Ultrastructural, Functional, and Transcriptional changes during AGE–induced aging to the RPE–Bruch’s membrane–choriocapillaris in the D–galactose treated mouse

Abstract

Abstract: : Purpose: To determine the ultrastructural, functional, and gene expression changes induced by advanced glycation endproduct (AGE) formation in the D–galactose (D–gal) aging mouse model. Methods: 5 mo Female C57Bl6 mice (n=8 per group) were given daily 50 mg/kg D–gal or PBS SQ injections for 8 wks. Electroretinography (ERG) was performed pre–treatment, 4 and 8 wks during treatment, and 1 and 3 mo post–treatment before animals were sacrificed. Eyes were fixed in 2.5% glutaraldehyde and prepared for electron microscopy, or cryopreserved, or the lens and RPE/choroid were immediately dissected. Samples were acid hydrolyzed and AGE fluorescence was assessed at ex=370/em=440, and ex=330/em=390. Total RNA from RPE/choroid was T7 amplified and analyzed using Affymetrix mouse U430A chip (n=3, each time point). Data were analyzed with DMT v3.0. Results: AGE fluorescence was increased in lens and RPE/choroid in D–gal treated animals compared to PBS controls at all time points (p<0.006). There was no significant difference between D–gal and PBS treated groups with regard to a– or b–wave amplitude and implicit time at any time point. Ultrastructural aging changes to D–gal treated eyes were progressive even after the treatment period which included: loss of RPE basolateral infoldings and cytoplasmic vacuole formation, disorganization of Bruch’s membrane with sub–RPE deposits, outer collagenous layer deposits, and choriocapillaris basement membrane duplication. Choriocapillaris endothelial fenestration loss was seen adjacent to large outer collagenous layer deposits. Microarray analysis of RPE/choroid showed differentially expressed genes related to cell structure (n=17), inflammation (n=14), and lipid metabolism (n=14) at mid–treatment; cell structure (n=12), extracellular matrix (n=12), and lipid metabolism (n=11) immediately after treatment; cell structure (n=11) and lipid metabolism (n=5) at 1 month post treatment; and cell structure (n=11), lipid metabolism (n=4) at 3 months post treatment. Conclusions: Ultrastructural aging developed in D–gal treated animals that show evidence of ocular AGE formation. Gene expression profiling suggests an early inflammatory response during treatment followed by altered expression of genes related to cell structure, extracellular matrix expansion, and lipid metabolism. These gene expression changes could contribute to AGE–related aging to the RPE–Bruch’s–choriocapillaris complex.

Keywords: aging • gene microarray • retinal pigment epithelium