Name: quantification of vascular parameters in whole mount retinas of mice with non-proliferative and proliferative retinopathies
Uploaded: 2022-03-12T15:00:00
Description: Watch this Scientific Journal Video about Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies at JoVE.com

我们感谢CEMINCO（Centro de Micro y Nanoscopía Córdoba，CONICET-UNC，Córdoba，阿根廷）的Carlos Mas，María Pilar Crespo和Cecilia Sampedro在共聚焦显微镜方面的协助，Soledad Miró和Victoria Blanco的专门动物护理以及Laura Gatica的组织学援助。我们还感谢Victor Diaz（FCQ机构传播副秘书）的视频制作和编辑，以及Paul Hobson对手稿的批判性阅读和语言修订。
本文由科尔多瓦国立大学（SECyT-UNC）Consolidar 2018-2021，Científica y Tecnológica（FONCyT），2015年第1314号（全部为M.C.S.）资助的资助。

C57BL / 6J小鼠根据ARVO声明在眼科和视力研究中使用动物的指南进行处理。实验程序由科尔多瓦国立大学化学科学学院的机构动物护理和使用委员会（CICUAL）设计和批准（Res. HCD 1216/18）。
1. 缓冲液和试剂的制备
<ol><li>制备1x磷酸盐缓冲盐水（PBS）：将8克氯化钠（NaCl），0.2克氯化钾（KCl），14.4克磷酸氢二钠二水合物（Na2HPO4 2H2O）和0.24克磷?...

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+associated+with+diabetic+retinopathy.">The pathology associated with diabetic retinopathy.</a> Vision Research. 139, 7-14 (2017).</li><li>Sun, Z., 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=angiography+metrics+predict+progression+of+diabetic+retinopathy+and+development+of+diabetic+macular+edema:+A+prospective+study.">angiography metrics predict progression of diabetic retinopathy and development of diabetic macular edema: A prospective study.</a> Ophthalmology. 126 (12), 1675-1684 (2019).</li><li>Jia, Y., 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=Quantitative+optical+coherence+tomography+angiography+of+vascular+abnormalities+in+the+living+human+eye.">Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (18), 2395-2402 (2015).</li><li>Pauleikhoff, D., Gunnemann, F., Book, M., Rothaus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+vascular+changes+in+macular+telangiectasia+type+2:+comparison+between+SD-OCT+and+OCT+angiography.">Progression of vascular changes in macular telangiectasia type 2: comparison between SD-OCT and OCT angiography.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 257 (7), 1381-1392 (2019).</li><li>Gammons, M. 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M., 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=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4 (11), 1565-1573 (2009).</li><li>Zarb, Y., 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=Ossified+blood+vessels+in+primary+familial+brain+calcification+elicit+a+neurotoxic+astrocyte+response.">Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response.</a> Brain. 142 (4), 885-902 (2019).</li><li>Smith, L. 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=Oxygen-induced+retinopathy+in+the+mouse.">Oxygen-induced retinopathy in the mouse.</a> Investigative Ophthalmology &amp; Visual Science. 35 (1), 101-111 (1994).</li><li>Subirada, P. V., 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=Effect+of+autophagy+modulators+on+vascular,+glial,+and+neuronal+alterations+in+the+oxygen-induced+retinopathy+mouse+model.">Effect of autophagy modulators on vascular, glial, and neuronal alterations in the oxygen-induced retinopathy mouse model.</a> Frontiers in Cellular Neuroscience. 13, 279 (2019).</li><li>Lutty, G. A., McLeod, D. 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视网膜病变的动物模型是研究血管发育、重塑或病理性血管生成的有力工具。这些研究在该领域的成功依赖于易于访问的组织，从而可以执行各种技术，从而提供来自 体内 和 死后 小鼠的数据26，27。此外， 在体内 研究和临床分析之间发现了很大的相关性，为这些模型提供了可靠的可追溯性和可靠性28。在本文中?...

