En protokol til evaluering og kvantificering af retinale pigmenterede epitelpatologier i musemodeller af aldersrelateret makuladegeneration
Summary
Musemodeller kan være nyttige værktøjer til at undersøge biologien af det retinale pigmenterede epitel (RPE). Det er blevet fastslået, at mus kan udvikle en række RPE-patologier. Her beskriver vi en fænotypeprotokol til belysning og kvantificering af RPE-patologier hos mus ved hjælp af lys, transmissionselektron og konfokal mikroskopi.
Abstract
Aldersrelateret makuladegeneration (AMD) er en invaliderende retinal lidelse i aldrende befolkninger. Det er almindeligt antaget, at dysfunktion af retinal pigmenteret epitel (RPE) er en vigtig patobiologisk begivenhed i AMD. For at forstå de mekanismer, der fører til RPE-dysfunktion, kan musemodeller bruges af forskere. Det er blevet fastslået ved tidligere undersøgelser, at mus kan udvikle RPE-patologier, hvoraf nogle observeres i øjnene hos personer diagnosticeret med AMD. Her beskriver vi en fænotypeprotokol til vurdering af RPE-patologier hos mus. Denne protokol omfatter forberedelse og evaluering af retinale tværsnit ved hjælp af lysmikroskopi og transmissionselektronmikroskopi såvel som RPE-fladmonteringer ved konfokal mikroskopi. Vi beskriver de almindelige typer murin RPE-patologier, der observeres ved disse teknikker og måder at kvantificere dem gennem upartiske metoder til statistisk testning. Som bevis på konceptet bruger vi denne RPE-fænotypeprotokol til at kvantificere RPE-patologierne observeret hos mus, der overudtrykker transmembranprotein 135 (Tmem135) og ældre vildtype C57BL/6J-mus. Hovedformålet med denne protokol er at præsentere standard RPE-fænotypemetoder med upartiske kvantitative vurderinger for forskere, der bruger musemodeller af AMD.
Introduction
Aldersrelateret makuladegeneration (AMD) er en almindelig blindende sygdom, der rammer befolkninger over 55år 1. Mange forskere mener, at dysfunktion i retinal pigmenteret epitel (RPE) er en tidlig og afgørende patobiologisk begivenhed i AMD2. RPE er et monolag af polariserede celler, der har til opgave at opretholde homeostase af nærliggende fotoreceptorer og choroidale blodkar3. Der findes en række modeller til at undersøge sygdomsassocierede mekanismer inden for RPE, herunder cellekulturmodeller4,5 og mus 6,7,8. En nylig rapport har beskrevet standardiserede protokoller og kvalitetskontrolkriterier for RPE-cellekulturmodeller4, men ingen rapport har forsøgt at standardisere fænotypningen af RPE i musemodeller. Faktisk mangler mange publikationer om musemodeller af AMD en komplet beskrivelse af RPE eller kvantificering af RPE-patologierne i dem. Det overordnede mål med denne protokol er at præsentere standard RPE-fænotypemetoder med upartiske kvantitative vurderinger for forskere, der bruger AMD-musemodeller.
Tidligere publikationer har bemærket tilstedeværelsen af flere RPE-patologier hos mus gennem tre billeddannelsesteknikker. For eksempel giver lysmikroskopi forskere mulighed for at se den grove morfologi af murine nethinden (figur 1A) og opdage RPE-patologier såsom RPE-udtynding, vakuolisering og migration. RPE-udtynding i en AMD-musemodel eksemplificeres ved en afvigelse i RPE-højden fra deres respektive kontroller (figur 1B). RPE-vakuolisering kan opdeles i to separate kategorier: mikrovakuolisering (figur 1C) og makrovakuolisering (figur 1D). RPE-mikrovakuolisering opsummeres ved tilstedeværelsen af vakuoler i RPE, der ikke påvirker dens samlede højde, mens makrovakuolisering indikeres ved tilstedeværelsen af vakuoler, der stikker ud i de ydre segmenter af fotoreceptorerne. RPE-migration kendetegnes ved pigmentets fokalaggregat over RPE-monolaget i et nethindetværsnit (figur 1E). Det skal bemærkes, at migrerende RPE-celler i AMD-donorøjne udviser immunreaktivitet over for immuncellemarkører, såsom klynge af differentiering 68 (CD68)9, og kan repræsentere immunceller, der opsluger RPE-affald eller RPE, der gennemgår transdifferentiering9. En anden billeddannelsesteknik kaldet transmissionselektronmikroskopi kan give forskere mulighed for at visualisere ultrastrukturen af RPE og dens kældermembran (figur 2A). Denne teknik kan identificere den fremherskende sub-RPE-aflejring hos mus, kendt som den basale laminære aflejring (BLamD) (figur 2B)10. Endelig kan konfokal mikroskopi afsløre strukturen af RPE-celler gennem billeddannelse af RPE-flade monteringer (figur 3A). Denne metode kan afdække RPE-dysmorfi, afvigelsen af RPE fra dens klassiske bikageform (figur 3B). Det kan også detektere RPE-multinucleation, tilstedeværelsen af tre eller flere kerner i en RPE-celle (figur 3C). For et resumé af de typer RPE-patologier, der findes i nuværende AMD-musemodeller, henviser vi forskere til disse anmeldelser fra litteraturen 6,7.
