This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

所提出的过程需要根据批准的学术医学中心的阿姆斯特丹，注册号NL41985.018的机构审查委员会研究方案的地方。书面知情同意从所有参与者必需的。 1.系统<ol><li>对于这个实验，使用傅立叶域OCT系统，在1,280-1,350纳米的波长带13运行。傅立叶域低相干干涉测量法允许连续扫描时相比第一代时域OCT系统，这增加了数据采集速度。注意：OCT系统在〜90°角接口与光纤探针，扫描螺旋形。它具有2.7F的外径（0.9毫米）和135厘米插入长度。探头连接到OCT控制台通过一个驱动电机和光学控制器（安装底座）为54毫米的范围内拉回。所获取的十月数据集包括541横截面图像（B扫描）瓦特第i个15微米的轴向分辨率（ 图1，2）。 </li><li>为了保证准确和可重复的衰减测量，通过测量校准μ 十月用于增加基于脂肪乳剂由Kodach 等人 14,15先前所描述的重量百分比，（ 例如脂肪乳）的浓度。 简而言之： <ol><li>稀释标准批量的20％脂肪乳剂用软化水的H 2 O来实现的0.125，0.250，0.5，1.0，2.0，4.0，10，15和20（股票）百分浓度。 <ol><li>放置在200毫升的脂肪乳剂的混合物在OCT探针和取得的OCT测量。 </li><li>交叉引用提取μ 十月与文献已知值的值。 </li></ol></li></ol></li></ol> 2.超时和患者定位<ol><li>在此之前启动的程序，执行“超时”检查姓名，出生，程序，PR日期ocedural侧，抗凝剂使用，和过敏。 </li><li>取决于肿瘤的位置，放置在患者中任俯卧或侧卧位。为患者提供足够的支持和验证，如果他/她希望能安心在这个位置在20到40分钟内。 </li><li>使用超声（美国）16，定位肿瘤，并标记与永久性墨水皮肤针切入点。 注：使用计算机断层扫描（CT），使用灵活的针指导模板本地化接入针的首选位置。 </li></ol> 3.消毒和无菌悬垂<ol><li>换上了手术帽口盖。 </li><li>清洗使用洗必泰/醇溶液围绕穿刺部位的皮肤上，注意不要取出先前放置针条目标记（步骤2.3）。消毒广域将防止的必要性的情况下意外访问针重新定位额外的清洁。 </li><li>其中r无菌内容egard，打开经皮穿刺组包含：10毫升注射器，钝抽吸针，21g的注射针，手术刀，一个15政同轴导引针，一个18G的套管针，和一个16摹核心活检枪。 </li><li>彻底洗净双手，将手消毒之后。换上了手术服和无菌手套。 </li><li>覆盖患者在无菌窗帘。 </li><li>应用无菌罩周围的超声探头和修复到位的导针。 </li></ol> 4.准备华侨城<ol><li>启动OCT控制台，然后输入使用控制台界面标记患者ID字段，姓氏，名字和出生日期（出生日期）病人的详细信息。 </li><li>用无菌的内容方面，解压缩包含的OCT探针，无菌的安装基座盖，和5ml的路厄锁注射器在OCT封装。 </li><li>适用于无菌盖华侨城控制台安装基座。引导非无菌安装基座需要的在助手的帮助。 </li><li>填写5毫升注射器用0.9％氯化钠并将其连接到冲洗端口。冲洗OCT探针直到水出现在探针盖的远端部分。 </li><li>加载华侨城探头插入安装底座。后装载探针将旋转并发射红光确认适当运作。离开探头在其保护盖冲洗和加载减少损失的风险中。 </li><li>从盖取下OCT探头。放置在坚硬的表面上的探针和使用手术刀缩短小费。切割，以尽量减少在光纤和棱镜在压力期间固定所述探针的远端部分。切断从棱镜5毫米远端，使用发射（红色）光的方向。 </li></ol> 5.穿刺<ol><li>麻醉皮肤和用2％利多卡因（20毫克/毫升）深的层。等待几分钟，允许利多卡因生效。询问患者是否有任何痛苦。 </li><li>使用针导向，放置为15G同轴引导针验证通过成像的位置。如果位置是令人满意的，除去闭孔（尖锐的针芯）。 </li><li>将18G的套管针通过引导针，刺入肿瘤。再次验证与成像针的位置。如果位置是令人满意的去除闭孔。 </li><li>饲料华侨城探头向上套管针，直到感觉阻力。 </li><li>同时固定在OCT探头，缩回套管针，露出在OCT探头至肿瘤组织。保持肿瘤内套针针尖时呼吸周期最小化在OCT探针的扭结。这降低了探头损坏的风险。 </li><li> OCT扫描： <ol><li>执行十月扫描，与控制台定为每541集B-扫描。这里使用的OCT系统将超过5.4厘米无需特定参数的调整的长度执行自动回调。 </li><li>检查的扫描质量，文物和实体组织的外观（图1a）。伪影最常出现圆形条带从正常十月图案（ 图1B）中脱颖而出。 </li><li>更换探头如果重新扫描后的文物仍然存在。 </li></ol></li><li>重复步骤5.6，直到至少10月3日的数据集被获取。 </li><li>除去在OCT探针和套管针，而使导引针就地。 </li><li>武装核心活检枪，并把它通过引导针，确认上成像的位置。 </li><li>如果定位是令人满意的，火活检枪。 </li><li>根据病理科的协议将活检材料的容器中。这里，地点活检上的培养皿用纸镶嵌，充分饱和的0.9％NaCl。 </li><li>检查活检的质量和，直到获得足够的材料重复步骤5.9和5.10。 </li></ol>

