Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells

PURPOSE: Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells. METHODS: Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP. RESULTS: In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor-sensitive manner. CONCLUSIONS: HB-EGF released on wounding acts as an autocrine-paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.     

Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats

PURPOSE. The purpose of the study was to investigate the effects of hyperglycemia on EGFR (epidermal growth factor receptor)-mediated wound response and signal transduction in the corneal epithelium of rats with type I diabetes mellitus (DM). METHODS. Corneal epithelia were removed from streptozotocin (STZ)- and weight-matched normal rats. Wound healing was monitored by fluorescein staining at 24 or 48 hours after epithelial debridement. Phosphorylation of EGFR, AKT, ERK, and BAD was determined by Western blot analysis. The distribution of phospho-AKT and proliferating cell nuclear antigen (PCNA) in rat corneas was examined by immunohistochemistry. Cell death was evaluated by TUNEL staining. RESULTS. A significant delay in corneal epithelial wound healing was observed 48 hours after wounding in the diabetic rats compared with the weight-matched control rats. In the DM rat corneas, epithelial cells demonstrated diminished responses to wounding, as assessed by the phosphorylation of EGFR and its downstream signaling molecules, AKT and ERK. Furthermore, although the distribution pattern of phospho-AKT suggested a role for AKT in epithelial migration and proliferation in the normoglycemic rat corneas, it was abrogated in the healing epithelia of the DM rats. Consistent with impaired AKT activity, the number of PCNA-stained cells was also greatly reduced in the healing corneas of the diabetic rats. Finally, decreases in pBAD (Ser(136) and Ser(112)) and increases in TUNEL-positive cells were observed in both the uninjured and healing corneal epithelia of the DM rats, but not of the control rats. CONCLUSIONS. In the corneas of SZT rats, EGFR-PI3K-AKT and ERK, as well as their downstream BAD signaling pathways in migratory epithelium, were altered, resulting in increased apoptosis, decreased cell proliferation, and delayed wound closure.     

Lysophosphatidic acid promoting corneal epithelial wound healing by transactivation of epidermal growth factor receptor

PURPOSE: To identify the underlying mechanisms by which lipid mediator lysophosphatidic acid (LPA) acts as a growth factor in stimulating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) during corneal epithelial wound healing. METHODS: Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence or absence of an epidermal growth factor receptor (EGFR) inhibitor (tyrphostin AG1478), a matrix metalloproteinase inhibitor (GM6001), or a heparin-binding EGF-like growth factor (HB-EGF) antagonist (CRM197) with or without LPA. EGFR activation was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibodies. Phosphorylation of ERK and AKT (a major substrate of PI3K) was analyzed by Western blotting with antibodies specific to the phosphorylated proteins. Wound- and LPA-induced shedding of HB-EGF was assessed by measuring the release of alkaline phosphatase (AP) in a stable THCE cell line that expressed HB-EGF with AP inserted in the heparin-binding site. RESULTS: In organ and cell culture models, LPA enhanced corneal epithelial wound healing. LPA-stimulated and spontaneous wound closure was attenuated by AG1478, GM6001, or CRM197. Consistent with the effects on epithelial migration, these inhibitors, as well as the Src kinase inhibitor (PP2), retarded LPA-induced activation of EGFR and its downstream effectors ERK and AKT in THCE cells. Unlike exogenously added HB-EGF, LPA stimulated moderate EGFR phosphorylation; the level of phosphorylated EGFR was similar to that induced by wounding. However, LPA appeared to prolong wound-induced EGFR signaling. The release of HB-EGF assessed by AP activity increased significantly in response to wounding, LPA, or both, and the release of HB-EGF-AP induced by LPA was inhibited by PP2 and GM6001. CONCLUSIONS: LPA accelerates corneal epithelial wound healing through its ability to induce autocrine HB-EGF signaling. Transactivation of EGFR by LPA represents a convergent signaling pathway accessible to stimuli such as growth factors and ligands of G-protein-coupled receptors in response to pathophysiological challenge in human corneal epithelial cells.     

Role of fibronectin and fibrinogen in healing of corneal epithelial scrape wounds

