Wound healing following anterior keratectomy and lamellar keratoplasty in the pig

PURPOSE: To examine corneal wound healing in an animal model of two types of mechanical lamellar keratectomy. METHODS: One eye from each of 28 pigs was studied. Using a motorized keratome, corneas were subjected to an anterior lamellar keratectomy with removal of anterior stroma and epithelium, or to automated lamellar keratoplasty (ALK) with reapposition of a corneal flap. The exposed stromal surfaces were labeled intraoperatively with a fluorescent dye (DTAF) to assess deposition of stromal components during subsequent wound healing. Examination before surgery and enucleation included measurement of corneal curvature and intraocular pressure, and assessment of corneal haze. Eyes were prepared for histological examination, fluorescence microscopy, and for fibronectin immunohistochemistry. RESULTS: Both keratectomy procedures produced flattening of corneas by up to 3.80 diopters, 28 days after surgery. Corneal haze was more pronounced in eyes from which epithelium was removed (anterior lamellar keratectomy group). The increased haze in this group was associated histologically with appearance of many reactive keratocytes and inflammatory cells, deposition of new stromal material, and more widespread appearance of fibronectin immunoreactivity. In the lamellar keratoplasty group, only the edges of the corneal wound showed significant reactivity, and included keratocyte activation and epithelial ingrowth. CONCLUSIONS: The pig provides a useful model for studies of refractive surgical techniques using procedures and instruments designed for use in humans. Mechanized keratectomy procedures that minimize disruption of the epithelium and Bowman's layer produce a less reactive corneal wound than procedures in which an expanse of epithelium and anterior stroma are removed.     

In vivo confocal microscopy findings in a patient with posterior amorphous corneal dystrophy

A 21-year-old man, with bilateral posterior amorphous corneal dystrophy, was studied by biomicroscopy, corneal topography and in vivo confocal microscopy. The best-corrected visual acuity was 6/21 in the right eye and 6/6.9 in the left eye. Biomicroscopy revealed bilateral, asymmetric, sheet-like opacification at the deep posterior stromal layer. The corneal topography displayed asymmetric against-the-rule astigmatism in the right eye and prominent steepening at the inferior paracentral cornea in both eyes. In vivo confocal microscopy of the corneas demonstrated microfolds and hyper-reflective layer at the posterior stroma just adjacent to the endothelial layer. The epithelium, Bowman's membrane, anterior stroma and the endothelial layer were normal. In vivo confocal microscopy is useful in evaluating the corneal dystrophies.     

颗粒状角膜营养不良活体共焦显微镜形态学研究

目的研究颗粒状角膜营养不良角膜各层组织的共焦显微镜形态改变。方法应用Confoscan2.0共焦显微镜对13例(26眼)颗粒状角膜营养不良患者的角膜进行扫描检查,记录与分析各层角膜图像。结果所有患眼前基质细胞及16/26眼后基质细胞结构不清,排列紊乱,并可见短棒状多形性强反光;6/26眼前弹力层不规则并增厚,神经纤维密度明显下降;6/26眼角膜上皮基底细胞层可见不定型的强反光;2/26眼角膜上皮细胞边界不清,排列呈疏松的蜂窝状,并出现不透明的强反光;所有患者角膜内皮细胞形态基本正常。视力0.3以下的患眼角膜上皮细胞层、上皮基底细胞层、前弹力层、后基质层发生形态异常的比例高于0.3以上的患眼(P<0.05)。结论1.共焦显微镜可活体检查颗粒状角膜营养不良角膜组织各层结构,起到类似病理组织切片的作用。2.前基质层形态异常可能是颗粒状角膜营养不良最基本的共焦显微镜形态特征,病情越重,前基质层以外的其它层次发生形态异常的可能性越大,但内皮细胞层一般不受累。3.共焦显微镜检查对颗粒状角膜营养不良手术方式的选择具有一定的参考价值。     

Histological comparison of corneal ablation with Er:YAG laser, Nd:YAG optical parametric oscillator, and excimer laser

