Adhesion molecules in normal and pathological corneas

Background: Adhesion molecules are cell surface receptors that are probably important in various cell-cell and cell-extracellular matrix interactions of the cornea. Method: In this immunohistochemical light-microscopic study we analyzed the expression pattern of adhesion molecules in normal and pathological human corneas (cases of corneal inflammation and degenerative disorders). The analyzed molecules included the 1 integrin or VLA family VLA-1-6, the 2 integrins or leukocyte integrins LFA-1, Mac-1, and p150,95, the immunoglobulins LFA-3, CD2, ICAM-1 and VCAM-1 and the selectins ELAM-1 and GMP-140. Results: Inflamed cornea (in contrast to normal cornea). On corneal epithelium, increased expression of the 2 subunit of VLA-2 was detected and ICAM-1 was induced on the basal epithelial cells. On corneal stromal keratocytes, LFA-3 was induced and expression of the subunits of VLA-1-6 and ICAM-1 was increased. On vascular endothelium, VCAM-1 and ELAM-1 were induced and ICAM-1 and GMP-140 expression was increased. On corneal endothelium, ELAM-1 was induced and increased levels of the 1 subunit of VLA-1 and GMP-140 were expressed. Degenerative disorders (in contrast to normal cornea): In corneas with degenerative disorders we found decreased expression of adhesion molecules. Conclusion: Inflammatory cytokines increase the expression of the adhesion molecules. Increased expression of the VLAs probably promotes cell-extracellular matrix and cell-cell interactions. ICAM-1, VCAM-1, LFA-3, ELAM-1 and GMP-140 expression was increased on vascular endothelium in inflamed corneas. Corresponding receptors on leukocytes probably enable a selective recruitment of different leukocyte populations in inflammatory corneal diseases. The decreased expression of adhesion molecules in corneal degenerative disorders is probably a sign of reduced cell-cell and cell-extracellular matrix interactions.     

人类白细胞抗原DR分子和细胞间粘附分子-1在正常和病变角膜的表达及意义

目的　探讨正常及病变角膜中人类白细胞抗原ＤＲ 分子和细胞间粘附分子１ 的表达及其在角膜炎症和移植排斥反应中的作用和影响。方法　应用免疫组化技术对１０ 例正常人及４８ 例患者的病变角膜标本进行人类白细胞抗原ＤＲ分子和细胞间粘附分子１ 的表达的检测。结果　正常人角膜无或轻微表达人类白细胞抗原ＤＲ 分子和细胞间粘附分子１ ;病变角膜,尤其是炎症角膜及角膜移植术后发生排斥反应的角膜,其阳性表达率明显增高。结论　人类白细胞抗原ＤＲ 分子和细胞间粘附分子１ 的异常表达与角膜炎性反应有关,并可促进移植排斥反应的发生     

Biochemical studies of normal and keratoconus corneas

Keratoconus corneas are characterized by a reduced mechanical stability. The mechanical strength depends primarily on the collagen. In the present study the cross-linking pattern is analysed because it is important for the mechanical properties; an analysis of the amino acids of the collagen is included. Surprisingly, we did not find any change in the cross-linking pattern of collagen from keratoconus corneas compared to normal corneas. Neither did we find any difference in the composition of amino acids of the collagens. Thus no alteration was found in the molecular structure which could explain the marked reduction in the mechanical stability of keratoconus corneas.     

Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

Videokeratography database of normal human corneas.

AIM: To form a database of videokeratography patterns and quantitative indices describing normal human corneas using the absolute scale. METHODS: Both eyes of 195 normal subjects were examined with a TMS-1 videokeratoscope. Videokeratographs were divided into 10 categories based on a classification scheme devised from the absolute scale and analysed with 10 quantitative indices devised to describe phenotypic features of keratoconus videokeratographs. Correlations were sought between videokeratograph patterns and quantitative indices. Additionally, data were analysed for differences in age, sex, and ethnicity. RESULTS: For symmetric videokeratography patterns, analysis in the absolute scale was similar to a previous study done in the normalised scale. In the asymmetric categories, analyses differed markedly. Using the absolute scale and our classification scheme more variation in normal videokeratography patterns could be appreciated. There was good correlation between quantitative indices and videokeratography patterns. Neither videokeratography patterns nor indices differed significantly between sex, ethnic groups, or age using two way analysis of variance. CONCLUSIONS: Pattern analysis of videokeratographs in the absolute scale using, a standard classification scheme, may be more useful in trying to determine whether a cornea is normal or represents subtle early disease than analysis in the normalised scale. Quantitative indices could remove the subjectivity from the decision making process thus facilitating universal reproducibility of videokeratography data interpretation.     

