Form deprivation modulates retinal neurogenesis in primate experimental myopia. Macaca mulatta

Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to the abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. By combining analysis of gene expression, injection of the thymidine analog 5-bromo-2'-deoxyuridine and immunocytochemistry, we show that the retinal periphery in both juvenile rhesus macaques and green monkeys harbors a population of mitotically active neuroprogenitor cells that proliferate when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with the axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. Keywords: disease state analysis Overall design: cDNA subtractions, construction of cDNA libraries and differential screening were performed as previously described (Tkatchenko et al, 2000). Two subtractions were carried out resulting in two cDNA libraries. One library was enriched in clones potentially up-regulated in the retina of the closed eye and the other was enriched in cDNAs potentially down-regulated. One thousand clones from each library were analyzed, and 535 differentially-expressed cDNAs were amplified by PCR and spotted onto glass slides coated with poly-L-lysine (five duplicates for each cDNA). The slides were then processed, hybridized with Cy3- and Cy5-labeled complex cDNA probes and washed in 0.2xSSC as described at http://cmgm.stanford.edu/pbrown/protocols/. Two hybridizations with color swap were carried for each monkey. After hybridization, slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA). The images were acquired and processed using the GenePix Pro 5.1 software package (Axon Instruments). The data obtained were normalized against GAPDH expression level and mean values (the reported values) and P-values (probability associated with a Student's t-test) were calculated for duplicate experiments (n = 10) with color swap using Microsoft Excel. Genes were defined as differentially regulated if P< 0.05. Cluster analysis was performed using the Genesis software package (provided by Alexander Sturn, Graz University of Technology, Austria).

幼年灵长类动物在眼睑融合导致视觉体验受损时会发生近视（myopia）。面对异常视觉输入，视网膜神经网络会引发眼球赤道后节段过度生长，但该眼轴延长的潜在机制尚不明确。本研究结合基因表达分析、胸苷类似物5-溴-2'-脱氧尿苷（5-bromo-2'-deoxyuridine）注射以及免疫细胞化学实验，证实幼年恒河猴与绿猴的视网膜周边区域存在一群有丝分裂活性神经祖细胞（mitotically active neuroprogenitor cells），当视觉体验因眼睑融合发生改变时，这群细胞会发生增殖。此外，分裂细胞的数量与眼轴延长及由此产生的近视屈光不正高度相关。由此可见，灵长类动物出生后眼球发育过程中，视网膜会发生活跃的生长。这种生长受视觉输入调控，在眼球发生轴性近视时会显著加速。
关键词：疾病状态分析（disease state analysis）
整体实验设计：
如前文所述（Tkatchenko等，2000），我们开展了cDNA消减杂交、cDNA文库构建以及差异筛选实验。本实验共完成两次消减杂交，构建了两个cDNA文库：其中一个文库富集了可能在闭合眼睑侧视网膜中上调的克隆，另一个则富集了可能下调的cDNA。我们对每个文库的1000个克隆进行了分析，通过PCR扩增得到535个差异表达cDNA，并将其点涂于包被多聚-L-赖氨酸（poly-L-lysine）的玻片上（每个cDNA设置5个重复孔）。
随后按照http://cmgm.stanford.edu/pbrown/protocols/ 所述方法处理玻片，用Cy3与Cy5标记的复合cDNA探针进行杂交，并在0.2×SSC溶液中洗涤。每只猴子均进行两次交换荧光标记的杂交实验。
杂交完成后，使用GenePix 4000B微阵列扫描仪（Axon Instruments，联合市，加利福尼亚州）扫描玻片图像，通过GenePix Pro 5.1软件包（Axon Instruments）进行图像采集与处理。
实验数据以甘油醛-3-磷酸脱氢酶（GAPDH）的表达水平作为参照进行标准化，针对带荧光标记交换的重复实验（n=10），使用Microsoft Excel计算平均值（即报告值）与P值（Student t检验对应的概率）。当P<0.05时，将基因定义为差异表达调控基因。
聚类分析使用Genesis软件包完成（由奥地利格拉茨科技大学Alexander Sturn提供）。