Expression of laminin-5 with amniotic membrane transplantation in excimer laser ablated rat corneas

PURPOSE: To investigate the expression of laminin-5 during epithelial healing and evaluate its expression in vivo using rat corneas on which amniotic membrane was applied to cover the wound after excimer laser photoablation. SETTING: Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. METHODS: Myopic photorefractive keratectomy (PRK) with a 100 microm deep ablation was performed in Sprague Dawley rats killed 6, 12, 24, 48, 72, and 96 hours after the procedure. In the first group of 30 rats, the excimer laser-ablated cornea was covered with amniotic membrane after PRK. Thirty other rats in which no amniotic membrane treatment was used served as controls. Immunohistochemical and immunofluoresce in techniques were used to monitor the expression of laminin-5, gamma2, and gamma1 in the rat corneas. Immunoblotting was used to compare the expression of laminin between the amniotic membrane group and the control group. RESULTS: In the immunoblotting study, laminin-5, alpha3, and gamma2 increased 24 hours after amniotic membrane treatment compared to the control group. At 12 hours, in vivo immunostaining of the corneas in both groups expressed laminin, but laminin-5 and gamma2 were more intensely expressed in the amniotic membrane group. This continued until reepithelialization. Expression of the gamma1 chain was not different between the 2 groups. CONCLUSION: With the use of amniotic membrane, the expression of laminin-5 and gamma2 was faster and more intense than in a control group during reepithelialization of excimer laser-ablated rat corneas.     

Electron microscopy of surface smoothness of porcine corneas and acrylic plates with four brands of excimer laser

PURPOSE: This study compares ablation smoothness patterns produced on four different excimer laser devices available for photorefractive surgery. METHODS: VISX calibration plastic and porcine cornea were ablated with standard -3.00-D, -6.00-D, and -9.00-D settings using four different excimer lasers: VISX S2 Smooth Scan, Nidek EC-5000, Autonomous Ladar Vision System, and Bausch and Lomb Technolas. Electron microscopy and laser interferometry were used for qualitative evaluation of the ablated surfaces. Corneal ablation surface smoothness was graded by ten independent observers. Calibration plastic ablated surfaces were evaluated quantitatively for smoothness by laser interferometry. RESULTS: The independent observer assessment of corneal ablation surface smoothness demonstrated that the Autonomous small spot Gaussian profile laser produced the smoothest ablation surfaces, followed by the other broad beam lasers. In comparing ablation smoothness among various refractive powers (-9.00 D, -6.00 D, and -3.00 D), a trend was observed that indicated a correlation of higher refractive settings with decreasing surface smoothness. However, this trend was not statistically significant. The quantitative laser interferometry measurements supported the independent observer ranking of the Autonomous flying small spot ablation profile as the smoothest. However, there were differences between the laser interferometry smoothness rankings and independent observer smoothness rankings. CONCLUSION: There were significant differences in ablation surface smoothness among the four excimer lasers tested.     

Mass spectroscopic analysis of excimer laser ablated material from human corneal tissue

The clinical feasibility of photorefractive keratectomy depends on the surface structure of the ablated cornea. Two factors that influence the remodeled surface are the homogeneity of the energy distribution and the properties of the laser source (energy and wavelength). Currently, the homogeneity of the beam is difficult to control. The second factor, laser source properties, was the focus of this study. We investigated the effect of laser wavelength and energy by analyzing the reaction products of photoablation. We monitored the fragments produced by UV-laser ablation of human corneas using mass spectroscopy in the range of 0 to 100 atomic mass units. At 248 nm (KrF), average photon energy was 5 mJ/cm2, increased to 90 mJ/cm2 by intervals of 5 mJ/cm2. At 193 nm (ArF), photon energy was increased by the same interval from 5 mJ/cm2 to 80 mJ/cm2. Our experiments showed that there was a fluence threshold of approximately 40 mJ/cm2 at 193 nm (ArF) and 50 mJ/cm2 at 248 nm (KrF). Exceeding this threshold led to sudden increases in the number and relative intensity of fragment peaks in mass spectroscopy. This indicates the onset of multiple-photon processes and effective photochemical breakdown. There was a significant difference between both wavelengths in the distribution of mass peaks, indicating higher ionization power at 193 nm.     

