Corneal Stromal Wound Healing Research Articles

Real‐time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

AbstractA modified tandem scanning confocal microscope was used for real‐time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

FGF promotes corneal stromal fibroblast motility.

It is well-known that growth factors accelerate wound healing by stimulating mitosis. Growth factors may also directly stimulate the motility of individual cells. We employed two different experimental methods to determine if fibroblast growth factor (FGF) enhances the individual motility of corneal stromal fibroblasts (independent of mitogenic effects). The effects of FGF on the motility of tissue-cultured rabbit corneal stromal fibroblasts were investigated in a modified Boyden chamber and by the agarose drop motility assay. Both assays showed a significant enhancement of stromal fibroblast motility by FGF at 100 ng/ml. It appears, therefore, that FGF may promote corneal stromal wound healing not only by increased cellular proliferation, but also by increased cellular motility.

Effect of growth factors with dexamethasone on healing of rabbit corneal stromal incisions

Treatment of rabbit corneal wounds with topical corticosteroid retards both epithelial regeneration and healing of penetrating stromal wounds. Currently, no clinical agent is available which accelerates the rate of stromal wound healing. Epidermal growth factor (EGF, 0·5 mg ml −1), fibroblast growth factor (FGF, 20 μg ml −1), and insulin (0·5 mg ml −1) were tested for their ability to accelerate healing of totally penetrating wounds in rabbit corneas when the hormones were administered alone or in combination with dexamethasone (1 mg ml −1). After 5 days of treatment with eye drops, the tensile strengths of corneal wounds treated with EGF (54±4 g mm −1) or treated with EGF and dexamethasone (32±9 g mm −1) were significantly higher than the tensile strengths of corneal wounds treated with only saline vehicle (3±1 g mm −1) or dexamethasone (1±0 g mm −1) ( P < 0·001). The combination of dexamethasone with EGF signficantly ( P < 0·025) reduced the strength of corneal wounds compared to treatment with EGF alone. Similarly, the tensile strength of corneal wounds after 5 days of insulin treatment alone (28±8 g mm −1) or in combination with dexamethasone (25±7 g mm −1) was significantly increased compared with saline- or dexamethasone-treated corneas ( P < 0·001). In the absence of dexamethasone, EGF increased the tensile strength of corneal wounds significantly better than insulin ( P < 0·01). However, when EGF or insulin were given in combination with dexamethasone there was no significant difference between the tensile strength produced by the peptide hormones. In comparison to the tensile strength of corneal wounds treated by EGF or insulin, treatment with FGF alone (5±4 g mm −1) or in combination with dexamethasone (2±1 g mm −1) produced poor wound healing. The in vitro actions of EGF or FGF alone or in combination with dexamethasone were tested for ability to stimulate [ 3H]-thymidine incorporation into pure cultures of human corneal fibroblasts (HCF) in defined culture medium. EGF (5 m m) or FGF (100 ng ml −1) alone stimulated [ 3H]-thymidine incorporation approximately 2·5-fold compared to control cultures, whereas in combination with dexamethasone (10 n m), the stimulatory action of FGF, but not EGF, was abolished. Dose-response curves indicated that HCF in culture were very sensitive to EGF, insulin, and FGF with maximum stimulation of [ 3H]-thymidine incorporation occurring at approximately 1 n m for EGF and insulin and at 100 μg ml −1 for FGF. Binding of 125I-EGF to HCF reached maximum after 2 hr at 37°C and was specific, saturable, and of high affinity (half saturation at 1 n m). These results indicate that EGF alone or in combination with dexamethasone is a potent accelerator of rabbit corneal stromal wound healing in vivo, and that EGF directly stimulates DNA synthesis of HCF in vitro presumably through specific high affinity receptors on corneal fibroblasts. EGF alone or in combination with corticosteroids may be useful in promoting healing in patients with corneal lacerations.

Synergistic growth stimulation of corneal fibroblasts by components of mesodermal growth factor from murine submaxillary glands.

Recycled mesodermal growth factor (R-MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R-MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is showing here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R-MGF has yielded several components, none of which has a greater biological potency than the parent R-MGF. In contrast, two components, MGF-I and -II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R-MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10-15 micrograms/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 micrograms of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R-MGF in vivo may also reflect synergistic action of more than one R-MGF component. Procedures for isolating gram quantities of R-MGF and for the purification of different R-MGF components by ion exchange chromatography are detailed.