Injury Of Rabbit Corneal Keratocytes After Epi-LASIK Or LASEK

Objectives To observe the injury of rabbit corneal keratocytes after epipolis laser in situ keratomileusis (Epi-LASIK) and 1aser-assisted subepithelial keratomileusis (LASEK).Methods 40 healthy Newzealand rabbits were randomly divided into Epi-LASIK or LASEK group, each has 20 rabbits. In each group, the twenty rabbits were divided into four subgroups (named A, B, C, D), each has five ones. One eye of each rabbit were treated with laser keratomileusis, while the fellow eye without any treatment (control eye). Different Diopters (D) of myopia correction were imitated in each keratomileusis group. i.e. -3.00D (A) , -9.00D (B) and -15.00D (C) for LASEK and Epi-LASIK. Rabbits in A, B and C group were observed for 24 hours. Five rabbits in each operation group were treated with -9.00D myopia correction and observed for 1 week. Rabbits were executed by aeroembolism, corneas of both operation and control eye were separated and divide to two parts along with the central line, one was fixed with formalin and imbedded with paraffin, and the other was fixed with glutaraldehyde and prepared for ultramicrocut. HE staining was use to observed histological changes of corneal tissue. Apoptosis of keratocyte were determined by TUNEL, and TUNEL positive cells in stroma were counted through high power lens to compare the difference between groups and operations. Besides, ultramicrostructure of keratocytes were observed through transmission electron microscopy.Results1. Histopathological changes of cornea:TUNEL positive cells were smaller and buffy stained. In normal cornea tissue, only epithelia layer exist such cells. There were positive cells in both stoma and epithelium after LASEK or Epi-LASIK.2. Change of ultrastructural organization of keratocytes: Under TEM, keratocytes were fusifom cells with intact mitochondria and nuclear. 24 h after operation, mitochondria appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae; endoplasmic reticulum was expended;chromatin was marginational.3. The cellular apoptosis level of the stroma after LASEK: 24h after LASEK:significant upgrade could be seen between all operated eyes and their control eyes(P <0.01);the level was significantly higher in -9.00 D and -15.00 D groups than that in -3.00 D group(P <0.01);there was no significance between -9.00 D and -15.00 D groups(P >0.05). 7d after LASEK:The difference between all operated eyes and their control eyes was insignificant(P >0.01); Compared with 24h group, the apoptosis level was significantly lessen.4. The cellular apoptosis level of the stroma after Epi-LASIK:24h after Epi-LASIK: The difference between operated eyes and their control eyes was insignificant in -3.00 D group(P >0.05)and significant in -9.00 D and -15.00 D groups (P <0.01); the level was significantly higher in -9.00 D and -15.00 D groups than that in -3.00 D group(P <0.01); there was no significance between -9.00 D and -15.00 D groups(P >0.05). 7d after Epi-LASIK: The difference between all operated eyes and their control eyes was insignificant(P >0.05); Compared with 24h group, the apoptosis level was significantly lessen.5. Between LASEK and Epi-LASIK groups: 24h after operation, the cellular apoptosis level of the stroma in LASEK group was significantly higher than that in Epi-LASIK group; after 7 days, there was no significant difference between the two groups. Conclusions Keratocytes apoptosis is an important form of acute phase of corneal response after LASEK or Epi-LASIK, and mitochondria damge existed universally in injured keratocyte. Level of apoptosis increased with the diopter of correction. Epi-LASIK caused less apoptosis compared to LASEK at the same diopter of correction.