Delayed wound closure and phenotypic changes in corneal epithelium of the spontaneously diabetic Goto-Kakizaki rat

PURPOSE: To characterize wound closure and phenotypic changes in the corneal epithelium of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. METHODS: Corneal wound healing was monitored by fluorescein staining after epithelial debridement. Tear secretion was measured with the Schirmer test, and corneal sensation was evaluated with an esthesiometer in 13- to 15-week-old GK and Wistar (control) rats. The distributions of cytokeratin 12 (K12), K14, and connexin43 in the corneal epithelium were examined by immunohistofluorescence analysis. The proliferation capacity of epithelial cells in the intact cornea and during wound healing was evaluated by immunostaining for Ki-67. RESULTS: Tear secretion, corneal sensation, and corneal epithelial wound closure rate were all decreased in GK rats compared with those in Wistar rats. Whereas connexin43, K14, and Ki-67 were all restricted to the single layer of basal cells in the corneal epithelium of Wistar rats, they were detected in the two layers of cells closest to the basement membrane in that of GK rats. The frequency of Ki-67-positive cells in the intact corneal epithelium was greater in GK rats than in Wistar rats, and it was increased to a greater extent in the peripheral cornea of GK rats than in that of Wistar rats during wound healing. CONCLUSIONS: Spontaneously diabetic GK rats manifest characteristics similar to those of diabetic keratopathy in humans, including delayed wound closure, and they exhibit phenotypic changes in corneal epithelial cells.     

Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats

PURPOSE: To understand the pathophysiology of the corneal basement membrane in diabetes, we compared the localization of laminin and type IV collagen in the epithelial basement membrane during corneal epithelial wound healing in diabetic and nondiabetic rats. METHODS: Streptozotocin was used to induce diabetes in half the rats. Two weeks later, the whole corneal epithelium was debrided. Diabetic and healthy rats (3-5 per group) were sacrificed before debridement and 1, 3, and 7 days and 1 month afterwards. The localization of laminin and type IV collagen was observed in cryosections by epifluorescence microscopy. RESULTS: In unwounded corneas of both diabetic and normal rats, laminin and type IV collagen were localized in the corneal epithelial basement. The intensity of fluorescence, however, was clearly stronger in the diabetic rats. In normal rats, wounding initially removed laminin and type IV collagen, but during healing these two proteins reappeared beneath the resurfacing corneal epithelium. Although similar results were observed in diabetic rats, the expression of laminin and type IV collagen was delayed, and their deposition was fragmented and irregular. CONCLUSIONS: These results suggest that delayed corneal epithelial wound healing in diabetes might involve delayed reappearance and abnormal reformation of epithelial basement membrane proteins.     

Advanced glycation end products in diabetic corneas

PURPOSE: Corneal complications are often associated with diabetes mellitus and can be vision threatening. Corneas in diabetic patients are exposed to increased glucose concentration despite cornea's avascular property, and this condition may contribute to the accumulation of advanced glycation end products (AGEs). The focus of this study was to examine the role of AGEs in the pathogenesis of diabetic keratopathy. METHODs: An anti-AGE monoclonal antibody (6D12), which recognizes a N(epsilon)-carboxymethyl lysine (CML)-protein adduct as an epitope, was prepared. Immunohistochemical localization of CML was examined in human age-matched diabetic and nondiabetic corneas (8 of each). In vitro, type I collagen-, type IV collagen-, or laminin-coated 96-well plates were glycated by glucose-phosphate. In some experiments, aminoguanidine was present in the incubation mixture. The amounts of CML-protein adducts in the extracellular matrix (ECM) were determined by enzyme-linked immunosorbent assay using 6D12. SV40-immortalized human corneal epithelial cells were seeded onto modified or unmodified ECM in 96-well plates and allowed to attach for 3 hours. Attached cells were fixed, and the areas of attached cells in each condition were measured. Attached cells without fixation were removed, and cell number was counted. RESULTS: In all of the 8 diabetic corneas, CML immunoreactivity was observed in the epithelial basement membrane, whereas CML immunoreactivity was not found in the corresponding area in 7 of 8 nondiabetic corneas. In vitro, nonenzymatic glycation of laminin on the culture dish attenuated adhesion and spreading of corneal epithelial cells. The presence of amninoguanidine in the incubation mixture during glycation inhibited CML formation and promoted the adhesion and spreading of corneal epithelial cells in a dose-dependent manner. CONCLUSIONS: The accumulation of AGEs on the basement membrane, particularly on laminin, may play a causative role in the corneal epithelial disorders of diabetic patients.     