PURPOSE: To use histological techniques to assess and compare the ablation depth, local damage, and surface quality of corneal ablations by a Q-switched Er:YAG laser, an optical parametric oscillator laser at 2.94 microm, a long pulse Er:YAG laser, and a 193-nm excimer laser. METHODS: Human cadaver eyes and in vivo cat eyes were treated with a 6.0-mm diameter, 30-microm-deep phototherapeutic keratectomy ablation and a 6.0-mm diameter, -5.00-D photorefractive keratectomy ablation. Human cadaver eyes were also treated with a 5.0-mm diameter, -5.00-D laser in situ keratomileusis (LASIK) ablation. Fluences and pulse widths used were 200 mJ/cm2 and 70 ns for the Q-switched Er:YAG, 150 mJ/cm2 and 7 ns for the optical parametric oscillator laser (OPO), 500 mJ/cm2 and 50 microseconds for the long pulse Er:YAG, and 160 mj/cm2 and 20 ns for the excimer laser. In the ablation rate study, 12 porcine eyes were ablated by the OPO laser with a range of layers and at different fluences ranging from 60 to 150 mJ/cm2, all using a 1.5-mm spot on the eye. The ablation depth of these acute ablations was evaluated by light microscopy examination. RESULTS: In the acute damage study, light microscopy showed a thin surface layer in all samples with minimal thermal damage except on the long pulse Er:YAG corneas. Transmission electron microscopy revealed less than 0.3-microm surface damage for all specimens of both the optical parametric oscillator and the excimer laser samples with no evidence of collagen shrinkage. Transmission electron microscopy showed damage layers of 0.5 to 3 microm for Q-switched Er:YAG and 3 to 10 microm for long pulse Er:YAG. Scanning electron microscopy showed smooth surfaces in all eyes, although the excimer was the roughest. In the porcine eye study, ablations were produced in both PTK and PRK modes with the ablation rate per layer increasing with the fluence. At 120 mJ/cm2, the average ablation rate was 1.9 microm per layer. CONCLUSIONS: The histology from the short pulse mid-infrared optical parametric oscillator laser at 2.94 microm was comparable to the 193-nm excimer with a smooth, damage-free, ablation zone when performing PRK and LASIK.     

圆锥角膜各期的共焦显微镜表现

目的评价共焦显微镜在圆锥角膜临床研究中的应用价值。方法应用共焦显微镜观察圆锥角膜患者32例（48眼）及正常对照组17例（28眼），分别比较早、中、晚期圆锥角膜与正常对照组的图像特点。结果早期圆锥角膜出现激活状态的角膜细胞、浅基质层的细小皱褶、深基质层的暗纹、部分内皮细胞异形性明显，中、晚期圆锥角膜出现角膜上皮细胞拉伸、细胞核皱缩；基质细胞排列紊乱、基质层暗纹；而对照组未发现上述表现。各期圆锥角膜的角膜基质层厚度、不同深度角膜基质细胞密度、内皮细胞密度与对照组之间差异有统计学意义（P〈0．05）。结论共焦显微镜对早期圆锥角膜的发现以及圆锥角膜病理发展的研究具有重要的临床价值。     

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.     

Reduction in corneal haze and apoptosis by amniotic membrane matrix in excimer laser photoablation in rabbits

PURPOSE: To determine whether preserved human amniotic membrane can reduce corneal haze and keratocyte apoptosis induced by excimer laser photoablation in rabbit corneas. METHODS: Excimer photoablation was performed bilaterally in 30 rabbits with a 6.0 mm ablation zone and 120 microm depth using the VISX Star laser with the phototherapeutic keratectomy (PTK) mode. One eye was randomly covered by preserved human amniotic membrane secured with 4 interrupted 10-0 nylon sutures, and the other eye served as the control. The amniotic membranes were removed at 1 week, and corneal haze was graded with slitlamp biomicroscopy by 3 masked corneal specialists biweekly for the ensuing 12 weeks until the rabbits were killed. Another 18 rabbits were divided into 4 subgroups and received PTK alone, PTK with membrane, PTK with sham sutures, or PTK with tarsorrhaphy. All eyes were studied histologically, and 3 eyes in each group were studied by in situ terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling assay at 1, 3, and 7 days and 12 weeks, respectively. RESULTS: A consistent grading of differences in corneal haze scoring between the control corneas and the amniotic-membrane-covered corneas was noted among the 3 masked observers. Organized reticular post-PTK corneal haze peaked at 7 weeks in both groups, and the corneal haze score in the amniotic-membrane-covered group was significantly less than in the control group from 7 to 12 weeks (all P < .001). Compared to the control corneas, the amniotic-membrane-covered corneas had less inflammatory response at 1 and 3 days, less keratocyte apoptosis in the ablated anterior corneal stroma at 1, 3, and 7 days (P < .001), and less stromal fibroblast cellularity and epithelial hyperplasia at 12 weeks. CONCLUSIONS: Amniotic membrane matrix introduced at an early stage of the corneal wound healing process effectively reduced corneal haze induced by excimer laser photoablation in rabbits. Studies linking suppression of apoptosis in the acute wound-healing process with reduction of subsequent corneal scarring may have useful clinical applications.     

