由瑞典医学研究理事会（批5942）的大部分研究提供财政支持。

1。动物<ol><li>小型啮齿动物，如大鼠，小鼠和豚鼠，已被用来作为动物模型。不幸的是，成年小鼠（许多小鼠品系已评估）往往缺乏或有肠系膜窗口内只有一个潜在的父微血管非常低的数量（可以从血管生成起源），这使得研究使用小鼠不可靠的。因此，法已开发主要是老鼠。 </li><li>老鼠，这是水土不服至少7天，从饲养员的交付标准的环境，通常是在对下一个12小时的光/暗周期免费使用水和颗粒封装在一个标准的笼子。每个动物标记。我们使用的大多是年轻的成年大鼠（通常7-10周的年龄不同菌株，但通常雄性Sprague - Dawley大鼠，大鼠比5-6岁的周年轻本土微血管的数量往往是低），从不同的育种者获得。在之前的实验，并在整个实验期间，动物称重每隔一天，总是在牺牲的日子。当动物被视为同时IP（诱导血管生成的，见下文）和SC或IV或PO，他们每天过磅。这使得等于体重的实验组和对照组在实验开始的设立，并允许监测治疗在实验过程中对人体体重增加的影响。由于成年雄性大鼠强劲增长，生理（获得约40-60克，每周根据应变式），体重增加迟缓是一个敏感的毒性替代标记，系统性福祉，厌食，并未能茁壮成长。 </li></ol>我们工作在一个高标准的动物护理设施和相当大的努力，以尽量减少动物所经历的压力水平。目前的道德准则是由工人体育等人建立的（在癌症研究中使用动物的福利和准则。溴。癌症研究102，1555年至1577年 ，2010 ）。哥德堡大学的动物伦理委员会批准了这些研究。 2。腹腔内促血管生成治疗<ol><li>要测试候选人促血管生成因子溶解和内毒素免费为病人输液用生理盐水稀释（即使在极低的水平，内毒素是促血管生成）。所有解决方案都新鲜一天注射，无菌过滤（0.22微米微孔过滤），中性pH值（7.1-7.3）检查，并在室温下使用。 测试剂注入浓度较低或很低的。例如，大鼠rVEGF 164（564-RV/CF;欧洲，R＆D系统有限公司，牛津郡，英国），这是主要的VEGF - A的异构体大鼠，稀释至96 pmole / ml的内毒素生理盐水，冻结和解冻，5毫升的体积注入大鼠的IP。我们用0.8毫米的针（绿针，21G）腹腔注射5毫升注射器。这种治疗，每天两次为4.5天， 即从星期一早上（0天）至周五（4日）上午，诱使一个健壮的萌芽在肠系膜测试组织血管生成的响应，在21天左右达到高峰。 管理解决方案，IP确保整个卷进入腹膜腔（而不是进入肠道，膀胱癌或其他器官）交付。 5毫升的迅速注入，允许运营商通过腹壁进入腹腔通过射流触觉确认。 以下，之前或同时与IP的促血管生成治疗，任何选择的测试剂，可管理的系统。 </li></ol> 3。全身的血管生成调节剂Administation <ol><li>可以使用IP路由比其他任何路线，因为IP的治疗，可能会影响正在进行的通过炎症或通过激活肥大细胞，在测试的组织，这可能会导致强烈的促血管生成响应可能诱导的血管生成反应。除了口服或单次注射SC或IV，我们经常使用SC管理使用渗透压minipumps（一个或多个不同卷和抽水时间），以恒定速率的解决方案能够为两个或几个星期。 </li><li> 持续皮下输注的测试剂 灌装和植入渗透minipumps 通常5天，即一个IP血管内皮生长因子治疗，渗透minipumps（例如，型号2ML2，不断抽水率5.0μL/ h的14-15天结束后的一天; Alzet渗透泵，加州山景城，美国）填充在无菌条件下的测试剂或相应的车辆。后存放在无菌0.9％（W / V）氯化钠一夜之间在37℃，泵（S）外科手术植入了腰板SC（一侧脊髓colu万肩胛区域的已使用异氟醚气体（Isoba兽医麻醉大鼠）;先灵葆雅动物保健公司，默克公司，怀特豪斯站，新泽西州，美国），最初是在一个密封的透明腔和随后通过口罩（尽量减少下列动物造成的压力，香港总商会是刷新前与空气重用）。立即植入后用丝线缝合皮肤切口泵植入。 由于动物体重增加在实验期间生理和测试代理管理在一个恒定的速率，实际用量为每公斤体重的试验药物选择是高于平均剂量在输液期间开始较低比在输液期间结束时的平均剂量。 连续输液，这里描述的，可以被看作是扩展形式的节律治疗。 </li></ol> 4。结束实验<ol><li>被放置在一个透明的密闭室，这是与CO 2气体，迅速 ​​杀死动物刷新一次的动物之一。商会与空气被刷新前被再次使用。 </li></ol> 5。收获的组织样本<ol><li>在DVD所示，腹壁被打开，小肠道肠系膜是确定的，包括最远端的一部分，到达回肠盲肠的阀门（小肠和大肠交界处）。四位于最远端的“窗口”（或以上）编号，并蔓延到客观的标准，未经处理的Superfrost加幻灯片（DAKO公司丹麦A / S公司，Glostrup，丹麦）。重要的是，如果可能，避免污染的血液，肠道内容或其他体液的样本，因为这些材料可非特异性染色，虽然这并不随后微血管的免疫组化可视化无效。 </li><li>肠系膜上附有小肠道段的每个窗口标本幻灯片上传播，并在室温下干燥20分钟。