August 1999
Volume 40, Issue 9
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Cornea  |   August 1999
Synchronization of the G1/S Transition in Response to Corneal Debridement
Author Affiliations
  • Eui–Hong Chung
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Audrey E. K. Hutcheon
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Nancy C. Joyce
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • James D. Zieske
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Investigative Ophthalmology & Visual Science August 1999, Vol.40, 1952-1958. doi:https://doi.org/
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      Eui–Hong Chung, Audrey E. K. Hutcheon, Nancy C. Joyce, James D. Zieske; Synchronization of the G1/S Transition in Response to Corneal Debridement. Invest. Ophthalmol. Vis. Sci. 1999;40(9):1952-1958. doi: https://doi.org/.

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

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Abstract

purpose. This study’s intention was to examine the progression of ocular surface epithelium through the G1/S transition of the cell cycle after corneal epithelial debridement.

methods. Three-millimeter debridements were made in central rat cornea and allowed to heal 4 to 48 hours in vivo. Unwounded contralateral eyes served as controls. Two hours before the animals were killed, 5-bromo-2-deoxyuridine (BrdU) was injected to detect S-phase cells. Incorporated BrdU was visualized by indirect immunofluorescence microscopy, and expression of G1 cell-cycle markers cyclins D and E was examined by indirect immunofluorescence and immunoblotting.

results. The number of BrdU-labeled cells in conjunctival, limbal, and peripheral epithelium peaked at 28 hours after wounding (3.9-, 4.5-, and 3.2-fold increases, respectively). In unwounded eyes, cyclin D showed diffuse cytoplasmic localization with occasional basal cells exhibiting a nuclear localization, while anti–cyclin E showed intense localization in limbal and conjunctival basal cells but only minimal labeling in corneal epithelium. Within 8 to 12 hours after wounding, the nuclei of most corneal basal cells outside the wound area were bound intensely by anti–cyclins D and E. Immunoblotting revealed that cyclin D and E protein levels increased 4.5- and 12.1-fold after wounding, respectively. Epithelium migrating into the wound area did not incorporate BrdU and did not exhibit nuclear localization of cyclins D and E.

conclusions. Corneal epithelial debridement stimulates basal cells outside the wound area to synchronously enter the cell cycle. However, cells migrating to cover the wound area do not progress through the cell cycle. These data suggest a compartmentalization of the proliferative and migratory phases of wound repair.

