作者感谢尼娜Bispham她的技术援助。这项工作是由美国心脏协会（12POST11920023）和美国国立卫生研究院（K23DK088833，K23DK087859）的支持。

此协议遵循科罗拉多多机构审查委员会（COMIRB）的指导方针。 1，用于测试会议筹备工作<ol><li>参与者应遵循这些限制的最精确的测量:12小时快速从食物和咖啡因，从运动，吸烟12小时克制，12小时克制（如适用），&gt; 4的半衰期从药物克制如果可能的话（可能是不可行的人群如慢性肾病患者）和绝经前妇女应在1-7天的月经周期，以尽量减少激素的影响进行测试。 </li><li>加入2毫升的0.5M乙二胺四乙酸（EDTA），0​​.05 g的肝素（180 USP单位/ mg）和2.5克牛血清白蛋白，以在476.8毫升磷酸盐缓冲盐水（PBS）中制备500mL的解离缓冲液的pH值为7.4。这可以被存储在4℃下几个月。 </li><li>打开超声波，电脑，以及非侵入血流动力学工作站（NIHem;动脉僵硬度的设备）。连接电缆输出超声波电脑R波触发盒。 </li></ol> 2，收集血管内皮细胞及处理<ol><li>一个训练有素的护士或医生进行收集（步骤2.2-2.5，2.7）和一个研究人员收集和处理的导线（步骤2.6 2.8-2.19） </li><li>与局部防腐剂PREP肘网站，应用止血带，找到静脉，导管插入带有18G的导管。放置一个heplock适配器上的第四末端。 </li><li>戴上无菌手套，并穿上无菌有窗的窗帘在现场。 </li><li>将2 J-导线上的窗帘。拉的"J"从两个电线解开的"J"形弧线。 </li><li>摘帽的heplock和饲料J形导丝插入静脉大约8 cm。卸下前导线来回推几次。避免毛血在电线上。 </li><li>用钢丝钳剪断电线，使他们适合在50ml锥形管中含有约30毫升分离缓冲液</li><li>重复第二线步2.5。 </li><li>重复第二线步2.6。返回管湿实验室。 </li><li>扣的导线与一对镊子和保持的导线管的内部，而是在溶液上方。 10分钟后，用电动吸移管从50ml锥形管中反复收集解离缓冲液，并使其向下穿过导线的长度，以冲洗和振动的电线，然后抖落多余的电线流体进入管释放。 </li><li>离心400×g离心7分钟，4°C。 </li><li>通过组合百毫升甲醛溶液+ 900毫升的PBS制备在金属箔覆盖管的甲醛溶液。 </li><li>慢慢地从离心分离机取出管，打开真空泵上，放置一个移液管尖的抽吸软管的端部，并离开〜400毫升的管中，抽真空脱休息，而不会干扰沉淀。 </li><li>盖上铝箔和吸管1毫升甲醛溶液成管固定样品。不要重悬。孵育在室温下10分钟。 </li><li>通过标记与主题和考察访问信息和每张幻灯片用PAP笔在画一个椭圆形的准备8幻灯片。 </li><li>加入15毫升的PBS，重悬，离心5分钟，在400 XG和4°C。 </li><li>重复步骤2.15，加上12毫升的PBS，重悬，离心6分钟，在400 XG和4°C。 </li><li>慢慢地从离心分离机取出管，打开真空泵上，放置一个移液管尖的抽吸软管的端部，并离开约2ml的管中，抽真空脱休息，而不会干扰沉淀。 </li><li>重悬和吸管均匀地分布在8幻灯片中的椭圆形区域。 </li><li>放置在培养箱中37℃，5小时，然后在-80℃下保存，直到准备分析（样本将被罚款多年）。 </li></ol>口蹄疫BA和aPWV 3。评估<ol><li>有研究课题换上一次性短裤和有HIM /她仰卧在一个安静，昏暗，气候控制的空间。 </li><li>将心电图的具体超声和动脉僵硬度的装置将适当数量（此过程使用非侵入血流动力学工作站[NIHem]测量动脉僵硬度，这需要4个电极），以及受血压袖带。 </li><li> 20分钟后，开始血压读数。执行至少为3，并重复直到测量是在5毫米汞柱，搁在每次读数之间持续2分钟。 </li><li>通过触诊的肱动脉搏动，并把眼压计使用的软件程序来记录肱波形开始眼压测量。 </li><li>重复桡，股动脉和颈动脉。 </li><li>测量每个从supersternal缺口用卷尺（肱动脉，桡动脉和）和自定义尺/卡尺（股）这些站点的距离。 </li><li>使用的软件程序计算颈臂，颈动脉 - 桡和颈股（aPWV）。 </li><li>地方前臂血压袖带只是远端尺骨鹰嘴过程和记录的基线肱动脉超声图像及血流速度的测量，至少10个心动周期，设置为触发模式下的血管的软件。一，机械手臂可用于如果需要以稳定的超声探头。 </li><li>前臂充气血压袖带至250毫米汞柱，并开始计时。指导参与者仍然一动不动。 </li><li>开始录制速度与血管的软件时设置的定时器读取4点45分触发模式。触发释放袖带5:00，改变超声记录B型（直径）的图像时，时钟读取5:10 </li><li>继续录制，直到时钟读取7:00 </li><li>基线肱动脉超声图像设置为触发模式下的血管的软件记录至少10个心动周期。 </li><li>取对象的血压。