Optical consequences of asymmetries in normal corneas

A survey of videokeratographs of normal corneas shows many with substantial peripheral asymmetries. For sufficiently large pupils (5.5 mm in this study) these asymmetries lead to coma-like axial aberrations large enough to produce measurable losses in vision in a number of cases. Starting from the output of a videokeratographic instrument, a method of estimating the optical effects of corneal asymmetries using Zernike circle polynomials is outlined. It is further shown that in a first approximation corneal asymmetries can be identified with the primary aberration coma and that this aberration is approximately due to a uniform gradient of refractive power across the cornea. Calculations for a representative case predict that a significant improvement in modulation transfer would follow from correction of this aberration.     

Comparison of mechanical properties of keratoconus and normal corneas

In order to measure more directly the physical properties of the corneoscleral shell in keratoconus, mechanical stress-strain tests were performed on strips of human cornea. Rectangular human corneal samples were glued to the arms of an extensiometer, a technique that avoids the problems of slippage and compression found with conventional clamps. Strain was measured using an electromagnetic displacement probe. The entire apparatus was immersed in a thermoregulated mineral oil bath to maintain constant corneal hydration. The resultant data obey the relation σ = A[e^α??1]. Stiffening constants α ranged from 34 to 82. We found that there are significant differences in the parameters for the two groups, with keratoconus corneas yielding lower α and higher A values than the normal controls. However, within the physiological range these differences offset so that there is no difference in the overall elastic behaviour of the keratoconus and normal corneas. Creep tests at constant load were also performed on normal and keratoconus corneas.     

Comparative studies of collagens in normal and keratoconus corneas

In this paper we present strong evidence that the aberrations in keratoconus corneas are not directly related to alterations in collagen composition and distribution. This conclusion is based on comparative studies of collagen types I, III, IV, V and the recently described collagen types VI and VII in keratoconus and normal corneas. The data are derived from biochemical analysis of collagen fractions sequentially extracted with pepsin and sodium-dodecylsulphate, from amino acid analysis of hydrolysates of entire corneal tissues as well as from immunoblotting of the extracted collagens with specific antibodies. These antibodies were also used to examine the distribution of the collagens in immunofluorescence experiments on corneal sections. The yields of the collagen extractions were demonstrated to be age dependent but were not altered in keratoconus samples. Apart from one case associated with osteogenesis imperfecta type I, comparative studies of keratoconus and normal corneas showed no differences in collagen composition of the extracts. This was confirmed by amino acid analysis of tissue-hydrolysates. The distributions of collagen types I, III, IV, V, VI and VII in corneal sections were in general unchanged in keratoconus corneas, the only differences being in scar tissues observed in the Bowman layer of some keratoconus samples.     

Human basement membrane components of keratoconus and normal corneas

Using immunofluorescence techniques, we analyzed the distribution of glycoproteins in normal and keratoconus corneas of humans. Laminin, bullous pemphigoid antigen, fibronectin, and fibrin/fibrinogen were all found in the epithelial basement membrane of normal corneas. Keratoconus corneas produced similar results, except that staining for fibrin/fibrinogen was weak. Fibrin/fibrinogen was absent from normal corneal basement membrane in animal models studied previously. Keratoconus may be the result of the lysis of fibrin or may involve impeded elaboration of fibrin.     

