Drusen Component Causes Overexpression Of Inflammatory Cytokines In Retina And RPE: An in vivo Study

Abstract

Purpose: : Amyloid-beta (Aβ) is present in drusen in patients with age-related macular degeneration (AMD). Our earlier study of Aβ stimulation caused dramatic upregulation of inflammatory cytokines and immune response genes in human RPE in vitro. This study investigates the effects of Aβ on immune response gene expression in RPE and neuroretina in an in vivo rat model.

Methods: : Fifteen, 5 month old male Long-Evans rats received an intravitreal injection of 5µL Aβ1-40 (1.4 µg/µL) in one eye. Control eyes received 5µL vehicle (PBS). Five animals were sacrificed at 1, 4 or 14 days post-injection. At each timepoint, retina and RPE-choroid tissues were dissected from three eyes while two eyes were sectioned for histology. Expression patterns of a select group of genes (based on in vitro work) in RPE-choroid and neuroretina were quantified by RT-PCR in triplicates via the ΔΔCt method and compared to those obtained from PBS-injected eyes. Fold change ≥1.5 and p<0.05 was considered significant. Microglia were identified using OX-42 immunochemistry and retinal thickness measurements were made in paraffin-embedded sections.

Results: : Aβ induced upregulation of genes in the RPE-choroid including IL-1β and TNF-α on day 1, IL-1β, IL-6, TNF-α, iNOS, XAF1, TRAIL, and RSAD2 on day 4. Largest fold increase was observed in IL-6 (9.03±0.61, mean±SE), TNF-α (5.56±0.76), iNOS (5.26±0.74), and IL-1β (5.06±0.74). By day 14 all genes returned to baseline level. Neuroretina showed significantly increased expression of IL-6 (2.02±0.29 fold) on day 1 and TNF-α (3.83±0.75 fold) on day 14. Both TNF-α and VEGF mRNA levels doubled from day 4 to day 14. Other genes did not show significant fold changes. OX-42 staining revealed significantly more activated microglia in the Aβ eyes on day 4. Retinal thickness was not markedly different between Aβ and control eyes at any point studied.

Conclusions: : The rat RPE-choroid and neuroretina respond robustly to Aβ, but with different patterns. Our results demonstrate that inflammatory genes are overexpressed, in RPE-choroid. Interestingly, prolonged Aβ stimulation of the neuroretina caused an upward trend in the expression of VEGF. Elevated levels of XAF1 and TRAIL warrant investigation into the activation of apoptotic pathways associated with Aβ stimulation. These results are consistent with earlier reports that AMD progression is associated with chronic, local inflammatory events in the outer retina, and point towards the causal role of drusen components in AMD progression.

Keywords: age-related macular degeneration • drusen • retinal pigment epithelium