如果收缩压&gt; 100毫米汞柱，将0.4毫克舌下含服硝酸甘油的主题下＆＃39;的舌头，并开始计时，除非患者有另一个禁忌。 </li><li>开始录制B型（直径图像）时，时钟读取3点00分设置为触发模式血管的软件。 </li><li>停止录制时，时钟读取8点。 </li><li>监测血压，直到它返回到基线</li></ol> 4，准备人脐静脉内皮细胞（HUVEC）控制幻灯片<ol><li>脐静脉内皮细胞生长到通道5-6和〜80％汇合。 </li><li>胰蛋白酶消化用3ml胰蛋白酶或什么是必要的菜/烧瓶。 </li><li>使用胰蛋白酶中和溶液等体积的中和胰蛋白酶。 </li><li>离心机在200 XG〜5分钟，用真空去除胰蛋白酶及中和的解决方案。 </li><li>重悬在约10毫升的PBS洗。 </li><li>离心机在200×g离心约5分钟。除去PBS。 </li><li>除去PBS中，在1800年PBS液+ 200μL甲醛固定。 </li><li>重新悬浮于PBS中（〜10毫升）中。 </li><li>离心机在200 XG〜5分钟。在一个适当的量以每年新增幻灯片〜200微升PBS中删除，重悬。 </li><li>店幻灯片在-80°C，直到准备分析（样本将被罚款多年）。 </li></ol>血管内皮细胞5。染色<ol><li>就拿滑出-80℃冷冻，等待5分钟，在室温（此程序是一批10张幻灯片，其中包括血管内皮细胞控制幻灯片）。 </li><li>擦去多余的水用一个棘手的任务擦拭（不要触摸滑动的中心）。 </li><li>重新滋润的幻灯片中加入PBS改变对每张幻灯片，让他们10分钟。 </li><li>而幻灯片站立，准备5％驴血清和其他解决方案。 <ol><li>准备5％驴血清中加入300微升驴血清至5700微升改变的PBS（至pH 7.4），最多10张幻灯片（增加这个金额以上）。 </li><li>淡化所关心的主要抗体在1,000微升5％血清。例如，硝基酪氨酸和NADPH氧化酶（1:300和1:1,500）可以作为氧化应激的标志物。 </li><li>通过稀释5微升AF568的1500μll5％血清的准备二次AF568。 </li><li> VE准备通过稀释2微升的钙粘蛋白VE钙粘到1000微升5％血清。 </li><li>准备AF488通过稀释5微升的VE钙粘到1000微升5％血清。 </li><li>保持AF568，VE在整个过程中根据箔cadherin和AF488，然后放在摇杆在4℃的冰箱而准备的幻灯片。 </li></ol></li><li>补液后10分钟，干片具有微妙的任务擦拭。 </li><li>添加5％驴血清60分钟，然后将一块塑料石蜡膜在圈定的区域，以确保该化学品的完全覆盖。 </li><li>一个棘手的任务丢弃塑料石蜡膜和干片擦拭， 不要冲洗 。加第一抗体60分钟，将一块塑料石蜡膜在圈定的区域，以确保完全覆盖了化学。 </li><li>丢弃塑料石蜡薄膜，改变PBS从喷瓶冲洗，浸泡在幻灯片列5分钟。而幻灯片浸泡，将它们移动到一个黑暗的房间。在黑暗中所有剩余的步骤工作。 </li><li>干片用的Kimwipes，45分钟添加AF568（二级抗体），并把一块塑料薄膜石蜡过的圆形区域，以确保化学品的全覆盖。从灯罩。 </li><li>丢弃在生物危险废物容器的塑料石蜡膜。用PBS改变从喷瓶冲洗，然后浸泡在列5分钟。 </li><li>干燥载玻片用的Kimwipes，再加入VE钙粘蛋白60分钟，将一块塑料石蜡膜在圈定的区域，以确保该化学品的完全覆盖。从灯罩。 </li><li>丢弃在生物危险废物容器的塑料石蜡膜。用PBS改变从喷瓶冲洗，然后浸泡在列5分钟。 </li><li>干片与一个棘手的任务擦拭，加AF488 30分钟，将一块塑料石蜡膜在圈定的区域，以确保该化学品的完全覆盖。从灯罩。 </li><li>丢弃在生物危险废物容器的塑料石蜡膜。用PBS改变从喷瓶冲洗，然后浸泡在列5分钟。 </li><li>干片具有微妙的任务擦拭，并允许片干燥20分钟。从灯罩。 </li><li>只添加含氟聚合物的一滴安装介质用4&#39;，6 - 二脒基-2 - 苯基吲哚盐酸盐（DAPI）对各滑动并覆盖每个盖玻片。 </li><li>放置在玻片4℃的冰箱用铝箔纸覆盖。成像需48小时内完成。 </li></ol> 6，成像和血管内皮细胞分析<ol><li>准备显微镜，用于根据特定的显微镜的式样成像染的内皮细胞。单盲法技术人员应该分析任何特定的蛋白质的蝙蝠通道细胞。 </li><li>系统扫描幻灯片。由阳性染色鉴定内皮细胞VE钙粘蛋白和阳性染色的DAPI确认核的完整性。 </li><li>每张幻灯片图片30细胞以供日后分析。重复的染色​​批次中的每个幻灯片，包括血管内皮细胞。 </li><li>分析染色的兴趣使用定性软件的主要抗体的强度。 </li><li>为了尽量减少不同的染色会议，报告值作为收集内皮细胞蛋白表达在血管内皮细胞相同的蛋白表达率之间的差异在强度染色的可能的混杂效应。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

