Increase in retinal ganglion cells' susceptibility to elevated intraocular pressure and impairment of their endogenous neuroprotective mechanism by age

Abstract

Purpose: To investigate age-associated changes in retinal ganglion cell (RGC) response to elevated intraocular pressure (IOP), and to explore the mechanism underlying these changes. Specifically, the effect of aging on inhibitor of apoptosis (IAP) gene family expression was investigated in glaucomatous eyes.

Methods: IOP was induced unilaterally in 82 Wistar rats using the translimbal photocoagulation laser model. IOP was measured using a TonoLab tonometer. RGC survival was evaluated in 3-, 6-, 13-, and 18-month-old animals. Changes in the RNA profiles of young (3-month-old) and old glaucomatous retinas were examined by PCR array for apoptosis; changes in selected genes were validated by real-time PCR; and changes in selected proteins were localized by immunohistochemistry.

Results: There were no significant IOP differences between the age groups. However, there was a natural significant loss of RGCs with aging and this was more prevalent in glaucomatous eyes. The number of RGCs in glaucomatous eyes decreased from 669±123 RGC/mm² at 3 months to 486±114 RGC/mm² at 6 months and 189±46.5 RGC/mm² at 18 months (n=4-8, p=0.048, analysis of variance). The PCR array revealed different changes in proapoptotic and prosurvival genes between young and old eyes. The two important prosurvival genes, IAP-1 and X-linked IAP (XIAP), acted in opposite directions in 3-month-old and 15-month-old rats, and were significantly decreased in aged glaucomatous retinas, while their expression increased significantly in young glaucomatous eyes. P53 levels did not vary between young glaucomatous and normal fellow eyes, but were reduced with age. B-cell leukemia/lymphoma 2 (Bcl-2) family members and tumor necrosis factor (TNF)-α expression were unaffected by age. Immunohistochemistry results suggested that the sources of changes in IAP-1 protein expression are RGCs and glial cells, and that most XIAP secretion comes from RGCs.

Conclusions: Decreased IAP-1 and XIAP gene expression in aged eyes may predispose RGCs to increased vulnerability to glaucomatous damage. These findings suggest that aging impairs the endogenous neuroprotective mechanism of RGCs evoked by elevated IOP.

Figure 1

Intraocular pressure in eyes of young and old rats. A and B: All experimental eyes had significantly elevated intraocular pressure (IOP) compared to their control fellow eyes. There was no significant difference in mean or peak IOP between young and old rats. Data presented as SEM, n=8, *=p<0.05.

Figure 2

Retinal ganglion cell loss increased with age in both glaucomatous and control fellow eyes. A: The mean retinal ganglion cell (RGC) survival 10 weeks after the induction of elevated intraocular pressure (IOP) is shown. There was a significant decrease in RGCs in the control fellow eyes with age (n=4–8 for each age group, data presented as SEM, p=0.002), as well as in the glaucomatous eyes (n=4–8, p=0.048). B: The amount of glaucomatous RGC loss increased with age (n=4–8, p=0.05). This progression in RGC loss due to age occurred under similar IOP levels. C-H: Representative fluorogold images of RGCs 10 weeks after induction of glaucoma in young and old eyes are shown. Magnification 40X. I: Labeled RGCs were counted with a 40 super wide field objective along two radii in four directions (i.e., superior, temporal, inferior, and nasal) centered on the position of the optic nerve head.

Figure 3

Effect of aging on selected proapoptotic and prosurvival genes. Real-time PCR analysis of selected genes was performed in young (3 months) and old (15 months) rats. The following genes are presented: Inhibitor of apoptosis (IAP)-1 gene (A), X-linked IAP (XIAP) gene (B); p53 gene (C), Bcl-2 gene (D), Bcl-xl gene (E), tumor necrosis factor (TNF)-α gene (F). Interestingly, IAP-1 and XIAP expression was significantly downregulated in the glaucomatous eyes of old rats, but upregulated in young rats. Data represent mean ± standard error of the mean (SEM); n=8; *p<0.05.

Figure 4

Immunohistochemistry for inhibitor of apoptosis 1, the retinal ganglion cell marker Thy 1, glial fibrillary acidic protein, and 4',6-diamidino-2-phenylindole in retinal cryosections of young and old rats at 8 days after induction of elevated intraocular pressure (IOP). The merged image shows colocalization of IAP with Thy 1 (yellow) and with glial fibrillary acidic protein (GFAP; purple), suggesting that the source for changes in IAP expression is from retinal ganglion cells (RGCs) and glial cells. A: In 3-months-old rats, IAP levels increased in glaucomatous eyes as well as staining for GFAP. B: IAP-1 staining decreased in old glaucomatous 13-month-old eyes as compared to fellow eyes. Magnification 40X, scale bars: all panels 20 μm.

Figure 5

Immunohistochemistry for X-linked inhibitor of apoptosis, Thy 1, glial fibrillary acidic protein, and 4',6-diamidino-2-phenylindole in retinal cryosections of young and old eyes at 8 days after induction of glaucoma. The merged image shows colocalization of X-linked inhibitor of apoptosis (XIAP) with Thy 1 (yellow), suggesting that the source for changes in XIAP expression is in the retinal ganglion cell (RGC) layer. A: In 3-month-old eyes, XIAP levels were increased as compared to fellow eyes. B: In old glaucomatous 13-month-old eyes, XIAP staining decreased in the RGC layer as compared to fellow eyes. Magnification 40X, scale bars: all panels 20 μm.