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（...

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 border="0" cellpadding="0" cellspacing="0" width="611">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:105px;">Name of the Material/Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:259px;">Comment/Description (optional)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:105px;">Multi Myograph</td>
			<td style="width:127px;">Danish Myo Technologies</td>
			<td style="width:120px;">610M</td>
			<td style="width:259px;">A myograph is critical to this bioassay, but there are other platforms available for use that will suffice.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">Powerlab 8/sp</td>
			<td style="width:127px;">ADIntruments</td>
			<td style="width:120px;">ML785</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">LabChart 7</td>
			<td style="width:127px;">ADInstruments</td>
			<td style="width:120px;">Version 7</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Force Calibration Kit</td>
			<td style="width:127px;">Danish Myo Technologies</td>
			<td style="width:120px;">100055</td>
			<td style="width:259px;">Specific to DMT myographs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Bottle-top Filter</td>
			<td style="width:127px;">Nalgene</td>
			<td style="width:120px;">595-4520</td>
			<td style="width:259px;">0.22 um pore size; 45 mm neck size</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">#5 Jewler&#8217;s Forceps</td>
			<td style="width:127px;">Miltex</td>
			<td style="width:120px;">555008FT</td>
			<td style="width:259px;">Any brand of forceps can be used</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Noyes Iris Scissors</td>
			<td style="width:127px;">Miltex</td>
			<td style="width:120px;">18-1510</td>
			<td style="width:259px;">Any brand of scissors can be used</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Dissecting Scope</td>
			<td style="width:127px;">Zeiss</td>
			<td style="width:120px;">Stemi 2000-C</td>
			<td style="width:259px;">Any brand of dissecting light microscope will suffice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:105px;">Adjustable Tissue Matrice</td>
			<td style="width:127px;">Braintree Scientific</td>
			<td style="width:120px;">TM C12</td>
			<td style="width:259px;">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 height="43">
			<td height="43" style="height:43px;width:105px;">Krebs-Hensleit Buffer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">K3753-10x1L</td>
			<td style="width:259px;">It is not necessary to buy Krebs, this can be made in house</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:105px;">Calcium chloride dehydrate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">C7902-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Sodium bicarbonate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">S5761-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Desipramine-HCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">D3900-5G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Propranolol-HCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">P0884-1G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">KCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">P9333-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:105px;">95% O2/5% CO2</td>
			<td style="width:127px;">Scott Gross</td>
			<td style="width:120px;">UN3156</td>
			<td style="width:259px;"></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...

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

Research

JoVE Journal

Medicine

9.6K 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

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

科研

医学

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成年小鼠静脉高压型号：颈总动脉，以颈外静脉吻合。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

颈动脉和颈静脉导管在有意识大鼠模型进行血流动力学措施，输液和血液采样

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

一个简单的设备，迅速准备小鼠肠的全坐骑

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...