Induction of Fibroblast Senescence During Mouse Corneal Wound Healing

Invest Ophthalmol Vis Sci. 2019 Aug 1;60(10):3669-3679. doi: 10.1167/iovs.19-26983.


Purpose: To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation.

Methods: An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and α-smooth muscle actin (α-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated β-galactosidase (SA-β-gal) staining and P16Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-β-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP).

Results: The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-β-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16Ink4a+ vimentin+ α-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-β-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models.

Conclusions: Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Animals
  • Apoptosis
  • Blotting, Western
  • Cell Proliferation
  • Cellular Senescence / physiology*
  • Corneal Injuries / metabolism
  • Corneal Injuries / physiopathology*
  • Fibroblasts / cytology*
  • Fibroblasts / metabolism
  • Flow Cytometry
  • Hydrogen Peroxide / toxicity
  • In Situ Nick-End Labeling
  • Ki-67 Antigen / metabolism
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Real-Time Polymerase Chain Reaction
  • Sodium Hydroxide / toxicity
  • Wound Healing / physiology*


  • Actins
  • Ki-67 Antigen
  • alpha-smooth muscle actin, mouse
  • Sodium Hydroxide
  • Hydrogen Peroxide