这项工作得到了国家，路易斯安那LEQSF（2009-12）的RD-A 19（有价证券：Murfee轮候册）的评议委员会和杜兰高血压和肾的卓越中心由NIH资助资助P20RR017659-08（有价证券L.加布里埃尔Navar）。

1。手术过程中设置说明<ol><li>手术前消毒手术用品和工具。每只大鼠的无菌手术中使用的物资包括1悬垂到layed的作为安置仪器的无菌表面，一个悬垂在中心约0.5×1.5预切孔被放置在老鼠和纱布。预切孔上的悬垂性，对大鼠切口对齐。手术所需的仪器，包括1标准对剪刀被用来切割缝合材料，两钳和罚款处理和揪心的缝合持针器，并用刀片的手术刀。我们已列出了具体手术材料和工具表在我们的实验室中使用的常用工具。然而，最终工具的选择取决于实验者的喜好。 </li><li>准备手术空间。加热垫置于100 mL 0.9％的无菌生理盐水袋，以确保接触瓦特的生理盐水随着肠系膜组织预先加热到大约37°C。作为替代生理盐水，你可以使用林格氏液，或其他生理缓冲。在一个易于访问的外化过程中的无菌的悬垂性，奠定了消毒的手术器械和用品。此外，您将需要无菌棉棒喷头和适当的3个类型的缝线（4-0，5-0，7-0）。使确保包预先打开，使物料可以用无菌处理访问。最后，在100％的乙醇浸泡至少5分钟消毒前的改性塑料阶段。舞台是100毫米Petri网盘中心（ 图1）剪一个椭圆形的孔。最初，如果有必要，扩大孔，可用于一个DREMEL工具。后孔，切割塑料的边缘是光滑用沙纸不同的晶粒。的阶段，然后洗净，最后，无论是惰性造型粘土（）或从当地的工艺品商店购买silic胶孔的边缘，以提供一个凸起，表面光滑。之前与组织联系，舞台将需要充分用无菌生理盐水冲洗。 </li><li>对于这种血管生成的模型，我们通常使用成年雄性Wistar大鼠（350±25克）。其他大鼠品系和年龄都可以使用。在我们的实验室，通过与氯胺酮（80毫克/公斤体重），甲苯噻嗪（8毫克/公斤体重），阿托品（0.08毫克/公斤体重）肌肉注射麻醉大鼠。大约5分钟后，麻醉效果证实，大鼠的腹部皮肤被剃光。 </li></ol> 2。大鼠肠系膜外化模型<ol><li>放置在其上一个加热垫背面的麻醉大鼠，以保持体温。在手术过程中使用无菌技术。戴无菌手套，不允许仪器及用品的接触大鼠组织以外的非无菌面。 </li><li>清洁腹部交替湿巾USI吴无菌纱布浸泡在70％异丙醇和碘。 </li><li>用手术刀刀片，使皮肤小（约0.75英寸）沿纵行切口，然后白线下方约1英寸的胸骨。 </li><li>将预切过腹节悬垂性的开放，使切口对齐。 </li><li>轻轻涂抹切口周围的压力。这通常会导致小肠，它可以很容易地确定曝光。 </li><li>使用的棉尖的喷头，轻轻拉出一段小肠和定位回肠。由于肠系膜被拉出，应轻轻layed在塑性阶段（ 图2）。 </li><li>确定6-8血管肠系膜窗口。一个的肠系膜窗口定义为喂养小肠（ 图2）之间的动脉/静脉双薄的半透明膜。 </li><li>记录的外化的起始时间。离开了mesenteRY部分形象化了20分钟。的外化期间，使用无菌注射器（5毫升），间歇性地在培养皿中补充，以确保该组织仍然沉浸并没有干出的盐水。 </li><li>在20分钟的曝光时间，庆祝位于市中心的无菌7-0缝合2肠系膜窗口。应在附近的肠道脂肪面积的痕迹。 </li><li>腹腔返回肠系膜形象化的地区。 </li><li>在中断模式用5-0单丝缝合关闭的腹部肌肉。 </li><li>在中断模式4-0单丝缝合关闭皮肤。 </li><li>交替使用无菌纱布浸泡在70％isopropoyl和碘的抹布擦拭缝合区。 </li><li>涂在大鼠双眼眼润滑油凝胶。润滑眼睛的目的是为了防止在手术和恢复角膜干燥。应适用于眼部润滑剂的应用前外科干预的开始。如果有必要，重新申请前眼部润滑剂回到复苏的老鼠笼子。 </li></ol> 3。组织收获和固定<ol><li>在一天的利息后的刺激，麻醉和安乐死大鼠。在我们的实验室中，大鼠麻醉通过肌肉注射氯胺酮（80毫克/公斤体重），甲苯噻嗪（8毫克/公斤体重），阿托品（0.8毫克/千克体重），然后通过腔内beuthanasia注射安乐死。 beuthanasia是1戊巴比妥钠/苯妥英的解决方案，可用于快速，无痛安乐死。