Comparison of Dextran Perfusion and GSI-B4 Isolectin Staining in a Mouse Model of Oxygen-induced Retinopathy

Purpose: Oxygen-induced retinopathy(OIR) is a robust and widely used animal model for the study of retinal neovascularization(NV). Dextran perfusion and Griffonia simplicifolia isolectin B4(GSI-B4) staining are two common methods for examining the occurrence and extent of OIR. This study provides a quantitative comparison of the two for OIR detection.Methods: At postnatal day 7(PN7), fifteen C57 BL / 6J mice were exposed to a 75% hyperoxic condition for 5 days and then returned to room air conditions. At PN17, the mice received intravitreal injection of GSI-B4 Alexa Fluor 568 conjugate. After 10 hours, they were infused with FITC-dextran conjugate via the left ventricle. Retinal flat mounts were photographed by confocal microscopy. Areas with fluorescent signals and the total retinal areas were quantified by Image J software.Results:Both GSI-B4 and dextran detected the peripheral neovascular area. The mean hyper fluorescence area was 0.33 ±0.14% of whole retinal area determined by GSI-B4 staining and 0.25±0.28% determined by dextran perfusion. The difference between the two measures was 0.08%(95% CI:-0.59%,0.43%)..The Pearson correlation coefficient between the two methods was 0.386,P =0.035..The mean coincidence rates were 14.3 ±13.4% and 24.9 ±18.5% for GSI-B4 and dextran staining, respectively.Conclusion:.Both methods can complement each other indemonstrating and quantitatively evaluating retinal NV. A poor agreement was found between the two methods;.GSI-B4 isolectin was more effective than FITC-dextran perfusion in evaluating the extent of retinal NV in a mouse model of OIR.

Expression of Sirt1 and Sirt2 in the Injured Optic Retina of Calorie-Restricted Rats

Purpose:To investigate retinal ganglion cell (RGC) survival after optic nerve injury in calorie-restricted (CR) rats,and analyze the potential role of Sirtuins.Methods:Ten-month old male Sprague-Dawley rats (n=14) were divided into calorie restricted (CR) and ad libitum (AL) groups.In the CR group (n=7),the rats were denied access to food every other day.Animals in the AL group (n=7) had had free access to food.PN-ON grafting was carried out on the right eye of all subjects after 5 months of feeding.Three weeks postoperatively,retina samples were collected,half of which were fixed in 4% paraformaldehyde (PFA) and subjected to standard immunofluorescence staining for TUJ-1.The remaining samples were subjected to total RNA analysis and RT-PCR for Sirt1 and 2 expression.Results:Comparing the amount of TUJ-1 staining between the groups,the mean density and the total number of RGCs showed no significant difference.RT-PCR results indicated that mRNA expression of Sirtuin2 in the CR group was significantly lower than that in the AL group,whereas no statistically significant difference was observed between the two groups regarding the mRNA expression of Sirt1.Conclusion:Calorie restriction had no impact on the survival of injured RGCs.The down-regulated mRNA expression of Sirt2 in the CR group may indicate an improved capacity for regeneration among these animals,but more work is needed to explore this possibility.