Corneal wound healing provides an excellent model to examine the regulation of cell migration and proliferation in vivo. In the unwounded cornea, basal cells across the cornea proliferate at approximately the same rate. 1 The overall proliferative rate in the limbus, however, is lower than that in the cornea, presumably since the limbus contains slow-cycling stem cells along with transient amplifying cells. In response to corneal wounding, these homeostatic rates of proliferation change rapidly. 2 3 4 5 6 7 In the migratory or reepithelialization phase of wound healing, cells adjacent to the wound flatten and migrate as a sheet to cover the wound area. This phase of healing is not dependent on cell proliferation 3 8 9 ; in fact, there appears to be a cessation of cell proliferation at the wound edge. 3 4 7 In the repopulation phase of wound repair, cells distal to the original wound proliferate to allow repopulation of the wound site. 3 4 5 6 7 Wounding of the corneal epithelium also appears to stimulate a proliferative response in the conjunctival epithelium. 6 7 10  
One of the intriguing questions regarding corneal and limbal epithelial cell proliferation is what mechanisms allow these cells to show differential rates of proliferation during homeostasis and wound repair. In initial experiments to examine this question, we surveyed the localization of several cell-cycle–associated proteins in human corneas. 11 In mammalian cells, the cell cycle is driven by a family of protein kinases referred to as cyclin-dependent kinases (CDKs), which are regulated by another family of proteins, termed cyclins. 12 Although the CDKs are present throughout the cell cycle, the synthesis of the cyclins is cell-cycle–dependent. There are four main groups of cyclins, termed D, E, A, and B. Cyclin D synthesis is stimulated by mitogenic signals, including growth factors. It appears early in G1 and is degraded in the S and G2 phases of the cell cycle. Three isoforms of cyclin D (termed 1, 2, and 3) are known. 13 In normal, nontumorous cells, the synthesis of cyclin E is initiated in mid-G1, its maximal expression occurs at the time of entry into S-phase, and its degradation occurs as the cell progresses through S-phase. 14 Accumulation of cyclins D and E is required for cells to progress through a point late in G1 termed the “restriction point.” Once cells pass through the restriction point, they are committed to progress through the remainder of the cell cycle. Thus, most of the regulation of the cell cycle occurs in G1, before the restriction point. 12 13 15 Cyclin A appears in S-phase and is important in DNA synthesis, 16 17 and B appears in G2 and is required for M-phase. 16 17 Thus, the presence or absence of the cyclins can be used as markers of the stages of the cell cycle. Before the initiation of our project on human corneas, we predicted that limbal basal cells were in a resting state, G0, and therefore would not express any of the cyclins at an elevated level. However, we observed high levels of cyclins E and D in the basal cell layer of the limbus 11 and conjunctiva (unpublished observation). We also found that basal cells in the central corneal epithelium expressed low levels of these cyclins and therefore appeared to be primarily in the G0-phase of the cell cycle. Suprabasal cells in both corneal and conjunctival epithelium appeared to have exited the cell cycle. 
In the current investigation, corneal epithelial debridement wounds in rat corneas were created to examine dynamic changes in cell proliferation. Using the thymidine analog 5-bromo-2-deoxyuridine (BrdU), we compared the kinetics of proliferation in the bulbar conjunctiva, the limbus, the peripheral corneal epithelium, and the leading edge of migrating epithelium. BrdU is stably incorporated into DNA as a substitute for thymidine during the S-phase of the cell cycle. In addition, we examined the localization of cyclins D and E as markers of the G1-phase of the cell cycle in peripheral corneal epithelium and in the leading edge. Most cell cycle studies have examined cells in culture, but few if any studies have examined alterations in cyclin expression and localization in an in vivo wound-healing model. 
Materials and Methods
Animal Model
All investigations described in this study conformed to the ARVO statement for the use of Animals in Ophthalmic and Vision Research. Male Sprague–Dawley rats, weighing 175 to 225 g each, were used in all studies. Rats were anesthetized by intramuscular injection of rodent anesthesia cocktail containing acepromazine, ketamine, and xylazine, with topical application of proparacaine hydrochloride. Three-millimeter central corneal debridement wounds were made in the right eyes of the rats and allowed to heal in vivo. 18 Antibiotic ointment was applied immediately after debridement. To control for the circadian variation in epithelial proliferation, untreated left eyes served as controls. Five rats were killed every 4 hours, from 4 to 48 hours after wounding. Two hours before the rats were killed, 10 mM BrdU (1 ml/100 g body wt; Sigma Chemical Company, St. Louis, MO) in sterile saline was injected subcutaneously (SC) in the dorsal subscapular region. In preliminary experiments, no differences in incorporation were detected when BrdU was injected SC or intraperitoneally. SC injection was chosen because it was less traumatic for the rats. 
Immunofluorescence Microscopy
After rats were euthanatized by an intraperitoneal injection of a lethal dose of sodium pentobarbital, globes were excised and frozen in Tissue Tek II OCT Compound (Laboratory Tek, Naperville, IL). Cryostat sections (6 μm) were placed on gelatin-coated slides, air-dried, rehydrated in phosphate-buffered saline (PBS), and blocked in 1% bovine serum albumin (BSA) for 10 minutes. 11 Slides used for cyclin D detection were fixed for 10 minutes in 4% paraformaldehyde at 4°C before rehydration in PBS. As a primary antibody, either anti-BrdU (Boehringer Mannheim, Indianapolis, IN), anti–cyclin D, or anti–cyclin E (Upstate Biotechnology, Inc., Lake Placid, NY) was placed on the slides and incubated in a moist chamber for 30 minutes at 37°C (anti-BrdU) or 1 hour at room temperature (anti–cyclin D or E). The slides were then rinsed with PBS and incubated in 1% BSA for an additional 10 minutes. Fluorescein-conjugated donkey anti-mouse IgG (anti-BrdU) or fluorescein-conjugated donkey anti-rabbit IgG (anti–cyclins D and E) (Jackson Immuno Research, West Grove, PA) was applied as a secondary antibody for 1 hour in a moist chamber at 37°C (anti-BrdU) or at room temperature (anti–cyclin D or E). After rinsing with PBS, the coverslips were mounted with a medium consisting of PBS, glycerol, and paraphenylene diamine. Negative control tissues prepared by omitting the primary antibody were routinely run with every antibody-binding experiment. Slides were viewed and photographed using a Zeiss Axiophot III microscope (Thornwood, NY). 
