Abstract
Purpose :
To characterize retinal degeneration (RD) phenotype in the aged CXCR5-/- mice, as well as to find mechanistic insights for the function of CXCR5 in the eye.
Methods :
Aged B6.129S2 (Cg)-Cxcr5tm1Lipp/J (CXCR5-/-) and C57bl/6j (C57) mice were used in this study. Fundus imaging was used to evaluate RD in vivo. Ex vivo study involved periodic acid Schiff (PAS) staining, immunofluorescence (IF) of histological sections, retina and RPE/choroidoscleral complexes (RCSC) flat mounts and transmission electron microscopy (TEM). Western blot (WB) analysis was performed on protein extracts from RCSC and retinas of CXCR5-/- mice and age-matched C57 controls. Mass spectrometry used to identify circulating autoantibody against known AMD related proteins in the serum of aged CXCR5-/- animals.
Results :
In vivo fundus images has demonstrated retinal degeneration with significant (p<0.001) increase of drusen-like deposits in the eyes of aged CXCR5-/- mice, compared with C57 controls.
Ex vivo PAS staining has revealed sub-RPE localization of deposits. Further analysis by immunofluorescence (IF) has detected the presence of Cryab, amyloid beta, and C3d within the deposits. These results were further supported by WB and has also indicated increase in COX-2 and Arg1 signaling within RCSC.
Peanut lectin stain has confirmed the significant (p<0.01) loss of photoreceptors further corroborated by a reduction in MAP2 signaling in CXCR5-/- mice compared with age-matched C57 mice. TEM has revealed the drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of blood-retinal barrier (BRB) integrity was demonstrated by the reduction of ZO-1 in RPE. CD4 immunostaining has revealed the accumulation of inflammatory cells in sub-RPE space and increase of IBA-1 positive microglia cells on the surface of RPE. Mass spec analysis of CXCR5 mice serum has identified autoantibodies against Cryab, Anxa2, Ubb, and others.
Conclusions :
Results suggest that with age CXCR5-/- mice develop microglia disfunction, progressive photoreceptor and neuron cell loss, as well as sub RPE deposition of cellular debris resulting in the production of immunogenic proteins such as Cryab and Anxa2. Chronic inflammation leads to the loss of tight junction proteins and the compromising of BRB integrity, resulting in autoantibody production, autoimmune-mediated inflammation and RD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.