Abstract
Purpose :
Mast cells (MCs) are known to be involved in the inflammatory process of AMD. MC degranulation can be triggered by many factors, including C-reactive protein (CRP). We have reported significantly increased CRP levels in AMD choroids. This study examined the effect of CRP on choroidal MCs and retinal pigment epithelial (RPE) cells.
Methods :
Recombinant rat CRP (1mg/mL) was delivered into the subretinal space (1uL) of Sprague Dawley rats. Rats were sacrificed at 1 and 2 weeks (WKs) post-injection. Choroids from one eye were stained for non-specific esterase (NSE) activity for counts of total and degranulated MCs. Choroids from the fellow eye were prepared for flat mount immunohistochemistry (IHC) and immunolabeled with antibodies against zonula occludens-1 (ZO-1) and Microphthalmia-associated transcription factor (MITF) to count degenerating RPE. Retinas were labelled with glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and ionized calcium binding adaptor molecule 1 (Iba1; microglia/macrophages). Images were collected under brightfield illumination for MC counts or using a Zeiss LSM 710 confocal microscope for RPE analysis and glial activation. MC and RPE counts were done using ImageJ.
Results :
At 1 & 2 WKS, CRP injected eyes had significantly more choroidal MCs than controls (p<0.05), MC degranulation was also significantly increased by CRP. At 2 WKs, CRP-injected eyes had 45% choroidal MC degranulation compared to 22.5% at 1WK (p<0.01) and 11.2% in controls. IHC revealed RPE degeneration scattered throughout the choroid in CRP injected eyes. In affected areas, 48.1% (at 1WK), 39.1% (at 2WK) of RPE cells were degenerated based on reduced or absent of ZO-1 staining in junctions. In these cells, MITF staining was cytoplasmic and absent from RPE nuclei, where it was prominently expressed in controls. At 2WKs, Müller cells throughout the retina expressed GFAP, indicating their activation. A majority of IBA-1+ microglia in the CRP injected area were activated based on their amoeboid morphology.
Conclusions :
Our results show that subretinal CRP directly causes degenerative changes in RPE and MC degranulation followed by activation of retinal glia. These findings support the evidence that CRP may be linked to AMD pathogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.