Expression of the receptor tyrosine kinases, epidermal growth factor receptor, ErbB2, and ErbB3, in human ocular surface epithelia

PURPOSE: To investigate the distribution and relative level of expression of the receptor tyrosine kinases, epidermal growth factor receptor (EGFR), ErbB2 and ErbB3, in human ocular surface epithelia. METHODS: Immunofluorescent staining was performed to identify expression of the EGFR, ErbB2 and ErbB3 in the corneal, limbal and conjunctival epithelium in tissue sections and impression cytologies taken from normal human eyes. Western blotting was undertaken to confirm the results of immunofluorescent staining. RESULTS: The three receptor tyrosine kinases, EGFR, ErbB2 and ErbB3, were detected in human corneal, limbal and conjunctival epithelia by immunofluorescent staining. Strong staining for the EGFR was observed in the basal epithelial cells at all 3 sites and throughout the corneal epithelium. Minimal or no staining for the EGFR was observed in the superficial conjunctival and limbal epithelia. The strongest staining for ErbB2 and ErbB3 was observed in the superficial ocular surface epithelium. All three receptors were detected in the corneal, limbal and conjunctival epithelium by western blot. CONCLUSION: EGFR, ErbB2 and ErbB3 are expressed by the ocular surface epithelia. EGFR is preferentially expressed by the basal epithelial cells that have the greatest proliferative potential. In contrast, ErbB2 and ErbB3 are preferentially expressed by the superficial differentiated ocular surface epithelia.     

Localization of lectin binding sites in human, cat, and rabbit corneas

Paraffin sections of human, cat, and rabbit corneas were stained with nine lectins, using an avidin-biotin-complex procedure to study glycoconjugates of the epithelium, keratocytes, and stromal matrix. Wheat germ agglutinin (WGA) stained plasma membranes of all epithelial cell layers of cat and human and superficial and wing cells of rabbit. Plasma membranes of superficial and wing cells of cat epithelium also stained with peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA-I). Human and cat keratocytes stained with WGA and RCA-I. Stromal matrices of all three species were stained with concanavalin A and lentil agglutinin. In neuraminidase-treated sections, the entire epithelium and keratocytes of all three species stained with PNA. Corneal sections from the three species did not stain with Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, Ulex europeus I, and Soybean agglutinin. These data suggest the presence of oligosaccharides with: N-acetylglucosamine/sialic acid residues in cell membranes of corneal epithelium of all species studied and in the keratocytes of human and cat; terminal beta-galactose residues in cat and human keratocytes, beta-galactose-galactosamine chains in cat epithelial cell membranes; and sialic acid-beta-galactose-galactosamine chains in epithelial cell membranes and keratocytes of all three species.     

角膜缘部干细胞对角膜上皮创伤愈合的影响

利用角膜缘上皮移植，结膜移植和非手术的方法分别对角膜上皮创伤伴随膜缘损伤的兔眼进行实验研究，结果表明角膜缘上皮移植组上皮愈合时间短（平均８．５天），且无杯状细胞，无或极少量新生血管；结膜移植组，上皮愈合时间延长（平均１１．５天），含有杯状细胞和新生血管，非手术组形成角膜血管翳性混浊。而不件随角膜缘损伤仅有角膜中央皮皮创伤的兔眼愈合时间最短（平均３．５天），愈合后无新生血管及怀状细胞。结果证实角膜部     

Cytoskeletal antigen expression in ocular mucosa-associated lymphoid tissue.

The human lacrimal gland (LG) and ocular surface contain discrete regions of epithelial cells with specific functions and at different stages of cellular differentiation. Epithelial cells contain cytoskeletal antigens that show a differentiation-dependent pattern of expression. The objective of this study was to characterize the various epithelial cell populations in normal human ocular mucosa-associated lymphoid tissue (MALT; LG, conjunctiva, and cornea) based on their immunohistochemical staining patterns with anticytoskeletal monoclonal antibodies (MoAbs) reactive with cytokeratins (AE-1, AE-2, AE-3, AE-5, AE-14, PKK1, and 34 beta E12), muscle-specific actin (HHF35), and vimentin. AE-1 stained LG (acini, ducts, and myoepithelia) and the full thickness of corneal and conjunctival epithelia. It stained only the superficial and basal limbal epithelium. AE-2 weakly stained all epithelia, except LG acini and proximal intralobular ducts. AE-3 and 34 beta E12 MoAbs had strong immunoreactivity with all MALT epithelia. AE-5 strongly stained the inner cells (suprabasal) of LG central intra- and interlobular ducts and the suprabasal epithelial layers of the cornea. It weakly stained LG myoepithelia and the superficial conjunctival epithelium. AE-14 stained the outer (basal) cells of LG central intra- and interlobular ducts, LG myoepithelia, basal epithelial layers of the limbus and conjunctiva, and all corneal epithelia. PKK1 stained all epithelia, except the basal limbus. HHF35 and the antivimentin MoAbs stained only the LG myoepithelia. The results of these studies indicate that the different epithelia in human ocular MALT may be differentiated by specific patterns of immunoreactivity with anticytoskeletal MoAbs. These MoAbs may be useful molecular markers for identifying ocular MALT epithelia.     