Non-enzymatic glycation in corneas from normal and diabetic donors and its effects on epithelial cell attachment in vitro.

BACKGROUND: Diabetic keratopathy manifests as persistent and recurrent erosions and delayed wound healing. To investigate the role of non-enzymatic glycation of proteins in the pathogenesis of diabetic keratopathy, we studied the presence of advanced glycation end products in corneas normal and diabetic donors, and the ability of human comeal epithelial cells to attach to non-enzymatically glycated extracellular matrix proteins. METHODS: Corneas from normal donors and donors with diabetes were sectioned and immunostaining was performed using a primary antibody that detects N(epsilon)-(carboxymethyl) lysine. Collagen I and fibronectin-coated tissue culture plates were non-enzymatically glycated by incubating with 500-mM glucose-6-phosphate for three weeks at 37 degrees C. To determine attachment corneal epithelial cells were allowed to adhere to glycated plates (1 to 4 hours, 37 degrees C). Non-adherent cells were removed by gentle washing and the number of attached cells was quantitated using Calcein-AM. RESULTS: Corneas from donors with diabetes showed more-intense immunostaining for N(epsilon)-(carboxymethyl) lysine than normal donors of similar ages. Non-enzymatic glycation of collagen I and fibronectin significantly reduced the ability of corneal epithelial cells to adhere to these extracellular matrix proteins. CONCLUSION: Our data suggest that non-enzymatically glycated proteins may interfere with attachment and hence migration of epithelial cells across the corneal surface, and therefore may contribute to some of the clinical manifestations of diabetic keratopathy. Diabetes leads to an accelerated aging process resulting in a cornea that may be much older than the chronological age of the patient. From a clinical perspective, particular care should be taken when considering the use of contact lenses and following corneal injury in these patients.     

Transmission electron microscopy and autofluorescence findings in the cornea of diabetic rats treated with aminoguanidine

BACKGROUND: The accumulation of advanced glycation end products (AGEs) has been implicated in the pathogenesis of diabetic keratopathy. The present study was aimed to understand if aminoguanidine (AG), an AGE inhibitor, was protective against the development of corneal complications in a diabetic rat model. METHODS: Wistar rats were divided into three experimental groups: control, diabetic, and AG-treated diabetic. Diabetes was induced in rats via a single intraperitoneal injection (60 mg/kg) of streptozocin (STZ) and AG was administered in drinking water at a dose of 1 g/L. All animals were sacrificed at the end of 10 weeks and corneas from diabetic and nondiabetic rats were analyzed via transmission electron microscopy (TEM). Corneal autofluorescence measurements were also performed in all experimental groups. RESULTS: Electron microscopic evaluation revealed that aminoguanidine treatment in diabetic rats prevented the formation of intracellular spaces between neighbouring cells in the superficial corneal epithelium. Hyperglycemia-induced degeneration of intracellular organelles and formation of cytoplasmic vacuoles in the corneal stroma was also prevented with the treatment of AG. Corneal autofluorescence detected in the diabetic group (5.98 +/- 2.17 Fi/mg protein) was found to be significantly greater than the control (3.92 +/- 0.56 Fi/mg protein) and the AG-treated diabetic group (4.18 +/- 0.59 Fi/mg protein) (p < 0.05). INTERPRETATION: The presented data provide evidence that AG is preventive against corneal alterations in experimental diabetes.     

