| There has been a renewed interest in surface ablation modalities like epipolis laser in situ keratomileusis (Epi-LASIK) over the past few years to avoid laser in situ keratomileusis (LASIK)-related complications, such as ectasia and dry eyes, and flap-related complications. However, the development of corneal Haze is an important factor to restrict the corneal surface refractive surgery. Haze is that of the corneal epithelial and subepithelial stroma appearing in the cut area of the cornea in a curtain period of time after performing the Photorefractive keratectomy, and also one of the main complication after performing the excimer laser surface ablation procedure. Different surface ablation modalities differ from each other in the incidence of Haze. Once the corneal epithelium is damaged by trauma or refractive surgery, a series of wound-healing processes begins, with cytokine-mediated interactions between epithelial cells and keratocytes. The initial stage of wound healing in the cornea involves apoptosis of the keratocytes , which occurs in response to release of various cytokines by the injuryed epithelium. After apoptosis, the remaining keratocytes begin to proliferate and migrate into the area of the healing wound, which may lead to myofibroblast differentiation. Reactivation and repopulation of keratocytes may affect surgical outcomes, including corneal Haze and myopic regression. Because of a variety of cells and factors involved in the regulation of Haze, Haze formation is the result from the common roles of various factors, and is, therefore, a complicated process of corneal wound healing responses. In recent years, the mechanism and prevention of Haze formation after performing the surface ablation procedure have become the hotspots of the studies on excimer laser refractive surgery. This thesis is, through setting up the animal models of Epi-LASIK, off-flap Epi-LASIK and photorefractive keratectomy (PRK), to make a study on the mechanism of Haze formation from the angle of histopathology and the level of molecular biology and, at the same time, makes comparisons between all the corneal wound healing responses and the differences of corneal Haze formation following different modalities of performing surface ablation procedure to provide the basis for the selection of a suitable surface ablation modality in photorefractive corrections.Partâ… Comparative study on the early corneal wound healing response following Epi-LASIK , off-flap Epi-LASIK and PRK in Rabbits .Objective:To evaluate the effect of different methods of the epithelial removal and different ways of the epithelial healing on the early corneal wound healing response by comparing between Epi-LASIK, off-flap Epi-LASIK and PRK.Methods:50 healthy New Zealand white rabbits (100 eyes) were used for corneal surface ablation, and 48 of them (96 eyes) were, at random, divided into 3 surgical groups of Epi-LASIK, off -flap Epi-LASIK and PRK, with 32 eyes in each, and the other 2 of them (4 eyes) without any surgery were used as the control group. All surgical procedures were performed by the same surgeon. Surgical technique in Epi-LASIK and off-flap Epi-LASIK groups, Epikeratome (Moria E3, France) was used for the creation of epithelial flap (9.0mm diameter), the epithelial flap was repositioned in Epi-LASIK group and was completely removed in off-flap Epi-LASIK group. In PRK group, a blunt spatula was used to remove the central corneal epithelium (9.0mm diameter). Excimer laser ablation (-10.0D) was performed using Bausch & Lomb Technolas 217Z (Germany) on all the eyes for experiment, with a 120μm ablation depth in a 6.0 mm ablation zone. The rabbits in each group were respectively put to death at four time points of 4 hours, 1 day, 3 days and 7 days after the surgery, and then their corneas were taken for experiment: 1 The light micrograph and transmission electron microphotograph were used to observe the healing ways of the corneal epithelial cells after Epi-LASIK, off-flap Epi-LASIK and PRK. 2 The immunohistochemical technique was used to detect the number of the inflammatory cells and apoptotic cells in the corneal stroma and the expressions of both IL-1βand proliferin Ki-67 in the corneal stroma.Results:1 Different feature of the epithelial healing ways after Epi-LASIK, off-flap Epi-LASIK and PRK were evaluated by light micrograph and transmission electron microscopy: (1) 4 hours after Epi-LASIK, edema of most cells appeared, together with slight injury in the epithelial surface and the combination of the epithelium and stroma were slightly loose. 1 day later, the epithelial flap was getting a little thinner, the microvilli on the epithelial surface reduced, a small number of the basal cells showed edema. The above indicated that the cell viability of epithelial flap was still better in a proper way. 3 days later, the edema of the epithelial flap could be seen. 7 days later, the edema of the epithelial flap subsided and the flap basically returned to the normal structure, but the number of the epithelial layer increased more than before (about 7-9 epithelial cell layers) and the microvilli on the epithelial surface were normal. (2) 4 hours after off-flap Epi-LASIK, no epithelium could be seen on the stromal surface in the corneal ablated zone. 1 day later, a layer of new corneal epithelial cells could be seen covering the ablated zone, and thereafter, the new corneal epithelium increased layer by layer to 7-8 epithelial cell layers 7 days after the surgery, and their structure become normal. (3) 4 hours after PRK, no corneal epithelium could be seen in the ablated zone. 1 day later , a single layer of epithelial cells appear expending towards the ablated zone from the margin of the ablated zone, but the bare stroma could still be seen there in the ablated area. Thereafter, the new corneal epithelium gradually covered the ablated area and their layers increased, but the arrangement of the corneal epithelial cells were in somewhat bigger disorder. 7 days later, the epithelium comed to 5-7 epithelial cell layers, and the epithelial cells were a little bit in disorder, the basement membrane is intact and some microvilli could be seen on the epithelial surface. 2 Corneal healing response: (1) 4 hours after Epi-LASIK, off-flap Epi-LASIK and PRK, the inflammatory cells and apoptotic keratocytes and the expression of IL-1βand proliferin ki-67 appeared in the anterior corneal stroma. The inflammatory cells and TUNEL-positive cells were most at 1 day and the expression of IL-βand Ki-67 reached a peak at 3 days after 3 surface ablation techniques. (2) At 7 days after surgery, the inflammatory cells were not found in Epi-LASIK and off-flap Epi-LASIK groups and they were scanty in PRK group, and there were less TUNEL-positive cells in corneal stroma and the expression of IL-βand Ki-67 began to descend in the 3 groups. (3) In PRK group, at every time point, the number of the inflammatory cells and apoptotic cells were more and the expression of IL-1βand Ki-67 in corneal stroma were notably higher than those in Epi-LASIK and off-flap Epi-LASIK group and the comparative differences between the 3 groups were significant statistically (p<0.05). At the different time points in Epi-LASIK group, the number of the inflammatory cells and apoptotic keratocytes were more and the expression of IL-1βand Ki-67 in corneal stroma were higher than those in off-flap Epi-LASIK group. The differences of the number of the inflammatory cells and apoptotic cells between Epi-LASIK and off-flap Epi-LASIK at 1 day and 3 days postoperatively were significant statistically (p<0.05). The significant differences were also found in the expression of IL-1βand Ki-67 between the two groups at 1 day, 3 days and 7days postoperatively (p<0.05).Conclusions:Different methods of the epithelial removal and different ways of the epithelial healing can cause differences in early corneal wound healing response after Epi-LASIK, off-flap Epi-LASIK and PRK, and off-flap Epi-LASIK has the slightest response. Therefore, off-flap Epi-LASIK possibly has a potential advantage over Epi-LASIK and PRK in reducing the early postoperative pain and the forward incidence of corneal Haze after surface ablation laser refractive surgery.Partâ…¡Comparative study on corneal Haze formation following Epi-LASIK , off-flap Epi-LASIK and PRK in RabbitsObjective:To explore the differences and causes in corneal Haze formation and development after Epi-LASIK, off-flap Epi-LASIK and PRK.Methods:47 healthy New zealand white rabbits (94 eyes) were ready for the experiment, and 45 of them (90 eyes) were randomly divided into 3 surgical groups of Epi-LASIK, off-flap Epi-LASIK and PRK, with 30 eyes in each , and the other two rabbbits (4 eyes) without any surgery were used as the control group. In the experiment , All surgical procedures were performed by the same surgeon. Surgical technique in Epi-LASIK and off-flap Epi-LASIK groups, Epikeratome (Moria E3, France) was used for the creation of epithelial flap (9.0mm diameter), the epithelial flap was repositioned in Epi-LASIK group and was completely removed in off-flap Epi-LASIK group. In PRK group, a blunt spatula was used to remove the central corneal epithelium (9.0mm diameter). Excimer laser ablation (-10.0D) was performed using Bausch & Lomb Technolas 217Z (Germany) on all the eyes for experiment, with a 120μm ablation depth in a 6.0mm ablation zone. Then, experimental observation was made as follows: 1 The time of corneal epithelial healing and corneal Haze were observed at regular intervals by slit-lamp microscope, and the time of corneal epithelial healing of the rabbits in every surgical group were recorded and the evaluation of Haze is made at different grades. 2 All the rabbits in every group were put to death at 3 time points of 1 week, 1 month and 3 months after the surgery, and then, their corneas were taken. Morphological changes of the cornea were observed by transmission electron microscope at the different time points after the surgeries.The experssion ofα-SMA and collagen typeâ…¢in the corneas, which were closely related to Haze formation, was evaluated by immuno- histochemistry analyses and Western blot analyses.Results:1 The time of corneal epithelial healing: The mean time of epithelial healing were respectively 4.1±1.0, 3.8±1.8 and 4.4±1.2 days in the treated eyes of Epi-LASIK,off-flap Epi-LASIK and PRK. No significant difference was noted in epithelial healing time between the 3 surgical groups (F=0.88, p>0.05). 2 Haze scores: (1) The incidence of Haze was 40% (6 eyes, grade 0.5 ; 2 eyes, grade 1) in the Epi-LASIK group and 35% (5 eyes, grade 0.5 ; 2 eyes, grade 1) in the off-flap Epi-LASIK group and 80% (8 eyes, grade 0.5; 5 eyes, grade 1) in the PRK group 1 month after surgery. There was a significant difference between the PRK group and the other two groups (p<0.05), and there was no statistically significant difference between Epi-LASIK and off-flap Epi-LASIK groups (p<0.05). (2) The incidence of Haze was 30% (3 eyes, grade 0.5) in the Epi-LASIK group and 30% (2 eyes, grade 0.5; 1 eye, grade 1) in the off-flap Epi-LASIK group and 60% (4 eyes, grade 0.5; 2 eyes, grade 1) in the PRK group 3 months after the surgery. There were no statistically significant differences between the 3 groups (p>0.05). 3 the observation by transmission electron microscopy: (1) 1 month after the surgery in the Epi-LASIK, off-flap Epi-LASIK and PRK groups , the stratification and integrity of the epithelial cells appeared morphological normal basically, the fibroblasts in the anterior corneal stroma increased obviously, and their morphous and arrangement were irregular, the nucleuses became larger, the nucleolus were clear, euchromatin and rough endoplasmic reticulum in the cells were rich, the metabolism was higher and the new collagen increased significantly and their arrangement were in disorder. The degree of the above was more serious in the PRK group than in the Epi-LASIK and off-flap Epi-LASIK groups, and there was no notable difference btween Epi-LASIK and off-flap Epi-LASIK. (2) 3 months after the surgery in the three groups , the number of fibroblasts in the corneal stroma started to reduce, their shapes and arrangement are more regular than before, the collagen arrangement remained slightly in disorder, and the degree of the above was a bit more serious and the collagen fibres were thicker and larger in the PRK group than in the other two groups. 4 The expression ofα-SMA in the cornea was evaluated by immunohistochemistry and Western blot analyses : The expression of a-SMA in the cornea started to increase at 1 week after Epi-LASIK, off-flap Epi-LASIK and PRK. 1 month later, the expression of a-SMA in the cornea reached the peak. The level of expression ofα-SMA in the cornea was PRK > Epi-LASIK > off-flap Epi-LASIK at 1 week, 1 month and 3 months after the surgery. The differences between the 3 groups and the control group were significant statistically (p<0.05), and between the 3 groups at the 3 time points were also significant statistically (p<0.05). 5 The expression of collagen typeâ…¢in the cornea was evaluated by immunohistochemistry and Western blot analyses: The expression of collagen typeâ…¢in the cornea increased significantly at 1 week and reached the peak at 1 month after Epi-LASIK, off-flap Epi-LASIK and PRK. The level of expression of collagen typeâ…¢in the cornea was PRK > Epi-LASIK > off-flap Epi-LASIK at 1 week, 1 month and 3 months after the surgery. The differences between the 3 groups and the control group were significant statistically (p<0.05), and between the 3 groups at the 3 time points were also significant statistically (p<0.05).Conclusions:There is no significant benefit in retaining the epithelial flap to promote corneal epithelial healing and reduce the development of corneal haze and postoperative pain in Epi-LASIK. Reducing corneal epithelial injury as many as possible is the key factor to prevent Haze formation in the surface ablation procedure. off-flap Epi-LASIK is hopefully to be the preferred corneal surface refractive surgery clinically because its postoperative cornceal epithelial healing is faster and corneal Haze response are less comparing with Epi-LASIK and PRK.Partâ…¢Expressions of TGF-β1 and CTGF in the cornea and their effects on corneal Haze formation after Epi-LASIK , off-flap Epi-LASIK and PRK in RabbitsObjective:To investigate the relationship between TGF-β1 and GTGF and their effect on corneal Haze formation and development by observing the experssion of TGF-β1 and GTGF in the corneas after Epi-LASIK, off-flap Epi-LASIK and PRK..Methods:47 healthy New zealand white rabbits (94 eyes) were ready for the experiment, and 45 of them (90 eyes) were randomly divided into 3 surgical groups of Epi-LASIK, off-flap Epi-LASIK and PRK, with 30 eyes in each , and the other two rabbbits (4 eyes) without any surgery were used as the control group. In the experiment , All surgical procedures were performed by the same surgeon. Surgical technique in Epi-LASIK and off-flap Epi-LASIK groups, Epikeratome (Moria E3, France) was used for the creation of epithelial flap (9.0mm diameter), the epithelial flap was repositioned in Epi-LASIK group and was completely removed in off-flap Epi-LASIK group. In PRK group, a blunt spatula was used to remove the central corneal epithelium (9.0mm diameter). Excimer laser ablation (-10.0D) was performed using Bausch & Lomb Technolas 217Z (Germany) on all the eyes for experiment, with a 120μm ablation depth in a 6.0mm ablation zone. Then, experimental observation was made as follows: 1 The time of corneal epithelial healing and corneal Haze were observed at regular intervals by slit-lamp microscope, and the time of corneal epithelial healing of the rabbits in every surgical group were recorded and the evaluation of Haze is made at different grades. 2 All the rabbits in every group were put to death at 3 time points of 1 week, 1 month and 3 months after the surgery, and then, their corneas were taken. After that, the analyses of immunohistochemistry and Western Blot are adopted to determine the experssion of the two main regulatory factors of TGF-β1 and GTGF in the cornea after the 3 surgeries.Results:1 The expression of TGF-β1 in the cornea was examined by immunohistochemistry and Western blot analyses: The expression of TGF-β1 in the control group was very weak and low. The expression of TGF-β1 notably increased at 1 week and reached the peak at 1 month and was still comparatively higher at 3 months after Epi-LASIK, off-flap Epi-LASIK and PRK. In the PRK group, the expression of TGF-β1 was obviously higher than in the Epi-LASIK and off-flap Epi-LASIK groups 1 week , 1 month and 3 months after the surgery, and the experssion of TGF-β1 in the Epi-LASIK group was slightly higher than in the off-flap Epi-LASIK group. The differences between the 3 groups and the control group were significant statistically (p<0.05) and the differences between the 3 surgical groups at the 3 time points were also significant statistically (p<0.05). 2 The expression of CTGF in the cornea was examined by immunohistochemistry and Western blot analyses : The expression of CTGF started to increase at 1 week and reached the highest point at 1 month and remained comparatively higher at 3 months after Epi-LASIK, off-flap Epi-LASIK and PRK. The expression of CTGF in the PRK group is obviously higher than in the Epi-LASIK and off-flap Epi-LASIK groups 1 week, 1 month and 3 months after the surgery (p<0.05). The expression of CTGF in the Epi-LASIK group is a little bit higher than in the off-flap Epi-LASIK group at the 3 time points after the surgery. The difference between the two groups were no significant statistically (p<0.05) at 1 week and the significant differences were found at 1 month and 3 months postoperatively (p<0.05). The differences between the 3 groups and the control group were significant statistically (p<0.05).Conclusions:TGF-β1 and GTGF mediated the coneal wound healing response following the corneal surface ablation, they have a synergistic effect and are closely related to Haze formation and development. The expression of TGF-β1 and GTGF in the off-flap Epi-LASIK group are notably lower than in Epi-LASIK and PRK groups at all the time points after the surgery, and the differences between the 3 groups are significant statistically (p<0.05). All of the above indicate that off-flap Epi-LASIK has an obvious advantage over Epi-LASIK, especially over PRK, in reducing corneal Haze formation after surface ablation refractive surgery.
|
PDF Full Text Request