The Study Of Haze From The Comparison Between Epi-LASIK And LASEK

PartⅠA comparative histopathological study between epithelial flaps made with KM-5000DE automatic rotation microepikeratome and alcohol in rabbitsObjective: To investigate the safety and stability of epithelial flaps made with KM-5000DE automatic rotation microepikeratome in rabbits, and to compare the epithelial flaps made with KM-5000DE and with those made with alcohol in histopathology. Methods: Epithelial flaps were randomly made with KM-5000DE automatic rotation microepikeratome in one eye and with 20% alcohol (35s) in the other eye in ten New Zealand white rabbits (20 eyes), and one rabbit (2 eyes) without any treatment was used as control. The facility of microepikeratome and situation of flaps were observed, and the samples of epithelial flaps were further studied by light microscope (PAS staining) and transmission electron microscope. Results: All performances of KM-5000DE microepikeratome are smooth, and all of these epithelial flaps were in good position. Free flap happened in one eye, and no disintegrated flap happened. The margin of these flaps was smooth. Light microscopic examination (PAS staining) revealed that there was no stroma tissue in corneal epithelial flaps, basement membrane was continuous and the conjuction between basal cells was tight. Transmission electron microscopic examination further showed the integrity of basement membrane with a normal appearing lamina densa, lamina lucida and hemidesmosomes, and the ultrastructure of basal cells was normal. All flaps created with alcohol showed the margin was irregular, and disrupted flap happened in two eyes. PAS staining showed there was significant interruption along the basal surface of the flaps, and the conjuction between basal cells was loose. Transmission electron microscopic examination further showed most of the basement membrane was absent, more vesicles were found in basal cells, the space between basal cells enlarged, and the basement membrane did not contain lamina densa and lamina lucida. Conclusions: It is safe, effective and easy to make corneal epithelial flap by KM-5000DE automatic rotation microepikeratome. The epithelial flaps made by this type contain complete basement membrane, and the conjuction and morphology of basal cells are normal. However, the flaps made with alcohol show that the conjuction between basal cells is loose, the basement membrane is incomplete, and the cleavage plane appears to be predominant between basal cell and basement membrane. KM-5000DE automatic rotation microepikeratome can be used for experimental study of epiplolis laser in situ keratomileusis (Epi-LASIK) on rabbits.PartⅡComparison of corneal wound healing between epipolis laser in situ keratomileusis and laser-assisted subepithelilal keratectomy in rabbitsObjective: To compare corneal wound healing processes after epipolis laser in situ keratomileusis (Epi-LASIK) and laser-assisted subepithelial keratectomy (LASEK) and to evaluate their effects on corneal haze histopathologically and its possible mechanism. Methods: Forty-eight New Zealand white rabbits (96 eyes) were randomly treated with Epi-LASIK in one eye and LASEK in the other, and 2 rabbits (4 eyes) without any treatment were used as control. Corneal haze was observed by slit-lamp microscope in 1 and 3 months postoperatively. Morphological changes of corneal stromal cells were observed by transmission electron microscope. Corneal stromal cell apoptosis was evaluated by terminal deoxyribonucleotidyl transferase-mediated deoxynuridine triphosphate nick end labeling (TUNEL) assay. Stromal cells proliferation and myofibroblasts generation, extracellular matrix (ECM) changes (collagen typeⅢ) and transforming growth factor-β1 (TGF-β1) were evaluated by immunocytochemical analyses of the expression of Ki-67, a-smooth muscle actin (α-SMA), collagen typeⅢand TGF-β1 respectively. Myofibroblast generation and the changes of collagen typeⅢwere further testified by Western blot analyses ofα-SMA and collagen typeⅢ. The expression of TGF-β1mRNA was further evaluated by real time RT-PCR. The number of TUNEL, Ki-67 andα-SMA positive cells and expression ofα-SMA, collagen typeⅢand TGF-β1mRNA was semi-quantitatively analyzed to explore their effects on corneal haze. Results: The coreal haze was heavier in Epi-LASIK group than in LASEK group in 1 and 3 months postoperatively. Transmission electron microscopic examination showed that the apoptotic cells were easily found in early stage after LASEK, metabolism of organell in stromal cells was more active in LASEK group than in Epi-LASIK group, and arrangement of collagen in LASEK group was less regular than in Epi-LASIK group in the same period. Many TUNEL-positive cells appeared in the central anterior stroma in early stages after both LASEK and Epi-LASIK, and the number of TUNEL-positive cells reached a peak 24 hours after either LASEK or Epi-LASIK. There were more TUNEL-positive cells found in LASEK group than that in Epi-LASIK group in 1 week postoperatively (P<0．