Fig 1.
Corneal epithelial defects after penetrating injury in each group.
A: normal control group, there was no fluorescein staining. B: At 3 days after injury, the corneal wound area was stained. C: At 1 week after injury, the staining area in the wound area decreased. D: At 2 weeks after injury, only a small area of fluorescein staining. E: At 3 weeks after injury, there was no fluorescein staining. F: At 1 month after injury, there was no staining.
Fig 2.
Corneal opacity under slit lamp microscopy in each experimental group.
After injury, corneal opacity appeared at 3 days, gradually became denser, and peaked at 1 month. Corneal opacity progressively subsided at 2 to 4 months and was stabilized at 3 to 4 months.
Fig 3.
Slit lamp examination after penetrating injury.
A: Assessment of corneal epithelial defect area. Compared with the control group, there were significant differences in the area of corneal epithelial defects at 3 days, 1 week, and 2 weeks after injury, but there was no significant difference at 3 weeks and 1 month after injury (**, p ≤ 0.01; ****, p ≤ 0.0001). B: Haze was evaluated by Fantes’ score within 4 months. Corneal opacity progressively alleviated 2-4 months following penetrating injury, and the degree of corneal opacity was significantly lower at 4 months than at 1 month post-injury (***, p ≤ 0.001; ****, p ≤ 0.0001).
Fig 4.
Hematoxylin and eosin (H&E) stained sections of unwounded and penetrated corneas 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, and 4 months after modeling. e
=epithelium, S=stroma, dotted rectangle=wounded area, *=unwounded area. (Scale bars: 100 μm).
Fig 5.
Immunofluorescent staining for collagen type I expression and localization in unwounded corneas and wounded areas following penetrating injury.
In each panel, e represents the epithelium, S represents the stroma, while blue represents the DAPI staining of all nuclei. A: In the unwounded cornea, collagen type I (green) was regularly and uniformly distributed in the full-thickness stroma. B: Three days after injury, collagen type I was not detected in the corneal wound area. C-D: 1-2 weeks after injury, collagen type I was disordered in the whole stroma. E-G: At 3 weeks to 2 months after injury, collagen type I was regularly distributed in the anterior stroma. H-I: Immunohistochemical staining revealed a homogeneous distribution of collagen type I expression throughout most of the stromal region. The red dotted rectangle indicates the wounded area, and the asterisk (*) marks the unwounded area. (Scale bar: 100 μm).
Fig 6.
Immunofluorescent staining for collagen type I expression and localization in control unwounded corneas and at specific time points after a penetrating injury in rabbits.
In each panel, e represents the epithelium, S represents the stroma, while blue represents the DAPI staining of all nuclei. In each instance, the depicted panel is representative of the results observed in two corneas at each time point per group. A: In the unwounded corneas, collagen type I was not detected in the wound area. B: At 3 days after injury, collagen type I was absent. C-D: At 1 to 2 weeks after penetrating injury, collagen type I (green) nearly filled the whole stromal thickness. E-I: At 3 weeks to 4 months after injury, collagen type I was observed in the posterior stroma. The red dotted rectangle=injured area, *=uninjured area. (Scale bar: 100 μm).
Fig 7.
Transmission electron microscopy (TEM) photographs.
A: Normal keratocytes were distributed parallel to the epithelium, collagen fibers formed lamellae, and the EBM had integrated lamina lucida and lamina densa (Magnification=30000). B-D: At 3 days to 2 weeks after injury, collagenous fibers and myofibroblasts significantly increased in the stroma, and the EBM remained absent (Magnification=7000). E: At 3 weeks after penetrating injury, collagenous fiber distribution was irregular and myofibroblasts increased further in the stroma while the defective EBM regenerated (Magnification=30000). F-H: One to 3 months after injury, collagenous fiber distribution became more and more regular, myofibroblasts decreased; the EBM was progressively restructured (Magnification=30000). I: Four months after penetrating injury, collagen fiber distribution was organized, the ECM was similar to the control group, myofibroblasts were practically undetectable, and the EBM had almost completely regenerated (Magnification=30000). e=epithelium, S=stroma, small arrow=myofibroblast, arrow=EBM.
Fig 8.
Ultrastructure of the stromal collagen fibers and DM during the later stage of corneal wound healing.
Note that certain fibrils are observed longitudinally, whereas others are observed in cross-section; in the control group, all the fibrils have an identical diameter (Fig.8A). A-C: The remodeling of the diameter, arrangement, and orientation of collagen in the anterior stroma at 2-4 months after full-layer corneal injury. (Magnification=30000) D-F: The reconstruction of collagen in the posterior stroma at 2-4 months after corneal injury. (Magnification=30000) G-I: The regeneration of DM at 2-4 months after corneal perforation injury. (Magnification=7000) e=epithelium, En=endothelium, S=stroma, *=DM layer, small arrow=scattered DM, arrow=EBM, circle=cross-section of collagen fibers, ▲=myofibroblast.