Generering af retinale skademodeller i Xenopus haletudser
Summary
Vi har udviklet flere protokoller til at fremkalde retinal skade eller retinal degeneration i Xenopus laevis haletudser. Disse modeller giver mulighed for at studere retinale regenereringsmekanismer.
Abstract
Retinale neurodegenerative sygdomme er de førende årsager til blindhed. Blandt mange terapeutiske strategier, der udforskes, viste stimulering af selvreparation sig for nylig som særlig tiltalende. En cellulær kilde til retinal reparation er Müller gliacellen, som har stamcellepotentiale og en ekstraordinær regenerativ kapacitet i anamnioter. Dette potentiale er dog meget begrænset hos pattedyr. Undersøgelse af de molekylære mekanismer, der ligger til grund for retinal regenerering i dyremodeller med regenerative evner, bør give indsigt i, hvordan man låser op for pattedyrs Müller-cellers latente evne til at regenerere nethinden. Dette er et vigtigt skridt i udviklingen af terapeutiske strategier inden for regenerativ medicin. Til dette formål udviklede vi flere retinale skadeparadigmer i Xenopus: en mekanisk nethindeskade, en transgen linje, der muliggør nitroreduktasemedieret fotoreceptorbetinget ablation, en retinitis pigmentosa-model baseret på CRISPR/Cas9-medieret rhodopsin-knockout og en cytotoksisk model drevet af intraokulære CoCl2-injektioner . Ved at fremhæve deres fordele og ulemper beskriver vi her denne serie protokoller, der genererer forskellige degenerative tilstande og tillader undersøgelse af retinal regenerering i Xenopus.
Introduction
Millioner af mennesker verden over er ramt af forskellige retinale degenerative sygdomme, der fører til blindhed, såsom retinitis pigmentosa, diabetisk retinopati eller aldersrelateret makuladegeneration (AMD). Hidtil er disse forhold stort set uhelbredelige. Nuværende terapeutiske tilgange under evaluering omfatter genterapi, celle- eller vævstransplantationer, neuroprotektive behandlinger, optogenetik og proteser. En anden ny strategi er baseret på selvregenerering gennem aktivering af endogene celler med stamcellepotentiale. Müller gliaceller, den vigtigste gliacelletype i nethinden, er blandt cellulære kilder af interesse i denne sammenhæng. Ved skade kan de dedifferentiere, proliferere og generere neuroner 1,2,3. Selvom denne proces er meget effektiv hos zebrafisk eller Xenopus, er den stort set ineffektiv hos pattedyr.
Ikke desto mindre har det vist sig, at passende behandlinger med mitogene proteiner eller overekspression af forskellige faktorer kan inducere pattedyrs Müller glia-cellecyklus genindtræden og i nogle tilfælde udløse deres efterfølgende neurogeneseforpligtelse 1,2,3,4,5. Dette er dog stort set utilstrækkeligt til behandlinger. Derfor er det nødvendigt at øge vores viden om de molekylære mekanismer, der ligger til grund for regenerering, for at identificere molekyler, der effektivt kan omdanne Müllers stamlignende celleegenskaber til nye cellulære terapeutiske strategier.
Med dette mål udviklede vi flere skadeparadigmer i Xenopus , der udløser retinal celledegeneration. Her præsenterer vi (1) en mekanisk nethindeskade, der ikke er celletypespecifik, (2) en betinget og reversibel celleablationsmodel ved hjælp af NTR-MTZ-systemet, der er målrettet mod stangceller, (3) en CRISPR / Cas9-medieret rhodopsin-knockout , en model af retinitis pigmentosa, der udløser progressiv stangcelledegeneration, og (4) en CoCl2-induceret cytotoksisk model, der ifølge dosis specifikt kan målrette kegler eller føre til bredere retinal celledegeneration. Vi fremhæver de særlige forhold, fordele og ulemper ved hvert paradigme.
