Non-Invasive Molecular Imaging of Selectin Ligands During Endotoxin-Induced Uveitis

Abstract

Purpose: : Uveitis is a devastating inflammatory disease of uncertain etiology. L-selectin on the surface of leukocytes regulates leukocyte recruitment to inflammatory sites in many vascular beds, however, the role of L-selectin in the eye has not been studied. Utilizing our novel molecular imaging technique we target ligands of L-selectin on the choriocapillaris endothelium to study the role of these molecules during disease.

Methods: : Uveitis was induced in Lewis rats by footpad injection of LPS. Fluorescent microspheres were conjugated to protein G and incubated with recombinant L-selectin or mAbs against the L-selectin ligands, MAdCAM-1 or CD34 (1 µg/ml). Microspheres were injected systemically into anesthetized rats and their adhesion in the choriocapillaris were investigated under physiologic flow conditions by scanning laser ophthalmoscopy (SLO). Subsequently, animals were perfused and retinal and choroidal flatmounts were prepared to count the number of firmly adhering microspheres. MAdCAM-1 and CD34 were assessed by immunohistochemistry and western blot (WB). The spatial relationship of the microspheres with the choriocapillaris endothelium was visualized by confocal microscopy.

Results: : In normal animals only a few L-selectin conjugated microspheres firmly adhered in the choriocapillaris (202.4±16.2, n=5). In contrast, 4 h after LPS, a significantly higher number of microspheres adhered to the choriocapillaris (2380±140.6, n=5, p<0.01), suggesting an upregulation of the expression of L-selectin ligands during uveitis. The microsphere interaction flux 24 h post LPS remained significantly higher in EIU animals compared to normal controls (p<0.01). MAdCAM-1 was upregulated in EIU animals 24 and 72 h after LPS as detected by WB and immunohistochemistry. Confocal microscopy indicated that the microspheres in the choriocapillaris were attached to the endothelium.

Conclusions: : To study uveitis, we utilize a novel non-invasive method for detection of choroidal endothelial surface molecules. We characterize the expression of L-selectin ligands in the endothelium of choriocapillaris during EIU. Our novel molecular imaging approach allows quantitative assessment of endothelial injury. This technique could be further developed to detect subclinical signs of ocular inflammation.

Keywords: imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • choroid • inflammation