我们的实验室通常每鼠注射0.1 - 0.2毫升。 </li><li>重新打开腹腔，轻轻取出小肠，并找到了两个显着的窗口。 </li><li>切出的形象化的的肠系膜使用镊子和微型剪刀窗口。包括脂肪每个窗口的边界是为以后组织到显微镜玻片上蔓延有利。立即，沉浸在PBS中的每一个窗口。切割时，尽量避免通过动脉/静脉双切割脂肪的边界内的窗户，以尽量减少潜在的血液，填补窗户。此外，尽量减少切割通过肠道，导致肠内容联系的窗口。 </li><li>使用镊子正电的载玻片上传播组织。两个组织可以安装在每张幻灯片。 </li><li>允许组织部分干燥，用手术刀刀片去除多余的脂肪。 </li><li>组织现在已经准备好要固定。典型的固定方法都可以使用。在我们的实验室中，我们通常固定在甲醇（-20℃）30分钟的幻灯片。不固定的组织也已进行成功的标签。固定方法可能取决于您的首选抗体。 </li></ol> 4。组织免疫标记<ol><li>组织现在一般可以根据指示，每抗体进行免疫组化标记。鉴于我们的利益，在确定的角色血管周细胞在血管生成过程中，我们的实验室常用标签的内皮细胞和血管周围细胞10，12，13。有用的标签，以确定沿血管内皮细胞的微血管网络的层次结构，是一种抗粘附分子抗体或BSI-凝集素。在这个组织中使用的标记，包括血管周围细胞NG2的，结蛋白，SMα-肌动蛋白，PDFGRβ，和III类β-微管蛋白。下面，我们在3.3节中，已列出比色法和荧光粘附分子免疫标记协议的协议。 </li><li>最初的主要抗体孵化之前，幻灯片是真空干燥以去除多余的缓冲溶液。此外，组织最初概述用腊笔，以防止​​不受控制的流走，从组织的抗体解决方案。最后，所有标签的​​步骤，其次是洗涤步骤。洗涤步骤，幻灯片放在内染色罐子。交换缓冲溶液中洗涤时间的3倍。 </li><li>内皮细胞抗体标记的程序：&lt;类p =“jove_step”的&gt;粘附分子色度Lableing <ol><li>主要抗体孵育：滴在上面每个组织约100-200毫升的主要抗体溶液（1:200小鼠单克隆生物素标记的CD31抗体稀释抗体缓冲区（在PBS 2％BSA的0.1％皂素））;确保整个组织覆盖抗体的解决方案。在室温下为1小时的潜伏期。 </li><li>用30分钟PBS 0.1％皂素组织。 </li><li>二次抗体孵育：滴灌二级抗体溶液（VECTASTAIN精英ABC媒介实验室的解决方案;链霉亲和素过氧化物酶二级抗体溶液）在组织之上。在室温下孵育1小时。 </li><li>用30分钟PBS 0.1％皂素组织。 </li><li>孵育15分钟媒介新星红组织。然后上升与水的组织。 </li><li>山幻灯片：幻灯片在95％乙醇浸10倍。浸泡2分钟的滑动在100％的乙醇。在另一个100％的乙醇浸泡幻灯片，以便lution为2分钟。然后浸泡在连续100％二甲苯的解决方案，每次2分钟的幻灯片。晾干并覆盖薄的VectaMount层和盖玻片的组织。 </li></ol>粘附分子荧光标记<ol><li>主要抗体孵育：滴在上面每个组织约100-200毫升的主要抗体溶液（1:200小鼠单克隆生物素标记的CD31抗体稀释抗体缓冲区（在PBS 2％BSA的0.1％皂素））;确保整个组织覆盖抗体的解决方案。在室温下为1小时的潜伏期。 </li><li>用30分钟PBS 0.1％皂素组织。 </li><li>二级抗体孵化：滴灌二级抗体溶液（顶部组织（1:100链霉亲和素-CY3抗体缓冲稀释在PBS +2％牛血清白蛋白（0.1％皂素）），室温孵育1小时。 </li><li>用30分钟PBS 0.1％皂素组织。 </li><li>山幻灯片：50:50 PBS甘油组织上滴。覆盖盖玻片。然后，使用指甲油密封盖玻片和幻灯片之间的差距。 </li></ol></li></ol> 5。代表结果粘附分子标记的大鼠肠系膜免疫组织的代表图像显示在图3。粘附分子的标签标识沿改造微血管网络的层次结构的所有船只类型，可以用来量化在特定时间点后刺激血管生成的指标。粘附分子标记也允许的动脉与静脉的决心。喂养动脉通常表现出更小的直径相比配对静脉（ 图4）和拉长内皮细胞形态。毛细血管和毛细血管豆芽可以根据他们的血管直径和网络内的相对位置确定。