笔者想感谢博士西尔维娅乙坎波斯Bilderback和乔治J罗德完成手术，放置静脉通路线。他们也想感谢萨拉É断奶维持慕尼黑的Wistar殖民地组成的Simonsen和Frömter的菌株。

1。共轭大鼠血清白蛋白磺酸罗丹明101磺酰氯（德州红） <ol><li>溶解100mg大鼠血清白蛋白（RSA）6.667毫升100毫米碳酸氢钠pH值9.0;终浓度为15毫克/毫升，50毫升锥形管。 </li><li>将溶液在冰/水的烧杯中，并冷却至0至4℃之间</li><li>高品质的无水二甲基甲酰胺（DMF）中加入200微升10毫克小瓶德克萨斯红磺酰氯（TRSC）培养基上，在15秒的涡流。 </li><li>的涡RSA解决方案培养基上，并添加溶解TRSC。 </li><li>余音50毫升​​锥形箔（封口膜可以用来保证形成紧密的密封），在水平方向冰桶/冰和摇杆慢慢搅拌溶液（避免形成气泡）的地方，使反应继续在0至4℃，1小时。 </li><li> 5升0.9％的生理盐水溶液，湿50 kDa的分子量切断腔室可以是一个）透析膜用夹子，二）二“辩管浮法一个仪，或c）滑一仪盒（适合除去未结合TRSC）。 </li><li>在透析室和5升的容器瓦特/ 0.9％的生理盐水溶液，将反应混合物在4℃透析过夜用温和的用搅拌棒搅拌。 </li><li>更改在早晨和傍晚5升透析液，直到孵育过夜结果几乎没有颜色的变化（这通常需要交流至少4〜48小时）。此外，用截留分子量为50 kDa的如此接近的采用RSA，分子量66kDa;可能永远不会产生澄清的溶液，这将依赖于膜的孔径分布在60小时和第5交流超过足够的时间。 </li><li>测量体积和划分的初始重量为100毫克的体积，得到浓度大约TR-RSA，典型的浓度范围为63-82毫克/毫升。最终染料：白蛋白比例应该是4:1，每1荧光15,00道尔顿的蛋白质兆瓦。储存在4℃;切勿冻结TR-RSA解决方案，产生的碎片可能发生改变渗透率值。 </li></ol> 2。倒置显微镜舞台成像/显微镜设置的准备<ol><li>位〜完美地堆叠在彼此的高压釜中带4-7枚（约¾英寸长）与40毫米的盖玻片的底部（盖玻片底菜）在一个50毫米的菜。这些应该放在靠近其中一个边缘，使得在纸带边缘接触与肾脏曲率的边缘，但不阻断的客观的光路（ 图1A和1B）;，较大的大鼠，将需要更多的空间之间的磁带和边缘菜。 </li><li>地点的2 Repti热垫下一阶段沿着50毫米的菜（ 图1A）。在舞台上放置变暖的水套毯。 </li><li>最大限度地提高效率时，采集图像，通过确保客观炮塔有10倍的空气Oŗ20X（空气或水浸泡）目标和更高的供电水浸泡客观定量采集图像。 </li><li>在成像过程中加水的目标是最有效的方式将其旋转到一边，加水用1毫升注射器PE-200管一根长长的，可以达到的目标的顶部。 </li><li>设置的激发光强度的激光10瓦特〜15％至20％的中性密度过滤器上的软件。砷化镓磷化的非退探测器设置为750至收集绿色排放，625收集红色排放。蓝色的发光（如核染色Hoechst 33342荧光）的被收集使用标准nondescanned multialkaline检测器750-800之间的设置。 </li><li>鲍曼的空间内，以保证适当的强度低排放的集合，确保探测器的下限设置为不排除这些值。视觉警告标志表示如果灵敏度设置太低，这些值是给定的强度值为零。 </li><li>加载〜5-8毫克的荧光白蛋白溶液稀释，用0.9％的无菌生理盐水，使总体积为1毫升1毫升注射器。 </li></ol> 3。暴露在慕尼黑Wistar大鼠肾脏活体2光子成像<ol><li>开始与预麻醉大鼠使用Pentabarbitol（50 mg / ml的溶液，120μl/100克体重），Inactin（130毫克/毫升的溶液，120克μ/100体重），或Isofluorane（5％的诱导，1.5维护2.5％），从正下方肋骨左大腿正上方的留置静脉通道线（或颈静脉或股静脉），和左翼剃光。 </li><li>将鼠平在其右侧，左，剃了头的一面朝上，确保它是平放在桌子上，其姿态拉长，而不是蹲下，用前爪抚摸对方后方的爪子抚摸对方（ 图2A）。 </李&gt; <li>非常轻轻触诊感觉肾脏，以确定它自然奠定腹膜内画一条直线平行于身体（从肋骨到大腿）使用一个骗子（ 图2A）。 </li><li>拿起一对齿镊皮肤，捏皮肤沿画线，用一对止血，，粉碎组织和防止出血。利用一对沿切口手术剪剪切。破碎外的皮肤和肌肉层切割前将大大减少，通常消除出血。 </li><li>重复此过程外肌肉层，这是薄。 </li><li>对于内部肌肉层，这将暴露腹膜切口进入，再触诊肾脏估计大小。捏住小于肾脏的切口线，保证切口仅仅是在肾脏。这是最好的，此切口太小，使其变大，如果需要。如果这是过大，缝合是必需的。这最后一个切口是至关重要的;太远在任何方向会影响舞台上的稳定性和诱发呼吸（最好的情况下）无论是运动伪影，或将延伸肾蒂不利减少肾脏灌注（最坏的情况）。 </li><li>找到（ 如图2B所示）和抓地力肾周围脂肪，使用镊子，一只手在手时尚肾脏的底部极努力。 </li><li>一旦到达肾脏下极，轻轻一拉肾脏通过切口，同时轻轻挤压低于肾脏exteriorize。如果切口过小，粉碎肌肉层切拓宽;重复的过程exteriorize肾。 </li></ol> 4。配售慕尼黑Wistar大鼠在舞台上成像<ol><li>肾脏放置走向与肾平皿盖玻片的边缘稍微旋转肾脏的腹侧向背侧（背面侧）的大鼠，以便我S制作接触与盖玻片底菜（ 图1A）。添加一个温暖，无菌的09％盐溶液的菜。 </li><li>翻阅10倍或20倍的目标和检查的议案。如果检测到运动，滚大鼠略有所以胸部比较远从盖玻片底部菜;确保肾脏尽可能靠近边缘的菜不拉伸肾蒂（ 图1B）。 </li></ol> 5。图像采集量化肾渗透白蛋白<ol><li>任何大分子计算肾小球通透性（Glomeruluar筛分系数; GSC）将荧光分子输液前需要采取个别肾小球的参考背景图像。如果您的显微镜配备有电动载物台的标记位置的能力，发现中心单个肾小球使用低功率的目标，并记下每一个位置。双通荧光/罗丹明萤光的过滤ter是这个程序的理想选择，虽然无论是发射滤波器（相衬或其他非荧光照明来源将无法正常工作）。肾小球会出现空的圆形结构由近端肾小管时，通过双通滤波器，其固有的橙黄色autoflourescence包围。 </li><li>如果您的显微镜不具有电动载物台，扫描的栅格图案中的肾，低功耗的目标，并位于单个肾小球的一个基本地图，依靠自然地标，例如大的浅表血管上方肾日本或脂肪补丁。 </li><li>炮塔切换到更高的功率水浸泡的目的和，拍摄3D数据集的每个肾小球毛细血管袢和鲍曼的空间都清晰可见的。使用伪彩色调色板（其他的东西比B / W），将有助于这些结构可视化。 </li><li>聚焦在浅表血管慢慢注入在t他荧光白蛋白确保时间，让全身分布。对于低GSC的分子，它是必不可少的，以最大限度地提高在血浆中的强度值，但没有达到的水平，将光检测器饱和，在显微镜。这增加过滤的分子的可探测性。注：通常是5-7秒延时荧光蛋白引入的时间之间的时间，屏幕上出现的（收购全帧，1帧/秒〜）。 </li><li>允许约10分钟，以使任何潜在的小分子量的碎片，以清除在获取三维体积在1微米的间隔被用在计算白蛋白GSC之前。通常情况下，西蒙森的应变慕尼黑Wistar大鼠肾小球少得多的表面，所以可以可视化成像和量化。的Frömter株具有非常大的号码，以便我们量化的数量限制在10〜。 </li><li>在研究结束时，将大鼠安乐死通过anesthet了过量的IC在研究中使用。双pneumothoracotamy以确保安乐死。 </li></ol> 6。三维卷荧光白蛋白计算GSC <ol><li>使用的MetaMorph图像处理软件加载的三维数据集的每个肾小球，无论是背景设置并设置输注后的荧光白蛋白。 </li><li>在体积含有荧光蛋白定位浅表毛细血管襻有足够的空间之间的边界和边缘的鲍氏囊空。 </li><li>背景音量，找到同一焦平面白蛋白含图像应该包含所有视觉线索。选择一个区域内的毛细血管襻还注意的平均强度读数。下一页鲍曼的空间内，并注意选择一个区域的平均强度读数。这些将被用来作为背景值。 </li><li>定量，选择类似的鲍曼的空间区域内的白蛋白containing图像。做到这一点的至少两个其他地区鲍曼的空间内的平均​​强度的平均值。 </li><li>选择毛细血管襻最亮的等离子强度和它周围绘制一个区域。其次，利用阈值函数，突出循环血浆内明亮的价值，避免循环RBC的暗条纹。请注意所选的等离子体空间的平均强度值。血浆中的明亮区域是很重要的优先选择，因为血液内的因素，只会导致血浆荧光水平的低估。 </li><li>使用Microsoft Excel电子表格中输入值来计算GSC： </li></ol><img alt="图1" fo:content-width="2.95in" fo:src="/files/ftp_upload/50052/50052eq1highres.jpg" src="/files/ftp_upload/50052/50052eq1.jpg" />