然后，肠残端被切断，而完整的膜肠系膜标本保存在-20 ° C长时间。这是显示在DVD。 </li></ol>应当提及的是它可以可视化的组织和专利血管收获前与透射光活体显微镜。 6。微血管的免疫组化染色的议定书<ol><li> 0天 标本被允许从-20 ° C解冻到室温，内阁在60 ° C干燥1小时，并保持在室温过夜。 </li><li> 第1天 放置在一个染色缸染色幻灯片持有人（在每一个第二轨道）的幻灯片，并在室温下10分钟两次用TBS（pH值7.8）。该解决方案将被丢弃，并与未来的解决方案所取代，等所有以下步骤。 </li><li> 胰蛋白酶处理 填充染色缸，将港口与胰蛋白酶溶液（25℃20分钟，C）染色幻灯片持有人。丢弃的解决方案和孵育2.5％的蔗糖溶液（室温每次10分钟）的两倍。丢弃的解决方案和冲洗水作空白时间（室温为每几分钟）夫妇。 </li><li> 封锁 填写的染色缸，0.5％H 2 O 2甲醇（室温30分钟）染色幻灯片持有人将海港。弃掉的解决方案，并以清水冲洗旱厕 DEST（室温下了几分钟） 。弃掉的解决方案，并与TBS（pH 7.4）中（室温为8分钟，每次）冲洗两次。 </li><li> 孵化 所有的孵化是在室温下湿盒。下面列出的解决方案，每年约150μL用于每张幻灯片。幻灯片放置在湿盒和吸管的解决方案是到标本（不需要的解决方案所涉及的非膜部分）。 对于下面的每一个步骤，仔细摆脱所有的解决方案，在随后的解决方案是添加。 <ol><li> 孵化正常（阻塞）血清 （马）（ABC试剂盒）3滴黄色溶液为20分钟/ 10毫升缓冲液稀释。 </li><li> 孵化与 ARU0181 主要抗体 ，这标签大鼠血管内皮细胞。抗体稀释缓冲液稀释（BIOSOURCE）（100μL稀释40倍[冻结]加900μL缓冲液稀释）和孵化1:600​​发生在4 ° C过夜</li></ol></li><li> 第2天 幻灯片从湿盒中删除，被丢弃的主要抗体，并幻灯片放置在染色幻灯片持有人，被淹没在染色缸，用TBS漂洗两次（pH值7.8）为8分钟。 <ol><li> 与生物素抗体孵化 （ABC试剂盒） 幻灯片横放在湿盒孵育1滴蓝溶液 / 10毫升缓冲液稀释30分钟。丢弃的解决方案和地方的幻灯片，在幻灯片淹没在染色缸染色持有和TBS（pH值7.4）冲洗两次为8分钟。 </li><li> 孵化与美国广播公司试剂 幻灯片再次在湿盒横放，和下面的解决方案是到标本的膜部分吸管：2 滴橙溶液加2 滴褐色的解决方案/ 10毫升TBS（pH值7.4），30分钟。混合小心使用前 30分钟！丢弃的解决方案和地方幻灯片，在幻灯片持有人在染色缸和TBS（pH值7.4）冲洗两次8分钟。 </li></ol></li><li> DAB染色（0.05％） 这一步必须在通风良好的化学罩！ 将幻灯片添加3,3 -二氨基联苯胺（DAB，Sigma - Aldrich公司）解决方案，使用吸管：0.1％，民建联的TBS溶液（pH值7.4）[冻结] 10μLH 2 O的混合物稀释1:1 2和7.5毫升TBS（pH值7.4）。这个解决方案是无菌的过滤，只是在使用前（以消除任何可能掩盖了微观图片的粒子）。 8分钟染色标本。丢弃的解决方案和地方的幻灯片，在幻灯片淹没在染色缸染色持有。下冲洗自来水和水作空白，然后在几分钟内。 </li><li> 脱水 幻灯片保存在染色幻灯片持有人。通过丢弃冲洗和加气站的染色缸， 含有水作空白 ，/乙醇：水作空白 ，2分钟，在每一个解决方案，70％的酒精，95％的酒精，无水酒精， 。 </li></ol> 7。成像和血管内皮细胞的免疫组织化学染色后评估每个肠系膜窗口标本的微血管网络在电影所示，微血管显微镜下可视化的完整组织原位 。 <ol><li>首先，我们使用绘图显微镜的放大倍率范围从14日至34（徕卡野生中号3Z）白纸上铅笔的整个窗户面积，以及整个血管化面积每肠系膜窗口（VA）。弗吉尼亚州，为整个区域的百分比，是很容易测量，使用电脑化或者非计算机化planimetry（或仅仅是切割和权重的整个窗口形象，吻合血管的部分，其所有的图像）。 </li><li>使用另一个绘图显微镜，个别的微血管型材，很容易识别，在白纸上的黑色墨水绘制在80随机选择的领域内的放大倍数。这是为随后的电脑形态评估微血管的长度（MVL），这基本上是一个微血管密度的测量起点。 </li><li>或者，微血管格局的形成有关的变量，以及评估微血管豆芽和个人（号SP和乐，SP，分别）微血管豆芽的长度的数量可以决定的。之间所描绘的黑色血管的结构和白色背景有利于大多数商业形态的电脑化计划的图像的详细分析最佳的对比度。 </li><li>在大多数情况下，主要的变量是VA和MVL，相乘产生的总微血管长度 （TMVL） 。 </li></ol> 8。统计分析我们通常使用的标准的非参数采用Mann - Whitney U检验分析未成（双尾）的意见。平均每只动物肠系膜四个窗口是用来作为独立的数据点肠系膜窗口中的每个变量。统计学意义的标准是P≤0.05。

