Real time imaging of retinal vascular inflammation

Abstract

Purpose : Retinal vascular inflammation plays a critical role in the pathogenesis of retinopathies, including diabetic retinopathy and age-related macular degeneration. Alteration of retinal inflammatory reactions is a key parameter when studying mechanisms of these diseases. Most of methods to analyze retinal vascular inflammation, such as determining the expression of inflammatory molecules and examining leukostasis after ex vivo perfusion, are only available for end-point determination and provide little information about the kinetics of inflammatory reactions in the same experimental animal. This study is to develop approaches to non-invasively examine retinal vascular inflammation in real-time in vivo

Methods : C57BL/6 mice were injected with lipopolysaccharide (LPS 4 mg/kg, i.p.) to induce retinal vascular inflammation. Two methods were used to label leukocytes in vivo: 1) Acradine orange (AO) was injected via tail vein (1 mg/kg); 2) Bone marrow from mice expressing green fluorescent protein (EGFP) was transplanted into wild type mice to generate chimeric mice (BM-EGFP) expressing GFP in leukocytes. Scanning laser ophthalmoscopy (SLO) was used to examine leukocyte trafficking in retinal vessels at 0-24 hours after LPS injection.

Results : LPS treatment induced retinal vascular inflammation as demonstrated by increases in leukocyte rolling and attachment along retinal vessels, which reached maximum at 6hours after LPS injection. In BM-EGFP mice, the fluorescence intensity of leukocytes was high and stable, producing high quality, sustained and repeatable imaging of leukocyte trafficking. AO injection could label total leukocytes. However, compared with the other BM-EGFP mice, the fluorescent signal of leukocytes was weak and transient, and therefore the quality of leukocyte imaging was low. Moreover, AO accumulated in the retina and produced high background, which further reduced the quality of imaging when repeated observation was needed.

Conclusions : BM-EGFP mice combined with SLO provide an excellent approach to examine retinal vascular inflammation in real-time in retinopathy.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.