眼睛由两个动脉 - 静脉系统滋养：脉络膜脉管系统，一种灌溉视网膜色素上皮和光感受器的外部血管网络;以及灌溉神经节细胞层和视网膜内核层的神经视网膜脉管系统1。视网膜脉管系统是一个有组织的血管网络，将营养物质和氧气输送到视网膜细胞并收集废物，以确保适当的视觉信号转导。这种脉管系统具有一些明显的特征，包括：缺乏自主神经支配，通过内在视网膜机制调节血管张力以及拥有复杂的视网膜 - 血液屏障2。因此，视网膜脉管系统一直是许多研究人员关注的焦点，他们不仅广泛研究了发育过程中的血管生成，还研究了这些血管在疾病中经历的改变和病理血管生成3。在视网膜病变中观察到的最常见的血管变化是血管扩张，新生血管形成，血管乔木化丧失和视网膜主血管变形，这使它们更加之字形4，5，6。所描述的一种或多种改变是临床医生最早发现的体征。血管可视化提供了一种快速、无创且价格低廉的筛查方法7。对在血管树中观察到的改变的广泛研究将确定视网膜病变是非增殖性的还是增殖性的，并进一步治疗。非增殖性视网膜病变可表现为血管形态异常，血管密度降低，无细胞毛细血管，包细胞死亡，黄斑水肿等。此外，增殖性视网膜病变还会导致血管通透性增加、细胞外重塑以及向玻璃体腔形成血管簇，这些簇容易分解或诱导视网膜脱离8。
一旦检测到，可以通过其血管变化来监测视网膜病变9，10。病理学的进展可以通过血管的结构变化来跟踪，这清楚地定义了疾病的阶段11。这些模型中血管改变的量化允许将血管变化和神经元死亡相关联，并测试疾病不同阶段患者的药物治疗。
鉴于上述陈述，我们认为血管改变的识别和定量是视网膜病变研究的基础。在这项工作中，我们将展示如何测量不同的血管参数。为此，我们将采用两种动物模型。其中之一是氧气诱导的视网膜病变小鼠模型12，它模仿早产儿视网膜病变和增殖性糖尿病视网膜病变的某些方面13，14。在这个模型中，我们将测量缺血区，新生血管区域以及主血管的扩张和弯曲。在我们的实验室中，已经开发了代谢综合征（MetS）小鼠模型，该模型可诱导非增殖性视网膜病变15。在这里，我们将评估血管密度和分支。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminuim foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck</td>
			<td>A4503</td>
			<td>quality</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Biopack</td>
			<td>9632.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope FV1200</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td>with motorized plate</td>
		</tr>
		<tr>
			<td>Covers</td>
			<td>Paul Marienfeld GmnH &#38; Co.</td>
			<td>111520</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>NIKON</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium-hydrogen-phosphate dihydrate</td>
			<td>Merck</td>
			<td>119753</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L&#160; tube</td>
			<td>Merck</td>
			<td>Z316121</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merck</td>
			<td>WHA5201090</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker GyroMini</td>
			<td>LabNet International</td>
			<td>S0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 Conjugate</td>
			<td>Invitrogen</td>
			<td>I21411</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol) (Mowiol 4-88)</td>
			<td>Merck</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>pHmeter</td>
			<td>SANXIN</td>
			<td>PHS-3D-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-dihydrogen phosphate</td>
			<td>Merck</td>
			<td>1,04,873</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Merck</td>
			<td>GE17-1321-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>X100-1GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Analysis Fiji software</td>
			<td>Mai Elfarnawany</td>
			<td></td>
			<td>https://imagej.net/Vessel_Analysis</td>
		</tr>
	</tbody>
</table>

quantification of vascular parameters in whole mount retinas of mice with non-proliferative and proliferative retinopathies

