的第一双海里的第二套的股动脉。第二套这种缝合线将用于攥在交易过程中的动脉。 </li><li>同样，扣人心弦的目的，闭塞近端与远端节的第一双海里的第二套的股动脉。 </li><li>断面的股动脉之间用细尖的棉签和一双弹簧剪刀的远端和近端节段。使用小心不要刺破股静脉壁。 </li><li>取出拉钩，用5-0薇乔缝线关闭切口。这些缝线不需要在以后的时间被删除，因为它们溶于自己。 </li><li>一旦关闭切口，放置在一个搭着回收笼在加热垫的顶部的动物和不断监视，直到清醒。 </li><li>动物后1小时恢复，进行激光多普勒血流灌注的步骤，以确认缺血诱导。 </li></ol> 2。激光多普勒血流灌注<ol><li>要开始的激光多普勒血流灌注一步，放入含有1-3％，在100％的氧气，在流速为1L/min时异氟醚麻醉诱导室的鼠标。 </li><li>留在鼠标感应室，直到它是对外界刺激的反应迟钝。然后从动物感应室。建议从包装盒中冲洗麻醉前，打开盖子，以减少操作人员暴露，以异氟醚。 </li><li>将连接到异氟醚的连续流前手术台动物。然后取出使用电动剃须刀，其次是必要的头发脱毛膏后肢头发。 </li><li>取出后的头发，放置在37℃加热5分钟下连续流异氟醚表面的动物。监控核心温度，以确保euthermia，随着温度的变化会显着影响灌注。 </li><li> 5分钟后，动物在无反射光吸收的表面，如绿色的布，连接到一个异氟醚的连续流动，仰卧位。扩展的后肢。 </li><li>接下来，打开激光多普勒成像仪和数据采集软件和初始化软件。指定大小的观点和解决的领域。这是最好的动物之间保持一致的观点和像素密度领域，以便使将来的分析更容易。 </li><li>打开一个新的文件。按开始着手获取的图像数据。一般来说，热像仪自动检测鼠标的距离，但如果出现提示，指定动物的距离的成像。激光将显示视野的边界，然后开始采集数据。 </li><li>收购完成后，图像将开始显示，灌注到腿的血液水平的指标的颜色范围。颜色可以设置到一个特定的灌注范围，以便更好地与动物之间进行比较的数据。 </li><li>当数据采集完成后，保存文件。 </li><li>然后返回的回收笼的动物和动物连续监测，直到清醒。 </li><li>的数据进行分析，利用分析软件的门槛调整，减去任何背景噪音。 0.2阈值通常是一个合理的值。选择2个区域的利益，或投资回报，其范围包括每个后肢面积。可用于各种地标，规范四肢和动物之间的投资回报率。然后确定的投资回报率平均灌注性和多变性。灌注之间的差异和肢体缺血和控制肢体血流比值，然后可以很容易地确定。 </li><li>重复此过程可遵循后肢灌注的变化，随着时间的推移。在所需的时间点，动物可以安乐死评估组织的功能和灌注图像数据相比。 </li></ol> 3。代表性的成果： 后肢血管的解剖结构显示在图 1 2。后肢股动脉explantation后的一个代表图如图2所示。为了确认后肢缺血诱导，激光多普勒血流灌注图像分析表明以肢体缺血的血流量急剧减少，在控制肢体比较，如图3所示。 <img src="/files/ftp_upload/old/1035_2009_1_26_9/figure1_1035.png" alt="图1" width="600" height="406" /> 图1。后肢血管解剖。星号表示，为肢体缺血诱导结扎的位置。 <img src="/files/ftp_upload/old/1035_2009_1_20_7/Niiyama_HLI_Fig2.png" alt="图片2" width="600" height="450" /> 图2显示后肢的解剖结构去除后，股动脉的近端和远端站点的股动脉结扎后的代表图。 <img src="/files/ftp_upload/old/1035_2009_1_20_8/Niiyama_HLI_Fig3.png" alt="图3" width="600" height="256" /> 图3激光多普勒图像显示缺血诱导的左后肢（箭头指示）之前和之后的血流量。