Using the Zeiss Axiophot III microscope, two observers (E–HC and JDZ) counted the number of BrdU-labeled epithelial cells in three microscopic vision fields: the bulbar conjunctiva adjacent to the limbus, the limbus, and the peripheral cornea. Each microscopic field corresponded to a linear distance of 280 μm. Four sections per eye were examined. Initially, it was planned to count the number of BrdU-labeled epithelial cells at the leading edge of migrating epithelium; however, the number of labeled cells dropped to zero after wounding. 
Definition of Conjunctival, Limbal, and Corneal Epithelial Zones
Limbal epithelium is easily distinguished from corneal epithelium by its subjacent blood vessels and loose stroma. Although the limbo-conjunctival transition can best be distinguished morphologically by the presence of goblet cells and biochemically by the presence of specific keratins, it is difficult to define the limbo-conjunctival junction with accuracy. As a counting unit, we used a microscopic vision field (280 μm), which included the whole limbal zone. The adjacent microscopic vision field was used as the counting unit of bulbar conjunctival epithelium. The epithelium, in one microscopic vision field centripetal to the limbo-corneal junction, was defined as peripheral cornea. The number of BrdU-labeled cells was analyzed statistically using a paired t-test, and P < 0.01 was considered significant. 
Electrophoresis and Immunoblotting
Western blot analyses were used to quantify cyclins D and E after debridement. A 3-mm trephine was used to demarcate the corneal epithelium, and then the epithelium inside the demarcation was scraped and collected as a control. The eyes were allowed to heal, and the rats were killed 4 to 48 hours after wounding. Limbus-to-limbus epithelial samples were harvested using a small scalpel and were solubilized in 1% sodium dodecyl sulfate containing phenylmethylsulfonyl fluoride (100 μg/ml) and aprotinin (63 μg/ml). Epithelium from four wounded corneas was pooled for each time point. Protein amounts were determined using the Pierce (Rockford, IL) bicinchoninic acid (BCA) protein assay. Equal amounts of total protein were loaded for each time point, electrophoresed on a 10% tris-glycine gel (Novex, San Diego, CA), and then electrophoretically transferred to nitrocellulose membrane (Bio-Rad Laboratory, Richmond, CA). The paper was placed in Blotto (5% dry milk in 150 mM NaCl; 10 mM Tris; 0.5% Tween-20, pH 7.5; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature to block nonspecific binding and then was incubated in anti–cyclin E diluted 1:500 or anti–cyclin D diluted 1:300 in Blotto for 1 hour. After washing with 0.5% Tween-20 in tris-buffered saline (150 mM NaCl; 10 mM tris, pH 7.5) two times for 7 minutes, the membrane was incubated for 1 hour with peroxidase-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories [KPL], Gaithersburg, MD) diluted 1:50,000 in Blotto. The membrane was soaked in chemiluminescent SuperSignal Substrate (KPL) for exactly 1 minute, exposed to Kodak Biomax MR film (Eastman Kodak, Rochester, NY), and developed using a Fuji x-ray film processor (Fuji Medical Systems, Stamford, CT). Band intensities were quantified using a scanning laser densitometer (model 300A; Molecular Dynamics, Sunnyvale, CA). 
Results
In an initial step to examine the kinetics of cell cycle progression in response to corneal debridement, the number of cells present in S-phase was quantified using BrdU. As seen in Figure 1 , BrdU was localized primarily in the basal cell layer in the peripheral, limbal, and conjunctival epithelia. After a central 3-mm debridement, the number of BrdU-labeled cells increased in all three fields, peaking 28 hours after wounding (Figs. 1 2) . In the peripheral corneal epithelium, a single peak of incorporation was observed 28 hours after wounding (Fig. 2A) indicating that wounding has synchronized the progression of these cells through the cell cycle. In the limbal epithelium, the number of BrdU-labeled cells appeared to increase as early as 12 hours after debridement and showed a small peak at 16 hours, with a second and larger peak at 28 hours (Fig. 2B) . In the conjunctival epithelium, the relative number of S-phase cells, compared to that in the contralateral eye, remained constant until 16 hours after debridement, when a large increase was observed (Fig. 2C) . The level of BrdU-labeled cells decreased until 28 hours, when another large peak was observed. 
In contrast to epithelium distal to the original wound area, the epithelium that migrated to cover the wound area did not show an increase in BrdU-labeling. In fact few, if any, cells at the leading edge incorporated BrdU (Fig. 1G) , indicating that these cells were not progressing through the cell cycle. To confirm this observation, wounded corneas were allowed to heal in organ culture, where BrdU was added to the medium to ensure that cells at the leading edge had access to BrdU. No BrdU-labeled cells were observed at the leading edge (Fig. 1H 1); however, BrdU-labeled cells were present in the peripheral cornea (Fig. 1H 2). These data suggest that different signals are received by cells migrating to cover the wound and by the cells outside the wound area. 
To further examine the G1/S transition in response to wounding, the localization and expression of cyclins D and E were examined. We previously demonstrated in human corneas that cyclins D and E are localized at high levels in the cytoplasm of limbal basal cells and at low levels in the remainder of the corneal epithelium. 11 Similar localizations were observed in the ocular surface of the rat. This intense cytoplasmic binding of anti–cyclins D and E was maintained at all times in the limbus and conjunctiva after corneal debridement, making the documentation of alterations in binding difficult. Therefore, we concentrated our observations on the peripheral corneal epithelium and cells at the leading edge of migrating epithelium. In unwounded corneas, cyclin D was localized in the nuclei of occasional basal cells across the cornea (Fig. 3 A). Low-level cytoplasmic localization also was observed. The number of cells exhibiting nuclear localization in the peripheral cornea appeared to increase as early as 4 hours after wounding, and by 8 hours most of the basal cells exhibited nuclear localization of cyclin D (Fig. 3B) . This nuclear localization was maintained until 32 hours after wounding (Figs. 3C 3D) and then became increasingly cytoplasmic, and by 48 hours no nuclear localization was seen (Fig. 3E) . In contrast, anti–cyclin D showed little if any nuclear binding in cells at the leading edge (Figs. 3F 3G)
To confirm the apparent increase in cyclin D levels, protein amounts were examined using Western blot analysis. As seen in Figure 4 , protein levels of cyclin D increased after wounding. In a representative experiment, cyclin D levels increased 0.5-, 1.7-, 2.1-, 3.9-, 4.5-, and 1.1-fold higher at 4, 8, 16, 24, 32, and 48 hours after wounding, respectively, compared to control. 
