Abstract
Purpose:
Our earlier studies demonstrated that a drusen component, Aβ, triggers a short lasting pro-inflammatory response in retinal pigment epithelium (RPE), both in vitro and in vivo, through the activation of NF-κB and NLRP3 inflammasome. Furthermore, we demonstrated that the inhibition of NF-κB activation abolished the Aβ induced caspase-1 cleavage and cytokine production in RPE in vivo. In this study, we prolonged the pro-inflammatory milieu in the outer retina by making multiple Aβ injections, in order to better model the chronic pro-inflammatory events proposed to underlie the pathogenesis of the dry form of age-related macular degeneration (AMD).
Methods:
Long-Evans rats were intravitreally injected with Aβ1-40 once every 4 days for a period of 12 days and then sacrificed. Next, the vitreous humor was collected and levels of secreted cytokines were assessed using a multiplex suspension array. Protein levels of the cleaved caspase-3, C5b-9, NF-κB and IL-18 were assessed in paraffin embedded retinal sections by immunohistochemistry. Morphometric measurements to analyze Aβ-induced changes in the RPE were obtained using custom algorithms designed for Photoshop.
Results:
Vitreous samples from Aβ injected eyes showed more than 1.5 fold increase in MIP-3α, IL-1β, VEGF, and MCP-1 compared to controls. Semi-quantitative analysis of RPE/choroid complex from Aβ injected eyes revealed an increased immunoreactivity of cleaved caspase-3, C5b-9 and IL-18, when compared with the controls (p<0.05). The percentage of NF-κB immunoreactive nuclei in both ONL and RPE layers suggested robust NF-κB activation in Aβ injected eyes compared to controls. Moreover, morphometric analysis showed enlarged, swollen RPE cell bodies in Aβ injected eyes, suggestive of multiple cell death mechanisms.
Conclusions:
Consistent with our previous studies, we continued to observe elevated pro-inflammatory cytokine secretion in vitreous, and enhanced NF-κB and inflammasome activation in RPE. The existence of swollen, dysfunctional RPE and the increased immunoreactivity of cleaved caspase-3 and C5b-9 in RPE/choroid suggest the involvement of different cell death mechanisms in response to prolonged Aβ exposure. Further investigations are warranted to fully characterize this model and its relationship to AMD.