Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (alpha SMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP + alpha SMA + myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45 + fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present m the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP + CD45 + cells were also alpha SMA + myofibroblasts. At 21 days after PTK, few GFP + alpha SMA + cells persisted m the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP + CD45 + SMA + cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature aSMA + myofibroblasts. Most GFP + CD45 + alpha SMA + myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGF beta requisite for myofibroblast survival.