<ol>
	<li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J. Cell. Mol. Med. 10, 588-612 (2006).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+testing+with+angiogenesis+models.">Drug testing with angiogenesis models.</a> Expert Opin. Drug. Discov. 3, 533-549 (2008).</li><li>Norrby, K., Jakobsson, A., Sorbo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast-cell-mediated+angiogenesis:+a+novel+experimental+model+using+the+rat+mesentery.">Mast-cell-mediated angiogenesis: a novel experimental model using the rat mesentery.</a> Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 52, 195-206 (1986).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+and+angiogenesis.">Mast cells and angiogenesis.</a> APMIS. 110, 355-371 (2002).</li><li>Albertsson, P., Lennernas, B., Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+meteronomic+chemotherapy:+modulation+of+angiogenesis+mediated+by+VEGF-A.">On meteronomic chemotherapy: modulation of angiogenesis mediated by VEGF-A.</a> Acta Oncol. 45, 144-155 (2006).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+apo-lactoferrin+enhances+angiogenesis+mediated+by+vascular+endothelial+growth+factor+A+in+vivo.">Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo.</a> J. Vasc. Res. 41, 293-304 (2004).</li><li>Norrby, K., Mattsby-Baltzer, I., Innocenti, M., &amp;amp, T. u. n. e. b. e. r. g., S, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+administered+bovine+lactoferrin+systemically+inhibits+VEGF(165)-mediated+angiogenesis+in+the+rat.">Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat.</a> Int. J. Cancer. 91, 236-240 (2001).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-molecular-weight+heparins+and+angiogenesis.">Low-molecular-weight heparins and angiogenesis.</a> APMIS. 114, 79-102 (2006).</li><li>Norrby, K., Nordenhem, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dalteparin,+a+low-molecular-weight+heparin,+promotes+angiogenesis+mediated+by+heparin-binding+VEGF-A+in+vivo.">Dalteparin, a low-molecular-weight heparin, promotes angiogenesis mediated by heparin-binding VEGF-A in vivo.</a> APMIS. 118, 949-957 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+and+de+novo+mammalian+angiogenesis.">Basic fibroblast growth factor and de novo mammalian angiogenesis.</a> Microvasc. Res.. 48, 96-113 (1994).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+and+de+novo+mammalian+angiogenesis.">Vascular endothelial growth factor and de novo mammalian angiogenesis.</a> Microvasc. Res. 51, 153-163 (1996).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+density+in+terms+of+number+and+length+of+microvessel+segments+per+unit+tissue+volume+in+mammalian+angiogenesis.">Microvascular density in terms of number and length of microvessel segments per unit tissue volume in mammalian angiogenesis.</a> Microvasc. Res. 55, 43-53 (1998).</li></ol>

