Study On Organization, Physiological Activity And Mechanism Of The Corneal Flap Striae In Rabbit Post-LASIK Eyes

Background: With the rapid development of refraction surgical technique, laser in situ keratomileusis(LASIK) is currently the most common refractive surgical procedure, and it acts as the most effective surgical procedure with satisfactory results, such as little or no significant corneal haze, less regression, short time recovery of vision, less pain, and good refractive predictability are the main advantages of the surgery. Corneal flap stria is one of the common complications in post-LASIK eyes. However, histopathological reports about corneal flap striae after LASIK are limited.Purpose: The aim of the study was to evaluate and compare the differences of histomorphology, enzyme activity, the expression of cytokines in corneal cells, as well as gene expression between the rabbit eyes received corneal flap striae after LASIK and the eyes received single LASIK. These results were expected to provide a theoretical foundation for the clinical therapy of corneal flap striae after LASIK.Methods: Fourteen adult New Zealand white rabbits were separated into 7 groups, two animals in a group. The right eye of each animal was received LASIK combined with complication of flap striae, the left one was received single LASIK. Two of these animals were killed randomly at 1 day, 3 days, 1 week, 2 weeks, 1 month, 3 months and 6 months postoperatively. The obtained corneal flaps were received histomorphometry observation using hematoxylin and eosin(HE), periodic acid-schiff(PAS) and Masson staining. Meanwhile, enzyme histochemical strain of frozen sections was applied to detect the enzyme activity of adenosine triphosphatase(ATPase) and glucose-6-phosphatase(G-6-Pase) of epithelial cells. The apoptotic cells and peripheral corneal epithelial proliferating cell nuclear antigen(PCNA)-positive cells were detected. Furthermore, the number of corneal stromal cells and the expression of interleukin 1 beta(IL-1β) and transforming growth factor beta 2(TGF-β2) were determined to evaluate the effect of corneal flap striae after LASIK on the early corneal wound healing response. Additionally, by the RNA-sequencing of rabbit cornea, new transcripts were predicted. Differentially expressed genes(DEGs) between the corneal flap striae and control groups were identified, and functions and interactions of DEGs were analyzed.Results: In the control corneas, the epithelial layers raised around the corneal flap edge adhesion, the arrangement of stroma collagen fibers was regular according to HE staining. PAS and Masson staining failed to show evident positive tinction. In the flap striae group corneas, the hyperplasia of epithelium was irregular in whole flap because the undulation of epithelial basal membrane in all time points. The epithelium was thin(about 2-3 layers) in striae ridge while was thick(about 8-12 layers) in striae trough. The 2/3 stroma collagen fibers was disarranged as the ‘wave-formed’ corneal flap striae after 2 weeks. PAS stain and Masson stain showed positive tinction in the area of adhesion between the corneal flap and stroma.ATP enzyme, G-6-P activity and the number of PCNA-positive cells of corneal epithelial cells in the striae group were not statistically significant comparing to the control group. Within one week after LASIK, the number of apoptotic corneal stromal cells was signifiacntly higher in the striae group. Furthermore, at day 5 and 7 after LASIK, the expression of IL-1β in corneal stromal cells was also distinctly higher in the striae group. Additionally, the difference of TGF-β2 expression in corneal stromal cells between the two groups was not significant.Furthermore, a total of 210 DEGs(98 upregulated genes and 112 downregulated genes) were identified between the corneal flap striae and control groups. Most of DEGs were involved in functions of binding, catalytic activity and celluar processes. According to the analysis of PPI network, some downregulated genes(e.g. ITGA2) and upregulated genes(e.g. EGFLAM、COL1A2 and COL1A1) may be closely associated with the occurrence of corneal flap striae after LASIK.Conclusions: The cells of corneal flap striae were changed on morphology, and corneal flap striae promoted corneal cells apoptosis, negatively affected the remodeling process of corneal cells, which inhibited the corneal wound healing. Besides, corneal flap striae changed expression of a set of genes.