微型角膜刀法准分子激光角膜上皮瓣下磨镶术后兔角膜上皮瓣结构及活性的研究

目的 探讨角膜微型刀上皮瓣下准分子激光原位角膜磨镶术（Epi-LASIK）术后上皮瓣形态结构、活性的变化、周边上皮（上皮瓣切口缘与角膜缘之间的区域）增生情况及其对细胞凋亡的影响。方法对29只新西兰白兔的双眼进行手术,28只眼行Epi—LASIK,24只眼行PRK,随机分为4组,在术后1、3、5、7d取标本,6只未手术眼作为空白对照组。采用透射电镜、光镜观察形态结构的变化;冰冻切片行酶组织化学检测上皮瓣细胞活性的变化;石蜡切片行凋亡及增殖细胞核抗原免疫组织化学检测。结果透射电镜发现KM5000D型上皮刀分离的上皮瓣基底膜完整,细胞之间结合紧密,术后上皮瓣与基质粘合牢固。1、3、5、7d上皮瓣细胞三磷酸腺苷酶和葡萄糖．6．磷酸酶活性（上皮瓣与周边上皮细胞酶活性比值）分别为79％、58％、69％、86％和79％、63％、77％、97％;各组Epi-LASIK眼周边上皮细胞活性与空白对照组差异无统计学意义（F=1．09,P〉0．05）。各组Epi-LASIK眼周边上皮增生与空白对照差异无统计学意义（F=1．10,P〉0．05）。Epi-LASIK和PRK组在术后1d基质细胞凋亡数为（3．429±1．693）和（3．796±1．998）个／10000μm^2,差异无统计学意义（t=-0．33,P〉0．05）;而Epi-LASIK眼术后3、5、7d基质细胞凋亡少于PRK,差异均有统计学意义（P〈0．01）。结论KM5000D型上皮刀分离的上皮瓣结构完整,细胞结合紧密,能保持较高活性,无明显刺激周边角膜上皮增生,有助于抑制凋亡的发生。（中华腠科杂志,2007,43：651-657）     

LASEK术后角膜上皮细胞的凋亡

目的探讨在准分子激光角膜上皮下磨镶术后,角膜上皮细胞凋亡的现象。方法采集行准分子激光角膜上皮下磨镶术后角膜上皮组织共100例,按制作上皮瓣时间5秒、10秒分两组各50例。用脱氧核苷酸末端转移酶缺口标记原位细胞检测法检测角膜上皮的凋亡细胞。并且观察术后的角膜上皮愈合、角膜上皮下雾样混浊分级和最佳矫正视力。结果在所有行准分子激光角膜上皮下磨镶术的患者角膜上皮均见有凋亡细胞,以基底细胞层多见。一组角膜上皮细胞的凋亡率为3.4%,二组的角膜上皮细胞的凋亡率为8%,两组比较P<0.05,差异有显著性。一组角膜上皮下雾样混浊haze术后10天和6月分别为20.99%、14.5%,而二组患者术后10天,6月上皮下雾样混浊为30%和20%。两组之间术后最佳矫正视力无差异。结论准分子角膜上皮下磨镶术后,角膜上皮细胞会发生凋亡现象,上皮细胞凋亡的轻重与上皮瓣制作的时间密切相关,且还与患者角膜上皮下雾样混浊产生有关。     

角膜上皮细胞基底膜的研究进展

角膜上皮细胞基底膜是一层很薄的组织,它的结构与其他部位的基底膜既有相似之处,也有独特的地方,在角膜创伤修复中有着重要的作用.基底膜组织的破坏会导致一系列棘手的角膜病变,而近来对角膜上皮细胞基底膜的研究使我们在这些病变的治疗方面有了新的人手点.就近年来角膜上皮细胞基底膜的研究进展做一综述.     

