SHG imaging microscopy of vehicle-treated excimer laser photoablated corneas showed a highly irregular organization of collagen fibers immediately underlying the corneal epithelium
(Fig. 3A) . This irregular organization at the most superficial level of the stoma appeared to form a honeycomb-like scaffolding (arrows) that surrounded clusters of basal epithelial cells identified by phalloidin and nucleic acid stain (Syto59; Molecular Probes;
Fig. 3B , arrows). These collagen scaffolds appeared to extend more deeply into the cornea and to merge with the underlying collagen to form thin, irregularly arranged collagen bundles (
Fig. 3A , arrowheads). Below the epithelium, the thin collagen bundles formed a highly irregular and interwoven collagen network with no particular organizational pattern
(Fig. 3C) . Although the thickness of this collagen layer varied between samples, the random organizational pattern was a consistent and characteristic feature that allowed for accurate determination of the thickness of this layer. Below this region, normal-appearing stromal collagen lamellae with parallel bundles of collagen fibers were detected, with regions of more irregular collagen from the region above appearing to extend between some lamellae (
Fig. 3D , asterisk). In MMC-treated corneas, the anterior stroma underlying the basal corneal epithelium appeared to contain disrupted normal corneal stromal collagen lamellae of varying thickness (
Fig. 3E , arrow). Occasional lamellae appeared to end abruptly (asterisk) in regions immediately adjacent to the overlying corneal epithelium, as identified in the same optical plane by phalloidin and nucleic acid stain (Syto59; Molecular Probes;
Fig. 3F , asterisk). Below this stromal-epithelial interface, the lamellar organization appeared to be normal and contained collagen fibers organized into broad lamellar bands (not shown).
Reconstruction of three-dimensional data sets along the
xz plane showed that the irregular collagen organization in the vehicle-treated eyes formed a layer of fibrotic tissue above the unablated corneal stroma (
Fig. 4A , top arrows). The average shrinkage-adjusted thickness of this region was 44.47 ± 1.57 μm (
n = 3), as measured from the basal epithelial cells to the interface between the irregular collagen network and the normal-appearing stromal lamellae. This region also appeared to contain a higher density of stromal cell nuclei detected in cross-section (
Fig. 4B , top arrows) and in optical sections through the irregular collagen network (
Figs. 4C 4D , same optical plane). The number of nuclei in a single optical plane through this region averaged 686 ± 104 cells/mm
2 (
n = 3). MMC-treated corneas showed no irregular collagen above the stromal interface in three-dimensional reconstructions along the
xz plane
(Fig. 5A) ; however, immediately below the corneal epithelium, a region apparently devoid of stromal cell nuclei was detected (
Fig. 5B , double-headed arrow). Optical sections through this region showed normal broad stromal lamellae with wide parallel bands of collagen fibers
(Fig. 5C)and occasional stromal cells that appeared isolated, with enlarged nuclei (
Fig. 5D , arrows), together with a few small cells, possibly macrophages (arrowheads). Although no fibrosis was detected in this experiment, disrupted collagen fiber organization at the epithelial stromal interface was detected and averaged 9.07 ± 3.70 μm in thickness. The cell density in these regions was also markedly below that observed in the vehicle-treated corneas and averaged 62 ± 10 cells/mm
2 (
n = 3).
Although MMC-treated corneal wounds showed no evidence of myofibroblasts, vehicle-treated corneas showed occasional areas where fibroblasts with abundant actin filament staining were detected 3 months after PRK
(Fig. 6) . These regions were located below the corneal epithelium (
Figs. 6A 6D , arrowhead) and immediately overlying regions of corneal fibrosis (
Figs. 6A 6D , brackets). SHG optical section through these regions showed small bundles of collagen fibers extending out from the corneal stroma, under the basal epithelial cells (
Fig. 6B 6E , arrows). Interestingly, these collagen fibers appeared to coalign with the actin filament bundles (
Figs. 6C 6F , arrows). Similar coalignment of collagen fibers and actin filament bundles have been noted in anti–type I collagen–stained corneal wounds that also showed colocalization with fibronectin fibrils and α5β1 integrin.
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