Name: highly sensitive measurement of glomerular permeability in mice with fluorescein isothiocyanate-polysucrose 70
Uploaded: 2019-08-09T13:00:00
Description: Watch this Scientific Journal Video about Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70 at JoVE.com

作者感谢克里斯蒂娜·施万特、布兰卡·杜夫尼亚克和尼古拉·库尔的出色技术援助,并感谢丹尼斯·索恩博士在荧光扫描方面给予的帮助。这项研究得到了德国福森斯格明舍夫特(DFG)SFB 612 TP B18的资助,并授予了洛杉矶和洛杉矶。在研究设计、数据收集和分析、决定出版或编写手稿方面,受资者没有作用。

调查是根据《实验室动物护理和使用指南》(美国国家卫生研究院出版物,1996年修订)中概述的准则进行的。所有动物实验均按照相关机构批准进行(州政府兰德萨姆特·法尔·纳图尔,乌姆韦尔特·德·维尔布劳赫舒茨[LANUV] 参考号84-02.04.2012.A397)。
1. 仪器、解决方案和设备的准备
<ol><li>用0.9%无菌氯化钠(NaCl)将FITC-聚糖70重组为10mg/mL的最终浓度(即100毫克的纳L)。</li>
	<li>Dialyze FITC-聚?...

<ol>
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An essential translational tool.</a> Acta Cirurgica Brasileira. 26, 106-110 (2011).</li><li>Konigshausen, E., 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=Angiotensin+II+increases+glomerular+permeability+by+beta-arrestin+mediated+nephrin+endocytosis.">Angiotensin II increases glomerular permeability by beta-arrestin mediated nephrin endocytosis.</a> Scientific Reports. 6, 39513 (2016).</li><li>Venturoli, D., Rippe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ficoll+and+dextran+vs.+globular+proteins+as+probes+for+testing+glomerular+permselectivity:+effects+of+molecular+size,+shape,+charge,+and+deformability.">Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability.</a> American Journal of Physiology-Renal Physiology. 288 (4), F605-F613 (2005).</li><li>Bohrer, M. 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提出的方法使研究者能够使用示踪剂以非常敏感的方式测试小鼠的球状渗透性。使用这种方法,只需少量尿液即可诊断肾小球渗透性的短期增加。成功掌握此技术最关键的步骤是:1) 发展小鼠手术的手动专业知识,特别是在中央静脉的罐中,2) 在不损害粘管的情况下放置导管,以及 3) 手动处理专业知识384 孔板,样品体积小。
放置中央静脉导管时,导管不得穿透静脉静脉。为了避免渗?...

球状过滤屏障内的功能或结构缺陷增加了白蛋白的球状渗透性,导致尿中白蛋白的检测(白蛋白)。白蛋白尿病预测心血管结果,是肾小球损伤的重要标志1。即使白蛋白尿水平较低,处于正常范围内,也与心血管风险增加1相关。
在生理条件下,白蛋白通过球蛋白过滤,在管状系统2,3中几乎完全重新吸收。在小鼠中,尿液中白蛋白的检测通常通过从24小时尿液收集中与白蛋白酶相关的免疫吸附剂测定(ELISA)进行。如果使用 24 小时尿收集或点尿的尿液,则由于测定敏感性问题,白蛋白浓度的微小差异可能会丢失。因此,大多数研究人员使用动物模型,其中白蛋白尿是由毒素、药物和肾脏手术引起的强健的肾损伤引起的。
因此,发现一种灵敏的方法来检测球状渗透性的微小和瞬态变化,对该领域非常重要。Rippe等人提出了一个大鼠模型,通过应用荧光标记的示踪剂,即FITC-聚糖70(即FITC-Ficoll 70),在白蛋白4的大小测试球状渗透性。示踪剂应用允许测试球状渗透性的短期变化(在几分钟内),并且非常敏感4。两项研究在小鼠5,6中使用了示踪法。尽管这种方法有其优点,但不幸的是,它的缺点:它在技术上非常复杂,放射性和侵入性。尿的进一步分析只能通过使用凝胶过滤或大小排除HPLC5,6完成。
在本文中,我们提出了一种替代的、灵敏的、非放射性的、快速的方法,用荧光标记的FITC-Polyucrose 70测量小鼠的球状渗透性。通过引入输尿管导管,尿液收集比膀胱穿刺、尿道和超尿管应用的侵入性小,并且允许至少每30分钟收集一次尿液。荧光板读取器。使用酶肌氨酸测定法,将尿液中的示踪剂浓度归化为尿液中的肌氨酸浓度。
因此,这种新方法为研究早期肾小球损伤和增加球状渗透性提供了一个敏感的工具。

