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Protocol
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Medicine

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

Published: November 12, 2020
doi:
10.3791/61865
, , , , , ,
1Laboratory of Photobiology,Keio University School of Medicine, 2Department of Ophthalmology,Keio University School of Medicine, 3Animal eye care,Tokyo Animal Eye Clinic, 4Tsubota Laboratory, Inc.

Summary

在这里，我们描述了一个鼠标模型的视网膜缺血通过瞬态的双边常见的胡萝卜动脉闭塞使用简单的缝合线和夹子。该模型可用于了解心血管异常引起的视网膜缺血的病理机制。

Abstract

多种血管疾病，如糖尿病视网膜病变、视网膜静脉或动脉闭塞以及眼缺血综合征可导致视网膜缺血。为了研究视网膜缺血的病理机制，需要开发相关的实验模型。从解剖学上讲，主要的视网膜供血血管是眼动脉 （OpA）， OpA 起源于普通胡萝卜动脉 （CCA） 的内胡萝卜动脉。因此，CCA的中断可以有效地导致视网膜缺血。在这里，我们建立了视网膜缺血的鼠标模型，通过瞬态的双边常见胡萝卜素动脉闭塞（tBCCAO），用6-0丝缝合右CCA，并通过夹子暂时将左CCA遮挡2秒，并表明tBCCAO可以诱发急性视网膜缺血，导致视网膜功能障碍。目前的方法通过仅使用手术针头和夹子来减少对手术器械的依赖，缩短了闭塞时间，以尽量减少意外的动物死亡，这在中脑动脉闭塞小鼠模型中经常看到，并保持常见视网膜缺血结果的可重复性。该模型可用于研究小鼠缺血视网膜病变的病理生理学，并进一步用于体内药物筛选。

Introduction

视网膜是一种神经感官组织，用于视觉功能。由于视觉功能需要大量的氧气，视网膜被称为身体中对氧气要求最高的组织之一。由于氧气通过血管输送，网膜易患血管疾病。各种血管疾病，如糖尿病视网膜病变和视网膜血管（静脉或动脉）闭塞，可诱发视网膜缺血。为了研究视网膜缺血的病理机制，认为有必要对视网膜缺血进行可重复和临床相关的实验模型。通过插入内脏丝进行中脑动脉闭塞（MCAO）是开发实验性脑缺血2、3体内啮齿动物模型的最普遍方法。由于眼动脉（OpA）接近MCA，MCAO模型也同时用于了解视网膜缺血的病理生理学4，5，6。为了诱导脑缺血和视网膜缺血，长丝通常通过普通胡萝卜动脉 （CCA） 或外部胡萝卜动脉 （ECA） 的切口插入。这些方法很难执行，需要很长时间才能完成手术（一只小鼠超过60分钟），并导致手术后结果的高变异性7。制定更好的模式以改善这些关切仍然很重要。

在这项研究中，我们只是使用短的瞬态双边CCA闭塞（tBCCAO）与针头和夹子诱导视网膜缺血在小鼠，并分析了视网膜缺血损伤的典型结果。在此视频中，我们将演示 tBCCAO 程序。

Protocol

这里描述的所有方法都得到了庆应大学医学院机构动物护理和使用委员会（IACUC）的批准。 1. 准备手术器械和动物 高压外科手术器械，并保持他们在70%的乙醇。在每一次新的外科手术之前，清洁手术器械小心使用70%的乙醇。 在特定无病原体 （SPF） 室中准备雄性 BALB/cAJc1 小鼠（6 周大，26-28 公斤），以便在手术前、手术期间和手术后保持无菌状态。 <p cl…

Representative Results

在FITC-dextran系统循环2分钟后，检查了假手术小鼠和tBCCAO手术小鼠左视网膜和右视网膜的视网膜血管（补充图1）。FITC-dextran在假手术小鼠的视网膜和tBCCAO操作的小鼠的左视网膜中完全可见，而在tBCCAO操作的小鼠的右视网膜中部分可检测到。 在tBCCAO之后，对眼睑下垂进行了检查（图2）。右眼显示温和（得分2;75%）和严重的眼睑（分数3和4：25%?…

Discussion

在这项研究中，我们已经表明，tBCCAO，使用简单的缝合和夹子，可以诱导视网膜缺血和伴随视网膜功能障碍。此外，我们已证明，我们目前的协议，开发视网膜缺血小鼠模型是更容易和更快相比，其他以前的协议，开发视网膜缺血损伤模型2，3，7。

从解剖学上讲，左右脑动脉可以通过后沟通动脉（PCAs）连…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了教育、文化、体育、科学和技术部（MEXT）的科学研究援助赠款（18K09424，东芝库里原和20K18393对三和裕一郎）的支持。

Materials

Atipamezole hydrochloride Zenoaq Antisedan For anti-anesthesia
Applied Biosystems 7500 Fast Applied Biosystems – For qPCR
Butorphanol tartrate Meiji Seika Pharma Vetorphale For anesthesia
BZ-II Analyzer KEYENCE – For an image merge
BALB/cAJc1 CLEA – Mouse strain
β-Actin (8H10D10) Mouse mAb CST 3700 For western blot
Clamp Forcep World Precision Instruments WPI 500451 For surgery
Dumont forceps #5 Fine Science Tools 11251-10 For surgery
DAPI solution Dojindo 340-07971 For IHC
Envisu SD-OCT system Leica R4310 For OCT
FITC-dextran Merk FD2000S For retinal blood perfusion
Fluorescence microscope KEYENCE BZ-9000 For fluorescence detection
Gatifloxacin hydrate Senju Pharmaceutical Gachifuro For anti-bacterial infection
GFAP Monoclonal Antibody (2.2B10) Thermo 13-0300 For IHC
Heating pad Marukan RH-200 For surgery
HIF-1α (D1S7W) XP Rabbit mAb CST 36169 For western blot
ImageQuant LAS 4000 mini GE Healthcare – For chemiluminescence
Midazolam Sandoz K.K SANDOZ For anesthesia
Microtome Tissue-Tek TEC 6 Sakura – For sectioning
Medetomidine Orion Corporation Domitor For anesthesia
Needle holder Handaya HS-2307 For surgery
PuREC MAYO Corporation – For ERG
Scissor Fine Science Tools 91460-11 For surgery
Sodium hyaluronate Santen Pharmaceutical Hyalein For eye lubrication
Tropicamide/Penylephrine hydrochloride Santen Pharmaceutical Mydrin-P For mydriasis
6-0 silk suture Natsume E12-60N2 For surgery
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Ischemic Retinal InjuryTransient Bilateral Common Carotid Artery OcclusionMurine ModelRetinal IschemiaPathophysiologyIschemic RetinopathiesIn Vivo Drug ScreeningVascular DiseasesDiabetic RetinopathyOcclusion Of Retinal Veins Or ArteriesOcular Ischemic SyndromeBALB/c MiceEyelid Drooping
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Lee, D., Miwa, Y., Jeong, H., Ikeda, S., Katada, Y., Tsubota, K., Kurihara, T. A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion. J. Vis. Exp. (165), e61865, doi:10.3791/61865 (2020).
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