As seen in Figure 5 A, high levels of cyclin E were present in the basal cells of the limbal and bulbar conjunctival epithelium, whereas little or no cyclin E was detected in corneal epithelium. As in human ocular surface, cyclin E was present primarily in the cytoplasm. The localization of cyclin E changed dramatically in peripheral cornea after central corneal debridement. As seen in Figure 5B , only occasional cells exhibited high levels of nuclear localization in unwounded peripheral epithelium. At 8 hours after debridement, nuclear localization began to increase, and by 12 hours almost all basal cells in peripheral epithelium showed intense binding of anti–cyclin E (Fig. 5C) . This binding was maintained until 24 hours after debridement (Fig. 5D) , decreased dramatically at 28 hours (Fig. 5E) , and returned to control levels by 36 hours. As seen with anti–cyclin D, little if any binding of anti–cyclin E was seen in epithelial cells at the leading edge (Fig. 5F)
To confirm the alterations in cyclin E levels, Western blot analysis was used to quantify protein levels after epithelial debridement. As seen in Figure 6 , the levels of cyclin E rapidly increased after wounding, reaching a maximum at 24 hours after debridement. In a representative experiment, levels were 2.1-, 2.8-, 8.5-, 12.1-, and 1.5-fold higher at 4, 8, 16, 24, and 48 hours, respectively, after debridement than those of unwounded controls. 
Discussion
In the current investigation, corneal epithelial debridement wounds were used to stimulate dynamic alterations in cell proliferation. Localization of BrdU, cyclin D, and cyclin E allowed an examination of the entry of the cells into the G1-phase of the cell cycle, progression through G1, and the subsequent G1/S transition. Our results indicate the following. 1 Peripheral corneal epithelium is stimulated by corneal debridement to progress synchronously through the cell cycle. 2 Epithelium migrating to cover the wound area does not progress through the cell cycle, suggesting that the cell cycle is inhibited in these cells to promote a migratory phenotype. 3 The synthesis and subsequent degradation of cyclins D and E in our in vivo wound model suggests that cells in the peripheral corneal epithelium respond to wounding in a similar manner as serum-starved cultured cells 13 14 15 stimulated to enter the cell cycle. 4 A portion of the conjunctival and limbal epithelial cells respond more rapidly to wounding than does peripheral epithelium. 5 The localization pattern of cyclin E is virtually identical in human and rat corneas. 
Our studies indicate that the peripheral corneal epithelial cells respond similarly to corneal debridement as do cells in culture stimulated by serum or growth factors. By 4 hours after wounding, cyclin D protein levels began to increase, indicating the cells have initiated their progression through G1. By 8 hours after wounding, most basal cells in the peripheral epithelium express nuclear cyclin D, and by 12 hours, most express cyclin E, indicating that they are progressing through the cell cycle in a synchronous manner. The levels of cyclin E increase until 24 hours after wounding and then begin to drop at 28 hours. This coincides with the entry of the peripheral cells into S-phase, as indicated by BrdU incorporation. Nuclear cyclin D was observed until 32 hours after wounding, after which it became primarily cytoplasmic. We were not able to make similar observations of nuclear cyclins D and E in the limbus and conjunctiva, because high levels of cytoplasmic cyclin D and E overwhelmed the nuclear localization. 
In contrast to epithelial cells outside the wound area, those migrating into the wound area do not incorporate BrdU or express nuclear cyclin D or cyclin E. Indeed, none of these markers were observed up to 48 hours after wounding. This confirms classical studies that cell proliferation decreases at the wound edge. 3 4 5 Our studies indicate that cells migrating into the wound area are receiving different signals than are cells outside the wound area. This can be best observed in cyclin D localization (Fig. 3G) —where cells at the leading edge do not exhibit nuclear localization—whereas cells away from the leading edge do. These data suggest that cells at the leading edge are not stimulated to enter the cell cycle or are blocked from progressing through it. This differential signaling may allow the creation of migratory and proliferative phenotypes. 
In our initial studies of human corneas, we observed that limbal basal cells expressed high levels of cytoplasmic cyclins D and E. 11 In the present study, we observed a similar localization of cyclins D and E in the rat limbal epithelium as well as in the bulbar conjunctiva. Other studies, in which transformed cell lines were used, have shown that overproduction of either cyclins D or E significantly shortens the cell cycle and that the cells enter S-phase more rapidly than do normal cells. 19 20 We hypothesized that a similar response might occur in the ocular surface. That is, after a mitogenic stimulation, limbal and conjunctival epithelia, which express high levels of cyclins D and E, would enter S-phase more rapidly than the corneal epithelial cells, which are in G0 and thus would have to synthesize the cell-cycle–associated proteins necessary to traverse G1 before entering S-phase. The quantitation of BrdU incorporation (Fig. 2) partially supports this hypothesis in that a portion of the limbal and conjunctival epithelial cells reach S-phase 16 hours after debridement, 12 hours before the peripheral epithelium. In the examination of BrdU incorporation, the limbal and conjunctival epithelium showed two peaks of incorporation at 16 and 28 hours after debridement. Further studies will be required to determine if these peaks represent two populations of cells with cell cycles of differing lengths or if limbal and conjunctival epithelia are not synchronized, by corneal debridement, to progress through the cell cycle to as great an extent as is peripheral corneal epithelium. 
Conjunctival epithelium, together with the limbal and corneal epithelia, comprise ocular surface epithelium. One of the intriguing aspects of this study is that conjunctival epithelium responded to corneal wounding. This has been reported by Danjo et al. 10 and is supported by a series of experiments performed by Haaskjold et al., 6 7 who observed that there were remarkable similarities between the cellular responses of corneal and conjunctival epithelia to a corneal wound. They suggested that these epithelia should be considered a functional unit and that conjunctival epithelium plays a role in corneal epithelial wound healing. It is unclear, however, as to the role that conjunctival epithelium plays in corneal wound healing. Potentially, the conjunctival epithelium could help to repopulate the corneal epithelium, as suggested by the findings of Chen and Tseng, 21 who observed that conjunctival epithelium migrated into the cornea when the superficial cells in the limbal epithelium were removed. Alternately, the conjunctival proliferation could be to replenish the goblet cell number, which decreases by up to 50% after corneal wounding. 22 Interestingly, our data (Figs. 1B 3D 5D) also suggest that cells residing in the stroma may show similar cell-cycle kinetics in response to a corneal debridement. Further studies will be required to examine the proliferative response of the stromal keratocytes. 
 
