Multimodal imaging of experimental choroidal neovascularization.

AIM To compare choroidal neovascularization (CNV) lesion measurements obtained by in vivo imaging modalities, with whole mount histological preparations stained with isolectin GS-IB4, using a murine laser-induced CNV model. METHODS B6N.Cg-Tg(Csf1r-EGFP)1Hume/J heterozygous adult mice were subjected to laser-induced CNV and were monitored by fluorescein angiography (FA), multicolor (MC) fundus imaging and optical coherence tomography angiography (OCTA) at day 14 after CNV induction. Choroidal-retinal pigment epithelium (RPE) whole mounts were prepared at the end of the experiment and were stained with isolectin GS-IB4. CNV areas were measured in all different imaging modalities at day 14 after CNV from three independent raters and were compared to choroidal-RPE whole mounts. Intraclass correlation coefficient (ICC) type 2 (2-way random model) and its 95% confidence intervals (CI) were calculated to measure the correlation between different raters' measurements. Spearman's rank correlation coefficient (Spearman's r) was calculated for the comparison between FA, MC and OCTA data and histology data. RESULTS FA (early and late) and MC correlates well with the CNV measurements ex vivo with FA having slightly better correlation than MC (FA early Spearman's r=0.7642, FA late Spearman's r=0.7097, and MC Spearman's r=0.7418), while the interobserver reliability was good for both techniques (FA early ICC=0.976, FA late ICC=0.964, and MC ICC=0.846). In contrast, OCTA showed a poor correlation with ex vivo measurements (Spearman's r=0.05716) and high variability between different raters (ICC=0.603). CONCLUSION This study suggests that FA and MC imaging could be used for the evaluation of CNV areas in vivo while caution must be taken and comparison studies should be performed when OCTA is employed as a CNV monitoring tool in small rodents

Systemic Lipopolysaccharide Exposure Exacerbates Choroidal Neovascularization in Mice.

This study aims to investigate the effect of a systemic lipopolysaccharide (LPS) stimulus in the course of laser-induced choroidal neovascularization (CNV) in C57BL/6 J mice. A group of CNV-subjected mice received 1 mg/kg LPS via the tail vein immediately after CNV induction. Mouse eyes were monitored in vivo with fluorescein angiography for 2 weeks. In situ hybridization and flow cytometry were performed in the retina at different time points. LPS led to increased fluorescein leakage 3 days after CNV, correlated with a large influx of monocyte-derived macrophages and increase of pro-inflammatory microglia/macrophages in the retina. Additionally, LPS enhanced Vegfα mRNA expression by Glul-expressing cells but not Aif1 positive microglia/macrophages in the laser lesion. These findings suggest that systemic LPS exposure has transient detrimental effects in the course of CNV through activation of microglia/macrophages to a pro-inflammatory phenotype and supports the important role of these cells in the CNV course

Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

BACKGROUND Clustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation. METHODS C57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre-posTlr4loxP/loxP) and TekCre-negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function. RESULTS Activation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre-negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge. CONCLUSIONS The findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia-endothelial cell interaction in inflammatory retinal disease