视网膜缺血再灌注损伤的小鼠模型通过眼压升高
Summary
本文介绍了在小鼠高眼压诱导视网膜缺血再灌注损伤的过程。视网膜缺血再灌注损伤的高眼压用于人类疾病视网膜特点是妥协的氧气和营养物质的交付模式，使研究人员能够检查视网膜神经血管单元的人类疾病的潜在细胞机制和治疗。
Abstract
视网膜缺血再灌注（I / R）是有助于在多个眼部病症，包括青光眼，糖尿病性视网膜病和视网膜血管阻塞的细胞损伤病理生理过程。 I / R损伤的啮齿动物模型中提供显著见解为人类I / R损伤的机制和治疗策略，特别是关于在视网膜神经血管单元神经变性损伤。这里介绍的是通过眼内压（IOP）升高诱导小鼠视网膜I / R损伤的协议。在这个协议中，眼部前房插管用针，通过该流动升高盐水储层的滴注。使用该滴提高眼压上述收缩动脉血压，从业者暂时停止内视网膜血流（缺血）。当循环被除去套管的恢复（再灌注），严重的细胞损伤随之而来，在视网膜的神经变性，最终导致。最近螺柱IES表明炎症，血管通透性和毛细管变性作为该模型的另外的元件。相比于替代视网膜I / R方法，如视网膜动脉结扎术，视网膜我/ R损伤的高眼压有优势在其解剖的特殊性，实验可追踪性和技术辅助，呈现自己作为一个有价值的工具，用于检查神经元病机及治疗视网膜神经血管单元。
Introduction
视网膜缺血再灌注（I / R）表征许多人视网膜病变，包括青光眼，糖尿病性视网膜病和视网膜血管闭塞1。在视网膜我/ R，血流量减少（缺血）在视网膜血管造成视网膜过敏的氧气和其他营养物质，沉淀严重的氧化和炎症损伤的状态时，循环后来又恢复（再灌注）2。视网膜神经似乎特别容易受到这些变化，患有视网膜神经变性的也许就是我/ R引起的损伤最鲜明的特点。这里介绍的是在小鼠模型视网膜I / R损伤的协议。这项技术使研究人员能够检查视网膜神经血管单元的人类疾病的潜在机制和治疗策略。
通过寻求理解手术贫血3的神经退行性后果外科医生于1952年首创，罗登ŧ视网膜的I /高眼压（IOP）R于1991年重新建立缺血损伤后4标准化神经退行性端点的目的。用盐水贮存提高眼压以上收缩压的滴，这些研究表明，加压眼部插管足以中止视网膜循环，并由此引发的神经元变性。使用视网膜我/ R高眼压最近的努力已开始拟订底层I / R引起的视网膜神经性变性5-12机制。多组报道额外的病理改变包括炎症13,14，血管通透性15,16，和毛细血管变性14,17。总之，这些研究已经更加普遍建立高眼压视网膜I / R损伤视网膜神经血管疾病的模型。
表征I / R损伤的机制是对VA的研究必不可少scular疾病。视网膜我/ R损伤的高眼压是众多缺氧性损伤模型，包括I / R损伤肺18，19心脏，脑20，21肝脏，肾脏22，和小肠23中的一个。这些模型已经在推进我们的血管疾病的认识及临床补救措施至关重要。通过扩展I / R进程眼部组织，视网膜我/ R损伤的高眼压的调查有助于绘制的这些相关条件，更全面的了解。
在临床视网膜神经退行性疾病，视网膜我/ R损伤的高眼压密切相应提出了兴趣探索缺血的发病机制研究的宝贵工具。本文描述的协议是有针对性的，听话的，并且可以访问。它是通过在神经元变性的端点，例如视网膜神经元的定量，视网膜厚度的测量，和电ř补充井视网膜神经元功能的ECORDING。这种模式已经证明了其在促进神经血管查询工具，它显示了在收入中的视觉医学研究的一个基础性协议状态的承诺。
Protocol
伦理学声明：所有程序均按照由约翰霍普金斯大学机构动物护理和使用委员会规定的准则进行。 注意：在拍摄过程中使用的小鼠是从杰克逊C57BL / 6小鼠，虽然也可以使用其他啮齿动物品系或物种。当使用其他菌株或物种，应注意麻醉剂量和伤害时间表可能会有所不同。它适应I / R的条件，以适应应变，物种和实验的变化是重要的。 1.准备麻醉鸡尾酒<…
Representative Results
由高眼压视网膜的I / R的神经变性的影响通常进行评价使用两个标准的方法。神经核的NeuN免疫标记已经揭示以下I / R损伤（ 图1）显著神经元细胞的损失。简单地说，眼睛剜7天I / R固定后，多聚甲醛，与神经细胞的NeuN标记标记和全装。图像用共焦显微镜拍摄的，并标有的NeuN细胞通过计数11定量。减少在神经节细胞层的神经元计数指示I / R诱导的细胞死?…
Discussion
视网膜我/ R损伤的高眼压已经证明了其在模拟细胞损伤和功能障碍，神经退行性疾病特别是在啮齿动物的视网膜神经血管单位效用。这个程序提供了一个强大的控制组织在技术成熟度而言很方便。它已经在这方面和其他I / R损伤模型，增加的压力和缺血的持续时间可能会增加损伤程度24被注意到。出于这个原因，一些从业者选择使用缺血性压力和持续时间与此处4,6-10,12提出不同。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
（; EJD EY022383和EY022683）和Core补助（P30EY001765），成像和显微镜核心模块这项工作是由美国国立卫生研究院的研究资助。
Materials
| Heparin Sodium Injection, USP | Abraxis Pharmaceutical Products | 1000 USP/mL | |
| BSS Sterile Irrigating Solution | Alcon Laboratories, Inc. | 9007754-0212 | 500 mL |