该实验于1986年3推出，并先后得到了进一步开发和完善了我们的实验室7,10-12。讨论审查文件（邀请）1，2，检测比较以及与其他哺乳动物体内血管生成的实验方面，尤其重要的功能，如成人的测试组织本身是吻合血管，缺乏生理性血管生成，最小的创伤，如果有的话，是组织施加的，真正的定量变量进行测量，允许健全的统计分析，剂量反应和分子活性的研究;萌芽血管生成的发生，这是在正常组织和肿瘤为主;和毒性数据很容易在大鼠的强劲增长，累计生理在成年期。不利的功能可能包括检测比较费时，尤其是当评估个别微血管段和微血管豆芽的数量和长度的事实，它不允许real-time/serial意见，这是很难适合对小鼠因为许多在小鼠的肠系膜窗口缺乏潜在的父微血管发起新的毛细血管。 然而，启用实时观测活体显微镜exteriorized但完整的肠系膜窗口是叉开显微镜阶段，而动物麻醉后， 在 1讨论。 角膜micropocket，鸡胚绒毛尿囊膜，和SC基质胶堵塞检测，这是有用的，但最终有限的工具，如使用的模型过去几十年中，在血管生成研究取得重大进展。未来抗血管生成和促血管生成疗法的临床应用，必须更加敏感和生物相关，真正定量的临床前模型，如在本报告所描述的，建立和验证。

缓冲液和试剂 0.5 M的Tris - HCl（pH值7.8）原液 三（羟甲基）aminomethan（60.57克）溶解在〜900毫升水作空白，实现pH值7.8，稀释至1公升水作空白，添加25％的盐酸（浓）〜45毫升。 的Tris缓冲液（TBS，pH值7.8 ） 添加18克氯化钠溶解在〜1800毫升水作空白，调整至7.8的pH值，使用原液，稀释至2公升水作空白，为200毫升的原液。 TBS（pH值7.4） 到200毫升的原液加入18 g氯化钠溶解在溶解〜1800毫升水作空白，调整至7.4的pH值，使用原液，稀释至2公升水作空白，。 胰蛋白酶溶液（0.05％胰蛋白酶，0.1％ 氯化钙 2 ） 含有胰蛋白酶在TBS（pH值7.8），200毫升0.1克和0.2克氯化钙 2 。 2.5％的蔗糖溶液 在TBS（pH值7.8），200毫升含有5克蔗糖。 联合解决方案的1％的TBS叠氮化钠（pH值7.4 ） 含有1克在100毫升叠氮化钠与TBS（pH值7.4）。 稀释缓冲液：0.1％BSA，0.01％NA -叠氮 （Vectastain ABC试剂盒用来稀释抗体）。包含0.1克牛血清白蛋白（BSA）和1毫升1％NA -叠氮，与TBS（pH值7.4）稀释至100 ml。 ABC试剂盒Vectastain PK 4002，ABC过氧化物酶的小鼠IgG 马的正常血清封闭血清， 黄色的解决方案：3 drops/10毫升缓冲液稀释。 生物素抗体，抗鼠IgG; 蓝溶液 1 drop/10毫升缓冲液稀释。 ABC试剂： 橙溶液 ，2滴，加上棕色溶液 2 drops/10毫升TBS（pH值7.4）。 搅拌20 分钟后方可使用这些解决方案！ 鼠标（单克隆）抗大鼠血管内皮细胞 （BIOSOURCE国际，Camarillo的，CA，美国） <table border="1"><tbody><tr><td>目录＃ </td><td> ARU0181 </td></tr><tr><td>克隆＃ </td><td> MRC OX - 43 </td></tr></tbody></table>这种抗体是针对所有血管内皮细胞在大鼠，在大脑中的异常。 Vectastain过氧化物酶亲和素生物素复合体（ABC）的试剂盒 该套件包含的主要抗体，生物素标记的第二抗体，抗生物素蛋白生物素化的酶复合物（ABC），和酶底物。 <a href="http://www.vectorlabs.com/">www.vectorlabs.com</a>或<a href="http://www.immunkemi.se/">www.immunkemi.se</a>协议。 牛血清白蛋白 （BSA）（Sigma - Aldrich公司，目录＃A - 4503）。 民建联 = 3,3&#39; -二氨基Isopac（Sigma - Aldrich公司，目录＃ð 9015）。 从牛胰腺胰蛋白酶 （Sigma - Aldrich公司，目录＃T 4665）。 Vectastain过氧化物酶ABC试剂盒 ，鼠IgG（Immunkemi F＆ð AB公司，Jarfalla，瑞典）

