Real-time, in vivo Imaging of Leukocyte-Endothelial Interaction in Allergic Conjunctivitis in the Rat: Paradoxical Cellular Infiltration Without a Concomitant Increase in Endothelial Adhesion

Abstract

Abstract: : Purpose: To investigate the interactions between leukocytes and microvascular endothelium in conjunctival tissue during the inflammation that occurs in allergic conjunctivitis. Methods: Lewis rats were immunized and boosted 3 weeks later with 100 µg ovalbumin (OVA) in alum. Immunized rats were challenged 2 weeks after the boost with either saline or OVA (50 mg/ml in saline) by injection of 4 µl into the tarsal conjunctival sac. At 0.25, 1, 6, 12, 24, 48 and 72 hours post challenge (n = 5-10 rats/time point), conjunctiva of rats were imaged by real-time intravital digital video microscopy. Just prior to imaging, we injected i.v. rhodamine 6G which fluorescently stains all blood leukocytes. Four or five 20-second recordings were made of non-overlapping regions of tissue per eye. The number of fluorescent rolling and sticking cells in each of these recordings was counted in a masked fashion. After microscopy, the rats were sacrificed and the eyes were enucleated with intact lids for histology. Three representative sections from each eye were stained with hematoxylin and eosin and with an esterase detection kit. The number of cells/mm₂ were quantified in tarsal conjunctiva. Results: A moderate conjunctivitis was induced as evidenced by tearing, redness and chemosis within 15 minutes post OVA challenge. Histology revealed the infiltration of leukocytes within 1 hour post challenge and peak infiltration at 6 hours. Approximately one-half of the infiltrating cells were esterase-positive neutrophils and a minority of cells were eosinophils. Leukocyte interactions (i.e., rolling and sticking) with endothelial cells in the microvasculature were evident, independent of the onset of conjunctivitis and there was no difference in the inflamed vs. uninflamed eyes. Conclusion: There was a significant increase in infiltration of leukocytes into the surrounding tissue without a corresponding increase in rolling or sticking of leukocytes in the conjunctival microvasculature. Thus, the quantitative monitoring of leukocyte rolling and sticking did not correlate with tissue infiltration. These data suggest that in conjunctiva, substantial basal adhesion between leukocytes and endothelial cells are sufficient to facilitate and sustain inflammation.

Keywords: 366 conjunctivitis • 431 imaging/image analysis: non-clinical • 437 inflammation