<p class="jove_content"这项工作是由美国国立卫生研究院授予P20RR018766和酒精研究从ABMRF /基金会授予的支持。</p

1。动物<ol><li>机构动物护理和使用委员会在路易斯安那州立大学健康科学中心的所有程序批准，并按照国家卫生研究所（NIH出版物，编号85-12，1996年修订）的指引执行。雄性SD大鼠（查尔斯河实验室，体重270-350克）被安置在一个控制温度（22℃）和控制的照明（12:12 h光照明暗周期）环境。抵达后，将大鼠提交了为期一周的驯化期间，提供了标准的大鼠议员（2018 Teklad全球18％的蛋白质鼠类饮食，哈伦）和水自由采食。 </li></ol> 2。实验设置<ol><li>是由安装淋巴管的玻璃微量硼硅玻璃与灯丝外径1.2毫米，身份证0.69毫米（萨特仪器高炉120-69-15）。拉移液器使用火焰/布朗的微管拖轮模型P - 97（萨特仪器和设备ENT），是一个微型磨床，如400（Narishige）获得一个〜60微米外径一个斜面尖端的斜面。 </li><li>山中分离出的船室（CH - 1模型，生命系统，伯灵顿，VT），微量。移液器应电阻匹配（相同大小的开口）。 </li><li>填充腔（5毫升）和微量白蛋白的生理盐溶液（APSS，见表1）。确保有微量或油管无气泡。 </li><li>准备上手眼科缝线和​​地方每个微节。 </li></ol> 3。收集淋巴隔离<ol><li>麻醉动物肌肉注射氯胺酮/甲苯​​噻嗪（毫克/千克，90％和9），使用一个BD注射器（1毫升）和针（BD皮锥26G3 / 8）。 </li><li>喷雾用70％酒精消毒腹部区域，进行中线剖腹探查，小肠和肠系膜exteriorize和消费。将IC组织E -冷APSS。安乐死与氯胺酮/甲苯​​噻嗪过量的动物和开放的胸部（每美国兽医医疗协会对安乐死的指引）确认死亡。 </li><li>针用冰冷的APSS填补了清扫室的肠系膜。仔细解剖立体显微镜的帮助下从周围的脂肪和结缔组织，收集淋巴管（80-200微米的内部直径和长度为1-2毫米）。使用解剖杜蒙钳INOX 55（精细的科学工具＃11255-20）和解剖春季剪刀（精细的科学工具＃15000-03）。使用隔离的船只，以确保在整个段的最佳压力控制在实验过程中只有一个阀门。 </li><li>孤立淋巴转移到隔离船室，含5毫升的APSS。搭售眼科缝合船只装载到两个电阻匹配的玻璃微量。 </li><li>室转移到一个倒置荧光显微镜（我们是一个NIKTE - 2000U）配备了氙气灯（萨特仪器LAMBDA LS2的300瓦），三百八十〇分之三百四十零轮过滤器系统（色度科技340和380 nm激发过滤萨特的Lambda 10-3），510 nm处分色发射器（色度技术D510的/80米），一个敏感的CCD相机（Photometrics 总部2），收购（尼康元素，AR）和软件（图1） 。 </li><li>将油管从原产微量可调水库或伺服反馈泵系统（生命系统的仪器仪表，伯灵顿VT），都可以用来改变压力和流量。 </li><li>室连接的供热单位（生活系统）至37 ° C，并设置洗澡</li><li>查看盖玻片直接通过淋巴室与10倍的目标（尼康10x/0.3福陆公司的计划，DIC的L/N1，∞/ 0.17，WD 16.0）的基础。使用图像采集软件（我们的系统尼康元素AR软件），可以取得快速的时间推移图像集。 </li></ol>2 +] i的<ol><li>的[Ca 2 +]我在孤立淋巴管平滑肌的Ca 2 +感应染料FURA 2 acetoxymethyl酯（上午）（分子探针，尤金，OR）来衡量。 </li><li>淋巴管负载交换浴APSS解决方案，包含30分钟FURA - 2 AM（2微米）和Pluronic酸（0.2％重量/体积）在37 ° C. FURA - 2 AM </li><li> 30分钟后，改变洗澡APSS的解决方案。冲洗2次，洗出液FURA - 2。平衡时间至少20分钟前离开该船只的[Ca 2 +] i的测量，以便重新建立自发性收缩。 </li><li>收集与收购协议，或者在340和380 nm波长照亮持续时间为50毫秒的比例FURA - 2在孤立淋巴管的测量。荧光灯通过分色镜（400纳米;色度TEchnology公司400DCLP）和宽波段发射滤光片（510纳米，80纳米带的宽度;色度科技公司D510/80m）和被收购Photometrics总部2相机。我们收集基线2厘米的H 2 O的腔内压力在逐步增加了2，4，6，8 2分钟的数据，并一〇厘米H 2 O </li><li>要分析平均的[Ca 2 +] i的，画的感兴趣区域（ROI），其中包括整个淋巴管及周边地区，使船只是完全放松和收缩（图2）期间跟踪。如果任一微管在视野是这些地区不应该包括在投资回报率。较小的投资回报率也可以选择，但潜在的问题是一个小的投资回报，血管壁可能的投资回报率在纵向方向移动，轻微血管收缩和放松。另一个潜在的问题是，一些船只在收缩扭曲，这些淋巴管不应该用于学习。钍Ë扭议案通常避免限制的孤立淋巴&lt;1毫米的长度。