中国人正常角膜的共焦显微镜检查

摘要目的探讨正常人共焦显微镜下角膜各层细胞的活体细胞形态学特征及密度.方法对28例(年龄36.6士14.5岁,范围19～68岁)41眼(男14眼,女27眼)正常人中央部角膜进行活体共焦显微镜检查,描述角膜各层结构的图象特点,并分析角膜各层细胞的密度与年龄的相关性.结果上皮表层细胞排列疏松,边界清楚,核反光较强;基底层细胞排列紧密,胞质和核反光弱,偶尔可见点状细胞核,细胞平均密度为5724.41士562.5个/mm2.Bowman's膜为无一定形状的膜状物,仅可分辨的结构是上皮下的念珠状神经纤维.基质层中可见相对较暗的背景下明亮的角膜细胞核.前基质内有放射状分布的神经纤维.前基质层角膜细胞平均密度为1099.10士164.90个/mm     

Glucose concentration profiles of normal and ultraviolet radiation-exposed rabbit corneas

The passage of glucose within the cornea has been thought to occur by passive diffusion processes. However, corneal glucose concentration profiles have not been established to support this notion. While microfluorometric methods of metabolite assay typically have been used as a means of assessing regional brain metabolism, this unique methodology of tissue isolation and metabolite determination has not previously been applied to the cornea. Since this technique permits metabolite quantification on microgram-sized tissue samples, a co-ordinated corneal glucose concentration profile can be obtained. Tissue preparation consisted of liquid nitrogen freezing, cryo-sectioning, and freeze-drying, with storage at -20 degrees C. The sections were thawed under vacuum pump, subsectioned, weighed, and assayed for glucose concentration (by dry weight). This study established a glucose concentration profile of the epithelium, anterior stroma, midstroma, posterior stroma, and endothelium for the normal pigmented rabbit cornea. A glucose concentration profile for UV radiation-exposed rabbit corneas also was documented. The UV radiation glucose profile data indicate the presence of an active transport mechanism capable of delivering glucose into the corneal epithelium against a concentration gradient. The presence of a transport system that 'pulls' glucose through the deeper corneal layers thus would make epithelial integrity important for the maintenance of overall corneal viability.     

Generation of complement-derived anaphylatoxins in normal human donor corneas

Complement-derived anaphylatoxins (C3a, C4a, and C5a) are potent, stable mediators of acute inflammation. Because human corneas contain functional complement, the authors subjected normal human donor corneas to various forms of immunologic or chemical injury to determine if the complement system could be activated and anaphylatoxins generated. The experimental cornea of each donor pair was injected with lipopolysaccharide (LPS) or immune complexes or injured by application of acid or alkali. The remaining cornea of each donor pair served as a control. After incubation of corneas in tissue culture media for 6 hours and elution in phosphate-buffered saline for 24 hours, C3a, C4a, and C5a were measured in corneal eluates by radioimmunoassay. Compared with control corneas, C3a levels were significantly increased in corneas injected with LPS or immune complexes and in corneas injured with acid or alkali. C4a levels were significantly elevated in corneas injected with immune complexes and in corneas injured with acid or alkali but not in corneas injected with LPS. C5a levels were detectable only in corneas injured with acid or alkali. These results suggest that immunologic reactions in the human cornea may activate the classic or alternative complement pathways and generate anaphylatoxins. Additionally, chemical injuries with acid or alkali generate anaphylatoxins in the cornea. Anaphylatoxins may participate in the acute inflammatory response of the human cornea to chemical or immunologic injury.     

A pathological study on rabbit corneas after laser in situ keratomileusis

PURPOSE: To investigate pathological changes in rabbit corneas after laser in situ keratomileusis (LASIK). MATERIALS AND METHODS: We performed LASIK on rabbit corneas to theoretically correct 10.0 diopters of myopia. The corneas were studied pathologically at day 0, and 3 days, 1 week, 3 weeks, 4 months, and 9 months after LASIK. RESULTS: At 3 days after LASIK, keratocytes in the ablated area changed morphologically into fibroblastic cells. And the structure of collagen fibers in the stroma was broken. These changes had disappeared almost entirely at 4 months after LASIK. There were no proliferative changes in the stroma of the ablated cornea 9 months after LASIK. No significant changes were observed in the endothelium. CONCLUSIONS: The damage to rabbit corneas induced by LASIK was mild to moderate under the present experimental conditions.     

Collagenolytic/gelatinolytic metalloproteinases in normal and keratoconus corneas.