Ophthalmic applications of atomic force microscopy

Applications of atomic force microscopy (AFM) to the research and development of novel contact lens material and better manufacturing techniques are discussed. It is demonstrated that the technique of AFM is a useful and novel tool with which to examine the surfaces of soft contact lenses in both the hydrated and the dehydrated state. Specifically, the results of ophthalmic studies designed to examine the manufacturing processes on the nanoscale, benchmark lens roughnesses, and clinical collaborations are presented.     

Histologic and ultrastructural changes in corneas with granular and macular dystrophy after excimer laser phototherapeutic keratectomy

PURPOSE: The histologic changes after phototherapeutic keratectomy (PTK) in corneas with granular and macular dystrophy were studied. METHODS: We studied 3 corneas of 2 patients (1 granular, 2 macular dystrophy), who underwent penetrating keratoplasty (PK) at 0.8, 2.16, and 3.25 years after PTK; and 11 corneas (controls) from 10 PK patients (5 granular, 6 macular dystrophy) by light microscopy and by transmission electron microscopy. PTK was performed by using the Asclepion-Meditec MEL 60 excimer laser. RESULTS: After PTK the epithelium (15-40 versus 5-100 microm), and the upper stromal collagen lamella thickness (50-75 versus 50-100 microm) were less irregular than for the controls. In 1 eye (macular dystrophy) 10 months after PTK an acid mucopolysaccharide-positive band was detected in the subepithelial stroma, which could be removed by hyaluronic acid digestion. This fact suggests that it was "haze" formed after PTK, rather than a subepithelial recurrence of the dystrophy. All PTK corneas had deposits in the mid- and posterior stroma. Concerning controls, deposits were detected under the epithelium in all corneas. Electron microscopy of the study corneas revealed a mostly continuous basal lamina, occasionally forming projections into the subepithelial stroma, and large numbers of well-developed hemidesmosomes (5.2 +/- 0.8 per microm membrane length) present at greater density than in the controls (3.5 +/- 0.8). CONCLUSIONS: In stromal dystrophies, PTK was effective in removing large subepithelial stromal plaques. There were no subepithelial recurrences, and hemidesmosome density was increased.     

原子力显微镜在眼组织结构观察及生物力学测量研究中的应用

原子力显微镜(atomic force microscope,AFM)能够在生理状态下获得样本表面结构的纳米级三维图像以及生物材料的力学性能,近年来被广泛用于生命科学研究领域,从单细胞到组织器官均取得了一定成果.眼球作为人体重要的器官组织,其结构和力学性能的变化与疾病的发生密切相关,AFM在对眼部各组织的超微结构观察以及生物力学测量为研究疾病发生机制做出了较大贡献.     

Analysis of intraocular lens surface properties with atomic force microscopy

PURPOSE: To analyze the surface optics of 4 currently available intraocular lenses (IOLs) with atomic force microscopy. SETTING: Licryl Laboratory, University of Calabria, Rende, Italy. METHODS: The surface roughness and topography of poly(methyl methacrylate) (PMMA), silicone, hydrophobic, and hydrophilic acrylic IOLs were evaluated with atomic force microscopy in contact mode. The analysis was performed in a liquid environment using cantilevers with a 0.01 Newtonw/meter nominal elastic constant. Measurements were made over areas of 10 microm2 on different locations of the posterior optic surface of the IOL. RESULTS: Atomic force microscopy permitted high-resolution imaging of IOL optic surface characteristics. Surface topography showed different features with respect to the lens biomaterial. The root-mean-square roughness of the IOL optic surface was significantly different between lenses of various materials (P < .001). The hydrophobic acrylic and silicone IOLs had the lowest mean surface roughness, 3.8 nm +/- 0.2 (SD) and 4.0 +/- 0.5 nm, respectively, and the 2 PMMA IOLs had the highest mean surface roughness, 6.6 +/- 0.3 nm and 7.0 +/- 0.6 nm. CONCLUSIONS: Atomic force microscopy was effective and accurate in analyzing IOL optics. The surface topography of IOLs may vary with different manufacturing processes.     

