Analysis of leukocyte populations and nerves in developing murine corneas

Abstract The avascular cornea is the most densely innervated tissue in the body and is endowed with resident bone marrow-derived cells. We have identified a novel subset of resident corneal plasmacytoid dendritic cells (pDCs). We have shown pDCs support corneal nerves through release of nerve growth factor (NGF). This study was to examine the presence of pDCs in the developing cornea. We examined corneas from E17, P2, and 8 week adult C57BL/6 mice. Corneas were stained for the pan-leukocyte marker CD45, the pDC marker plasmacytoid dendritic cell antigen-1 (PDCA-1), the pan-neuronal marker bIII-tubulin. Corneal CD45+ and PDCA-1+ cells and nerves were quantified. Significance was assigned by ANOVA and post-hoc Tukey test. All data is reported as mean ± standard deviation. Corneal CD45+ leukocyte density increased from E17 (65.4±14 cells/mm2) to P2 pups (301.9±19 cells/mm2, p<0.05). Both E17 and P2 corneas had lower CD45+ density when compared to adults (345±34 cells/mm2, p<0.05). We observed pDCs in the anterior stroma proximal to corneal nerves in E17, P2, and adult corneas. In E17, few pDCs were present in the peripheral cornea (1.5±2.6 cells/mm2), with none in the central cornea. In P2, pDCs were found in the periphery (68.19±8.99 cells/mm2), but not central corneas. The density of pDCs in adult peripheral (82.2±5.1 cells/mm2) and central (51.6±4.9 cells/mm2) corneas was greater than E17 (p<0.001) and P2 (p<0.05) corneas. Nerve density increased from E17 (45.43±6.75 mm/mm2) when compared to P2 (83.15±.21.6 mm/mm2 p<0.05) and to adult (151.6±3.25 mm/mm2 p<0.05) corneas. Our data show pDC density increased from embryonic, to neonatal, and adult corneas. Concurrently corneal nerve density increased during development from embryonic to adult mice.