Immunohistochemical localization of NQO1 in epithelial dysplasia and neoplasia and in donor eyes

PURPOSE: To examine the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase), a potential bioactivating enzyme for mitomycin C in corneal and conjunctival epithelial dysplasia and neoplasia and in normal tissues from human donor eyes, by immunohistochemistry. METHODS: Formalin-fixed, paraffin-embedded sections of human donor eyes and tissue sections with histologic diagnoses of corneal and conjunctival epithelial dysplasia and neoplasia from the Eye Pathology Laboratory, University of Colorado Health Sciences Center were analyzed. Detection of NQO1 in tissues was performed using standard immunohistochemical techniques with monoclonal antibodies against NQO1 and immunoperoxidase staining. RESULTS: All 20 tumors stained positive for NQO1. In seven eyes from four donors, positive staining for NQO1 was detected in all epithelial and endothelial layers, in fibroblasts, in all retinal layers except the photoreceptor outer segments, and in the fascicles and arachnoid of the optic nerve. Only minimal staining was detected in the photoreceptor outer segments and the optic nerve pia and dura. Immunostaining was markedly reduced in all tissues in both eyes from donor 5. Genetic analysis confirmed that this individual was homozygous for a polymorphism in NQO1 (NQO1*2). CONCLUSIONS: NQO1 was detected by immunohistochemistry in every examined section of corneal and conjunctival epithelial dysplasia and neoplasia, suggesting that NQO1 may play a role in the bioactivation of mitomycin C in these tumors. However, the presence of NQO1 in the corneal, conjunctival, and ciliary epithelium; the retinas; and the optic nerves of donor eyes may indicate the potential for mitomycin C toxicity, particularly at higher doses.     

糖尿病患者泪膜及角结膜上皮稳定性的改变

目的了解糖尿病患者眼表上皮及泪膜稳定性改变情况。方法选50名糖尿病患者100眼,52名健康对照者104眼,性别年龄成组匹配,分别进行角膜知觉检查、基础泪液分泌试验(SchirmerⅠ)、泪膜破裂时间检查(BUT)、角膜荧光素染色、结膜印迹细胞学检查、糖尿病眼底检查、眼底照相或荧光造影,观察杯状细胞及结膜化生情况,问卷调查干眼。结果糖尿病组角膜知觉下降(P<0.05),SchirmerⅠ试验值、BUT值均下降(P<0.05);角膜荧光素染色阳性增加(P<0.05);杯状细胞丢失、结膜鳞状化生明显(P<0.05)。糖尿病眼底病变程度与SchirmerⅠ试验、BUT呈负相关,与角膜荧光素染色、结膜鳞状化生等级呈正相关(P<0.05),与病程无相关性(P>0.05)。结论糖尿病可导致患者泪液分泌减少、泪膜稳定性下降及角结膜上皮的损害,在伴周围神经病变的患者中表现尤其明显,且损害程度与糖尿病视网膜病变程度具有相关性。     

Paracellular permeability of corneal and conjunctival epithelia

The paracellular permeability of normal rabbit cornea and conjunctiva was studied in vivo and in vitro. After intravenous administration, horseradish peroxidase was found to percolate to the intercellular space of conjunctival epithelia and was restricted by the tight junctions of the superficial epithelium. Only minimal tracer was present in the limbus and cornea. The difference between corneal and conjunctival paracellular pathways was further compared in vitro by tissue perfusion studies using various tracers from subepithelial space to apical surface. The intact full-thickness cornea was permeable to mannitol (MW 182) but not to inulin or dextran. The conjunctiva was permeable to mannitol, inulin and FITC-dextran (MW 20,000). The quantitative permeability to 3H-mannitol (X10(-8) cm/sec) of adult rabbit cornea was 0.12 +/- 0.02, which is about 55-fold and 50-fold lower than that of conjunctiva (6.78 +/- 0.21) and peritoneum (6.12 +/- 0.63), respectively. Removal of the corneal epithelium increased the permeability 40-fold; however, removal of the endothelium had little effect on the solute permeation. When both corneal epithelium and endothelium were debrided, the bare stroma became edematous and the permeability increased 70-fold. The permeability of 1-week-old rabbit cornea was 1.32 +/- 0.18, which decreased to 0.46 +/- 0.06 in 2-week-old rabbits, and became similar to the adult level at 4 weeks of age. When Tenon's capsule was included in the perfusion, the conjunctival permeability decreased 2.5-fold. With the apposition of bare corneal stroma to the conjunctiva and Tenon's capsule, the permeability decreased further (4-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

