作者想承认DRS。黄保罗和德米特里Atochin的的使用DMT的压力肌动描记和DRS。冰蓝羽和庄磊老鼠喂食高脂肪的饮食或饮食标准。 资金来源这项工作是支持的科学家发展格兰特10SDG2610313从美国心脏协会（ES收购），埃莉诺和万里海岸50周年纪念奖学金计划学者在哈佛医学院医学（ES收购）。

1。溶液的制备<ol><li> 500X EDTA股票：称取500毫克EDTA钠2·2H 2 O，溶于50毫升去离子水。在室温下保存。 </li><li>氯化钾去极化的解决方案。 <ol><li>准备K10X原液：3.69克氯化钠，氯化钾，18.64克0.36克硫酸镁无水，0.41克KH 2 PO 4，0.46克CaCl 2·2H 2 O溶解在250毫升的去离子水。在室温下保存。 </li><li>对于100毫升最终的氯化钾溶液1X去极化：将0.21的NaHCO 3，0.18克葡萄糖90毫升去离子水溶解。加入10毫升的K10X股票溶液。 500X EDTA溶液中加入95μl。拌匀。贮存于4℃一周内使用。 </li></ol></li><li>准备新鲜HEPES-PSS解决方案在当天的实验（1升）。 <ol><li> 6.96克氯化钠，氯化钾，0.35克0.288克硫酸镁4·7H 2 O，0.1605克KH 2 PO 4，2.38克HEPES。溶解在100毫升去离子水中。标记为烧瓶A. </li><li> 0.312克氯化钙2·2H 2 O溶解在100毫升的去离子水。标记为B瓶中。 </li><li> 1.25克的NaHCO 3，葡萄糖0.991克。溶于98毫升去离子水。标记为烧瓶C. </li></ol></li><li>添加2毫升500X EDTA烧瓶C.在一个大瓶700毫升去离子水。连续的震荡的大瓶中，倒入烧瓶A，B和C的内容。用NaOH将pH调节至7.4。 </li></ol> 2。使用的药物<ol><li>去氧肾上腺素（10 -2 M）：20.37毫克溶于10毫升去离子水。分装1毫升和储存在-80°C。串行稀释，得到10 -3，10 -4，10 -5，10 -6 mol / L的天实验。 </li><li>乙酰胆碱（10 -2 M）：18.17毫克溶于10毫升去离子水。分装1毫升和储存在-80°C。串行稀释，得到10 -3，10 -4，10 -5，10 -6mol / L的天实验。 </li></ol> 3。小鼠整个研究研究男WT和sGCα1 - / -小鼠对S6和B6背景。 sGCα1 - / -产生如前面所述4,5。住房和程序，涉及实验动物（小鼠），马萨诸塞州的波士顿马萨诸塞州总医院的研究动物护理小组委员会批准。 4。肠系膜动脉血管反应性测量<ol><li>安乐死小鼠戊巴比妥钠（200毫克/千克，IP）。打开腹腔及肠系膜组织解剖。隔离和三阶免费的结缔组织和脂肪组织的解剖肠系膜上动脉，动脉放置在冰冷的HEPES-PSS解决方案预平衡95％O 2/5％CO 2为15分钟。切2-3毫米长，没有从肠系膜分支环大动脉。时，有没有血液在血管的动脉和静脉之间微血管水平的差异是有问题的。因此，我们建议识别船只，而它在动物解剖出来之前，仍然完好无损。 </li><li>填写在HEPES-PSS溶液与10毫升注射器的帮助下，通过入口阀和出口阀的肌动描记器的压力腔室（DMT型号110P 11P版本1.31， 图2）与在玻璃插管。