人结膜杯状细胞和粘液的形态—扫描电镜研究

研究人结膜杯状细胞和粘液的形态。方法８例正常成年人眼标本，年龄１８－４２岁，男７人，女１人，将每个标本的结膜分为１６个组织块，用日立Ｓ－５２０型扫描电镜进行观察。结果人结膜杯状细胞有两种分布形式：（１）散在于结膜上皮细胞间并直接开口于结膜表面；（２）位于Ｈｅｎｌｅ隐窝内，其分泌物通过隐窝的腔隙再排出到结膜表面。     

Histochemical analysis of secretory vesicles in nongoblet conjunctival epithelial cells

An Alcian-blue/Periodic acid Schiff reagent (AB/PAS) pH dependent staining method was used to identify the mucus content of secretory vesicles of non-goblet epithelial cells of the human conjunctiva. Twenty subjects were selected for biopsy of the midcentral upper tarsal conjunctiva: 10 subjects had no contact lens wearing experience, and 10 subjects wore contact lenses. Human respiratory epithelium was used as a control for the staining procedure. Four-micron paraffin sections of conjunctival and control tissues were stained with Alcian blue at pH 2.5 and 1.0 and counterstained with PAS. Non-goblet epithelial cell secretory vesicles contained neutral mucin, sialmucin, and sulphomucin. This study provides evidence for a 'second' mucus system of the conjunctiva, that this system is in the non-goblet epithelial cells, and that the mucus of this system has sulphomucins, sialomucins and neutral mucins.     

Demonstration of the mucous layer of the tear film by electron microscopy

The mucous layer on the ocular surface maintains the stability, spread, and coherence of the tear film and is essential for normal vision. In spite of its importance, the precise thickness and localization of mucus on the surface of the eye are not known because it is not preserved in conventional electron-microscopic preparations. The authors used two different methods to show mucus on the guinea pig cornea and conjunctiva. First, the authors precipitated mucous glycoproteins by adding a quaternary ammonium compound, either cetylpyridinium chloride or hexadecyltrimethylammonium bromide, to aldehyde fixatives. This procedure stabilized the mucus over the goblet cells and adjacent epithelium, although the mucous layer was not preserved uniformly in other areas. Tannic acid intensely stained mucus precipitated by these methods and showed it to be 0.8 micron thick on the cornea and 1.4 micron thick on the conjunctiva. To confirm these results, the authors also prepared specimens of cornea and conjunctiva by freeze substitution. This technique preserved the mucus in a smooth, uninterrupted layer. The thickness of the mucus was somewhat variable; it measured 1.0 micron over the cornea and varied from 2.0 to 7.0 micron over the conjunctiva because of the greater irregularity of the tissue. The authors' results show that mucus constitutes a considerable part of the precorneal tear film and is thicker than was recognized formerly.     

Ultrastructural localization of lectin-binding sites on the surface of the guinea pig conjunctival epithelium

The cell-surface binding sites of two lectins, concanavalin A (Con A) and wheat-germ agglutinin (WGA) in the guinea pig conjunctiva were investigated at the ultrastructural level by means of pre-embedding staining of the tissue with lectin-colloidal gold complexes. Wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, showed prominent and fairly uniform binding to the microvilli. The binding was markedly increased in the vicinity of the goblet cells, indicating that the same carbohydrate ligands were also present in the mucus. In contrast, no binding of concanavalin A, which recognizes mannose and N-glucose, was observed. The results suggest the presence of sialic acid and galactose as the constituent carbohydrates of glycoconjugates in the surface membrane of conjunctival epithelial cells as well as in the mucus produced by the goblet cells.This investigation was supported by grants from the Swedish Medical Research Council (Project 12X-734) and from the Research Committee of Östergbtlands Läns Landsting     

Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques

Conjunctival biopsies from patients with cicatricial pemphigoid affecting the conjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva.     

Characteristics of a glycoprotein in the ocular surface glycocalyx.