Figure 1.
 
Immunolocalization of BrdU in the peripheral cornea (A, B), limbal region (C, D), bulbar conjunctiva (E, F), and leading edge of migrating epithelium (G, H) in contralateral unwounded eyes (A, C, E), 28 hours after debridement (B, D, F), and 16 hours after debridement (G, H). (G) Leading edge of 16-hour debridement; note lack of BrdU localization. (H 1 ) Leading edge of 16-hour debridement allowed to heal in organ culture. (H 2 ) Peripheral epithelium of 16-hour debridement allowed to heal in organ culture; note localization of BrdU. In (H), 3-mm debridements were made in situ, and corneas were allowed to heal in organ culture as previously described. 9 BrdU (final concentration 100 μM) was added 2 hours before termination of experiment. Arrows indicate BrdU-labeled cells in the stroma. Bar, 50 μm.
Figure 1.
 
Immunolocalization of BrdU in the peripheral cornea (A, B), limbal region (C, D), bulbar conjunctiva (E, F), and leading edge of migrating epithelium (G, H) in contralateral unwounded eyes (A, C, E), 28 hours after debridement (B, D, F), and 16 hours after debridement (G, H). (G) Leading edge of 16-hour debridement; note lack of BrdU localization. (H 1 ) Leading edge of 16-hour debridement allowed to heal in organ culture. (H 2 ) Peripheral epithelium of 16-hour debridement allowed to heal in organ culture; note localization of BrdU. In (H), 3-mm debridements were made in situ, and corneas were allowed to heal in organ culture as previously described. 9 BrdU (final concentration 100 μM) was added 2 hours before termination of experiment. Arrows indicate BrdU-labeled cells in the stroma. Bar, 50 μm.
Figure 2.
 
