Kenneth C Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97239, USA.
Invest Ophthalmol Vis Sci. 2010 Jun;51(6):3003-18. doi: 10.1167/iovs.09-4663. Epub 2010 Jan 13.
To determine and compare gene expression patterns in the whole retina and retinal ganglion cell layer (RGCL) in a rodent glaucoma model.
IOP was unilaterally elevated in Brown Norway rats (N = 26) by injection of hypertonic saline and monitored for 5 weeks. A cDNA microarray was used on whole retinas from one group of eyes with extensive optic nerve injury and on RGCL isolated by laser capture microdissection (LCM) from another group with comparable injury, to determine the significantly up- or downregulated genes and gene categories in both groups. Expression changes of selected genes were examined by quantitative reverse transcription-PCR (qPCR) to verify microarray results.
Microarray analysis of the whole retina identified 632 genes with significantly changed expression (335 up, 297 down), associated with 9 upregulated and 3 downregulated biological processes. In contrast, the RGCL microarray yielded 3726 genes with significantly changed expression (2003 up, 1723 down), including 60% of those found in whole retina. Thirteen distinct upregulated biological processes were identified in the RGCL, dominated by protein synthesis. Among 11 downregulated processes, axon extension and dendrite morphogenesis and generation of precursor metabolism and energy were uniquely identified in the RGCL. qPCR confirmed significant changes in 6 selected messages in whole retina and 11 in RGCL. Increased Atf3, the most upregulated gene in the RGCL, was confirmed by immunohistochemistry of RGCs.
Isolation of RGCL by LCM allows a more refined detection of gene response to elevated pressure and improves the potential of determining cellular mechanisms in RGCs and their supporting cells that could be targets for enhancing RGC survival.
在啮齿动物青光眼模型中确定并比较全视网膜和视网膜神经节细胞层（RGCL）中的基因表达模式。
通过向棕褐色挪威大鼠（N = 26）的眼内注射高渗盐水来单侧升高眼内压，并在 5 周内进行监测。使用 cDNA 微阵列对一组具有广泛视神经损伤的眼睛的整个视网膜以及另一组通过激光捕获显微切割（LCM）分离的 RGCL 进行分析，以确定两组中显著上调或下调的基因和基因类别。通过定量逆转录-PCR（qPCR）检查选定基因的表达变化，以验证微阵列结果。
全视网膜微阵列分析确定了 632 个表达显著改变的基因（335 个上调，297 个下调），与 9 个上调和 3 个下调的生物学过程相关。相比之下，RGCL 微阵列产生了 3726 个表达显著改变的基因（2003 个上调，1723 个下调），其中包括在全视网膜中发现的 60％。在 RGCL 中鉴定出 13 个独特的上调生物学过程，其中以蛋白质合成为主导。在 11 个下调的过程中，轴突延伸和树突形态发生以及前体代谢和能量生成在 RGCL 中是唯一确定的。qPCR 证实了在全视网膜中有 6 个选定的消息和在 RGCL 中有 11 个消息的显著变化。在 RGCL 中最上调的基因 Atf3 通过 RGC 的免疫组织化学得到证实。
通过 LCM 分离 RGCL 可以更精细地检测升高的压力对基因的反应，并提高确定 RGC 和其支持细胞的细胞机制的潜力，这些机制可能成为增强 RGC 存活的靶标。