此外，有时阀两侧段不收缩/放松同步。在这种情况下，因为这些细分市场代表不同的功能lymphangions，淋巴应研究只有一个阀门的一侧，或每个lymphangion可以放在一方的投资回报率分别研究。使用10倍的目标时，应保持血管壁的重点在阶段性收缩，但是如果有外出期间的收缩，这些重点领域，不应该包括在投资回报率的墙壁的任何部分。一个背景的投资回报率也应放在远从容器（例如，在图像的角落）。在三百八十零分之三百四比例的增加，显示的[Ca 2 +] i的增加。管腔直径在不同的时间点，也可以使用该软件的测量功能进行测量或跟踪随着时间的推移，血管壁（见第6节）。/ LI&gt; </ol> 5。压力控制协议<ol><li>为学习压力独立施加流，同样的压力，必须适用于每个微量伺服空反馈泵。每个微管是通过一个T型连接器共同管装泵连接。初步确定在2厘米H 2 O的腔内压力为45-60分钟，这是足够的时间，使淋巴自发性收缩的发展。 </li><li>学习使用平衡时间内，满足下列条件的唯一船只：（1）船只发展自发音，H 2 O 2厘米（2）船只发展常规，整个船只的长度合理统一的自发性收缩。通常从一个阀门区下游收缩比其他船只，这些船只将被用来只要有证据，其余的船只显示收缩活动。 </li><li>时间推移快速的图像记录在压力步骤程序。对于每个压力将开始在H 2 O 2厘米，30秒，然后提出在一个步骤是由2，4，6，8，或一分钟一零厘米H 2 O，并降低回H 2 O为30秒至2厘米</li><li>的压力步骤系列完成后，改变液一个+免费APSS在37 CA 2 ° C，来衡量上述管腔压力最大的被动直径（ 最大四 ） 和 Ca 2 +荧光测量。最大D是用来计算的色调和正常化不同大小的淋巴系统之间的数据。 </li></ol> 6。淋巴管收缩参数<ol><li>使用对象追踪工具在大多数的图像分析软件包，可确定血管直径随时间的变化。这可以通过使用唯一的渠道之一（我们使用的340纳米通道，但380通道也可以用来）。对比阈值是应用程序谎称图像的堆栈，使血管壁的突出显示，然后投资回报包括每个墙，随着时间的推移，他们被吸引到轨道（图3）。内部直径可通过测量每面墙的重心和计算两个质心之间的距离决定，减去每个墙厚度的一半。 第二个选项时，是很难跟踪每一个强烈的信号，在管腔面积的墙体由于是测量外径，并使用修正系数基础上获得内部直径的血管壁的静态测量。在这种情况下，投资回报率跨越整个容器外径为跟踪（图二 ）。这种方法也可以用来判断淋巴整个可视横截面面积（如图3架C）。由于lymphangion的典型形状，后一种选择，可以提供一个更好的估计流体的实际体积每次收缩泵在选定的部分的直径比使用该船只。 </li><li>从时间推移的研究，确定淋巴管腔直径测量4,12,13以下参数： 收缩频率（CF），通过计算每分钟的阶段性收缩的数目确定。 舒张末期内径（EDD），这是只是一个阶段性的收缩之前的直径。 收缩末直径（ESD），直径在一个阶段性的收缩结束。 收缩幅度（AMP =的EDD，ESD），简化的搏出量指数。 每个压力最大的被动直径（ 最大D，确定在Ca 2 +条件），可用于确定由淋巴管平滑肌生成的基调，也可用于不同直径的淋巴系统之间的数据正常化，或同样的淋巴研究在不同的管腔压力。 EDD 1，这是先收缩后压力步骤相关的EDD。时这是作为一个起点决定在5管腔压力的逐步增加生肌一个淋巴管收缩。 生肌收缩后压力步骤孤立淋巴代表EDD的正常化变化，压力逐步增加= 100 *（EDD - EDD 1）/ 最大 ð 。 可以计算，但讨论的细节4的其他变量是： 音= 100 *（最大D - EDD）/最大ð，规范化的安普AMP /最大ð 每搏输出量指数（SVI）=π（EDD的2 - ESD 2） 射血分数（EF）= SVI /（πEDD2） 体积流量指数（VFI）= CF x的SVI。 </li></ol> 7。代表性的成果： 在每个阶段性收缩的开始，有一个短暂升高的[Ca 2 +] i的（图4） 。此外，后管腔压力的一步增加，频率的 Ca 2 +瞬变和Phasic收缩在同步增加。这些意见是类似以前的发现，使用安装在一个14丝肌动描记和支持以前的数据表明振荡的Ca 2 +释放内部商店在底层的阶段性收缩周期15机制的核心作用胸导管。 我们也调查是否的[Ca 2 +]在舒张过程中， 我 （之间的 Ca 2 +瞬变和阶段性收缩）相关的淋巴音生肌收缩时施加压力的一步增加（图5 ）拉伸造成的。后，立即在4厘米H 2 O和更高的[Ca 2 +] 我在舒张稳步增长，较基线前的压力一步（图5B）压力的一步增加。此外，初始直径增加，因为，在压力（EDD 1）步增加后，有生肌收缩（图余吕杏茜5C），定义为从1 EDD减少5,12以前的报告在EDD。 EDD的平均规范化的变化，在不同压力步骤进行比较时，，有显著更多的收缩后增加的8一零厘米H 2 O相比，二厘米H 2 O的（ 图 6） 。在稳步上升的[Ca 2 +] 我也与压力，一步增加+6，+8和一〇厘米， 造成钙的变化显着高于H 2 O +比二厘米H 2 O（图6 乙 ）。