桑迪普库拉纳是由美国国立卫生研究院（K08DKO81479）的支持。维克兰特Rachakonda由（T32DK067872）的支持。

这里显示的例子是来自佐治亚大学校务经IACUC实验 - 协议编号：＃2011-0408 1.准备试剂<ol><li>准备剥离液货：500毫升股票清扫液（5倍），溶解21.18克氯化钠，0.875克氯化钾，0.739克硫酸镁 ，1.049克MOPS和450毫升的Milli-Q水0.019克EDTA。调节pH至7.3-7.4用1N NaOH洗涤。补足体积至500ml用Milli-Q水。原液可以存储多达7-10天。请参考表1化学品及其供应商的列表。 见表2;以毫米浓度。 </li><li>准备工作夹层解决方案：准备新的工作夹层解决方案的每一天。 100 ml工作溶液，溶解0.091克葡萄糖，0.016克的NaH 2 PO 4和0.022克丙酮酸钠在79.8毫升的Milli-Q水中。中加入0.2ml 1M 氯化钙和20毫升夹层解决方案的股票带来VOLU我到100毫升。 </li><li>制备生理盐溶液（PSS）：制备千毫升PSS，溶解0.365克氯化钾，6.545克氯化钠，0.296克硫酸镁 ，0.163克KH 2 PO 4，2.072克葡萄糖，2.184克碳酸氢钠和2.383在950毫升ģHEPES的Milli-Q水。调节pH至7.3-7.4用1N NaOH洗涤。补足体积至1000毫升Milli-Q水。除去加入2ml溶液，并将其用2ml的1M的CaCl 2代替。 （PSS鲜需要每天准备） </li><li>准备钙（Ca 2+）免费PSS：对于100mL来说PSS无钙 ，溶解0.036克氯化钾，0.654克氯化钠，0.029克硫酸镁 ，0.016克磷酸二氢钾 ，0.207克葡萄糖，0.218克碳酸氢钠 ，0.238克HEPES，0.015克EGTA和0.0026克硝普钠（SNP）的95毫升Milli-Q水中。调节pH至7.3-7.4用1N NaOH洗涤。补足体积至100毫升用Milli-Q水。 </li></ol> 2.制备玻璃插管<ol><li>拉玻璃吸管生成用吸管拉马按照生产商的指导100-150微米放倒插管。 </li><li>伞用微电极beveller玻璃套管末端，火擦亮他们用热探针由〜45°弯曲的玻璃套管末端。 </li><li>加载到插管微量座和附加的微支架上的灌注腔。 </li></ol> 3.准备灌注腔<ol><li>冲洗灌注腔用Milli-Q水，然后用解剖溶液，每次5分钟。加载室用2ml解剖溶液。 </li><li>用10毫升注射器，并认真填写了整个套管和油管连接没有任何气泡通过插管吸解剖的解决方案。适用于吸轻轻地防止产生气泡。 </li><li>准备两缝线用半结利用每个钝钳。由于眼用单丝尼龙缝线（10-0，0.2公吨）用于制备只属于节直径1-2毫米，解剖显微镜，可能需要。 </li><li>解剖显微镜下可视化，用解剖钳来加载两个套管与局部封闭缝线结稍微远离尖。后来这些节将精心滑动到空心动脉末端和完全关闭。 </li></ol>从SD大鼠4.收集肠系膜动脉街机<ol><li>进行这些实验之前征得当地机构动物护理和使用委员会（IACUC）的批准。房子的动物在控制温度和照明，并允许自由饮水和商业啮齿动物食物的动物设施中。 </li><li>通过腹膜内注射氯胺酮（80毫克/千克）和赛拉嗪（10毫克/千克）麻醉大鼠。经确认脚趾捏，如果需要额外的管理麻醉深度麻醉。 </li><li>确认手术麻醉后，用断头安乐死的动物。遵循UTI AAALAC指引lizing适当的方法对动物实施安乐死。 </li><li>用剪刀解剖和镊子从骨盆到胸骨进行中线剖腹手术。这样做是在两个步骤：首先，切开皮肤和第二，切开下面的肌肉层。必须注意不要伤及腹内脏器。 </li><li>切肠接近幽门和所述远端靠近回肠盲肠结的近端。