纤维蛋白胶对眼表的作用

目的观察以纤维蛋白胶（FG）为载体构建兔角膜上皮、基质和内皮细胞片,以及FG对兔结膜和角膜的黏合作用。方法在培养板孔底制作薄层和厚层FG,将培养兔角膜3种细胞分别接种于FG表面,观察细胞生长情况。FG对兔结膜的黏合研究：分为A组（结膜原位粘合组）,B组（结膜移位粘合组）,对照组（单纯切除结膜植片）,每组5只眼,观察结膜组织黏合情况。FG对兔角膜的黏合研究：3例兔前板层角膜移植术,先用10—0尼龙线间断缝合4针固定植片,再用FG黏合,将植片固定于植床。结果角膜3种细胞在薄层和厚层FG表面生长良好：角膜上皮细胞可形成单层和复层;角膜基质细胞间网状连接;角膜内皮细胞排列紧密。对兔结膜的黏合研究：A组和B组植片与植床对位良好,紧密黏合。组织切片显示术后第4周A组和B组结膜上皮完整,炎症基本消退。对照组结膜上皮有局部脱落区,纤维层局部显示瘢痕组织结构特征。兔前板层角膜移植术后3个月,植片与受体角膜愈合良好。结论FG可作为角膜3种细胞的生长载体构建细胞片。FG对兔结膜和角膜组织有良好的黏合作用。     

Expression patterns of sialylated epitope recognized by KL-6 monoclonal antibody in ocular surface epithelium of normals and dry eye patients

PURPOSE: To determine the association of the KL-6 epitope (sugar moiety) with MUC1 and its distribution on the ocular surface of human non-dry and dry eyes. METHODS: The human ocular surface was examined immunohistochemically and immunoelectron microscopically using monoclonal antibody (mAb) KL-6, which recognize a carbohydrate epitope of MUC1. The expression patterns of KL-6 epitope in corneal and conjunctival cells were examined by impression cytology from 24 non-dry eye volunteers and 43 dry-eye patients. RESULTS: In the cornea, bulbar conjunctiva, and limbus epithelium, mAb KL-6 reacted to the apical cell membrane of superficial cells and the intercellular space of superficial and wing cells. No immune reactivity of mAb KL-6 was observed in the basal plasma membrane of basal epithelial cells. Results of impression cytology indicated that the corneal epithelium of 13 of 24 non-dry eyes was weakly stained by mAb KL-6, whereas 42 of 43 dry eyes showed a mosaic pattern. In non-dry eyes, 19 of 24 bulbar conjunctival epithelia expressed the KL-6 epitope in a honeycomb pattern. In mild (17/19) and moderate (17/17) dry eye conjunctiva, the KL-6 epitope showed a mosaic pattern. However, the expression of KL-6 epitope decreased in severe dry eyes, showing a mosaic pattern in three of seven patients and labeling a few cells weakly in four of seven patients. CONCLUSIONS: These findings suggest that there is an upregulation of the sialylated KL-6 epitope of MUC1 by apical corneal and conjunctival cells in mild and moderate dry eyes. This upregulation may in part alleviate the consequences caused by goblet cell mucin dysfunction in dry eyes. It is noteworthy that the KL-6 epitope is downregulated in the conjunctiva of severe dry eyes, a phenomenon that may be explained in part by the malfunction of conjunctival epithelial cells.     

