Name: A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration (Video) | JoVE Journal
Uploaded: 2023-03-10T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Learn a protocol to evaluate retinal pigmented epithelium pathologies in mouse models of age-related macular degeneration using light, transmission electron, and confocal microscopy. This method enables quantitative assessments for statistical comparisons.

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The authors would like to acknowledge Satoshi Kinoshita and the University of Wisconsin (UW) Translational Research Initiatives in Pathology laboratory (TRIP) for preparing our tissues for light microscopy. This core is supported by the UW Department of Pathology and Laboratory Medicine, University of Wisconsin Carbone Cancer Center (P30 CA014520), and the Office of The Director-NIH (S10OD023526). Confocal microscopy was performed at the UW Biochemistry Optical Core, which was established with support from the UW Department of Biochemistry Endowment. This work was also supported by grants from the National Eye Institute (R01EY022086 to A. Ikeda; R01EY031748 to C. Bowes Rickman; P30EY016665 to the Department of Ophthalmology and Visual Sciences at the UW; P30EY005722 to the Duke Eye Center;NIH T32EY027721 to M. Landowski; F32EY032766 to M. Landowski), Timothy William Trout Chairmanship (A. Ikeda), FFB Free Family AMD Award (C. Bowes Rickman); and an unrestricted grant from the Research to Prevent Blindness (Duke Eye Center).

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The authors of this protocol have no disclosures and conflicts of interest.

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<su...

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 models4,5 and mice6,7,8. A recent report has described standardized protocols and quality control criteria for RPE cell culture models4, yet no report has attempted to standardize the phenotyping of the RPE in mouse models. In fact, many publications on mouse models of AMD lack a complete description of the RPE or quantification of the RPE pathologies in them. The overall goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using AMD mouse models.
Previous publications have noted the presence of several RPE pathologies in mice through three imaging techniques. For instance, light microscopy allows researchers to view the gross morphology of the murine retina (Figure 1A) and detect RPE pathologies such as RPE thinning, vacuolization, and migration. RPE thinning in an AMD mouse model is exemplified by a deviation in the RPE height from their respective controls (Figure 1B). RPE vacuolization can be divided into two separate categories: microvacuolization (Figure 1C) and macrovacuolization (Figure 1D). RPE microvacuolization is summarized by the presence of vacuoles in the RPE that do not affect its overall height, whereas macrovacuolization is indicated by the presence of vacuoles that protrude into the outer segments of the photoreceptors. RPE migration is distinguished by the focal aggregate of pigment above the RPE monolayer in a retinal cross-section (Figure 1E). It should be noted that migratory RPE cells in AMD donor eyes exhibit immunoreactivity to immune cell markers, such as cluster of differentiation 68 (CD68)9, and could represent immune cells engulfing RPE debris or RPE undergoing transdifferentiation9. Another imaging technique called transmission electron microscopy can permit researchers to visualize the ultrastructure of the RPE and its basement membrane (Figure 2A). This technique can identify the predominant sub-RPE deposit in mice, known as the basal laminar deposit (BLamD) (Figure 2B)10. Lastly, confocal microscopy can reveal the structure of RPE cells through imaging RPE flat mounts (Figure 3A). This method can uncover RPE dysmorphia, the deviation of the RPE from its classic honeycomb shape (Figure 3B). It can also detect RPE multinucleation, the presence of three or more nuclei within an RPE cell (Figure 3C). For a summary of the types of RPE pathologies present in current AMD mouse models, we refer researchers to these reviews from the literature6,7.
Researchers studying AMD should be aware of the advantages and disadvantages of using mice to investigate RPE pathologies prior to the phenotyping protocol. Mice are advantageous because of their relatively short life span and cost-effectiveness, as well as their genetic and pharmacologic manipulability. Mice also exhibit RPE degenerative changes, including RPE migration, dysmorphia, and multinucleation, that are observed in AMD donor eyes11,12,13,14,15,16,17; this suggests that similar mechanisms may underly the development of these RPE pathologies in mice and humans. However, there are key differences that limit the translatability of mouse studies to human AMD. First, mice do not have a macula, an anatomically distinct region of the human retina necessary for visual acuity that is preferentially affected in AMD. Second, some RPE pathologies in mice, like RPE thinning and vacuolization, are not typically seen in AMD donor eyes18. Third, mice do not develop drusen, a hallmark of AMD pathology19. Drusen are lipid- and protein-containing deposits with very few basement membrane proteins that form between the RPE basal lamina and the inner collagenous layer of Bruch&#39;s membrane (BrM)19. Drusen differ from BLamD, the common sub-RPE deposit in mice, in both their composition and anatomical location. BLamDs are age- and stress-dependent extracellular matrix-enriched abnormalities that form between the RPE basal lamina of BrM and the basal infoldings of the RPE20. Interestingly, BLamDs have a similar protein composition and appearance in both mice and humans6,10,21. Recent work suggests BLamDs may act in the pathobiology of AMD by influencing the progression of AMD to its later stages18,22; thus, these deposits may represent diseased RPE in the mouse retina. Knowledge of these benefits and limitations is critical for researchers interested in translating results from mouse studies to AMD.
In this protocol, we discuss the methods to prepare eyes for light, transmission electron, and confocal microscopy to visualize RPE pathologies. We also describe how to quantify RPE pathologies in an unbiased manner for statistical testing. As proof of concept, we utilize the RPE phenotyping protocol to investigate the structural RPE pathologies observed in transmembrane protein 135- (Tmem135) overexpressing mice and aged wild-type (WT) C57BL/6J mice. In summary, we aim to describe the phenotyping methodology to characterize the RPE in AMD mouse models, since there are currently no standard protocols available. Researchers interested in examining and quantifying pathologies of the photoreceptors or choroid, which are also affected in AMD mouse models, may not find this protocol useful for their studies.

