作者承认瑞恩·卓别林和肯塔基州肉类实验室和动物与食品科学系大学的格雷格伦特福罗博士提供的机会来收集本文采用实验的组织。

在这项研究中所使用的程序并不需要来自肯塔基州的动物护理和使用委员会的批准，大学，因为没有活的动物中使用。取货前这里所使用的任何样品，所有动物都惊呆了紧固螺栓和放血。这是在肯塔基大学的联邦检查屠宰场设施进行。美国农业部食品安全检验局的官方代表指出，处理尸体的活的动物和处理的所有活动。 1，准备仪器仪表<ol><li>以最小化的组织样本的集合之间的时间量，以实验的开始，设置了设备和事先准备的缓冲区来收集实验组织（或者如果额外的人员是可用的，这些任务可以同时进行）。 注意:这不仅提供了时间，该组织保持生存能力，进行实验的最大量，但有些实验可用于长时间运行（或者有一个愿望，完成了一天多次实验），因此通常希望以开始早在可行的。 </li><li>上电相关的计算机，数据采集设备，水浴（缓冲区（S））和肌动描记装置（S）。将校准设备的肌动描记装置（S）上，并开启肌动描记热（预设为37.5℃），并允许单元和校准设备升温至预设温度。 <ol><li>打开电脑上先前创建的图表软件的设置文件，并开始运行（而不是采集数据）。 </li></ol></li><li>预热设备10分钟。 <ol><li>开始采集数据。 </li><li> ZERO的所有通道。 </li><li>检查，并进行校准（如果有必要），每个通道（4通道每multimyograph）上进行标准化，从与该信道，以由经认证的重量提供一个2克的力相关联的相应的力传感器所提供的电信号。 </li><li>校准和单位转换完成后，应将所有后续数据为克。相应地更改基于每个独立实验室的欲望;的设备和软件允许使用其它单位，如毫牛顿和伏特。 </li></ol></li><li>确保天然气管道的畅通无阻，并打开气源（95％O 2/5％的CO 2），以肌动描记（S）为单位。连续气在整个过程中的所有缓冲区（中肌动描记室）。 </li><li>填写所有肌动描记室用70％乙醇浸泡10分钟，除去乙醇，并重复了第二个10分钟浸泡。 <ol><li>除去乙醇后，冲洗三次以去离子水，然后冲洗三次以制备缓冲液（见第2部分为缓冲液配制），留在腔室中的第三另外的缓冲区。 注意:此时的设备可以保持空闲状态在这种状态下，直到缓冲区和组织准备和工作人员已准备好继续进行experimen吨。 </li></ol></li></ol> 2，缓冲溶液之制备<ol><li>准备用于运输和组织样品的处理，以达到最终浓度为11.1 mM的D-葡萄糖的1L的Krebs-Henseleit缓冲溶液; 1.2毫米硫酸镁4; 1.2mM的KH 2 PO 4; 4.7毫米氯化钾; 118.1毫米氯化钠; 3.4毫米氯化钙2; 24.9毫米碳酸氢钠3。 <ol><li>混合9.6克克雷布斯盐成一个搅拌盘约900毫升去离子水。 </li><li>拌入0.373克氯化钙脱水后每升缓冲所需为2.1g碳酸氢钠。 </li><li>为20分钟，用95％O 2/5％CO 2气体的缓冲溶液。 </li><li>放气后，调节pH值至7.05（缓冲过滤增加了pH值，最终目标pH值应为7.4），调整音量至1L。 </li><li>过滤灭菌缓冲到一个干净的高压灭菌的1L媒体瓶并储存缓冲液中于4℃下，直到准备使用。 </li></ol></li><li>准备单独克雷布斯-H在为交通克 - 亨氏缓冲带，涉及到的收缩试验的另外的化合物的步骤2.1描述的肌动描记实验使用enseleit缓冲溶液。 注意:通常2升将足以为本文中所描述的实验，但这个量可能需要增加或根据实验的长度减少，肌动描记腔室，并且数缓冲器的替代值（相对于治疗的添加）的数量。 <ol><li>在此之前的充气步骤（步骤2.1.3），加入9.1毫克（每升缓冲区正在准备），去甲丙咪嗪，盐酸缓冲。 </li><li>制备普萘洛尔盐酸的1.0μM溶液和加入1 ml（每升缓冲器正在编写）将该溶液的的Krebs-Henseleit缓冲溶液。就使用的一天，这个缓冲区，并保持在肌动描记工作温度（即产生了37.5℃的温度，在肌动描记的温度）。 注:地昔帕明添加抑制生物胺再摄取机制并允许从缓冲试验增加洗澡的血管发生更迅速的清除。加入普萘洛尔防止非特异性的治疗化合物结合于β-肾上腺素能受体。 </li></ol></li></ol> 3，收集和血管系统的准备<ol><li>只要可能的话，除去从胎体的胃肠道。一旦动物不再出现任何不自主反射，除去头和隐藏，然后抬起尸体垂直。去除尸体胃肠内脏（食管到肛门）应由批准的屠宰场人员（&lt;20分钟的时间惊人的）来完成。 请注意:如何快，通常受限于其中的肠道被从与标准作业程序或联邦要求的程序接着设施人员收集的位置/设施。 <ol><li>在大多数设施，进行胃肠道的操作在从注定要进入食品供应的尸体处理一个单独的位置。使用附近的一个便利的位置，胃肠道可采取摊开检查。 </li></ol></li><li>一旦在胃肠道被分散开来，确定小肠，回盲部折连接小肠到盲肠和回肠凸缘延伸的近端到回盲倍（ 图1A）。 <ol><li>用手术刀或刀，很轻易使切口在回肠法兰中心肠系膜膜。用两根食指，毫不客气地解剖掉脂肪和结缔组织暴露肠系膜血管（ 图1B）。 </li></ol></li><li>从该凸缘或隆起的肠系膜，解剖出多个分支（〜2厘米的长度），该支持回肠（ 图1C）的这一部分的露出肠系膜动脉和静脉束。 <ol><li>如果妳星钳把握组织，注意不要直接掌握或拉的血管，而在所有消除他们的任何形式的延伸会损害与组织绩效14负效应。血管去除是最好的做法，通过在部分的每个端部切割要除去，然后切割沿着或平行于现在分离部。为了方便，去掉一些周围组织与血管提供一些组织掌握了钳子。 </li><li>有一对钳子的，淹没组织样品中含有的冰冷的Krebs-Henseleit缓冲液的管和储存在冰上直到处理可以发生在实验室。 </li></ol></li><li>放置组织样品上的切割表面或在培养皿中，并在冰冷的克 - 亨氏部分地淹没。 <ol><li>使用＃5珠宝商镊子，诺伊斯虹膜剪刀和放大镜灯或解剖范围（2.5〜5.0X放大倍率就足够了），仔细解剖掉周围的脂肪和connecti已经组织并分离动脉和静脉。识别所述容器开口的部分的一端，并仔细把握周围用钳子容器内的筋膜上。 </li><li>使平行于所述容器通过滑动剪刀的尖端的凸起面板下方的切口用剪刀。初始切口后，将脂肪和结缔组织，可进一步从该容器通过切割下来两侧用剪刀剥离。确保船只尽可能干净，同时尽量减少时间的船花了板凳上的金额。 </li><li>返回血管的新鲜的Krebs-Henseleit缓冲和存储管，在4℃（样品可保持存储采集后长达24小时，并仍能产生有效的收缩数据）。 </li><li>用剃刀刀片，以便在肌动描记成2毫米的部分所需的数被使用切片器（它是有帮助的使用组织切片机，以获得一致的横截面）。 </li><li>审查根据解剖上海合作组织各部分PE（12.5X放大率），以确保各部分没有异常，树枝，阀门，或浅表损伤解剖和清洗过程中无意中进行。更换容器部分，如果有任何异常情况，分支机构，阀门，或表面损伤。 </li></ol></li><li>店上可接受的切片器切片（浸没于Krebs-Henseleit缓冲液）在冰上或在4℃下，直到准备用于安装在肌动描记腔（通常&lt;30分钟）。 </li><li>轻轻地安装在肌动描记的船只通过管腔插入支架，并增加张力（使用于肌动描记的微定位器），注意不要伸展超过2到3G阅读的船只。 </li><li>一旦所有的腔室都包括在内，真空从所有腔室中的缓冲和重新注入5.0缓冲液中的溶液，并开始一个15分钟的定时器，开始平衡期和实验。 </li></ol> 4，实验<ol><li>平衡各船节1.5小时，实现了稳定的水库挺紧张1.0克。 <ol><li>更换克 - 亨氏孵育缓冲液每隔15分钟。 </li><li>期间的平衡，不断调整的血管部分中的张力上升至2g，然后允许放松下来大约0.80克。尽量不要让血管放松太多（他们可能会滑落的坐骑）。努力实现一个稳定的基本张力，那里的船只持有1克紧张，而无需调整。 </li></ol></li><li>期间的平衡，制备所用的KCl水溶液1.32摩尔溶液作为参考。 </li><li>在完成一个令人满意的平衡，加500微升1.32摩尔KCl溶液以产生在5.5 ml的溶液洗澡容器中的0.12M的溶液。 <ol><li>接着加入1.32摩尔KCl，不手动调整张力从该添加的最大反应是用来评价组织活力并可以用来归一化处理的数据（ 例如，浓度反应出了gonist）。 </li><li>代替在15分钟间隔的缓冲区中，直到张力返回到1.0克的基线值。 </li></ol></li><li>一旦基本达到，改变缓冲，同时开始1分钟定时器。同时这样做对所有室，避免了惊人的启动时间。 <ol><li> VORTEX要添加的标准，1分钟倒计时过程中，准备室1评论。 </li><li>在25μl的等份加入标准的每个腔室的实验使然（此保持低于总体积的0.5％的处理）。 </li><li>当最后一个标准增加了，开始9分钟计时器。 </li><li>在9分钟的标准孵育结束时，从室中取出含处理缓冲器和加入5.0毫升新鲜缓冲液中，并开始2.5分钟定时器。 </li><li>重复步骤4.4.4。 </li><li>在第二次2.5分钟冲洗结束时，真空室，加上新的缓冲区，并启动1分钟计时器倒计时的塞康开始D标准的补充。 </li></ol></li><li>继续这个循环对所有当天的标准添加的。 </li><li>在最后的加标和随后的9分钟孵化准备的运行参考剂量加入500微升结束的1.32米氯化钾参照物。 </li><li>继1分钟间隔的最终处理除后，加入氯化钾，并开始9分钟计时器。 注:氯化钾除了是确认组织活力在实验的结论，是有益的，如果施用的治疗，导致忽略不计的回应。 </li><li>在最后的9分钟参考化合物温育结束时，真空或从所有的腔室移除缓冲。不要添加新鲜的缓冲液。总结实验。 </li><li>保存图表文件，删除的血管段和处置得当，并按照清理方案（由制造商提供，并且可以指定实验室）的肌动描记设备。 </li></ol>