应该做填充套管轻轻地，仔细，因为压力过大可能会损坏脆弱的传感器连接到套管。灌装后插管进，出口阀门紧紧关闭。 </li><li>船只到右套管的一端安装，并仔细配合它与尼龙缝合线的细线之一。借助注射器，冲洗，并填写在血管与的HEPES解决方案通过进气阀。在左侧的套管带来接近容器，容器的另一端安装到其上。把它绑尼龙缝合。填室HEPES溶液10毫升。 HEPES轻轻推解决方案的帮助下，用注射器通过进气阀泄漏检查。 </li><li>安装室肌动描记下的视频摄像头。室开始的氧气和热量（37℃）。 HEPES溶液容器从第一管P1的入口阀连接，而在该阀关闭。让管任何气泡和解决方案，通过在管内，直到没有气泡。打开阀门。 </li><li>将左侧室管P2来自压力调节阀。再次检查任何气泡。不应该有任何气泡或在整个系统中的泄漏。 </li><li>压力菜单上肌接口的面板或在软件中打开泵，同时关闭流。 </li><li>打开程序，然后点击索取。船舶应出现在屏幕上（ 图3）。该容器是可视化用照相机装在显微镜，允许monitoring和分析血管腔，血管直径和壁厚。 </li><li>肌接口面板或程序选择P1压力逐步提高的interluminal通过P1阀的压力和输入的压力值如下5毫米汞柱→10→20→40→60毫米汞柱。船只的某个时候容易扭曲或卷积的压力增加时，调整张力或垂直或纵向微定位（ 图2）的帮助下，与相 ​​应的应变。平衡的容器中，在60毫米汞柱和37℃下至少45分钟。更换槽液一次预热的HEPES在平衡。 </li><li>应用10毫升的KCl去极化溶液，在37℃下预热，浴完全去极化平滑肌细胞和达到最大收缩。收缩稳定后，冲洗HEPES溶液3次，每次10分钟洗澡。 </li><li>稳定与生殖血管的内皮细胞的完整性检查的可行性此外去氧肾上腺素（10 -5 M）和乙酰胆碱的IBLE反应（10 -5 M）。冲洗3次，用HEPES溶液每10分钟。 </li><li>添加脱羟肾上腺素（10 -5 M）以preconstrict容器，并且得到稳定的收缩时，执行的累积浓度-的血管舒张响应曲线（CRC）顺序加入增加剂量的乙酰胆碱（10 -9 3×10 - 9，10 -8 3×10 -8，10 -7 3×10 -7，10 -6 3×10 -6，10 -5 M）。最后一剂后，HEPES溶液冲洗3次，每10分钟才开始新的CRC。 </li><li>应用的Ca 2 + PSS的含有2mM EGTA确定被动管腔的直径。从所记录的描记测量管腔直径为每个剂量响应（ 图4），这将被用于计算的所有参数。 </li><li> PERC表达所有的乙酰胆碱的舒张反应entage管腔直径去氧肾上腺素preconstriction后的变化相比，苯肾上腺素收缩后的直径无钙和直径之间的差异，使用下面的公式： </li><li>扩百分率= 100％×[（深x - D i的）/（D 钙自由 - D i的）]，其中D是测量的管腔直径和X，I，和无钙激动剂在每个剂量表示动脉直径（x）的初始直径以下福林收缩部（ⅰ），和的Ca 2 +的缓冲液（无钙）。 </li></ol> 5。统计分析所有连续测量平均值±SEM表示。肠系膜动脉血管反应性重复测量双向方差分析。在所有情况下，P &lt;0.05被认为是统计学意义。