获得准确的结果口蹄疫BA和aPWV需要获得高质量的超声图像和压力波形，分别。中央的，这是由操作员44适当并继续训练每种技术和使用。此外，重要的是，以控制可能通过标准化测试会话（ 例如 ，前12小时快，气候受控的房间等 ）44,45影响结果的尽可能多的外部变量。如上所述，使用商用的R波门控采集软件和边缘检测软件，强烈建议，以尽量减少在测量4...

慢性肾脏病（CKD）是已经达到了流行病的程度，影响〜人口的11.5％，在美国就有1一个重要的公共卫生问题。 CKD患者心血管死亡或心血管事件的风险显著增加，与普通人群相比2-4。虽然CKD患者表现出传统的心血管危险因素的患病率较高，这只能说明他们增加的心血管疾病发生率（CVD）5的一部分。血管功能障碍是获得在肾脏病6-9领域日益认识到一个重要的非传统的心血管危险因素。 尽管许多变化可能的动脉功能障碍的发展做出贡献，在那些最令人关注的是血管内皮功能障碍的发展，最常用评估为减值内皮依赖性舒张功能（EDD）和LA的硬化RGE弹力型动脉10。各种技术的存在是为了评估EDD和大的弹性动脉僵硬，但最常用的是分别肱动脉血流介导的舒张口蹄疫BA和主动脉脉搏波速度（aPWV）。另一种常用的技术评估EDD是测量前臂血流反应使用静脉闭塞体积描记法11,12药理剂，如乙酰胆碱。然而，这种方法需要肱动脉，这比口蹄疫BA更具侵入性的，可能在CKD患者是禁忌的导尿。一种替代技术来评估动脉僵硬度是测量颈总动脉的局部动脉顺应性（刚度的倒数），虽然这是不广泛使用或证实与临床终点作为aPWV 13。 慢性肾脏病患者宣扬口蹄疫受损14-16 学士学位 ，并增加主动脉脉搏波速度APWV 13,17,18，甚至需要透析前。重要的是从临床的角度来看，两者的血管功能障碍这些非侵入性的措施是未来心血管事件和死亡率无论是在CKD患者19-21，以及在其他人群中22-26的独立预测因素。这些技术可用于研究各种人群心血管疾病的风险，包括慢性肾脏病患者。 其中CKD中动脉功能不全发展的确切机制尚未完全了解;然而，一氧化氮减少（NO）的生物利用度是一个关键贡献者27-30和两个EDD受损的共同机制和动脉僵硬度增加10,31。在CKD中，氧化应激增加，并有助于在NO生物利用度的32-34的减少。氧化应激被定义为活性氧（ROS）的相对于抗氧化防御系统过度的生物利用度。生理刺激，INC泸定炎症信号，促进氧化酶系统产生活性氧，包括超氧阴离子（O 2● - ）（ 例如 ，氧化剂酶NADPH氧化酶）35。超氧化物产生最终导致降低一氧化氮（NO）的生物利用度。 减值NO生物利用度可能反过来导致慢性肾脏病的发展，血管内皮功能障碍是事件的CKD 36的独立预测因素。这与动物实验数据表明eNOS的抑制引起的高血压（全身和肾小球）一致，肾小球缺血，肾小球硬化和肾小管-间质损伤37。事实上，为减少实验肾脏疾病，模仿人类疾病的发生和发展，显然有必要NO的生物利用度，提示内皮功能障碍的人CKD 38,39了关键作用。 血管氧化应激的标志物可以在v中进行评估血管性从人类研究对象收集内皮细胞，采用最初由科伦坡等 40开发和改性密封件等 41-43的技术。使用2个无菌的J-丝，细胞是从肘前静脉收集，回收，固定和购买积极鉴定为内皮细胞和使用免疫目的蛋白质的表达进行分析。 我们在此提供这样的方法，可以用来向）测量FMD BA的讨论;二）测量aPWV; C）测量氧化应激标志物的血管内皮细胞蛋白的表达。重点是慢性肾脏病患者，而不是需要长期透析治疗。

