PURPOSE: Keratoconus is a bilateral noninflammatory ecstatic disease of cornea. Clinical manifestations and treatments are well-described , but the exact pathophysiology has many debates. There are many reports on pathologic abnormalities of keratoconus, but few reports on epithelial adhesion complex. The authors investigated the abnormalities in epithelial adhesion complex of keratoconus. METHODS: Using 4 corneas from 4 recipients of penetrating keratoplasty, examination was done with transmission electron microscope (Hitachi-600, Japan) after proper fixation and staining. Central and peripheral portion of each corneal tissues were examined. RESULTS: In two tissues, severe degeneration of basement membrane and Bowman's layer were found. Some degree of abnormalities was found in other tissues, which had minimal change. Some of hemidesmosomes, the most distinct part of adhesion complex, were found only in well-maintained tissue but the distribution was abnormal. CONCLUSIONS: The fact that basal plasma membrane had selectively more degenerations and changes than intercellular plasma membrane implies pathophysiology of keratoconus on adhesion complex, basal plasma membrane, basement membrane and Bowman's layer. Further study on this issue will reveal more information as to its pathophysiology.
Basement Membrane ; Cell Membrane ; Cornea ; Hemidesmosomes ; Keratoconus* ; Keratoplasty, Penetrating

PURPOSE: To investigate the effects of an amniotic membrane patch on corneal epithelial thickness and formation of hemidesmosomes during corneal stromal wound healing. METHODS: A stromal wound 9 mm in diameter and 130 microm in depth was created on rabbit cornea using a microkeratome. The changes in corneal epithelial thickness and hemidesmosome formations were compared between the amniotic membrane, contact lens, and control groups. Changes in the corneal epithelium were examined using H&E staining and hemidesmosome formation was examined using an electron microscope at 2 and 4 weeks after flap removal. RESULTS: Two weeks after treatment, the corneal epithelial thickness was 95.3 +/- 6.3 microm in the amniotic membrane group being significantly thicker than 76.4 +/- 5.1 microm in the contact lens group and 68.3 +/- 6.1 microm in the control group. Furthermore, more hemidesmosome formations were observed in the amniotic membrane group compared to the other 2 groups. However, there were no significant differences in corneal epithelial thickness or hemidesmosome formation among the 3 groups at week 4. CONCLUSIONS: The amniotic membrane group showed a thicker corneal epithelium and more hemidesmosome formation than the other 2 groups 2 weeks after flap removal. Thus, the use of an amniotic membrane patch appears to be effective in the early stages of corneal stromal wound healing.
Amnion ; Cornea ; Electrons ; Epithelium, Corneal ; Hemidesmosomes ; Wound Healing

This study was performed to elucidate the structural changes of the attachment complex in the cultured cornea. The three normal corneal tissues were used in this study. The ultrastructural changes of the attachment complex were observed by electron microscopy in one corneal tissue which was not cultured and cultured for 6 days and two corneal tissues which were cultured 10 days and 20 days, respectively. The cornea cultured for 6 days showed changes in the electron density of the hemidesmosome. The basal lamina was focally detached from the cytoplasmic wall of the basal epithelial cells in the cornea cultured for 10 days. The anchoring fibrils within the nuded corneal stroma, which was cultured for 20 days, were markedly decreased in number. These findings suggest that the normal basal epithelial cell, hemidesmosomes and basal lamina might be the essential factors to maintain the networks of normal anchoring fibrils in corneal stroma.
Basement Membrane ; Cornea ; Corneal Stroma ; Cytoplasm ; Epithelial Cells ; Hemidesmosomes ; Microscopy, Electron

The effect of 1% Na-Hyaluronan(Na-HA) on the morphogenesis of microvilli in superficial epithelial cells and of hemidesmosomes in basal epithelial cells together with the organization of superficial stromal collagen were evaluated in n-heptanol induced corneal epithelial wounds. Epithelial wounds were produced by applying a 5.5mm round filter paper, soaked in n-heptanol, on the central cornea for 60 seconds. 1% Na-HA in phosphate buffered saline(PBS) or PBS alone were instilled 4 times a day for 3 days. Epithelial healing rates determined during the first two days were not altered by Na-HA. The number of microvilli in superficial epithelial cells and of collagens fibers in superficial stroma were approximately the same between two groups. However, the number of hemidesmosome in the central cornea, which was counted in 2micrometer length of the basement membrane, significantly increased by the treatment with 1% Na-HA, being 10.0+/-1.1 in the 1% Na-HA treated group and 6.5+/-2.5 in the control group. The results suggest that topically applied 1% Na-HA may enhance the formation of hemidesmosome in n-heptanol wounded cornea.
Basement Membrane ; Collagen ; Cornea ; Epithelial Cells* ; Hemidesmosomes ; Heptanol ; Microvilli ; Morphogenesis* ; Wounds and Injuries*