分别配合两端，防止食糜和粪便从而避免细胞外液沐浴污染的泄漏。切开肠系膜在馈送脉管即 ，肠系膜上动脉接近其底部和整个小肠肠系膜床转移到50ml烧杯含冰冷夹层溶液。 </li><li>允许收获的组织留在冰冷的夹层中5分钟，用新鲜的解剖溶液冲洗摆脱血液。 </li></ol> 5.分离的第 4 次插管</SUP&gt;订单肠系膜动脉<ol><li>牵制在右手侧以SYLGARD涂层盘肠的近端。延长剩余肠中逆时针路径，钉扎的段向下传播肠系膜和暴露的血管（ 图1）。注意：我们分离在室温下动脉段。否则，我们把这道菜含冰肠系膜商场。一些实验室，包括在我们的机构，用冷水机组解剖动脉在4℃。 </li><li>在立体声变焦显微镜解剖了3 次和4 阶小肠系膜动脉（〜260微米）平行于用小剪刀小肠。首先剖析了所有的覆盖脂肪。然后解剖出的静脉和隔离带V形分支点的动脉。要小心，不要刺破所选段。开始解剖脂肪附近2阶分支，找到办法3 次或4 次 ORD呃船只。 <ol><li>注意：动脉和静脉可以基于他们的壁厚来区分 - 动脉壁比静脉的厚。此外，当相邻的结缔组织轻轻拉动垂直于血管，静脉容易崩溃，而动脉没有。由于动脉管腔直径&lt;400微米是全身血管阻力主要场所，对于这个协议，我们采用4 阶大鼠肠系膜动脉（管腔直径&lt;300微米）。 </li></ol></li><li>隔离动脉平行的4-5毫米部到小肠。想象所有的5 阶分支嵌入到小肠，切成稍微远离分支的起源和保存的部分。分支的这些保留的部分作为保持部位（与解剖钳）用于传送的动脉段到灌注室，并随后引导插管。 </li><li>然后通过切口2远端切断动脉段在动脉上的每一侧的5 阶分支并将其转移到灌注腔（ 见图1C和说明）。 </li><li>使用镊子剥离按住动脉段与解剖钳提示：（100-150微米直径）的玻璃微的一个导管插入血管的一端。以前加载的局部封闭缝合滑入空心端和固定。注意：在动脉的近端可以被插入套管上，其连接到伺服控制压力调节装置的原位环境模仿玻璃套管。 </li><li>附加一个夹层溶液内有10毫升注射器连接到这个套管，使得在连接所述插管和旋塞油管夹层溶液合并与该注射器中的活塞。轻轻抬起注射器。在溶液中的重力将去除帧内血管血液从容器的开口端。除去动脉血液后，关闭水龙头。 注意：可替代地，附加的活栓到压力控制器，其打开和压力轻轻增加至5-10毫米汞柱，以达到相同的结果。 </li><li>通过仔细使其他套管尽可能接近到动脉段的解开端扎血管的远端上的第二玻璃套管。以前加载的局部封闭缝合滑入空心端和固定。必须注意不能拉拽或拉动脉段。确保连接到两个插管旋塞关闭。 </li><li>转让灌注腔倒置显微镜配备了实时视频录制的阶段。 </li><li>连接套管连接到动脉段的近端到一个伺服控制压力调节装置的活塞，并确保活塞附连到其它插管保持关闭，以维持稳定的管腔内的压力。 </li><li>接着，附着在真空管连接到抽吸口的次的灌注管向腔室的灌注端口。 注：斜口针端口用于抽吸和钝针端口用于灌注。 </li><li>通过单列直插溶液加热器启动容器的灌注用温的PSS（37℃，平衡的气体混合物：5％的CO 2，5％O 2和90％的N以保持中性pH和充分氧合17）以2毫升/分钟使用蠕动泵。打开真空上也是如此。放置一个热敏电阻在室内连续监测温度。 </li><li>由于PSS在炉膛内的温度接近〜37℃（通常在5分钟内），慢慢增加腔内的压力为20〜100毫米汞柱，并检查血管是否有泄漏。这是通过使用压力调节器的自动压力设置。丢弃船只泄漏并更换另一段。与泄漏的容器将不能够保持压力。 </li><li>评估动脉段弯曲，同时保持在100毫米汞柱的压力。使用螺丝杆，将套管挺起动脉段。不要过度拉伸动脉段;我们的目标是在体内动脉段长度来模仿。 </li><li>降低压力至70毫米汞柱（ 在在肠系膜拱廊18 体内压力模仿），并允许在动脉段稳定和发展生肌基调。 （40-70毫米汞柱）根据实验策略和血管床的动脉可以可变加压。以前发布的审查提供可变性的极好审查MT来自不同血管床动脉8段。 </li></ol> 6.测量动脉直径<ol><li>在配备有一个单色视频电荷耦合器件照相机的显微镜视图动脉在10X物镜。利用视频图像采集和实时边缘检测系统测量管腔直径。 表3中提供了使用的设备的列表。 </li><li>监视和记录器Ðiameter不断。 </li><li>为观察MT的发展。注意：我们观察到，在大鼠肠系膜动脉阻力，在70毫米汞柱，MT的发展的特征在于，约20％的减少的直径。 MT根据血管床和动物物种而异。 </li><li>确认通过评估血管收缩和血管舒张反应为1μM苯福林（苯丙氨酸）和1μM的乙酰胆碱（ACh）的血管的生存能力。 </li><li>在每个实验结束时，确定被动直径（PD），通过将动脉在无Ca 2+的PSS 20分钟。 </li></ol> 7.生肌响应<ol><li>降低压力至20毫米汞柱，并允许直径稳定。增加在增量步骤（20，40，60，80和100）的管腔内的压力，并在各压力步允许动脉以实现稳定的直径（通常在5分钟内）。 </li><li>降低腔内压力到20毫米汞柱和孵化2+的 PSS含0.39毫米的Ca动脉段EGTA和0.1mM的SNP。允许动脉直径稳定（通常为15分钟）。 </li><li>重复在含0.39毫EGTA和0.1mM的SNP的Ca 2+ -free PSS中的压力阶跃响应。 </li></ol> 8.解释成果数据，并计算<ol><li>计算的MT与根据以下计算直径观察的Ca 2+的百分差值含与无Ca 2+的PSS在每个压力： <img alt="式（1）" fo:src="/files/ftp_upload/50997/50997eq1highres.jpg" src="/files/ftp_upload/50997/50997eq1.jpg" /></li><li>对动脉进行血管舒缩直径可以通过平均平顶阶段1分钟计算。根据关系表示所收集的数据作为最大松弛（％PD）的百分比：％PD = 100×[ΔD/ PD]; ΔD是前和添加任何试验化合物（ 例如苯丙氨酸）的后直径之间的差异; PD为被动直径（也最大直径）。 </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+ischemia+impairs+middle+cerebral+artery+myogenic+responses+in+the+pregnant+rat.">Placental ischemia impairs middle cerebral artery myogenic responses in the pregnant rat.</a> Hypertension. 58, 1126-1131 (2011).</li><li>Bagi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+2+diabetic+mice+have+increased+arteriolar+tone+and+blood+pressure:+enhanced+release+of+COX-2-derived+constrictor+prostaglandins.">Type 2 diabetic mice have increased arteriolar tone and blood pressure: enhanced release of COX-2-derived constrictor prostaglandins.</a> Arterioscler Thromb Vasc Biol. 25, 1610-1616 (2005).</li></ol>