<ol>
	<li>Bridges, L. E., Williams, C. L., Pointer, M. A., Awumey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+artery+contraction+and+relaxation+studies+using+automated+wire+myography.">Mesenteric artery contraction and relaxation studies using automated wire myography.</a> J. Vis .Exp. (55), e3119 (2011).</li><li>Arribas, S. M., Daly, C. J., McGrath, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+vascular+remodeling+by+confocal+microscopy.">Measurements of vascular remodeling by confocal microscopy.</a> Methods Enzymol. 307, 246-273 (1999).</li><li>Lei, C., Yu, B., 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=Inhaled+Nitric+Oxide+Attenuates+the+Adverse+Effects+of+Transfusing+Stored+Syngeneic+Erythrocytes+in+Mice+with+Endothelial+Dysfunction+after+Hemorrhagic+Shock.">Inhaled Nitric Oxide Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock.</a> Anesthesiology. , (2012).</li><li>Buys, E. S., Cauwels, A., 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=sGC&#945;1&#946;1+attenuates+cardiac+dysfunction+and+mortality+in+murine+inflammatory+shock+models.">sGC&#945;1&#946;1 attenuates cardiac dysfunction and mortality in murine inflammatory shock models.</a> Am. J. Physiol. Heart Circ. Physiol. 297 (2), H654-H663 (2009).</li><li>Buys, E. S., Sips, P., 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=Gender-specific+hypertension+and+responsiveness+to+nitric+oxide+in+sGC&#945;1+knockout+mice.">Gender-specific hypertension and responsiveness to nitric oxide in sGC&#945;1 knockout mice.</a> Cardiovasc. Res. 79 (1), 179-186 (2008).</li><li>Panza, J. A., Quyyumi, A. A., Brush, J. E., Epstein, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+endothelium-dependent+vascular+relaxation+in+patients+with+essential+hypertension.">Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension.</a> N. Engl. J. Med. 323 (1), 22-27 (1990).</li><li>Huang, P. L., Huang, 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=Hypertension+in+mice+lacking+the+gene+for+endothelial+nitric+oxide+synthase.">Hypertension in mice lacking the gene for endothelial nitric oxide synthase.</a> Nature. 377 (6546), 239-242 (1995).</li><li>Friebe, A., Koesling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+NO-sensitive+guanylyl+cyclase:+what+we+can+learn+from+genetic+mouse+models.">The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models.</a> Nitric Oxide. 21 (3-4), 149-156 (2009).</li><li>Ehret, G. B., Munroe, P. B., 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=Genetic+variants+in+novel+pathways+influence+blood+pressure+and+cardiovascular+disease+risk.">Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.</a> Nature. 478 (7367), 103-109 (2011).</li><li>Buys, E. S., Raher, M. 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=Genetic+modifiers+of+hypertension+in+soluble+guanylate+cyclase+alpha1-deficient+mice.">Genetic modifiers of hypertension in soluble guanylate cyclase alpha1-deficient mice.</a> J. Clin. Invest. 122 (6), 2316-2325 (2012).</li><li>Kauffenstein, G., Laher, I., Matrougui, K., Guerineau, N. C., Henrion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+G+protein-coupled+receptors+in+microvascular+myogenic+tone.">Emerging role of G protein-coupled receptors in microvascular myogenic tone.</a> Cardiovascular Research. 95 (2), 223-232 (2012).</li><li>Ruilope, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension+in+2010:+Blood+pressure+and+the+kidney.">Hypertension in 2010: Blood pressure and the kidney.</a> Nat. Rev. Nephrol. 7 (2), 73-74 (2011).</li><li>Michael, S. K., Surks, H. K., 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=High+blood+pressure+arising+from+a+defect+in+vascular+function.">High blood pressure arising from a defect in vascular function.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (18), 6702-6707 (2008).</li><li>Mendelsohn, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+hypertension,+the+kidney+is+not+always+the+heart+of+the+matter.">In hypertension, the kidney is not always the heart of the matter.</a> J. Clin. Invest. 115 (4), 840-844 (2005).</li></ol>