重塑网络的典型特征，包括增加毛细血管发芽，血管密度，血管区和小静脉tortuosITY。各种血管生成的指标量化识别网络增长（ 图5）的时间过程。毛细管从发芽前的现有船舶，山峰之间的第3天及5天，10天的无刺激水平的回报。其次是增加血管密度和血管面积在萌芽这短暂的增加。作为证据为重塑较大的船只，在这个模型中，小动脉和小静脉段的人数也增加了时间当然。 在我们的实验室中，这种模式已被用于确定在特定时间点的细胞表型的改变，在这个重塑的过程10，11。例如，第三类β-微管蛋白标识沿着新生血管周细胞（ 图6）。在未受刺激的组织，Ⅲ类β-微管蛋白表达神经具体。相反，在毛细血管发芽的高峰期，Ⅲ类β-微管蛋白表达的血管周围细胞。这种类型的结果凸显了这个简单而强大的血管模型，以确定新的血管生成过程中所涉及的细胞类型。 <img alt="图1" src="/files/ftp_upload/3954/3954fig1.jpg" /> 图1。塑料阶段前和修改后的图片。修改前的阶段，是一个100毫米的培养皿。修改包括在该中心的椭圆孔切，随后除了造型粘土或硅胶孔的边缘，为创造一个凸起，表面光滑。这表面提供了一个内部​​边界灌流有助于形象化肠系膜窗口。比例尺为1厘米。 <img alt="图2" src="/files/ftp_upload/3954/3954fig2.jpg" /> 图2。形象化的肠系膜区域的图像。被定义为薄的半透明膜之间的动脉/静脉喂养对小肠肠系膜窗口。在“外化持续时间，肠系膜区域布局，并在盐碱地改良培养皿内浸泡。惰性黄色的橡皮泥，提供了一个通过预切孔拉肠系膜表面光滑。比例尺为1厘米。 <img alt="图3" src="/files/ftp_upload/3954/3954fig3.jpg" /> 图3。无刺激的组织和组织，在3和10天的后外化肠系膜肠系膜微血管网络的代表图像。粘附分子标记的微血管网络的层次结构，包括动脉（一），静脉（V）和毛细血管（三）确定。邮政刺激，微血管网络显示在毛细管发芽（箭头元首）和血管密度的增加。比例尺是100微米。 <img alt="图4" src="/files/ftp_upload/3954/3954fig4.jpg" /> 成年大鼠肠系膜微血管网络内动脉/小静脉对代表影像图4。第动脉（一）在两个图像，可从静脉（五）有区别的基础上相对较小的直径和内皮细胞的形态拉长。标尺是20微米。 <img alt="图5" src="/files/ftp_upload/3954/3954fig5.jpg" /> 图5。以上的的微血管增长后肠系膜外化过程中的血管生成指标代表量化。一）血管面积每组织区域。毛细管豆芽每血管面积数）。 c）血管总长度每血管面积。 *代表未刺激组相比显着性差异。统计数据进行了比较唐恩的测试采用单因素方差分析。 （P &lt;0.05）。联合国代表无刺激。 <img alt="图6" src="/files/ftp_upload/3954/3954fig6.jpg" /> 图6。代表的荧光图像肠系膜微血管网络，无刺激的组织和组织在3日和10天的肠系膜的外化。免疫粘附分子标记（红色）标识的内皮细胞，Ⅲ类β-微管蛋白标签（绿色）标识神经（箭头元首）和血管周围细胞（箭头）。血管周围细胞瞬时上调Ⅲ类β-微管蛋白在毛细血管发芽。在未受刺激的微血管网络，第三类β-微管蛋白是特定的神经和血管周围细胞无法识别。后3天的刺激下，β-微管蛋白Ⅲ类积极标签沿微血管血管周围细胞。第10天，第三类β-微管蛋白的表达模​​式，开始返回到刺激时的场景。比例尺为50μm。 <img alt="图7" src="/files/ftp_upload/3954/3954fig7.jpg" /> 图7。图像支持跟踪预先标记的本地应用细胞在微血管肠系膜外化的网络刺激增长的可行性。细胞灌流超过mesenterIC窗口在20分钟的外化。 1天，手术后，DiI标记细胞（红色），观察在圣母院焦平面与粘附分子阳性微血管（绿色）。 ，A，B）DiI标记的骨髓细胞的例子展示圆形和拉长的形貌。在某些情况下，被拉长沿微血管细胞（箭头）。 c）标记的乙酰集群为例毛细管萌芽（箭头）的尖端附近的间质干细胞。