Corneal thickness and axial length

PURPOSE: A thin central cornea has been reported to be a risk factor for developing primary open-angle glaucoma among ocular hypertensive eyes. A thin scleral bed of lamina cribrosa seen in deeply excavated optic nerves in glaucomatous eyes is a quintessential finding in advanced glaucomatous eyes. Association between thin cornea and weak sclera contributing to vulnerability of lamina cribrosa has been postulated. The purpose of this study is to determine whether there is an association between corneal thickness and axial length of human eyes in a clinical setting. DESIGN: This is an observational, retrospective cross-sectional study. METHODS: The ocular parameters of 1,084 consecutive eyes with both corneal thickness and axial length measurements were analyzed and compared by age, gender, and race. RESULTS: In the total patient study group, there was no statistically significant association between central corneal thickness and axial length. Subgroup analysis by age, gender, and race also failed to show an association. CONCLUSIONS: Central corneal thickness and axial length are independent occurrences. Thin corneas are not associated with longer eyes.     

Keratin-like proteins in corneal and conjunctival epithelium are different

Using SDS polyacrylamide slab-gel electrophoresis, water-insoluble (keratin-like) proteins in normal and regenerated ocular surface epithelium from rabbits were studied. The results indicated that keratin-like proteins from corneal and conjunctival epithelia in vivo distinctly different. Regenerated epithelia from either source retained their original keratin characteristics for at least 10 days after healing over the cornea, but at very early stages of healing migrating and regenerated epithelia showed either an extra band or a prominent band in addition to the original keratin-like proteins. Three months after healing, however, regenerated conjunctival epithelium on the cornea had changed its keratin characteristics, and resembled, but was not identical to, corneal epithelium.     

Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium

Previous studies have shown that using n-heptanol to create a total corneal epithelial defect beyond the limbus results in two different healing patterns with an unpredictable incidence. Between 14-68% of these wounded rabbit corneas (n = 287, combining various reports) showed extensive vascularization and conjunctivalization, whereas the remaining were not vascularized and had conjunctival transdifferentiation with a cornea-like epithelium. To investigate the role of the limbal epithelium in these two healing patterns, the authors treated rabbit eyes for various durations with n-heptanol and additional scraping. Histology showed that treatment for up to 120 seconds removed both the corneal and conjunctival epithelia but left the limbal basal cells intact. To prove viability, they cultured the treated limbal explants on collagen gel. After 14 days of culture, increased stratification of the limbal epithelium and an epithelial outgrowth onto the corneal stroma was observed. The latter was proven to be of corneal origin (positive to AE-5 but negative to AM-3 monoclonal antibody staining). The authors then surgically removed the entire limbal zone including 2 mm of peripheral cornea and 3 mm of adjacent conjunctiva in addition to n-heptanol debridement of the entire corneal epithelium in 54 rabbit eyes and observed a high incidence (96%) of corneal vascularization and conjunctivalization of the resultant epithelial phenotype (positive to AM-3, but negative to AE-5 monoclonal antibody staining). These results support the hypothesis that corneal epithelial stem cells are located in the limbus and indicate that an incomplete removal of the basal limbal epithelium by n-heptanol leads to unvascularized corneas with conjunctival transdifferentiation. Conversely, complete removal of such cells results in corneal vascularization and conjunctivalization.     

角膜的结膜瓣遮盖术实验研究

目的 探讨碘酊烧灼联合机械刮除角膜上皮行角膜全结膜遮盖手术的效果。方法 对实验组２０只纯种新西兰大耳白兔分组进行单纯机械刮除角膜上皮（单纯法）及碘酊烧灼联合机械刮除角膜上皮（综合法）角膜全结膜瓣遮盖术。结果 单纯法１０只眼,３眼结膜与角膜愈着,手术成功,７眼未成功。综合法１０眼,８眼手术成功,２眼未成功,２组成功率有显著差异（Ｐ〈０．０５）。结论 综合法能更有效地毁损角膜上皮及角膜干细胞,提高手术     

Immunohistochemical and ultrastructural analysis of dysplastic epithelium of human ocular surface: basement membrane and intermediate filament.

PURPOSE: The dysplastic corneal epithelium is characterized by the abnormal proliferation of epithelial cells. The phenotypes of these cells have not been elucidated. We investigated whether such epithelium expresses the phenotypes of corneal or conjunctival epithelial cells. METHODS: The corneas and conjunctivae from four normal subjects and from one patient with epithelial dysplasia of the central cornea were immunostained for IV and VII collagens and for cytokeratins. Monoclonal antibodies against collagen IV reacted to the [alpha1(IV)]2alpha2(IV) or alpha5(IV) molecule. Anti-cytokeratin antibodies were used to define epithelial cell types. The ultrastructure of the basement membrane (BM) of each specimen also was examined. RESULTS: Type VII collagen immunoreactivity was detected in all the specimens of epithelial BM. The anti-collagen IV [alpha1(IV)]2alpha2(IV) antibody labeled the conjunctival BMs, not the BMs of the corneal epithelia, of each subject. The normal corneal epithelial BM, not the BM of the conjunctival or dysplastic corneal epithelium, was immunolabeled with anti-alpha5(IV) antibody. The pattern of cytokeratin expression in the corneal epithelial dysplasia resembled that seen in the normal conjunctivae. Small breaks in the BM of dysplastic corneal epithelium were ultrastructurally revealed. The number of hemidesmosomes in the dysplastic corneal epithelium was decreased as compared with that in the normal BM. CONCLUSION: The composition of collagen types within the BM and the cellular phenotype of the dysplastic epithelium in the cornea resembled those of conjunctival epithelium, not of the cornea.     