<ol>
	<li>Huang, N. F., Niiyama, H., De, A., Cooke, J. P., , . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+non-invasive+tracking+of+embryonic+stem+cell-derived+endothelial+cells+for+treatment+of+hindlimb+ischemia.">Transplantation and non-invasive tracking of embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia.</a> J Vis Exp. , (2008).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The anatomy of the laboratory mouse. , (1976).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Dokun, A. O., Keum, S., Hazarika, S., Li, Y., Lamonte, G. M., Wheeler, F., Marchuk, D. A., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117, 1207-1215 (2008).</li></ol>

有一些变异来源的考虑，同时规划和执行后肢缺血模型。首先，缺血的水平可能会有所不同，根据结扎偏科的位置。因此，动物模型的一致性，应接受动脉结扎的同一水平。缺血性恢复的变化的另一个来源是动物的年龄有关，与年轻的动物，具有更快和更完整的回收率比年长的动物（8-10个月大的）（6-8周龄），作为评估血流动力学（即激光多普勒血流灌注）或功能（如跑步机测试）的措施。其中之一是评估血管生成剂的研究，它可能是最好使用旧的动物，因为群体之间有较大的差异，可能与干预治疗观察。相反，研究，其中之一是评估一个抗血管生成的因素，它可能是可取的使用年轻的动物，最大限度地发挥规模效应3。 如果正确执行，应该有最小的并发症如出血，感染，或死亡率等。如果出血发生意外中断，通过股静脉或其他船只，适度的压力，用无菌棉放倒撒施或纱布，应适用于出血部位，直到出血停止。动物感染的迹象，应与抗感染药物治疗。动物表现出显着性坏疽可能需要beeuthanized。这种并发症是较常见的老年动物，并在某些菌株，如BALB C小鼠4。此外，后肢缺血模型可能会导致轻度至中度疼痛。因此，止痛药，如丁丙诺啡或卡洛芬应管理作为疼痛治疗的需要，根据IACUC的建议。 总之，我们已经证明了一个简单和可重复性的方法研究在小鼠后肢缺血模型的PAD。后肢缺血模型，用激光多普勒成像分析是一个优秀的系统，为研究血管病症和评估治疗候选人。

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murine model of hindlimb ischemia 

在美国，周围动脉疾病（PAD）影响约10万个人，是全世界也普遍存在。为缓解症状的药物治疗是有限的。手术或血管内介入一些个人是有用的，但长期的结果往往令人失望。因此，有需要开发新的疗法来治疗PAD。小鼠后肢缺血准备的PAD的典范，是用于测试新疗法。当相比，其他车型的组织缺血，如冠状动脉或脑动脉结扎，结扎股动脉提供了一个简单的模型缺血组织。这个模型的其他优点是易于访问股动脉和低死亡率。 在这段视频中，我们展示了单方面hindimb缺血小鼠模型的方法。将描述创建和评估模型的具体材料和程序，包括肢体灌注的激光多普勒成像技术评估。此协议也可以用于移植和非侵入性的细胞，这是由黄等证明。的跟踪。

Watch this Scientific Journal Video about Murine Model of Hindlimb Ischemia at JoVE.com

Murine Model of Hindlimb Ischemia

单侧后肢缺血诱导的外科手术，激光多普勒血流灌注显像缺血确认证明。

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 ...

Research

JoVE Journal

Biology

Murine Model of Hindlimb Ischemia 

57.3K 次观看.  Stanford University. 单侧后肢缺血诱导的外科手术，激光多普勒血流灌注显像缺血确认证明。

视频: Murine Model of Hindlimb Ischemia

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

围产期缺血缺氧缺血性脑病模型

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

科研

生物学

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

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 ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

传统美国宇航局后肢卸小鼠模型的一个交替的方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

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 ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

用异体皮肤移植重建的烧伤伤口的穆林模型

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

ApoE小鼠后肢缺血的改进手术模型-/- 使用微型切口

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...