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在这种生物测定的发展，最初的挑战是建立一个可重复的集合网站肠系膜血管。示例站点的一致性是至关重要的，因为从通过空肠回肠，因此肠系膜进展过程中的一些小肠变化的功能各不相同的类似的模式。肠系膜上动脉和静脉的回肠分公司分别通过解剖标志是最容易识别。通过定位在盲肠和以下的回盲部折叠到其末端上肠系膜的左侧，这是接近颅肠系膜动脉15的终止。再肠系膜的面积（称为凸缘）和回盲部的折叠的延伸（独特的牛类动物）在众多动物非常可重复的16条作出的采样。之一的，可以作出较大的缺陷是在生物测定的这一阶段。机械拉伸对后续船P A大的负面影响erformance 14和研究者会注意到被去除和随后的清洗过程中过度地操纵或拉伸的容器将不被使用的参照化合物反应。这个不幸的是，要真正评估血管制剂的质量在功能水平，不以氯化钾反应，不应再用于生成可靠的数据被认为是血管的唯一途径。 其他两个方法验证步骤（未示出），导致了本文中所呈现的最后一种方法是使用其它的化学品作为参比化合物和试验血管下列24小时存储的生存能力。去甲肾上腺素和血清素的评价采用与参考化合物，而是由肠系膜动脉和静脉的反应过于可变跨容器类型和整个动物对它们可以成功地使用。相反，增加了120毫米氯化钾导致在两个动脉和静脉的准备很重复响应。 áLSO，动脉和静脉的响应被收集的当天进行评价和重新评价后24小时收集并有在由任一动脉或静脉激动剂反应没有差异。这是生物测定的一个重要方面，因为经常有大量的治疗（或多种治疗的组合），其可被应用，但研究人员通过对肌动描记和时间空间的限制。通过从集合延伸的生存能力的期间在24小时的时间，以获得更多的实验性大大增加。 它为研究人员理解施加到这个生物测定处理结构的灵活性是很重要的。一个研究人员可以添加任何化合物的温育缓冲液，他或她的爱好有长期暴露血管以控制一些生物相关性，或作为治疗（在克茨等人的情况下，17，这是与实现侧隐静脉持续暴露于磕tanserin，其中拮抗感兴趣的血清​​素受体）。肠系膜血管可预处理之前在进行收缩反应的实验兴趣的化合物。生物测定，如呈现，旨在进行累积浓度响应试验看着麦角生物碱的激动剂和是否事先膳食暴露受影响的血管反应18麦角生物碱。代表性的结果表明，该方法可以产生可测量的血管反应和仅四种浓度被用来实现这一目的。在Egert 等人 18的情况下，有用于构建响应曲线多达10个不同的浓度。此方法是一种在时间的小窗口来评估大量不同的化合物或受体对血管从单个动物。