Corneal complications in streptozocin-induced type I diabetic rats

PURPOSE. This study seeks to characterize corneal functions and complications in a streptozocin (STZ)-induced rat model of type I diabetes mellitus (DM) and to understand the pathogenesis of diabetic keratopathy. METHODS. DM was induced via STZ injection in Sprague-Dawley rats. Body weight, length, and corneal size were measured and compared with the age-matched normal controls. Corneal morphology and histology were evaluated with slit lamp, digital confocal microscopy and hematoxylin and eosin staining. Tear secretion was measured with cotton threads, and corneal sensitivity was determined with an esthesiometer. Protein expression and distribution were assessed with Western blotting and immunohistochemistry. Wound healing was determined using an in vivo corneal epithelial debridement model. RESULTS. Compared with the normal control rats, STZ rats had reduced body weight, and body length, but minimally affected corneal size. No significant changes in ocular surface regularity, corneal thickness, and morphology were noted in diabetic corneas. STZ rats showed stronger Rose Bengal staining, decreased tear secretion, slightly attenuated sensitivity, less innervation, delayed epithelial wound healing, and impaired epidermal growth factor receptor signaling in their corneas. While the expression of adherens junction protein β-catenin, and tight junction proteins occludin and ZO-1 was unchanged, the formation of these junctions after wound closure was delayed. CONCLUSIONS. Pathogenesis of diabetic keratopathy involves multiple tissues and/or cell types and several events including reduced tear secretion, impaired innervation, weakened cell junction, and altered wound responses. These insights may prove useful for the clinical translation of evolving strategies for the management and treatment of diabetic corneal complications.     

The contribution of blood flow by the anterior ciliary arteries to the anterior segment in the primate eye

The rate and mode of corneal wound healing in severely diabetic rats were studied by light microscopy and scanning electron microscopy. Diabetes mellitus was induced in 52 rats by alloxan injection, and 52 nondiabetic rats were used as controls. After 3 weeks, a nonpenetrating razor-blade wound was made in the central cornea of both eyes in 48 diabetic and 48 normal rats. The incision passed through the epithelium and into the stroma. The effects of diabetes on the unwounded cornea were observed by comparison with corneas from eight unwounded rats (four diabetic and four normal). Whole corneas from wounded diabetic and normal rats were studied at 0, 1, 3, 6, 12 and 24 hr and at 2–7 days after wounding. The rate and mode of healing were not found to differ between diabetics and normals. The surfaces of corneal wounds in both groups appeared to be completely healed and indistinguishable from the surrounding unwounded epithelium after 24 hr. The epithelial cells involved in the initial healing process were derived primarily from the layer of wing cells which progressed across the wound close to the connective-tissue base. Only in the final stages of healing, after the wound had been filled by the deeper epithelial cells, did superficial epithelial cells migrate. There appeared to be more exfoliating superficial epithelial cells over the entire cornea in diabetic rats than in normals. Because the healing of central corneal incisions occurs initially and primarily by sliding of the deeper epithelial cells, and because the diabetic condition appears to be associated with increased exfoliation of surface cells, the healing of central incisions may be less affected by diabetes than the healing of defects of the whole corneal surface, where the superficial epithelial cells have been reported to be the main migratory cells in the initial healing process and where healing in diabetics is delayed.     

Electron microscopic study of anterior lens capsule allotransplants in chronic corneal ulcers

PURPOSE: This study provides ultrastructural morphology and quantitative analysis of allotransplants of anterior lens capsule in the treatment of recurrent corneal ulcers. METHODS: Mechanical ulcers of uniform size were created with a 6-mm corneal trephine in 9 eyes at one-third corneal depth in 6 white New Zealand male rabbits. Following initial epithelial regrowth, an identical injury was created in the same area of each cornea a second time. In 6 eyes (treated group), an anterior lens capsule from a healthy donor rabbit was sutured into the ulcer bed, followed by antibiotic/steroid drops 3 times daily for 1 week. The remaining 3 control eyes were allowed to heal without surgical intervention using the same antibiotic/steroid drops only. Slit-lamp examination, histopathology, and electron microscopic findings with hemidesmosome counts and size were recorded over a 3-month follow-up period. RESULTS: The control injured group had discontinuous epithelial basement membrane with significant reduction in hemidesmosome count compared with noninjured controls (P<0.0001). The treated subgroup 7 days after surgery showed linear basement membrane with identifiable lamina lucida and densa and newly-formed hemidesmosomes, which were significantly more numerous than in the injured but nontreated group (P<0.0001). Numerous hemidesmosomes and firm adhesion of epithelium to underlying stroma were seen in this group with lens capsule allografts 3 months after surgery. The number and size of hemidesmosomes did not significantly differ between groups 7 days and 3 months after surgery. CONCLUSIONS: On the ultrastructural level, anterior lens capsule allotransplants in mechanically induced corneal ulcer heal with continuously formed epithelial basement membrane and numerous hemidesmosomes. This enables significant reformation of hemidesmosomes that are identical in size to hemidesmosomes in healthy corneas and helps forming a stronger apposition for epithelial cells to underlying structures in eyes where epithelial basement membrane has been destroyed from chronic ulceration.     