Quantitation of BrdU-labeled cells in peripheral epithelium (A), limbal epithelium (B), and bulbar conjunctival epithelium (C) after debridement (gray) and in contralateral unwounded eyes (cross-hatched). Values are indicated as the mean ± SEM. *Statistically different from that in contralateral eye; P < 0.01.
Figure 2.
 
Quantitation of BrdU-labeled cells in peripheral epithelium (A), limbal epithelium (B), and bulbar conjunctival epithelium (C) after debridement (gray) and in contralateral unwounded eyes (cross-hatched). Values are indicated as the mean ± SEM. *Statistically different from that in contralateral eye; P < 0.01.
Figure 3.
 
Immunolocalization of cyclin D in peripheral cornea (A, B, C, D, E) and leading edge of migrating epithelium (F, G) of unwounded (A) and wounded corneas 8 hours (B, F, G), 16 hours (C), 32 hours (D), and 48 hours (E) after debridement. At lower magnification (G), the transition of basal cells expressing nuclear cyclin D outside the wound area (bracket) to migratory cells lacking nuclear cyclin D can be seen clearly. Bars, 50 μm.
Figure 3.
 
Immunolocalization of cyclin D in peripheral cornea (A, B, C, D, E) and leading edge of migrating epithelium (F, G) of unwounded (A) and wounded corneas 8 hours (B, F, G), 16 hours (C), 32 hours (D), and 48 hours (E) after debridement. At lower magnification (G), the transition of basal cells expressing nuclear cyclin D outside the wound area (bracket) to migratory cells lacking nuclear cyclin D can be seen clearly. Bars, 50 μm.
Figure 4.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, 32, and 48 hours. The blot was reacted with anti–cyclin D as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Anti-human cyclin D, according to manufacturer’s specifications, recognizes p36 cyclin D1 and cross-reacts with p34 cyclin D2, which has a highly homologous epitope. Arrow indicates band corresponding to known molecular mass of cyclin D1. The relative amount of the lower molecular weight band corresponding to cyclin D2 was not altered during wound repair. Data are representative of three separate experiments.
Figure 4.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, 32, and 48 hours. The blot was reacted with anti–cyclin D as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Anti-human cyclin D, according to manufacturer’s specifications, recognizes p36 cyclin D1 and cross-reacts with p34 cyclin D2, which has a highly homologous epitope. Arrow indicates band corresponding to known molecular mass of cyclin D1. The relative amount of the lower molecular weight band corresponding to cyclin D2 was not altered during wound repair. Data are representative of three separate experiments.
Figure 5.
 