To evaluate the effects of interleukin-6(IL-6) on epithelial migration in corneal epithelial wound healing process after excimer laser surface ablation, the morphologic changes as well as the healing rate of the defect area were observed on 30 rabbits(60 eyes). The animals were divided into three groups of twenty each: the control group and the two IL-6 treated groups(eye dropping with 0.1mg/L and 1.0mg/L of IL-6). On light microscopic examination, after 9 hours, centripetal migration of epithelial cells and covering of bare stroma with monolayered epithelial cells were more remarkable in the 1.0mg/L group than in the control group, reaching the peak at 18 hours. On transmission electron microscopic examination after 18 hour, the 1.0mg/L group showed the loss of hemidesmosome beneath the leading edge of sliding epithelial cell, other intracellular structures were well preserved. On scanning electron microscopic examination, epithelial migration started at 9 hour in all three groups and the epithelial migration was more rapid in the 1.0mg/L group than in the control group. Fifteen hours and afterwards, the healing rate was significantly accelerated in the 1.0mg/L group than in the control group. In view of the above findings, it was speculated that IL-6 could be used effectively in the early treatment of excimer laser keratectomy and other corneal epithelial injury.
Animals ; Epithelial Cells ; Hemidesmosomes ; Interleukin-6* ; Lasers, Excimer* ; Rabbits* ; Wound Healing ; Wounds and Injuries*

BACKGROUND: Matrix metalloproteinases (MMPs) participate in extracellular matrix degradation and may play an important role in basal membrane damage in many dermatologic diseases. Recent studies implicated the importance of MMP-9 in the pathogenesis of bulla formation of bullous pemphigoid (BP). Various autoimmune bullous diseases are strongly associated with desmosome or hemidesmosome pathologies, and show an increased level of lesional MMP and exposed autoantigens from these structures. OBJECTIVE: This study evaluated the level of MMP-2, -3, and -9 in three types of autoimmune bullous disease [BP, pemphigus vulgaris (PV), pemphigus foliaceus (PF)] with the aim of investigating the role of MMPs in the pathogenesis of autoimmune bullous diseases. METHODS: Sample specimens were obtained from skin lesions of patients with BP (n=12), PV (n=10), and PF (n=12), and from normal controls (n=8). The immunohistochemical expression of MMP-2, -3, and -9 was analyzed and serum levels of MMP-2, -3, and -9 were measured by enzyme-linked immunosorbant assay (ELISA). The results were analyzed with reference to graded levels of clinical severity. RESULTS: Expression of dermal MMP-2, -3, and -9 were increased in BP, PV, and PF (p=0.036, 0.022, and 0.015, respectively). However, decreased expression of the three MMPs in the epidermis of skin lesions may have resulted from epidermal destruction. ELISA-determined serum levels of MMP-2, -3, and -9 increased in BP, PV and PF. Interestingly, MMP-2 was significantly increased in the sera of BP patients (p=0.015), consistent with the previous studies concerning the role of gelatinase (MMP-2 and -9) in the pathogenesis of BP. In BP patients, clinical severity was proportional to increased levels of MMP-2 in both skin lesions and and sera. CONCLUSION: The increased expression of MMP-2, -3, and -9 in skin lesions and sera may reflect the involvement of these enzymes in the mechanism of bulla formation in autoimmune bullous diseases including BP. In addition, expression of MMP and clinical severity may be closely connected.
Autoantigens ; Blister ; Desmosomes ; Epidermis ; Extracellular Matrix ; Gelatinases ; Hemidesmosomes ; Humans ; Matrix Metalloproteinases ; Membranes ; Pemphigoid, Bullous ; Pemphigus ; Skin

Epidermolysis bullosa (EB) comprises a collection of clinically diverse inherited blistering diseases that affect the skin and, in some subtypes, mucous membranes and other organs. Currently classified into four main subtypes (EB simplex, junctional EB, dystrophic EB, and Kindler syndrome, mainly based on the level of skin cleavage), the spectrum of EB extends to more than 30 clinical subtypes with pathogenic mutations in at least 18 distinct genes. This review focuses on three recent additions to variants of EB: all are autosomal recessive, and result from mutations in either DST-e (coding for epidermal dystonin, also known as the 230 kDa bullous pemphigoid antigen, BP230), EXPH5 (coding for exophilin-5, also known as Slac2-b), or ITGA3 (coding for the integrin alpha-3 subunit). Each of these new forms of EB is reviewed with respect to the initial gene discovery, clinical features, the current mutation database, and skin pathology. Awareness of these recently described forms of EB is helpful in the clinical evaluation of patients with EB and in defining genotype-phenotype correlation for inherited blistering skin diseases.
Basement Membrane ; Blister ; Epidermolysis Bullosa* ; Genetic Association Studies ; Hemidesmosomes ; Humans ; Mucous Membrane ; Pathology ; Pemphigoid, Bullous ; Skin ; Skin Diseases ; Transport Vesicles

