Summary

评估和量化年龄相关性黄斑变性小鼠模型中视网膜色素上皮病理的协议

Published: March 10, 2023
doi:

Summary

小鼠模型可以成为研究视网膜色素上皮(RPE)生物学的有用工具。已经确定小鼠可以发展出一系列RPE病理。在这里,我们描述了一种表型方案,以使用光,透射电子和共聚焦显微镜阐明和量化小鼠的RPE病理。

Abstract

年龄相关性黄斑变性(AMD)是老年人群中一种使人衰弱的视网膜疾病。人们普遍认为,视网膜色素上皮 (RPE) 功能障碍是 AMD 的关键病理生物学事件。为了了解导致RPE功能障碍的机制,研究人员可以利用小鼠模型。以前的研究已经确定,小鼠可以发展为RPE病理,其中一些是在被诊断患有AMD的个体的眼睛中观察到的。在这里,我们描述了一种表型方案来评估小鼠的RPE病理。该协议包括使用光学显微镜和透射电子显微镜制备和评估视网膜横截面,以及通过共聚焦显微镜制备和评估RPE平面安装。我们详细介绍了这些技术观察到的小鼠RPE病理的常见类型,以及通过无偏的统计测试方法量化它们的方法。作为概念验证,我们使用这种RPE表型方案来量化在过表达跨膜蛋白135(Tmem135)和老年野生型C57BL / 6J小鼠中观察到的RPE病理。该协议的主要目标是为使用AMD小鼠模型的科学家提供具有无偏定量评估的标准RPE表型方法。

Introduction

年龄相关性黄斑变性(AMD)是一种常见的致盲性疾病,影响55岁以上的人群1。许多研究人员认为,视网膜色素上皮(RPE)功能障碍是AMD2的早期和关键的病理生物学事件。RPE是单层极化细胞,其任务是维持邻近光感受器和脉络膜血管的稳态3。存在多种模型来研究RPE内的疾病相关机制,包括细胞培养模型45和小鼠678最近的一份报告描述了RPE细胞培养模型4的标准化方案和质量控制标准,但没有报告试图标准化小鼠模型中RPE的表型。事实上,许多关于AMD小鼠模型的出版物缺乏对RPE的完整描述或对其中RPE病理的量化。该协议的总体目标是为使用AMD小鼠模型的科学家提供具有无偏定量评估的标准RPE表型方法。

以前的出版物已经通过三种成像技术注意到小鼠中存在几种RPE病理。例如,光学显微镜允许研究人员查看小鼠视网膜的粗大形态(图1A)并检测RPE病理,例如RPE变薄,空泡化和迁移。AMD 小鼠模型中的 RPE 变薄示例是 RPE 高度与其各自对照的偏差(图 1B)。RPE空泡化可分为两类:微空泡化(图1C)和大空泡化(图1D)。RPE微空泡化总结为RPE中存在不影响其总高度的液泡,而大空泡化则表现为存在突出到感光器外部部分的液泡。RPE迁移的特征在于视网膜横截面中RPE单层上方色素的焦点聚集体(图1E)。应该注意的是,AMD供体眼睛中的迁移性RPE细胞对免疫细胞标志物表现出免疫反应性,例如分化簇68(CD68)9,并且可能代表免疫细胞吞噬RPE碎片或RPE经历转分化9。另一种称为透射电子显微镜的成像技术可以让研究人员可视化RPE及其基底膜的超微结构(图2A)。该技术可以识别小鼠中主要的亚RPE沉积物,称为基底层流沉积物(BLamD)(图2B10。最后,共聚焦显微镜可以通过成像RPE平面支架揭示RPE细胞的结构(图3A)。这种方法可以发现RPE畸形,即RPE与其经典蜂窝形状的偏差(图3B)。它还可以检测RPE多核,即RPE细胞内存在三个或更多细胞核(图3C)。有关当前AMD小鼠模型中存在的RPE病理类型的摘要,我们请研究人员参考文献67中的这些评论。

研究AMD的研究人员应该意识到在表型方案之前使用小鼠研究RPE病理的优缺点。小鼠是有利的,因为它们的寿命相对较短,成本效益,以及它们的遗传和药理可操作性。小鼠还表现出RPE退行性变化,包括RPE迁移,畸形和多核,在AMD供体眼睛中观察到111213,14151617;这表明类似的机制可能是小鼠和人类这些RPE病理发展的基础。然而,存在一些关键差异,限制了小鼠研究对人类AMD的可转化性。首先,小鼠没有黄斑,黄斑是人类视网膜中视力所必需的解剖学上独特的区域,在AMD中优先受到影响。其次,小鼠的一些RPE病理,如RPE变薄和空泡化,在AMD供体的眼睛中并不常见18。第三,小鼠不会发育玻璃疣,这是AMD病理学的标志19。玻璃膜疣是含有脂质和蛋白质的沉积物,在RPE基底层和布鲁赫膜(BrM)的内部胶原层之间形成很少的基底膜蛋白19。玻璃膜疣与BLamD不同,BLamD是小鼠中常见的亚RPE沉积物,无论是它们的组成还是解剖位置。BLamD 是年龄和应激依赖性细胞外基质富集异常,在 BrM 的 RPE 基底层和 RPE20 的基底折叠之间形成。有趣的是,BLamD在小鼠和人类中具有相似的蛋白质组成和外观6,1021最近的研究表明,BLamDs可能通过影响AMD进展到后期来影响AMD的病理生物学1822;因此,这些沉积物可能代表小鼠视网膜中的患病RPE。了解这些益处和局限性对于有兴趣将小鼠研究结果转化为AMD的研究人员至关重要。

