A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Summary

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Abstract

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Introduction

Age-related macular degeneration (AMD) is a common blinding disease that affects populations over the age of 55¹. Many researchers believe that dysfunction within the retinal pigmented epithelium (RPE) is an early and crucial pathobiological event in AMD². The RPE is a monolayer of polarized cells tasked with maintaining the homeostasis of neighboring photoreceptors and choroidal blood vessels³. A variety of models exist to investigate disease-associated mechanisms within the RPE, including cell culture models^4,5 and mice^....

Protocol

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison, and are in adherence with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Evaluation of mouse RPE by light microscopy

1. Make a fixative buffer that has a final concentration of 2% paraformaldehyde and 2% glutaraldehyde at room temperature (RT) in a glass bottle.
   NOTE: This protocol requires, at most, 14 mL of fixative buffer per mouse.
   CAUTION: Paraformaldehyde and g....

Results

Completion of the RPE phenotyping protocol described in this article provides a quantitative analysis of the structural RPE abnormalities commonly observed in mouse models of AMD. To confirm the effectiveness of this protocol, we used it in mice that are known to display RPE pathologies, including transgenic mice that overexpress WT Tmem135 driven by the chicken beta-actin promoter (Tmem135 TG)³⁰ and aged C57BL/6J mice^31,32

Discussion

In this article, we introduced a phenotyping protocol for assessing the structural RPE pathologies of mouse models. We described the steps required for processing the eyes for various imaging techniques including light, transmission electron, and confocal microscopy, as well as the quantitation of typical pathologies observed via these imaging methods. We proved the effectiveness of our RPE phenotyping protocol by examining Tmem135 TG and 24-month-old WT mice, since these mice display RPE pathologies

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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