Collagen pleomorphism in Descemet's membrane of streptozotocin-induced diabetic rats: an electron microscopy study

BACKGROUND: Diabetes mellitus causes ultrastructural changes in various basement membranes. These changes include increase in collagen biosynthesis rate and production of altered collagen. PURPOSE: To evaluate the ultrastructural changes in the corneas of streptozotocin- induced diabetic rats, focusing on Descemet's membrane. METHODS: Sprague-Dawley rats were sacrificed at different intervals after induction of diabetes mellitus by streptozotocin. Ten animals were sacrificed 1, 2, 3, 4, 6, 8, and 12 months after the injection of streptozotocin. Five untreated rats of the same age were used as normal controls and were sacrificed at the same intervals. Ultrathin sections were obtained from the corneas and were examined by transmission electron microscopy. RESULTS: Unusual 120-nm-spaced collagen fibril bundles were found in Descemet's membrane of the diabetic rats as early as the second month following the induction of diabetes. Their concentration and size increased gradually over the follow-up period of 12 months. CONCLUSIONS: Changes in the morphologic features of the collagen within Descemet's membrane may occur in diabetic-induced rats. The appearance of wide-spaced collagen fibrils in Decemet's membrane may represent alteration in collagen biosynthesis by the endothelial cells in diabetes or altered assembly of collagen due to increased glycosylation of normal formed collagen.     

Pathologic findings in postmortem corneas after successful laser in situ keratomileusis

PURPOSE: To examine the histologic and ultrastructural features of human corneas after successful laser in situ keratomileusis (LASIK). METHODS: Corneas from 48 eyes of 25 postmortem patients were processed for histology and transmission electron microscopy (TEM). The 25 patients had LASIK between 3 months and 7 years prior to death. Evaluation of all 5 layers of the cornea and the LASIK flap interface region was done using routine histology, periodic acid-Schiff (PAS)-stained specimens, toluidine blue-stained thick sections, and TEM. RESULTS: In patients for whom visual acuity was known, the first postoperative day uncorrected visual acuity was 20/15 to 20/30. In patients for whom clinical records were available, the postoperative corneal topography was normal and clinical examination showed a semicircular ring of haze at the wound margin of the LASIK flap. Histologically, the LASIK flap measured, on average, 142.7 microm (range, 100-200). A spectrum of abnormal histopathologic and ultrastructural findings was present in all corneas. Findings at the flap surface included elongated basal epithelial cells, epithelial hyperplasia, thickening and undulations of the epithelial basement membrane (EBM), and undulations of Bowman's layer. Findings in or adjacent to the wound included collagen lamellar disarray; activated keratocytes; quiescent keratocytes with small vacuoles; epithelial ingrowth; eosinophilic deposits; PAS-positive, electron-dense granular material interspersed with randomly ordered collagen fibrils; increased spacing between collagen fibrils; and widely spaced banded collagen. There was no observable correlation between postoperative intervals and the severity or type of pathologic change except for the accumulation the electron-dense granular material. CONCLUSIONS: Permanent pathologic changes were present in all post-LASIK corneas. These changes were most prevalent in the lamellar interface wound. These changes along with other pathologic alterations in post-LASIK corneas may change the functionality of the cornea after LASIK.     

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.     

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.     

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.     

Elevated expression of O-GlcNAc-modified proteins and O-GlcNAc transferase in corneas of diabetic Goto-Kakizaki rats

PURPOSE: The hexosamine biosynthetic pathway is one of the possible mechanisms involved in diabetic keratopathy. The purpose of this study was to examine the role of O-glycoside-linked N-acetylglucosamine (O-GlcNAc) modification of proteins in the pathogenesis of diabetic keratopathy in the Goto-Kakizaki (GK) rat, which has spontaneous development of diabetes. METHODS: An anti-O-GlcNAc antibody, an anti-O-GlcNAc transferase antibody, and digoxigenin (DIG)-labeled cRNA probes were used to examine the localization of O-GlcNAc-modified proteins, O-GlcNAc transferase protein and mRNA in the corneas of diabetic GK rats and in those of nondiabetic Wistar rats. The corneas from Wistar rats were organ cultured in control medium or in medium containing 100 micro M O-(2-acetamide-2-deoxy-D-glucopyranosylidene) amino-N-phenyl-carbamate (PUGNAc), an inhibitor of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins. The morphologic changes were examined by electron microscopy. RESULTS: In normal corneas, immunoreactive O-GlcNAc and O-GlcNAc transferase were observed in the epithelial, endothelial, and stromal cells. In the diabetic corneas, their immunoreactivities in the epithelium were increased in intensity. Morphologically, the number of hemidesmosomes in the epithelial basal cells was lower than that in those cells from the nondiabetic rats. In some areas, the basement membrane had detached from the epithelial basal cells. The PUGNAc treatment of Wistar rat corneas increased the level of O-GlcNAc immunoreactivity and caused a decrease in the number of hemidesmosomes and the detachment of corneal epithelial cells from the basement membrane. CONCLUSIONS: The elevated expression of O-GlcNAc-modified proteins and O-GlcNAc transferase may play a causative role in the corneal epithelial disorders of diabetic GK rats.     