标尺是50微米（一），20微米（乙，丙）。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Mesentery+Angiogenesis+Assay.">Rat Mesentery Angiogenesis Assay.</a> J. Vis. Exp. (52), e3078 (2011).</li><li>Murfee, W. L., Rehorn, M. R., Peirce, S. M., Skalak, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+cells+along+venules+upregulate+NG2+expression+during+microvascular+remodeling.">Perivascular cells along venules upregulate NG2 expression during microvascular remodeling.</a> Microcirculation. 13, 261-273 (2006).</li><li>Stapor, C. P., Murfee, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+class+III+&#946;-tubulin+as+a+marker+of+angiogenic+perivascular+cells.">Identification of class III &#946;-tubulin as a marker of angiogenic perivascular cells.</a> Microvascular Research. , (2011).</li><li>Yang, M., Aragon, M., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+Mesenteric+Microvascular+Networks+from+Spontaneously+Hypertensive+Versus+Normotensive+Rats.">Angiogenesis in Mesenteric Microvascular Networks from Spontaneously Hypertensive Versus Normotensive Rats.</a> Microcirculation. , (2011).</li><li>Robichaux, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic/Blood+endothelial+cell+connections+at+the+capillary+level+in+adult+rat+mesentery.">Lymphatic/Blood endothelial cell connections at the capillary level in adult rat mesentery.</a> Anat. Rec. (Hoboken). 293, 1629-1638 (2010).</li><li>Amos, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+The+role+of+human+adipose-derived+stromal+cells+in+inflammatory+microvascular+remodeling+and+evidence+of+a+perivascular+phenotype.">IFATS collection: The role of human adipose-derived stromal cells in inflammatory microvascular remodeling and evidence of a perivascular phenotype.</a> Stem Cells. 26, 2682-2690 (2008).</li></ol>

