Mapping the corneal sub-basal nerve plexus in keratoconus by in vivo laser scanning confocal microscopy

PURPOSE: To produce a two-dimensional reconstruction map of the living corneal sub-basal nerve plexus in keratoconus with in vivo confocal microscopy. METHODS: Four eyes of four subjects with keratoconus were examined by slit lamp biomicroscopy, Orbscan II slit-scanning elevation topography (Bausch & Lomb Surgical, Rochester, NY), and laser scanning in vivo confocal microscopy with the Heidelberg Retina Tomograph II, Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany). Subjects were asked to fixate on targets arranged in a grid to enable in vivo confocal microscopy of the cornea in a wide range of positions. RESULTS: A mean of 402 +/- 57 images were obtained for each cornea, to create confluent montages. The mean dimensions of the corneal areas mapped were 6.60 +/- 0.70 mm horizontally and 5.91 +/- 0.72 mm vertically. All corneas exhibited abnormal sub-basal nerve architecture compared with patterns previously observed in normal corneas. At the apex of the cone, a tortuous network of nerve fiber bundles was noted, many of which formed closed loops. At the topographic base of the cone, nerve fiber bundles appeared to follow the contour of the base, with many of the bundles running concentrically in this region. Central sub-basal nerve density was significantly lower in keratoconus corneas (10,478 +/- 2,188 microm/mm2) compared with normal corneas (21,668 +/- 1,411 microm/mm2; Mann-Whitney; P < 0.01). CONCLUSIONS: This is the first study to elucidate the overall distribution of sub-basal nerves in the living central to midperipheral human cornea in keratoconus, using laser scanning in vivo confocal microscopy.     

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。     

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.     

圆锥角膜的共焦显微镜表现临床分级

目的 观察临床上不同阶段圆锥角膜的共焦显微镜图像，推测圆锥角膜的病理发展过程，并进行圆锥角膜的共焦显微镜分级。方法 采用共焦显微镜（Confoscan 2．0），观察24例24眼不同发展阶段（lawless分期）圆锥角膜患者的角膜共焦显微镜图像，进行分析并提出焦显微镜下的临床分分期。结果 共焦显微镜下圆锥角膜首先出现了排列规则的裂隙状暗纹，随着病情的不断进展，病变逐渐由角膜后弹力层向前发展，逐渐累及角膜的后基质和前基质，同时角膜基质细胞核拉长、排列出现紊乱，前后基质细胞失去了原有特征。急性圆锥角膜的患者角膜基质细胞出现水肿，角膜瘢痕在共焦显微镜下呈强反光的无细胞样结构。讨论 共焦显微镜下圆锥角膜的病理发展过程，是从角膜后弹力层开始，由后向前发展，逐渐累及角膜的后基质、前期基质，最后角膜破裂引起急性圆锥角膜，角膜出现水肿，愈合后遗留瘢痕。据此提出圆锥角膜共焦显微镜分期将为四个阶段：第一阶段、角膜裂隙样暗纹累及角膜后弹力层；第二阶段、角膜裂隙样暗纹累及角膜后后基质；第三阶段、角膜裂隙样暗纹累及角膜前基质；第四阶段、出现角膜基质水肿或者瘢痕。     

In vivo corneal confocal microscopy in keratoconus

PURPOSE: To evaluate the corneas of keratoconic subjects using in vivo confocal microscopy. METHODS: Slit scanning confocal microscopy was used to evaluate the central cornea of one eye of each of 29 keratoconic subjects (mean age 31 +/- 10 years; range 16-49 years). Quantitative aspects of corneal morphology were compared against data from control subjects. RESULTS: Compared with normal control corneas, epithelial wing cell nuclei were larger (p < 0.0001) and epithelial basal cell diameter was larger (p < 0.05) in the keratoconic cornea. Many of the keratoconic corneas investigated showed increased levels of stromal haze and reflectivity, which appeared to be related to the presence of apical scarring on slit lamp examination. A grading scale was devised to quantify the levels of haze. This scale was shown to provide a measure of the level of scarring present. The anterior keratocyte density (AKD) and posterior keratocyte density were 19% lower (p < 0.0001) and 10% lower (p = 0.004) than in controls, respectively. The reduction in AKD was significantly associated with three factors: a history of atopy, eye rubbing and the presence of corneal staining. The mean endothelial cell density in keratoconus was 6% greater than that of normal controls (p = 0.05). The level of endothelial polymegethism was shown not to be different between keratoconic subjects and matched controls (paired t-test: t = 1.82, p = 0.08). CONCLUSIONS: Confocal microscopy demonstrates significant quantitative alterations of corneal morphology in keratoconus.     