在血液流向组织床的改变可能对器官功能产生很大的影响。小肠的主要功能是营养物质的吸收。动脉血流到肠道的吸收表面所需的营养物质的吸收和血流量的增加，以在营养吸收有助于沿面1食糜移动。血流量的减少会导致营养吸收减少，由于在跨膜梯度2的下降。除了营养物，小肠，也可以接触到的次级代谢产物，药物或毒素施加于肠系膜局部血流量的效果。在反刍动物的情况下，化合物可从饲料中解放出来（ 例如 ，营养物质如氨基酸，或毒素，例如麦角生物碱）通过前肠的发酵工艺。如果这些化合物生存瘤胃发酵的微生物新陈代谢，它们现在可以吸收或相互作用，因为它们通过在动物的胃肠道中行进。 有许多不同的方法可用于测量体内的血流量（ 例如 ，多普勒超声，留置血流量计，放射性微球，和指示剂稀释技术），其允许的各种实验方案或疗法的评价。然而，为了获得关于血管平滑肌的机械或药理学特性的信息，方法仍然有限，以大型船舶，直至Mulvany和哈尔3发表了一篇文章，描述使用电线的技术安装在肌动描记血管环的准备。由于该技术的发展，改进继续进行到允许各种不同的应用为管状结构的评价相关联的肌动描记系统。该系统也被改编利用固定杆，用于安装大型船舶4，其中灌注技术是不希望的。 因为在不同的解剖起源和区别在来自不同物种的动物的，数据从容器和动物型相同船只船只相异的不能容易地在不同的容器或在不同种类动物的5相同的容器中推断出来。因此，独立的生物鉴定必须制定并随时验证这些方面发生变化。最近几个生物测定已经开发了这些技术用于牛外侧隐静脉，右瘤胃动脉和静脉6,7。 这个生物测定的开发是为了专门调查效果麦角生物碱对血管支撑小肠。据报道，供给生物碱的50-60％出现在皱胃内容，但只有5％被回收粪便中8。斯特里克兰等 9麦角碱的审查规定，也可用数据sugg估该小肠可以是用于ergopeptine吸收的最重要位点。埃克特等 10回顾生物制药麦角生物碱方面表示，一旦他们穿过上皮屏障，麦角生物碱要么运经淋巴系统向锁骨下静脉或经肠系膜静脉和门静脉进入血液。罗德等人 11报道了减少血液流向十二指肠和结肠公牛消耗高的内生真菌感染（高麦角生物碱）的饮食。使用正确的瘤胃动脉和静脉的生物测定，Foote 等12表明，麦角生物碱是血管瘤胃液中的血管。 Foote 等13后来证明在体内的瘤胃接触麦角生物碱导致降低瘤胃上皮细胞的血流量。这减少血流量，瘤胃的吸收表面伴随造成养分（挥发性脂肪酸）的磁通的降低。鉴于曲麦角生物碱传递来自前肠小肠antity;有人推测会发生在小肠血管和营养吸收同样的效果。这需要牛近端回肠肠系膜上动脉和静脉的生物测定的发展。