然而，压力一步的Ca 2 +的关系出现高原以及这些压力。这些结果表明，钙2 +依赖的机制与淋巴响应舒展生肌收缩。然而，在高原的压力一步的Ca 2 +的关系，提出一种机制， 增加的Ca 2 +的敏感性可能也有助于增加生肌constriction在较高的压力步骤。 <img alt="图1" src="/files/ftp_upload/3438/3438fig1.jpg" /> 图1用于测量显微镜系统的原理图的[Ca 2 +]我在一个孤立的淋巴。孤立淋巴腔放置在倒置显微镜下阶段。试样在340和380 nm，或者亮起和淋巴发出的荧光信号与CCD相机收集并存储在个人电脑中。空伺服反馈系统控制的淋巴腔内压力。用户设置为系统的压力，压力传感器继电器行与淋巴腔内压力的反馈系统，可以动态地提高或降低通过泵的压力。 <img alt="图2" src="/files/ftp_upload/3438/3438fig2.jpg" /> 感兴趣区域（RO示例图2。我）FURA - 2的比例在孤立淋巴管成像研究的选择。选定该地区包括大部分的船只（感人的微量的地区除外）及周边地区，以确保船只将整个时间过程跟踪。一个背景的投资回报率（右上角）也被用来消除非特异性信号从周围浴。 <img alt="图3" src="/files/ftp_upload/3438/3438fig3.jpg" /> 图3。跟踪抽水淋巴的方法。随着时间的推移，使用的投资回报A.内部直径可确定血管壁的轨道运动。 B.当对比度阈值设置为包括全船，外径可以跟踪一个单一的投资回报率。纠正壁厚内径可从这些测量估计，C。另一种选择是该地区所涵盖的船只，它可用于纠正壁厚计算量和跟踪假设淋巴圆柱几何。 <img alt="图4" src="/files/ftp_upload/3438/3438fig4.jpg" /> 图4之间的关系的[Ca 2 +] i和淋巴管的直径 。代表从时间推移FURA - 2在一个孤立的淋巴强度（热图格式）视频图像。在每幅图像的箭头大纲的船只外径。第1张图片显示在舒张期相对较低的[Ca 2 +] i的淋巴。图像2，三百八十零分之三百四十〇在血管壁的比例增加强度，只是阶段性收缩，在图像3-5发生之前。 4通过图像，瞬态的Ca 2 +已经结束，并通过图像5，收缩阶段已经结束，船只回到静止状态，B。的[Ca 2 +] i和淋巴管腔直径的阴谋显示，在短暂升高的[Ca 2 +] i的发生前每个阶段性收缩。 <img alt="图5" src="/files/ftp_upload/3438/3438fig5.jpg" /> 图5。淋巴变化的[Ca 2 +] i和淋巴响应收缩周期，加强压力增大。显示压力步协议（一），三百八十零分之三百四十零比数据，代表的Ca 2 +] I（B），并随着时间的推移（三）在淋巴直径的变化。 A.基线管腔压力是2厘米的H 2 O和淋巴管受到步骤2增加，4，6，8，和十厘米的H 2 O彼此之间的压力分步录音的时间间隔为1-2分钟。每个压力步骤造成双方在短暂增加的频率增加的[Ca 2 +] I（B）和阶段性收缩（C ）淋巴管的直径也增加了每个管腔压力的逐步增加，4至+10 厘米 H 2 O压力的步骤，有一个初步的EDD减少发生AFEDD1之三（三），以前称为淋巴生肌收缩描述。还有一个稳步增长的基础[的Ca 2 +] 我之间的瞬态后立即每个压力逐步增加（二）。生肌收缩，具有较大的压力步骤更为显着，但在基础[ 的 Ca 2 +]增加，我是大约为4至10厘米，H 2 O压力步骤（b）相同。先前报告中AMP代偿性增加也很明显，后6至10厘米H 2 O（三）压力步骤。 <img alt="图6" src="/files/ftp_upload/3438/3438fig6.jpg" /> 图6。生肌淋巴管收缩和自由逐渐增加的[Ca 2 +] i的在舒张期后不久，在压力的一步增加 。 30-50小号之间的EDD平均后的EDD 1 乙是用来计算归EDD的压力为每一步的变化。。除以平均三百八十零分之三百四十零在基线的比例（2厘米H 2 O）的三百八十○分之三百四十○比例30-50小号后的EDD 1力度。 * P &lt;0.05 - 2厘米的 H 2 O压力步骤。 N = 4时船舶研究。 电影1。例如研究使用的船只在一个孤立的淋巴阶段性收缩。管腔压力定为2厘米H 2 O所用时间和比例尺显示在底部。时间推移图像收集与10X使用透射光的目标<a href="http://www.jove.com/files/ftp_upload/3438/3438movie1.avi">。点击这里观看影片</a> 。 电影2。成比例FURA - 2成像在孤立淋巴抽水。热地图的比例尺显示在左上角和所用的时间和比例尺的底部。初步定在2厘米H 2 O的压力，提高到8厘米H 2 O，0.5分和returne开始d至2厘米，在1.5分钟H 2 O <a href="http://www.jove.com/files/ftp_upload/3438/3438movie2.avi">。点击这里观看电影</a> 。

