Normal aging involves modulation of specific inflammatory markers in the rat retina and choroid

Abstract

Recent work has suggested that inflammation is a common component of a number of age-related diseases. The hypothesis of the present study was that normal aging of the retina and choroid would increase levels of inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), and tumor necrosis factor alpha (TNF-alpha). To investigate this hypothesis, gene expression and protein analyses were completed on retinal and choroidal samples from Fischer 344 x Brown Norway F1 hybrid rats at 8, 22, and 32 months of age. Aging of the choroid produced significant increases in PGE2, with decreased TNF-alpha protein. Protein levels and messenger RNA of iNOS and TNF-alpha protein levels were significantly decreased in the aging retina in contrast to PGE2 protein activity, which was increased with age in the retina. These results suggest that PGE2 is likely involved in the aging process in both the retina and choroid, whereas iNOS plays a role predominantly in the retina.

Figure 1.

Gene and protein levels of inducible nitric oxide synthase (iNOS) in the aged retina. (A) Bar graph of the steady-state messenger RNA expression determined using real-time polymerase chain reaction (PCR) for iNOS in the aging retina. (B) Representative image of the Western blots obtained for iNOS. (C) Bar graph of the mean densitometry numbers as a percentage of the 8-month values for all iNOS Western blot results. *p < .05 versus 8 months, N = 4 for both real-time PCR and Western blot analysis.

Figure 2.

Enzyme-linked immunosorbent assay (ELISA) and Western blot results for prostaglandin E2 (PGE2) and its receptor PGE2-EP2 in the aging retina. (A) Bar graph of the ELISA results for PGE2 completed on aging retinal samples expressed as a percentage of the 8-month values. (B and C) The representative image and bar graph of the Western blot analysis for the EP2 subtype of the PGE2 receptor. *p < .05, N = 5 for ELISA analysis and N = 4 for the Western blot analysis.

Figure 3.

Tumor necrosis factor alpha (TNF-α) messenger RNA (mRNA) expression and protein levels in the aged retina. (A) Bar graph of the steady-state mRNA expression for TNF-α in the aging retina. (B) Results from the enzyme-linked immunosorbent assay (ELISA) analysis. Because the same amount of protein is loaded into all wells, the results are presented as mean absorbance as compared with the 8-month values. *p < .05 versus 8 months, N = 5 for real-time polymerase chain reaction and N = 4 for ELISA analysis.

Figure 4.

Messenger RNA (mRNA) expression and protein levels of inducible nitric oxide synthase (iNOS) in the aging choroid. (A) Bar graph of the steady-state mRNA expression determined using real-time polymerase chain reaction (PCR) for iNOS in the aging choroid. (B) Representative image of the Western blots obtained for iNOS. (C) The bar graph of the mean densitometry numbers expressed as a percentage of the 8-month values for all iNOS Western blot results. N = 5 for real-time PCR and N = 4 for Western blot analysis.

Figure 5.

Protein analysis of prostaglandin E2 (PGE2) and its receptor subtype PGE2-EP2 in the aged choroid. (A) The bar graph of the enzyme-linked immunosorbent assay (ELISA) results for PGE2 completed on aging choroidal samples. Because the same amount of protein is loaded into all wells, the results are presented as mean absorbance relative to the 8-month values. (B and C) The representative image and bar graph of the Western blot analysis for the EP2 subtype of the PGE2 receptor. *p < .05, N = 4 for both ELISA analysis and Western blot analysis.

Figure 6.

Gene expression and protein activity of tumor necrosis factor alpha (TNF-α) in the aged choroid. (A) The bar graph of the steady-state messenger RNA expression for TNF-α in the aging choroid. (B) The results from the enzyme-linked immunosorbent assay (ELISA) analysis. Because the same amount of protein is loaded into all wells, the results are presented as mean absorbance as a percentage of the 8-month values. *p < .05 versus 8 months, N = 5 for real-time polymerase chain reaction and N = 4 for ELISA analysis.