<table border="0" cellpadding="0" cellspacing="0" style="width:736px;" width="736">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:109px;">Company</td>
			<td style="width:244px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">J-wire</td>
			<td style="width:109px;">St. Jude</td>
			<td style="width:244px;">404584</td>
			<td>2 per collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Disposable shorts (MediShorts)</td>
			<td>Quick Medical&#160;</td>
			<td>4507</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Non-invasive hemodynamic workstation (NIHem)</td>
			<td style="width:109px;">Cardiovascualr Engineering</td>
			<td style="width:244px;">N/A</td>
			<td>Includes custom ruler.&#160; An alternate system is the Sphygmocor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ultrasound</td>
			<td style="width:109px;">G.E.</td>
			<td>Model: Vivid7 Dimension</td>
			<td>We use a G.E., but there are many companies and models</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vascular software (Vascular Imager)&#160;</td>
			<td style="width:109px;">Medical Imaging Applications</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">R-wave trigger box</td>
			<td style="width:109px;">Medical Imaging Applications</td>
			<td>N/A</td>
			<td>custom made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rapid Cuff Inflation System</td>
			<td style="width:109px;">Hokanson</td>
			<td style="width:244px;">Model: Hokanson E20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Forearm blood pressure cuff</td>
			<td style="width:109px;">Hokanson</td>
			<td style="width:244px;">N/A</td>
			<td>custom cuff with 6.5 x 34 cm bladder&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HUVECs</td>
			<td style="width:109px;">Invitrogren&#160;</td>
			<td>C-015-5C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Donkey serum</td>
			<td style="width:109px;">Jackson&#160;</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Pap pen</td>
			<td style="width:109px;">Research Products International</td>
			<td>195505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">VE Cadherin</td>
			<td style="width:109px;">Abcam</td>
			<td style="width:244px;">ab33168</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AF568</td>
			<td style="width:109px;">Life Technologies</td>
			<td>A11011</td>
			<td>depends on specifications of microscpe&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AF488</td>
			<td style="width:109px;">Life Technologies</td>
			<td>A11034</td>
			<td>depends on specifications of microscpe&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Nitrotyrosine antibody&#160;</td>
			<td style="width:109px;">Abcam</td>
			<td>ab7048</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NADPH oxidase antibody</td>
			<td>Upstate</td>
			<td>07-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DAPI&#160;</td>
			<td style="width:109px;">Vector</td>
			<td style="width:244px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Delicate task wipe (Kimwipe)&#160;</td>
			<td style="width:109px;">Fisher Scientific</td>
			<td>06-666-A&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic paraffin film (parafilm)&#160;</td>
			<td style="width:109px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Confocal microscope&#160;</td>
			<td style="width:109px;">Olympus&#160;</td>
			<td>Model: FV1000 FCS/RICS</td>
			<td>many options exist&#160;</td>
		</tr>
	</tbody>
</table>