PURPOSE: LASEK is a newly developed refractive surgery technique that can make up for the complications from PRK and LASIK. The most unique procedures in LASEK is covering of the cornea with epithelial flap after keratectomy. We examined the effect of corneal epithelial flap on the wound healing of canine cornea. METHODS: Operation was performed in eyes from 12 dogs, and the 12 eyes were recovered with epithelial flap and the remaining 12 eyes were recovered without epithelial flap. Wound healing process was compared using fluorescein staining, light and transmission electron microscopic examination. RESULTS: Fluorescein stained area of the cornea was reduced with time in both groups, and from 9 hours after the operation, it was significantly reduced in the group with epithelial flap compared with those of the group without epithelial flap (p< 0.05). On light microscopic examination of the group with epithelial flap, and normal epithelial structure was found at 24 and 48 hours, respectively. However, in the group without epithelial flap, no complete reepithelialization had occurred on center at 48 hours after the operation. On transmission electron microscopic examination, eyes of the group with epithelial flap showed hemidesmosomes in the area where epithelial flap was closely contacted with the stroma at 24 hours, and they were completely developed at 48 hours. On the other hand, in the group without epithelial flap, hemidesmosomes developed only in the proximal portion but not at the leading edge even at 48 hours. CONCLUSIONS: These results suggest that corneal epithelial flap accelerate the wound healing process of the cornea and the wound healing process depend on the vitality of the epithelial flap.
Animals ; Cornea ; Dogs ; Fluorescein ; Hand ; Hemidesmosomes ; Keratectomy, Subepithelial, Laser-Assisted ; Keratomileusis, Laser In Situ ; Refractive Surgical Procedures ; Wound Healing* ; Wounds and Injuries*

PURPOSE: To investigate adhesion complex formation in cultivated human limbal epithelium after transplantation into the chemical burn model. METHODS: human limbal epithelial cells were cultured on amniotic membrane that had not undergone dispase treatment. Laminin V was evaluated using immunohistochemistry. The adhesion complex was examined by electron microscopy. Cultured epithelium was transplanted into limbal deficient rabbits induced by chemical burn and mechanical limbal removal. The transplanted rabbits and the controls with mechanical wounding were sacrificed at 1, 2, 3, and 4 weeks. The adhesion complex was examined by electron microscopy. RESULTS: Linear staining was observed against laminin V at 4-week culture but matured adhesion complex was not found. Graft failure developed in 3 rabbits (25%) after transplantation. Morphologically identifiable hemidesmosomes appeared at 1 week and matured adhesion complex with continuous basement membrane was found at 3 weeks. The mean numbers of hemidesmosomes/2.25 micro meter were 2.3 +/- 0.9, 2.5 +/- 0.5, 5.2 +/- 1.0, and 4.0 +/- 0.9 at 1, 2, 3, and 4 weeks, respectively. The adhesion assembly nearly recovered to the level of that in the human cornea (3.7 +/- 60.11) at 3 weeks. CONCLUSIONS: Adhesion complex of cultivated limbal epithelium did not developed in vitro, but the assembly was almost completed at 3 weeks after transplantation in vivo.
Amnion* ; Basement Membrane ; Burns, Chemical* ; Cornea ; Epithelial Cells ; Epithelium* ; Hemidesmosomes ; Humans ; Immunohistochemistry ; Laminin ; Microscopy, Electron ; Rabbits ; Transplants ; Wounds and Injuries

PURPOSE: In LASEK models, the viability of corneal epithelial cells after ethanol treatment was investigated in addition to morphological analysis. METHODS: Cell viability was assayed using a confocal laser scanning microscopy(CLSM) and MTT assay for in vivo and cultured rabbit corneal epithelial cells after treatment with various concentrations of ethanol. Ethanol treated human corneas were compared with control in terms of structural changes. RESULTS: CLSM assay showed that cells were viable at the ratio of 78.5, 75.0, 57.3, 43.3, 23.9, 4.3% for 0, 10, 20, 30 40, 60 % ethanol groups, respectively. The relative survival rates in comparison to 0% ethanol-treated (control) corneas were 95.5(10%), 72.9(20%), 55.2(30%), 30.4(40%), 5.5(60%). On MTT assay after 10, 20, 25, 30, 40, 60% ethanol treatment, cultured epithelial cells were still alive at the percentage of 92.7, 92.2, 36.6, 30.7, 5.4, 5.1% (20 sec), 90.8, 57.1, 29.0. 28.6, 5.0, 4.7% (40 sec) at 0 hr with decreasing cell survival over time in 20% ethanol group. TEM showed multiple vacuole-like loosenings along the intercellular junction of superficial squamous and wing cells. The loss and break-up of hemidesmosome and basement membrane were also demonstrated in conjunction with the loosening of sub-basal interface. CONCLUSIONS: After 20% ethanol exposure for 40 seconds, 72.9% and 57.1% of in vivo and cultured rabbit corneal epithelial cells were in viable state with decreasing cell survival over increasing ethanol concentration and time. The loss and break-up of hemidesmosome and basement membrane, resulting in loosening of sub-basal interface, might be the mechanism for the detachment of LASEK flap en bloc.
Basement Membrane ; Cell Survival ; Cornea ; Epithelial Cells* ; Epithelium, Corneal ; Ethanol* ; Hemidesmosomes ; Humans ; Intercellular Junctions ; Keratectomy, Subepithelial, Laser-Assisted ; Survival Rate