在该协议中,我们讨论了为光,透射电子和共聚焦显微镜准备眼睛以可视化RPE病理的方法。我们还描述了如何以无偏的方式量化RPE病理以进行统计测试。作为概念验证,我们利用RPE表型方案来研究在跨膜蛋白135-(Tmem135)过表达小鼠和老年野生型(WT)C57BL / 6J小鼠中观察到的结构RPE病理。总之,我们的目标是描述表征AMD小鼠模型中RPE的表型方法,因为目前没有可用的标准协议。有兴趣检查和量化光感受器或脉络膜病理的研究人员,这些病理在AMD小鼠模型中也受到影响,可能发现该协议对他们的研究没有用。

Protocol

所有涉及动物受试者的程序均已获得威斯康星大学麦迪逊分校机构动物护理和使用委员会的批准,并符合视觉和眼科研究协会 (ARVO) 关于在眼科和视力研究中使用动物的声明。 1.光学显微镜评估小鼠RPE 在玻璃瓶中制备终浓度为2%多聚甲醛和2%戊二醛的固定缓冲液,在室温(RT)下。注意:该方案每只小鼠最多需要14 mL固定缓冲液。注意:多聚甲醛和?…

Representative Results

本文中描述的RPE表型方案的完成提供了对AMD小鼠模型中常见的结构RPE异常的定量分析。为了确认该协议的有效性,我们在已知显示RPE病理的小鼠中使用它,包括由鸡β-肌动蛋白启动子(Tmem135 TG)30和老年C57BL / 6J小鼠31,32驱动的过表达WT Tmem135的转基因小鼠。这些实验的目的是向刚接触小鼠模型的研究人员展示使用本协议?…

Discussion

在本文中,我们介绍了一种表型协议,用于评估小鼠模型的结构RPE病理。我们描述了处理各种成像技术(包括光,透射电子和共聚焦显微镜)的眼睛所需的步骤,以及通过这些成像方法观察到的典型病理的定量。我们通过检查Tmem135 TG和24个月大的WT小鼠证明了我们的RPE表型方案的有效性,因为这些小鼠显示RPE病理303132

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者要感谢Satoshi Kinoshia和威斯康星大学(UW)病理学实验室转化研究计划(TRIP)为我们的组织准备用于光学显微镜。该核心由威斯康星大学Carbone癌症中心(P30 CA014520)和NIH主任办公室(S10OD023526)支持。共聚焦显微镜在UW生物化学光学核心进行,该光学核心是在UW生物化学系的支持下建立的。这项工作还得到了国家眼科研究所的资助(R01EY022086给A. Ikeda;R01EY031748 至 C. 鲍斯·里克曼;P30EY016665 到华盛顿大学眼科和视觉科学系;P30EY005722到杜克眼科中心;NIH T32EY027721 至 M. Landowski;F32EY032766 至 M. Landowski),蒂莫西·威廉·特劳特主席(A. 池田),FFB 自由家庭 AMD 奖(C. Bowes Rickman);以及预防失明研究(杜克眼科中心)的无限制资助。