<table><tbody><tr><td>0.1 M Cacodylate Buffer pH7.2</td><td>PolyScientiifc R&amp;amp;D Company</td><td>S1619</td><td></td></tr><tr><td>100 Capacity Slide Box</td><td></td><td></td><td>Two are needed for this protocol (one for H&amp;amp;E-stained slides and one for RPE flat mounts.)</td></tr><tr><td>100% Ethanol&amp;nbsp;</td><td>MDS Warehouse</td><td>2292-CASE</td><td>Can be used to make diluted ethanol solutions in this protocol.</td></tr><tr><td>1-Way Stopcock, 2 Female Luer Locks</td><td>Qosina</td><td>11069</td><td></td></tr><tr><td>1x Phosphate Buffer Solution (PBS)</td><td></td><td></td><td>Premade 1x PBS can be used in this protocol.&amp;nbsp;</td></tr><tr><td>2.0 mL microtubes</td><td>Genesee Scientific&amp;nbsp;</td><td>24-283-LR</td><td></td></tr><tr><td>24 Cavity Embedding Capsule Substitute Mold</td><td>Electron Microscopy Sciences</td><td>70165</td><td></td></tr><tr><td>24 inch PVC Tubing with Luer Ends</td><td>Fisher Scientific</td><td>NC1376778</td><td></td></tr><tr><td>400 Mesh Gilder Thin Bar Square Mesh Grids</td><td>Electron Microscopy Sciences</td><td>T400-Cu</td><td></td></tr><tr><td>95% Ethanol</td><td>MDS Warehouse</td><td>2293-CASE</td><td></td></tr><tr><td>Absorbent Underpads with Waterproof Moisture Barrier (23 inches by 24 inches)</td><td>VWR</td><td>56616-031</td><td></td></tr><tr><td>Adjustable 237 ml&amp;nbsp; Spray Bottle</td><td>VWR</td><td>23609-182</td><td></td></tr><tr><td>Alexa Fluor488 Conjugated Donkey anti-Rabbit IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A-21206</td><td></td></tr><tr><td>Aluminum Foil</td><td></td><td></td><td></td></tr><tr><td>BD Precision glide 19 Gauge Syringe Needle</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Z192546</td><td></td></tr><tr><td>Bracken Forceps; Curved; Fine Cross Serrations; 4&quot; Length, 1 mm Tip Width</td><td>Roboz Surgical Instrument</td><td>RS-5211</td><td>Known as curved forceps in this protocol.</td></tr><tr><td>Camel Hair Brush</td><td>Electron Microscopy Sciences</td><td>65575-02</td><td></td></tr><tr><td>Carbon Dioxide Euthanasia Chamber</td><td></td><td></td><td></td></tr><tr><td>Carbon Dioxide Flow Meter</td><td></td><td></td><td></td></tr><tr><td>Carbon Dioxide Tank</td><td></td><td></td><td></td></tr><tr><td>Castaloy Prong Extension Clamps</td><td>Fisher Scientific&amp;nbsp;</td><td>05-769-7Q</td><td></td></tr><tr><td>Cast-Iron L-shaped Base Support Stand</td><td>Fisher Scientific&amp;nbsp;</td><td>11-474-207</td><td></td></tr><tr><td>Cell Prolifer Program</td><td></td><td></td><td>Available to download: https://cellprofiler.org/releases</td></tr><tr><td>Clear Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Colorfrost Microscope Slides Lavender</td><td>VWR</td><td>10118-956</td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542-5MG</td><td></td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;0</td><td></td><td></td><td>Water from Milli-Q Purification System was used in this protocol.</td></tr><tr><td>Dumont Thin Tip Tweezers; Pattern #55</td><td>Roboz Surgical Instrument</td><td>RS-4984</td><td>Known as fine-tipped forceps in this protocol, and 3 are needed for this protocol (two for dissections and one for electron microscope processing).