视网膜病变是一组影响眼睛神经感觉组织的异质性疾病。它们的特征是神经变性，胶质病和血管功能和结构的进行性变化。虽然视网膜病变的发病以视觉感知的微妙障碍为特征，但血管丛中的修饰是临床医生检测到的第一个迹象。新生血管形成的缺失或存在决定了视网膜病变是被归类为非增殖性 （NPDR） 还是增殖性 （PDR）。从这个意义上说，一些动物模型试图模仿每个阶段的特定血管特征，以确定内皮改变，神经元死亡和视网膜中发生的其他事件所涉及的潜在机制。在本文中，我们将提供在产后一天测量成人和早产小鼠视网膜血管参数所需的程序的完整描述（P）17。我们将详细介绍在整个支架中使用Isolectin GSA-IB4进行视网膜血管染色的方案，以便以后进行显微镜观察。还提供了使用Image J Fiji软件进行图像处理的关键步骤，因此，读者将能够测量血管密度，直径和曲折度，血管分支以及缺血和新生血管区域。这些工具对于评估和量化非增殖性和增殖性视网膜病变中的血管改变非常有帮助。

如方案部分所述，从单个荧光染色测定中，您可以获得血管形态并定量评估感兴趣的几个参数。特定改变的搜索将取决于所研究的视网膜病变的类型。本文在增殖性视网膜病变小鼠模型中评估了缺血和新生血管区域、曲折性和扩张，而在MetS小鼠模型中分析了血管分支和密度，该模型诱导了非增殖性视网膜病变。
在第一个实验示例中，采用了氧诱导视网膜病变（OIR）小鼠模型?...

Watch this Scientific Journal Video about Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies at JoVE.com

