The corneal endothelium in an endotoxin-induced uveitis model: correlation between in vivo confocal microscopy and immunohistochemistry

PURPOSE To analyze the involvement of the corneal endothelium in uveitis to better understand the formation mechanisms and the keratic precipitate composition. In vivo confocal microscopy images were correlated with ex vivo immunostaining of corneal endothelium from rat eyes with endotoxin-induced uveitis (EIU). METHODS EIU was induced in Lewis rats by lipopolysaccharide (LPS) injection. Slit-lamp examination and in vivo confocal microscopy were performed 6, 24, 48, 72, and 96 h after the LPS injection. One group of rats was killed at 24 h and the other rats at 96 h. Immunohistochemistry on corneal endothelium using antibodies to intercellular adhesion molecule (ICAM-1), phalloidin, CD68 (anti-macrophage), MCA967 (anti-granulocyte), T cell receptor alpha/beta (TCR alpha/beta; anti-lymphocyte), zonula occludens-1 (ZO-1), and occludin was performed on flat-mount corneas and was analyzed using a three dimensional (3D) laser confocal microscope. RESULTS In vivo confocal microscopy showed numerous hyperreflective round dots on the corneal endothelium, in the anterior chamber, and in the anterior stroma corresponding to inflammatory cells with a maximum at 24 h after the injection and detectable until the 96th hour. Upon immunostaining, corneal endothelial cells in rats with EIU overexpressed ICAM-1. ZO-1 and occludin had a lower endothelial expression and more heterogeneous distribution in EIU rats than in controls, showing disruption of endothelial cell junctions. Compared to controls, CD68, MCA967, and TCR alpha/beta expression was observed in corneas in rats with EIU. The two techniques showed a circular peripheral network of corneal vessels derived from a large circumferential vascular structure resembling the major arterial circle of iris where the inflammatory cells marginalized to infiltrate the anterior stroma. CONCLUSIONS The correlation between in vivo confocal microscopy and ex vivo immunostaining helped to better understand in vivo confocal microscopy images. The two new techniques applied here were very effective and complementary in evaluating the corneal endothelium involvement in EIU. Based on these findings, in vivo confocal microscopy in clinical practice could be very helpful to better analyze keratic precipitates and corneal modifications in patients with uveitis.