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The authors of this article have nothing to disclose.

在本出版物中，我们报告了经皮，针根据肾，华侨城的可行性。这是华侨城发展的至关重要的第一步成肿瘤的分化临床应用技术，称为“光活检”。我们的第25例患者表现出经皮华侨城是一个简单而安全的过程。光学活检有两个好处比传统的核心活检。首先，将实时采集的OCT数据和分析将提供即时诊断的结果，相对于5-10天的常规病理学的处理时间。第二，OCT具有减​​少的非诊断的程序，这是常?...

在过去几十年已经表明肾群众1,2的发病率稳步上升。到现在为止，肾传治疗决策已经作出主要根据MRI和CT成像特性，年龄和合并症的基础。然而，这些诊断方法和临床参数缺乏技巧，真正检测肾质量的恶性潜能。核心活检或细针穿刺有足够的组织进行病理评估（诊断）提供了客观的肿瘤分化程度与敏感性和特异性在95-100％3的范围内。因此活检获得接受可疑肾群众4,5的评价。然而，活组织检查没有足够的组织建立了诊断或具有在10-20％的速度正常肾实质（非诊断）发生整体，甚至高达30％的在肾脏小块（&lt;4厘米，的SRM），延迟由于频繁必要性额外诊断过程活检手术3,5。 光学相干断层扫描（OCT）是一种新型的成像模态具有克服在肾传分化上述障碍的可能性。根据近红外光的散射，十月提供了一个15微米的轴向分辨率的图像在2-3毫米的有效组织渗透（ 图1，2）。每组织渗透，组织特异性光散射的所得中，毫米波信号强度的损失被表示为衰减系数（μ 十月 ：毫米-1）所描述的费伯等人6。组织学特征可以与可μ 十月值提供用于组织的分化（ 图3）的定量参数。 期间发生，恶性细胞显示增加的数目，以及更大和更不规则形状的核<html>更高的折射率，更积极的线粒体。由于这种过表达细胞成分，在μ 十月的变化是比较恶性肿瘤良性肿瘤或未受影响的组织7时可以预期的。 最近我们研究了浅表华侨城良性和恶性肾群众8,9区分的能力。在16名患者，使用外部放置十月探针获得肿瘤组织的手术中十月测量。该控制臂由未受影响的组织中的同一患者的OCT测量。正常组织呈显著较低位数衰减系数相比恶性组织，证实OCT的肿瘤分化的潜力。这种定量分析是在一个类似的方式向分级其他类型的恶性组织中，被施加如尿路上皮癌10,11和外阴上皮瘤分化12。 ENT“&gt;我们的目标是发展成为十月的光学活检，提供实时影像结合在现场肿瘤分化。目前研究的目的是来形容一个经皮，针基础，华侨城患者的方法诊断为固体增强肾质量，这种方法的描述是，就我们所知，在第一评估针的肾肿瘤的可能性基于十月。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

中的第一25肿瘤（23例），一共有24个成功的OCT程序进行。在一种情况下，一个探测器故障导致无法获得的OCT扫描。两个不良事件（AE）发生，其中详细讨论部分中描述。一般患者的特征见于表1。 华侨城控制台已预装软件获得的数据集的直接定性分析提供实时OCT图像。进一步分析和衰减测量，华侨城的数据可以导出为原始数据，TIFF，DICOM和AVI格式。 μ的华侨城<...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

in-vivo-percutaneous-needle-based-optical-coherence-tomography-renal

Research

JoVE Journal

Medicine

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

8.8K 次观看.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

视频: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

多普勒光学相干断层扫描视网膜循环

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

科研

医学

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

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 ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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 ...

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

在体内，经皮，基于针，肾群众的光学相干断层扫描