</td></tr><tr><td>Electron Microscopy Grid Holder</td><td>Electron Microscopy Sciences</td><td>71147-01</td><td></td></tr><tr><td>EPON 815 Resin</td><td>Electron Microscopy Sciences</td><td>14910</td><td></td></tr><tr><td>Epredia Mark-It Tissue Marking Yellow Dye</td><td>Fisher Scientific&amp;nbsp;</td><td>22050460</td><td>Please follow manufacturer&#039;s protocol when using this tissue marking dye.&amp;nbsp;</td></tr><tr><td>Epredia Mounting Media</td><td>Fisher Scientific</td><td>22-110-610</td><td>Use for mounting H&amp;amp;E slides.&amp;nbsp;</td></tr><tr><td>Fiber-Lite Mi-150 Illuminator Series,150 w Halogen Light Source</td><td>Dolan-Jenner Industries</td><td>Mi-150</td><td>Light source for dissecting microscope.</td></tr><tr><td>Fiji ImageJ Program</td><td></td><td></td><td>Available to download: https://imagej.net/downloads</td></tr><tr><td>Flexaframe Castaloy Hook Connector</td><td>Thermo Scientific&amp;nbsp;&amp;nbsp;</td><td>14-666-18Q</td><td></td></tr><tr><td>Fume hood</td><td></td><td></td><td></td></tr><tr><td>Glutaraldehyde 2.5% in Phosphate Buffer, pH 7.4, 32%</td><td>Electron Microscopy Sciences</td><td>16537-05</td><td></td></tr><tr><td>JEM-1400 Transmission Electron Microscope (JEOL) with an ORIUS (1000) CCD Camera</td><td></td><td></td><td></td></tr><tr><td>Laboratory Benchtop Shaker</td><td></td><td></td><td>Two are needed for these experiments. One should be at room temperature while the other should be in a 4 degree Celsius cold room.</td></tr><tr><td>Laser Cryo Tag Labels</td><td>Electron Microscopy Sciences</td><td>77564-05</td><td></td></tr><tr><td>Lead Citrate</td><td>Electron Microscopy Sciences</td><td>17800</td><td></td></tr><tr><td>Leica EM UC7Ultramicrotome</td><td></td><td></td><td></td></tr><tr><td>Leica Reichert Ultracut S Microtome</td><td></td><td></td><td></td></tr><tr><td>LifterSlips</td><td>Thermo Fisher Scientific</td><td>22X22I24788001LS</td><td>Use these coverslips for the RPE flat mounts as they have raised edges and accommodate the thickness of the RPE.</td></tr><tr><td>Mayer&#039;s Hematoxylin</td><td>VWR</td><td>100504-406</td><td></td></tr><tr><td>McPherson-Vannas Micro Dissecting Spring Scissors</td><td>Roboz Surgical Instrument</td><td>RS-5600</td><td>Known as micro-dissecting scissors in protocol.&amp;nbsp;</td></tr><tr><td>Methanol</td><td>Fisher Scientific&amp;nbsp;</td><td>A412-4</td><td></td></tr><tr><td>Mice</td><td></td><td></td><td>Any AMD mouse model and its respective controls can work for this protocol.</td></tr><tr><td>Micro Dissecting Scissors; Standard Version; Curved; Sharp Points; 24 mm Blade Length; 4.5&quot; Overall Length</td><td>Roboz Surgical Instrument</td><td>RS-5913</td><td>Known as curved scissors in this protocol.</td></tr><tr><td>Microsoft Excel</td><td></td><td></td><td></td></tr><tr><td>Microtube racks</td><td></td><td></td><td></td></tr><tr><td>Nikon A1RS Confocal Microscope</td><td></td><td></td><td></td></tr><tr><td>Normal Donkey Serum</td><td>SouthernBiotech</td><td>0030-01</td><td></td></tr><tr><td>Number 11 Sterile Disposable Scalpel Blades</td><td>VWR</td><td>21909-380</td><td></td></tr><tr><td>Osmium Tetroxide&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>19150</td><td></td></tr><tr><td>Paraformaldehyde, 