Compositional differences between infant and adult human corneal basement membranes

PURPOSE: Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS: Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS: Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS: The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.     

Human diabetic corneas preserve wound healing,basement membrane,integrin and MMP-10 differences from normal corneas in organ culture

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.     

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.     

Matrix metalloproteinase 2 activation in cultured corneas

PURPOSE: Corneas that are maintained in tissue culture medium shed their epithelial cells and repopulation following graft surgery is an essential facet of the healing process. Failure to do so may be a result of structural damage to the epithelial basement membrane of a donor cornea. The purpose of the present investigation was to ascertain whether MMP-2, the matrix metalloproteinase produced by corneal keratocytes, may be activated during storage and hence cleave the type IV collagen component of the epithelial cell basement membrane. METHODS: Fresh and transplant rejected corneas that had been stored in culture medium for varying time periods and of known donor age were collected. The soluble protein fractions of these corneas were obtained. Their MMP-2 proteins were visualised by zymography on SDS gelatin polyacrylamide gels and assayed for activity against nitrophenyl acetate and denatured [(3)H]type I collagen. RESULTS: The stromal tissue of fresh, normal corneas produced inactive MMP-2 of M(r) 66,000. Although the cultured corneas did not up-regulate MMP-2 production, they contained additional MMP-2 activities of M(r) 62,000 and M(r) 43,000. The appearance of these additional MMP-2 activities correlated with corneal culture time but not donor age. The ability to cleave denatured [(3)H]type I collagen correlated with the appearance of the M(r) 43,000 activity but not the M(r) 62,000 activity. CONCLUSION: Activated MMP-2 is produced in cultured corneas. For this reason the corneas donated for all graft procedures should not be held in culture medium for periods exceeding 4 weeks.     

Sloughing of corneal epithelium and wound healing complications associated with laser in situ keratomileusis in patients with epithelial basement membrane dystrophy

PURPOSE: To report sloughing of corneal epithelium during laser in situ keratomileusis and subsequent wound healing complications in patients with epithelial basement membrane dystrophy. METHODS: In a retrospective study, the surgical procedures, postoperative course, and visual acuities of 16 eyes of nine patients with epithelial basement membrane dystrophy who underwent laser in situ keratomileusis complicated with epithelial sloughing at three centers were reviewed. The mean follow-up period was 23 weeks (range, 4 to 52 weeks). RESULTS: In 13 (81%) of 16 eyes with epithelial basement membrane dystrophy, epithelial sloughing occurred during laser in situ keratomileusis. In eight of the 13 eyes, epithelial growth beneath the flap was observed. The flap was lifted and the interface epithelium scraped in six eyes. Flap melt or keratolysis occurred in four eyes. At the last follow-up visit, 13 of 16 eyes had an uncorrected visual acuity of 20/30 or better, and all eyes had a best-corrected visual acuity of 20/30 or better. CONCLUSIONS: Patients with epithelial basement membrane dystrophy have poorly adherent corneal epithelium and are predisposed to epithelial sloughing during the microkeratome pass of laser in situ keratomileusis. This may lead to flap distortion, interface epithelial growth, flap keratolysis, and corneal scarring. It is not recommended that laser in situ keratomileusis be performed in patients with classic, symptomatic epithelial basement membrane dystrophy. In patients who present with mild and asymptomatic epithelial basement membrane dystrophy, laser in situ keratomileusis should be performed with caution, or photorefractive keratectomy may be the preferred refractive procedure.     

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.     

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.     

Anterior stromal micropuncture electron microscopic changes in the rabbit cornea

Anterior stromal micropuncture has become an effective treatment for recurrent erosion. The healing process in rabbit corneas was investigated. Following micropuncture of the corneal surface with a 27-gauge needle knife, electron microscopy was carried out at regular intervals from time 0 through 5 months. The corneal incisions began to fill with epithelium by day 1. Activated keratocytes were adjacent to the basement membrane defect by 7 days. The basement membrane appeared to be healed at 2 and 4 weeks. Epithelial projections into the stromal incisions with underlying mature basement membrane persisted at 5 months postsurgery. Basement membrane reproduction occurred much more rapidly following needle puncture than after microdiathermy. This was thought to occur because the corneal epithelial cell was immediately exposed to type I collagen, whereas following microdiathermy, new type I collagen must be secreted on the necrotic collagen before the corneal epithelium will secrete basement membrane.     