<table><tbody><tr><td>Multi Myograph</td><td>Danish Myo Technologies</td><td>610M</td><td>A myograph is critical to this bioassay, but there are other platforms available for use that will suffice.&amp;nbsp;</td></tr><tr><td>Powerlab 8/sp</td><td>ADIntruments</td><td>ML785</td><td></td></tr><tr><td>LabChart 7</td><td>ADInstruments</td><td>Version 7</td><td></td></tr><tr><td>Force Calibration Kit</td><td>Danish Myo Technologies</td><td>100055</td><td>Specific to DMT myographs</td></tr><tr><td>Bottle-top Filter</td><td>Nalgene</td><td>595-4520</td><td>0.22 &amp;#956;m pore size; 45 mm neck size</td></tr><tr><td>#5 Jewler&amp;rsquo;s Forceps</td><td>Miltex</td><td>555008FT</td><td>Any brand of forceps can be used</td></tr><tr><td>Noyes Iris Scissors</td><td>Miltex</td><td>18-1510</td><td>Any brand of scissors can be used</td></tr><tr><td>Dissecting Scope</td><td>Zeiss</td><td>Stemi 2000-C</td><td>Any brand of dissecting light microscope will suffice</td></tr><tr><td>Adjustable Tissue Matrice</td><td>Braintree Scientific</td><td>TM C12</td><td>This is not critical to the assay, but greatly reduces section to section variation in length and speeds up the slicing process greatly</td></tr><tr><td>Krebs-Hensleit Buffer</td><td>Sigma-Aldrich</td><td>K3753-10x1L</td><td>It is not necessary to buy Krebs, this can be made in house</td></tr><tr><td>Calcium chloride dehydrate</td><td>Sigma-Aldrich</td><td>C7902-500G</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Desipramine-HCl</td><td>Sigma-Aldrich</td><td>D3900-5G</td><td></td></tr><tr><td>Propranolol-HCl</td><td>Sigma-Aldrich</td><td>P0884-1G</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Scott Gross</td><td>UN3156</td><td></td></tr></tbody></table>

isolating and using sections of bovine mesenteric artery and vein as a bioassay to test for vasoactivity in the small intestine

哺乳动物的胃肠道系统经常暴露于化合物（期望的和不希望），可以对血液流动的效果，并从该系统中。血流变化到小肠可导致对器官的吸收功能的作用。从饲料通过发酵和消化过程中释放毒素，特别关注已经发展反刍动物的地方生产效率可以改进的地方。与本文相关的视频描述了体外生物测定发展到屏幕化合物vasoactivity牛肠系膜动脉孤立的横截面，静脉使用multimyograph。一旦血管安装和平衡的肌动描记，生物测定本身可用于:作为筛选工具，以评估收缩反应或感兴趣的化合物vasoactivity;确定由药理学靶向受体的特异性激动剂的受体类型的存在S;确定与一个或一个以上的拮抗剂存在的受体的作用;或确定与拮抗剂的感兴趣的化合物的潜在相互作用。通过这一切，数据采集的实时性，组织从一个单一的动物收集的可暴露于大量的不同实验处理（ 在体外的优势），并表示对毛细血管床，以提供精确的任一侧的脉管图象的什么可以发生在传入和传出的血液供应支撑小肠。