| SC-2kg Digital Pocket Scale | American Weigh Scales, Inc. | SC-2kg | |
| Tropicamide Ophthalmic Solution USP 1% | Bausch + Lomb | 1% (10 mg/mL) | |
| Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% | Bausch + Lomb | 0.5% (5 mg/mL) | |
| INTRAMEDIC Polyethylene Tubing | Becton Dickinson and Company | 427400 | Inner diameter: 427400 |
| 30G1/2 PrecisionGlide Needles | Benton Dickinson and Company | 305106 | |
| BC 1mL TB Syringe, Slim Tip with Intradermal Bevel Needle, 26G x 3/8 | Benton Dickinson and Company | 309625 | |
| BD 60mL Syringe Luer-Lok Tip | Benton Dickinson and Company | 309653 | |
| Zeiss OPMI Visu 200/S8 Microscope | Carl Zeiss AG | 000000-1179-101 | |
| Sterile Syringe Filter | Corning Inc. | CLS431224 | 0.20 µm |
| Durasorb Underpads | Covidien | 1038 | 23 x 24 inches |
| Alcohol Prep | Covidien | 6818 | 2 Ply, Medium |
| Student Dumont #5 Forceps | Fine Science Tools | 91150-20 | |
| Hartman Hemostats | Fine Science Tools | 13002-10 | |
| Primary Set, Macrobore, Prepierced Y-Site, 80 Inch | Hospira | 12672-28 | |
| Phosphate Buffered Saline pH 7.4 (1X) | Invitrogen | 10010-049 | 500 mL |
| Distilled water | Invitrogen | 15230-204 | 500 mL |
| C57BL/6J Mice | The Jackson Laboratory | 664 | |
| AnaSed Injection: Xylazine Sterile Solution | LLOYD, Inc. | 20 mg/mL | |
| Lubricating Jelly, Water Soluble Bacteriostatic | MediChoice | 3-Gram Packet | |
| NAMIC Angiographic Pressure Monitoring Manifold | Navilyst Medical, Inc. | 70039355 | 5-Valve Manifold with Seven Female Ports |
| Goniosoft, Hypromellose 2.5% Ophthalmic Demulcent Solution: Hydroxypropyl Methylcellulose | OCuSOFT, Inc. | 2.5% (25 mg/mL) | |
| Ketaset CIII: Ketamine Hydrochloride | Pfizer, Inc. | 100 mg/mL | |
| Trans-Pal I.V. Stand | Pryor Products | 372 | Furnished with a home-constructed 60-cm stainless steel extension |
| Acepromazine: Acepromazine Maleate Injection, USP | Vet One | 10 mg/mL | |
| V-Top Surgery Table/Adjustable Hydraulic | VSSI | 100-4041-21 | |
| Tube Fitting Luer Male to Luer Male | Warner Instruments | 64-1579 |
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Citazione di questo articolo
Hartsock, M. J., Cho, H., Wu, L., Chen, W., Gong, J., Duh, E. J. A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure. J. Vis. Exp. (113), e54065, doi:10.3791/54065 (2016).