Abstract
Purpose :
Claudin-5, a tight junction protein enriched in retinal capillary endothelial cells, is a key modulator of inner blood-retina barrier (iBRB) integrity and paracellular permeability. Persistent suppression of claudin-5 in mice fed a high cholesterol diet (HCD) for several weeks has been shown to induce a geographic atrophy (GA) like phenotype. Here, we aim to characterise a potential new model of GA and further investigate the role of claudin-5 cycling at the iBRB. Claudin-5 expression levels were disrupted by inducing heterozygosity at the Cldn5 locus in endothelial cells using the Cre-loxP system.
Methods :
Cldn5Flx/wt; Tie2-cre+ mice were generated using the Cre-loxP system. Specific expression of Cre recombinase, driven by the endothelial cell promoter, Tie2, allows Cldn5 heterozygosity to be induced at the iBRB. Heterozygous mice were fed a normal or HCD, and after 6-10 weeks retinal cryosections were imaged and compared to wild-type littermate controls. Additionally, retinal macrophages and microglia were examined using immunohistochemistry and detected using F4/80, Cd68, and Iba1 staining. ImageJ software was used to quantify the relative level of staining in wild-type and heterozygous animals.
Results :
Retinal claudin-5 was found to be decreased and an accompanying increase in macrophage markers were detected in the heterozygous mice. Histological analysis showed an accumulation of subretinal cells in heterozygous animals fed a HCD for 10 weeks. Additionally, some of these subretinal cells were CD68+, potentially indicating the presence of subretinal immune cells in these mice. A notable disruption of the RPE was evident.
Conclusions :
Our results show that the integrity of the iBRB may influence macrophage recruitment to the retina. Claudin-5 has been shown to cycle under the control of the circadian clock transcription factor, BMAL1. It is hypothesised that disruption of this cycling pattern, a process that may occur with age or due to genetic risk factors, could perturb iBRB function, eventually leading to GA. Subretinal immune cell accumulation is another known hallmark of AMD. Therefore, this study further illustrates the important role of claudin-5 cycling and how a disruption in this process may be a key initiating factor in the development of AMD.
This is a 2021 ARVO Annual Meeting abstract.