32%</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td></td></tr><tr><td>Pencil</td><td></td><td></td><td></td></tr><tr><td>Petri Dish</td><td>VWR&amp;nbsp;</td><td>21909-380</td><td></td></tr><tr><td>Pipette Tips</td><td></td><td></td><td></td></tr><tr><td>Pipettes&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Polyclonal Anti-ZO-1 Antibody</td><td>Thermo Fisher Scientific</td><td>402200</td><td></td></tr><tr><td>Propylene Oxide</td><td>Electron Microscopy Sciences</td><td>20412</td><td></td></tr><tr><td>Razor Blade</td><td>VWR</td><td>10040-386</td><td></td></tr><tr><td>Shallow Tray for Mouse Perfusions</td><td></td><td></td><td></td></tr><tr><td>Shandon Eosin Y Alcoholic</td><td>VWR</td><td>89370-828</td><td></td></tr><tr><td>Sharpie Ultra Fine Tip Black Permanent Marker</td><td>Staples</td><td>642736</td><td></td></tr><tr><td>Slide Rack for Staining</td><td>Grainger</td><td>49WF31</td><td></td></tr><tr><td>Squared Cover Glass Slips</td><td>Fisher Scientific&amp;nbsp;</td><td>12-541B</td><td></td></tr><tr><td>Staining Dish with Cover</td><td>Grainger</td><td>49WF30</td><td>Need 15 for H&amp;amp;E staining procedure.</td></tr><tr><td>Target All-Plastic Disposable Luer-Slip 50 mL Syringe&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>S7510-50</td><td>Use only the syringe barrel.</td></tr><tr><td>Timer</td><td>Fisher</td><td>1464917</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Electron Microscopy Sciences</td><td>22400</td><td></td></tr><tr><td>Vacuum Oven</td><td></td><td></td><td></td></tr><tr><td>Vectashield Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td>Use for mounting RPE flat mounts.&amp;nbsp;</td></tr><tr><td>Xylene</td><td>Fisher Scientific&amp;nbsp;</td><td>22050283</td><td></td></tr><tr><td>Zeiss Axio Imager 2 Light Microscope</td><td></td><td></td><td>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.&amp;nbsp;</td></tr><tr><td>Zeiss Stemi 2000 Dissecting Microscope</td><td>Electron Microscopy Sciences</td><td>65575-02</td><td></td></tr></tbody></table>

a protocol to evaluate and quantify retinal pigmented epithelium pathologies in mouse models of age-related macular degeneration

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

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.

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 mice31,32</su...

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.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

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Research

JoVE Journal

Genetics

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Retinal Pathophysiological Evaluation in a Rat Model

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and α/β C-termini, can be quantified.

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Learn a protocol to evaluate retinal pigmented epithelium pathologies in mouse models of age-related macular degeneration using light, transmission electron, and confocal microscopy. This method enables quantitative assessments for statistical comparisons.

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration (Video) | JoVE Journal

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

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