<ol>
	<li>Russo, L. M., 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=The+normal+kidney+filters+nephritic+levels+of+albumin+retrieved+by+proximal+tubule+cells;+retrieval+is+disrupted+in+nephritic+states.">The normal kidney filters nephritic levels of albumin retrieved by proximal tubule cells; retrieval is disrupted in nephritic states.</a> Kidney International. 71, 504 (2007).</li><li>Russo, L. M., 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=Impaired+tubular+uptake+explains+albuminuria+in+early+diabetic+nephropathy.">Impaired tubular uptake explains albuminuria in early diabetic nephropathy.</a> Journal of the American Society of Nephrology. 20 (3), 489 (2009).</li><li>Sandoval, R. M., 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=Multiple+factors+influence+glomerular+albumin+permeability+in+rats.">Multiple factors influence glomerular albumin permeability in rats.</a> Journal of the American Society of Nephrology. 23 (3), 447 (2012).</li><li>Tojo, A., Endou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarenal+handling+of+proteins+in+rats+using+fractional+micropuncture+technique.">Intrarenal handling of proteins in rats using fractional micropuncture technique.</a> American Journal of Physiology. 263, 601 (1992).</li><li>Asgeirsson, D., 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=Glomerular+sieving+of+three+neutral+polysaccharides,+polyethylene+oxide+and+bikunin+in+rat:+Effects+of+molecular+size+and+conformation.">Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat: Effects of molecular size and conformation.</a> Acta Physiologica. 191 (3), 237 (2007).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+endocytosis+in+vivo+using+intravital+two-photon+microscopy.">Quantifying endocytosis in vivo using intravital two-photon microscopy.</a> Methods in Molecular Biology. 440, 389 (2008).</li><li>Dunn, K. W., 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=Live-animal+imaging+of+renal+function+by+multi-photon+microscopy.">Live-animal imaging of renal function by multi-photon microscopy.</a> Curr. Protoc. Cytom. Chapter 12, Unit 12.9 (2007).</li></ol>

什么都没有透露。

这里强调的步骤代表我们的感受是，将产生一致和准确的渗透值，因为他们规避以下陷阱： <ol><li>散射：使用红色发光的荧光基团可以更有效地收集光，因为较长的波长的光子是不容易分散。使用绿色或蓝色发光荧光团将在GSC的引入更大的变化，因为增加了毛细血管袢和鲍曼的空间变异强度值。 </li><li>图像深度：尽管深通常是收集3D数据集（通常是总深度在30-45微米之间）;高10-15微米代表震源?...

