August 2000
Volume 41, Issue 9
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Cornea  |   August 2000
Coordinated Reassembly of the Basement Membrane and Junctional Proteins during Corneal Epithelial Wound Healing
Author Affiliations
  • Katsuyoshi Suzuki
    From the Department of Ophthalmology, Yamaguchi University School of Medicine, Ube City, Japan.
  • Toshiro Tanaka
    From the Department of Ophthalmology, Yamaguchi University School of Medicine, Ube City, Japan.
  • Miho Enoki
    From the Department of Ophthalmology, Yamaguchi University School of Medicine, Ube City, Japan.
  • Teruo Nishida
    From the Department of Ophthalmology, Yamaguchi University School of Medicine, Ube City, Japan.
Investigative Ophthalmology & Visual Science August 2000, Vol.41, 2495-2500. doi:https://doi.org/
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      Katsuyoshi Suzuki, Toshiro Tanaka, Miho Enoki, Teruo Nishida; Coordinated Reassembly of the Basement Membrane and Junctional Proteins during Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2495-2500. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To characterize changes in the localizations of the basement membrane protein laminin-1 and of adhesion proteins of intercellular junctions during wound healing after epithelial ablation in the rat cornea.

methods. Epithelial ablation was performed with an excimer laser. Rats were killed immediately, 12 hours, 24 hours, 3 days, or 4 weeks after ablation, and corneal cryosections were subjected to two-color immunofluorescence staining with antibodies to laminin-1 and antibodies to connexin43 for gap junctions, desmoglein 1 or 2 (desmoglein 1 + 2) for desmosomes, or E-cadherin for adherens junctions. Sections were also stained with antibodies to occludin for examination of tight junctions.

results. Laminin-1 was detected in the basement membrane, connexin43 in the basal cell layer, desmoglein 1 + 2 in the wing cell layer, E-cadherin in all cell layers, and occludin in the wing and superficial cell layers of the intact corneal epithelium. Laminin-1 immunostaining was not detected at the leading edge of migrating epithelial cells until 24 hours after ablation. Expression of connexin43 and desmoglein 1 + 2 coincided with the reappearance of laminin-1, whereas that of E-cadherin and occludin was apparent regardless of the absence or presence of laminin-1. Epithelial remodeling was complete after 4 weeks. The basement membrane was re-established, and the expression patterns for all the adhesion proteins were identical with those characteristic of the intact cornea.

conclusions. Actively migrating epithelial cells no longer manifested gap junctions and desmosomes in the wounded area with no basement membrane. Re-establishment of the basement membrane coincided with reassembly of these intercellular junctions, suggesting that the presence of the basement membrane may be required for their reformation in the rat cornea.

