Erythrocyte (RBC) extravasation in the abraded mouse cornea

Abstract

Purpose : In the mouse, a central corneal epithelial abrasion initiates an inflammatory cascade involving neutrophil (PMN), platelet, and RBC extravasation at the limbus. Corneal abrasion in hypomorphic mutant mice expressing low levels of the leukocyte CD18 integrin (CD18_hypo) have marked reductions in platelet extravasation. In the present study, we sought to define the role of CD18 on RBC extravasation in this model of inflammation, focusing on two relevant cell types that express CD18: PMNs and mast cells.

Methods : C57BL/6J wildtype (WT), CD18_hypo, and mast cell deficient mutant KitW-sh/W-sh (C-Kit) mice aged 6-8 wks received a 2mm central corneal epithelial abrasion. Prior to wounding, some WT mice received cromolyn or ketotifen intraperitoneal injections to inhibit mast cell degranulation. Injured corneas were harvested 6-36h later and immunostained with fluorescent antibodies against CD41 (platelets), Ly6G (PMNs), CD31 (vessels), and TER-119 (RBCs), FITC-Avidin (mast cells), and DAPI (nuclei). Stained corneas were imaged on a Delta Vision microscope and analyzed using ImageJ software. Some corneas were processed for Serial Block-Face Scanning Electron Microscopy (SBF-SEM) and 3D reconstruction. Data were analyzed using student’s t-test or a one or two-way ANOVA with appropriate post-hoc tests. Significance was set at p≤0.05.

Results : CD18_hypo mice showed reduced RBC and platelet extravasation (55% and 80% reductions, respectively, p<0.05) and reduced mast cell degranulation and vessel engorgement 24h post-abrasion (p<0.05). The c-kit mice showed similar reductions in extravasated RBCs and platelets (55%, 60%, respectively, p<0.05); while PMN extravasation was delayed (12-18h; p<0.05) but not reduced. SBF-SEM imaging of WT corneas showed that perivascular macrophages phagocytosed extravasated RBCs (erythrophagocytosis); PMNs rarely phagocytosed RBCs.

Conclusions : PMNs, CD18, and mast cell degranulation are required for RBC extravasation in the abraded cornea. Vessel engorgement resulting from mast cell degranulation is associated with RBC extravasation. The significance of macrophage erythrophagocytosis remains to be determined in the injured mouse cornea.

This is a 2020 ARVO Annual Meeting abstract.