Optical and atomic force microscopy of an explanted AcrySof intraocular lens with glistenings

PURPOSE: To assess the surface morphology and cause of glistenings in an explanted AcrySof intraocular lens (IOL). SETTING: Shakai Hoken Kobe Central Hospital, Kobe, Japan. METHODS: A 63-year-old Japanese man had implantation of an AcrySof IOL in the capsular bag. One month postoperatively, he had a neodymium:YAG laser capsulotomy for posterior capsule opacification, which changed the IOL's position in the capsular bag. A few months later, the patient developed disabling night glare from intralenticular glistenings and progressive hyperopic refractive error. The IOL was explanted and then analyzed by optical microscopy and atomic force microscopy (AFM). Laboratory analysis of control AcrySof IOLs kept in a balanced salt solution at steady room and body temperature for 2 months was also performed to evaluate the cause of the glistenings observed clinically. RESULTS: Optical microscopy showed that the explanted AcrySof IOL had several microvacuoles; no abnormalities were observed in the control AcrySof IOLs before or after folding at the room and body temperatures. The AFM analysis showed a significant change in the surface morphology of the explanted IOL, including vacuolar formations in the posterior surface as well as numerous anterior surface irregularities. No microvacuoles or surface morphology alterations were observed in the control AcrySof IOLs by AFM analysis. CONCLUSIONS: The glistenings in the explanted AcrySof IOL were likely caused by temperature changes and not mechanical stress from folding.     

The applications of atomic force microscopy to vision science

The atomic force microscope (AFM) is widely used in materials science and has found many applications in biological sciences but has been limited in use in vision science. The AFM can be used to image the topography of soft biological materials in their native environments. It can also be used to probe the mechanical properties of cells and extracellular matrices, including their intrinsic elastic modulus and receptor-ligand interactions. In this review, the operation of the AFM is described along with a review of how it has been thus far used in vision science. It is hoped that this review will serve to stimulate vision scientists to consider incorporating AFM as part of their research toolkit.     

正常中国汉族儿童角膜的共焦显微镜研究

目的采用角膜激光共焦显微镜对正常中国汉族儿童活体角膜各层组织结构进行观察,分析正常中国汉族儿童角膜各层细胞的活体细胞形态学特征及密度。方法 49例6～13岁正常中国汉族儿童中央部角膜进行活体共焦显微镜检查,研究角膜各层结构的图象特点,并分析角膜各层细胞的密度,与成年人进行对比。结果中国汉族儿童正常角膜上皮表层细胞排列疏松,边界清楚,胞体较大,核反光较强,又称翼状细胞;基底层细胞排列紧密,呈规则的蜂巢状,胞质反光弱,正常情况下未见细胞核,细胞平均密度为5947.58±769.3个/mm2。前弹力膜为无细胞结构的膜状物,可见串珠状的神经纤维走行。基质层中可见在相对较暗的背景下明亮的角膜基质细胞核。角膜基质内有神经纤维分布。前基质层角膜细胞平均密度为1621.12±123.26个/mm2,后基质层角膜细胞平均密度为984.71±113.17个/mm2,前后基质层细胞密度有显著性差别（P〈0.05）。后弹力层中无细胞结构,为均一无结构组织。内皮细胞层表现为规则的六边形结构,细胞结构清晰,细胞平均密度为3682.38±251.87个/mm2。左右眼及男女之间角膜各层细胞密度的差异无统计学意义（P=0.341～0.611和P=0.194～0.855）。各层角膜细胞的面积和密度与性别和眼别无差异。中国汉族儿童角膜各层细胞的密度较正常成人高。结论角膜激光共焦显微镜能够在实时、活体和三维空间从细胞水平清晰观察中国汉族儿童活体角膜各层细胞的形态结构,可以对角膜各层结构进行定性和定量分析,在儿童角膜病的研究和临床诊断方面是十分有用的工具。     