The corneal epithelium is exposed to the ever-changing external environment and serves as the frontal barrier of the whole eyeball. For clear vision, it is important that the surface of the corneal epithelium remain smooth, so that light is uniformly refracted, and that defects be resurfaced by an active repair process. Cell–cell and cell–matrix interactions play important roles in the maintenance of the stratified structure of the corneal epithelium. In response to epithelial damage, remaining epithelial cells migrate to cover the defective area. The condition of the basement membrane is an important factor during wound healing in the corneal epithelium. 1 2 The basement membrane is reformed as epithelial defects heal. 3 4 Thus, the interactions between epithelial cells and the underlying matrix are a critical determinant of successful repair of epithelial defects. Cell–cell interaction is also an important factor in the repair process. Thus, if epithelial cells adhere tightly to each other, then they may not be able to migrate to the damaged area. The migration of cells probably requires that they undergo cycles of de-adhesion and re-adhesion. Examination of changes in the localization of intercellular junctions would thus probably shed light on the mechanism of corneal epithelial wound healing. 
Four types of intercellular junctions have been identified in the corneal epithelium: gap junctions, desmosomes, adherens junctions, and tight junctions. 5 Gap junctions mediate intercellular signaling and thereby allow functional synchronization among neighboring cells, and they are composed of the protein connexin. 6 Desmosomes and adherens junctions serve to anchor cells together and are formed by members of the cadherin family of adhesion proteins. 7 Desmoglein is one of the adhesion proteins of desmosomes, 8 and E-cadherin is the adhesion protein of adherens junctions. 7 9 Tight junctions serve a barrier function in epithelia and are composed of the adhesion protein occludin. 9  
Corneal epithelial wound healing has been studied extensively at the histologic and immunohistologic levels. Various types of wounding have been applied to the epithelium, including mechanical abrasion with a razor blade or a scalpel and chemical abrasion with iodine or n-heptanol. 10 11 12 13 Recent advances in laser technology have allowed application of the excimer laser for epithelial abrasion. The use of the excimer laser for the study of epithelial wound healing has the advantages that the depth and size of the resultant wounds are uniform and reproducible and that the associated inflammatory reactions, including leukocyte infiltration, are less pronounced than are those induced by other techniques. 14  
To elucidate the mechanisms of corneal epithelial wound healing with the use of immunofluorescence staining and confocal laser microscopy, we have now characterized the excimer laser ablation–induced changes in the localizations of the basement membrane protein laminin-1 and of four adhesion proteins of intercellular junctions 1 : connexin43 of gap junctions, 2 desmoglein 1 or 2 (desmoglein 1 + 2) of desmosomes, 3 E-cadherin of adherens junctions, and 4 occludin of tight junctions. 
Methods
Materials
Male Wistar rats (body mass, 200–250 g) were obtained from Seac Yoshitomi (Fukuoka, Japan). Pentobarbital sodium was obtained from Abbott Laboratories (North Chicago, IL), and 0.4% (wt/vol) oxybuprocaine hydrochloride and 0.3% (wt/vol) ofloxacin ophthalmic solutions were from Santen Pharmaceutical (Osaka, Japan). Mouse monoclonal antibodies to connexin43 were obtained from Chemicon (Temecula, CA), mouse monoclonal antibodies to desmoglein 1 + 2 from Progen Biotechnik (Heidelberg, Germany), mouse monoclonal antibodies to E-cadherin from Transduction Laboratories (Lexington, KY), rabbit polyclonal antibodies to occludin from Zymed (South San Francisco, CA), rabbit polyclonal antibodies to laminin-1 from LSL (Tokyo, Japan), tetramethylrhodamine isothiocyanate (TRITC)-conjugated antibodies to rabbit IgG and whole rabbit serum from Cappel Organon Teknika (Durham, NC), fluorescein isothiocyanate (FITC)-conjugated antibodies to either mouse or rabbit IgG from ICN (Aurora, OH), and BALB/c mouse control ascites fluid from Cedarlane (Hornby, Ontario, Canada). Silane-treated slides were obtained from Dako (Carpinteria, CA), optimal temperature cutting (OCT) compound from Sakura Finetechnical (Tokyo, Japan), bovine serum albumin (BSA) fraction V from Nacalai Tesque (Kyoto, Japan), and Vectashield mounting medium from Vector Laboratories (Burlingame, CA). 
Excimer Laser Photoablation
Wister rats (n = 20) were anesthetized by an intraperitoneal injection of pentobarbital sodium (10 mg/kg body mass), and 0.4% oxybuprocaine hydrochloride ophthalmic solution was applied to the cornea as a topical anesthetic. Photoablation in photorefractive keratectomy mode (80 μm in depth, 3 mm in diameter) was performed on the right eye of each rat with an excimer laser (EC-5000; Nidek, Gamagori, Japan); immediately after the surgery, 0.3% ofloxacin ophthalmic solution was applied to the eye. The left eye of each animal served as an unwounded control. The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Yamaguchi University School of Medicine and was performed in accordance with the Guidelines for Animal Experiments of Yamaguchi University School of Medicine, Law No. 105 and Notification No. 6 of the Japanese Government, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Two-Color Immunofluorescence Staining and Laser Confocal Microscopy
Rats were killed immediately or 12 hours, 24 hours, 3 days, or 4 weeks after excimer laser–induced ablation by an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The eyes were enucleated, embedded in OCT compound, frozen in acetone and dry ice, and stored at− 80°C. Cryostat sections (6 μm) were cut with a microtome (HM505N; Microm, Walldorf, Germany) and transferred to silane-treated slides. 
