基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析
Summary
描述了成年斑马鱼的机械脑损伤模型，以研究调节其高再生能力的分子机制。该方法解释了在多种小鱼的视顶上造成刺伤，以使用荧光免疫染色评估再生反应。
Abstract
虽然斑马鱼具有卓越的中枢神经系统（CNS）再生能力，但青鳉鱼具有较低的CNS再生能力。在斑马鱼和青鳉鱼的成年视顶中建立了脑损伤模型，并进行了比较组织学和分子分析，以阐明调节这些鱼类中该组织高再生能力的分子机制。这里提出了成人视顶的刺伤损伤模型，使用针头和组织学分析来分析神经干细胞（NSC）的增殖和分化。手动将针插入视顶的中心区域，然后对鱼进行心内灌注，并解剖它们的大脑。然后对这些组织进行冷冻切片，并使用针对适当的NSC增殖和分化标志物的免疫染色进行评估。这种顶盖损伤模型在斑马鱼和青鳉鱼中提供了可靠且可重复的结果，可以比较受伤后的NSC反应。这种方法可用于小型硬骨鱼，包括斑马鱼、青鳉鱼和非洲鳉鱼，使我们能够比较它们的再生能力并研究独特的分子机制。
Introduction
与其他哺乳动物相比，斑马鱼（Danio rerio）具有更高的中枢神经系统（CNS）再生能力1，2，3。最近，为了更好地了解这种再生能力增加的分子机制，已经使用下一代测序技术对组织再生进行了比较分析4，5，6。斑马鱼和四足动物的大脑结构完全不同7，8，9。这意味着已经开发了几种使用具有相似大脑结构和生物学特征的小鱼的脑损伤模型，以促进对有助于增加再生能力的潜在分子机制的研究。
此外，青鳉鱼（Oryzias latipes）是一种受欢迎的实验动物，与斑马鱼相比，心脏和神经元再生能力低10，11，12，13。斑马鱼和青鳉鱼具有相似的大脑结构和成体神经干细胞（NSC）的生态位14，15，16，17。在斑马鱼和青鳉鱼中，视顶包括两种类型的NSC，神经上皮样干细胞和径向神经胶质细胞（RGC）15，18。先前开发了成年斑马鱼视顶的刺伤损伤，该模型用于研究调节这些动物脑再生的分子机制19，20，21，22，23。这种年轻成年斑马鱼刺伤损伤模型诱导了RGC19，24，25的再生神经发生。这种视顶刺伤是一种稳健且可重复的方法13，19，20，21，22，23，24，25。当相同的损伤模型应用于成年青鳉鱼时，通过对损伤后RGC增殖和分化的比较分析，揭示了青鳉鱼视顶RGCs的低神经源性能力13。
木乃伊模型26 中也开发了视盖中的刺伤损伤模型，但与端脑损伤27 相比，顶盖损伤的细节尚未得到充分记录。使用斑马鱼和青鳉鱼在视顶的刺伤损伤可以研究具有不同再生能力的物种之间的差异细胞反应和基因表达。该协议描述了如何使用注射针在视顶进行刺伤。这种方法可以应用于斑马鱼和青鳉鱼等小鱼。本文介绍了使用荧光免疫组织化学和冷冻切片进行组织学分析以及细胞增殖和分化分析的样品制备过程。
Protocol
所有实验方案均由国家先进工业科学技术研究所的机构动物护理和使用委员会批准。斑马鱼和青鳉鱼按照标准程序进行饲养28. 1.成人视顶刺伤 准备0.4%（w / v）三卡因储备溶液进行麻醉。对于 100 mL 储备溶液，将 400 mg 甲磺酸三卡因（参见 材料表）溶解在 90 mL 蒸馏水中，并使用 1 M Tris HCl 缓冲液 （pH 9.0） 将 pH 值调节至 7.0。调…
Representative Results
使用针插入右半球的视顶刺伤（图1，图 4A和 图5A）诱导各种细胞反应，包括径向神经胶质细胞（RGC）增殖和新生神经元的产生。同样，斑马鱼和青鳉鱼的老年种群被用来抵消再生反应中的任何衰老影响。然后对冷冻切片进行荧光免疫染色，分析斑马鱼和青鳉鱼顶盖损伤后的RGC增殖和分化（图4-5?…
Discussion
这里描述了一组方法，可用于利用针头诱导视盖中的刺伤，以促进脑损伤后RGC增殖和分化的评估。针头介导的刺伤是一种简单、高效实施的方法，可以使用一套标准工具应用于许多实验样品。斑马鱼大脑几个区域的刺伤损伤模型已经开发出来3，19，29。视顶是大脑中最大的部分之一，易于操作。此外，与端脑相比，视顶中的大?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了JSPS KAKENHI授权号18K14824和21K15195以及日本AIST的内部资助的支持。
Materials
| 10 mL syringe | TERUMO | SS-10ESZ | |
| 1M Tris-HCl (pH 9.0) | NIPPON GENE | 314-90381 | |
| 30 G needle | Dentronics | HS-2739A | |
| 4% Paraformaldehyde Phosphate Buffer Solution | Wako | 163-20145 | |
| Aluminum block | 115 x 80 x 37 mm (W x D x H) is enough size to freeze 6 cryomolds | ||
| Anti-BLBP | Millipore | ABN14 | 1:500 |
| Anti-BrdU | Abcam | ab1893 | 1:500 |
| Anti-HuC | Invitrogen | A21271 | 1:100 |
| Anti-PCNA | Santa Cruz Biotechnology | sc-56 | 1:200 |
| Brmodeoxyuridine | Wako | 023-15563 | |
| Confocal microscope C1 plus | Nikon | ||
| Cryomold | Sakura Finetek Japan | 4565 | 10 x 10 x 5 mm (W x D x H) |
| Cryostat | Leica | CM1960 | |
| Danio rerio WT strains RW | |||
| Extension tube | TERUMO | SF-ET3520 | |
| Fluoromount (TM) Aqueous Mounting Medium, for use with fluorescent dye-stained tissues | SIGMA-ALDRICH | F4680-25ML | |
| Forceps | DUMONT | 11252-20 | |
| Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | Invitrogen | A32723 | |
| Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 | Invitrogen | A11035 | |
| Hoechst 33342 solution | Dojindo | 23491-52-3 | |
| Hydrochloric Acid | Wako | 080-01066 | |
| Incubation Chamber for 10 slides Dark Orange | COSMO BIO CO., LTD. | 10DO | |
| MAS coat sliding glass | Matsunami glass | MAS-01 | |
| Micro cover glass | Matsunami glass | C024451 | |
| Microscopy | Nikon | SMZ745T | |
| Normal horse serum blocking solution | VECTOR LABRATORIES | S-2000-20 | |
| O.C.T Compound | Sakura Finetek Japan | 83-1824 | |
| Oryzias latipes WT strains Cab | |||
| PAP Pen Super-Liquid Blocker | DAIDO SANGYO | PAP-S | |
| Phosphate Buffered Saline (PBS) Tablets, pH 7.4 | TaKaRa | T9181 | |
| Styrofoam tray | 100 x 100 x 10 mm (W x D x H) styrofoam sheet is available as tray | ||
| Sucrose | Wako | 196-00015 | 30 % (w/v) Sucrose in PBS |
| Tricaine (MS-222) | nacarai tesque | 14805-24 | |
| Trisodium Citrate Dihydrate | Wako | 191-01785 | |
| Triton X-100 | Wako | 04605-250 |
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Citazione di questo articolo
Shimizu, Y., Kawasaki, T. Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity. J. Vis. Exp. (180), e63166, doi:10.3791/63166 (2022).