Tandem-scanning (confocal) microscopy of the full-thickness cornea

We have utilized a radically new type of optical scanning microscope to study the full-thickness morphology of the intact cornea in an excised human eye bank eye and in freshly sacrificed rabbit eyes in situ. This technology enables one to study corneal morphology layer by layer in extremely thin sections, only disturbing the tissue with an applanating tip. We have demonstrated the cells of the corneal surface, subsurface cells, the topography of Bowman's membrane, corneal lamellae, stromal keratocytes, and the corneal endothelium. The application of this technology lends itself to the in vivo examination of the human cornea. This should aid us greatly in the study of normal morphology, disease states, and the reaction of the cornea in wound healing.     

Central corneal thickness correlated with glaucoma damage and rate of progression

PURPOSE: To evaluate whether the amount of glaucomatous optic nerve damage at presentation of the patient and the rate of progression of glaucoma during follow-up are related to central corneal thickness. METHODS: The prospective observational clinical study included 861 eyes of 454 white subjects (239 normal eyes of 121 subjects, 250 ocular hypertensive eyes of 118 patients, 372 eyes of 215 patients with chronic open-angle glaucoma). For 567 eyes (304 patients) with ocular hypertension or chronic open-angle glaucoma, follow-up examinations were performed, with a mean follow-up time of 62.7 +/- 33.2 months (median, 60.8; range, 6.2-124.9). All patients underwent qualitative and morphometric evaluation of color stereo optic disc photographs and white-on-white visual field examination. Central corneal thickness was measured by corneal pachymetry. RESULTS: Central corneal thickness correlated significantly (P < 0.001) and positively with the area of the neuroretinal rim and negatively with the loss of visual field. Development or progression of glaucomatous visual field defects detected in 119 (21.0%) eyes was statistically independent of central corneal thickness, in univariate (P = 0.99) and multivariate Cox regression analyses (P = 0.19). CONCLUSIONS: At the time of patient referral, the amount of glaucomatous optic nerve damage correlated significantly with a thin central cornea. Progression of glaucomatous optic nerve neuropathy was independent of central corneal thickness, suggesting that central corneal thickness may not play a major role in the pathogenesis of progressive glaucomatous optic nerve damage.     

Conjunctival and corneal findings in bleb‐associated endophthalmitis: an in vivo confocal microscopy study

Purpose: To report the conjunctival and corneal findings in delayed onset glaucoma filtering bleb‐associated endophthalmitis (BAE), by using in vivo confocal microscopy (IVCM). Methods: This was an observational case series. Four eyes of four glaucomatous patients who previously underwent mytomicin C augmented filtering surgery and affected with delayed onset BAE, underwent IVCM of conjunctival bleb and cornea at diagnosis, after 2 and 8 weeks of therapy. The inflammatory status of the conjunctival epithelium and sub‐epithelium was microscopically investigated. Corneal epithelial cells, stromal and endothelial morphology were also evaluated. A group of eight patients with functioning conjunctival filtering bleb was used as control. Results: At diagnosis, a diffuse inflammatory cell infiltration within the conjunctival epithelium presenting evident microcysts was found; conversely, there were no such alterations in the sub‐epithelium. An evident stromal oedema, keratocytes activation and diffuse endothelial inflammatory precipitates were the major corneal hallmarks. After 2 weeks of therapy, besides a remarkable improvement of epithelial inflammation and an evident reduction in endothelial precipitates, dendritic cells appeared within conjunctival sub‐epithelium and corneal epithelium showed aspects of cellular disruption. After 8 weeks, the conjunctival and corneal features consistently improved, except for the endothelium which still presented high‐reflective residual precipitates. Conclusions: In vivo confocal microscopy proved valuable in the analysis of conjunctival bleb and cornea in patients affected with delayed onset BAE, permitting an evaluation of the course of the disease, the response to therapy and the modulation of dose regimen.