Immunolocalization of cyclin E in unwounded (A, B) and wounded corneas (C, D, E, F). (A) Low magnification micrograph showing the localization of cyclin E in the basal cells of conjunctival and limbal epithelia. conj, conjunctiva; lim, limbus; cor, cornea. (B) Higher magnification of unwounded peripheral corneal epithelium. Peripheral cornea 12 hours (C), 24 hours (D), and 28 hours (E) after debridement. (F) Leading edge of migrating epithelium 20 hours after debridement. Note lack of nuclear localization of cyclin E. Bars, 50μ m.
Figure 5.
 
Immunolocalization of cyclin E in unwounded (A, B) and wounded corneas (C, D, E, F). (A) Low magnification micrograph showing the localization of cyclin E in the basal cells of conjunctival and limbal epithelia. conj, conjunctiva; lim, limbus; cor, cornea. (B) Higher magnification of unwounded peripheral corneal epithelium. Peripheral cornea 12 hours (C), 24 hours (D), and 28 hours (E) after debridement. (F) Leading edge of migrating epithelium 20 hours after debridement. Note lack of nuclear localization of cyclin E. Bars, 50μ m.
Figure 6.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, and 48 hours. The blot was reacted with anti–cyclin E as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Arrow indicates band corresponding to known molecular mass of cyclin E. The bands around 116 kDa and other minor bands also were observed in control blots, where the primary antibody was omitted. Data are representative of three separate experiments.
Figure 6.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, and 48 hours. The blot was reacted with anti–cyclin E as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Arrow indicates band corresponding to known molecular mass of cyclin E. The bands around 116 kDa and other minor bands also were observed in control blots, where the primary antibody was omitted. Data are representative of three separate experiments.
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Figure 1.
 
Immunolocalization of BrdU in the peripheral cornea (A, B), limbal region (C, D), bulbar conjunctiva (E, F), and leading edge of migrating epithelium (G, H) in contralateral unwounded eyes (A, C, E), 28 hours after debridement (B, D, F), and 16 hours after debridement (G, H). (G) Leading edge of 16-hour debridement; note lack of BrdU localization. (H 1 ) Leading edge of 16-hour debridement allowed to heal in organ culture. (H 2 ) Peripheral epithelium of 16-hour debridement allowed to heal in organ culture; note localization of BrdU. In (H), 3-mm debridements were made in situ, and corneas were allowed to heal in organ culture as previously described. 9 BrdU (final concentration 100 μM) was added 2 hours before termination of experiment. Arrows indicate BrdU-labeled cells in the stroma. Bar, 50 μm.
Figure 1.
 
Immunolocalization of BrdU in the peripheral cornea (A, B), limbal region (C, D), bulbar conjunctiva (E, F), and leading edge of migrating epithelium (G, H) in contralateral unwounded eyes (A, C, E), 28 hours after debridement (B, D, F), and 16 hours after debridement (G, H). (G) Leading edge of 16-hour debridement; note lack of BrdU localization. (H 1 ) Leading edge of 16-hour debridement allowed to heal in organ culture. (H 2 ) Peripheral epithelium of 16-hour debridement allowed to heal in organ culture; note localization of BrdU. In (H), 3-mm debridements were made in situ, and corneas were allowed to heal in organ culture as previously described. 9 BrdU (final concentration 100 μM) was added 2 hours before termination of experiment. Arrows indicate BrdU-labeled cells in the stroma. Bar, 50 μm.
Figure 2.
 
Quantitation of BrdU-labeled cells in peripheral epithelium (A), limbal epithelium (B), and bulbar conjunctival epithelium (C) after debridement (gray) and in contralateral unwounded eyes (cross-hatched). Values are indicated as the mean ± SEM. *Statistically different from that in contralateral eye; P < 0.01.
Figure 2.
 
