这项工作是支持的卡罗林斯卡研究所的KID资金，瑞典辐射保护局，卡罗林斯卡医学院眼科研究基金会，Konung古斯塔夫枪OCH贝蒂尔Stohnes Stiftelse，圣Erik的眼科医院研究基金会，Ögonfonden:秒OCH Drottning Viktorias Frimurarstiftelse。

1。暴露于紫外线辐射<ol><li>前15分钟的曝光，麻醉一个雌性Sprague-Dawley大鼠用90毫克/千克Ketalar（氯胺酮）和10毫克/公斤Rompun（甲苯噻嗪）通过腹膜内注射的混合物。 </li><li>将动物在大鼠阻挡，并拧紧而不会造成一个中继线挤压13，直到固定大鼠的皮带。 </li><li>灌输Mydriacyl（托），10毫克/毫升，在两只眼睛的大鼠诱导散瞳。 </li><li>将动物，使一只眼睛被靠住一个狭窄的光束的紫外线辐射在300 nm 14与10nm的半最大值全宽和屏蔽对侧眼用黑胶带。 </li><li>公开大鼠单方面亚阈值剂量的1千焦/米2 UVR-B在300 nm为15分钟，15。 </li></ol> 2。解剖<ol><li>预先确定的暴露后的时间间隔（1，5，24和120小时）后，牺牲的6周龄大鼠（重量&lt;200克）的碳水化合物Ň二氧化碳窒息和颈椎脱位。 </li><li>摘除细胞核的眼睛。此后，把眼睛角膜下，后侧。然后，冲头通过施加的27口径，插管和推切向巩膜，以避免损坏，这是非常接近的巩膜透镜，一个最小的孔。然后用一对眼科手术剪刀切圆周背后的后部巩膜缘，直到可以升空。然后，用钝头弯钳提起的镜头。 </li><li>在显微镜下从透镜赤道移除残余的睫状体，保持透镜的平衡盐溶液中不再比5-10分钟。 </li></ol> 3。定量RT-PCR <ol><li>将镜头到350微升RA1裂解缓冲液（NucleoSpin RNA总RNA提取试剂盒II）和3.5μLβ-巯基乙醇2 ml Eppendorf管中的混合。 </li><li>在此混合，在室温下的30分钟期间保持镜头。 </li><li>均质的透镜与只有硬核的镜头的针，直到剩下从透镜（皮层和胶囊的镜头的裂解缓冲液中裂解）。从组合中移除的核心。 </li><li>店内样品立即在-70°C。 </li><li>解冻的样品，并按照协议"总RNA纯化培养的细胞和组织"（NucleoSpin RNA总RNA提取试剂盒II）。 </li><li>专区RNA样品在-70℃下</li><li>要验证足够去除DNA，从每个样品中，运行一个PCR在以下条件下（第1步:95℃2分钟;步骤2中进行40个循环:95℃20秒，55℃下持续20秒，72° ℃20秒;步骤3:72℃，7分钟）与p53 DNA的特异性引物，正向5&#39;-ACCCTCTGACCTTTTTCCCA-3&#39;和逆5&#39;-TGCTGGGATCTTAGGCACTC-3&#39;和Taq DNA的聚合酶（dNTPack）的，根据制造协议。预期的PCR产物为243个碱基对。 </li><li>运行的1.5％琼脂糖凝胶电泳以检测DNA中的243个碱基对的特异性PCR产物。为了准备在TBE缓冲液中的1.5％琼脂糖凝胶3.75克琼脂糖，TBE缓冲液在250毫升的解决这个问题。加入溴化乙锭（0.5毫克/毫升）500微升在500毫升1.5％的琼脂糖凝胶溶液。加热该混合物，在一个微波欧文和搅拌解决琼脂糖。 </li></ol>样品没有发现任何DNA特异的PCR产物在1.5％的琼脂糖凝胶电泳。 <ol start="9"><li> RNA样品（1微升），测量的浓度中的NanoDrop ND-1000分光光度计在260 nm（RNA）和280 nm（蛋白质）的样品的吸光度的比例。如果RNA蛋白质的RNA样品的吸光度的比率是2.0或以上，则RNA样品是纯的。如果RNA样品的吸光度的比例低于2.0，重做RNA纯化步骤3.5。 </li><li>以对应的1微克RNA样品的体积，并合成cDNA后，协议第一链cDNA合成试剂盒（ 第一链cDNA合成试剂盒用于RT-PCR）。 </li><li>在-20°C贮存cDNA样本（周期为1以上，储存在-70℃下）。 </li><li>运行实时定量PCR的iCycler MyiQ™实时单色实时PCR检测系统。的负载一式三份于96孔板与1μlcDNA样本，TaqMan基因表达的TaqMan母液，半胱天冬酶3的基因表达分析，根据生产指令。