The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy

PURPOSE: Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. METHODS: Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of < or =270 microm in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 +/- 5.05%) and lowest in the central stroma (9.31 +/- 1.09%). In the anterior stroma, volume density was found to be 10.19 +/- 4.37%. CONCLUSION: Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.     

In-vivo slit scanning confocal microscopy of normal corneas in Indian eyes

OBJECTIVE: To study the cellular populations of healthy corneas of Indian eyes using confocal microscopy and to evaluate the correlation with age, gender and laterality. METHODS: The central corneas of 100 eyes of 50 healthy subjects were examined using an in-vivo slit scanning confocal microscope (Confoscan 2). Images were analysed for cell densities of the epithelium, stroma and endothelium. RESULTS: Good quality images enabling analysis of all cell layer populations were obtained in 74 eyes of 43 healthy subjects (22 males and 21 females) with a mean age of 31.89 +/- 13.47 (range 19-71 years). The basal epithelial cell density was 3601.38 +/- 408.19 cells/mm2 (range 3017.3-4231.1 cells/mm2). The mean keratocyte nuclei density in the anterior stroma was 1005.02 +/- 396.86 cells/mm2 (range 571.6-1249.6 cells/mm2) and in the posterior stroma was 654.32 +/- 147.09 cells/mm2 (range 402.6-1049.1 cells/mm2). Posterior keratocyte nuclei density was 30.76% less than the anterior stromal keratocyte nuclei density. The difference in keratocyte nuclei density was statistically significant (P=0.001). The mean endothelial cell density was 2818.1 +/- 361.03 cells/mm2 (range 2118.9-4434 cells/mm2) and the mean endothelial cell area was found to be 385.44 +/- 42.66 mm2 (range 268.9-489.2 mm2). Hexagonal cells formed 22.5-69.4% of the endothelial cell populations (mean 42.04 +/- 11.81%). Mean coefficient of cell size variation was 32.29 +/- 3.06 (range 27.2-39.2). No statistically significant differences were found in cell densities of any corneal layer either between female and male patients or between right and left eyes. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r=0.12, 0.07, -0.12 respectively) (P=0.001). There was a statistically significant negative correlation between mean endothelial cell density and increase in age (r=-0.42, P=0.001). Coefficient of cell size variation and age were positively correlated (r=0.73, P=0.001). CONCLUSION: In-vivo slit scanning confocal microscopy is useful for the study of corneal cell populations. Our study provides normative data of these cell populations.     

圆锥角膜行去上皮角膜胶原交联术后角膜微结构的变化

目的：采用共焦显微镜观察进展期圆锥角膜行去上皮角膜胶原交联术后角膜微结构的变化。

方法：选取2016-02/2017-02于我院行上皮角膜胶原交联术治疗的进展期圆锥角膜患者11例15眼,分别于手术前后行共焦显微镜检查,观察角膜微结构变化。

结果：术后早期角膜上皮下神经纤维显著减少或消失; 角膜前基质呈蜂窝状,几乎无典型的角膜基质细胞,术后3mo基质细胞开始出现,术后12mo基质细胞数量几乎恢复到术前水平,但角膜上皮下神经仍稀疏,未达到术前水平; 术后后部角膜基质细胞和内皮细胞大小及形态未受影响。