A monoclonal antibody has been produced that binds to the apical squames (flattened cells) of the rat ocular surface epithelium and to the goblet cells of the conjunctiva. Immunoelectron microscopic localization of the antigen indicates that in apical cells it is present along the apical-microplical membrane in the region of the glycocalyx. In subapical squames, the antigen is in cytoplasmic vesicles. In some goblet cells, the antigen is in the Golgi network, and in others, it is located primarily in the membrane of the mucous granules. SDS-PAGE and immunoblot analysis demonstrate that the molecular weight of the antigen is greater than 205 kD, and the electrophoretic band stains with Alcian blue followed by silver stain. Periodate oxidation of immunoblots and cryostat sections removes antibody binding. Neuraminidase treatment of cryostat sections does not remove antibody binding, whereas N-glycanase does. Taken together, these data indicate that the antigen recognized by the monoclonal antibody is a carbohydrate epitope on a high-molecular-weight, highly glycosylated glycoprotein in the glycocalyx of the ocular surface epithelium and goblet cell mucin granule membrane. The antigen appears to be stored within cytoplasmic vesicles and reaches the glycocalyx when cells differentiate to the apical-most position where the glycocalyx interfaces with the mucin layer of the tear film.     

SDS-gradient polyacrylamide gel electrophoresis of individual ocular mucus samples from patients with normal and diseased conjunctiva

Individual mucus samples were collected from normal individuals and from patients with ocular cicatricial pemphigoid (CP), Stevens-Johnson syndrome (SJS), and various types of conjunctival inflammation (rosacea, meibomianitis, atopy, keratoconjunctivitis sicca, etc.). The mucus samples were dissolved in sample buffer containing 8M urea, 2% SDS and 5% 2-mercaptoethanol and were electrophoresed on gradient 2-16% polyacrylamide gels. Four glycoproteins with molecular weights greater than 200,000 daltons were consistently observed in both individuals with normal conjunctiva and patients with CP, SJS, and other diseases exhibiting conjunctival inflammation. The amounts of each glycoprotein appeared to vary from one individual to another; however, the presence or absence of specific glycoproteins could not be correlated with the different ocular diseases. The techniques described for mucus analysis offer advantages over previously published techniques since improved resolution of the mucous glycoproteins can be achieved by electrophoresis on 2-16% gradient gels, and individual samples can be analyzed. Our results suggest that substantial amounts of ocular mucous glycoprotein are present in the eyes of patients with CP and SJS, diseases which have been previously described as mucin-deficient dry eye syndromes.     

Amniotic membrane transplantation to reconstruct the conjunctival surface in cases of chemical burn

PURPOSE: To evaluate the efficiency of preserved human amniotic membrane transplantation for conjunctival surface reconstruction in cases of chemical burn. METHODS: Preserved amniotic membrane transplantation was performed in 6 patients (6 eyes) having symblepharon and fornix insufficiency due to chemical burn. Amniotic membrane was sutured to the intact conjunctiva after the fibrotic tissue was excised. The fornix was reconstructed in cases having fornix insufficiency. These cases were followed up for 4-24 months. RESULTS: During the mean follow-up period (10+/-7.37 months) adequate bulbar conjunctiva and fornix depth was achieved in 5 patients without recurrence and with mild fibrosis. In 1 patient who did not have a healthy conjunctiva preoperatively, conjunctival fibrosis and symblepharon recurred. CONCLUSIONS: Preserved human amniotic membrane transplantation is a good alternative treatment method for conjunctival surface reconstruction in those cases with some healthy peripheral conjunctival tissue.     

Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva

Paraffin-embedded and frozen biopsies from the human conjunctival epithelium were examined by fluorescence microscopy after labeling with eight fluorescein-conjugated lectins: wheat germ agglutinin; soybean agglutinin; peanut agglutinin; Ricinus communis agglutinin-1; Limulus polyphemus agglutinin; Ulex europaeus agglutinin-1; Dolichos biflorus agglutinin; concanavalin A. The lectins were used as specific molecular probes to detect carbohydrate composition of glycoproteins secreted from the conjunctival goblet cells. The labeling pattern of goblet cells and conjunctival epithelial surfaces with various lectins suggested that N-acetyl-glucosamine, galactose, N-acetyl-galactosamine and sialic acid are contained in goblet cells and contribute to the formation of tear mucus glycoprotein. Fucose and mannose, which are present in the tear mucus, were not detectable in goblet cells.     

