Platelet recruitment promotes keratocyte repopulation following corneal epithelial abrasion in the mouse

Abstract

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and neutrophil (PMN) recruitment after corneal abrasion is beneficial to epithelial wound healing, we wanted to determine if these cells play a role in regulating keratocyte repopulation after epithelial abrasion. A 2 mm diameter central epithelial region was removed from the corneas of C57BL/6 wildtype (WT), P-selectin deficient (P-sel-/-), and CD18 hypomorphic (CD18hypo) mice using the Algerbrush II. Corneas were studied at 6h intervals out to 48h post-injury to evaluate platelet and PMN cell numbers; additional corneas were studied at 1, 4, 14, and 28 days post injury to evaluate keratocyte numbers. In WT mice, epithelial abrasion induced a loss of anterior central keratocytes and keratocyte recovery was rapid and incomplete, reaching ~70% of uninjured baseline values by 4 days post-injury but no further improvement at 28 days post-injury. Consistent with a beneficial role for platelets and PMNs in wound healing, keratocyte recovery was significantly depressed at 4 days post-injury (~30% of uninjured baseline) in P-sel-/- mice, which are known to have impaired platelet and PMN recruitment after corneal abrasion. Passive transfer of platelets from WT, but not P-sel-/-, into P-sel-/- mice prior to injury restored anterior central keratocyte numbers at 4 days post-injury to P-sel-/- uninjured baseline levels. While PMN infiltration in injured CD18hypo mice was similar to injured WT mice, platelet recruitment was markedly decreased and anterior central keratocyte recovery was significantly reduced (~50% of baseline) at 4-28 days post-injury. Collectively, the data suggest platelets and platelet P-selectin are critical for efficient keratocyte recovery after corneal epithelial abrasion.

Fig 1. Morphometric analysis of keratocytes, PMNs, and platelets.

(A) A morphometric number estimation box to count keratocyte nuclei through the thickness of the stroma. Non-FITC stained cells with blue fluorescing nuclei were considered keratocytes and were counted if the entire nucleus was contained within the box or on the accepted lines (dashed green lines; top and right). Nuclei that contacted the forbidden lines (solid red lines; bottom and left) were excluded. Scale bar = 10 μm. (B) A 3 x 3 morphometric number estimation grid used to determine PMN density (PMN/mm²). Each grid square had an area of 625 μm². All PMN nuclei within the grid box or on the accepted line were counted (white arrowheads) whereas PMN nuclei outside the grid boxes or on the forbidden line were not counted (red asterisk). Scale bar = 25 μm. (C) Upper panel: a 10 x 6 number estimation grid was oriented perpendicular to the major axis (arrow, top panel) of the limbal vessels. Lower panel: extravascular platelets (arrows) were identified and enumerated to determine platelet density. Scale bar = 50 μm.

Fig 2. Anterior central keratocyte recovery is incomplete after corneal epithelial abrasion.

(A) Anterior keratocyte counts in the central region (solid line) dropped and remained below baseline values out to 28 days post-injury (*p≤0.05), whereas posterior central keratocyte counts (dashed line) increased after injury at 4 days (⁺p≤0.05) before returning to baseline levels. (B) Anterior paralimbal keratocytes (solid line) decreased initially (*p≤0.05), but returned to baseline by 28 days. Posterior paralimbal keratocytes (dashed line) were elevated by 4 and 14 days (⁺p≤0.05), but returned to baseline by 28 days. Results are mean percentages of baseline keratocyte counts ± S.E.M. N = 5 mice per time point.

Fig 3. Platelet P-selectin is necessary for keratocyte recovery after corneal epithelial abrasion.

Anterior central keratocyte counts (expressed as percent of baseline) in P-sel^-/- mice at 4 days post-injury were decreased (*p≤0.05). Infusion of WT platelets prior to injury increased keratocyte recovery at 4 days post-injury compared to no infusion (⁺p≤0.05). In contrast, P-sel^-/- mice receiving P-sel^-/- platelets had significantly reduced keratocyte recovery compared to baseline (*p≤0.05), similar to mice receiving no infusion, and this was significantly lower than keratocyte recovery in mice receiving WT platelets (⁺p≤0.05). N = 3–10 mice per group.

Fig 4. PMN recruitment is normal in mice expressing low levels of CD18.

The recruitment of PMNs is similar between CD18^hypo (x) and WT (o) mice in the center, paralimbus, and limbus regions of the cornea from 0 to 48h after injury. N = 3–6 mice per genotype per time point.

Fig 5. CD18 is necessary for platelet recruitment to the injured cornea.

Platelet counts were low and similar between uninjured corneas from CD18^hypo and WT mice. In WT mice at 12 and 24h post-injury, there was a significant increase in recruited platelets at the limbus (*p≤0.05), but not in CD18^hypo mice. N = 3–5 mice per genotype per time point.

Fig 6. CD18^hypo mice have impaired keratocyte recovery after corneal epithelial abrasion.

(A) Anterior central keratocyte recovery (solid line) in CD18^hypo mice is only 50% of baseline values by 28 days post-injury, whereas the posterior (dashed line) region was not significantly different from baseline. (B) In the paralimbal region, where there was no direct epithelial injury, anterior keratocyte counts decreased by 40% and were not recovered at 28 days post-injury. *p≤0.05 compared to uninjured levels. N = 3–5 per time point.