The Study For Early Corneal Wound Healing After Epipolis Laser In-Situ Keratomileusis On Rabbits Eyes

Objective To evaluate the changes of morphology,histological ultrastructure and viability of corneal epithelial flap cells and its effects to the early corneal wound healing after epipolis laser in-situ keratomileusis (Epi-LASIK)surgery on rabbits eyes. In order to reveal characteristic of corneal wound healing of Epi-LASIK.The study includes two partsExperiment 1 To study the changes of morphology,histological ultrastructure and viability of corneal epithelial flap cells after Epi-LASIK surgery on rabbits eyes.Experiment 2 To study the effects of keeping corneal epithelial flap to the early changes of corneal wound healing after Epi-LASIK surgery on rabbits eyes.MethodsExperiment 1 Epi-LASIK was performed on New Zealand rabbits eyes, all treated-eyes were randomly divided into four groups and were humanly killed at 1,3,5,7 days postoperatively, And rabbits eyes without treatment were served as blank controls. Histological structure were examined by light,trasmission electron microscopy(TEM ) on Epi-LASIK specimens. Epithelial cells viability were assessed by enzymohistochemistry. Immunohistochemistry staining PCNA were performed to detect proliferation of peripheral corneal epithelial cells. Apoptotic cells were detected by TUNEL (TdT-mediated dUTP nick-end labeling).Experiment 2 Epi-LASIK or photorefractive keratectomy(PRK) was performed on New Zealand rabbits eyes, treated-eyes were randomly divided into four groups(1d,3d,5d,7d ) and rabbits eyes without treatment were served as blank controls. To compare the changes of corneal wound healing between Epi-LASIK groups and PRK groups,including keratocytes apoptosis,the number of keratocytes,the number of inflammatory cells in the central anterior stroma and expression of Interleukin (IL-1β),expression of transforming growth factor(TGF-β2)for ablation area of corneal epithelium and stroma .ResultsExperiment 1(1)Morphology :Epithelial flap separated by KM5000D type epi-keratome retained its typical stratification and integrity and the basement membrane including lamina densa and lamina lucida by TEM. Epithelial flap keeped normal morphology and compaginated with stroma at 7 days Epi-LASIK postoperatively.(2)Cell enzyme viability :ATP enzyme viability of epithelial cells (enzyme viability of epithelial flap cells to peripheral epithelium ) were79%,58%,69%,86% and G-6-P enzyme viability were79%,63%,77%,97% at 1,3,5,7days Epi-LASIK postoperatively respectively. These data indicate viability of epithelial cells is high at 1 day,lower at 3 days,upgrade at 5 days , recovers normal viability at 7 days postoperatively.(3)Cell proliferation and apoptosis :There was no statistically significant difference in the number of PCNA of peripheral epithelial cells among four Epi-LASIK groups and control group(F=1.10,P>0.05). No epithelial flap cell apoptosised . Reposition of epithelial flap didn't stimulate peripheral epithelial cells to proliferate and epithelial cells to apoptosis. The results indicate that epithelial flap cells keep normal physiological functions.Experiment 2(1)Keratocyte apoptosis: No significant difference in TUNEL positive cells between Epi-LASIK group and PRK group at 1 day postoperatively (t=-0.33,P>0.05); At 3,5,7days postoperatively, there was a significant increase in keratocyte apoptosis in PRK groups to Epi-LASIK groups(t=-7.05,t=-5.93,t=-0.33,均P<0.01). These data confirm that less keratocyte apoptosis in the Epi-LASIK groups compared with PRK groups .(2)The number of inflammatory cells: There was a significant difference from the number of inflammatory cells increased in the corneal stroma of PRK groups compared with Epi-LASIK groups at 1,3 days postoperatively(t=12.42,P<0.01;t=4.57,P<0.05),and no inflammatory cell infiltrated for PRK,Epi-LASIK groups at 5,7 days postoperatively. We consider that less inflammatory cells infiltrated for Epi-LASIK groups compared with PRK groups.(3)The number of keratocytes: The number of keratocytes were 18.33±7.03,19.20±3.03 pieces in 400 magnification light microscope for 1d,3d post-Epi-LASIK groups respectively and no significant difference compared with the control group(t=0.19,t=-0.24, P>0.05). There was a significant difference from less the number keratocytes of Epi-LASIK groups compared with PRK groups at 1,3 days postoperatively(t=2.59,P<0.05,t=3.32,P<0.01). The number keratocytes were 33.57±10.03,42.00±9.72 pieces in 400 magnification light microscope for 5d,7d post-Epi-LASIK groups respectively and there was statistically significant difference compared with the control group(t=3.67,t=-7.99,均P<0.01). The number keratocytes was no significant difference at 5 days between post-Epi-LASIK and post-PRK(t=0.22,P>0.05). And there was a significant difference less at 7 days post-Epi-LASIK(t=4.35,P<0.01 ) . The number keratocytes were no changes at 1,3 days postoperatively and increased at 5,7 days for post-Epi-LASIK groups. These data indicate that corneal wound healing response is low and is beneficial to recover post- Epi-LASIK.(4)Morphology at 7 days postoperatively: Epi-LASIK treated eyes: Epithelial flap compaginated with stroma , collagen fibers keeped well-arranged and corneal stroma cells keeped resting state. PRK treated eyes: Epithelial flap pasted the stroma, collagen fibers presented derangement, karyoplasm proportion were get largerer and cellular organ were abundance, corneal stroma cell presented proliferation state. These data indicate that epithelium cohered with stroma tighter,collagen fibers keeped more well-arranged,more keratocytes to stand still in Epi-LASIK than PRK .(5)Expression of IL-1βand TGF-β2 in the corneaNo significant difference from the expression of TGF-β2 in the corneal epithelium between Epi-LASIK and PRK groups at 3,5,7days postoperatively respectively(t=0.06,t =-2.0,t =-1.8,均P>0.05)and the same to IL-1β(t =0.29,t =-1.29,t =-0.31,均P>0.05). And there was a significant difference from the expression of IL-1βand TGF-β2 decrease in the corneal stroma for Epi-LASIK compared with PRK at 1,3days postoperatively(TGF-β2 : t =-8.4,t =-3.1,均<0.05;IL-1β:t =-2.3,t =-2.7,均P<0.05) and no significant difference at 5,7days postoperatively(TGF-β2 :t =-1.9,t =-2.0,均P>0.05;IL-1β:t =1.8, t =-0.6均P>0.05). The expression of IL-1βand TGF-β2in the corneal stroma is low for post-Epi-LASIK groups compared with PRK, And its major mechanism may be the barrier of epithelial flap keeps integrity.Conclusion1,Epithelial flap separated by KM5000D type epi-keratome retained its typical stratification and integrity and the basement membrane including lamina densa and lamina lucida. And epithelial flap keeped normal morphology.2,Epithelial flap cells keeped high viability,normal morphology and physiological functions post- Epi-LASIK.3,The early corneal wound healing response were lower for post-Epi-LASIK groups compared with PRK . Its mechanism may be the organism emergency reaction and corneal epithelium-strom reaction were lower, Because of epithelial flap cells keeped high viability and may be barrier of integrity epithelial flap .