Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age‐related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H₂O₂) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H₂O₂. The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE‐19 cells from apoptosis through downregulation of caspase‐3 protein activation which was controlled by the upstream proteins Bcl‐2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE‐19 cells by promoting the expression of p‐Akt.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

BMI and retinal vascular caliber in children

Objective: In adult populations, changes in retinal vascular caliber have been linked with obesity and metabolic syndrome. We examined the association of BMI and weight with retinal vascular caliber in children. Research Methods and Procedures: This was a school‐based, cross‐sectional study of 768 children, 7 to 9 years old, randomly sampled from the Singapore Cohort Study of the Risk Factors for Myopia. Participants had digital retinal photographs. Retinal vascular caliber was measured using a computer‐based program and combined to provide average calibers of arterioles and venules in that eye. Weight and height were measured using standardized protocol. These data were used to calculate BMI. Results: In this population, the mean retinal arteriolar and venular calibers were 156.40 μm [95% confidence interval (CI), 155.44 to 157.36] and 225.43 μm (95% CI, 224.10 to 226.74) respectively. After controlling for age, gender, race, parental monthly income, axial length, birth weight, and birth length, each 3.1 kg/m² (standard deviation) increase in BMI was associated with a 2.55‐μm (95% CI, 1.21 to 3.89; p < 0.001) larger retinal venular caliber. In multivariable analysis, greater weight was also significantly associated with larger retinal venular caliber. BMI and weight were not associated with retinal arteriolar caliber. Height was not significantly associated with retinal arteriolar or venular caliber. Discussion: Greater BMI and weight are associated with larger retinal venular caliber in healthy children.     

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.