结论：角膜胶原交联术后角膜微结构发生变化最明显的是上皮下神经纤维和前基质细胞,但随着随诊时间的延长,这种变化呈逐渐减弱趋势。     

Protective role of corneal epithelium against ultraviolet radiation damage

PURPOSE: It is known that the corneal epithelium strongly absorbs ultraviolet radiation (UVR). The aim of the present study was to examine the protective role of corneal epithelium against UVR damage by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas. METHODS: Six New Zealand albino rabbit corneas were exposed to UVR centred around 280 nm at a dose that causes biomicroscopically significant keratitis (012 J/cm(2)). Three corneas underwent manual de-epithelialization prior to UVR exposure. A control group of three rabbits underwent only manual de-epithelialization. The animals were killed 76 hours after treatment. The corneas were stained with haematoxylin and evaluated by light microscopy. RESULTS: Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area. No damage to keratocytes or the stroma was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells, and also keratocytes in the anterior quarter of the corneal stroma. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes disappeared through the entire thickness of the stroma in the UVR-exposed area. CONCLUSION: Exposure to UVR at 280 nm alone does not result in any deep damage to the corneal stroma and keratocytes. Manual de-epithelialization causes the disappearance of anterior keratocytes. However, the stromal damage caused by UVR in the de-epithelialized corneas was very deep. The corneal epithelium serves to protect the deeper corneal structures against UVR damage, probably by absorbing a substantial amount of the UVR energy applied to the eye.     

Comparative study of human keratocyte density after corneal grafting by using confocal microscopy in vivo

PURPOSE: In a case control study we determined keratocyte density and size of nuclear area in the central segment of the corneal stroma. METHODS: We compared 20 corneas after keratoplasty with 24 eyes of normal healthy individuals. Both groups were matched according to age. Keratocyte density and nuclear area were analyzed using Nidek Confoscan 2 separately for each group. In corneal graft recipients we studied how the mentioned variables were influenced by age of corneal graft donor (ranged from 4- to 71-years old) and by the time from surgery (8 to 77 months). RESULTS: The comparison of healthy controls and patients with keratoplasty revealed no changes in keratocyte density and the size of nuclear area in central stromal layers. In patients after keratoplasty donor age influenced an increase in keratocyte nuclei area only in the posterior stroma layer (p = 0.042). No such changes were observed in anterior and midstroma layers. Donor age was not found to be significant for keratocyte density in any of the layers. Time from surgery neither influenced changes in keratocyte density nor in keratocyte nuclei area. CONCLUSIONS: In our study using confocal microscopy in vivo we found that corneal grafting does not influence keratocyte density and nuclear area in individual layers of the central corneal stroma segment (anterior, midstroma and posterior layers). Donor age influenced an increase in keratocyte nuclei area only in the posterior stroma layer.     

In vivo confocal laser-scanning microscopy to characterize wound repair in rabbit corneas after collagen cross-linking

Background: Collagen cross‐linking using the photosensitizer riboflavin combined with ultraviolet A light was developed to stiffen the cornea by increasing its mechanical and biochemical stability. Investigation of post‐treatment events, such as wound healing, is important to evaluate possible risks and to optimize treatment protocols. This in vivo confocal laser‐scanning microscopy study in rabbits was conducted to provide a quantitative and qualitative analysis of corneal wound repair over 16 weeks following collagen cross‐linking. Methods: Six New Zealand White rabbits underwent riboflavin/ultraviolet A cross‐linking. In vivo confocal laser‐scanning microscopy using a Heidelberg Retina Tomograph equipped with a Rostock Cornea Module was performed preoperatively and at 2, 4, 8, 12 and 16 weeks postoperatively. Results: From 2 weeks onwards the epithelium demonstrated no abnormalities. Evidence of inflammation was visualized in the intermediate, basal cells and Bowman's membrane. Nerve fibre regeneration was first noted at 12 weeks. Keratocyte activation and hyperreflective extracellular matrix were observed consistently, but by 16 weeks keratocyte activation was diminished, and extracellular matrix resumed normal reflectivity. Cell density in the posterior stroma and endothelium regained preoperative values by 4 weeks, although anterior stroma keratocyte cell density was still reduced by about 10% at 16 weeks. Conclusions: Complete qualitative and quantitative characterization of corneal wound repair was achieved by in vivo confocal laser‐scanning microscopy over 16 weeks following collagen cross‐linking in rabbits. In terms of assessing the ever‐increasing range of cross‐linking protocols, in vivo confocal laser‐scanning microscopy may contribute to minimizing the number of experimental animals, because multiple examinations of the same cases are possible over time.     