<table border="0" cellpadding="0" cellspacing="0" style="width:671px;" width="669">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Motic SMZ168 BL</td>
			<td style="width:77px;">Motic</td>
			<td style="width:101px;">SMZ168BL</td>
			<td style="width:235px;">microscope for mouse surgery</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:257px;">KL1500LCD</td>
			<td style="width:77px;">Pulch and Lorenz microscopy</td>
			<td style="width:101px;">150500</td>
			<td>light for mouse surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Microfederschere</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">FD100R</td>
			<td>fine scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Durotip Feine Scheren</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BC210R</td>
			<td>for neck cut</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Anatomische Pinzette</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BD215R</td>
			<td>for surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Pr&#228;parierklemme</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">BJ008R</td>
			<td>for surgery&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Seraflex</td>
			<td style="width:77px;">Serag Wiessner</td>
			<td style="width:101px;">IC108000</td>
			<td>silk thread</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Ketamine 10%</td>
			<td style="width:77px;">Medistar</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/100</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/200</td>
			<td style="width:235px;">Catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Harvard apparatus 11 Plus</td>
			<td style="width:77px;">Harvard Apparatus</td>
			<td style="width:101px;">70-2209</td>
			<td>syringe pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;"></td>
			<td style="width:77px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">BD Insyte Autoguard</td>
			<td style="width:77px;">BD</td>
			<td style="width:101px;">381823</td>
			<td>&#160;urinary catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Multimode Detector DTX 880</td>
			<td style="width:77px;">Beckman Coulter</td>
			<td style="width:101px;"></td>
			<td>plate reader</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:257px;">384 well microtiterplate</td>
			<td style="width:77px;">Nunc</td>
			<td style="width:101px;">262260</td>
			<td style="width:235px;">384 well platte</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Creatinine Assay Kit</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">MAK080</td>
			<td>to measure creatinine concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">96 well plate</td>
			<td style="width:77px;">Nunc</td>
			<td style="width:101px;">260836</td>
			<td>for creatinine assay&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">FITC-labeled polysuccrose 70</td>
			<td style="width:77px;">TBD Consultancy</td>
			<td style="width:101px;">FP70</td>
			<td>FITC-ficoll</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Angiotensin II</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9525</td>
			<td>used to test glomerular permeability</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BP-98A</td>
			<td style="width:77px;">Softron</td>
			<td style="width:101px;"></td>
			<td>for blood pressure measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">OTS 40.3040</td>
			<td style="width:77px;">Medite</td>
			<td style="width:101px;">01-4005-00</td>
			<td>heating plate for mouse surgery</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Instillagel 6mL</td>
			<td style="width:77px;">Farco-Pharma GmbH</td>
			<td style="width:101px;"></td>
			<td>for urinary catheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Exacta</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">GT415</td>
			<td>shaver</td>
		</tr>
	</tbody>
</table>

highly sensitive measurement of glomerular permeability in mice with fluorescein isothiocyanate-polysucrose 70

尿液中白蛋白的流失(白蛋白尿)预测心血管结果。在生理条件下,少量的白蛋白被球蛋白过滤,并在管状系统中重新吸收,直到达到吸收极限。病理白蛋白过滤的早期增加可能错过通过分析白蛋白尿。因此,使用示踪剂来测试球状的渗透率似乎具有优势。荧光标记示踪剂氟辛等子素(FITC)-多糖(即FITC-纤维素),可用于研究球状渗透率。FITC-多糖分子被球蛋白自由过滤,但不在管状系统中重新吸收。在小鼠和大鼠中,使用技术复杂的程序(即放射性测量、高性能液相色谱[HPLC]、凝胶过滤)在球状渗透性模型中对FITC-Polyucrose进行了研究。我们修改并促进了基于FITC-多糖示踪剂的协议,以测试小鼠中FITC-Polyucrose 70(白蛋白大小)的球状渗透性的早期和微小增加。此方法允许重复尿分析与小尿体积 (5 μL)。该协议包含有关追踪器 FITC-Polyucrose 70 如何静脉注射和通过简单的尿导管收集尿液的信息。尿通过荧光板读取器进行分析,并归化为尿浓度标记(肌氨酸),从而避免技术上复杂的程序。

如图2所示,测试小鼠球状渗透性的方法分三个阶段建立。第一阶段称为准备阶段,其中放置尿导管和中央静脉导管。第二阶段称为平衡阶段,从静脉注射FITC-Polyucrose 70开始,然后持续输注FITC-Polyucrose 7060分钟。最后一个阶段称为实验阶段。在此阶段,继续注入FITC-多糖70,并可以测试药物或其他物质影响球状渗透性。尿在每个阶段结束时收集。
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Watch this Scientific Journal Video about Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70 at JoVE.com

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

在这里,我们提出了一个协议,用高度灵敏的非放射性示踪剂测试小鼠的球状渗透性。此方法允许重复尿分析与小尿量。

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 ...