Alcohol debridement of the corneal epithelium in PRK and LASEK: an electron microscopic study

PURPOSE: To determine the plane of cleavage of the corneal epithelium and smoothness of underlying stroma, after alcohol debridement in photorefractive keratectomy (PRK) and laser subepithelial keratectomy (LASEK). METHODS: The epithelial flap from six patients undergoing alcohol delamination of corneal epithelium before PRK and the epithelium and stroma from three eye bank donor eyes were fixed and processed for transmission (TEM) and scanning electron microscopy (SEM). The smoothness of the underlying stroma was studied by SEM and the plane of cleavage was determined by morphologic examination and morphometric measurements of basement membrane attached to the epithelial flap, using image-analysis software. RESULTS: A very smooth stromal bed, ideal for PRK was seen in the stroma of all three eye bank donor eyes after alcohol delamination. The plane of cleavage was determined to be at the hemidesmosomal attachments, including the most superficial part of the lamina lucida of the basement membrane. CONCLUSIONS: Alcohol delamination of the corneal epithelium before PRK or LASEK consistently results in a very smooth cleavage at the level of the hemidesmosomal attachments, including the superficial lamina lucida. It leaves behind a very smooth surface, which is ideal for PRK. It also allows for an intact epithelial flap to be lifted as a sheet from the corneal surface and hence is ideally suited for the LASEK technique.     

In vivo laser confocal microscopic analysis of human corneal epithelial sheet cultured on amniotic membrane.

BACKGROUND AND OBJECTIVE: Corneal epithelial sheet cultured on amniotic membrane ex vivo has been developed for the treatment of severe ocular surface disorders. In this study, human corneal epithelial sheets cultured on amniotic membranes were analyzed using a laser scanning confocal microscope. MATERIALS AND METHODS: Human corneal epithelial cells were cultured on four sheets of amniotic membrane, and they were examined layer by layer using the laser scanning confocal microscope. RESULTS: The superficial layer cells were mosaic in appearance. In the superficial layer, focal areas of slender and elongated cells were noted. In addition, highly reflective materials were noted. Basal epithelial cells of the corneal epithelium sheet at a slightly deeper plane displayed mosaic pattern with smaller cells. At a slightly deeper plane, the basement membrane of amnion showed relatively bright homogeneity. CONCLUSION: The laser scanning confocal microscope can examine the morphology of human corneal epithelial cells cultured on amniotic membranes.     

乙醇对角膜上皮瓣活性及基底膜分离定位研究

目的 探讨不同乙醇作用时间对角膜上皮瓣活性的影响,并确定角膜上皮瓣的解剖分离层面.方法 按标准准分子激光角膜上皮瓣下磨镶术(LASEK)方法制备7只尸体眼角膜上皮瓣,其中对照组1只眼,其余6只分为A、B、C 3个组,每组2只眼,乙醇浸润时间分别为20、30和40 S.对7只眼进行HE、增殖细胞核抗原(PCNA)、层黏连蛋白免疫组化及Ⅶ型胶原免疫荧光染色,评价角膜上皮瓣细胞活性,确定LASEK手术中角膜上皮瓣的解剖分离层面,采用X2检验分析.结果 角膜上皮瓣为复层上皮细胞组织,没有Bowman's层和角膜基质成分,PCNA阳性细胞率为A组B组C组,其laminin染色在角膜床和上皮瓣基底膜一侧均有呈片段样棕色条纹状,其collagenⅦ染色阳性反应呈一条绿色线形,而且只存在于角膜床,上皮瓣基底膜侧不着色.结论 角膜上皮瓣活性随乙醇浸润时间延长而降低;乙醇浸润法制备角膜上皮瓣的组织分离层面位于角膜上皮基底膜内,并且定位于基底膜的透明层和致密层之间.     