Double immunostaining was performed to localize epithelial basement membrane and adhesion proteins of intercellular junctions with the combination of antibodies to laminin-1 and antibodies to either connexin43, desmoglein 1 + 2, or E-cadherin. Immunostaining for occludin was not combined with that for laminin-1. In brief, after the sections were rinsed three times (5 minutes each time) in phosphate-buffered saline (PBS), they were incubated for 1 hour at room temperature with 1% (wt/vol) BSA in PBS to block nonspecific binding. The sections were incubated with primary antibodies to adhesion proteins for 1 hour in a moist chamber at room temperature, rinsed three times in PBS (5 minutes each time), and incubated for 1 hour in a moist chamber at room temperature with corresponding FITC-conjugated secondary antibodies (which generate green fluorescence). After they were rinsed three times in PBS (5 minutes per rinse), the sections exposed to antibodies to either connexin43, desmoglein 1 + 2, or E-cadherin were incubated for 1 hour in a moist chamber at room temperature with antibodies to laminin-1. The sections were rinsed three times in PBS (5 minutes per rinse) and incubated for 1 hour in a moist chamber at room temperature with corresponding TRITC-conjugated secondary antibodies (which generate red fluorescence). After three final rinses in PBS, the sections were mounted (Vectashield). 
For incubation with sections, the primary antibodies were used at the following dilutions prepared with 1% BSA in PBS: connexin43, 1:200; desmoglein 1 + 2, 1:100; E-cadherin and occludin, 1:500; and laminin-1, 1:1000. Secondary antibodies were also diluted with 1% BSA in PBS as follows: FITC-conjugated antibodies to mouse IgG, 1:500; FITC-conjugated antibodies to rabbit IgG, 1:2000; and TRITC-conjugated antibodies to rabbit IgG, 1:1000. For control staining, sections were incubated with whole rabbit serum or BALB/c mouse ascites fluid, both at 1:1000 dilutions prepared with 1% BSA in PBS, in place of the corresponding primary antibodies. 
All slides were observed under a laser confocal microscope (Fluoview; Olympus, Tokyo, Japan). Fluorescence images combined with Nomarski differential interference contrast (DIC) images were obtained from three different regions of the cornea: the central region, where the corneal epithelium was abraded; the mid-periphery, which constituted the transition zone between the abraded area and the uninjured area; and the periphery, which was not abraded by the excimer laser. Representative images are presented. 
Results
Immunolocalization of Laminin-1 and Junctional Adhesion Proteins in the Intact Cornea
The immunolocalization of laminin-1, connexin43, desmoglein 1 + 2, E-cadherin, and occludin in the intact rat cornea is shown in Figure 1 . A linear pattern of fluorescence specific for laminin-1 was detected in the basement membrane region beneath the epithelium. A punctate pattern of fluorescence specific for connexin43 was apparent in the basal cell layer of the corneal epithelium. Specific fluorescence for desmoglein 1 + 2 was apparent at the apical surface of basal cells and in the wing cell layer but was not detected in the superficial cell layer. In contrast, E-cadherin–specific fluorescence was detected at the surface of cells in all layers. Dots or short linear stretches of occludin-specific fluorescence were apparent in the wing and superficial cell layers of the epithelium. 
Protein Localization Immediately after Laser Photoablation
The epithelium, basement membrane, and superficial layer of the stroma in the central region of the cornea were abraded by excimer laser irradiation. In the treated region, no specific immunofluorescence for laminin-1 was detected immediately after photoablation (Fig. 1 , lower panels). Nomarski DIC images also revealed the absence of epithelial cells and cell debris in this region. The border of the abraded area and the remaining epithelium was well defined in the midperipheral region of the cornea by immunostaining for laminin-1. Whereas laminin-1 was absent from the abraded area, a smooth line of laminin-1 fluorescence was apparent beneath the epithelium in the remainder of the cornea. Nomarski DIC images revealed no sign of epithelial migration. In the untreated region of the corneal epithelium, the localizations of connexin43, desmoglein 1 + 2, E-cadherin, and occludin were also identical with those apparent in the intact cornea, and they did not change during the wound-healing process (data not shown). 
Protein Localization during Wound Healing
The localizations of laminin-1, connexin43, desmoglein 1 + 2, E-cadherin, and occludin during wound healing after excimer laser photoablation are shown in Figures 2 and 3 . Twelve hours after photoablation, the epithelial defect remained apparent in the central region of the cornea (data not shown). However, the remaining epithelial cells of the untreated region appeared to have begun migrating toward the defective area. Laminin-1 was not detected in the portion of the epithelium containing the migrating cells; moreover, of the four adhesion proteins examined, only occludin was apparent in this region. Occludin staining was observed in the superficial and subsuperficial cell layers of the migrating epithelium. 