作者没有任何经济利益冲突。

关键步骤，故障排除和修改在典型的等压容器的准备，动脉被加压至灌注用温水（37℃）的PSS两个玻璃插管之间70毫米汞柱。后30-45分钟，动脉开发MT，其特征在于，稳定在20-30分钟的直径自发下降。从各种血管床的阻力动脉开发可变MT。例如大鼠阻力肠系膜动脉开发的MT〜PD的25％，而cremastric动脉可能实现的PD的MT〜40％。动脉没有在60分钟内开发MT应该被丢弃;这个持续时间可以根据...

小阻力动脉是SVR的主要决定因素，并在许多疾病1,2的病理生理学中发挥重要作用。条件如糖尿病3，妊娠4，缺血再灌注5，肥胖和高血压6,7-经常与改变微血管功能相关联。血管肌动描记不仅可以提供重要的见解在各种疾病中的变化微血管功能也有助于确定治疗目标和评估血管活性化合物的功效。血管的功能已经被使用等距或等压容器的条件下，8孤立的小动脉的影响。等距肌动描记的详细描述在其他地方提供了9。不过，也有来自等距获得与等压制剂10-12数据的差异。因为加压动脉制剂允许微血管功能的研究在近生理条件下，该得到的结果可能会与相关的血管床8,13 体内的行为更好。 1902年，拜里斯首次描述血管直径14透压力的影响。他观察到在从兔，猫，狗的各种血管床中压力的降低随后的血管舒张小阻力动脉，并增加了压力，随后由血管收缩。这种现象被称为肌应答。贝利斯和随后的调查发现，在等压条件下阻力小动脉持续发展的收缩称为MT 15,16。既生肌响应和MT可以通过使用压力肌动描记（PM）的技术进行评估。 PM主要用于确定小动脉，静脉等血管血管活性。除了评估血管直径血管活性化合物的作用，PM - 顾名思义 - 用来评估血管内压力介导的CH安格斯对血管的直径。在过去的几十年中前进在计算机软件，这增强视频显微镜和玻璃吸管拉动，取得了下午更容易执行。然而，小血管存活完好分段解剖仍然繁琐，有时具有挑战性。在这里，我们勾勒出一个详细的协议，以研究在小肠系膜动脉阻力来自大鼠分离生肌响应。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Chemical</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma Aldrich</td>
			<td>A6625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G5767</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene diamine tetra acetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E9884</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3784</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M5162</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Sigma Aldrich</td>
			<td>P6126</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate (NaHCO3 )</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside</td>
			<td>Sigma Aldrich</td>
			<td>13451</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P8574</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1.</td>
		 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>
 <table border="1">
		<tbody>
		 <tr>
			<td>Physiological salt solution (1000 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.365 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>6.545 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.296 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.163 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>2.072 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>2.184 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>2.383 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>998 ml</td>
		 </tr>
		 <tr>
			<td>Ca2+ free physiological salt solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.036 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>0.645 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.029 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>0.207 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>0.218 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>0.238 g</td>
		 </tr>
		 <tr>
			<td>EGTA </td>
			<td>0.39</td>
			<td>0.015 g</td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside </td>
			<td>0.1</td>
			<td>0.0026 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>100 ml</td>
		 </tr>
		 <tr>
			<td>Dissection solution, stock (500 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>145</td>
			<td>21.18 g</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.7</td>
			<td>0.875 g</td>
		 </tr>
		 <tr>
			<td>MgSO4</td>
			<td>1.2</td>
			<td>0.739 g</td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>2</td>
			<td>1.049 g</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>0.02</td>
			<td>0.019 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>500 ml</td>
		 </tr>
		 <tr>
			<td>Working dissection solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection solution stock</td>
			<td>20 ml</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>1.2</td>
			<td>0.091 g</td>
		 </tr>
		 <tr>
			<td>NaH2PO4</td>
			<td>5</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>2</td>
			<td>0.022 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>0.2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>79.8 ml</td>
		 </tr>
	 </tbody>
</table>
 </td>
	 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>Table 2. Composition of Experimetnal solutions</td>
	 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>CCD Monochrome Camera</td>
			<td>The imaging Source</td>
			<td>DMK 21AU04</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Single inline solution heater</td>
			<td>Warner Instruments</td>
			<td>64-0102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thermistor</td>
			<td>Warner Instruments</td>
			<td>64-0108</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual automatic temperature controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instruments </td>
			<td>P-97</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fluorescence System Interface</td>
			<td>IonOptix</td>
			<td>model FSI-700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps and scissors </td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ion Wizard-Core and Analysis</td>
			<td>IonOptix</td>
			<td>Ion Wizard 6.0</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Laboratory tubing</td>
			<td>Silastic </td>
			<td>508-005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Male Sprague Dawley rat</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Master flex console drive</td>
			<td>Cole-parmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Milli-Q Plus Ultrapure Water System</td>
			<td>Millipore</td>
			<td>ZD5211584</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophthalmic monofilament nylon suture </td>
			<td>Ethicon </td>
			<td>9007G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photometry and Dimensioning Microscope</td>
			<td>Motic</td>
			<td>AE31</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pressure Servo Controller with peristaltic pump and pressure transducer</td>
			<td>Living Systems Instrumentation</td>
			<td>PS-200</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereomicroscope</td>
			<td>Nikon Instruments Inc</td>
			<td>SMZ660</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Vessel Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection dish</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-90-S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW120-6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microforge</td>
			<td>Stoelting</td>
			<td>51550</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3.</td>
		 </tr>
	 </tbody>
</table>