负载三份在另一96孔板中与1μlcDNA样品，TaqMan基因表达母液，TaqMan基因表达为18秒的测定，根据生产指令（TaqMan基因表达含量议定书）。在系列稀释cDNA的3个随机选择的非暴露镜头的一小部分。运行串行稀释液一起从样品中的96 - 孔板的cDNA。 </li><li>使用标准曲线法进行定量的结果。的周期的数量在阈值荧光用作MyiQ™实时软件中的测量。在相对集中的校准功能的阈值的标准曲线表示的周期数是ES建立起来，在每块板中的系列稀释液。 cDNA的未经处理的镜片样品用于校准。对每个样品的周期相关的阈值数的标准曲线，得到样品的cDNA的测量的相对浓度。最后，得到的靶基因的表达水平，由有关的内部控制，18S基因的cDNA的目标相对集中。 </li></ol> 4。免疫组化染色<ol><li>固定术<ol><li>解剖的眼睛在PBS（磷酸盐缓冲液，pH 7.4）中。 </li><li>将眼球的管中填充用4％的多聚甲醛（PFA）在冰冷的20分钟。准备PFA 4％的前一天。储存于4℃直至使用。 PFA是新鲜的大约一个星期。 </li><li>微量移除PFA。然后，20分钟，用冰冷的PBS洗涤。 </li><li>把的眼睛蔗糖的30％在4°C过夜。 </li><li>把眼睛中充满了最佳切削温度（OCT）介质一杯（组织-TEK）。在一个合适的位置，切片放在眼。 </li><li>干冰冻结OCT介质。 </li><li>储存于-70℃直至使用。 </li></ol></li><li>切片<ol><li>冰冻切片在适当的平面上放置的眼睛。 </li><li>切三5μm厚的矢状面中的部分从每个透镜的中心部分。放弃至少6节之间连续的部分，以免弄脏相同的细胞核中不同的部分。 </li><li>一旦部分已被切断，轻轻地放在一张幻灯片，在它的上面。本节应坚持的幻灯片。 </li><li>离开幻灯片，空气干燥，使用前。幻灯片可以储存在-20℃，直到需要。 </li></ol></li><li>荧光免疫组化<ol><li>让样品达到室温（5-10分钟）。 </li><li>绘制一个PAP笔（Invitrogen公司）的标本与周围的边缘。 </li><li>让边境干燥的一段时间（10分钟）。 </li><li>标签的显微镜幻灯片。 </li><li>补充水分，1×PBS 15分钟。 </li><li>通透标准块溶液中30分钟。 </li><li>加入一抗（兔多克隆抗体，切割的caspase-3抗体Asp175 966​​1;细胞信号技术公司）稀释1:3,000标准块的解决方案。 </li><li>储存在湿盒在4°C过夜。 </li><li>通过移液（3×5分钟），或通过浸渍在一个大的浴（&gt; 15分钟，1-2的变化），在PBS中清洗。 </li><li>添加在标准块溶液1:300稀释的第二抗体（抗兔第二抗体与一个特定的吸收/发射光谱）。 </li><li>通过移液（3×5分钟），或通过浸渍在一个大的浴（&gt; 15分钟，1-2的变化），在PBS中清洗。 </li><li>擦拭掉PAP笔涂片。 </li><li>存储在一个加湿室中在室温搅拌3小时。避免暴露在光线下。 </li><li>添加一个的Vectashield下降，并在幻灯片上放置盖玻片。避免气泡。 </li><li>让Vectashield变硬（〜20分钟）。 </li><li>保持第In是黑暗的，直到分析。 </li><li>所有的控制部的处理的情况下的初级抗体。 </li><li>查找的结果，在荧光显微镜下在几个小时内。 </li><li>计数上皮细胞的细胞核从一个侧面核弓对方核弓的每一个镜头。应用标准的蓝色滤色器看到所有镜头上皮细胞核在蓝色和一个标准的过滤器适合的二次抗体的发射绿色看到的caspase-3阳性细胞核。 </li><li>记录全部晶状体上皮细胞核和阳性细胞核的数目的数目。计数细胞3次的每个部分。 </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+exposure+time+for+UV+radiation-induced+cataract.">Influence of exposure time for UV radiation-induced cataract.</a> Investigative Ophthalmology and Visual Science. 41, 3539-3543 (2000).</li></ol>

