[Effects of amniotic extraction on epithelial wound healing and stromal remodelling after excimer laser keratectomy in rabbit cornea].

OBJECTIVE: To investigate the effects and mechanism of amniotic extraction on corneal healing after photorefractive keratectomy (PRK).

METHODS: Experimental Study. Thirty-six New Zealand rabbit corneas were performed with PRK models (-10 diopters, 6.5 mm diameter). According to random number table, all eyes were divided into three groups, including treated with amniotic extraction, 0.1% dexamethasone and excipient respectively after operation. Clinical and histopathologic examinations were taken by slit-lamp microscope and light microscope. Corneal epithelium reparation was observed by fluorescent staining. Corneal stroma cell apoptosis was evaluated by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Myofibroblast generation was evaluated by immunofluorescence checking the expression of alpha-smooth muscle actin (α-SMA). The number of TUNEL and α-SMA positive cells was analyzed to explore the effects on corneal haze. The haze grading was compared between groups using Kruskal-Wallis H test. Mean values for each experiment were compared between groups using a one-way analysis of variance and LSD-t test.Spearman rank analysis was used to evaluate the correlation between the haze grading and the expression of TUNEL positive cells and α-SMA.

RESULTS: The corneas of seventy-two eyes reepithelialized in 6 days after operation. The average epithelium repair time of the AE group was (4.12 ± 0.62) d, the dexamethasone group was (5.25 ± 0.78) d, and the excipient group was (4.96 ± 0.73) d. The progression of reepithelialization was significantly faster in the AE group than the other two groups (F = 14.144, P < 0.01). The haze appeared in the first week after the PRK in all three groups, increased after 3-4 weeks, and relieved after 8 weeks. The degree of haze was significantly lower in the AE group than the other two groups in the first week (Vs. dexamethasone group, H = 3.995, P < 0.05; vs. excipient group, H = 12.77, P < 0.01), in the 4th week (Vs. dexamethasone group, H = 4.468, P < 0.05;vs. excipient group, H = 9.003, P < 0.01), and 8th week (Vs dexamethasone group, H = 4.397, P < 0.05;vs. excipient group, H = 5.744, P < 0.05) after PRK. The TUNEL-positive cells appeared in the central anterior stroma at the first week after surgery. And the number of TUNEL-positive cells in the AE group was (2.2500 ± 0.3750) cells/HP, the dexamethasone group was (4.5000 ± 0.7500) cells/HP, and the excipient group was (7.1250 ± 0.9063) cells/HP. The number of TUNEL-positive cells in AE group was less than those in the other two groups (Vs. dexamethasone group, t = 4.26, P < 0.01; vs. excipient group, t = 8.13, P < 0.01). The TUNEL-positive cells were only found in the excipient group (2.8750 ± 0.6563)/HP in 4th week after operation.It was significantly different between the dexamethasone group and the excipient group (t = 9.01, P < 0.01). There were no significant TUNEL-positive cells in 8th weeks in all three groups.α-SMA-positive cells started to appear apparently at the first week after surgery in the dexamethasone and excipient groups, and the peaks appeared at the 4th week after treatments, and there were still a lot of α-SMA-positive cells in corneal stroma at the 8th week after operation in both groups.On the contrary, there were no significant α-SMA-positive cells in the AE group all the time after surgery. The statistical significant difference can be found between the AE group vs. the dexamethasone and excipient groups in the first week (t = 28.62, 36.55;P < 0.01), in the 4th week (t = 30.40, 35.96; P < 0.01), and in the 8th week (t = 34.02, 38.32; P < 0.01).Spearman rank analysis demonstrated that the formation of haze was proportional to the expression of TUNEL positive cells (r = 0.881, P < 0.01) and α-SMA (r = 0.710, P < 0.01).

CONCLUSION: Amniotic extraction can reduce the formation of haze, which was more effective than 0.1% dexamethasone.It might release certain factors which were transported into corneal matrix, then affected the healing of epithelial cell by interacting with the corneal cell factors, reducing the cell apoptosis, corneal wound healing response and rebuilding the corneal matrix with less myofibroblast, collagen and scar and finally reduce the formation of haze.