Keratocyte density in keratoconus. A confocal microscopy study(a)

PURPOSE: To estimate keratocyte density in human corneas with keratoconus by confocal microscopy. DESIGN: Prospective, observational cohort study. METHODS: Twenty-nine unscarred corneas of 19 patients with keratoconus and 29 corneas of 19 controls matched for age (+/-3 years) and contact lens wear were examined by using confocal microscopy. Images were recorded from the full-thickness central cornea. A masked observer manually counted bright objects (keratocyte nuclei) in images without motion blur. Cell densities in anteroposterior stromal layers of keratoconus corneas were compared with densities in corresponding layers of control corneas. RESULTS: In keratoconus patients, age 40 +/- 15 years (mean +/- standard deviation), keratocyte density was 19% lower in those who wore contact lenses (16,894 +/- 4032 cell/mm(3), n = 12) than in those who did not wear contact lenses (20,827 +/- 4934 cell/mm(3), n = 17, P =.03). In control patients, age 39 +/- 16 years, there was no difference in keratocyte density between those who wore contact lenses (n = 12) and those who did not wear contact lenses (n = 17, P =.80). Among contact lens wearers, keratocyte density was 25% lower in keratoconus corneas (16,894 +/- 4, 032 cell/mm(3), n = 12 [9 = rigid gas-permeable lenses, 3 = soft lenses]) than in control corneas (22,579 +/- 2, 387 cell/mm(3), n = 12 [3 = rigid gas-permeable lenses, 9 = soft lenses], P =.002), the result of cell density being lower in the most anterior keratocyte layer (P =.001) and the layers between 0% to 10% (P <.001), 67% to 90% (P <.001), and 91% to 100% (P <.001) of stromal thickness. Among noncontact lens wearers, there was no difference in cell density between keratoconus and controls (P =.41). CONCLUSION: Keratocyte density is decreased in the anterior and posterior stroma of keratoconus patients who wear contact lenses.     

Superoxide dismutase isoenzymes in the normal and diseased human cornea

PURPOSE: The human cornea, a tissue much exposed to oxidative stress, is rich in extracellular superoxide dismutase (SOD). In this study, the contents and distributions of the SOD isoenzymes in the normal human cornea were compared with those in corneas affected by keratoconus and bullous keratopathy. METHODS: The central and peripheral parts of normal human corneas were analyzed separately. Central corneal buttons were obtained from patients with keratoconus and bullous keratopathy who were undergoing primary keratoplasty or retransplantation. SOD enzymatic activities were determined by a direct spectrophotometric method, and extracellular SOD and the cytosolic Cu- and Zn-containing SOD (CuZn-SOD) proteins were determined with ELISA and studied with immunohistochemistry. RESULTS: The total SOD content, and particularly the extracellular SOD content, was lower in the central than in the peripheral normal cornea. CuZn-SOD and extracellular SOD were demonstrated in all three corneal layers. CuZn-SOD was found in cells, whereas extracellular SOD appeared to be localized on cell surfaces, in basal membranes, and in the stroma. In keratoconus, corneal levels of extracellular SOD were half those in the control corneas, whereas CuZn-SOD and the mitochondrial Mn-containing SOD levels were normal. In bullous keratopathy, apart from edematous dilution, SOD isoenzyme levels were essentially normal. In a remarkable finding, the same pattern in SOD isoenzyme levels as in the original disease was also found at retransplantation. CONCLUSIONS: Extracellular SOD and CuZn-SOD show markedly different distribution patterns within the human cornea. Extracellular SOD activity in the central cornea is halved in keratoconus, compared with that in normal control corneas. The finding of a similar reduction at retransplantation in keratoconus suggests reduced corneal extracellular SOD synthesis in cells of the host as a cause of the low enzyme levels.     

Determination of corneal volume from anterior topography and topographic pachymetry: application to healthy and keratoconic eyes