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

本文介绍了一种成熟且可重复的凝集素染色测定，用于整个支架视网膜制剂，以及定量测量增殖性和非增殖性视网膜病变中经常改变的血管参数所需的方案。

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

在视网膜病变中，血管改变是临床医生发现的第一个体征。他们跟随疾病的进展，并根据这些变化选择治疗方法。为了研究病理学的不同阶段，已经开发了几种动物模型。在这项工作中，我们将展示如何测量不同的血管改变。为此，我们将采用两种动物模型。其中之一是氧气诱导的视网膜病变小鼠模型，它再现了早产儿的视网膜病变和增殖性糖尿病视网膜病变的某些方面。在这里，我们将测量缺血区，新生血管区以及小动脉的扩张和曲折。在我们的实验室中，开发了一种代谢综合征小鼠模型，该模型诱导非增殖性视网膜病变。在这项工作中评估了血管密度和分支。在小鼠被处死后，用剪刀去核。在室温下用新鲜制备的多聚甲醛固定去核的眼睛一小时。在解剖显微镜下，用剪刀沿着边缘切除角膜，并解剖整个视网膜。丢弃眼睛的前段，然后将视网膜与RPE脉络膜分开。将视网膜置于200微升管中。然后在6小时内以4度阻断并渗透100微升含有5%牛血清白蛋白Triton的TBS中的视网膜，并在摇摇杯中轻轻搅拌。然后倒置管子将视网膜放入杯中，并用移液管取出阻塞溶液。加入含有异凝素的100微升溶液。用铝箔包裹管子，以保护样品免受光照。用凝集素溶液在四度下孵育视网膜过夜，并在摇摇杯中轻轻搅拌。用100微升TBS-Triton清洗视网膜20分钟，在摇摇杯中轻轻搅拌。重复此步骤三次。将视网膜放入载玻片中，然后添加一滴PBS。在解剖显微镜下，从视网膜边缘向视神经进行四次等距离的切口。要展开视网膜的花瓣，请使用镊子或小块滤纸。确保感光器侧朝下。用滤纸从载玻片上取下剩余的 PBS。然后添加安装中位数和盖玻片。让它在室温下干燥一小时。将载玻片存放在四度，避光。在共聚焦显微镜下，将载玻片面朝下放在板中。聚焦视网膜脉管系统的上神经丛，然后选择一个区域开始图像采集。在软件中设置一般激光参数。在 X、Y 和 Z 轴中设置坐标。初始化扫描。扫描过程完成后，打开图像并使用首选扩展名保存。在 ImageJ FIJI 软件中，转到菜单栏，然后单击文件、打开。选择要处理的图像。在"生物格式导入选项"窗口中，选择"显示 OME-XML 元数据"，然后单击 OK.In"OME 元数据"窗口中，搜索像素大小信息。复制此数据。对于图像校准，请转到菜单栏，然后选择图像属性。复制信息，然后单击确定。为图像指定首选的 lut。要在单个图像中可视化所有堆栈，请转到菜单栏并选择图像，堆栈，Z项目。在显示的投影窗口中，选择可视化船只的所有堆栈。在投影类型中，选择平均强度，然后单击确定。转到菜单栏，图像，调整，亮度/对比度。单击应用并保存更改。复制为亮度和对比度选择的参数。对于所有图像，这些图像必须相同。在菜单栏中，选择"魔杖"工具。选择缺乏形成血管的视网膜区域。按键盘上的 T 以在 ROI 管理器中记录所选区域。单击 ROI 管理器中的测量以获取缺血区域信息。重复这些步骤以测量视网膜的总面积。在菜单栏中，选择"魔杖"工具。放大图像以更好地可视化新血管。使用魔杖工具逐个选择新卫星。按键盘上的 T 字母以在 ROI 管理器中记录所选区域。单击 ROI 管理器中的测量以获取面积数据。在菜单栏中，选择直线工具。放大图像以更好地可视化容器壁。在第一个分支之前对主容器执行三条横向线。按键盘上的 T 以在 ROI 管理器中记录所选区域。单击 ROI 管理器中的测量以获取距离数据。在菜单栏中，用鼠标右键单击直线工具图标，然后选择"分割线"。在血管内从视神经开始画一条线，直到第一个血管分支，遵循血管形状。按键盘上的 T 字母以在 ROI 管理器中记录所选区域。在菜单栏中，选择直线工具。从视神经开始画一条线，直到第一个血管分支。按键盘上的 T 字母以在 ROI 管理器中记录所选区域。单击 ROI 管理器上的测量以获取距离数据。使用菜单栏的"椭圆"工具，定义一个同样适用于所有实验条件的区域。所选区域必须是围绕视神经绘制的同心圆。按键盘上的 T 字母以在 ROI 管理器中记录所选区域。手动计算选择区域内主船产生的主要分支数量。将图像转换为 8 位模式。转到菜单栏中的插件，选择血管分析，血管密度。使用菜单栏的"正方形"工具定义要测量容器密度的区域。单击"确定"。程序将显示一个新窗口，其中包含所需的信息。在第一个实验示例中，采用了氧诱导的视网膜病变小鼠模型。在出生后第12天进行玻璃体内注射，以确定药物作为抗血管生成的有效性。使用用载体注射的小鼠作为对照。缺血区被定义为缺乏视网膜血管的区域。从获得的图像中，血管区域可以量化为无血管区域的总和除以视网膜的总面积。机械损坏的区域也显示没有船只。要识别受损区域，请使用Hoechst染色分析神经元层的完整性。Neovessels是小口径血管，起源于先前存在的血管上血管。我们通过量化视网膜总面积所占据的视网膜新生血管面积来计算新血管簇占据的面积。由于新卫星的形状和大小是可变的，因此偶尔，它们看起来类似于碎片和伪影。为了区分新血管，请验证簇是否从成熟的容器中生长。为了分析膨胀，我们测量了第一个分支之前的三个高度的主血管直径。研究人员必须定义一个预定的距离来测量容器直径，以减少结果之间的差异。理想情况下，距视神经最远的点应在300微米左右。我们建议进行分析，然后，每种情况至少平均六只动物。关于曲折性，我们测量主血管按照其形状行进的距离，相对于视神经和第一分支点之间的直线距离。正如我们在图像中看到的，主船的性存在异质性。为了获得具有代表性的结果，必须对每种情况进行不少于六只小鼠的定量。在表现出非增殖性视网膜病变的代谢综合征模型中分析了血管分支和密度的最新参数。为了测量血管分支，我们通过绘制同心圆来定义定量区域，然后逐个计算主中央血管产生的初级分支。从获得的图像中，血管密度被量化为血管占用的区域除以ROI的面积，ROI位于每个显微照片中的不同位置。避免量化具有机械损坏的区域。如果有两个以上的区域有穿刺，则必须丢弃视网膜。总之，在本文中，我们展示了经典的，成熟的和可重复的技术来量化临床实践中考虑的一些最相关的参数。

Research

JoVE Journal

Neuroscience

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...