没有利益冲突的声明。

<html>本文介绍的方法，使学习在UVR-B引起的白内障发生的分子事件。 考虑，大多数信息可用于在体内的紫外线辐射性白内障是来自实验白化只SD大鼠7日，16日，17日，18日，19后，我们决定在目前的研究中使用的白化SD大鼠。年龄的影响是6周大的。的性别是女性，因为在对男性相比，女性较少有过敏尿。此外，有没有性别差异的严重程度的紫外线辐射性白内障20。 所有的动物都被保存和处理根据ARVO声明眼科和视觉研究中使用动物的。伦理许可获得的来自乌普萨拉动物实验伦理委员会，协议号C 29/10。大鼠从商业饲养员（Taconic公司，丹麦）得到。 帐篷"&gt;甲窄带紫外线辐射源，以便选择要频谱定义良好的紫外线辐射。大鼠透镜到UVR-B的最大灵敏度大约是300纳米。1千焦耳/米2的剂量被选择为低于阈值的剂量15，15分钟的曝光时间被选择诱导的透镜21的最严重的损坏。暴露到UVR-B的剂量和后曝光时间可以有所不同，根据实验设计。 成对器官的眼睛在动物研究中使用的统计和道德方面具有明显的优势。单侧曝光的眼睛能够使用一侧露出，因此，在一个动物的另一侧作为控制它减少了一半的动物数量未成对器官相比。 在这个协议中，我们使用了麻醉作为用于动物固定方法的。然而，其他的替代，大鼠阻挡器13，它在我们的实验室中，我们设计了可以用于固定unanaesthetized动物。 ŕ安非他明类兴奋剂有空调对大鼠限制器前的UV曝光。由于其副作用，麻醉时，不建议使用此装置可有效地控制重复风险。这里，我们使用大鼠作为麻醉动物的定位和保持装置，用于阻挡。 大鼠限制器是用木头做的，是在我们的实验室在几个项目。我们清理我们的束缚设备，在每个动物的位置上。木块，淌着和木材站作为富集的老鼠笼。这种富集批准乌普萨拉动物实验伦理委员会和联邦的欧洲实验动物科学协会（FELASA）。 在短的时间内（5-10分钟），该透镜具有在BSS被解剖。这个限制允许的镜头留下清晰和透明。 定量RT-PCR 保持镜头的RA1裂解缓冲液中的30分钟的时间是enough，破坏晶状体囊膜和皮质。晶状体核仍然难以在30分钟内。晶状体核，或细胞器 - 自由区域被认为是一种转录非活性部分的镜头。因此，从样品中除去细胞核，以增加mRNA的表达比的信号/噪声。 用于RNA的纯度（有无的DNA）控制的特定的DNA引物中任意选择的是p53的引物。可以选择任何普遍发生的经过编码的序列，特别是动物基因组DNA的。 PCR，每曝光后间隔为10只和每个镜头两款镜头进行了分析，在三个独立的测量，caspase-3的RNA含量的测定。 RT-PCR，mRNA的利益来衡量。转换的mRNA逆转录成互补的DNA，然后通过PCR扩增。使用通过放大连续稀释的样品的cDNA的int得到的标准曲线进行定量测量erest。应用标准曲线的优点是标准曲线提供了一种可靠的方法来计算的不确定性浓度的标准曲线的mRNA的利益提供了定量测量。可选地，感兴趣的mRNA的相对含量，可以通过直接比较所需要的周期，以获得一个标准化的荧光信号，的Ct法的数目估计。校准曲线的主要缺点是，它需要使用更多的基因组的Ct方法的产品比。 免疫组化染色用免疫组化研究晶状体上皮细胞的活性caspase-3在空间分布。 的眼睛立刻被冻结到-70°C至停止所有正在进行的生物化学。的眼睛，然后将其存储在相同温度下保存。 免疫组化与两个一般亲张国才，陈浩，大学物理COLLEGE PHYSICS;初级抗体的特异性和非特异性结合的抗体。 ，应该获得一个很好的控制源，从而保证了特异性抗体。如果可能的话，应含有的抗原表位的组织染色，作为阳性对照。为了outrule非特异性染色，如果可能的话，应阻止的抗原表位，然后再与特定的抗体，阴性对照染色。此外，非特异性染色，可以最小化，通过建立的最优的抗体浓度和反应时间。通过两个组织暴露于紫外线辐射和未暴露于紫外线辐射的组织染色，它是可能的建立紫外线辐射诱导的表位，在这里的caspase-3。为了方便的点caspapse-3信号的细胞表达的荧光显微镜图像的数字记录。为了最大限度地减少背景噪声的变化，我们使用了固定的设置，所有的显微镜照片。 背景荧光信号的强度变化在一个共同ntinuous规模。因此，显著染色的阈值已被设置。然而，可能会发生变化的平均荧光观察使其难以显著染色使用绝对阈值的段内的空间。因此，通常的判断，具体的染色依赖于一个经验丰富的观察员和特殊染色结果的绝对依赖于经验丰富的观察员认为，但该观察员将是一致的。出于这个原因，使用了只有一个有经验的观察者。为了提高信号的实验变量，暴露于紫外线辐射引起的，作为噪声相比，每个部分的照片进行计数三次。 如果可能的话，免疫组织化学，应验证通过免疫印迹，从而确认与预期的分子大小的抗原决定基的存在。