New insights into the surface cells and glands of the conjunctiva and their relevance to the tear film.

BACKGROUND: A number of features of the conjunctiva are highlighted clinically, but there are limited data on the ultrastructure underlying these features. A series of systematic studies have been undertaken to address this, using both scanning electron microscopy (SEM) and transmission electron microscopy (TEM). METHODS: Tissue from pigmented rabbits (2 to 2.5 kg) was prepared for electron microscopy, with special attention being paid to the preparation of the palpebral or bulbar conjunctival tissue by stretching it to its fully extended configuration. The fixative (2% glutaraldehyde in 80 mM cacodylate, pH 7.2 to 7.4, 320 to 340 mOsm/kg) has been previously validated in studies on corneal tissue. RESULTS: Both SEM and TEM reveal the presence of a substantial network of surface pores and underlying ducts associated with the acini of the tarsal accessory gland of Wolfring. These are predominantly found close to the tarsal-orbital boundary of the palpebral conjunctiva. The ductal system is lined with cells containing prominent secretory granules and has features that suggest that it readily collapses if the tissue is not adequately prepared. The same preparative technique also shows the presence of large and open lymphatic vessels in the stroma under the bulbar conjunctiva. Secretory granules and vesicles can also be found associated with the surface ducts and with the surface cells of the palpebral conjunctiva. Studies on the goblet cells located within the conjunctival cell layers also show that the shape of the cells can change substantially with the preparative technique. CONCLUSIONS: The outcome of these studies indicate that there is still much to be learned concerning the contribution of the accessory lacrimal glands, secreted mucins, the goblet cells, and the lymphatic system to the maintenance of a normal healthy ocular surface.     

Treatment of large conjunctival nevus by resection and reconstruction using amniotic membrane

Background Nevus of the bulbar conjunctiva is a benign pigmented lesion of the ocular surface. Unless a biopsy is required for ruling out malignant melanoma, a nevus is usually excised for cosmetic reasons only. However, with large lesions involving two or more quadrants of the bulbar conjunctiva, a total resection can be difficult. In this report, we present a case of a huge nevus treated by surgical excision and amniotic membrane transplantation for reconstruction of the bulbar conjunctiva.Methods A 54-year-old Japanese man was referred to our clinic with suspected malignant tumor of the bulbar conjunctiva in his right eye. A large and diffuse pigmented tumor with numerous small cysts was present mainly on the upper bulbar conjunctiva. Resection of the conjunctival tumor and amniotic membrane transplantation for reconstruction of the bulbar conjunctiva were performed.Results The histopathological diagnosis was conjunctival nevus. Epithelialization of the bulbar conjunctiva over the amniotic membrane sheet was completed 4 weeks after resection. At 44-month follow-up, there was no recurrence or any postoperative complication.Conclusion Surgical resection combined with reconstruction by amniotic membrane transplantation is effective for the treatment of large conjunctival nevus.     

Transplantation of amniotic membrane for patients with bullous keratopathy and chemical and thermal burns

The aim of this paper is, to evaluate the efficacy of amniotic membrane transplantation for ocular surface reconstruction in patients with bullous keratopathy and chemical and thermal burns of cornea and conjunctiva. Amniotic membrane is a thin, semitransparent tissue forming an innermost layer of the fetal membrane, which contains a thick basement membrane with a single layer of epithelium and avascular matrix. This transplantation promotes normal conjunctival epithelization while suppressing fibrosis formation. Amniotic membrane transplant may be considered as an alternative method for treating ocular surface reconstruction in patients with thermal and chemical burns. Authors suggest that this method of treatment is not efficient in patients with bullous keratopathy.     