用于生成包括结果的血管从6荷斯坦公牛（425±8千克）3周的时间间隔内被收集。一个典型的肠系膜静脉的收缩反应，以KCl和治疗增加在浓度增加的一个例子示于图2。响应的幅度将随一些与容器（与供体动物的大小相关）的大小，但也可以的影响（负面的）操作不当，收集和清洗过程中（拉伸）的船只。因此，观察到在容器暴露于氯化钾Â显著收缩反应是很重要的。如果可以忽略的响应是在这点上观察到的，这是不可取的，以进一步继续进行任何与该容器中的过程。如可见于图2中的9分钟温育期间，有在反应逐渐增加，随着浓度的增加相对应。虽然希望这个particular实验设计，它可能不总是发生或追捧与其他治疗方法。氯化钾此外在实验结束时，虽然在数据的收集，处理不使用时，是非常重要的，因为它证实了组织的生存力，在实验期结束时（变得更重要，如果容器不响应任何治疗加法）。 在图2中给出的数据被记录在克的张力。它正常化这些数据，以尽量减少任何动物对动物和容器到容器中的变化是很重要的。因此，除了被用作存活力的指标，最大氯化钾反应被用于标准化所有治疗相加。这会产生收缩反应数据为氯化钾最高的百分比。这是如何肠系膜动脉和静脉的响应曲线，以5-羟色胺浓度的增加（血清素; 图3）和去甲肾上腺素（ <str翁&gt;图4）给出。在这些图中的数据已使用非线性回归得到拟合S形浓度响应曲线。 肠系膜静脉响应于两个5-羟色胺（ 图3）和去甲肾上腺素（ 图4）开始是负值。这是在修正之前由最初的氯化钾除了记录基线张力均9分潜伏期记录的最大张力的结果。此基线校正，确保在张力的任何微小的差别不包括在分析中，并允许提供的数据，以反映在张力只发生了变化。负值结果从血管松弛和滴下面的预氯化钾基线水平的后续治疗加成之前。这是静脉血样品，其中所述治疗或激动剂的初始浓度过低，会引起收缩反应中尤其普遍。这不是对我的情况下<html> senteric动脉响应于任一生物胺，它保持张力高于基线直到容器开始到治疗增加作出反应。这个差别体现在毛细管床的两侧和为什么他们都视为本生物测定法的一部分，在船只上的差异。 这个生物测定可用于评价对血管供给营养，小肠以及携带吸收养分远离小肠血管化合物vasoactivity。在图3和图4的数据是如何可以实现简单的例子。在5-羟色胺（ 图3）的情况下，有没有在动脉或静脉的收缩反应的差。相反地​​，通过肠系膜静脉增加去甲肾上腺素浓度的收缩反应明显大于肠系膜动脉。 gure 1"FO:内容宽度="三合一"SRC ="/文件/ ftp_upload / 52020 / 52020fig1highres.jpg"WIDTH ="300"/&gt; 图1:用于肠系膜血管源牛肠道的一个例子的红色星号表示回盲折叠在所有三个窗格作为一个参考点的可见部分。 （ 一）全系有红色的椭圆形表示，其中血管的采样发生回肠法兰。 （二）曝光的肠系膜血管床。 （C）虹膜剪将指向肠系膜静脉（可见）和动脉（不可见）只之前收集的暴露的分支。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52020/52020fig2highres.jpg" width="500" /> 图2:肠系膜静脉的去甲肾上腺素的浓度渐增的浓度响应的实施例 （1×10 -7至1×10 -4 M）。在跟踪开始的时候和结束的大事件是响应于0.12米氯化钾补充。红色的矩形表示对应的9分钟潜伏期为每个治疗加成的数据收集区。遵循这一点，3个细长的尖峰是由缓冲替换生成的构件，不包括在分析中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52020/52020fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52020/52020fig3highres.jpg" width="500" /> 图3:平均数肠系膜动脉（MA）和静脉的收缩反应（MV;每组6阉牛），以5-羟色胺（血清素）浓度的增加表明，使用非线性回归分析对数据拟合到S形曲线生成线条。浓度Ř其中利用下面的公式esponse曲线:Y =底+（顶-底）/（1 +10（logEC 50 - X））]，其中顶部和底部是120毫米氯化钾的最大收缩反应的百分比在高原和欧共体50生物碱生产氯化钾最大响应的50％的摩尔浓度。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52020/52020fig4highres.jpg" width="500" /> 图4:平均数肠系膜动脉（MA）和静脉的收缩反应（MV;每组6阉牛），以增加去甲肾上腺素的浓度示出了使用非线性回归分析对数据拟合到利用S形浓度响应曲线生成线条。下面的方程:Y =底部+（顶部-底部）/（1 +10（logEC 50 - X））]，其中顶部和底部是120毫米氯化钾最大c中的百分比ontractile响应于高原，以及EC 50是生物碱生成的氯化钾的最大反应的50％的摩尔浓度。

Watch this Scientific Journal Video about Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine at JoVE.com

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

小肠经常暴露于毒素可影响血液流动和养分吸收的负面影响。使用multimyograph和肠系膜动脉和静脉分离物，化合物或感兴趣的毒素可筛选vasoactivity。

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

isolating-using-sections-bovine-mesenteric-artery-vein-as-bioassay-to

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K 次观看.  USDA-Agricultural Research Service. 小肠经常暴露于毒素可影响血液流动和养分吸收的负面影响。使用multimyograph和肠系膜动脉和静脉分离物，化合物或感兴趣的毒素可筛选vasoactivity。 

视频: Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

肠系膜动脉收缩和舒张的研究，使用自动线Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

科研

医学

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

体外肠囊评估黏膜通透性在胃肠道疾病模型

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

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 ...

生物学

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

靶向大鼠的小肠:长期输注到肠系膜上动脉

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

生物工程

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

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 ...