我们什么都没有透露。

据报道，2006年的外化模型和改编自以前的机械性损伤大鼠肠系膜血管生成4-7模型，并产生类似的结果，以建立腹腔注射模型，利用大鼠肠系膜9。实验确定的外化时间20分钟，以产生一种强大的血管反应。而这段时间内可以改变，但它允许本地应用血管生成抑制剂的机理研究和直接应用外源性细胞的细胞系的研究4。支持将重塑肠系膜组织细胞纳入可行性初步研究，使用预先标记的骨髓细胞和骨髓基质干细胞（ 图7），在我们的实验室，并成功地调查了人体脂肪基质细胞注射IP 14的命运。在我们的实验室中，我们用这个模型来识别周细胞PHEN在血管反应时间10课程及评估血管的潜力，在病理条件下，如高血压12 otypic变化。血管反应和细胞表型相关的变化，这种模式也可以观察大鼠肠系膜血管模型，包括慢性低氧暴露10，11。 的外化模型的局限性，是引发血管生成的确切机制尚不清楚。肠系膜的外化与肥大细胞脱颗粒和增加6组胺水平，还需要作进一步调查，以获得更深入的了解。血管生成的刺激无疑是多因子，产生跨层次的微血管网络的强大的重塑反应。而未知的机制仍然是这种模式的主要批评，其重现性和简单性使其更具有吸引力识别细胞动力学国际富豪VED在本质上是复杂的毛细血管的出芽过程。支持该模型的可重复性（Wistar雄性和雌性SD）在先前发表的研究报告，12个实验室10多个大鼠株微血管网络增长的时间当然比血管可比指标。由于大多数成年大鼠肠系膜组织的血管，​​该模型还允许多个检查每个动物的组织。不幸的是，这个模型是没有明显适用于基因小鼠模型，小鼠肠系膜窗口少本地血管和，在我们的经验，普遍缺乏观察分支网络。未来的应用，包括使用在特定的时间点内的重要显微镜血管生成过程中的血管功能的调查，调查有关在淋巴管和神经细胞动力学。虽然每本地血管肠系膜窗口的程度似乎是柔ghly比例随着年龄的增长，我们已经观察到分枝4-5周岁的年轻雄性Wistar大鼠微血管网络。这些结果表明，外化模型也可以用于比较不同年龄的血管差异。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 附加说明 </td></tr><tr><td>悬垂</td><td>卡地纳健康</td><td> 4012 </td><td> 12“X12”生物盾定期消毒包装纸</td></tr><tr><td>诺伊斯微型剪刀</td><td> roboz手术仪器</td><td> RS-5677 </td><td>诺伊斯微弹簧剪刀解剖; 直，夏普钝点;13毫米切割边缘​​;尖端宽度0.25毫米，4 1/2“的总长度</td></tr><tr><td>格雷夫夹钳</td><td> roboz手术仪器</td><td> RS-5135 </td><td>微解剖钳;锯齿轻微的曲线;尖端宽度0.8毫米; 4“长度</td></tr><tr><td>格雷夫夹钳</td><td> roboz手术仪器</td><td> RS-5130 </td><td>微解剖钳;锯齿，直;尖端宽度0.8毫米; 4“长度</td></TR&gt; <tr><td> 4-0缝合</td><td>爱惜康</td><td> 699G </td><td> （1.5公吨）ETHILON尼龙缝线黑色抽丝</td></tr><tr><td> 5-0缝合</td><td>爱惜康</td><td> 8556 </td><td> （1.0公吨）普理灵Polyprolene缝合蓝抽丝</td></tr><tr><td> 7-0缝合</td><td>爱惜康</td><td> 1647G </td><td> （0.5公吨）ETHILON尼龙缝线黑色抽丝</td></tr><tr><td> castroviejo微持针器</td><td>罚款科学工具</td><td> 12060-02 </td><td>提示宽度：0.6 夹紧长度：5mm 长：9cm 直尖</td></tr><tr><td> castroviejo针持有人</td><td>罚款科学工具</td><td> 12565-14 </td><td>笔尖形状：直 提示宽度：1.5毫米 夹紧长度：10MM 剪刀：无 锁：有 长度：14cm 锯齿：是</td></tr><tr><td>手术刀手柄</td><td> ROBOž手术器械</td><td> RS-9843 </td><td>手术刀手柄，＃3;固体; 4“长度</td></tr><tr><td>无菌手术刀片</td><td>辛辛那提手术</td><td> 0110 </td><td>不锈钢;尺寸10 </td></tr><tr><td>培养皿</td><td> Fisher Scientific则</td><td> 08-757-13 </td><td>斜角岭，Slippable </td></tr></tbody></table> 表的具体手术的材料和工具。 <table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 附加说明 </td></tr><tr><td> beuthanasia </td><td>先灵葆雅动物保健公司联盟（MWI的兽医供应有序） </td><td> MWI的＃：011168 </td><td>活性成分：每100毫升，390毫克苯巴比妥钠，50毫克的苯妥英钠</td></tr><tr><td>氯胺酮</td><td>道奇堡动物健康（从MWI的兽医供应有序） </td><td> MWI的＃：000680 </td><td> kateset 100毫克/毫升</td></tr><tr><td>甲苯噻嗪</td><td>劳埃德。公司 （MWI的兽医供应有序） </td><td> MWI的＃：009307 </td><td> Anased 100毫克/毫升</td></tr><tr><td>盐水</td><td>赫士睿公司</td><td> 94-217-托德</td><td></td></tr><tr><td> PBS </td><td>西格玛</td><td> 011M8207 </td><td></td></tr><tr><td>皂甙</td><td>西格玛</td><td> BCBB4080 </td><td></td></tr><tr><td>粘附分子（CD31的） </td><td>屋宇署Pharmingen公司</td><td> 553371 </td><td></td></tr><tr><td>链霉亲和素-Cy3标记</td><td>杰克逊ImmunoResearch </td><td> 016-160-084 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>杰克逊ImmunoResearch </td><td> 096555 </td><td></td></tr><tr><td> VECTASTAIN精英ABC </td><td>媒介实验室</td><td> PK-6100 </td><td></td></tr><tr><td>矢量新星红</td><td>媒介实验室</td><td> SK-4800 </td><td></td></tr><tr><td> VectaMount </td><td>媒介实验室</td><td>的H-500 </td><td></td></tr></tbody></table> 特定试剂表