Protocol
Representative Results
Discussion
Fordele og ulemper ved forskellige retinale skadeparadigmer i Xenopus haletudser
Mekanisk retinal skade
Forskellige kirurgiske skader på nethinden er blevet udviklet i Xenopus haletudser. Den neurale nethinde kan enten fjernes helt15,16 eller kun delvist udskæres16,17. Den mekaniske skade, der præsenteres her, involverer…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af bevillinger til MP fra Association Retina France, Fondation de France, FMR (Fondation Maladies Rares), BBS (Association du syndrome de Bardet-Biedl) og UNADEV (Union Nationale des Aveugles et Déficients Visuels) i partnerskab med ITMO NNP (Institut Thématique Multi-Organisme Neurosciences, sciences cognitives, neurologie, psychiatrie) / AVIESAN (Alliance Nationale pour les sciences de la vie et de la santé).
Materials
| 1,2-Propanediol (propylène glycol) | Sigma-Aldrich | 398039 | |
| Absolute ethanol ≥99.8% | VWR chemicals | 20821-365 | |
| Anti-Cleaved Caspase 3 antibody (rabbit) | Cell signaling | 9661S | Dilution 1/300 |
| Anti-GFP antibody (chicken) | Aveslabs | GFP-1020 | Dilution 1/500 |
| Anti-M-Opsin antibody (rabbit) | Sigma-Aldrich | AB5405 | Dilution 1/500 |
| Anti-mouse secondary antibody, Alexa Fluor 594 (goat) | Invitrogen Thermo Scientific | A11005 | Dilution 1/1,000 |
| Anti-Otx2 antibody (rabbit) | Abcam | Ab183951 | Dilution 1/100 |
| Anti-rabbit secondary antibody, Alexa Fluor 488 (goat) | Invitrogen Thermo Scientific | A11008 | Dilution 1/1,000 |
| Anti-rabbit secondary antibody, Alexa Fluor 594 (goat) | Invitrogen Thermo Scientific | A11012 | Dilution 1/1,000 |
| Anti-Recoverin antibody (rabbit) | Sigma-Aldrich | AB5585 | Dilution 1/500 |
| Anti-Rhodopsin antibody (mouse) | Sigma-Aldrich | MABN15 | Dilution 1/1,000 |
| Anti-S-Opsin antibody (rabbit) | Sigma-Aldrich | AB5407 | Dilution 1/500 |
| Apoptotis detection kit (Dead end fluorimetric TUNEL system) | Promega | G3250 | |
| Benzocaine | Sigma-Aldrich | E1501 | Stock solution 10% |
| bisBenzimide H 33258 (Hoechst) | Sigma-Aldrich | B2883 | Stock solution 10 mg/mL |
| Butanol-1 ≥99.5% | VWR chemicals | 20810.298 | |
| Calcium chloride dihydrate (CaCl2, 2H2O) | Sigma-Aldrich (Supelco) | 1.02382 | Use at 0.1 M |
| Cas9 (EnGen Spy Cas9 NLS) | New England Biolabs | M0646T | |
| Clark Capillary Glass model GC100TF-10 | Warner Instruments (Harvard Apparatus) | 30-0038 | |
| Cobalt(II) chloride hexahydrate (CoCl2, 6H2O) | Sigma-Aldrich | C8661 | Stock solution 100 mM |
| Coverslip 24 x 60 mm | VWR | 631-1575 | |
| Dako REAL ab diluent | Agilent | S202230-2 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
| Electronic Rotary Microtome | Thermo Scientific | Microm HM 340E | |
| Eosin 1% aqueous | RAL Diagnostics | 312740 | |
| Fluorescein lysine dextran | Invitrogen Thermo Scientific | D1822 | |
| Fluorescent stereomicroscope | Olympus | SZX 200 | |
| Gentamycin | Euromedex | EU0410-B | |
