Wounded embryonic corneas exhibit nonfibrotic regeneration and complete innervation

Abstract

Purpose: Wound healing in adult corneas is characterized by activation of keratocytes and extracellular matrix (ECM) synthesis that results in fibrotic scar formation and loss of transparency. Since most fetal wounds heal without scaring, we investigated the regenerative potential of wounded embryonic corneas.

Methods: On embryonic day (E) 7 chick corneas were wounded by making a linear incision traversing the epithelium and anterior stroma. Wounded corneas were collected between E7 and E18, and analyzed for apoptosis, cell proliferation, staining of ECM components, and corneal innervation.

Results: Substantial wound retraction was observed within 16-hours postwounding (hpw) and partial re-epithelialized by 5-days postwounding (dpw). Corneal wounds were fully re-epithelialized by 11 dpw with no visible scars. There was no difference in the number of cells undergoing apoptosis between wounded and control corneas. Cell proliferation was reduced in the wounded corneas, albeit mitotic cells in the regenerating epithelium. Staining for alpha-smooth muscle actin (α-SMA), tenascin, and fibronectin was vivid but transient at the wound site. Staining for procollagen I, perlecan, and keratan sulfate proteoglycan was reduced at the wound site. Wounded corneas were fully regenerated by 11 dpw and showed similar patterns of staining for ECM components, albeit an increase in perlecan staining. Corneal innervation was inhibited during wound healing, but regenerated corneas were innervated similar to controls.

Conclusions: These data show that minimal keratocyte activation, rapid ECM reconstruction, and proper innervation occur during nonfibrotic regeneration of the embryonic cornea.

Keywords: corneal development; corneal wound healing; extracellular matrix; innervation.

Figure 1

Wound healing of embryonic corneas. (A) Comparison of wounded and nonwounded stage-matched corneas between 0 and 11 dpw. Arrowheads indicate the extent of wound caused by linear incision. (B) Cross-sections through wounded corneas immunostained with laminin showing the wound at 0 dpw, wound retraction at 3 dpw, and the re-epithelialized cornea at 11 dpw. Brackets and asterisks denote wounded region. (C) Analysis of the wound size at different time points. Wound size was measured using ImageJ (National Institutes of Health, Bethesda, MD) and percent wounded area was calculated by dividing the area of the wound by the total area of the cornea. ep, epithelium; st, stroma; en, endothelium. Scale bars: 1 mm (A), 100 μm (B).

Figure 2

Quantification of cell death and proliferation during wound healing of the embryonic cornea. The number of (A) cas3A-positive cells or (D) pH3-positive cells were counted on seven random sections and averaged for three wounded and nonwounded corneas at each time point. *P < 0.05. (B, C) Cas3a staining (arrows) in representative sections of (B) 16 hrpw and (C) stage-matched control corneas. (E, F) Localization of pH3-postive cells (arrowheads) in the corneal epithelium (E) at the wound margin at 5 dpw, and (F) in stage-matched control. Brackets denote wound region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 50 μm.

Figure 3

Expression of α-SMA during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing: (A, B) α-SMA-positive staining in the wound at 16 hrpw (arrowhead) and 3 dpw. Note, the cornea endothelium stains positive for α-SMA at 16 hrpw. (C) Alpha–smooth muscle actin staining is not detected at 5 dpw, and (D) after re-epithelialization. Brackets and asterisks denote wounded region. Dotted lines demarcate the cornea epithelium/stroma boundary. Scale bars: 100 μm.

Figure 4

Expression of fibronectin and tenascin during wound healing of the embryonic cornea. Cross-sections through embryonic corneas immunostained for fibronectin and tenascin showing: (A–C) Nonoverlapping staining for fibronectin and tenascin localized in the anterior region of the wound between 2 and 5 dpw. (A'–C') Staining for fibronectin and tenascin in stage-matched control corneas. (D) At 11 dpw, fibronectin is downregulated in the re-epithelialized wound and tenascin is expressed in the anterior–posterior gradient similar to stage-matched control (D'). Brackets and asterisks denote wounded region. Scale bars: 100 μm.

Figure 5

Procollagen I expression in healing embryonic corneal wounds. Cross-sections through embryonic corneas showing expression of procollagen I in: (A–D) wounded, and (A'–D') stage-matched controls. Arrowheads indicate procollagen staining in regenerated epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm.

Figure 6

Expression of perlecan in the embryonic cornea. During (A) cornea development and (B) wound healing. Arrowheads in (A) indicate localization of perlecan to the anterior and posterior basement membranes. Arrowheads in (B) indicate perlecan staining in the anterior basement membrane during re-epithelialization. Brackets and asterisks denote wounded region. Scale bars: 100 μm; inset 50 μm.

Figure 7

Expression of KSPG during wound healing of the embryonic cornea. Cross-sections through embryonic corneas showing expression of KSPG at: (A–A'') 2 dpw, (B–B''') 5 dpw, and (C–C') 11 dpw. Control staining is from nonwounded stage-matched cornea. Arrowheads indicate regenerating epithelium. Brackets and asterisks denote wounded region. Scale bars: 100 μm (A–C), 50 μm (A'–C', Control).

Figure 8

Innervation of wounded embryonic corneas. Whole-mount immunostaining with anti–β-neurotubulin antibody (TUJ1) showing nerve projections into (A, C) nonwounded and (B, D) wounded corneas. (B'') Optical section of the wound region shown in (B'). Bracket (B) indicates regenerating epithelium. White dotted line (B, B') demarcates the wound boundary. Yellow dotted line (B'') designates epithelial–stromal boundary. Arrows and arrowheads (B', B'') indicate stromal nerve and epithelial nerve leashes, respectively. (E) Quantitation of Sema3A transcripts. The values shown at each time point are the ratios of fold expression levels between wounded and nonwounded corneas.