Scanning electron microscopy of conjunctival surfaces in patients with ocular cicatricial pemphigoid

The conjunctival surfaces of ten patients with active, ocular cicatricial pemphigoid, three patients with drug-controlled ocular cicatricial pemphigoid, and six patients with normal conjunctivas were studied using scanning electron microscopy. A homogeneous granular sheet of amorphous mucin-like material was observed covering extensive areas of the conjunctiva in eight of ten patients with active ocular cicatricial pemphigoid. This sheet of amorphous material was absent on drug-controlled ocular cicatricial pemphigoid and normal conjunctival specimens. Our study demonstrates that patients with active ocular cicatricial pemphigoid possess ocular surface mucus that appears thicker and more continuous than normal ocular mucus when observed with scanning electron microscopy. This observation is in agreement with clinical observations of thick mucus strands in the inferior fornix of patients with active ocular cicatricial pemphigoid.     

Reconstruction of conjunctiva with amniotic membrane after excision of large conjunctival melanoma: a long-term study

PURPOSE: To evaluate, on a long- term basis, the role of amniotic membrane in the reconstruction of large conjunctival defects after excision of large conjunctival melanoma. METHODS: Four consecutive patients with diffuse conjunctival melanoma involving both bulbar and palpebral conjunctiva were studied. Conjunctival melanoma was completely excised (with wide clinically disease-free margins) and amniotic membrane immediately sutured to the surrounding conjunctiva and sclera to cover the conjunctival defect. Minimum follow-up was 48 months. RESULTS: Successful conjunctival surface reconstruction and physiologic fornical depth were achieved in all patients within 6 weeks. No recurrence of primary melanoma was observed during long-term follow-up. CONCLUSIONS: Amniotic membrane transplantation is an effective alternative in ocular surface repairing surgery after removal of large conjunctival tumors.     

Goblet cell population among patients with inactive trachoma

Trachoma is a chlamydial disease that affects millions of people each year, particularly in developing countries. In the chronic phase, inflammation causes scarring of the conjunctiva followed by dry eye which can result in blindness. Trachoma may cause dryness of the eye by decreasing mucus production and aqueous secretions. Conjunctival impression cytology was carried out to determine the goblet cell population among patients with trachoma.We performed impression cytology on 32 patients with inactive trachoma and 31 age and sex matched controls. Impression cytology showed that the nasal conjunctiva contains the greatest number of goblet cells. Trachoma patients with severe scarring had significantly less goblet cell counts than those with mild scarring (p<0.05). In the group often patients with severe trachoma and keratinization, there was marked reduction or absence of goblet cells. Trachoma appears to initiate a viscious cycle of conjunctival scarring, mucus deficiency, and chronic conjunctival inflammation.     

In vivo findings of the bulbar/palpebral conjunctiva and presumed meibomian glands by laser scanning confocal microscopy

OBJECTIVE: To report in vivo laser scanning confocal microscopic findings of the normal human conjunctiva and presumed meibomian glands and to investigate the potential application of this confocal microscope (HRT II Rostock Cornea Module) as a diagnostic device for normal and abnormal ocular surface structures. METHOD: Four healthy volunteers (51-, 49-, 31-, and 30-year-old men) participated in this study. The temporal bulbar conjunctiva, approximately 5 mm away from the limbus, and the upper tarsus conjunctiva of each patient were examined in vivo by a laser confocal microscope. Also, presumed meibomian glands beneath the tarsal conjunctiva were examined. RESULTS: In all subjects, the in vivo laser confocal microscope obtained similar images. In the superficial epithelial cell layer of the bulbar conjunctiva, numerous bright nuclei were observed. We also observed many Langerhans cells with characteristic dendritic morphology and goblet cells with relatively homogeneous brightness in the bulbar conjunctiva. Most interestingly, the palpebral conjunctival epithelium with invaginations or crypts was observed. In addition, weblike structures, presumably meibomian glands, were observed beneath the palpebral conjunctival epithelium. CONCLUSIONS: These results indicate that in vivo laser confocal microscopy can be used not only to visualize the bulbar conjunctiva but the upper tarsus conjunctiva and possibly subconjunctival meibomian glands. Further investigations of conjunctival and meibomian gland pathologies may further elucidate the potential of this device in clinical ophthalmology.     