Twenty-four hours after photoablation, epithelial migration had further progressed to cover the damaged area. Weak staining for laminin-1 was apparent beneath the front edge of the migrating epithelium, whereas intense and irregular staining for laminin-1 was observed at the border of the region of photoablation. Although connexin43 was not detected at the front edge of the migrating epithelium, faint staining for this protein was apparent in the basal cell layer of the back end of the migrating epithelium. Desmoglein 1 + 2 was detected in the subsuperficial cell layer of the migrating epithelium at the border of the region of ablation; however, staining was only weak at the front end of the migrating epithelium. Upregulation of E-cadherin expression in all cell layers was observed at both the front and back ends of the migrating epithelium. Similar upregulation of occludin expression was also apparent in the superficial and subsuperficial cell layers of the migrating epithelium. 
Three days after photoablation, the epithelial defect was completely covered by the migrating epithelium. The regenerated epithelium showed a normal thickness, well-defined layered structure, and smooth apical surface; however, its interface with the stroma appeared serrated. Laminin-1 was detected beneath both the regenerated epithelium and the nonabraded epithelium. Whereas the pattern of lamnin-1 staining was smooth and linear in the nonabraded area, it was jagged in the regenerated region. The staining patterns of the junctional adhesion proteins in the abraded region were virtually identical with those observed in the nonabraded region of the epithelium: Connexin43 was expressed in the basal cell layer, desmoglein 1 + 2 in the wing cell layer, E-cadherin in all cell layers, and occludin in the wing and superficial cell layers. 
Four weeks after photoablation, no marked differences in Nomarski DIC images or in the staining patterns of laminin-1, connexin43, desmoglein 1 + 2, E-cadherin, and occludin were apparent between the treated region and the untreated region of the cornea (data not shown). 
Discussion
We have described changes in the localizations of the basement membrane component laminin-1 and of four adhesion proteins of intercellular junctions (connexin43, desmoglein 1 + 2, E-cadherin, and occludin) during corneal epithelial wound healing after photoablation. Laminin-1 appeared beneath the migrating epithelium by 24 hours after injury. Redistribution of connexin43 and desmoglein 1 + 2 coincided with this expression of laminin-1. The expression of E-cadherin and occludin, however, appeared constitutive regardless of the absence or presence of laminin-1 beneath the migrating epithelium. 
The basement membrane functions as a dynamic structure in tissue morphology, differentiation, and maintenance. 15 Laminin-1, a major constituent of the basement membrane, regulates various cell processes, including adhesion, proliferation, and differentiation. 16 17 18 19 Remodeling of the basement membrane is thus likely to be an important event during the wound healing process. Our present data suggest that migrating epithelial cells synthesize and deposit laminin-1 beneath themselves within 24 hours after photoablation. Expression of connexin43 and desmoglein 1 + 2 increased at approximately the same time as did that of laminin-1, whereas E-cadherin and occludin were expressed constitutively in the migrating epithelium. This observation that the re-establishment of the basement membrane coincided with the reassembly of two of the four types of intercellular junctions examined suggests that the basement membrane may affect the expression of junctional adhesion proteins in corneal epithelial cells. 
Our data also demonstrate that each layer of the intact corneal epithelium expresses a different combination of intercellular junctions. Gap junctions (connexin43) are present in the basal cell layer, desmosomes (desmoglein 1 + 2) in the wing cell layer, adherens junctions (E-cadherin) in all cell layers, and tight junctions (occludin) in the wing and superficial cell layers of the epithelium. These results suggest that each cell layer plays a distinct role in maintenance of the structure and function of the corneal epithelium as a result of the expression of specific types of junctional adhesion proteins. Our results are consistent with those of previous immunohistochemical or electron microscopic studies on the localizations of intercellular junctions in the rabbit corneal epithelium. 5 10 12 20  
Our data are also consistent with the previous observation 10 that migrating epithelial cells no longer express connexin43 after wounding of the rabbit corneal epithelium. Expression of connexin43 and desmoglein 1 + 2 in the rat cornea was upregulated 3 days after epithelial ablation at a time when the basement membrane was reassembled, and the defect was completely covered. 
The localization of E-cadherin in all cell layers of the corneal epithelium during wound healing was previously shown not to differ from that observed in the intact rabbit cornea. 12 However, in the present study, E-cadherin was not detected at the leading edge of the migrating epithelium 12 hours after excimer laser ablation; upregulation of E-cadherin expression, preceding that of connexin43 and desmoglein 1 + 2, was apparent 24 hours after photoablation. 
In the present study, the tight junction protein occludin was present in the wing and superficial cell layers of the corneal epithelium, regardless of whether the epithelium was stationary or actively migrating. Previous electron microscopic observations have shown that tight junctions reform rapidly after wounding of the rabbit corneal epithelium, 20 and immunohistochemical data have shown that ZO-1, a protein closely associated with tight junctions, is induced by removal of superficial cells of the rabbit corneal epithelium. 21  
In summary, our results indicate that the condition of the basement membrane is closely associated with that of the corneal epithelium, specifically with the expression of adhesion proteins of intercellular junctions. Further investigations are required to clarify the molecular nature of the regulatory interactions between corneal epithelial cells and their underlying basement membrane. 
 
