罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型
Summary
氧气诱发的视网膜病变 （OIR） 可用于模拟缺血性视网膜疾病，如早熟和增殖性糖尿病视网膜病变的视网膜病变，并作为概念验证研究的模型，用于评估神经血管疾病的抗角原药物。OIR 在可量化的网膜中诱导强健和可重复的神经血管化。
Abstract
缺血性视网膜病变的常用模型之一是氧诱发的视网膜病变 （OIR） 模型。在这里，我们描述了OIR模型诱导的详细协议，以及它在小鼠和大鼠身上的读数。视网膜神经血管化通过让啮齿动物幼崽暴露在高氧（小鼠）或交替的超氧和缺氧水平（大鼠）中诱导。这些模型的主要读数是视网膜内血管 （NV） 和血管 （AVA） 区域的大小。这种前体模型可用于评估潜在抗血管生成药物的疗效，或使用基因操纵的动物解决特定基因在视网膜血管生成中的作用。该模型在 OIR 感应中具有一些应变和供应商特定的变化，在设计实验时应考虑这些变化。
Introduction
需要可靠和可重复的实验模型来研究血管原性眼病背后的病理学，并开发新的治疗这些破坏性疾病的方法。病理血管生成是湿年龄相关的黄斑变性（AMD）和许多缺血性视网膜疾病的标志，其中包括早产（ROP）、增殖性糖尿病视网膜病变（PDR）和视网膜静脉闭塞（RVO）1，2，3，4。人类和啮齿动物视网膜遵循类似的发展模式，因为人类和啮齿动物视网膜都是最后被血管化的组织之一。在视网膜血管完全发育之前，视网膜从透明体血管中接收其营养供应，当视网膜血管开始发育1、2时，直肠血管会退步。在人类中，视网膜血管发育在出生前完成，而在啮齿动物中，视网膜血管的生长发生在出生后。由于视网膜血管发育发生在啮齿动物产后，它提供了一个理想的模型系统来研究血管生成2，3。新生啮齿动物有一个血管性网膜，逐渐发展，直到到第三产后第4周结束时完成血管网膜发育。新生儿小鼠的血管生长是塑料的，它们在高氧刺激5期间会经历回归。
ROP是西方国家儿童失明的主要原因，因为它影响近70%的出生体重在1250克6，7以下的早产儿。ROP 发生在早产儿中，这些婴儿在视网膜血管完成正常生长之前出生。ROP进展分两个阶段：在第一阶段，早产延迟视网膜血管生长，在第二阶段之后，发育中的视网膜未完成血管化导致缺氧，从而诱发血管生长因子的表达，刺激新的和异常的血管生长8。OIR模型是研究ROP和其他缺血性视网膜病理学以及测试新药候选者2、3、9的一个广泛使用的模型。它被广泛认为是进行眼部和非眼部疾病潜在抗心原药物概念验证研究的可重复模型。两种啮齿动物模型，即小鼠和大鼠OIR在诱导模型和疾病表型上有所不同。大鼠模型更准确地模仿ROP表型，但小鼠模型为视网膜神经化（NV）提供了一个更坚固、更快、更可重复的模型。在鼠标模型中，NV 发展到中央视网膜。这种病理读出对于许多缺血性视网膜病变（如 PDR、RV 和渗出性 AMD）以及癌症等非眼性血管病的药理疗效研究非常重要。此外，基因操纵（转基因和淘汰）小鼠的可用性使小鼠OIR模型成为更受欢迎的选择。然而，无论是小鼠还是大鼠OIR模型都没有产生视网膜纤维化，这在人类疾病中是典型的。
认识到高氧水平有助于在20世纪50年代发展ROP10，11导致动物模型的发展。关于氧气对视网膜血管影响的第一批研究是在1950年12月12日、13日、14日进行的，直到20世纪90年代，OIR模型还进行了许多改进。史密斯等人在1994年的研究为目前的小鼠OIR模型设定了一个标准，该模型将透明质病与视网膜病变15分开。康纳等人广泛采用的血管抹杀和病理NV量化方法（2009年）进一步增加了其受欢迎程度16.在这个模型中，小鼠被放置在75%的氧气（O2）在P7 5天，然后5天在正常条件下。从 P7 到 P12 的超氧会导致视网膜血管在中央视网膜中退步。回到正常状态后，血管视网膜变得缺氧（图1A）。由于血管中央视网膜的缺氧刺激，一些视网膜血管发芽向视网膜，形成前视网膜NV，称为前视网膜塔夫茨2，3。这些塔夫特是不成熟的，而且透水性很强。NV 峰值量在 P17，之后会退步。视网膜完全重新血管化，NV由P23-P25（图2A）2，3完全回归。
大鼠OIR模型（使用不同级别的O2）在20世纪90年代首次描述，显示不同的O2水平在80%和40%导致更明显的NV比低于80%O2恒定接触17。后来发现间歇性缺氧模型，其中O2循环从超氧（50%）到缺氧（10-12%），导致更多的NV比80/40%O2模型18。在50/10%的模型中，大鼠幼崽在24小时内接触50%，随后在10%O2中接触24小时。这些周期一直持续到P14，当老鼠幼崽回到正常状态（图1B）。与人类ROP患者一样，在大鼠模型中，由于视网膜血管丛不成熟，血管区域发展到视网膜外围（图3）。
在这两种型号中，通常量化的主要参数是 AVA 和 NV 的大小。这些参数通常从视网膜平面坐骑中分析，其中内皮细胞被标记为4，16。以前，通过计数血管或血管细胞核延伸至内限膜上方的静脉，从视网膜横截面评估了视网膜NV的量。这种方法的主要局限性是无法量化 AVA。
Protocol
此处描述的议定书已得到芬兰国家动物伦理委员会的批准（协议编号ESAVI/9520/2020和ESAVI/6421/04.10.07/2017）。 1. 实验动物和鼠标OIR模型感应 注：使用时间适应的动物，例如常用的C57BL/6J小鼠，让幼崽在同一天出生。使用培养水坝，例如129株（129S1/SvImJ或129S3/SvIM）哺乳水坝，在诱发超氧期间和之后给幼崽喂奶。或者，确保有额外的哺乳水坝可用的情况下，护理水…