rat mesentery angiogenesis assay

成年大鼠肠系膜窗口血管生成的实验是生物学适当的，是特别适合发芽体内的血管生成研究[见审查文件，这是人类肿瘤和非肿瘤组织血管生成的主导形式，邀请审查讨论论文1,2。在膜的肠系膜部分腹腔（IP）注射一个促血管生成因子诱导血管生成的可调制皮下（SC），静脉注射（IV）或口服（PO）与治疗的选择改性剂。每个肠系膜膜是半透明的，由脂肪组织框架，给它一个窗口一样的外观。 该试剂盒具有以下优点：（一）测试组织本身血管化，虽然人烟稀少，因为它是非常薄，微血管网络几乎是二维的，这使得整个网络在原位显微镜进行评估 ; （ ii）在成年鼠的测试组织缺乏显着的生理性血管生成，其中大多数正常成年哺乳动物组织的特征;原生血管的程度，但是， 与年龄，在1讨论;（三）对创伤诱导的血管生成忽略不计的水平（V），确保高灵敏度;（四）检测复制的临床情况，作为血管生成调节试药管理系统和观察到的反应，反映了所有的代谢，细胞，并诱导治疗的分子变化的净效应;测定，以使评估的客观，定量的，不偏不倚的变量微血管的空间扩展，密度，和网络格局的形成，以及毛细血管发芽，从而使强大的剂量效应和分子结构-活性关系的统计分析;及（ vi ）具有灵敏度高，检测结果显示，在生理体增重率下降方面治疗的有毒或有害的影响，作为成年大鼠强劲增长。 肥大细胞介导的血管生成是首次证明，使用这种检测 3,4 。该模型演示了高剂量 - 效应关系和系统管理密切相关的化学或功能的药物和蛋白质，包括测量效果的歧视水平：（一）低剂量，metronomically管理标准化疗的收益率不同，具体药物的效果（ 即血管生成抑制，中性或刺激血管生成活动 5），（二）自然不饱和铁人乳铁蛋白，刺激的VEGF - A介导的血管生成 6，和自然铁不饱和牛的乳铁蛋白，从而抑制血管内皮生长因子- A - 7介导的血管生成;及（iii）由8,9各种手段生产的低分子量肝素的分数。此外，该法是非常适合的血管生成，并发或连续的方式管理系统的代理商的综合影响的研究。 这个视频的想法源于已故的犹大福克曼博士，当他参观我们的实验室，目睹了正在演示的方法。 审查文件（特邀）讨论和评价检测 Norrby，K 在体内血管生成模型。研究细胞。 MOL。 MED。 10，588-612（2006年）。 Norrby，K.与血管生成的模型药物测试。专家OPIN。药物。发现了。 3，533-549（2008）。

Watch this Scientific Journal Video about Rat Mesentery Angiogenesis Assay at JoVE.com

Rat Mesentery Angiogenesis Assay

正常成人的吻合血管的哺乳动物组织，缺乏生理性血管生成和尚未暴露手术干预是用来研究：（一）发起和血管生成测试剂腹腔给药后的发展;及（ii）全身给药的血管生成的修改选定的测试剂。

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 ...

Research

JoVE Journal

Biology

13.8K 次观看.  University of Gothenburg. 正常成人的吻合血管的哺乳动物组织，缺乏生理性血管生成和尚未暴露手术干预是用来研究：（一）发起和血管生成测试剂腹腔给药后的发展;及（ii）全身给药的血管生成的修改选定的测试剂。

视频: Rat Mesentery Angiogenesis Assay

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

器官型E18大鼠脑切片文化

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

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

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

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 ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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 ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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.