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Physiol. 297, 726-726 (2009).</li><li>Karaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements,+distribution,+and+functions+in+smooth+muscle.">Calcium movements, distribution, and functions in smooth muscle.</a> Pharmacol. Rev. 49, 157-157 (1997).</li><li>Ratz, P. H., Berg, K. M., Urban, N. H., Miner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+smooth+muscle+calcium+sensitivity:+KCl+as+a+calcium-sensitizing+stimulus.">Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.</a> Am. J. Physiol. Cell. Physiol. 288, C769-C769 (2005).</li><li>Somlyo, A. P., Somlyo, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sensitivity+of+smooth+muscle+and+nonmuscle+myosin+II:+modulated+by+G+proteins,+kinases,+and+myosin+phosphatase.">Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.</a> Physiol. Rev. 83, 1325-1325 (2003).</li><li>Souza-Smith, F. M., Kurtz, K. M., Molina, P. E., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+mesenteric+collecting+lymphatic+pump+function+following+acute+alcohol+intoxication.">Adaptation of mesenteric collecting lymphatic pump function following acute alcohol intoxication.</a> Microcirculation. 17, 514-514 (2010).</li><li>Breslin, J. W., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-C+alters+barrier+function+of+cultured+lymphatic+endothelial+cells+through+a+VEGFR-3-dependent+mechanism.">VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism.</a> Lymphat. Res. Biol. 5, 105-105 (2007).</li><li>Shirasawa, Y., Benoit, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-induced+calcium+sensitization+of+rat+lymphatic+smooth+muscle.">Stretch-induced calcium sensitization of rat lymphatic smooth muscle.</a> Am. J. Physiol. Heart. Circ. Physiol. 285, H2573-H2573 (2003).</li><li>Imtiaz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaking+through+Ca2++stores+interacting+as+coupled+oscillators+via+membrane+depolarization.">Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.</a> Biophys. J. 92, 3843-3843 (2007).</li><li>Muller, J. M., Davis, M. J., Kuo, L., Chilian, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+coronary+endothelial+cell+Ca2++concentration+during+shear+stress-+and+agonist-induced+vasodilation.">Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.</a> Am. J. Physiol. 276, 1706-1706 (1999).</li><li>Ferrusi, I., Zhao, J., van Helden, D., von der Weid, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopiazonic+acid+decreases+spontaneous+transient+depolarizations+in+guinea+pig+mesenteric+lymphatic+vessels+in+endothelium-dependent+and+-independent.">Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent.</a> 286, H2287-H2287 (2004).</li></ol>

