Benefits of genetic and immunohistochemical markers in understanding abnormalities in aging retina

Abstract

The aim of the study was to better understand the interplay between genetic factors and the aging process in the human retina through mapping complement factor H (CFH) and related proteins. Two human eyes, from 92-and 64-year-old donors, were genotyped for the expression of CFH-related 1 (CFHR1) and CFH-related 3 (CFHR3) genes. Deoxyribonucleic acid (DNA) was extracted and analyzed for concentration and purity with a spectrophotometer, at 260 nm. The results showed a DNA concentration of 469.17 ng/μL in the aged retina and of 399.20 ng/μL in the younger one. Through polymerase chain reaction (PCR) genotyping, the DNA CFHR1 and CFHR3 were visible as bands of 175 bp and 181 bp. Immunohistochemistry by immunofluorescence method was used with a panel of specific antibodies for CFH, CFHR1, CFHR3 and GFAP, a marker for Müller cells. All the samples were examined, and images captured using confocal microscopy. In the younger retina, CFH was localized in the inner plexiform layer and below the outer nuclear layer, while in the aged retina, it was found in the photoreceptors. CFH was also detected in the choriocapillaris and within the end-feet of the Müller cells. Our controls showed autofluorescence of the retinal pigment epithelium shedding light on a false positive CFH immunostaining of this layer. GFAP immunoreactivity highlighted an increased gliosis within the aged retina. CFHR3 signal was found in the microglia, while CFHR1 was detected in the choriocapillaris. In summary, underpinning the expression of these components can show the potential involvement of these modulators in implementing new treatment strategies.