Stromal haze, myofibroblasts, and surface irregularity after PRK

The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups: [see table in text]. Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin alpha4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2+/-0.2 cells/400x field) and group V (1.8+/-0.4), with significantly more in groups II (3.5+/-1.8), III (6.8+/-1.6), VII (7.9+/-3.8), IV (12.4+/-4.2) and VI (14.6+/-5.1). The screened groups were significantly different from each other (p < 0.05), with myofibroblast generation increasing with higher surface irregularity in the -4.5 diopter PRK groups. The -9.0 diopter PRK group VI had significantly more myofibroblast generation than the -9.0 diopter PRK with PTK-smoothing group VII (p < 0.01). Areas of basement membrane disruption were demonstrated by staining corneas for integrin alpha4 and were prominent in corneas with grade I or higher haze. SMA-positive myofibroblasts tended to be present sub-adjacent to basement membrane defects. Late apoptosis was detected at 1 month after surgery within clusters of myofibroblasts in the sub-epithelial stroma. In conclusion, these results demonstrated a relationship between the level of corneal haze formation after PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane regeneration and increased epithelium-derived TGFbeta signalling to the stroma that increases myofibroblast generation. Late apoptosis appears to have a role in the disappearance of myofibroblasts and haze over time.     

Transmission electron microscopy study of corneal epithelial flaps following removal using mechanical scraping, alcohol, and epikeratome techniques

PURPOSE: To evaluate various methods of surface ablation by comparing the integrity of basal epithelial cells and the basement membrane using transmission electron microscopy. METHODS: Corneal epithelial flaps were created using four approaches: mechanical removal (using a blunt spatula after application of 0.5% proparacaine, mechanical group); conventional LASEK (after application of 20% alcohol for 20 seconds, LASEK group); epi-LASIK using PMMA separator (PMMA separator group); or epi-LASIK using metal separator (metal separator group). Each group comprised epithelial sheets taken from five eyes of five patients. The sheets were immediately fixed in glutaraldehyde following removal. The flaps were examined using both light and transmission electron microscopy by three independent observers blinded to the creation methods, who assessed the adherence and integrity of the basement membrane to the basal epithelial layer. RESULTS: In the mechanical and PMMA separator groups, > 70% of flap areas had an intact basement membrane and normal basal cell integrity. Such integrity was present in < 30% of the flap areas in the LASEK group, and in 30% to 70% of the flap areas in the metal separator group. Different epikeratomes caused different amounts of damage. CONCLUSIONS: Mechanical scraping and epi-LASIK were superior to LASEK in terms of preservation of the epithelial basement membrane and basal epithelial cells. Preservation ratio may differ depending on the type of epikeratome used.     

Effects of cryopreservation on histology and viability of cultured corneal epithelial cell sheets in rabbit

PURPOSE: To increase the availability of cultured corneal epithelial cell sheets as a replacement for corneal limbal allograft, the effects of cryopreservation on viability of cultured corneal epithelial cells were investigated. METHODS: Normal rabbit corneal limbal tissue was excised, and the cells were cultured with mitomycin C-treated 3T3-J2 cells as a feeder layer. Stratified corneal epithelial cell sheets were obtained within 20 days. All sheets with a diameter of 8 mm were punched out with a biopsy punch and stored in either 10% glycerol or dimethyl surfoxide (DMSO) at -80 degrees C or -196 degrees C for 4 or 12 weeks. After thawing, the sheets were evaluated histologically, and cell viability was analyzed by colorimetric cell viability assay using a tetrazolium salt. RESULTS: Structural damage such as vacuolar degeneration was more clearly observed in the corneal epithelial cell sheets cryopreserved with DMSO than those with glycerol, especially at -80 degrees C, whereas only minor morphologic changes were observed in the corneal epithelial cell sheets cryopreserved in glycerol at both temperatures. Colorimetric cell viability assay revealed that the storage conditions at the lower temperature (-196 degrees C) showed higher cell survival than those at the higher storage temperature (-80 degrees C). The difference between the two cryoprotectants, however, was not significant. Among the conditions used in this study, the samples cryopreserved with glycerol at -196 degrees C showed the highest cell survival rate (70.3 +/- 8.3% and 66.4 +/- 14.7% for 4 and 12 weeks, respectively). The difference between this group and those stored at -80 degrees C was significant for both 4 weeks and 12 weeks of storage using either glycerol or DMSO. Although the cryopreserved cell sheets could not maintain their original layered structure after thawing, viable cell sheets could be regenerated. CONCLUSIONS: The cell survival rates obtained after freezing storage were reasonable; however, the cryopreserved sheets could not maintain their original layered structure. More work needs to be done to better preserve the corneal epithelial cell sheets.     