<table border="1">
 <tbody>
 <tr>
 <td>Olympus Floview 1000 confocal/Multi-photon microscope</td>
 <td>Olympus America</td>
 <td></td>
 <td>Filters for detectors: Blue 430/100, Green 525/50, Red 605/90</td>
 </tr>
 <tr>
 <td>Mode-Locked Ti:Sapphire Mai Tai Laser</td>
 <td>Spectra-Physics</td>
 <td></td>
 <td>Tunable excitation wavelengths: ~750-1150 nm</td>
 </tr>
 <tr>
 <td>Gallium arsenide phosphide photodetectors</td>
 <td>Hamamatsu Corp</td>
 <td></td>
 <td>Note: Front or Side mounted configurations available.</td>
 </tr>
 <tr>
 <td>Metamorph Image processing Software</td>
 <td>Molecular Dynamics</td>
 <td></td>
 <td>Note: Version 6.1r1</td>
 </tr>
 <tr>
 <td>Microsoft Excel</td>
 <td>Microsoft Corportation</td>
 <td></td>
 <td>2007 version</td>
 </tr>
 <tr>
 <td>Handling Forceps</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78266-04</td>
 <td></td>
 </tr>
 <tr>
 <td>Mayo Dissecting Scissors</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72962</td>
 <td></td>
 </tr>
 <tr>
 <td>CA Micro scissors Model 1C300</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78180-1C3</td>
 <td></td>
 </tr>
 <tr>
 <td>Kelly Hemostatic Forceps (straight)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72930</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Jacket Blanket + Heating Pad</td>
 <td>Gaymar</td>
 <td></td>
 <td>T/Pump PN 11184-000 Blanket-66N111CC</td>
 </tr>
 <tr>
 <td>Repti-Therm Under Tank Heater</td>
 <td>ZooMed</td>
 <td></td>
 <td>RH-4</td>
 </tr>
 <tr>
 <td>Texas Red Sulfonyl Chloride</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>Cat# T-353</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat Serum Albumin</td>
 <td>Sigma Aldrich</td>
 <td>Cat# A-6272</td>
 <td></td>
 </tr>
 <tr>
 <td>High Quality Anhydrous DMF</td>
 <td>Sigma Aldrich</td>
 <td>Cat# 270547</td>
 <td></td>
 </tr>
 <tr>
 <td>Strate-Line Autoclave Tape</td>
 <td>Fisher Scientific</td>
 <td>Cat# 11-889-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Willco-dish Coverslip Bottom Dishes (50 mm/40 mm coverslip)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 70665-07</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in munich wistar rats

肾疾病，尿中丢失大必不可少的大分子，如血清白蛋白，一直被认为由足细胞的通透性屏障，血管内皮细胞，和以协调方式工作的基底膜的改变引起的。数据从我们的实验室中使用活体双光子显微镜揭示一个更具渗透性的肾小球滤过屏障（GFB），比以前认为的生理条件下，过滤白蛋白发生在早期的子细胞，称为肾小管上皮细胞（PTC）1,2检索， 3。 用以前的技 ​​术研究肾小球滤过和建立滤过屏障特性，包括微注射，这些早期的管状部分的内腔中的流体含量的采样和分析4。这些研究确定管腔液中的白蛋白浓度几乎不存在相应密切通常是在尿中检测到。然而，葡聚糖聚合物，用这种技术定义的大小的表征显示大小类似血清白蛋白的管状腔体和尿中有更高的水平;提示通透性增加5。 本文是一个详细的轮廓，所使用的技术，直接可视化和量化肾小球荧光体内白蛋白通透性 。这种方法可以检测过滤白蛋白整个鲍曼的空间（初始尿过滤室）的滤过屏障进入，也允许由近端肾小管重吸收白蛋白定量和随后的白蛋白transcytosis的6可视化。荧光白蛋白的情况下购买沿管状段途中膀胱突出检索通路在较早的近端小管段的效率。此外，当这种技术用于测定渗透率葡聚糖具有类似大小白蛋白几乎相同的渗透率值报告2。这些意见直接支持需要扩大许多蛋白尿的肾脏疾病的重点包括改变近端小管细胞填海的。

图3显示拍摄的图像，例如，从一个表面肾小球慕尼黑WistarFrömter的大鼠和所采取的步骤，以确定荧光蛋白的渗透性。 GSC的白蛋白的值0.0111来自这个人肾小球下降的慕尼黑Wistar大鼠的供给条件3时，该菌株在看到的范围内。在这些图像中看到的稳定性是由于仔细地规划和在图1和图2中所示的指令的执行。如前所述，切口的位置外肾是最关​​键的一步，在生产稳定?...

Watch this Scientific Journal Video about Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats at JoVE.com

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

利用高分辨率intavital 2 - 光子显微镜的技术直接可视化和量化表面肾小球过滤gloemrular。此方法允许在正常和疾病状态的大分子渗透特性的直接测定。

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

quantifying-glomerular-permeability-fluorescent-macromolecules-using

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Medicine

10.4K 次观看.  Indiana University School of Medicine. 利用高分辨率intavital 2 - 光子显微镜的技术直接可视化和量化表面肾小球过滤gloemrular。此方法允许在正常和疾病状态的大分子渗透特性的直接测定。

视频: Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

科研

医学

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

一种使用加速度计量化上肢表现在日常生活的方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

体内Menière 病耳和正常听觉的磁共振成像对人颅神经的形态学分析

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

小鼠肾小球滤过率的透皮测定

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 &#181;L). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

高灵敏度测量与氟素等西洋盐-聚糖小鼠的球状渗透性 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

小鼠窦房和房室结的全卡口免疫荧光染色、共聚焦成像和 3D 重建

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

小鼠窦房和房室结常驻心脏巨噬细胞的分离和培养

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

分析长期心电图记录以检测小鼠心律失常

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...