assessing myogenic response and vasoactivity in resistance mesenteric arteries using pressure myography

阻力小动脉收缩，并分别以响应增加或减少腔内压力扩张;这种现象被称为肌源性反应是局部血流量的重要调节器。在等压条件下阻力小动脉持续养成收缩称为肌音（MT），这是全身血管阻力（SVR）的主要决定因素。因此，阻力小动脉体外加压制剂的主要工具来研究微血管功能在近生理状态。为了实现这一目标，一个小的阻力动脉（直径〜260微米）的新鲜分离的完好分段被安装到两个小玻璃套管并加压。这些动脉制剂保留大部分体内特性和血管张力的实时许可证评估。在这里，我们为评估在大鼠加压阻力小肠系膜动脉血管活性详细的协议;这些动脉开发持续的血管收缩 - 最大直径的约25％ - 当在70毫米汞柱压力。这些动脉制剂可用于研究研究化合物对动脉内压力和血管活性之间关系的影响，并确定在各种疾病的动物模型的改变微血管功能。

一个典型的压力肌动描记设置示意图示于图1。将容器的两端插管用玻璃微并固定在两侧的缝合线。通过管道和开口旋塞，一个插管连接至伺服控制压力调节器;另一个套管连接到一个封闭的活栓。腔灌注有通过倒置显微镜相连的CCD摄像机观察PSS和血管直径的变化。 加压，在70毫米汞柱的动脉区段孵育在新鲜制备的温暖的PSS，流经动脉腔在2-4毫升/分钟，吸出。动脉?...

Watch this Scientific Journal Video about Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography at JoVE.com

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

压力肌动描记用于评估小动脉的持续发展压力收缩时血管活性。这份手稿提供了一个详细的协议在小肠系膜动脉大鼠，血管活性和腔内压力对血管直径的影响隔离段评估。

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 ...

assessing-myogenic-response-vasoactivity-resistance-mesenteric

Research

JoVE Journal

Medicine

16.6K 次观看.  Georgia Regents University. 压力肌动描记用于评估小动脉的持续发展压力收缩时血管活性。这份手稿提供了一个详细的协议在小肠系膜动脉大鼠，血管活性和腔内压力对血管直径的影响隔离段评估。 

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

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

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

硬膜外颅内压测量中使用的光纤压力传感器的大鼠

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

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

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for 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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

小梁响应压升高在活的人类眼睛

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

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 ...

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 ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Cardiac Response to &amp;#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

通过压力-体积环路分析确定心脏对β肾上腺素能刺激的反应

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...