Materials

0.1 M Cacodylate Buffer pH7.2 PolyScientiifc R&D Company S1619
100 Capacity Slide Box Two are needed for this protocol (one for H&E-stained slides and one for RPE flat mounts.)
100% Ethanol  MDS Warehouse 2292-CASE Can be used to make diluted ethanol solutions in this protocol.
1-Way Stopcock, 2 Female Luer Locks Qosina 11069
1x Phosphate Buffer Solution (PBS) Premade 1x PBS can be used in this protocol. 
2.0 mL microtubes Genesee Scientific  24-283-LR
24 Cavity Embedding Capsule Substitute Mold Electron Microscopy Sciences 70165
24 inch PVC Tubing with Luer Ends Fisher Scientific NC1376778
400 Mesh Gilder Thin Bar Square Mesh Grids Electron Microscopy Sciences T400-Cu
95% Ethanol MDS Warehouse 2293-CASE
Absorbent Underpads with Waterproof Moisture Barrier (23 inches by 24 inches) VWR 56616-031
Adjustable 237 ml  Spray Bottle VWR 23609-182
Alexa Fluor488 Conjugated Donkey anti-Rabbit IgG  Thermo Fisher Scientific A-21206
Aluminum Foil
BD Precision glide 19 Gauge Syringe Needle Sigma-Aldrich  Z192546
Bracken Forceps; Curved; Fine Cross Serrations; 4" Length, 1 mm Tip Width Roboz Surgical Instrument RS-5211 Known as curved forceps in this protocol.
Camel Hair Brush Electron Microscopy Sciences 65575-02
Carbon Dioxide Euthanasia Chamber
Carbon Dioxide Flow Meter
Carbon Dioxide Tank
Castaloy Prong Extension Clamps Fisher Scientific  05-769-7Q
Cast-Iron L-shaped Base Support Stand Fisher Scientific  11-474-207
Cell Prolifer Program Available to download: https://cellprofiler.org/releases
Clear Nail Polish Electron Microscopy Sciences 72180
Colorfrost Microscope Slides Lavender VWR 10118-956
Computer
DAPI Sigma-Aldrich D9542-5MG
Distilled H20 Water from Milli-Q Purification System was used in this protocol.
Dumont Thin Tip Tweezers; Pattern #55 Roboz Surgical Instrument RS-4984 Known as fine-tipped forceps in this protocol, and 3 are needed for this protocol (two for dissections and one for electron microscope processing).
Electron Microscopy Grid Holder Electron Microscopy Sciences 71147-01
EPON 815 Resin Electron Microscopy Sciences 14910
Epredia Mark-It Tissue Marking Yellow Dye Fisher Scientific  22050460 Please follow manufacturer's protocol when using this tissue marking dye. 
Epredia Mounting Media Fisher Scientific 22-110-610 Use for mounting H&E slides. 
Fiber-Lite Mi-150 Illuminator Series,150 w Halogen Light Source Dolan-Jenner Industries Mi-150 Light source for dissecting microscope.
Fiji ImageJ Program Available to download: https://imagej.net/downloads
Flexaframe Castaloy Hook Connector Thermo Scientific   14-666-18Q
Fume hood
Glutaraldehyde 2.5% in Phosphate Buffer, pH 7.4, 32% Electron Microscopy Sciences 16537-05
JEM-1400 Transmission Electron Microscope (JEOL) with an ORIUS (1000) CCD Camera
Laboratory Benchtop Shaker Two are needed for these experiments. One should be at room temperature while the other should be in a 4 degree Celsius cold room.
Laser Cryo Tag Labels Electron Microscopy Sciences 77564-05
Lead Citrate Electron Microscopy Sciences 17800
Leica EM UC7Ultramicrotome
Leica Reichert Ultracut S Microtome
LifterSlips Thermo Fisher Scientific 22X22I24788001LS Use these coverslips for the RPE flat mounts as they have raised edges and accommodate the thickness of the RPE.
Mayer's Hematoxylin VWR 100504-406
McPherson-Vannas Micro Dissecting Spring Scissors Roboz Surgical Instrument RS-5600 Known as micro-dissecting scissors in protocol. 
Methanol Fisher Scientific  A412-4
Mice Any AMD mouse model and its respective controls can work for this protocol.
Micro Dissecting Scissors; Standard Version; Curved; Sharp Points; 24 mm Blade Length; 4.5" Overall Length Roboz Surgical Instrument RS-5913 Known as curved scissors in this protocol.
Microsoft Excel
Microtube racks
Nikon A1RS Confocal Microscope
Normal Donkey Serum SouthernBiotech 0030-01
Number 11 Sterile Disposable Scalpel Blades VWR 21909-380
Osmium Tetroxide  Electron Microscopy Sciences 19150
Paraformaldehyde, 32% Electron Microscopy Sciences 15714-S
Pencil
Petri Dish VWR  21909-380
Pipette Tips
Pipettes 
Polyclonal Anti-ZO-1 Antibody Thermo Fisher Scientific 402200
Propylene Oxide Electron Microscopy Sciences 20412
Razor Blade VWR 10040-386
Shallow Tray for Mouse Perfusions
Shandon Eosin Y Alcoholic VWR 89370-828
Sharpie Ultra Fine Tip Black Permanent Marker Staples 642736
Slide Rack for Staining Grainger 49WF31
Squared Cover Glass Slips Fisher Scientific  12-541B
Staining Dish with Cover Grainger 49WF30 Need 15 for H&E staining procedure.
Target All-Plastic Disposable Luer-Slip 50 mL Syringe  Thermo Scientific  S7510-50 Use only the syringe barrel.
Timer Fisher 1464917
Uranyl Acetate Electron Microscopy Sciences 22400
Vacuum Oven
Vectashield Mounting Medium Vector Laboratories H-1000 Use for mounting RPE flat mounts. 
Xylene Fisher Scientific  22050283
Zeiss Axio Imager 2 Light Microscope This microscope has the capacity to generate stitched 20x images. If a light microscope does not have this capacity, then take images of the entire retina that are slightly overlapping each other. Use Adobe Photoshop to stitch these images together. Please refer to the manuals of the Adobe Photoshop program for image stitching. 
Zeiss Stemi 2000 Dissecting Microscope Electron Microscopy Sciences 65575-02

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Landowski, M., Grindel, S., Hao, Y., Ikeda, S., Bowes Rickman, C., Ikeda, A. A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration. J. Vis. Exp. (193), e64927, doi:10.3791/64927 (2023).

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