Name: in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy
Uploaded: 2017-11-11T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

这项工作得到了瑞士国家科学基金会 (SNSF; #320030_156019) 的资助。作者获得德国海德堡工程有限公司的非金融支持。

在所有过程中, balb/c/c 的成年雄性和雌性小鼠在 "Cx3cr1" 的启动子下表达 gfp, 使用 24 。根据《关于在眼科和视力研究中使用动物的帕特声明》对小鼠进行了治疗, 所有的程序都是瑞士政府根据《联邦瑞士动物福利条例》批准的。通过皮下注射盐酸 medetomidine (0.75 毫克/千克) 和氯胺酮 (45 毫克/千克) 麻醉小鼠。通过监测呼吸速率和动物和 #39 对尾部捏的反射, 确定了适当的麻醉。...

<ol>
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本篇文章展示了一个协议为获取视网膜 B 扫描和成像的 gfp 阳性小胶质细胞分布在老鼠视网膜在同一成像会议。SD OCT 和斯洛伐克越来越多地用于视网膜疾病的动物模型, 提供信息的视网膜改变的时间10,14,17,18,21。使用此协议, Cx3cr1gfp/gfp 或Cx3cr1gfp/+ </em...

在实验性眼科, 视网膜病理学的检查通常使用组织学技术进行评价。然而, 组织学要求动物 euthanization, 并可能导致改变的实际性质的组织。SD OCT 和斯洛伐克是常规用于临床眼科诊断目的和监测的一些视网膜疾病, 如糖尿病性黄斑水肿9, 前缺血性视神经病变10, 或视网膜色素变性11.SD OCT 和斯洛伐克是非侵入性的技术, 产生高分辨率的图像的视网膜, 这是可视化通过扩张瞳孔不进一步干预。SD OCT 提供视网膜结构和视网膜厚度的信息, 通过收集后向散射数据来创建视网膜的横断面图像, 而斯洛伐克收集荧光数据以产生视网膜的立体高对比度图像。现在, 这两种技术都越来越多地用于实验性眼科使用小鼠12,13,14,15 (甚至斑马鱼16,17), 可以提供定性和定量信息12、17、18、19、20、21。
内源性荧光如 lipofuscins 的积累或视网膜疣的形成可通过斯洛伐克作为自动荧光信号进行可视化。该功能使斯洛伐克成为诊断和监测视网膜疾病, 如年龄相关的黄斑变性或视网膜色素变性的有价值的技术22,23。在实验性眼科, 自动荧光成像 (AF) 可用于检测特定的细胞类型在记者的鼠标线。例如, 在Cx3cr124的启动子下, 杂表达 gfp 的小鼠对在正常视网膜中胶质细胞的可视化以及对胶质细胞/巨噬生物的研究是有利的。视网膜疾病的动态21。小胶质细胞是视网膜的常驻巨噬生物, 在损伤的组织稳态和组织修复中起关键作用1,25,26。小胶质细胞活化在视网膜已报告视网膜损伤, 缺血, 和变性, 提示在视网膜疾病的这些单元的作用2,3,4,5,6。
本议定书的目的是描述一个相对简单的方法, 视网膜成像和测量视网膜厚度使用 SD OCT, 并可视化 gfp 阳性小胶质细胞在Cx3cr1gfp/gfp 鼠标视网膜使用斯洛伐克 (海德堡 Spectralis HRA + OCT 系统)。本协议可用于各种小鼠行中健康或病变视网膜的成像和厚度测量。此外, 还可以使用斯洛伐克21对视网膜小胶质细胞数和微胶质细胞的活化进行形态学分析。小胶质细胞与中枢神经系统 (CNS) 的退行性疾病有关, 包括视网膜27,28,29。因此, 结合本协议中使用的两种方法, 可以使小胶质细胞分布与视网膜变性的相关性, 从而有助于监测疾病的严重性或治疗方法的有效性在体内。

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

光谱领域光学相干层析成像 (SD OCT) 和扫描激光眼底 (斯洛伐克) 广泛应用于实验眼科。在本协议中, 在Cx3cr1的启动子 (balb/c/c-Cx3cr1gfp/gfp) 下, 使用表达绿色荧光蛋白 (gfp) 的小鼠在视网膜中对胶质细胞进行了体内的图像。小胶质细胞是视网膜的常驻巨噬, 并有牵连在几个视网膜疾病1,2,3,4,5,6。该协议提供了一个详细的方法来生成视网膜 B 扫描, 与 SD OCT, 和成像的小胶质细胞分布在Cx3cr1gfp/gfp 鼠与斯洛伐克在体内, 使用眼科成像平台系统。该协议可用于几个记者鼠标线。但是, 这里提出的协议有一些限制。首先, 斯洛伐克和 SD OCT, 当用于高分辨率模式, 收集数据具有较高的轴向分辨率, 但横向分辨率较低 (3.5 µm 和6µm, 分别)。此外, 斯洛伐克的焦点和饱和度高度依赖于参数的选择和眼睛的正确对准。此外, 使用设计的人的病人在小鼠是具有挑战性的, 由于更高的总光功率的鼠标眼睛相比, 人眼;这可能导致侧向放大率不准确7, 这也取决于鼠标镜头在其他方面的放大倍数。然而, 尽管轴向扫描位置取决于横向放大率, 轴向 SD OCT 测量是准确的8。

使用这里提供的协议, SD OCT 扫描和斯洛伐克图像是从Cx3cr1gfp/gfp 鼠标在同一成像会话中获得的。图 3包括具有代表性的 SD OCT 单扫描, 它获得了30°或55°透镜 (图 3A) 和具有55°或102°透镜的具有代表性的斯洛伐克图像, 其中 gfp 阳性小胶质细胞是可视化的。与55°透镜相比, 在30°的 SD OCT 扫描中观察到脉络膜的高反射率?...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

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

本协议描述了如何利用眼科成像平台系统, 在小啮齿动物中利用光谱域光学相干层析成像和扫描激光眼底等高分辨率成像技术, 获取有关视网膜厚度和胶质细胞分布, 分别。

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 ...

Research

JoVE Journal

Medicine

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

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

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Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

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 ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice 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

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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 ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

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无创成像和脑缺血分析生活中的大鼠使用正电子发射断层扫描<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

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结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

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Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

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纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...