Synergistic effect of ethanol and mitomycin C on corneal stroma

PURPOSE: To investigate the combined effects of ethanol and mitomycin C (MMC) application on the corneal stroma of rabbits that underwent photorefractive keratectomy (PRK). METHODS: Twenty-four rabbits (24 eyes) underwent PRK to correct -9.00 diopters of myopia. Twelve eyes had ethanol application before removing the epithelium and 12 eyes had the epithelium manually removed without ethanol. Eyes in both groups had topical MMC 0.02% application for 12 seconds immediately after excimer laser ablation. Twelve rabbits were sacrificed at two time points -4 hours and 4 weeks after surgery--and immunohistochemistry was performed with TUNEL assay, alpha-smooth muscle actin (alpha-SMA), and DAPI. RESULTS: More TUNEL-positive cells were observed in the ethanol-treated group compared to the mechanical debridement group at 4 hours after surgery (P<.01). No significant difference in alpha-SMA-positive cells was detected between the two groups at 4 weeks after sugery. However, decreased keratocyte density in the anterior stroma was more pronounced in the ethanol-treated group compared to the mechanical debridement group (P<.02). CONCLUSIONS: Ethanol application for epithelial removal during PRK seems to produce a synergistic effect with MMC, resulting in fewer keratocytes in the anterior stroma of rabbit corneas treated with MMC and ethanol than in corneas treated with MMC alone after PRK.     

Corneal epithelial healing after photorefractive keratectomy: analytical study

PURPOSE: To characterize the velocity of epithelial migration after photorefractive keratectomy (PRK) with 3 different corneal ablation patterns. SETTING: Department of Ophthalmology, Catholic University of Rome, Rome, Italy. METHODS: Fifteen patients (30 eyes) with mild to moderate myopia and with simple to compound myopic astigmatism were enrolled for this study. The surgical procedure consisted of standardized PRK with final smoothing performed using the Technolas Keracor 217C excimer laser. The reepithelialization process was evaluated at 0 hours, 20 hours, 40 hours, and 60 hours after surgery using a digital photo camera and custom software for measurement. Digital analysis of the images was performed. Corneal topographies were taken at 1 month, 3 months, 6 months, and 12 months after PRK. RESULTS: The mean speed of radial migration in the 10 eyes (33%) in the low spherical ablation group was 0.087 mm/h +/- 0.008 (SD). This was significantly higher than that found in the 10 eyes (33%) in the high spherical ablation group (mean speed 0.078 +/- 0.007 mm/h; P<.001) and in the 10 eyes (33%) in the cross-cylinder ablation group (mean speed 0.055 +/- 0.014 mm/h; P<.001). CONCLUSION: Analysis of the data shows that epithelial migration along the photoablated corneal surface depends on the ablation pattern. The epithelial sliding is highly influenced by local variations in the curvature of the stromal surface. The data demonstrate that faster epithelial wound healing after PRK is predictive of optimal visual performance.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

Novel enzymatic isolation of an entire viable human limbal epithelial sheet

OBJECTIVE. To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS. Human pigmented limbus was incubated at 4 degrees C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS. Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% +/- 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS. An intact limbal epithelial sheet can be consistently and reproducibly isolated and contains stem cell characteristics in the basal epithelium by degrading laminin 5 and part of collagen IV, and disassembling collagen VII.     

