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Jiangyuan Gao, Ruozhou Tom Liu, Sijia Cao, Eleanor To, Aikun Wang, Jing Z Cui, Joanne A Matsubara; Chronic Exposure to a Drusen Component Causes a Sustained Pro-inflammatory Milieu in Rodent Outer Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3775.
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© ARVO (1962-2015); The Authors (2016-present)
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).
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.
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.
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.
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