rat mesentery exteriorization: a model for investigating the cellular dynamics involved in angiogenesis

微血管网络的发展和重塑，是伤口愈合，炎症，糖尿病视网膜病变，肿瘤的生长和其他疾病的条件1，2的关键方面。通常网络的增长归因于血管生成，定义为新船的预现有船只的增长。血管生成过程中也可以直接联系到小动脉，毛细血管的血管周围细胞涂层和船只扩大收购定义。不用说，血管生成是复杂的，涉及多个玩家在细胞和分子水平3。了解如何微血管网络的发展需要确定的时空动态，以及在血管生成的时间当然网络的层次结构。此信息旨在操纵血管生长疗法的发展是至关重要的。 在这篇文章中描述的外化模型代表一个简单的，重复性的刺激模型需接上大鼠肠系膜血管生成。它改编自伤口愈合，在大鼠肠系膜4-7的模型，是另一种刺激，通过腹腔注射促血管生成剂8，9肠系膜血管生成。外化模式是有吸引力的，因为它需要最小的手术治疗，并在整个微血管网络三维可视化的组织，允许在一个相对短的时间过程中产生的戏剧性，在毛细管豆芽，血管面积和血管密度增加重复性单细胞水平上。刺激增长反映没有外国血管分子干扰的自然生理环境中的血管反应。采用免疫组化标记方法，这种模式已被证明非常有用的，在确定参与血管生成的新细胞事件。研究人员可以很容易地关联时间当然随着时间的推移SP重塑过程中的血管生成的指标ecific动态，如细 ​​胞表型的改变或细胞相互作用4，5，7，10，11。

Watch this Scientific Journal Video about Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis at JoVE.com

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

本文介绍了一种刺激的大鼠肠系膜血管生成的简单模型。该模型产生毛细血管发芽，血管面积和血管密度超过在组织允许EN面对整个微血管网络的可视化的单细胞水平的相对较短的时间当然急剧增加。

大鼠肠系膜外化：调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

rat-mesentery-exteriorization-model-for-investigating-cellular

Research

JoVE Journal

Biology

19.0K 次观看.  Tulane University. 本文介绍了一种刺激的大鼠肠系膜血管生成的简单模型。该模型产生毛细血管发芽，血管面积和血管密度超过在组织允许EN面对整个微血管网络的可视化的单细胞水平的相对较短的时间当然急剧增加。

视频: Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

科研

生物学

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

One-channel Cell-attached Patch-clamp Recording

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

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

单通道细胞贴附膜片钳记录

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

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...