Keratocyte activation and apoptosis in transplanted human corneas in a xenograft model

PURPOSE: To study keratocyte activation and cellular apoptosis in transplanted human corneas during the early postoperative period. METHODS: Ten human donor corneas preserved for 6 days at 4 degrees C were transplanted into the eyes of 10 adult cats. After confocal and specular microscopy in vivo 1 week after keratoplasty, the cats were killed, and the fixed corneas were examined by TUNEL assay and by scanning (SEM) and transmission electron microscopy (TEM). RESULTS: Abnormal keratocytes, in which portions of cell bodies and processes as well as nuclei were visible, were present in all corneas and occupied the anterior 16 to 562 microm of the stroma. By TEM in the same corneas, these abnormalities represented keratocytes that were activated to a repair phenotype. Only 0% to 1% of all corneal cells were apoptotic by TUNEL assay, except for the donor keratocytes near the wound, where 7% were apoptotic. The midstromal keratocyte density was decreased at 13,936 +/- 5,910 cells/mm(3) (mean +/- SD), and the endothelial cell density was 2,298 +/- 688 cells/mm(2), representing an endothelial cell loss of 7% +/- 16%. CONCLUSIONS: Substantial keratocyte activation and low levels of cellular apoptosis occur 1 week after human corneal transplantation. The human-to-cat xenograft model of corneal transplantation demonstrated endothelial cell loss and other clinical findings similar to human allografts. The model will be useful for preclinical testing of new methods of long-term corneal preservation and of donor endothelial cell augmentation, as well as the study of human corneal wound healing and keratocyte replacement during the early postoperative period.     

Corneal hysteresis, resistance factor, topography, and pachymetry after corneal lamellar flap

PURPOSE: To measure prospectively the early changes in corneal hysteresis, topography, and pachymetry after the creation of a stromal flap cut without laser photoablation. METHODS: A 37-year-old man was referred for a bioptic procedure to correct for compound myopic astigmatism in the left eye. A 159-microm-thick 8x8.5-mm superior hinged flap was created with a mechanical microkeratome in the left cornea. Changes in the corneal hysteresis, corneal resistance factor, Goldmann correlated intraocular pressure (lOP), corneal compensated IOP, anterior and posterior topography, and optical and ultrasound pachymetry were monitored prospectively before and at 1 hour, 1 day, 5 days, and 25 days after flap creation. The right eye served as a control. RESULTS: In the left eye, corneal hysteresis and corneal resistance factor decreased immediately after the flap cut and remained lower than preoperatively at 1 hour, 1 day, 5 days, and 25 days. Corneal compensated IOP varied significantly less than Goldmann correlated IOP in both eyes. Central flattening of the horizontal meridians was observed on the difference topography maps. The values of the left eye posterior best fit sphere increased after the flap cut. Increased central corneal thickness occurred immediately after the flap cut and decreased over time without returning to its preoperative value. CONCLUSIONS: The creation of a stromal flap can modify the biomechanical properties of the cornea, including a reduction in corneal hysteresis. The topographic changes were consistent with previously reported cases of flap cut in normal corneas.     

Confocal microscopic observations of the human cornea following overnight contact lens wear

Background: Striae and folds are observed with a slidamp biomicroscope in the cornea following overnight contact lens wear. These phenomena are poorly understood. The aim of this study is to employ confocal microscopy to observe and document these and other morphological changes in die human cornea following overnight contact lens wear. Methods: Slitlamp biomicroscopy, slit‐scanning confocal microscopy and ultrasonic pachometry were performed on both eyes of 13 subjects (3M, 10F, age 24 ± 3 years) before and after eight hours overnight wear of a ‐3.00 D Bausch & Lomb one day disposable soft contact lens (Dk/t = 15.1 times 10^‐9 (cm/sec) x {ml O²/ml x mmHg)) in one eye; the other non‐lens‐wearing eye acted as a control. Results: Following sleep, both corneas were swollen (lens‐wearing eye 11.8 ± 3.8 per cent; control eye 2.1 ± 1.9 per cent) and the stroma of both corneas displayed an apparent reduction in keratocyte density (lens‐wearing eye 21 per cent; control eye 10 per cent). Folds were observed with the slidamp biomicroscope and long, straight, dark, orthogonal lines were observed widi die confocal microscope, in the posterior stroma of the oedematous lens‐wearing eyes. Such features were not observed in die control eyes. The keratocytes appeared less distinct with greater levels of corneal oedema. Conclusion: The apparent loss of keratocytes following overnight lens wear is an optical artefact that can be explained in terms of corneal oedema causing volumetric tissue expansion and a loss of optical clarity, which hampers keratocyte detection. These findings place the onus on researchers postulating a loss of stromal keratocytes following clinical interventions, such as contact lens wear, to account for the effects of oedema.     