Purpose: To describe a method to measure corneal volume from topography and pachymetry, and test its clinical use on a sample of healthy human subjects and a case of circumscribed posterior keratoconus. Methods: Corneal curvature (PCT 200^® corneal topography system; Optopol Technology SA, Zawiercie, Poland) and ultrasonic topographic pachometry on 25 points (Ophthasonic^® A‐Scan/Pachometer III; Teknar Inc., St Louis, MO, USA) were measured on each of 12 young healthy corneas and one cornea suffering from circumscribed posterior keratoconus. Topography and pachymetry data were used to calculate the coordinates for the corresponding points on the posterior surface of the cornea. TableCurve 3D software (Systat Software Inc., Chicago, IL, USA) was used to fit a surface to those points measured. Integration of the surface fitted to the data points, corresponding to the anterior and posterior corneal surfaces, was used to calculate the volume underneath each of them. Subtraction of volumes underneath anterior and posterior surfaces, taking into account an axial offset equal to the central corneal thickness, rendered corneal volume for the central 6 mm of the cornea. Results: Central corneal thickness ranged from 520 to 630 μm for the healthy corneas. Corneal volumes for this sample analyzed averaged 18.66 ± 1.15 mm³ (range 17.25–20.53 mm³). For the posterior keratoconic cornea, the affected area was located at about 1.5–2 mm from the corneal center on the 135° hemimeridian of the right eye, observed through topographic pachymetry. Calculated corneal volume for the central 6 mm was 16.072 mm³, noticeably lower than those found in the sample without pathology, but within the range for corneas presenting with keratoconus. Conclusions: Corneal volume is a useful parameter for characterising dystrophic corneas and can aid in the detection of rare anomalies which are hardly detected with corneal topography and/or central corneal thickness evaluation. A potentially useful measure of corneal volume can be calculated from anterior corneal topography and topographic pachymetry data. Values obtained are in good agreement with previous studies using corneal tomography techniques. The methodology has been shown to have potential for retrospective analysis of data, or where no access is available to tomographical techniques.     

Expression of betaig-h3 is lower than normal in keratoconus corneas but increases with scarring.

PURPOSE: Keratoconus is a progressive ectatic disease of the cornea. Despite extensive clinical and laboratory investigations, its pathogenesis remains unclear. In this study, we examined the localization of betaig-h3, a recently described extracellular matrix protein in keratoconus corneas both in the absence and presence of subepithelial scarring. METHODS: Two normal corneas and central corneal buttons of 10 patients with keratoconus were excised during perforating keratoplasty and examined, including one case with acute corneal hydrops. In one case, keratoconus was associated with Down syndrome. Immunodetection was done with an antipeptide antibody reacting with the N-terminal part of betaig-h3. RESULTS: We found decreased betaig-h3 levels in the basal epithelial layer and keratocytes of keratoconus corneas. In the scarred corneas, however, betaig-h3 levels were increased in the basal epithelial layers and in activated keratocytes at the places of scarring. In the cornea of the patient with Down syndrome, we found an additional betaig-h3-positive zone in the anterior stroma. CONCLUSIONS: The decreased levels of betaig-h3 corneas seem to be specific for keratoconus. Considering the putative role of betaig-h3 as a cellular-attachment protein, paucity of betaig-h3 in the corneal stroma may lead to decreased mechanical stability and contribute to the development of keratoconus.     

圆锥角膜的光密度分析

目的 使用Pentacam对圆锥角膜和健康角膜的光密度进行测量,比较健康角膜与不同等级圆锥角膜的透明度.方法 使用Pentacam眼前节分析系统测量对照组(30例30眼正常角膜)以及圆锥角膜组(36例36眼圆锥角膜)的光密度,以角膜顶点为中心,分别测量0～2 mm、2～6 mm、6～ 10 mm、10 ～ 12 mm直径范围的前层(120μm)、后层(60 μm)以及中间层的角膜光密度.使用Amsler-Krumeich分级方法对圆锥角膜进行分级,对比不同等级光密度差异.分析圆锥角膜光密度与厚度、陡峭K值的相关性.结果 总直径的全层角膜光密度圆锥角膜组(17.96±3.23)和对照组(17.39±1.95)之间差异无统计学意义(P =0.124);但是在0～2 mm直径范围内的前层、中层、全层,2～6mm直径范围内的前层以及总直径的前层中,圆锥角膜组的光密度均显著高于对照组(均为P＜0.05).按照圆锥角膜等级分组,对照组角膜光密度与轻度圆锥角膜组各层比较,差异均无统计学意义(均为P＞0.05),但对照组角膜光密度与中度圆锥角膜组0～2mm直径范围内的前层比较差异有统计学意义(P＜0.05).重度圆锥角膜在0～2 mm直径范围内各层光密度较对照组、轻度圆锥角膜组、中度圆锥角膜组均显著增高(均为P＜0.05),在2～6 mim直径范围和总直径的前层、中层和全层与其余三组相比均显著增加(均为P＜0.05).圆锥角膜组的陡峭K值与0～2 mm直径范围的总角膜光密度显著相关(P＜0.05).圆锥角膜组的角膜顶点以及角膜最薄点的厚度与光密度也存在明显的相关性(均为P＜0.05).结论 相比正常角膜,轻度圆锥角膜光密度不发生改变,中度圆锥角膜光密度增高主要发生于角膜前层0～2 mm直径范围内,重度圆锥角膜光密度增高主要发生于角膜前层0～6 mm直径范围内和角膜中层2 mm直径范围内,而且圆锥角膜光密度随疾病发展而增高.角膜光密度是圆锥角膜的一个重要参考指标,可以用来检测圆锥角膜发病进程以及治疗效果.     