Representative Results
模型的主要结果是血管表型：AVA的大小和NV的量。在小鼠OIR模型中，血管抹杀发生在中央视网膜（图2A），而在大鼠模型中，它在外围发育，即类似于人类ROP22（图3A）。这是因为当小鼠暴露于高氧时，表面血管丛已经发育，而在大鼠模型中，网膜在OIR诱导（P0）时是血管性的。前视网膜血管化在血管区域附近发展，即小鼠的中央视网膜…
Discussion
疾病表型的严重程度取决于小鼠和大鼠OIR模型23中的菌株甚至供应商。这表明病理学发展存在广泛的基因变异性。一般来说，色素啮齿动物比白化病的啮齿动物发展更严重的表型。例如，白化病BALB/c的视网膜血管在多发性缺氧后迅速重新血管化，24日不会发展为NV。同样，在大鼠中，色素棕色挪威大鼠表现出比白化病更严重的病理学（SD）大鼠25。</s…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢玛丽安·卡尔斯伯格、安妮·玛丽·哈帕涅米、佩维·帕塔宁和安妮·坎库宁提供出色的技术支持。这项工作由芬兰科学院、皮维基和萨卡里·索尔伯格基金会、坦佩雷结核病基金会、芬兰医学基金会、皮尔坎马医院地区研究基金会和坦佩雷大学医院研究基金资助。
Materials
| 33 gauge, Small Hub RN Needle | Hamilton Company | 7803-05, 10mm, 25°, PS4 | For intravitreal injection |
| Adobe Photoshop | Adobe Inc. | For image analysis | |
| Air pump air100 | Eheim GmbH & Co. KG. | 143207 | For inhalation anaesthesia |
| Anaesthesia unit 410 AP | Univentor Ltd. | 2360309 | For inhalation anaesthesia |
| AnalaR NORMAPUR Soda lime | VWR International Ltd | 22666.362 | For CO2 control during model induction |
| Attane Vet 1000 mg/g | VET MEDIC ANIMAL HEALTH OY | vnr 17 05 79 | For inhalation anaesthesia |
| Brush | For preparation of flat mounts | ||
| Carbon dioxide gas | For sacrifice | ||
| Celeris D430 ERG system | Diagnosys LLC | 121 | For in vivo ERG |
| Cell culture dishes | Greiner Bio-One International GmbH | 664 160 | For preparation of flat mounts |
| Cepetor Vet 1 mg/mL | VET MEDIC ANIMAL HEALTH OY | vnr 08 78 96 | For anaesthesia |
| Cover slips | Thermo Fisher Scientific | 15165452 | For preparation of flat mounts |
| O2 Controlled InVivo Cabinet, Aninal Filtrarion System and Dehumidifier | Coy Laboratory Products | Closed system for disease model induction, optional for semi-closed system | |
| E702 O2 sensor | BioSphenix, Ltd. | E207, 1801901 | For oxygen level measurement |
| Envisu R2200 Spectral Domain Optical Coherence Tomograph (SD-OCT) | Bioptigen, Inc. | BPN000668 | For in vivo imaging |
| Eye spears | Beaver-Visitec International, Inc. | 0008685 | For intravitreal injection and in vivo imaging |
| Flexilux 600LL Cold light source | Mikron | 11140 | For intravitreal injection or tissue collection |
| Fluorescein sodium salt | Merck KGaA | F6377-100G | For in vivo imaging |
| Gas Exhaust unit (+Double 3-way valve, mouse and rat face masks, UNOsorb filter) | UNO Roestvaststaal BV | GEX 17015249 | For inhalation anaesthesia |