Preliminary in vitro study of the histological effects of low fluence 193-nm excimer laser irradiation of corneal tissue

PURPOSE: To determine if moderate numbers of low fluence, 193-nm excimer laser pulses modify or damage the corneal stroma. METHODS: The corneal epithelium of fresh bovine eyes was scraped off and the exposed stroma was irradiated with 200 low fluence laser pulses from an argon fluoride excimer laser. This process was performed on five eyes each at two laser fluences, 10 mJ/cm2 and 30 mJ/cm2. The ten irradiated and three control (unirradiated) corneas were sectioned and studied by electron microscopy. The maximum and minimum thickness of the anterior layer of randomly oriented collagen fibers was measured using electron microscopy. RESULTS: The mean maximum thickness of the anterior randomly oriented layer of collagen was 1.23 +/- 0.45 microm in the control corneas, 0.67 +/- 0.32 microm in the corneas irradiated at 10 mJ/cm2, and 0.10 +/- 0.12 microm in the corneas irradiated at 30 mJ/cm2. The mean thickness of corneal stroma removed was 0.7 microm at a fluence of 10 mJ/cm2 and 1.1 microm at a fluence of 30 mJ/cm2. A thin, electron-dense pseudomembrane was present at both fluences. CONCLUSION: We report removal of bovine corneal stroma at 10 mJ/cm2--below the previously reported ablation threshold of 20 mJ/cm2.     

Long-term microstructural changes following epikeratophakia: in vivo confocal microscopy study

The unilateral epikeratophakic eye of a 20-year-old woman with a history of congenital cataracts was examined using laser scanning in vivo confocal microscopy 17 years after transplantation. In vivo confocal microscopy demonstrated a reduced keratocyte density in the grafted lenticule and the host stroma, with unusual elongated and tortuous hyperreflective branching structures in the anterior stroma of the host cornea. The sub-basal nerve plexus was present in the lenticule, although with a reduced nerve density. The appearance of the host endothelium was similar to that observed in Fuchs endothelial dystrophy. Dramatic microstructural changes were observed in almost all layers of the cornea 17 years after epikeratophakia. Although no longer performed as routine practice, in vivo confocal microscopy examination of epikeratophakia has provided fascinating insight into the potential corneal adaptations at a cellular level.     

Iron deposits in cornea in confocal microscope

PURPOSE: The study aimed to evaluate the iron deposits in corneas in confocal microscope. MATERIAL AND METHODS: The material comprised 16 eyes which underwent photorefractive keratectomy (PRK) procedure. The structure of corneas was evaluated between 3-10 years after PRK. The visual acuity after PRK was the same as the best corrected visual acuity before the procedure. The structure of corneas was evaluated in vivo using scanning slit confocal microscopy. The confocal images of corneas in patients after PRK were compared with confocal corneal images of patients with corneal scars (2 eyes), keratoconus (2 eyes), after radial keratotomy (RK) (2 eyes) and healthy patients. RESULTS: Within the central part of corneal epithelium and anterior part of stroma, the clusters of iron deposits were observed. They were round and produced different shapes. In the paracentral and peripheral part of corneas the subepithelial nerve plexus was detected. Beneath, the pattern of keratocytic nuclei, characteristic for state after PRK, was detected. In patients with corneal scars, keratoconus and after RK, the same clusters of deposits were detected. In cases of corneal scars, additionally high reflectivity of corneal structure was observed. CONCLUSIONS: The iron deposits in corneal structure arise in epithelium and anterior part of corneal stroma. The iron deposits which produce different shapes have no influence on visual acuity.     

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.