Corneal Wound Healing: Cells and Molecules

Date

2016-08

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Abstract

Purpose: Corneal abrasion elicits an acute inflammatory response involving neutrophil (PMN) and platelet extravasation from the limbus. While these cells deliver important growth factors that promote wound healing, resolution of the inflammatory response (i.e., PMN clearance) is also needed to restore corneal homeostasis. This dissertation evaluated mechanisms regulating platelet recruitment after corneal injury, PMN migration in the abraded mouse corneal stroma and PMN clearance during the resolution of inflammation.

Methods: Adult C57BL/6J mice were anesthetized and a 2mm central corneal abrasion was made using a golf-spud/Alger brush. As a means of dysregulating platelet extravasation, approaches were used that build on the established observation that platelet recruitment is linked to PMN recruitment. Some mice received an intraperitoneal injection of anti-Ly6G antibody to deplete circulating PMNs while others received topical applications of recombinant interleukin-20 (rIL-20) to blunt inflammation. To determine if platelet extravasation was dependent on CD18 expression levels, two groups of mutant mice were studied, one with intermediate-high CD18 expression (CD18hypo (I-H)) and one with low-intermediate CD18 expression (CD18hypo (L-I)). To evaluate inflammation, excised corneas were immunolabelled and PMNs, platelets, mast cells, and degranulated mast cells were counted and limbal vessel diameters were measured. To evaluate PMN surface contacts with other cells and the extracellular matrix, excised corneas were imaged using serial block-face scanning electron microscopy. To study PMN migration in vivo, time-lapse sequences were collected using the Heidelberg Retinal Tomographer III with Rostock Cornea module (HRT-RCM).

Results: Following a central corneal abrasion, IL-20-treated mice and anti-Ly6G treated mice showed reduced PMN and platelet extravasation. PMN extravasation in CD18hypo (I-H) mice was similar to WT mice while platelet extravasation was diminished (~80%) only in CD18hypo (L-I) mice. Passive transfer of WT PMNs into CD18hypo (L-I) mice did not restore platelet extravasation to WT levels. In all cases, mice with diminished platelet recruitment showed diminished limbal vessel dilatation and reduced mast cell degranulation (p≤ 0.05).

8h post-injury, majority (70%) of paralimbal PMNs close to the limbus moved circumferentially with an average speed of 3.1±0.4µm/min, significantly less than the 6.1±0.3µm/min speed of PMNs at the paralimbus/closer to the center of the cornea where majority (60%) of these PMNs showed oriented migration toward the wound (p≤0.05). Serial block-face data provided evidence for a circumferential orientation of corneal cells and collagen at the paralimbus. Here, PMN surfaces predominantly contacted collagen (~60%), and keratocytes (~21%). At the center of the cornea, PMN numbers were maximal at 24h and markedly reduced by 72h post-injury. 24h post-injury, 64% of central PMNs were migrating away from the center which was significantly higher than that observed at 48h post-injury (p≤0.05). 52% of parawound PMNs were observed migrating away from the center of the cornea and by 72h this percentage dropped to 25%. 30h-72h post-injury macrophage numbers increased and macrophage phagocytosis of PMNs was clearly evident at the limbus.

Conclusion: While PMNs and platelets share a strong co-dependence in their recruitment after corneal abrasion, it appears that vessel dilatation is only required for platelet extravasation. Vessel dilatation appears to be mediated by mast cell degranulation which seems to require PMN CD18. The observation that many extravascular PMNs at the limbus are not oriented toward to the wound but are instead oriented circumferentially is novel. Given that the mouse cornea possesses an annular ring of collagen at the paralimbus, and collagen accounts for the majority of PMN surface contact, this collagen ring may account for the observed circumferential migration pattern of limbal PMNs during corneal inflammation. The clearance of PMNs during the resolution of inflammation appears to involve migration away from the center of the cornea and removal via macrophage phagocytosis.

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Keywords

Corneal wound healing, Reverse migration, CD18, Platelet extravasation, IL-20, Ly6G, Mast cells, Neutrophils

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