Morphologic evaluation of Descemet's membrane and corneal endothelium in diabetes mellitus

Diabetes mellitus is characterized by abnormally thickened basement membranes throughout the body and the eye. In addition, abnormal corneal endothelial cell morphology has been found in eyes of diabetics. The authors examined Descemet's membrane (the basement of the corneal endothelium) and the endothelial cells in 19 diabetic corneas and 19 age-matched nondiabetic normal corneas in a masked fashion by transmission electron microscopy and specular microscopy. No statistically significant quantitative or qualitative differences were found between the two groups.     

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.     

Immunofluorescent characteristics of the diabetic cornea

In view of the increasing awareness of corneal abnormalities in diabetes, five diabetic and five nondiabetic post-mortem corneas were investigated. Indirect fluorescent antibody techniques were used for the detection of immunoglobulin, complement components, fibrinogen, and fibronectin, as some of these had already been found in the kidney, pancreas, and skin of diabetic patients. There was no obvious difference in the deposition of IgG, IgA, IgM, fibrinogen, or fibronectin between diabetic and control corneal specimens. Five diabetic specimens versus three control specimens demonstrated positive staining at 1:50 dilution for complement components Clq, C3, and C4. This staining was primarily present in the epithelium. Of greater interest was the finding that complement components were present in the basement membrane of two diabetic patients, but were not found in nondiabetic patients. In conclusion, diabetic corneas demonstrate unusual staining for complement components in the epithelial basement membrane.     

Inner blood-retinal barrier GLUT1 in long-term diabetic rats: an immunogold electron microscopic study

PURPOSE: The GLUT1 glucose transporter mediates glucose entry into the endothelial cells of the inner blood-retinal barrier (BRB). In many cell types, exposure to high glucose concentrations or diabetes downregulates GLUT1. To determine whether long-standing diabetes alters the expression and distribution of inner BRB GLUT1, changes in immunoreactive retinal endothelial cell GLUT1 were studied in Goto-Kakizaki (GK) rats, an animal model of type 2 diabetes. METHODS: Immunogold staining for GLUT1 was performed on ultrathin sections of retinal specimens obtained from 1-year-old GK rats and age-matched Wistar controls. Retinal capillary endothelial cells were visualized by transmission electron microscopy, and GLUT1 immunogold was quantified on the luminal and abluminal membranes of endothelial cells from digital microphotographs of individual vessels by computer. RESULTS: Forty-one microvessels from six diabetic rats and 43 microvessels from six nondiabetic Wistar control rats were analyzed. Densitometric quantification revealed an asymmetry of GLUT1 distribution between luminal and abluminal membranes of both diabetic and nondiabetic rats, with a luminal-to-abluminal ratio of approximately 1 to 3. The distribution pattern and density of retinal endothelial GLUT1 immunoreactivity were not significantly different between the diabetic and control rats. CONCLUSIONS: As determined by GLUT1 immunogold distribution, there is no compensatory downregulation of GLUT1 on the inner BRB in an animal model of long-standing diabetes.     

