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Neuroscienze

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

Published: March 12, 2022
doi:
10.3791/63126
, , , , ,
1Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas,Universidad Nacional de Córdoba, 2Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI),Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 3Vitreo-retinal Department,Centro Privado de Ojos Romagosa-Fundación VER, 4Unidad de Investigación y Desarrollo en Tecnología Farmacéutica (UNITEFA),Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 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?…

Representative Results

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?…

Discussion

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?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Keywords: Vascular ParametersWhole Mount RetinasNon-proliferative RetinopathyProliferative RetinopathyOxygen-induced RetinopathyMetabolic SyndromeVascular AlterationsAvascular AreasNeovascular AreasArteriole DilatationArteriole TortuosityVascular DensityVascular BranchingRetinal DissectionImmunohistochemistryIsolectin Staining
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Subirada, P. V., Paz, M. C., Vaglienti, M. V., Luna, J. D., Barcelona, P. F., Sánchez, M. C. Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies. J. Vis. Exp. (181), e63126, doi:10.3791/63126 (2022).
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