In vivo investigations of the corneal epithelium with the confocal Rostock Laser Scanning Microscope (RLSM)

BACKGROUND: The confocal tandem scanning microscope was first used in 1985 by Lemp et al for in vitro and in 1990 by Cavanagh et al for in vivo investigation of human eyes. The aim of this study was to investigate the cells of the central and the peripheral portions of the corneal epithelium and to measure corneal epithelium thickness and the total thickness of the corneas of our volunteers with the new Rostock Laser Scanning Microscope. MATERIAL AND METHODS: A Heidelberg Retina Tomograph (HRT II) was used in combination with a water contact microscope lens (Zeiss, x63, 0.95), the Rostock cornea module (RCM) developed at our institute for the in vivo examination of the cornea. In this study, 92 eyes of 68 subjects between the ages of 15 and 88 years were examined. RESULTS: At the superficial cell layer, the average cell density in the central cornea was 840 +/- 295 cells/mm2, and in the periphery it was 833 +/- 223 cells/mm2. At the wing cell layer, the average cell density rises to 5070 +/- 1150 cells/mm2 in the central and to 5582 +/- 829 cells/mm2 in the peripheral cornea. At the basal cell layer, the cell density rises further to 8996 +/- 1532 cells/mm2 in the central and 10,139 +/- 1,479 cells/mm2 in the peripheral corneal epithelium. The average corneal thickness in the central region was found to be 545 +/- 25 microm, and 652 +/- 75 microm in the periphery. The average epithelium thickness was determined centrally to be 54 +/- 7 microm, and peripherally 61 +/- 5 micrim. CONCLUSIONS: The Rostock Scanning Laser Microscope offers a standardized, reproducible, safe, and fast diagnostic procedure for the evaluation of the corneal epithelium. This technology allows better image quality compared with confocal-slit scanning microscopes and produces a precise depth measurement.     

Observations of banding patterns (Vogt striae) in keratoconus: a confocal microscopy study

PURPOSE: To investigate Vogt striae in keratoconus using confocal microscopy. METHODS: The central cornea of 51 eyes of 29 subjects with keratoconus was observed using a slit-lamp biomicroscope, slit-scanning confocal microscope (TOMEY Confoscan 1), and corneal topographer (EyeSys 2000). RESULTS: Alternating dark and light bands were seen in the stromal images of 23 eyes examined. The bands corresponded with the appearance of Vogt striae on slit-lamp biomicroscopy examination. Bands were found most commonly in the posterior stroma. Posterior bands varied in width, ran mainly in a nearly vertical direction, and appeared to run a straight course through individual image frames. Keratocyte nuclei were located in between the bands. Posterior keratocyte density was unaffected by the presence of bands. Nerve fibers appeared to run a straight course through the bands. When present, bands in the anterior stroma showed greater variability in width and direction within a single frame. Bands were only present in the anterior stroma in more severe levels of keratoconus. The difference in banding pattern noted between the anterior and posterior stroma parallels the known collagen fiber arrangement in the anterior and posterior stroma. CONCLUSIONS: The bands apparent on confocal microscopy of the stroma of the keratoconic cornea correspond with Vogt striae on slit-lamp microscopy. It appears that these bands (and hence Vogt striae) represent collagen lamellae under stress. The stress pattern appears to radiate from the center of the cone and is consistent with the direction of striae when viewed with the confocal microscope.     

Corneal topography of posterior keratoconus.

Posterior keratoconus is an unusual abnormality of the cornea generally classified as one of the anterior chamber cleavage anomalies. It is characterized clinically by the presence of a circumscribed or generalized corneal thinning with posterior depression of the cornea and is considered distinct from keratoconus. Although patients with posterior keratoconus may have visual complaints clearly related to their abnormal corneas, the surface topography of these corneas has not been studied in detail. Keratometry and photokeratoscopy provide an incomplete picture of the surface geometry of posterior keratoconus. We utilized computer assisted topographic analysis to study the cornea of a patient with posterior keratoconus. The Topographic Modeling System demonstrated that the patient's cornea showed a central steepened "cone" coincident with the area of circumscribed posterior keratoconus as well as paracentral flattening. This report documents the topographic abnormality in this rare disorder.     

