Differential gene expression in Danio rerio during optic nerve regeneration. Danio rerio

To gain a better understanding of the factors necessary for successful CNS regeneration, a temporal analysis of the changes in gene expression in the eye caused by optic nerve injury was conducted. Dual color oligonucleotide microarrays were used to compare total RNA harvested from the eyes of sham-operated and optic nerve-injured fish at 3, 24, and 168 hours following surgery. Optic nerve injured fish are compared to sham-operated fish in order to eliminate gene expression due to non-neuronal damage and inflammatory response. Statistical analyses identified 131 genes with a 2.0-fold or greater difference in expression. Overall design: Wild type zebrafish were obtained from a local pet store. Optic nerve injury was conducted using a severing model accomplished as follows. Zebrafish were anesthetized in 0.2% MS-222 dissolved in tank water. The muscles surrounding the eye were cut and the eye angled rostrally to expose the nerve. The optic nerve was then severed using microsissors without damaging the ophthalmic artery. In sham operated fish the muscles surrounding the eye were severed but the nerve was not damaged. RNA was extracted from the eye at three time points following surgery 3 hours, 24 hours, and 168 hours. RNA was pooled from multiple fish to achieve 10 ug total RNA. Samples were collected in triplicate per time point. Gene expression was analyzed on a dual color oligonucleotide array where the optic nerve injured fish were compared to sham-operated fish. Four samples of RNA were also collected from control fish and compared to each other on the microarray to confirm that processing did not create expression differences.

为深入解析中枢神经系统（Central Nervous System，CNS）成功再生的关键调控要素，本研究针对视神经（optic nerve）损伤引发的眼部基因表达变化开展了时序分析。本研究采用双色寡核苷酸微阵列（dual color oligonucleotide microarrays），对术后3、24及168小时时，假手术组与视神经损伤组斑马鱼眼部提取的总RNA进行比对分析。通过将视神经损伤组与假手术组进行比对，可排除非神经元损伤及炎症反应所导致的基因表达变化。经统计分析，共鉴定出131个表达差异倍数达2.0倍及以上的基因。
整体实验设计如下：野生型斑马鱼购自当地宠物店。视神经损伤采用切断模型，具体操作流程如下：将斑马鱼置于溶于饲养水的0.2% MS-222溶液中进行麻醉。剪断眼部周围肌肉，将眼球向吻侧牵拉以暴露视神经，随后使用显微剪切断视神经，且确保不损伤眼动脉（ophthalmic artery）。假手术组斑马鱼仅剪断眼部周围肌肉，不对视神经造成损伤。
分别在术后3、24及168小时三个时间点从斑马鱼眼部提取RNA。将多条斑马鱼的RNA混合，以获得总量为10 μg的总RNA。每个时间点设置三次生物学重复样本。采用双色寡核苷酸微阵列分析基因表达，将视神经损伤组与假手术组进行比对。此外，还采集了4份对照组斑马鱼的RNA样本，将其相互比对以确认实验处理过程未引入人为表达差异。