Figure 1.
 
Immunolocalization of laminin-1 and junctional adhesion proteins in the intact and wounded cornea. The photomicrographs represent digital overlays of Nomarski DIC images and immunofluorescence images. Upper left: Red immunofluorescence image of laminin-1 and green immunofluorescence image of connexin43 (Cx43); upper right: red immunofluorescence image of laminin-1 and green immunofluorescence image of desmoglein 1 + 2 (Dsg 1 + 2); middle left: red immunofluorescence image of laminin-1 and green immunofluorescence image of E-cadherin (E-cad); middle right: green immunofluorescence image of occludin (Oc); bottom: red immunofluorescence images of laminin-1 (LN) in the middle (left) and at the edge (right) of the damaged region of the cornea immediately after excimer laser photoablation. The arrowhead indicates the margin of the wounded area in the midperipheral region of the cornea. Scale bars, 50μ m.
Figure 1.
 
Immunolocalization of laminin-1 and junctional adhesion proteins in the intact and wounded cornea. The photomicrographs represent digital overlays of Nomarski DIC images and immunofluorescence images. Upper left: Red immunofluorescence image of laminin-1 and green immunofluorescence image of connexin43 (Cx43); upper right: red immunofluorescence image of laminin-1 and green immunofluorescence image of desmoglein 1 + 2 (Dsg 1 + 2); middle left: red immunofluorescence image of laminin-1 and green immunofluorescence image of E-cadherin (E-cad); middle right: green immunofluorescence image of occludin (Oc); bottom: red immunofluorescence images of laminin-1 (LN) in the middle (left) and at the edge (right) of the damaged region of the cornea immediately after excimer laser photoablation. The arrowhead indicates the margin of the wounded area in the midperipheral region of the cornea. Scale bars, 50μ m.
Figure 2.
 
Immunolocalization of connexin43 (left) and desmoglein 1+ 2 (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images, red immunofluorescence images of laminin-1, and green immunofluorescence images of connexin43 (Cx43) or desmoglein 1 + 2 (Dsg 1 + 2) at 12 hours, 24 hours, and 3 days after photoablation, as indicated. Center, the central region of the cornea, which was subjected to photoablation; margin, the midperipheral region of the cornea, containing the edge of the wounded region (arrowhead). Scale bar, 50 μm.
Figure 2.
 
Immunolocalization of connexin43 (left) and desmoglein 1+ 2 (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images, red immunofluorescence images of laminin-1, and green immunofluorescence images of connexin43 (Cx43) or desmoglein 1 + 2 (Dsg 1 + 2) at 12 hours, 24 hours, and 3 days after photoablation, as indicated. Center, the central region of the cornea, which was subjected to photoablation; margin, the midperipheral region of the cornea, containing the edge of the wounded region (arrowhead). Scale bar, 50 μm.
Figure 3.
 
Immunolocalization of E-cadherin (left) and occludin (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images and either red immunofluorescence images of laminin-1 and green immunofluorescence images of E-cadherin (E-cad; left), or green immunofluorescence images of occludin (Oc; right). The cornea was examined 12 hours, 24 hours, or 3 days after photoablation, as indicated. Center and margin: see Figure 2 . Scale bar, 50 μm.
Figure 3.
 