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Ketalar </td><td>辉瑞公司</td><td> 150086 </td><td> 50毫克/毫升</td></tr><tr><td> Rompun </td><td>拜耳</td><td> 022545 </td><td> 20毫克/毫升</td></tr><tr><td>眼膏单</td><td> Apoteket，瑞典</td><td> 336164 </td><td> 5克</td></tr><tr><td> Mydriacyl </td><td>爱尔康</td><td> 00352 </td><td> 10毫克/毫升</td></tr><tr><td>平衡盐溶液</td><td>爱尔康</td><td> 0007950055 </td><td></td></tr><tr><td> 3.5 ULβ-巯基乙醇</td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II总RNA提取试剂盒</td><td> MACHEREY-NAGEL GMBH＆CO，德国Düren </td><td> 740955.50 </td><td></td></tr><tr><td> p53的DNA特异引物</td><td> biomers.net有限公司</td><td>订制</td><td></td></tr><tr><td> Taq DNA聚合酶，dNTPack </td><td>罗氏公司</td><td> 04 728 866 001 </td><td></td></tr><tr><td>琼脂糖</td><td>西格玛</td><td> A5093 </td><td></td></tr><tr><td>溴化乙锭溶液0.5毫克/毫升</td><td>西格玛</td><td> E1385 </td><td></td></tr><tr><td>纳米滴ND-1000分光光度计</td><td>的NanoDrop产品</td><td></td><td></td></tr><tr><td> 第一链cDNA合成试剂盒用于RT-PCR（AMV） </td><td>罗氏诊断公司</td><td> 11 483 188 001 </td><td></td></tr><tr><td>单色的iCycler MyiQ™实时实时PCR检测系统</td><td> Bio-Rad公司实验室</td><td></td><td></td></tr> &lt;TR&gt; <td> 96 - 孔板</td><td>萨尔斯塔特</td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan基因表达预混</td><td>美国应用生物系统公司</td><td> 4369016 </td><td></td></tr><tr><td> caspase 3的TaqMan基因表达分析</td><td>美国应用生物系统公司</td><td> Rn00563902_m1 </td><td></td></tr><tr><td>对于18岁的TaqMan基因表达分析</td><td>美国应用生物系统公司</td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ™实时软件</td><td> Bio-Rad公司实验室</td><td></td><td></td></tr><tr><td>劈开的caspase-3（Asp175） </td><td>细胞信号技术</td><td> 9661 </td><td></td></tr><tr><td>抗兔IgG </td><td> Abcam公司</td><td> Ab6798 </td><td></td></tr><tr><td>蔗糖</td><td> Sigma Aldrich公司</td><td> 84097 </td><td></td></tR&gt; <tr><td> Vectashield </td><td>矢量实验室</td><td></td><td></td></tr><tr><td>万能工具显微镜Axioplan 2影像</td><td>卡尔·蔡司</td><td></td><td></td></tr><tr><td>多聚甲醛</td><td> Sigma Aldrich公司</td><td> 441244 </td><td></td></tr><tr><td> TBE缓冲液10倍</td><td> Promega公司</td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