assessment of vascular function in patients with chronic kidney disease

慢性肾脏病（CKD）相比，总人口显著增加心血管疾病（CVD）的风险，这是由传统的心血管病危险因素只能部分解释。血管功能障碍是一种重要的非传统的危险因素，其特征在于，血管内皮功能障碍（最常见的评估为减值内皮依赖性扩张[EDD]）和大的弹性动脉的硬化。虽然各种技术存在来评估的EDD和大的弹性动脉僵硬，最常用的是肱动脉血流介导的舒张（FMD BA）和主动脉脉搏波速度（aPWV），分别为。两者的血管功能障碍这些非侵入性的措施是患者和无肾脏疾病的未来心血管事件的独立预测因素。慢性肾脏病患者宣扬受损的口蹄疫学士学位 ，并增加aPWV。而其中的确切机制血管功能障碍德韦洛普斯在CKD的不完全理解，增加的氧化应激和随后的还原中一氧化氮（NO）的生物利用度是非常重要的贡献者。在氧化应激的细胞的变化可以通过从肘前静脉收集血管内皮细胞和测定用免疫荧光氧化应激标志物的蛋白的表达进行评估。我们在这里提供的这些方法的讨论来衡量口蹄疫BA，aPWV和血管内皮细胞蛋白的表达。

口蹄疫BA被量化为以下反应性充血的最大变化在肱动脉的直径。因此，在其余的直径相比，直径的5分钟，血压袖带闭塞期间（ 图1）的端部以下。图A显示肱动脉的代表超声图像，和图B显示在直径以下的R波门控变化从袖口释放到2分钟的图形，如用市售软件获得。由于变化往往相当少（在图1中的变化是4.8％），在测量小的差异对结果的影响较大。使用市售的自动边...

Watch this Scientific Journal Video about Assessment of Vascular Function in Patients With Chronic Kidney Disease at JoVE.com

Assessment of Vascular Function in Patients With Chronic Kidney Disease

血管功能障碍和促进生理机制的程度可以在慢性肾脏病通过测量肱动脉血流介导的舒张，主动脉脉搏波速度和血管内皮细胞的蛋白表达进行评估。

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

assessment-vascular-function-patients-with-chronic-kidney

Research

JoVE Journal

Medicine

16.2K 次观看.  University of Colorado, Denver. 血管功能障碍和促进生理机制的程度可以在慢性肾脏病通过测量肱动脉血流介导的舒张，主动脉脉搏波速度和血管内皮细胞的蛋白表达进行评估。 

视频: Assessment of Vascular Function in Patients With Chronic Kidney Disease

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

情节记忆的特点，采用事件相关电位研究在阿尔茨海默氏症患者

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

科研

医学

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

利用重复经颅磁刺激改善脑卒中患者的语言功能与慢性非流利性失语

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

使用小动物看更衣室反转恢复（SALLI）的MRI大鼠心脏功能和心肌形态学评估

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th联合切除术，高盐饮食和一氧化氮合酶抑制剂诱导Lewis大鼠慢性肾脏病

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

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 ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

[18f] fdg 示踪剂在重型颅脑损伤患者中的半定量评价

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

Interventional Diagnostic Procedure: A Practical Guide for the Assessment of Coronary Vascular Function

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery disease (ANOCA). Abnormal coronary function underlies coronary vasomotion syndromes including coronary endothelial dysfunction, microvascular angina, vasospastic angina, post-PCI angina and myocardial infarction with no obstructive coronary arteries (MINOCA). Each of these endotypes are distinct subgroups, characterized by specific disease mechanisms. Diagnostic criteria and linked therapy for these conditions are now established by expert consensus and clinical guidelines.
Coronary function tests are performed as an adjunctive interventional diagnostic procedure (IDP) in appropriately selected patients during coronary angiography. This aids differentiation of patients according to endotype. The IDP includes two distinct components: a diagnostic guidewire test and a pharmacological coronary reactivity test. The tests last approximately 5 minutes for the former and 10-15 minutes for the latter. Patient safety and staff education are key.
The diagnostic guidewire test measures parameters of coronary flow limitation (fractional flow reserve [FFR], coronary flow reserve [CFR], microvascular resistance [index of microvascular resistance (IMR)], basal resistance index, and vasodilator function [CFR, resistive reserve ratio (RRR)]).
The pharmacological coronary reactivity test measures the vasodilator potential and propensity to vasospasm of both the main coronary arteries and the micro-vessels. It involves intra-coronary infusion of acetylcholine and glyceryl trinitrate (GTN). Acetylcholine is not licensed for parenteral use and is therefore prescribed on a named-patient basis. Vasodilatation is the normal, expected response to infusion of physiological concentrations of acetylcholine. Vascular spasm represents an abnormal response, which supports the diagnosis of vasospastic angina.
The purpose of this practical guide is to provide information on the preparation and administration of the IDP in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success. The IDP supports stratified medicine for a personalized approach to health and wellbeing.

The purpose of this practical guide is to provide information on the preparation and administration of an interventional diagnostic procedure in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success.

介入诊断程序:冠状动脉血管功能评估实用指南

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery ...