The Study Of Corneal Wound Healing And Mechanism After Femtosecond Laser-assisted Refractive Lenticule Extraction

PartⅠ The study of corneal wound healing after femtosecond laser-assisted refractive lenticule extractionPurpose:To evaluate the morphological changes during corneal wound healing after femtosecond laser-assisted refractive lenticule extraction (ReLEx). Methods: Twenty-one New Zealand white rabbits were divided into group A of12rabbits and group B of9rabbits. Group A underwent small incision lenticule extraction (SMILE) and group B underwent femtosecond laser-asssisted laser in situ keratomileusis (LASIK) with the same refractive corrections of-6.00DS/-1.00DC. The surface regularity on the refractive corneal lenticules and flap was evaluated by scanning electron microscopy. Corneas were given a slit-lamp examination postoperatively. Light microscopy and transmission electron microscopy was applied to observe changes in the cornea at postoperative week1, months1, and3. The opposite eye of three rabbits served as a control. Results:All the operations were sucessful and the corneal healing postoperatively was uneventful in both groups. The recovery of corneas was faster in SMILE group by slit-lamp examination. Light microscopy confirmed the mild edema of comeal stroma with clear intrastromal crevice in SMILE group and the adhesion of the flap and stromal bed in LASIK group at postoperative week1. The filling of epithelial cells at the flap margin and the irregular arrangement of collagens were demonstrated in both groups. At postoperative month1, stromal edema was dissipated and collagens become more regular in both groups. The intrastromal crevice disappeared in SMILE group at postoperative month3. Transmission electron microscopy also revealed that the edema of keratocyte mitochondrion within one month and the keratocyte recovered to normal in month3. The surface regularity of the front surface was better than that of the back. Conclusions:Smooth intrastromal lenticule can be completed safe by femtosecond laser. The corneal healing in ReLEx group was better than that in LASIK group. Early intrastromal crevice should be pay attention to clinically.Part Ⅱ The study on the keratocyte apoptosis in the stroma after femtosecond laser-assisted refractive lenticule extraction Purpose:To determine the characteristics of keratocyte apoptosis after femtosecond laser-assisted ReLEx. Methods:Twenty-four New Zealand white rabbits were divided into group A of12rabbits and group B of12rabbits. Group A underwent SMILE and group B underwent femtosecond laser-asssisted LASIK with the same refractive corrections of-6.00DS/-1.00DC. Keratocyte apoptosis was evaluated by TUNEL and transmission electron microscopy at postoperative4th and24th hour. The opposite eye of six rabbits served as a control. Results:TUNEL positive cells can be abserved in both experimental groups but not detected in control group. At postoperative4h and24h, there were statistically less TUNEL positive cells in SMILE group (P<0.01). Transmission electron microscopy demonstrated the apoptosis or injury of keratocytes. Conclusions:ReLEx can trigger less keratocyte apoptosis and small incision and free of opening corneal cap may take account.Part Ⅲ The study of keratocyte proliferation and inflammation after femtosecond laser-assisted refractive lenticule extractionPurpose:To determine the characteristics of keratocyte proliferation and inflammation after femtosecond laser-assisted ReLEx. Methods:Fourty-eight New Zealand white rabbits were divided into group A of24rabbits and group B of24rabbits. Group A underwent small incision lenticule extraction (SMILE) and group B underwent femtosecond laser-asssisted laser in situ keratomileusis (LASIK) with the same refractive corrections of-6.00DS/-1.00DC. The corneas were subjected to immunofluorescent staining for Ki-67and CD11b at postoperative day1and3, week1, month1. The opposite eye of six rabbits served as a control. Results:Immunostaining of Ki-67showed statistically less expression in corneas that underwent ReLEx than LASIK(P<0.05). The expression of Ki-67was highest at postoperative day3and little at postoperative month1. Less CD11b-positive cells were also detected in ReLEx group(P<0.01). Conclusions:ReLEx may trigger less keratocyte proliferation and inflammation and small incision and free of opening corneal cap may take account.Part IV The study of collagen cross-linking with riboflavin in a femtosecond laser-created pocketPurpose:To determine the characteristics of corneal healing after collagen cross-linking (CXL) with a femtosecond laser. Methods:The right eyes of10male New Zealand white rabbits were treated with CXL with a femtosecond laser. A femtosecond laser was used to create an intrastromal pocket with80-μm depth and7-mm diameter. Intrastromal administration of0.1%riboflavin solution was made into the femtosecond laser-created pocket and the cornea was irradiated with3mW/cm2ultraviolet A light of mean370nm wavelength for10minutes. Corneas were given a slit-lamp microscopy postoperatively. The corneal topography, pachymetry and Scheimpflug images were evaluated by the Pentacam preoperatively and at postoperative day1, week1, week2, and months1,3, and6. Light microscopy was applied to observe pathological changes in the cornea at postoperative month6. Results:Cornea healing postoperatively was uneventful in all cases. Stromal pocket was apparently visible shortly after the operation and disappeared within days. In8of10cases, a demarcation-line-like change in the stroma was visible over the whole cornea by slit-lamp microscopy as early as2weeks after CXL treatment. The central corneal thickness (CCT) in the treated rabbit cornea reached the peak value at postoperative day1, which descended gradually to the minimum at month1and returned to a high level at postoperative months3and6. The CCT data postoperatively was significantly thicker than that preoperatively, except at postoperative month1(P>0.05). Steepening of the operative area was found at the corneal front at postoperative day1, which recovered dramatically after only1week. Then the treated area gradually flattened over the course of follow-up. In8of10rabbits, a demarcation line-like change in the stroma was visible in the Scheimpflug image at slightly increased contrast as early as1month after CXL treatment. The micromorphologic examination also confirmed the existence of the demarcation line and change. Crystalline lens transparency remained unchanged all the time. Conclusions:CXL with a femtosecond laser appears to be safe. The cornea can realize a faster, uneventful recovery. The stromal demarcation line may be a direct clinical sign to detect an effective corneal cross-linking during the early phase postoperatively.PartⅤ Clinical Study of femtosecond laser-assisted refractive lenticule extractionPurpose:To evaluate the clinical efficacy and safety of after femtosecond laser-assisted ReLEx. Methods:This is a prospective clinical trial involved12cases (12eyes). The patients aged from18years old to53years old (an average of32.7± 10.5years old). Their corneal thickness were488μm～578μm (an average of528.92μm±26.64μm). ReLEx was used to correct myopia of-4.88DS to-9.50DS of spherical equivalence (an average of-6.97±1.36DS). The patients were followed up for6to10months with visual acuity, manifest refraction, slit-lamp microscopy, intraocular pressure, wavefront aberration, comeal topography. Comeal wound healing was evaluated by optical coherence tomography (OCT) and confocal microscope postoperatively. Results:The procedure was completed sucessfully in every patients. Cornea healing postoperatively was uneventful in all cases. The patients’ UCVA improved to0.6-1.2while BCVA improved0.7-1.2postoperatively. BCVA improved more than2lines in2eyes whereas1line in3eyes. No eye lost BCVA. The difference between achieved and target refraction postoperatively was within±0.75DS. OCT showed that mild stromal edema can be detected at postoperative day1and recovered dramatically at postoperative week1. The stability of corneas was achieved at postoperative month1and3. In vivo confocal microscopy showed light-scattering particles at the interface. The epithelial plug at the flap margin and microfolds at the lenticule margin were also confirmed. At month1, secondary fibrosis could be observed at flap edge. Conclusions:ReLEx appears to be efficiency and safety for myopia. And postoperative cornea healing may be uneventful and corneal stability can be achieved in one month.