01) . There were a lot of Ki-67-positive cells in anterior corneal stroma after both LASEK and Epi-LASIK, but the peak appeared at 72 hours after the treatments. The Significant difference was also found in the number of Ki-67-positive cell between the two treatments in 1 week postoperatively (P<0．01) .α-SMA-positive cells started to appear apparently at 1 week after both LASEK and Epi-LASIK, and both the peaks appeared at 1 month after the treatments, and there were still a lot of a-SMA-positive cells in corneal stroma at 3 months after LASEK. There were more a-SMA-positive cells found after 1 week in LASEK group than that in Epi-LASIK group (P<0．01) . Both immunocytochemical analyses and western blot analyses showed that the expression of collagen typeⅢwas higher in LASEK group that that in Epi-LASIK group (P<0．01) , and the peak was also in 1 month postoperatively. Immunocytochemical analyses and real time RT-PCR of TGF-β1 also showed the same changes, and the difference between two surgical methods was significant statistically (P<0．01) . Conclusions: There are less stromal cell apoptosis, proliferation and myofibroblast generation, expression of collagen typeⅢand TGF-β1 in Epi-LASIK than that in LASEK. Therefore, comparing with LASEK, Epi-LASIK induces less corneal haze response in correcting myopia. Intact corneal epithelium may alleviate the degree of corneal haze after excimer laser refractive surgery.PartⅢEffects of corneal epithelial flaps with microepikeratome and alcolol on corneal keratocytes in vitroObjective: To compare the effects of corneal epithelial flaps made with microepikeratome and alcohol on corneal keratocytes in vitro, and to explore the mechanism of the intact corneal epithelial flap on corneal haze. Methods: Rabbit corneal keratocytes were isolated by enzyme digestion. Six groups are involved in this study, including blank control(1% serum DMEM), positive control(1ng/ml TGF-β1) , co-culture with corneal epithelial flap made by microepikeratome (Epi-LASIK method), co-culture with corneal epithelial flap made with alcohol (LASEK method), and two other co-culture groups with anti-TGF-β1 antibody added, respectively. Morphological changes of keratocytes were observed by phase contrast microscope. Keratocytes proliferation and myofibroblasts generation were evaluated by immunocytochemical analyses of the expression of Ki-67 and a-smooth muscle actin (α-SMA). Myofibroblast generation was further testified by Western blot analysis ofα-SMA. Both the number of Ki-67-positive cells and the expression ofα-SMA were semi-quantitatively analyzed. Results: Dendritic quiescent keratocytes in early stage underwent morphological changes characterized by marked cell spreading, and these changes were more obvious in two co-culture groups with epithelial flaps by microepikeratome and alcohol. Three days after co-culture, the number of Ki-67-positive cells in two co-culture groups with Epi-LASIK and LASEK was more than in blank control group (P<0．01) , and so were the two co-culture groups with Epi-LASIK and LASEK with anti-TGF-β1 antibody added (P <0．05 ) , however there was no statistical difference between two co-culture groups with Epi-LASIK and LASEK (P>0．05) , and so did the co-culture groups with Epi-LASIK and LASEK with anti-TGF-β1 antibody added (P>0．05) . There was also no statistical difference between the two co-culture groups added anti-TGF-β1 antibody and the blank control (P>0．05) . Immunocytochemical analyses of a-SMA showed that both cell skeleton and intensity of fluorescence were stronger in the two co-culture groups with Epi-LASIK and LASEK, and Western blots analyses of a-SMA further testified the same changes were founded in immunocytochemical analyses of Ki-67．Conclusions: It is the good barrier of intact corneal epithelium which separates corneal stroma from tears may be the main reason of haze difference between Epi-LASIK and LASEK. TGF-β1 probably plays an important role on corneal haze. However, further study is necessary to illustrate the exact mechanism.