什么都没有透露。

老鼠是许多研究者的实验模型的选择，部分是因为引进遗传修饰小鼠模型，从而产生人类生理的可能性。作用于血管的阻力小的状态，但并不代表较大的导管容器主要确定了整个血管系统11的血流量的调节。动脉阻力的大小，在小动物，如小鼠，可防止使用钢丝肌动描记研究微血管功能。压力myographs不仅克服这个限制，但也允许在接近生理条件下，血管功能的测量。 ?...

压力肌动描记系统用来测量小动脉，静脉和其他血管的生理功能和属性。动脉或静脉的一个完整的小部分被安装到两个小玻璃插管，加压至一个合适的腔的压力，使容器保持其大部分在体内的特征（ 图1和2）。在压力肌动描记接近生理条件反映了体内的血管床的行为，允许调查孤立船只的内在属性（ 如生肌音）。压力myography超过的线myography，肌肉收缩的评估是通过直接的机械耦合的力换能器1的一些优点，包括（i）微动脉阻力，它定义了在血管系统的整体电阻，可以研究，而丝肌动描记限于较大的动脉导管，（ⅱ）吨帽子来损伤血管内皮细胞的风险被降低了，因为没有电线需要通过血管腔，（ⅲ），更好地保持该船只的自然形状，及（iv）可以在范围广泛的研究，容器尺寸压力或剪切力2。 研究微船舶可以比学习更大的动脉导管，以帮助了解病理生理学和分子机制，有助于心血管疾病如高血压血管张力改变更多的信息。例如，8周高脂肪的饮食喂养小鼠血管内皮功能相关的减值可以证明在第二阶肠系膜动脉，但不是在主动脉环（ 图3）。压力myography的另一个优点是，内在生肌收缩的压力容器是否存在，并可以研究，在这种现象中的血管内皮细胞的作用和功能。在这里，我们DESCRIBE使用压力肌动描记小鼠肠系膜三阶阻力动脉血管反应性研究中设置的障碍一氧化氮（NO）-cGMP信号。

<table border="1">
		<tbody>
		 <tr>
			<td>Name</td>
			<td>Company</td>
			<td>Cataloge No.</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
		 </tr>
		 <tr>
			<td>CaCl2 (2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
		 </tr>
		 <tr>
			<td>MgSO4 </td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
		 </tr>
		 <tr>
			<td>KH2PO4 </td>
			<td>Sigma</td>
			<td>P3786</td>
		 </tr>
		 <tr>
			<td>NaHCO3 </td>
			<td>Fisher Scientific</td>
			<td>BP328</td>
		 </tr>
		 <tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318</td>
		 </tr>
		 <tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP121</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Acros Organics</td>
			<td>AC20724</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma</td>
			<td>A6625</td>
		 </tr>
		 <tr>
			<td>Pressure Myograph System</td>
			<td>DMT</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

assessing murine resistance artery function using pressure myography

压力肌动描记系统是精美实用的小动脉功能评估，加压到一个合适的透压力。接近生理条件实现压力myography的允许深入刻画的内在反应的药理和生理刺激，可以外推到在体内行为的血管床。压力肌动描记有几个优点，比传统的线myographs。例如，较小的阻力血管，可以研究在严格控制和生理相关的腔内压力。在这里，我们学习三阶肠系膜动脉（长3-4毫米），预先使用新福林，血管放松对乙酰胆碱的反应能力。肠系膜动脉插管连接到加压和密封系统，保持在恒定的压力为60毫米汞柱的安装。容器的内腔和外直径是后续的狡黠的记录，可使用视频摄像头，允许实时定量去氧肾上腺素和乙酰胆碱的血管收缩和血管舒张，。 为了演示的适用性的压力myography研究心血管疾病的病因学，我们评估了在小鼠模型的全身性高血压的内皮依赖性的血管功能。高血压缺陷小鼠可溶性鸟苷酸环化酶（sGCα1 - / - ）的α亚基上129S6（S6）的背景（sGCα1 -/-S6），但不是当上C57BL / 6（B6）背景（sGCα 1 -/-B6）。使用压力myography，我们证明了sGCα1缺乏业绩受损的内皮依赖性血管舒张。血管功能障碍更加明显在sGCα1 -/-S6比1sGCα-/-B6小鼠，可能贡献的纳克较高的血压比sGCα1 -/-B6小鼠在sGCα1 -/-S6。 压力myography是一个比较简单，但敏感机制有用的技术，可用于评估各种刺激血管的收缩和舒张的效果，从而增强我们深入了解心血管疾病的机制。

NO的中心参与血压体内平衡维持在6，在动物模型中7人类。控制血管平滑肌松弛的能力NO介导的可溶性鸟苷酸环化酶（SGC），含血红素的异二聚体酶生成cGMP的8。最近，血压修改遗传变异被确定轨迹包含sGCα1和1sGCβ基因，说明在人类9调节血压相关的SGC。有趣的是，雄性小鼠缺乏SGC（sGCα1 - / - ）的α亚基上129S6（S6）的背景（sGCα1</...