Forskere, der studerer AMD, bør være opmærksomme på fordele og ulemper ved at bruge mus til at undersøge RPE-patologier inden fænotypeprotokollen. Mus er fordelagtige på grund af deres relativt korte levetid og omkostningseffektivitet samt deres genetiske og farmakologiske manipulerbarhed. Mus udviser også RPE-degenerative ændringer, herunder RPE-migration, dysmorfi og multinucleation, der observeres i AMD-donorøjne 11,12,13,14,15,16,17; dette tyder på, at lignende mekanismer kan ligge til grund for udviklingen af disse RPE-patologier hos mus og mennesker. Der er dog vigtige forskelle, der begrænser oversætteligheden af musestudier til human AMD. For det første har mus ikke en makula, en anatomisk distinkt region af den menneskelige nethinde, der er nødvendig for synsstyrke, der fortrinsvis påvirkes i AMD. For det andet ses nogle RPE-patologier hos mus, som RPE-udtynding og vakuolisering, typisk ikke i AMD-donorøjne18. For det tredje udvikler mus ikke drusen, et kendetegn ved AMD-patologi19. Drusen er lipid- og proteinholdige aflejringer med meget få kældermembranproteiner, der dannes mellem RPE basal lamina og det indre kollagenøse lag af Bruchs membran (BrM)19. Drusen adskiller sig fra BLamD, den almindelige sub-RPE-aflejring i mus, både i deres sammensætning og anatomiske placering. BLamD’er er alders- og stressafhængige ekstracellulære matrixberigede abnormiteter, der dannes mellem RPE basal lamina af BrM og de basale indfoldninger af RPE20. Interessant nok har BLamD’er en lignende proteinsammensætning og udseende hos både mus og mennesker 6,10,21. Nyligt arbejde tyder på, at BLamD’er kan virke i AMD’s patobiologi ved at påvirke udviklingen af AMD til dets senere stadier18,22; således kan disse aflejringer repræsentere syg RPE i musens nethinden. Viden om disse fordele og begrænsninger er afgørende for forskere, der er interesserede i at oversætte resultater fra musestudier til AMD.
I denne protokol diskuterer vi metoderne til at forberede øjnene på lys, transmissionselektron og konfokal mikroskopi for at visualisere RPE-patologier. Vi beskriver også, hvordan man kvantificerer RPE-patologier på en upartisk måde til statistisk testning. Som bevis på konceptet bruger vi RPE-fænotypeprotokollen til at undersøge de strukturelle RPE-patologier, der observeres i transmembranprotein 135- (Tmem135), der overudtrykker mus og ældre vildtypemus (WT) C57BL/6J-mus. Sammenfattende sigter vi mod at beskrive fænotypemetoden til at karakterisere RPE i AMD-musemodeller, da der i øjeblikket ikke er nogen standardprotokoller tilgængelige. Forskere, der er interesseret i at undersøge og kvantificere patologier af fotoreceptorerne eller choroid, som også påvirkes i AMD-musemodeller, finder muligvis ikke denne protokol nyttig til deres studier.
Protocol
Representative Results
Discussion
I denne artikel introducerede vi en fænotypeprotokol til vurdering af musemodellernes strukturelle RPE-patologier. Vi beskrev de trin, der kræves til behandling af øjnene til forskellige billeddannelsesteknikker, herunder lys, transmissionselektron og konfokal mikroskopi, samt kvantificering af typiske patologier observeret via disse billeddannelsesmetoder. Vi beviste effektiviteten af vores RPE-fænotypeprotokol ved at undersøge Tmem135 TG og 24 måneder gamle WT-mus, da disse mus viser RPE-patolog…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne anerkende Satoshi Kinoshita og University of Wisconsin (UW) Translational Research Initiatives in Pathology laboratory (TRIP) for at forberede vores væv til lysmikroskopi. Denne kerne understøttes af UW Department of Pathology and Laboratory Medicine, University of Wisconsin Carbone Cancer Center (P30 CA014520) og Office of The Director-NIH (S10OD023526). Konfokal mikroskopi blev udført på UW Biochemistry Optical Core, som blev etableret med støtte fra UW Department of Biochemistry Endowment. Dette arbejde blev også støttet af tilskud fra National Eye Institute (R01EY022086 til A. Ikeda; R01EY031748 til C. Bowes Rickman; P30EY016665 til Institut for Oftalmologi og Visuelle Videnskaber ved UW; P30EY005722 til Duke Eye Center; NIH T32EY027721 til M. Landowski; F32EY032766 til M. Landowski), Timothy William Trout formandskab (A. Ikeda), FFB Free Family AMD Award (C. Bowes Rickman); og en ubegrænset bevilling fra Research to Prevent Blindness (Duke Eye Center).
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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