白内障是导致失明的世界1。世界卫生组织定义白内障混浊的眼睛，阻碍了光传输的镜头。白内障是一种多因素的疾病，与糖尿病，吸烟，紫外线辐射（UVR），酒精，电离辐射，类固醇和高血压有关。有强烈的实验2-4和流行病学证据5,6，紫外线辐射会导致白内障。我们开发了两种麻醉和非麻醉动物的紫外线B诱导白内障的动物模型。 唯一的治疗白内障的手术治疗，但这种治疗方法是无法访问的所有。据估计10年白内障的发病延迟可以减少白内障手术的需要的50％9。为了延缓白内障的发生率，它是需要了解白内障的形成机制和有效的预防地层几种学习策略。白内障发展的机制之一，细胞凋亡起着至关重要的作用在启动白内障在人类和动物的10。最近我们的重点是在镜头凋亡的机制白内障的发展8,11,12。据预测，更好地了解紫外线辐射的影响，对细胞凋亡通路的发现新的药物，以防止白内障的可能性。 在这篇文章中，我们将介绍如何白内障可以通过实验诱导体内暴露于UVR-B。进一步的RT-PCR和免疫组化被作为工具来研究UVR-B诱发白内障的分子机制。

测量中的变化的各种来源，估计与方差分析，并发现，考虑三个测量每只动物的方差用于测量的时间是在15％的动物的顺序。因此，考虑到整个透镜分析，这是不可能的，以增加的精度。正交试验阐明了统计上显着的对比度，120小时之间延迟的时间间隔与较短的延迟时间间隔的caspase-3的消息。 在体内紫外线辐射暴露诱导的caspase-3的表达。 <img alt="图1" fo:content-width="4.5in" fo:src="/files/ftp_upload/4016/4016fig1highres.jpg" src="/files/ftp_upload/4016/4016fig1.jpg" /> 图1。暴露于紫外线辐射的流动的示意图。 <img alt="图2" src="/files/ftp_upload/4016/4016fig2.jpg" /> 图2。演变CASPASE-3（CASP3）mRNA表达的晶状体后在体内曝光1千焦/米2，紫外线辐射波长为300 nm。误差线为95％的置信区间为平均比的的CASP3 mRNA/18s rRNA基因之间暴露的镜头和对侧没有暴露的镜头。的装置的上方和下方的黑线，1相对。单元，分别表示向上和向下调节的CASP3基因。 <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/4016/4016fig3highres.jpg" src="/files/ftp_upload/4016/4016fig3.jpg" /> 图3，Caspase-3表达（A）中的露出的透镜，曝光24小时后，和（B）在一个未曝光的透镜。箭头显示标记的细胞。

Watch this Scientific Journal Video about Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract at JoVE.com

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

白内障是导致失明的世界。太阳紫外线辐射（UVR）是白内障发展的主要危险因素。远紫外线B诱导白内障动物模型的开发。在这篇文章中，我们描述了白内障的形成进行调查:暴露于紫外线辐射，定量RT-PCR和免疫组化的方法。

表征的分子机制<em在体内</em&gt;紫外线辐射诱发白内障

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

characterization-molecular-mechanisms-vivo-uvr-induced

Nanyang Technological University

Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K 次观看.  Karolinska Institutet. 白内障是导致失明的世界。太阳紫外线辐射（UVR）是白内障发展的主要危险因素。远紫外线B诱导白内障动物模型的开发。在这篇文章中，我们描述了白内障的形成进行调查:暴露于紫外线辐射，定量RT-PCR和免疫组化的方法。

视频: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

回肠致胆汁积滞在小鼠肠道中

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

表征的分子机制

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体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

在体内 ^19女性 MRI对细胞跟踪

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和

化疗引起的血管毒性 - 实时

在多囊肾小型啮齿动物模型采用超高场磁共振成像

的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型

建立和UTI和CAUTI表征在小鼠模型

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

回肠致胆汁积滞在小鼠肠道中