没有利益冲突的声明。

一个新颖的方法组合研究内在的淋巴管抽水。能够同时测量变化的[Ca 2 +] i和抽水淋巴管的直径将允许的 Ca 2 +依赖和 Ca 2 +敏的信号通路在整体管收缩淋巴循环的机制。相对贡献的研究淋巴管收缩周期由过音叠加的阶段性收缩。数据表明，每一个阶段性的收缩与中的[Ca 2 +] i的瞬态上升，这表明一个核心作用?...

<table border="1"><tbody><tr><td> 1。林格5倍股 </td></tr><tr><td化学</td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>氯化钠</td><td>电解二氧化锰</td><td> SX0420 - 3 </td><td> 35摹</td></tr><tr><td>氯化钾</td><td> JT贝克</td><td> 3040 </td><td> 1.75摹</td></tr><tr><td>氯化钙</td><td>西格玛</td><td> C - 3881 </td><td> 1.47摹</td></tr><tr><td>硫酸镁</td><td>西格玛</td><td> M - 9397 </td><td> 1.44摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td>进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2。 MOPS缓冲 </td><td></td><td></td><td></td></tr><tr><td> 化学 </td><td> 公司 </TD&gt; <td> 产品编号 </td><td> 金额 </td></tr><tr><td> MOPS </td><td>西格玛</td><td> M3183 </td><td> 125.6摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td>进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3。白蛋白的生理盐溶液（APSS） </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>林格股票（5X） </td><td> N / A </td><td> N / A </td><td> 200毫升</td></tr><tr><td> MOPS缓冲</td><td> N / A </td><td> N / A </td><td> 5毫升</td></tr><tr><td>磷酸钠</td><td>西格玛</td><td> S - 9638 </td><td> 0.168摹</td></tr><tr><td>丙酮酸钠</td><td>西格玛</td><td> P5280 </td><td> 0.22摹</td></tr><tr><td> EDTA钠SAL吨</td><td>西格玛</td><td> ED2SS </td><td> 0.0074摹</td></tr><tr><td>血糖</td><td>西格玛</td><td> G7528 </td><td> 0.901摹</td></tr><tr><td>白蛋白，牛</td><td> USB </td><td> 10856 </td><td> 10克</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td>调整pH至7.4，在37 °，然后放入高压灭菌瓶和储存在4℃的无菌过滤° C。 </td></tr></tbody></table> 表1具体所用试剂。所有存储在4 ° C。

