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).

<ol>
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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

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...

Watch this Scientific Journal Video about A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration at JoVE.com

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

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

1.5K Views.  University of Wisconsin-Madison. Dit protocol is het eerste dat beschrijft hoe de muisgrootte voor RP-pathologieën kan worden verwerkt en geëvalueerd met behulp van lichttransmissie-elektronen- en confocale microscopie. Het belangrijkste voordeel van dit protocol is de opname van kwantitatieve beoordelingen van de RP-pathologieën, met behulp van deze drie beeldvormingstechnieken voor statistische vergelijkingen. De technieken die in dit protocol worden beschreven, kunnen ook helpen om onze kennis van moleculaire routes di...

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

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Neuroscience

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

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

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

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.

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

Chemistry

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

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

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.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

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.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Medicine

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

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

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

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.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

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.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Biology

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

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

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.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

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.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

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

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

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.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Author Spotlight: Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...

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

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