Quantitation of BrdU-labeled cells in peripheral epithelium (A), limbal epithelium (B), and bulbar conjunctival epithelium (C) after debridement (gray) and in contralateral unwounded eyes (cross-hatched). Values are indicated as the mean ± SEM. *Statistically different from that in contralateral eye; P < 0.01.
Figure 3.
 
Immunolocalization of cyclin D in peripheral cornea (A, B, C, D, E) and leading edge of migrating epithelium (F, G) of unwounded (A) and wounded corneas 8 hours (B, F, G), 16 hours (C), 32 hours (D), and 48 hours (E) after debridement. At lower magnification (G), the transition of basal cells expressing nuclear cyclin D outside the wound area (bracket) to migratory cells lacking nuclear cyclin D can be seen clearly. Bars, 50 μm.
Figure 3.
 
Immunolocalization of cyclin D in peripheral cornea (A, B, C, D, E) and leading edge of migrating epithelium (F, G) of unwounded (A) and wounded corneas 8 hours (B, F, G), 16 hours (C), 32 hours (D), and 48 hours (E) after debridement. At lower magnification (G), the transition of basal cells expressing nuclear cyclin D outside the wound area (bracket) to migratory cells lacking nuclear cyclin D can be seen clearly. Bars, 50 μm.
Figure 4.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, 32, and 48 hours. The blot was reacted with anti–cyclin D as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Anti-human cyclin D, according to manufacturer’s specifications, recognizes p36 cyclin D1 and cross-reacts with p34 cyclin D2, which has a highly homologous epitope. Arrow indicates band corresponding to known molecular mass of cyclin D1. The relative amount of the lower molecular weight band corresponding to cyclin D2 was not altered during wound repair. Data are representative of three separate experiments.
Figure 4.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, 32, and 48 hours. The blot was reacted with anti–cyclin D as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Anti-human cyclin D, according to manufacturer’s specifications, recognizes p36 cyclin D1 and cross-reacts with p34 cyclin D2, which has a highly homologous epitope. Arrow indicates band corresponding to known molecular mass of cyclin D1. The relative amount of the lower molecular weight band corresponding to cyclin D2 was not altered during wound repair. Data are representative of three separate experiments.
Figure 5.
 
Immunolocalization of cyclin E in unwounded (A, B) and wounded corneas (C, D, E, F). (A) Low magnification micrograph showing the localization of cyclin E in the basal cells of conjunctival and limbal epithelia. conj, conjunctiva; lim, limbus; cor, cornea. (B) Higher magnification of unwounded peripheral corneal epithelium. Peripheral cornea 12 hours (C), 24 hours (D), and 28 hours (E) after debridement. (F) Leading edge of migrating epithelium 20 hours after debridement. Note lack of nuclear localization of cyclin E. Bars, 50μ m.
Figure 5.
 
Immunolocalization of cyclin E in unwounded (A, B) and wounded corneas (C, D, E, F). (A) Low magnification micrograph showing the localization of cyclin E in the basal cells of conjunctival and limbal epithelia. conj, conjunctiva; lim, limbus; cor, cornea. (B) Higher magnification of unwounded peripheral corneal epithelium. Peripheral cornea 12 hours (C), 24 hours (D), and 28 hours (E) after debridement. (F) Leading edge of migrating epithelium 20 hours after debridement. Note lack of nuclear localization of cyclin E. Bars, 50μ m.
Figure 6.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, and 48 hours. The blot was reacted with anti–cyclin E as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Arrow indicates band corresponding to known molecular mass of cyclin E. The bands around 116 kDa and other minor bands also were observed in control blots, where the primary antibody was omitted. Data are representative of three separate experiments.
Figure 6.
 
Western blot of unwounded corneal epithelium (C) and corneal epithelium from tissues allowed to heal 4, 8, 16, 24, and 48 hours. The blot was reacted with anti–cyclin E as described in the Materials and Methods section. Molecular masses (in kilodaltons) determined from standard proteins are noted. Each lane was loaded with 20 μg of total protein. Arrow indicates band corresponding to known molecular mass of cyclin E. The bands around 116 kDa and other minor bands also were observed in control blots, where the primary antibody was omitted. Data are representative of three separate experiments.
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