"去上皮"羊膜对体外培养结膜上皮细胞的作用

目的 :探索羊膜基底膜对结膜上皮细胞形态功能的影响。方法 :把“去上皮”羊膜按照基底膜或实质面向上分为两组 ,接种结膜上皮细胞 ,同样条件下培养。取培养 14天的两组标本进行组织学 (HE和PAS染色 ) ,透射电镜 ,以及免疫组化检查 (包括抗角蛋白和抗波形蛋白染色 )。结果 :细胞 72小时内贴附在羊膜上。基底膜面接种的上皮细胞顶端见微绒毛 ,单层排列 ,细胞间有丰富桥粒 ,基底膜完整 ,可见半桥粒 ,抗角蛋白染色阳性 ,抗波形蛋白染色阴性 ;实质面接种的上皮细胞胞浆内有大量脂滴 ,表面未见微绒毛 ,细胞松散堆积 ,与羊膜连接不紧密 ,未见桥粒和半桥粒 ,抗角蛋白染色弱阳性 ,抗波形蛋白染色强阳性。结论 :羊膜基底膜是结膜上皮细胞体外培养的良好载体 ,有利于上皮细胞生长、粘附、分化 ,并能减缓上皮细胞老化。     

Expression of Delta Np63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane

PURPOSE: To evaluate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression of Delta Np63 in human limbal epithelial cells (HLECs) during ex vivo expansion on amniotic membrane (AM). METHODS: Primary HLECs were cultured either on AM or plastic surfaces and were treated with 1 micro g/mL PMA for 24 hours. Expression of Delta Np63 and the differentiation-associated gap junctional protein connexin 43 (Cx43) were studied by laser scanning microscopy. RESULTS: The labeling index (LI) of Delta Np63 was higher in HLECs cultured on AM than in HLECs grown on plastic (81.4% +/- 12.2% and 66.6% +/- 16.5%, respectively; P < 0.001). After PMA treatment, Delta Np63 expression in HLECs on plastic dramatically decreased to 20.4% +/- 11.4%. However, HLECs cultured on AM showed only a moderate decrease in Delta Np63 expression (56.4% +/- 10.9%, P < 0.001) after PMA treatment. It was also observed that 72.8% +/- 17.5% of the Delta Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% +/- 3.7% of the Delta Np63-positive cells in HLECs cultured on AM (P < 0.001). The latter indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype. CONCLUSIONS: Delta Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports Delta Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.     

Mitomycin C-induced reduction of keratocytes and fibroblasts after photorefractive keratectomy

PURPOSE: To investigate the effects of mitomycin C (MMC) on the number of keratocytes and the proliferation of fibroblasts after photorefractive keratectomy (PRK) and exposure to ultraviolet B (UV-B) irradiation. METHODS: The right eyes of New Zealand White rabbits in Groups 1, 2, and 3 (n = 18 each) underwent PRK to correct -10 diopters with 5 mm optical zone. Sponges soaked with 0.02% MMC were applied to the right eyes of Group 1 rabbits for 2 minutes. Antibiotic ointment was applied daily to all rabbits until the epithelium healed completely, after which 0.02% MMC eye drops were applied twice daily to the right eyes in Group 2 until 4 weeks after PRK. Three weeks after PRK, the right eyes of all the remaining rabbits were exposed to 100 mJ/cm2 C UV-B radiation. Corneal haziness was assessed biomicroscopically using the Fantes scale every 3 weeks. Six eyes of each group were each enucleated 3, 6, and 12 weeks after PRK, and tissue specimens were stained with hematoxylin and eosin and with TUNEL stain. The tissues were evaluated immunohistochemically with antibody to alpha-smooth muscle actin (SMA). Cellular changes in the anterior stroma and epithelial basement membrane were evaluated by electron microscopy. RESULTS: Corneal haze was observed after PRK and was aggravated by UV-B irradiation. A single intraoperative application of MMC immediately after PRK induced opacity and apoptosis of keratocytes. Twelve weeks after PRK, MMC significantly reduced corneal haze, the number of keratocytes, apoptotic cells, and fibroblasts, even after UV-B irradiation. Relatively large numbers of apoptotic and SMA-positive cells were found only in PRK-treated, non-MMC treated rabbits (Group 3), even after 12 weeks. Three weeks after PRK, dying stromal cells showed cell shrinkage, and chromatin condensation was observed in all treated groups by electron microscopy. Twelve weeks after PRK, fewer keratocytes and inflammatory cells were observed just beneath the epithelial layer in Group 1 than in any of the other groups. CONCLUSIONS: MMC is a potent inhibitor of corneal haze induced by PRK. MMC reduced the number of keratocytes and fibroblasts after PRK and UV-B irradiation. Although MMC would improve the clinical results of PRK, it has significant toxicity on corneal keratocytes, which did not disappear until 3 months after PRK.     