该方法有助于回答肾病学领域有关肾病肾功能渗透作用的关键问题。该技术的主要优点是，使用荧光铅标记示踪剂可渗透性小而瞬态增加。麻醉后，在雌性小鼠中放置尿导管。将鼠标放在背背中。紧固下腹部，找到尿道骨。获取 22 G 血管导管的塑料部分，然后用木质凝胶润滑。使用左食指小心地将张力放在下腹部，将导管引入三毫米的尿道骨中，同时与半边尿道轴平行。将血管导管顶部转动 180 度，将尖端保持在尿道骨内并保持尿道轴。然后，将导管进一步引入小鼠7毫米，以便它被放置在膀胱内。将1.5毫升棕色管放在血管导管顶部以收集尿液。涂抹一毫升0.9%氯化钠皮，以提高尿液生产。剃掉老鼠的脖子，然后用头朝外科医生放置。用胶带将鼠标头超长。用70%异丙醇消毒颈部。用钳子和一把剪刀在下颚下做一个五毫米的皮肤切口。切皮约一厘米的胸骨方向，直到胸骨中间达到。用一把剪刀。小心解剖颈部右侧的皮肤。在右侧对皮肤进行矩形切口，以露出颈部的软组织。小心不要损坏血管。用一把剪刀和一把钳子用两个夹子固定皮肤皮瓣。使用细钳的尖端，通过钝处理仔细暴露壶静脉。如有必要，用细剪刀取出纸巾。避免对静脉分支造成伤害。将一根丝线放在可见壶脉的后半部分，即朝向鼠标头处，用丝线放置并关闭连字。用胶带固定丝线，使连结张紧。这确保了壶脉上的轻微张力。在血管的近位部分周围准备一个连字。之后，用平衡和融合溶液填充导管。用胶带固定导管，使导管对齐导管静脉。控制气泡以避免空气栓塞。在插入位置用细钳子抬起壶静脉。将管子平行于壶静脉，并刺穿针对流明的壶状静脉。插入约两到四毫米平行的壶脉轴。避免任何轻快的动作。关闭连字以修复导管。通过显微镜仔细观察，控制连字的紧固性。在手术现场放一个潮湿的棉签。将尾袖放在鼠标尾底，而鼠标则躺在背背中。开始测量，每个时间点重复十次。如果血压测量看起来不正确，并且产生了错误数据，请调整尾袖口的位置。在平衡阶段之前，更换尿液收集管并放在冰上。折叠一个拭子，并把它放在小血管导管周围，被引入到血管静脉。在拭子上放置一个夹子，以消除逆行血流或空气栓塞。将 27 G 针头与装满 FITC 螺栓的 1 毫升注射器连接到小血管导管的端。打开夹具并应用 100 微升 FITC 螺栓。再次关闭夹具，将较大的导管与较小的导管连接。重新打开夹子，以每分钟 0.008 毫升的流量启动注射器泵。继续输液60分钟。在这个阶段，对球状膜选择性的有效药物进行了调查。将尿管形成时间点零分钟更改为另一个 1.5 毫升棕色管。在小容器导管周围放一个拭子，然后断开大导管与小容器导管的连接。用实验阶段溶液填充注射器。将它放入注射器泵中，让输液泵运行，使大导管充满实验阶段溶液。重新连接导管，同时避免空气栓塞并重新打开夹具。以每分钟 0.008 毫升的流量启动注射器泵。继续运行注射器泵60分钟，然后收集尿管内的尿液。将尿管放在冰上。在这项研究中，本地小鼠尿液的荧光扫描显示峰值为395纳米。FITC多糖70在小鼠尿液中每毫升浓度为40微克，最大荧光量为525纳米。小鼠尿液不会干扰 FITC 聚糖 70 的荧光测量。在525纳米的入院中，FITC聚糖70的浓度增加，表明荧光强度增加。此方法还能够检测球状渗透性的差异。在实验阶段血管紧张素II刺激开始前，白柱代表70级。白柱代表任何水平。剂量60分钟后，与对照组相比，球状渗透性增加。血管紧张素II洗掉降低球状渗透性。FITC多糖70水平下降后，额外的60分钟的血管紧张素II刺激或60分钟的血管紧张素II洗净。血管紧张素II导致的球状渗透性增加可能被血管紧张素II受体阻滞剂阻断。用尾袖口法监测的血压没有显示对照组与血管紧张素II治疗组之间的显著差异。在尝试此程序时，在更换输液溶液时避免空气栓塞非常重要。

Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

科研

医学

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

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 ...

An in vivo Assay to Test Blood Vessel Permeability

An in vivo Assay to Test Blood Vessel Permeability

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Surgical Models of Gastroesophageal Reflux with Mice

胃食管反流与小鼠手术模型

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

自由活动小鼠使用遥测长期血压测量

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Assessment of Kidney Function in 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 ...