Abnormal extracellular matrix in corneas with pseudophakic bullous keratopathy

The present study describes biochemical and morphological differences of pseudophakic bullous keratopathy (PBK) corneas as compared with normal corneas. At the ultrastructural level, all PBK corneas studied had abnormal fibrillar material posterior to the Descemet's membrane. In addition, two of the six PBK buttons had subepithelial fibrocellular materials disrupting the epithelial basement membrane and Bowman's layer. Aggregates of collagen fibrils with 110 nm periodicity were occasionally seen within the stroma of the PBK corneas. Isolation and purification of the collagen from the Descemet's membrane/posterior collagenous layer (DM/PCL) showed an increased amount of material with molecular weight in the range of 50-60K daltons (presumably type VIII collagen) and decreased amounts of higher molecular weight, disulfide-bonded collagenous materials (presumably type IV collagen) as compared with normals. Sugar-specific lectin studies showed an increased deposition of peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA120) in the DM/PCL of the PBK corneas. Our data suggest that the DM/PCL of PBK corneas have an increased accumulation of terminal B-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues and altered ratios of low and high molecular weight collagenous proteins.     

Immunohistochemical evaluation of two corneal buttons with post-LASIK keratectasia

PURPOSE: To examine immunohistochemically 2 human corneal buttons after corneal transplantation for post-laser in situ keratomileusis (LASIK) keratectasia. METHODS: Two ectatic corneas after penetrating keratoplasty and 2 postmortem control corneas from a patient after uncomplicated LASIK were used. Cryostat sections were stained by immunofluorescence for >30 extracellular matrix (ECM) components and proteinases. RESULTS: The ratios of distance between LASIK flap interface and the upper epithelial layer to total corneal thickness were 0.27-0.34 in all cases. The whole flap interface was positive only for total and cellular fibronectin. Stromal types VI and XIV collagen, fibrillin-1, tenascin-C, and vitronectin were unchanged with no evidence of fibrosis. In ectasia cases, keratocytes adjacent to the flap did not express nidogens. Staining for type IV collagen alpha5 chain, nidogen-2, chains of laminin-8, and laminin-10 was weak and discontinuous in the epithelial basement membrane (EBM). Type IV collagen alpha1/alpha2 chains were found in the EBM of ectasia cases only. Matrix metalloproteinase (MMP)-10 showed increase in the epithelium, and MMP-3, in some keratocytes near the flap interface of ectatic corneas. Also, cathepsin F was seen at the flap margin only. Staining for limbal basal epithelial marker, alpha-enolase, was mostly absent in the ectatic cases, suggesting largely normal epithelial differentiation. CONCLUSIONS: Abnormal EBM structure similar to that previously observed in keratoconus and bullous keratopathy and an increase in certain proteinases suggest ongoing EBM lysis and remodeling. Immunohistochemical staining for fibronectin may be used to reveal the position of flap interface.     

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.     

Cleavage of corneal basement membrane components by ethanol exposure in laser-assisted subepithelial keratectomy

PURPOSE: To determine the anatomic cleavage plane after exposure to 20% ethanol for approximately 20 to 25 seconds to create an epithelial flap in laser-assisted subepithelial keratectomy (LASEK). SETTING: Ocular Surface Research & Education Foundation, Miami, Florida, and Hermann Eye Center Refractive Surgery Center, Houston, Texas, USA. METHODS: Immunofluorescence staining using monoclonal antibodies against laminin 5, collagen VII, and integrins beta(1) and beta(4) was performed to determine the anatomic location of the cleavage plane in an epithelial flap created by 20-second exposure to 20% ethanol in cadaver eyes and in epithelial flaps obtained from LASEK patients. RESULTS: Immunofluorescence staining to laminin 5 and integrin beta(4) was patchy in the lifted flap and the remaining corneal basement membrane. Immunostaining to collagen VII, the main component of anchoring fibrils, remained exclusively in the corneal bed. Immunostaining to integrin beta(1), present in the pericellular location of all epithelial cell layers, remained exclusively in the epithelial flap. This finding was consistent in cadaver corneas and LASEK epithelial flaps. CONCLUSIONS: The cleavage plane of the ethanol-induced corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane, where integrin beta(4) interacts with laminin 5 to form hemidesmosomes.