Regeneration of Defective Epithelial Basement Membrane and Restoration of Corneal Transparency After Photorefractive Keratectomy

Abstract

PURPOSE To study regeneration of the normal ultrastructure of the epithelial basement membrane (EBM) in rabbit corneas that had -9D photorefractive keratectomy (PRK) and developed late haze (fibrosis) with restoration of transparency over one to four months after surgery and in corneas that had incisional wounds. METHODS Twenty-four rabbits had one of their eyes included into one of the two procedure groups (-9D PRK or nearly full-thickness incisional wounds), while the opposite eye serving as unwounded controls. All corneas were evaluated with slit lamp photos, transmission electron microscopy and immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin and collagen type III. RESULTS In the ‘-9D PRK group’, corneas at one month after surgery had dense corneal haze and no evidence of regenerated EBM ultrastructure. By two months after surgery, however, small areas of stromal clearing began to appear within the confluent opacity (lacunae), and these corresponded to small islands of normally-regenerated EBM detected within larger area of the excimer laser-ablated zone with no evidence of normal EBM. By four months after surgery, the EBM was fully-regenerated and the corneal transparency was completely restored to the ablated zone. In the ‘Incisional wound group’, the two dense, linear corneal opacities were observed at one month after surgery and progressively faded by two and three months after surgery. The EBM ultrastructure was fully regenerated at the site of the incisions, including around epithelial plugs that extended into the stroma, by one month after surgery in all eyes. CONCLUSIONS In the rabbit model, spontaneous resolution of corneal fibrosis (haze) after high correction PRK is triggered by regeneration of EBM with normal ultrastructure in the excimer laser- ablated zone. Conversely, incisional wounds heal in rabbit corneas without the development of myofibroblasts because the EBM regenerates normally by one month after surgery.

Epithelial basement membrane injury and regeneration modulates corneal fibrosis after pseudomonas corneal ulcers in rabbits

“…The large numbers of myofibroblasts generated after the severe keratitis likely develop from both bone marrow-derived and keratocyte-derived precursor cells (Barbosa et al, 2010; Singh et al, 2014). Also, similar to the rabbit PRK model (Marino et al, in press), regeneration of normal EBM lamina lucida and lamina densa at two months to four months after pseudomonas keratitis is associated with the disappearance of anterior stromal α-SMA+ myofibroblasts. We hypothesize that these anterior to mid-stromal myofibroblasts die by apoptosis after normal EBM regenerates and decreases the penetration of epithelium-derived TGFβ and PDGF into the stroma.…”

“…We hypothesize that these anterior to mid-stromal myofibroblasts die by apoptosis after normal EBM regenerates and decreases the penetration of epithelium-derived TGFβ and PDGF into the stroma. Subsequently, the stroma is repopulated with keratocytes that may facilitate EBM regeneration through the production of EBM components (Santhanam et al, 2017; Marino et al, in press). Thus, keratocyte contributions of EBM components such as laminin α-3 and nidogen-2 may be critical to the EBM regeneration process following corneal injury (Santhanam et al, 2017).…”

Fibrocytes, Wound Healing, and Corneal Fibrosis

“…Fibrocytes produce noninterstitial collagens, perlecan, versican, and hyaluronan, and consequently contribute to the timely regeneration of basement membranes that has been associated with the resolution of fibrosis in some tissues. 71,81 The MMPs secreted by fibrocytes may participate in the degradation of collagens in fibrotic tissues. 39 Bianchetti et al 71 also show that fibrocytes degrade collagenous proteins through an Endo180mediated pathway that contributes to collagen turnover and ECM remodeling.…”

Injury and defective regeneration of the epithelial basement membrane in corneal fibrosis: A paradigm for fibrosis in other organs?

“…In these clear areas, normal keratocytes have repopulated the stroma, mature EBM has regenerated, and the underlying myofibroblasts that are deprived of TGFβ and PDGF have undergone apoptosis, whereas the EBM continues to be morphologically and functionally defective in adjacent scarred areas where myofibroblasts persist (Fig. 2C and 3C) [40]. Over time, these lacunae tend to enlarge and coalesce as more surrounding EBM regenerates and full transparency of the cornea can be restored.…”