| Glycerin albumin acc. Mallory | Diapath | E0012 | Use at 3% in water |
| Hematoxylin (Mayer's Hemalun) | RAL Diagnostics | 320550 | |
| HEPES potassium salt | Sigma-Aldrich | H0527 | |
| Human chorionic gonadotropin hormone | MSD Animal Health | Chorulon 1500 | |
| Hydrochloric acid fuming, 37% (HCl) | Sigma-Aldrich (SAFC) | 1.00314 | |
| L-Cysteine hydrochloride monohydrate | Sigma-Aldrich | C7880 | Use at 2% in 0.1x MBS (pH 7.8 – 8.0) |
| Magnesium Sulfate Heptahydrate (MgSO4, 7H2O) | Sigma-Aldrich (Supelco) | 1.05886 | |
| Metronidazole | Sigma-Aldrich (Supelco) | M3761 | Use at 10 mM |
| Microloader tips | Eppendorf | 5242956003 | |
| Micropipette puller (P-97 Flaming/Brown) | Sutter Instrument Co. | Model P-97 | Program : Heat 700 / Pull 100 / Vel 75 / Time 90 / Unlocked p = 500 |
| Mounting medium to preserve fluorescence, FluorSave Reagent | Millipore | 345789 | |
| Mounting medium, Eukitt | Chem-Lab | CL04.0503.0500 | |
| MX35 Ultra Microtome blade | Epredia | 3053835 | |
| Needle Agani 25 G x 5/8'' | Terumo | AN*2516R1 | |
| Nickel Plated Pin Holder | Fine Science Tools | 26016-12 | |
| Nylon filtration tissue (sifting fabric) NITEX, mesh opening 1,000 µm | Sefar | 06-1000/44 | |
| Paraffin histowax without DMSO | Histolab | 00403 | |
| Paraformaldehyde solution (32%) | Electron Microscopy Sciences | EM-15714-S | Use at 4% in 1x PBS pH 7.4 |
| Peel-A-Way Disposable Embedding Molds | Epredia | 2219 | |
| Pestle | VWR | 431-0094 | |
| Petri Dish 100 mm | Corning Gosselin | SB93-101 | |
| Petri Dish 55 mm | Corning Gosselin | BP53-06 | |
| Phosphate Buffer Saline Solution (PBS) 10x | Euromedex | ET330-A | |
| PicoSpritzer Microinjection system | Parker Instrumentation Products | PicoSpritzer III | |
| Pins | Fine Science Tools | 26002-20 | |
| Polysucrose (Ficoll PM 400 ) | Sigma-Aldrich | F4375 | Use at 3% in 0.1x MBS |
| Potassium chloride (KCl) | Sigma-Aldrich | P3911 | |
| Powdered fry food : sera Micron Nature | sera | 45475 (00720) | |
| Scissors dissection | Fine Science Tools | 14090-09 | |
| Slide Superfrost | KNITTEL Glass | VS11171076FKA | |
| Slide warmer | Kunz instruments | HP-3 | |
| Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | |
| Sodium citrate trisodium salt dihydrate (C6H5Na3O7, 2H2O) | VWR chemicals | 27833.294 | |
| Sodium hydrogen carbonate (NaHCO3) | Sigma-Aldrich (Supelco) | 1.06329 | |
| Sodium hydroxide 30% aqueous solution (NaOH) | VWR chemicals | 28217-292 | |
| Stereomicroscope | Zeiss | Stemi 2000 | |
| Syringes Omnifix-F Solo Single-use Syringes 1 mL | B-BRAUN | 9161406V | |
| trans-activating crRNA (tracrRNA) | Integrated DNA Technologies | 1072533 | |
| Triton X-100 | Sigma-Aldrich | X-100 | |
| Tween-20 | Sigma-Aldrich | P9416 | |
| X-Cite 200DC Fluorescence Illuminator | X-Cite | 200DC | |
| Xylene ≥98.5% | VWR chemicals | 28975-325 |
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