Immunolocalization of E-cadherin (left) and occludin (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images and either red immunofluorescence images of laminin-1 and green immunofluorescence images of E-cadherin (E-cad; left), or green immunofluorescence images of occludin (Oc; right). The cornea was examined 12 hours, 24 hours, or 3 days after photoablation, as indicated. Center and margin: see Figure 2 . Scale bar, 50 μm.
The authors thank Kiyoe Takeuchi for technical assistance. 
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Figure 1.
 
Immunolocalization of laminin-1 and junctional adhesion proteins in the intact and wounded cornea. The photomicrographs represent digital overlays of Nomarski DIC images and immunofluorescence images. Upper left: Red immunofluorescence image of laminin-1 and green immunofluorescence image of connexin43 (Cx43); upper right: red immunofluorescence image of laminin-1 and green immunofluorescence image of desmoglein 1 + 2 (Dsg 1 + 2); middle left: red immunofluorescence image of laminin-1 and green immunofluorescence image of E-cadherin (E-cad); middle right: green immunofluorescence image of occludin (Oc); bottom: red immunofluorescence images of laminin-1 (LN) in the middle (left) and at the edge (right) of the damaged region of the cornea immediately after excimer laser photoablation. The arrowhead indicates the margin of the wounded area in the midperipheral region of the cornea. Scale bars, 50μ m.
Figure 1.
 
Immunolocalization of laminin-1 and junctional adhesion proteins in the intact and wounded cornea. The photomicrographs represent digital overlays of Nomarski DIC images and immunofluorescence images. Upper left: Red immunofluorescence image of laminin-1 and green immunofluorescence image of connexin43 (Cx43); upper right: red immunofluorescence image of laminin-1 and green immunofluorescence image of desmoglein 1 + 2 (Dsg 1 + 2); middle left: red immunofluorescence image of laminin-1 and green immunofluorescence image of E-cadherin (E-cad); middle right: green immunofluorescence image of occludin (Oc); bottom: red immunofluorescence images of laminin-1 (LN) in the middle (left) and at the edge (right) of the damaged region of the cornea immediately after excimer laser photoablation. The arrowhead indicates the margin of the wounded area in the midperipheral region of the cornea. Scale bars, 50μ m.
Figure 2.
 
Immunolocalization of connexin43 (left) and desmoglein 1+ 2 (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images, red immunofluorescence images of laminin-1, and green immunofluorescence images of connexin43 (Cx43) or desmoglein 1 + 2 (Dsg 1 + 2) at 12 hours, 24 hours, and 3 days after photoablation, as indicated. Center, the central region of the cornea, which was subjected to photoablation; margin, the midperipheral region of the cornea, containing the edge of the wounded region (arrowhead). Scale bar, 50 μm.
Figure 2.
 
Immunolocalization of connexin43 (left) and desmoglein 1+ 2 (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images, red immunofluorescence images of laminin-1, and green immunofluorescence images of connexin43 (Cx43) or desmoglein 1 + 2 (Dsg 1 + 2) at 12 hours, 24 hours, and 3 days after photoablation, as indicated. Center, the central region of the cornea, which was subjected to photoablation; margin, the midperipheral region of the cornea, containing the edge of the wounded region (arrowhead). Scale bar, 50 μm.
Figure 3.
 
Immunolocalization of E-cadherin (left) and occludin (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images and either red immunofluorescence images of laminin-1 and green immunofluorescence images of E-cadherin (E-cad; left), or green immunofluorescence images of occludin (Oc; right). The cornea was examined 12 hours, 24 hours, or 3 days after photoablation, as indicated. Center and margin: see Figure 2 . Scale bar, 50 μm.
Figure 3.
 
Immunolocalization of E-cadherin (left) and occludin (right) in the cornea at various times after excimer laser photoablation. The photomicrographs represent digital overlays of Nomarski DIC images and either red immunofluorescence images of laminin-1 and green immunofluorescence images of E-cadherin (E-cad; left), or green immunofluorescence images of occludin (Oc; right). The cornea was examined 12 hours, 24 hours, or 3 days after photoablation, as indicated. Center and margin: see Figure 2 . Scale bar, 50 μm.
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