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Summary
Abstract
Introduction
Protocol
Resultados
Discussion
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Neurociencias

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Published: October 24, 2013
doi:
10.3791/51017
,
1Department of Anatomy and Cell Biology,Wayne State University School of Medicine, 2Department of Opthalmology,Wayne State University School of Medicine

Summary

多个光损伤协议已损坏感光细胞，从而诱发成年斑马鱼视网膜的再生反应。此协议描述了一种改进的方法，可以用在着色的动物，并在整个视网膜杆和视锥细胞，损害绝大多数。

Abstract

光诱导的视网膜变性（LIRD）是常用的啮齿动物损伤杆和视锥细胞和斑马鱼。在成年斑马鱼，光感受器变性触发穆勒胶质细胞重新进入细胞周期和产生瞬态放大祖​​。这些前体细胞继续增殖，他们迁移到受损区域，他们最终产生新感光。目前，有两个广泛使用LIRD的范例，其中每个感光损失和相应的差异在再生反应在不同程度的结果。随着遗传学和药理学工具可用于测试单个基因在再生过程中的利益的作用，有必要制定一个强大的LIRD范式。这里我们描述一个LIRD协议，导致在杆和锥感光体，其中结合使用的两个以前建立LIRD技术的广泛和一致的损失。此外这个协议可以延长使用色素的动物，从而消除了需要保持白化背景LIRD研究的转基因株系的利息。

Introduction

光诱导的视网膜变性（LIRD）是常用的啮齿动物损伤杆和视锥细胞和斑马鱼。在成年斑马鱼，光感受器变性触发穆勒胶质细胞重新进入细胞周期和产生瞬态放大祖​​。这些祖细胞继续增殖，因为他们迁移到受损区域，在那里他们最终产生新的光感受器。目前，有两个广泛使用LIRD的范例，其中每个感光损失和相应的差异在再生反应在不同程度的结果。随着遗传学和药理学工具可用于测试单个基因在再生过程中的利益的作用，有必要制定一个强大的LIRD范式。这里我们描述一个LIRD协议，导致在杆和锥感光体，其中结合使用的两个以前建立LIRD技术的广泛和一致的损失。此外这个协议可以延长使用色素的动物，从而消除了需要保持白化背景LIRD研究的转基因株系的利息。

Protocol

在韦恩州立大学医学院动物使用委员会批准，在本协议中所述的所有程序。 1。暗适应转让〜10成年白化或色素的鱼，从正常的住房制度进入一个黑暗的外壳。如果可用，建成斑马鱼的正常水流通过水箱外壳模块，它允许使用一个黑暗的外壳。 （如果这样的系统不可用，放置鱼缸在一个完全黑暗的外壳，确保通气鱼与氧气）。 保持10天的鱼在黑暗中。当?…

Representative Results

此前所述的光治疗方案相比，每一个人的LIRD方法。在暗处理成年的白化动物（ 图3-5），个人光疗法造成重大损失棒（ 图3）和锥感光器（ 图4）。然而，无论是单独的治疗方法主要损坏感光体在背侧视网膜一半，留下相对保护的光的治疗方法（ 图3和4）的腹侧视网膜。此外，相比与卤素灯处理，UV光处理损坏的视锥细胞在背视?…

Discussion

在这里，我们提供持续的强光曝晒普遍的感光损失和强大的再生反应的结果，结合短的紫外线照射。这个相结合的方法相比与个人LIRD方法，也是最有效的协议，损害双方的两部分的视网膜杆和视锥。重要的是，这种治疗是有效的颜料的动物，以及患白化病的动物。

虽然我们提供的证据表明与个别LIRD方法相比更为广泛和一致的损伤合并协议结果，科学的考虑，应该讨论…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

笔者想感谢西峡罗优良的鱼类养殖和技术支持。这项工作是由美国国立卫生赠款R21EY019401（RT）和P30EY04068（RT），和启动资金RT，包括无限制的授予从韦恩州立大学，眼科防盲研究。 JT是由托马斯·C.隆隆韦恩州立大学研究生院提供的奖学金支持。

Materials

UV light source Leica EL600
Glass Petri dish (150 x 20 mm) Sigma-Aldrich/Pyrex CLS3160152BO
250 ml glass beaker Sigma-Aldrich/Pyrex CLS1000250
4 L glass beaker Sigma-Aldrich/Pyrex CLS10004L
Aluminum foil Fisher 01-213-105
250 W halogen lamps Workforce 265-669
1.8 L clear acrylic tanks Aquaneering ZT180T
1.8 L clear acrylic tank lids Aquaneering ZT180LCL
Fan Honeywell HT-900
Aerator Tetra 77853-900
Thermometer Cole-Parmer YO-08008-58
Bent forceps (5/45) World Precision Instruments 504155

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Referencias

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Tags

Light-induced Retinal DegenerationLIRD ParadigmPhotoreceptor DegenerationMüller Glial CellsRetinal RegenerationAdult ZebrafishRod And Cone PhotoreceptorsTransient-amplifying ProgenitorsPigmented Animals

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Thomas, J. L., Thummel, R. A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish. J. Vis. Exp. (80), e51017, doi:10.3791/51017 (2013).
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