Reconstruction of the Periocular Mucous Membrane by Autologous Conjunctival Transplantation

We have extended the concept of autologous conjunctival transplantation for corneal resurfacing as recommended by Thoft¹ to reconstruction in 14 patients with unilateral abnormalities of the bulbar and palpebral conjunctiva caused by alkali burns (2), irradiation (2), neoplasms (3), degenerative diseases (5), trauma (1). and developmental anomalies (1). Large, free conjunctival grafts from bulbar and forniceal donor sites were used. No complications have been noted at the recipient or donor sites. Grafts of normal conjunctiva provided intact basement membrane, goblet cells, and epithelium that help restore normal ocular and lid surfaces. The use of free conjunctival grafts provides significant advantage over the use of buccal mucous membrane grafts. The techniques and results of our experience with free conjunctival grafts in 14 patients are discussed.     

翼状胬肉上结膜反向移植联合羊膜移植治疗复发胬肉

目的：观察胬肉上结膜反向移植联合羊膜移植治疗复发性翼状胬肉的疗效。

方法：回顾性系列病例研究。回顾2009-01/2012-01本院确诊的复发性翼状胬肉26例26眼,均为经历一次手术,手术方法为翼状胬肉切除联合自体角膜缘干细胞移植术,复发1a以上病例。本次研究中,切除胬肉组织时,将其上结膜充分游离后切下保存,羊膜覆盖胬肉切除后裸露巩膜面,再将保留的胬肉上的结膜植片反向覆盖羊膜上缝合。术后1,2wk; 1mo及3mo观察结膜植片愈合情况、羊膜溶解吸收时间、角膜创面修复时间、角膜新生血管及胬肉复发率。术后随访12mo。

结果：结膜植片愈合时间6.03±2.76d,羊膜溶解吸收时间14.26±3.64d,角膜创面修复时间5.42±1.58d。术后3mo,胬肉切除后裸露巩膜面新生结膜及结膜植片愈合良好,无明显胬肉术后并发症。术后1a,再度复发2例,复发率8%,治愈率92%。

结论：对于复发性翼状胬肉,因传统自体角膜缘干细胞移植已经创伤部分角膜缘及眼表结膜瘢痕,单纯结膜移植,或单纯羊膜移植,术后胬肉复发率偏高,本方法取胬肉上结膜反向移植,避免大面积创伤眼表,取材容易,而且良好控制术后翼状胬肉复发问题,不失为一种解决复发性翼状胬肉的良好选择。     

In vivo confocal microscopy of the bulbar conjunctiva

Background:  The aim of this work is to develop a more complete qualitative and quantitative understanding of the in vivo histology of the human bulbar conjunctiva.
Methods:  Laser scanning confocal microscopy (LSCM) was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 healthy human volunteer subjects.
Results:  The superficial epithelial layer of the bulbar conjunctiva is seen as a mass of small cell nuclei. Cell borders are sometimes visible. The light grey borders of basal epithelial cells are clearly visible, but nuclei can not be seen. The conjunctival stroma is comprised of a dense meshwork of white fibres, through which traverse blood vessels containing cellular elements. Orifices at the epithelial surface may represent goblet cells that have opened and expelled their contents. Goblet cells are also observed in the deeper epithelial layers, as well as conjunctival microcysts and mature forms of Langerhans cells. The bulbar conjunctiva has a mean thickness of 32.9 ± 1.1 µm, and a superficial and basal epithelial cell density of 2212 ± 782 and 2368 ± 741 cells/mm², respectively. Overall goblet and mature Langerhans cell densities are 111 ± 58 and 23 ± 25 cells/mm², respectively.
Conclusions:  LSCM is a powerful technique for studying the human bulbar conjunctiva in vivo and quantifying key aspects of cell morphology. The observations presented here may serve as a useful marker against which changes in conjunctival morphology due to disease, surgery, drug therapy or contact lens wear can be assessed.