在 非洲爪蟾 蝌蚪中生成视网膜损伤模型
Summary
我们已经开发了几种方案来诱导 非洲爪 蟾蝌蚪的视网膜损伤或视网膜变性。这些模型为研究视网膜再生机制提供了可能性。
Abstract
视网膜神经退行性疾病是导致失明的主要原因。在正在探索的众多治疗策略中，刺激自我修复最近显得特别有吸引力。Müller神经胶质细胞是视网膜修复的一个感兴趣的细胞来源，它具有干细胞的潜力和非凡的再生能力。然而，这种潜力在哺乳动物中非常有限。在具有再生能力的动物模型中研究视网膜再生的分子机制应该有助于了解如何释放哺乳动物Müller细胞再生视网膜的潜在能力。这是再生医学治疗策略发展的关键一步。为此，我们在 非洲爪蟾中开发了几种视网膜损伤范式：机械性视网膜损伤、允许硝基还原酶介导的光感受器条件消融的转基因系、基于 CRISPR/Cas9 介导的视紫 红质 敲除的视网膜色素变性模型，以及由眼内注射 CoCl2 驱动的细胞毒性模型。为了强调它们的优点和缺点，我们在这里描述了这一系列的方案，这些方案会产生各种退行性条件，并允许研究 非洲爪蟾的视网膜再生。
Introduction
全世界有数百万人患有各种导致失明的视网膜退行性疾病，例如视网膜色素变性、糖尿病视网膜病变或年龄相关性黄斑变性 （AMD）。迄今为止，这些疾病在很大程度上仍然无法治愈。目前正在评估的治疗方法包括基因治疗、细胞或组织移植、神经保护治疗、光遗传学和假肢装置。另一种新兴策略是基于通过激活具有干细胞潜力的内源性细胞进行自我再生。Müller 神经胶质细胞是视网膜的主要神经胶质细胞类型，是在这种情况下感兴趣的细胞来源之一。受伤后，它们可以去分化、增殖并产生神经元 1,2,3。虽然这个过程在斑马鱼或非洲爪蟾中非常有效，但在哺乳动物中却在很大程度上是低效的。
尽管如此，已经表明，用有丝分裂蛋白进行适当的治疗或各种因子的过表达可以诱导哺乳动物 Müller 神经胶质细胞周期重新进入，并且在某些情况下，触发其随后的神经发生承诺 1,2,3,4,5。然而，这在很大程度上仍不足以进行治疗。因此，增加我们对再生分子机制的了解对于确定能够有效地将Müller干细胞样特性转化为新的细胞治疗策略的分子是必要的。
为此，我们在 非洲爪蟾 中开发了几种触发视网膜细胞变性的损伤范式。在这里，我们提出了 （1） 非细胞类型特异性的机械性视网膜损伤，（2） 使用靶向杆细胞的 NTR-MTZ 系统的条件性和可逆细胞消融模型，（3） CRISPR/Cas9 介导的视 紫红质 敲除，触发进行性杆细胞变性的视网膜色素变性模型， 以及 （4） CoCl2诱导的细胞毒性模型，根据剂量可以特异性靶向视锥细胞或导致更广泛的视网膜细胞变性。我们强调每种范式的特殊性、优点和缺点。
Protocol
动物护理和实验是根据机构许可A91272108根据机构准则进行的。该研究方案已获得机构动物护理委员会 CEEA #59 的批准，并获得了 Direction Départementale de la Protection des Populations 的授权，参考编号为 APAFIS #32589-2021072719047904 v4 和 APAFIS #21474-2019071210549691 v2。有关这些方案中使用的所有材料、仪器和试剂的详细信息，请参阅 材料表 。 1.机械性视网膜损伤 <p cl…
Representative Results
机械性视网膜损伤遭受协议第 1 节中描述的机械损伤的蝌蚪视网膜切片显示，视网膜病变包含组织的所有层，同时仅限于穿刺部位（图 2A，B）。 使用 NTR-MTZ 系统的条件杆细胞消融如方案第2节所述，在立体显微镜下分析经MTZ处理的麻醉Tg（rho：GFP-NTR）转基因蝌蚪的眼睛（图3A，B<str…
Discussion
非洲爪蟾蝌蚪各种视网膜损伤范式的优缺点
机械性视网膜损伤
在非洲爪蟾蝌蚪中已经发展出各种神经视网膜的手术损伤。神经视网膜可以完全切除 15,16 或仅部分切除16,17。这里介绍的机械损伤不涉及任何视网膜切除术，而是我们之前在非洲爪?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了法国视网膜协会、法国基金会、FMR（罕见病基金会）、BBS（Bardet-Biedl 综合征协会）和 UNADEV（国家 Aveugles et Déficients Visuels）的赠款，并与 ITMO NNP（多生物体神经科学、认知科学、神经学、精神病学研究所）/AVIESAN（国家生命和健康科学联盟）合作。
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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Citazione di questo articolo
Parain, K., Donval, A., Chesneau, A., Lun, J. X., Borday, C., Perron, M. Generating Retinal Injury Models in Xenopus Tadpoles. J. Vis. Exp. (200), e65771, doi:10.3791/65771 (2023).