Keratinocyte Growth Factor-2 on the Proliferation of Corneal Epithelial Stem Cells in Rabbit Alkali

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Purpose: To determine whether the topical application of keratinocyte growth factor-2 (KGF-2) can enhance corneal epithelial healing in rabbit alkali burned cornea. In addition, the distribution and proliferation of corneal epithelial stem cells in KGF-2-treated and control corneas were investigated to explain their mechanisms of effects on the epithelium. Methods: Twenty-four New Zealand eyes were divided into four groups, treated with KGF-2 solution (1, 50, 100 μg/ml) and PBS solution. Eighth millimeter filter paper discs, produced by standard paper punch, were soaked for 15 sec in 0.5N NaOH solution. The alkali-soaked discs were applied to the central cornea, centered on the pupil and held gently in position with forceps for 1 min. The cornea was finally irrigated over 1 min with 100 ml balanced salt solution (BSS). Keratinocyte growth factor-2 was then applied topically three times a day. The phosphate-buffered saline (PBS) group was served as a control. Each corneal epithelial defect was subsequently photographed every 24 hours with a slit lamp and was measured by computer-assisted digitizer. In each group, two rabbits were sacrificed for light microscopic examination after the interval of 7, 14 and 21 days. Meanwhile, the cornea epithelium was examined by immunohistochemistry for P63, AE5, EGFR. Results: Topical application of 10 μg/ml to 100 μg/ml KGF-2 significantly accelerated corneal epithelial wound healing when compared with controls. After 24 hours, epithelial healing rate of the 100 μg/ml KGF-2 group and the PBS treated group was (74±6)% and (40±8)% (P < 0.05). After 48 hours, the rate of the C group was (94±6)%, whereas in the control group it was (73±12)% (P < 0.05). Epithelial defects were often recurrent, which happened only two times in the 100 μg/ml KGF-2-treated group, but many times in the control group. In the corneal epithelial stem cell analysis, the number of the P63 positive cells was higher in the KGF-2-treated corneal epithelium than in the controls. The P63 positive cells in the alkali burned epithelium were found not only in the limbal area but also in the central cornea. In addition, the number of stem cells in each group came to the maximum on the 14th day. For example, on the 7th day after alkali injury, it was 40.3±2.1 NPC in the non-limbal area of 50 μg/ml KGF-2-treated group; whereas, it was 84.8±2.7 NPC on the 14th day(P = 0.000). Conclusions: From the daily evaluation of the corneal surface as well as the microscopic examinations at the end of the three periods of observation, we concluded that KGF-2 provided a beneficial effect in the treatment of alkali burns of the cornea. Furthermore, the results of epithelial stem cell analysis demonstrated that KGF-2 accelerated the corneal epithelial healing by markedly stimulating epithelial stem cells proliferation and making them migrate to the central cornea. Purpose: To determine whether the topical application of keratinocyte growth factor-2 (KGF-2) can enhance corneal epithelial healing in rabbit alkali burned cornea. In addition, the distribution and proliferation of corneal epithelial stem cells in KGF-2-treated and control corneas were investigated to explain their mechanisms of effects on the epithelium. Methods: Twenty-four New Zealand eyes were divided into four groups, treated with KGF-2 solution (1, 50, 100 μg / ml) paper discs, produced by standard paper punch, were soaked for 15 sec in 0.5 N NaOH solution. The alkali-soaked discs were applied to the central cornea, centered on the pupil and held gently in position with forceps for 1 min. The cornea was Finally irrigated over 1 min with 100 ml of balanced salt solution (BSS). Keratinocyte growth factor-2 was then applied topically three times a day. The phosphate-buffered saline (PBS) group was served as a control. Each corneal epithelial de The subjects were photographed every 24 hours with a slit lamp and was measured by computer-assisted digitizer. In each group, two rabbits were sacrificed for microscopic examination after the interval of 7, 14 and 21 days. Meanwhile, the cornea epithelium was by immunohistochemistry for P63, AE5, EGFR. Results: Topical application of 10 μg / ml to 100 μg / ml KGF-2 significantly accelerated corneal epithelial wound healing when compared with controls. After 24 hours, the epithelial healing rate of the 100 μg / ml The KGF-2 group and the PBS treated group was (74 ± 6)% and (40 ± 8)% (P <0.05). After 48 hours, the rate of the C group was (94 ± 6)%, The control group it was (73 ± 12)% (P <0.05). Epithelial defects were often recurrent, which happened only two times in the 100 μg / ml KGF-2-treated group, but many times in the control group. corneal epithelial stem cell analysis, the number of the P63 positive cells was higher in the KGF-2-treated corneal epithelium than inthe controls. The P63 positive cells in the alkali burned epithelium were found not only in the limbal area but also in the central cornea. In addition, the number of stem cells in each group came to the maximum on the 14th day. For example, on the 7th day after alkali injury, it was 40.3 ± 2.1 NPC in the non-limbal area of ​​50 μg / ml KGF-2-treated group; Conclusions: From the daily evaluation of the corneal surface as well as the microscopic examinations at the end of the three periods of observation, we said that KGF-2 provided a beneficial effect in the treatment of alkali burns of the cornea. of epithelial stem cell analysis of that KGF-2 accelerated the corneal epithelial healing by markedly stimulating epithelial stem cells proliferation and making them migrate to the central cornea.
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