Epithelial healing and clinical outcomes in excimer laser photorefractive surgery following three epithelial removal techniques: mechanical, alcohol, and excimer laser

PURPOSE: To evaluate epithelial healing, postoperative pain, and visual and refractive outcomes after photorefractive keratectomy (PRK) using three epithelial removal techniques. DESIGN: Prospective, nonrandomized, comparative trial. METHODS: SETTING: Department of Ophthalmology, Yonsei University College of Medicine and Balgensesang Ophthalmology Clinic, Seoul, Korea. INTERVENTIONS: For the PRK procedure, the corneal epithelium was removed in one of three ways: mechanically (conventional PRK [PRK]) in 88 eyes of 44 patients; using excimer laser (transepithelial PRK [tPRK]) in 106 eyes of 53 patients; or using 20% diluted alcohol, laser-assisted subepithelial keratomileusis (LASEK) in 106 eyes of 53 patients. MAIN OUTCOME MEASURES: Epithelial healing, postoperative pain, uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), and remaining refractive error. RESULTS: The mean postoperative pain scores were 4.84 +/- 1.45 for PRK, 4.71 +/- 1.62 for tPRK, and 4.63 +/- 1.52 for LASEK (P = .125). The mean epithelial healing rates were 12.3 +/- 4.6 for PRK, 15.2 +/- 4.9 for tPRK, and 18.1 +/- 5.2 mm2/day for LASEK (P < .001). The postoperative 6-month remaining mean spherical equivalents (diopters) were -0.46 +/- 1.01 for PRK, 0.18 +/- 0.91 for tPRK, and -0.82 +/- 1.18 for LASEK (P = .01). The LASEK group showed less favorable UCVA than other groups. There was no significant difference in BSCVA between the groups. CONCLUSIONS: Postoperative pain, subepithelial opacity and BSCVA were similar regardless of the epithelial removal procedure. A faster epithelial healing rate did not result in better visual or refractive outcomes. Using the same nomogram, tPRK resulted in a slight overcorrection, and LASEK resulted in a slight undercorrection.     

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

AIM:To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.METHODS: Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.RESULTS:HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.CONCLUSION: Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.     

Preoperative evaluation of cultured human corneal limbal epithelium on amniotic membrane by confocal microscopy

PURPOSE: Although sheet transplantation with cultured corneal limbal epithelium has been widely performed as a strategy for ocular surface reconstruction, there has been no optimal method for evaluating the morphology of these sheets prior to transplantation. We propose the use of in vivo confocal microscopy as a novel method for the evaluation of limbal corneal epithelium cultured on amniotic membrane. METHODS: Human limbal epithelial sheets were grown on amniotic membranes by following a standard protocol and were stained with hematoxylin and eosin. Morphology was studied using in vivo confocal microscopy for cultured corneal epithelium on amniotic membrane, human intact amniotic membranes, and epithelium-denuded human amniotic membranes. RESULTS: Histologic examination showed a stratified corneal epithelium sheet by the fourth week of culture. The surface and basal layers of the cultured limbal epithelium and amniotic membrane were clearly distinguished by in vivo confocal microscopy. A monolayer of amniotic epithelial cells was observed on the intact amniotic membrane, but not on the epithelium-denuded human amniotic membrane. CONCLUSIONS: Our findings support the use of in vivo confocal microscopy as a valid technique for the preoperative evaluation of cultured corneal limbal epithelial cell sheets on amniotic membrane.     

Evaluation for safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C

PURPOSE: To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD: Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS: Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS: Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.