Cells of keratoconus corneas have been reported to produce higher levels of collagenolytic/gelatinolytic enzymatic activities than do cells of normal corneas. The current study investigates the contribution of 1) specific enzyme gene products, and 2) the degree to which these proteins are present in the activated forms, to the increased enzymatic activities. We demonstrate that two neutral gelatinolytic enzymes, a 66/59 kD form and a 92 kD form, can be directly extracted from both normal and keratoconus corneas. These enzymes are identified as the pro- and activated forms of MMP-2 and as the pro-form of MMP-9, specific members of the matrix metalloproteinase family. Normal and keratoconus corneas show no significant differences in amounts or types of extractable neutral gelatinases, nor in the amounts or types that they synthesize in culture. Furthermore, in both the normal and keratoconus corneas, gelatinases are found primarily in the inactive form. These studies suggest the possible importance of changes in proteinase inhibitor levels to the characteristic biochemical features of keratoconus corneas.     

Ⅰ型前弹力层角膜营养不良患者角膜与正常角膜蛋白的差异表达

目的 探讨中国人Ⅰ型前弹力层角膜营养不良(corneal dystrophy of Bowman laver type 1,CDB1)患者角膜与正常角膜的蛋白差异表达.方法 取1个CDB1家系3例患者外周血,提取基因组DNA,PCR扩增TGFBI基因第4、11、12、14外显子片段并进行直接测序,证实其突变位点.取板层或穿透性角膜移植术后的病变角膜,部分标本行HE染色、阿辛蓝、PAS、刚果红、Masson三色染色后光学显微镜观察,剩余标本提取组织蛋白后进行双向凝胶电泳.以3例正常角膜组织为对照.结果 3例CDBI患者均发现TGFBI基因R124L突变.HE染色证实病变主要累及前弹力层,PAS、刚果红、Masson三色染色阳性,阿辛蓝染色阴性.在PI 4-7,相对分子质量7×103～30×103之间病变与正常角膜蛋白表达存在27个差异点.结论 中国人CDB1患者以R124L基因突变为主.CDB1角膜异常沉着性质为细胞外淀粉样纤维蛋白沉着.在PI 4-7,相对分子质量7×103～30×103之间的差异蛋白可能在CDB1发病过程中起着重要作用.     

A pathological study on rabbit corneas after laser In situ keratomileusis

Purpose: To investigate pathological changes in rabbit corneas after laser in situ keratomileusis (LASIK).Materials and Methods: We performed LASIK on rabbit corneas to theoretically correct 10.0 diopters of myopia. The corneas were studied pathologically at day 0, and 3 days, 1 week, 3 weeks, 4 months, and 9 months after LASIK. Results: At 3 days after LASIK, keratocytes in the ablated area changed morphologically into fibroblastic cells. And the structure of collagen fibers in the stroma was broken. These changes had disappeared almost entirely at 4 months after LASIK. There were no proliferative changes in the stroma of the ablated cornea 9 months after LASIK. No significant changes were observed in the endothelium. Conclusions: The damage to rabbit corneas induced by LASIK was mild to moderate under the present experimental conditions.     

Distribution of catecholaminergic nerve fibers in normal and alkali-injured rabbit corneas

The alkali-burned corneas of 12 rabbits were studied with fluorescence microscopy 1 week, 3 weeks, and 6 months after injury to identify the arrangement of catecholaminergic nerve fibers (CNF) and related levels of norepinephrine. In the wounded corneas, CNF were reduced at both 1 and 3 weeks and were restored by 6 months, as shown by histofluorescent staining. Biochemical results showed that norepinephrine also passes through surviving, degenerating, and regenerating phases.     

I型和II 型胶原在正常角膜和圆锥角膜中的表达

目的　观察正常人及圆锥角膜中 I、II型的表达 ,探讨胶原 I、II型在圆锥角膜病变中的变化。方法　采用免疫组织化学 - SA BC法检测角膜组织中胶原 I、II型胶原的表达。结果　胶原 I型在正常角膜和圆锥角膜的上皮层、实质层及内皮层均有阳性表达 ,但圆锥角膜比正常角膜的表达弱 ;而胶原 II型在正常角膜和圆锥角膜组织的基质层和 Bow m an层均可检测到 ,但圆锥角膜中 II型胶原表达减低 ,在圆锥角膜基质斑块状瘢痕区 II型胶原呈强阳性表达。结论　 I、II型胶原的减少会导致角膜的稳定性降低 ,使角膜的机械抵抗力减弱 ,并使角膜前凸变薄。