Electron microscopy of the epithelial flap created by ethanol treatment in the rabbit corneal epithelium

PURPOSE: To assess the effect of corneal debridement with 25% ethanol on rabbit corneal epithelium by electron microscopy. SETTING: Department of Ophthalmology, Osaka University School of Medicine, Osaka, Japan. METHODS: Rabbit corneas were deepithelialized by applying 25% ethanol for 3 minutes, and a hinged epithelial flap was created and repositioned. Ten corneas were evaluated immediately after the epithelial debridement procedure and at 1, 3, and 7 days. Histological changes were evaluated using scanning and transmission electron microscopy. RESULTS: Corneas treated with ethanol showed a decrease in microvilli, breaks in intercellular junctions, epithelial cell edema, and damage in basement membrane hemidesmosomes. These toxic changes recovered to an almost normal appearance after 24 hours and rapidly improved over the following week. An increase in desquamated cells was observed after debridement; this returned to normal after 1 week according to the normal cellular cycle. Wing cells showed no damage at any stage. CONCLUSIONS: Although ethanol appeared to have a toxic effect on rabbit epithelial cells, the effect did not persist over time. The first signs of recovery were observed 24 hours after debridement, and full recovery was observed over the following week. The ethanol path to the central basement membrane appeared to be centripetally from a round cut through the basement membrane, leaving the wing cells intact.     

Alterations of epithelial adhesion molecules and basement membrane components in lattice corneal dystrophy (LCD)

Background

The aim of the study was to investigate the histopathological and ultrastructural correlate of delayed epithelial healing in eyes with lattice corneal dystrophy (LCD).

Materials and Methods

Corneal buttons from 4 patients with LCD (two with subepithelial, two with stromal amyloid deposits) and 2 control corneas were examined. Cell-matrix adhesion molecules and basement membrane components of the corneal epithelium were analyzed by immunohistochemistry and hemidesmosomes between epithelium and stroma were quantified by transmission electron microscopy (TEM).

Results

By TEM well-developed hemidesmosomes anchored the basal epithelial cells to the underlying basement membrane in all normal and LCD corneas. Hemidesmosome density was not significantly different in subepithelial (224.7?±?34.1/100 µm) and stromal (234.3?±?36.3/100 µm) LCD compared to controls (241.3?±?26.8/100 µm). The basement membrane was interrupted in subepithelial, but continous in stromal LCD. Integrin α6 and ß4 staining formed a continous line along the basal surface of the corneal epithelium in control corneas, whereas it appeared discontinous and patchy both in subepithelial and stromal forms of LCD. Staining for αV integrin showed irregular staining patterns, i.e. enhanced labelling intensity in subepithelial and interrupted pattern in stromal LCD, respectively. Integrins α3, ß1, ß2, and ß5, dystroglycan, and plectin were not markedly different in dystrophic corneas. Type VII collagen showed a discontinuous staining in subepithelial forms of LCD. In stromal forms of LCD, type VII collagen staining occurred in additional patches underneath the epithelial basement membrane zone. Type XVII collagen staining was reduced in subepithelial LCD. Laminin-1, laminin-5 and laminin γ2 showed variable irregular staining patterns in dystrophic corneas with focal interruptions, focal thickenings, and reduplications of basement membrane. Some irregularities in corneas with subepithelial amyloid were observed for collagen types IV, V, and XVIII, laminin α1, α3, and γ1, nidogen-1 and -2, perlecan, fibrillin-1.

Conclusions

Immunohistochemical and electron microscopic evidence of structural alterations was found in LCD compared to normal corneas concerning cell-matrix adhesion molecules and basement membrane components. These alterations were more pronounced in dystrophic corneas with subepithelial amyloid deposits than in those with stromal deposits. Histopathological findings may correspond to reduced cell-matrix interactions and partly explain delayed epithelial healing in patients with lattice corneal dystrophy.     

Outgrowth of cells from human conjunctival explants onto cornea in vitro.

The epithelium was removed from human corneas, and samples of conjunctival tissue were cultured as explants on the denuded corneal surface for 1 and 2 weeks. Cells migrating from the conjunctival explants onto the corneal surface produced a multilayer where cells on the surface generally showed a flattened appearance. The apical membrane of these cells demonstrated villi as well as microplicae. Surface projections were also detected on cells in the deeper layers of the epithelium. Neighbouring cells were connected by junctional complexes. After 2 weeks, however, a lack of intercellular junctions in some areas resulted in the formation of intraepithelial cystoid spaces. Basal cells were connected to the underlying basement membrane by hemidesmosomes. Although transdifferentiation of the cells into a corneal epithelium was not observed within the 2 weeks, the present system provides a tool for studies on factors affecting reepithelialization of corneal epithelial defects by conjunctival cells.     

Ultrastructural changes in corneas of diabetic patients: an electron-microscopy study

BACKGROUND: Although diabetic retinopathy has been thoroughly studied, little attention has been given to the corneal changes of diabetic patients. Pathophysiologic and clinical findings may be related to the ultrastructural changes found in these corneas. PURPOSE: To investigate the ultrastructural corneal changes of diabetic patients. PATIENTS AND METHODS: Transmission electron microscopic ultrathin sections were prepared from corneas of 16 noninsulin-dependent diabetic patients (mean age, 65 years; range, 40-82 years) who suffered from the disease for a mean period of 22 years (range, 10-30 years). We used 16 corneas from healthy age-matched donors as normal controls. RESULTS: In addition to the epithelial changes that include accumulation of glycogen granules, occasional focal epithelial cell degeneration, and irregular thickening and multilamination of the epithelial basement membrane, unusual 120-nm wide-spaced collagen fibril bundles were observed scattered among both Descemet's membrane and stromal matrix. CONCLUSIONS: The aggregates of wide-spaced collagen fibrils, which have not been described in other basement membranes of diabetic patients, may reflect an excessive glycosylation rate.     

角膜上皮细胞基底膜的研究进展

角膜上皮细胞基底膜是一层很薄的组织,它的结构与其他部位的基底膜既有相似之处,也有独特的地方,在角膜创伤修复中有着重要的作用.基底膜组织的破坏会导致一系列棘手的角膜病变,而近来对角膜上皮细胞基底膜的研究使我们在这些病变的治疗方面有了新的人手点.就近年来角膜上皮细胞基底膜的研究进展做一综述.     

Expression of K12 keratin in alkali-burned rabbit corneas.

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.     

Extracellular matrix and Na+,K+-ATPase in human corneas following cataract surgery: comparison with bullous keratopathy and Fuchs' dystrophy corneas.

PURPOSE: To examine the distribution of extracellular matrix (ECM) and basement membrane (BM) components and of Na+,K+-ATPase in postcataract surgery (PCS) corneas. These corneas were from patients who never developed pseudophakic or aphakic bullous keratopathy (PBK/ABK) after cataract surgery. PCS corneas were compared with PBK/ABK and Fuchs' dystrophy corneas. METHODS: The distribution of PBKIABK ECM and BM markers and of all three Na+,K+-ATPase alpha subunits was studied by immunofluorescence in 10 healthy, 11 PCS, 16 PBK/ABK, and 12 Fuchs' dystrophy corneas. RESULTS: Fibrotic ECM proteins, tenascin-C and fibrillin-1, were found in only 1 of 10 healthy and in 2 of 11 PCS corneas. In contrast, these proteins were expressed in all PBK/ABK and more than half of the Fuchs' dystrophy corneas. BM components in PCS corneas were altered to a greater extent (40-60%), especially fibronectin and laminin-10. A decreased epithelial immunostaining for Na+,K+-ATPase alpha subunits was seen in approximately 40% of PCS corneas and in approximately two thirds of PBK/ABK and Fuchs' dystrophy corneas. However, the endothelial staining was normal in all groups. CONCLUSIONS: Because tenascin-C and fibrillin-1 were mostly found in diseased but not in PCS corneas, their expression may be related to later, clinical stages of corneal edema development. However, BM components abnormal in PBK/ABK and Fuchs' dystrophy corneas were also altered in PCS corneas without clinical evidence of ocular disease. This may result from subclinical corneal changes resulting from cataract surgery, lens removal, exposure to the intraocular lens, or endothelial cell damage. Alterations of epithelial Na+,K+-ATPase point to the importance of epithelial changes in the development of corneal edematous diseases.