Kustermann Stefan, Schmid Susanne, Biehlmaier Oliver, Kohler Konrad
Division of Experimental Ophthalmology, Centre for Ophthalmology, Roentgenweg 11, 72076 Tübingen, Germany.
Cell Tissue Res. 2008 May;332(2):195-209. doi: 10.1007/s00441-008-0589-5. Epub 2008 Mar 12.
Over the last 20 years, the zebrafish has become an important model organism for research on retinal function and development. Many retinal diseases do not become apparent until the later stages of life. This means that it is important to be able to analyze (gene) function in the mature retina. To meet this need, we have established an organotypic culture system of mature wild-type zebrafish retinas in order to observe changes in retinal morphology. Furthermore, cell survival during culture has been monitored by determining apoptosis in the tissue. The viability and excitability of ganglion cells have been tested at various time points in vitro by patch-clamp recordings, and retinal functionality has been assessed by measuring light-triggered potentials at the ganglion cell site. Since neurogenesis is persistent in adult zebrafish retinas, we have also monitored proliferating cells during culture by tracking their bromodeoxyuridine uptake. Reverse genetic approaches for probing the function of adult zebrafish retinas are not yet available. We have therefore established a rapid and convenient protocol for delivering plasmid DNA or oligonucleotides by electroporation to the retinal tissue in vitro. The organotypic culture of adult zebrafish retinas presented here provides a reproducible and convenient method for investigating the function of drugs and genes in the retina under well-defined conditions in vitro.
在过去20年里，斑马鱼已成为视网膜功能与发育研究的重要模式生物。许多视网膜疾病直到生命后期才会显现。这意味着能够在成熟视网膜中分析（基因）功能很重要。为满足这一需求，我们建立了成熟野生型斑马鱼视网膜的器官型培养系统，以观察视网膜形态的变化。此外，通过测定组织中的凋亡情况来监测培养过程中的细胞存活。通过膜片钳记录在体外不同时间点测试神经节细胞的活力和兴奋性，并通过测量神经节细胞部位的光触发电位来评估视网膜功能。由于成年斑马鱼视网膜中的神经发生持续存在，我们还通过追踪它们对溴脱氧尿苷的摄取来监测培养过程中的增殖细胞。目前尚无用于探究成年斑马鱼视网膜功能的反向遗传学方法。因此，我们建立了一种快速便捷的方案，通过电穿孔将质粒DNA或寡核苷酸导入体外视网膜组织。本文介绍的成年斑马鱼视网膜器官型培养提供了一种可重复且便捷的方法，用于在体外明确的条件下研究药物和基因在视网膜中的功能。