佩戴角膜接触镜后角膜变化的激光共焦显微镜观察

目的 应用激光共焦显微镜对长期佩戴角膜软性接触镜的患者活体角膜的组织结构变化进行观察.方法 用激光共焦显微镜对长期佩戴角膜软性接触镜的15例患者进行检查,并选择未戴角膜接触镜者11例进行对照,对两组结果进行比较.结果 1.佩戴角膜软性接触镜组与对照组相比,基底上皮细胞密度减少,为3705.00±447.62个/mm2(P＜0.05),角膜上皮层厚度变薄,为54.3±8.44μm(P＜0.05),并有部分剥脱.2.佩戴角膜软性接触镜组角膜内出现白色点状物,朗汉氏细胞数目多,成树枝状改变.3.角膜软性接触镜组与对照组相比神经纤维数量及密度无明显变化(P＞0.05),但曲折度增加,有分支出现(P＜0.05).结论 佩戴角膜软性接触镜,角膜组织可发生一系列改变,激光共焦显微镜与传统光学共焦显微镜相比,图象清晰,深度定位准确,在疾病的早期诊断、治疗和研究中将起重要作用.     

Application of atomic force microscopy (AFM) in ophthalmology

Atomic force microscopy (AFM) allows to examine surface of different biological objects in the nearly physiological conditions at the nanoscale. The purpose of this work is to present the history of introduction and the potential applications of the AFM in ophthalmology research and clinical practice. In 1986 Binnig built the AFM as a next generation of the scanning tunnelling microscope (STM). The functional principle of AFM is based on the measurement of the forces between atoms on the sample surface and the probe. As a result, the three-dimensional image of the surface with the resolution on the order of nanometres can be obtained. Yamamoto used as the first the AFM on a wide scale in ophthalmology. The first investigations used the AFM method to study structure of collagen fibres of the cornea and of the sclera. Our research involves the analysis of artificial intraocular lenses (IOLs). According to earlier investigations, e.g. Lombardo et al., the AFM was used to study only native IOLs. Contrary to the earlier investigations, we focused our measurements on lenses explanted from human eyes. The surface of such lenses is exposed to the influence of the intraocular aqueous environment, and to the related impacts of biochemical processes. We hereby present the preliminary results of our work in the form of AFM images depicting IOL surface at the nanoscale. The images allowed us to observe early stages of the dye deposit formation as well as local calcinosis. We believe that AFM is a very promising tool for studying the structure of IOL surface and that further observations will make it possible to explain the pathomechanism of artificial intraocular lens opacity formation.     

Atomic force microscopic study of the human cornea following excimer laser keratectomy

The aim of this study was to examine the corneal surface structures with a new investigative method, the atomic force microscope following 193 nm excimer laser photoablation. Fresh human corneas were irradiated in vitro with an increasing number of impulses emitted by a 193 nm ArF laboratory excimer laser in order to produce either smooth flat surfaces or stair-like formations within the cornea. The corneas were investigated in a Topometrix(R) atomic force microscope in their native state. For comparison, three corneas were fixed with glutaraldehyde and processed for scanning electron microscopy.Atomic force microscopy and scanning electron microscopy revealed the same surface characteristics of photoablated corneas, though the preparation for scanning electron microscopy induced considerable shrinkage of the tissues. The layers of the cornea could be distinguished from each other and deeper ablations of the stroma produced a rougher surface. On the lateral walls of ablated stairs small droplets of ejected material could be seen with scanning electron microscope.Atomic force microscope produces three-dimensional images of the scanned native corneal surfaces and it could be a valuable tool to investigate the corneal smoothness. Our investigations have provided similar results as those obtained with scanning electron microscopy showing that the laser-ablated corneal surface remains relatively smooth. We suggest that the formation of condense droplets of ejected materials is based on hydrodynamic motions induced by boiling water solutions.