measurement of cytosolic ca2+ in isolated contractile lymphatics

淋巴管组成一个多功能的交通运输系统保持流体的动态平衡，血脂中央流通，作为一个潜在的有害抗原的监控系统，优化粘膜免疫和适应性免疫反应1。从组织间液进入盲端的初始淋巴管，然后是对运输压力梯度较大的收集淋巴管形成淋巴。每个收集淋巴是由一系列称为lymphangions段，二尖瓣阀门，防止回流隔开。每个lymphangion拥有一个收缩的周期，推动反压力梯度向中央循环 2淋巴结。这种阶段性的收缩模式类似心动周期，收缩压和舒张压阶段，并具有较低的收缩频率4。此外，淋巴管平滑肌产生提示音并显示生肌的收缩和扩张，我ñ回应管腔压力的增加和减少，分别为5。因此，建议同时支持淋巴管的阶段性和补品收缩的分子机制的混合体。 平滑肌收缩一般是受细胞质的Ca 2 +浓度（内[Ca2 +] i的），加上灵敏度的Ca 2 +，在周围的细胞6环境变化的响应收缩元素。的[Ca 2 +] 我是运动的Ca 2 +通过质膜配体或电压门控钙离子通道的释放和吸收的Ca 2 +从内部商店相结合确定。胞质的Ca 2 +结合到钙调蛋白和酶的激活，如肌球蛋白轻链（MLC）的激酶（MLCK），这反过来又磷酸化MLC的肌动蛋白-肌球蛋白介导的收缩 8 。然而，这种通路的敏感性钙<sup&gt; 2 +可调节的MLC磷酸酶（MLCP）9。 MLCP活性受Rho激酶（ROCK）和肌球蛋白磷酸酶抑制蛋白CPI - 17。 在这里，我们目前超过隔离，淋巴管灌注时间的方法，以评估变化的[Ca 2 +] 我为了研究的Ca 2 +依赖和Ca 2 +敏淋巴管平滑肌收缩机制。采用离体大鼠肠系膜淋巴管收集，我们研究的[Ca 2 +]拉伸引起的变化我和收缩活动。孤立淋巴模型提供了优势的压力，流量，和化学成分的沐浴液，可以严格控制。的[Ca 2 +] 我是通过加载比例的Ca 2 +结合染料FURA - 2淋巴确定。这些研究将提供新的方法来学习不同的分子机制，规范阶段性的更广泛的问题进补收缩与淋巴管平滑肌的收缩。

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

我们介绍的方法来评价细胞质CA<sup&gt; 2 +</sup&gt;孤立淋巴集中在研究CA<sup&gt; 2 +</sup&gt;依赖和Ca<sup&gt; 2 +</sup&gt;敏淋巴管平滑肌收缩机制。

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

measurement-of-cytosolic-ca2-in-isolated-contractile-lymphatics

Research

JoVE Journal

Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

13.5K 次观看.  Louisiana State University Health Sciences Center. 我们介绍的方法来评价细胞质CA 2 +孤立淋巴集中在研究CA 2 +依赖和Ca 2 +敏淋巴管平滑肌收缩机制。

视频: Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

科研

免疫与感染

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

测量抗体乳鼠心肌细胞对细胞功能的影响

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

细菌性前列腺炎小鼠模型的测量触觉异常

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

尺寸事项:新生隐球菌中胶囊直径的测定

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

生物样品中赤子酶活性的测定

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

实时分析离体淋巴管中的钙和收缩力

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

作者聚焦：淋巴水肿和高血压研究的创新方法

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

实时评估隔离、加压淋巴管中的绝对、胞质、游离 Ca2+ 和相应的收缩力

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...