| Glass syringe, Model 65 RN | Hamilton Company | 7633-01 | For intravitreal injection |
| HRA2 Retina angiograph (FA) | Heidelberg Engineering GmbH | Spec-KT-05488 | For in vivo imaging |
| Isolectin GS-IB4, Alexa Fluor 488 Conjugate | Thermo Fisher Scientific | I21411 | For labeling retinal vasculature on flat mounts |
| Ketaminol Vet 50 mg/mL | Intervet International B.V. | vnr 51 14 85 | For anaesthesia |
| Medicinal Oxygen gas | For disease model induction | ||
| Mice C57BL/6JRj | Janvier Labs | Also other strains possible | |
| Microscope slides | Thermo Fisher Scientific | J1800AMNZ | For preparation of flat mounts |
| Minims Povidone Iodine 5% (unit) | Bausch & Lomb U.K Limited | vnr 24 11 304 | For intravitreal injection |
| Nitrogen gas | For disease model induction (rat) | ||
| Oftan Chlora 10 mg/g | Santen Pharmaceutical Co., Ltd. | vnr 55 01 11 | For intravitreal injection |
| Oftan Metaoksedrin 100 mg/ml | Santen Pharmaceutical Co., Ltd. | vnr 55 03 43 | For in vivo ERG |
| Oftan Obucain 4 mg/ml | Santen Pharmaceutical Co., Ltd. | vnr 55 03 50 | For intravitreal injection |
| Oftan Tropicamid 5 mg/ml | Santen Pharmaceutical Co., Ltd. | vnr 04 12 36 | For in vivo imaging |
| ProOx Model 110 O2 controller and animal chamber | BioSphenix, Ltd. | 803 | For disease model induction, semi-closed system, optional for closed system |
| ProOx Model P360 O2 controller and animal chamber | BioSphenix, Ltd. | 538 | For disease model induction, semi-closed system, optional for closed system |
| Rats CD(SD) | Charles River Laboratories | Also other strains possible | |
| Revertor 5 mg/mL | VET MEDIC ANIMAL HEALTH OY | vnr 13 04 97 | For anaesthesia reversal |
| Silica gel | For humidity control during model induction | ||
| Systane Ultra 10ml | Alcon | Tamro 2050250 | For hydration of the eye |
| Systane Ultra unit 0.7ml | Alcon | Tamro 2064871 | For hydration of the eye |
| Transfer pipette | Thermo Fisher Scientific | 1343-9108 | For preparation of flat mounts |
| VENTI-Line VL 180 PRIME Drying oven | VWR | VL180S 170301 | For drying silica gel |
| VisiScope SZT350 Stereomicroscope | VWR | 481067 | For intravitreal injection or tissue collection |
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Citer Cet Article
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