Increased endothelial cell density in the paracentral and peripheral regions of the human cornea

PURPOSE: To systematically investigate the central, paracentral, and peripheral endothelial cell density (ECD) in normal human corneas. DESIGN: Observational case series and experimental study. METHODS: Noncontact specular microscopy was undertaken to determine the ECD of the central, paracentral (2.7 +/- 0.2 mm from center) and peripheral (4.7 +/- 0.2 mm from center) regions of the cornea of 48 normal eyes. The ECDs of central and peripheral regions were also determined with contact specular microscopy in 21 normal eyes and a group of 30 Optisol-GS eye bank corneas were evaluated with alizarin red stain. Histologic ECD of 13 Optisol-GS stored corneas were also determined. RESULTS: Paracentral and peripheral ECD measured with the noncontact specular microscope were 5.8% (P <.01) and 9.6% (P <.001) increased compared with central ECD. Superior peripheral ECD was increased compared with the other three peripheral quadrants (P <.05) and was 15.9% higher than central ECD. Contact specular microscopy showed an increase of 8.9% in the peripheral ECD from the center. Alizarin red stained corneas confirmed the specular microscopy numbers with a 9.2% increase in the paracentral region, and a 17.2% increase in the peripheral region. Histological cross sections of human corneas also showed a 22.9% increase in peripheral ECD compared with the central region. CONCLUSIONS: The human cornea has an increased ECD in the paracentral and peripheral regions of cornea compared with the central region. The superior peripheral region of the corneal endothelium has the largest increase in ECD. These data on normal endothelial cell distribution in the human cornea are especially significant as they relate to new surgical techniques and endothelial wound repair.     

圆锥角膜的免疫组织化学研究

目的：了解圆锥角膜基底膜免疫组化的变化,以发现圆锥角膜可能的特异性改变,为探讨其发病机制提供依据。方法：收集圆锥角膜在我院眼科行穿透性角膜移植术的角膜片,用链霉亲和素－生物素法（SP法）检测圆锥角膜、角膜白斑、正常角膜基底膜Ⅳ型胶原、Fn、Ln表达的异同,并进行统计学分析。结果：圆锥角膜基底膜Ⅳ型胶原,Fn染色较正常角膜显著升高,但与角膜白斑比较无显著性差异。结论：圆锥角膜基底膜的改变可能与创伤愈合有关。提示圆锥角膜原发病变可能在角膜基质细胞。眼科学报2000;17：65－67。     

Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy

PURPOSE: Evaluation of a new method to visualize distribution and morphology of human corneal nerves (Adelta- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. METHODS: Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 microm thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope "Odyssey XL," step size between 0.5 and 1 microm, and optical sections were digitally 3D-reconstructed. RESULTS: Immediate staining of explanted corneas by the Life/Dead-Kit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, Adelta- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. Adelta-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, Adelta-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. CONCLUSIONS: Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.     

Quantitative analysis of immunogold labellings of collagen types I,III, IV and VI in healthy and pathological human corneas

Background: We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. Methods: Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. Results: Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowman's layer. In pathological corneas with a collagenous layer posterior to Descemet's membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. Conclusion: The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.     

圆锥角膜中细胞凋亡及Fas-L蛋白的表达

目的 探讨凋亡与圆锥角膜发病的关系及凋亡相关蛋白Fas-L的表达.方法 对20例圆锥角膜及5例正常角膜用原位末端标记法(TUNEL)检测凋亡,用免疫组织化学SP法检测Fas-L蛋白的表达;透射电镜观察凋亡细胞的形态学变化.结果 TUNEL染色示圆锥角膜组中上皮层、基质层及内皮层中细胞凋亡与正常角膜组比较差异均有统计学意义(P＜0.05);免疫组织化学示圆锥角膜组与正常角膜组基质层间Fas-L表达比较差异有统计学意义(P＜0.05);透射电镜可见圆锥角膜中存在凋亡特征的细胞.结论 圆锥角膜中存在凋亡,Fas-L蛋白的表达存在异常,Fas-FasL系统可能在圆锥角膜细胞凋亡中发挥了重要作用.     

Two cases of posterior keratoconus

We studied 3 eyes from 2 patients with posterior keratoconus. One patient (a 17-year-old female) had posterior keratoconus bilaterally and the other (a 8-year-old male) had posterior keratoconus in one eye and Peters' anomaly in the contralateral eye. Slit-lamp biomicroscopy disclosed localized thinning with stromal haze underlying the endothelium in the central cornea. Photokeratoscopy revealed no abnormalities suggestive of anterior keratoconus. Wide-field specular microscopy showed the endothelial cells with normal cell density (approximately 3500 cells/mm2) and morphology in both the peripheral and central corneas (adjacent to the hazy areas).     

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.