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Assessing Murine Resistance Artery Function Using Pressure Myography

在压力myography，一艘船一个完整的小部分安装到两个小插管，加压到一个合适的管腔压力。在这里，我们描述的方法来衡量鼠标3的血管舒张反应<sup&gt; RD</sup为了在C57和sGCα的肠系膜动脉<sub&gt; 1</sub<sup&gt; - / -</sup&gt;小鼠使用压力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  ...

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Biology

21.9K 次观看.  Massachusetts General Hospital, Harvard Medical School. 在压力myography，一艘船一个完整的小部分安装到两个小插管，加压到一个合适的管腔压力。在这里，我们描述的方法来衡量鼠标3的血管舒张反应 RD

视频: Assessing Murine Resistance Artery Function Using Pressure Myography

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

使用卷压力记录，一条尾巴袖套法小鼠的血压测量

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

科研

生物学

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

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 ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; Effective dampening of flow against pressure changes occurs over periods as short as ~15 sec and becomes progressively greater over longer time periods. Thus, slower changes in blood pressure are effectively blunted and faster changes or fluctuations pass through to cerebral blood flow relatively unaffected. The primary difficulty in characterizing the frequency dependence of cerebral autoregulation is the lack of prominent spontaneous fluctuations in arterial pressure around the frequencies of interest (less than ~0.07 Hz or ~15 sec). Oscillatory lower body negative pressure (OLBNP) can be employed to generate oscillations in central venous return that result in arterial pressure fluctuations at the frequency of OLBNP. Moreover, Projection Pursuit Regression (PPR) provides a nonparametric method to characterize nonlinear relations inherent in the system without a priori assumptions and reveals the characteristic non-linearity of cerebral autoregulation. OLBNP generates larger fluctuations in arterial pressure as the frequency of negative pressure oscillations become slower; however, fluctuations in cerebral blood flow become progressively lesser. Hence, the PPR shows an increasingly more prominent autoregulatory region at OLBNP frequencies of 0.05 Hz and below (20 sec cycles). The goal of this approach it to allow laboratory-based determination of the characteristic nonlinear relationship between pressure and cerebral flow and could provide unique insight to integrated cerebrovascular control as well as to physiological alterations underlying impaired cerebral autoregulation (e.g., after traumatic brain injury, stroke, etc.).

Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.

通过振荡下体负压和投影寻踪回归评估脑自动调节

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

用奔放的整体体积描记测量呼吸功能的影响

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

心脏压力容量环分析小鼠使用电导导管

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Intracavernosal 压力记录对啮齿动物勃起功能的评价

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and disease. EIM is obtained by applying a low-intensity, directionally focused, electrical current to a muscle of interest across a range of frequencies (i.e., from 1 kHz to 10 MHz) and recording the resulting voltages. From these, several standard impedance components, including the reactance, resistance, and phase, are obtained. When performing ex vivo measurements on excised muscle, the inherent passive electrical properties of the tissue, namely the conductivity and relative permittivity, can also be calculated. EIM has been used extensively in animals and humans to diagnose and track muscle alterations in a variety of diseases, in relation to simple disuse atrophy, or as a measure of therapeutic intervention. Clinically, EIM offers the potential to track disease progression over time and to assess the impact of therapeutic interventions, thus offering the opportunity to shorten the clinical trial duration and reduce sample size requirements. Because it can be performed noninvasively or minimally invasively in living animal models as well as humans, EIM offers the potential to serve as a novel translational tool enabling both preclinical and clinical development. This article provides step-by-step instructions on how to perform in vivo and ex vivo EIM measurements in mice and rats, including approaches to adapt the techniques to specific conditions, such as for use in pups or obese animals.

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals, pups).

在啮齿动物中进行体内和离体电阻抗肌图

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...