A provisional fibronectin (Fn)-fibrinogen (Fg) matrix forms de novo on the bare basement membrane (BM) surface of superficial epithelial scrape wounds in rabbit and guinea pig (GP) corneas. We determined whether such a substrate is essential for epithelial cell adhesion and migration, using three different approaches. (1) Polyclonal and monoclonal IgG reactive against GP Fn were topically administered to inhibit Fn formation in a GP epithelial scrape wound model. Immunofluorescence studies showed that deposition of both Fn and Fg was inhibited in antibody-treated corneas. During the first 38 hr after wounding, the healing rates were 0.46 +/- 0.06 mm2/hr in control eyes, 0.43 +/- 0.05 mm2/hr in those treated with rabbit polyclonal IgG anti-GP Fn and 0.45 +/- 0.09 mm2/hr in those treated with murine monoclonal IgG anti-GP Fn (P greater than 0.4). (2) In a rabbit epithelial scrape wound model, ancrod was administered intravenously to induce systemic Fg depletion. Fg deposition was completely inhibited on the wound surface, but Fn deposition was not suppressed. The healing rate was 1.24 +/- 0.41 mm2/hr in ancrod-treated corneal wounds and 1.19 +/- 0.29 mm2/hr in control eyes during the first 48 hr after wounding (P greater than 0.5). These data from the antibody and ancrod inhibition indicate that Fg binds to Fn and that Fn binds to components other than Fg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corneal endothelial healing rate and the effect of topical retinoic acid

These studies were undertaken to evaluate wound healing rates of the corneal endothelium in vivo. After insertion of a 26-gauge needle into the anterior chamber of the rabbit eye through the limbus, a 5-0 nylon monofilament was introduced through the needle, and endothelial wounds were made by scratching the cells with the filament. The wounds were photographed with a wide-field specular microscope at various intervals. Montages of the wounds were made, and the areas of the wounds were determined by planimetry. Wound closure occurred rapidly in a linear manner during the first 6 hr after wounding, after which the rate of cell migration decreased. Healing rates (micron2/hr) during the first 6 hr were calculated by linear regression analysis. There was a direct linear correlation between the healing rate and initial wound area. The slope of this line for nine normal (untreated) corneas was 0.093 hr-1. Nine corneas were treated with 0.1% retinoic acid in petrolatum ointment, while eight control corneas received vehicle alone. The slope of healing rate versus initial wound area for treated corneas (0.11 hr-1) was significantly greater than control (0.097 hr-1). This was interpreted as a stimulation of corneal endothelial migration during healing by retinoic acid. As a result of this study, a method for analysis of corneal endothelial healing rate has been developed which can be used for comparison of healing rates among treatments when initial wound area cannot be standardized.     

Role of fibronectin in the healing of superficial keratectomies in vitro

A fibronectin (Fn)-fibrinogen (Fg) surface matrix is not essential for epithelial cell migration in a corneal epithelial scrape wound model, in which the basement membrane is preserved. We have therefore tested whether such a provisional scaffolding becomes more critical in a superficial keratectomy model, when the basement membrane is surgically removed. Exogeneous Fn at 0.3 mg/ml was added to the medium of organ cultures of rabbit superficial keratectomies. At 48 hr after wounding, the healing rate was 1.12 +/- 0.03 mm2/hr in control corneas and 1.11 +/- 0.03 mm2/hr in those cultured with Fn. At 64 hr after wounding, the healing rates were also not significantly different (P greater than 0.5). Immunofluorescence studies showed that Fn was not detectable on the surface of control corneas but could be depicted under the migrating epithelium. In corneas cultured with Fn, it diffused throughout the entire stroma but did not deposit as a surface matrix. We therefore attempted to obtain formation of a provisional Fn-Fg surface matrix before establishment of the in vitro organ culture by leaving the superficial keratectomies in vivo for 8 hr, 24 hr or 64 hr. At 64 hr after wounding, their healing rate was 0.80 +/- 0.04 mm2/hr, 0.86 +/- 0.04 mm2/hr and 0.85 +/- 0.06 mm2/hr, respectively, which were not significantly different from that of contralateral ex vivo-wounded cultured corneas (0.83 +/- 0.04 mm2/hr, P greater than 0.5). Immunofluorescence studies revealed a Fn matrix on the bare surface of in vivo-wounded specimen, which was not detectable on ex vivo-wounded cultured corneas; there was also no diffuse stromal Fn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corneal epithelial wound healing in partial limbal deficiency.

Previous studies have shown that the corneal epithelial stem cells are located at the limbal basal layer. The limbal stem cells are regarded as the ultimate source for corneal epithelial cell proliferation and differentiation. This paper examines epithelial wound healing in rabbit corneas with partial limbal deficiency (PLD), which was created by the surgical removal of two-thirds of the limbal zone (superior and inferior). Four to eight months after PLD creation, all corneas appeared normal, without vascularization. The residual stem cell capacity then was challenged by two sizes of corneal epithelial debridement created with combined n-heptanol and mechanical scraping. In the first group, two consecutive 6-mm defects were created 1 month apart. After the first wounding, three of eight PLD corneas had delayed wound healing and two of the three had vascularization, as compared to controls (n = 7). After the second wounding, both controls (n = 7) and the remaining PLD (n = 5) corneas showed similar rapid healing. In the second group, a large defect of up to 1 mm within the limbus was created. Healing was completed in 25-40 days in PLD (n = 6) corneas, a more marked delay compared to the 10-12 days for controls (n = 6) (P = 0.001). In addition, all PLD corneas showed increased vascularization and had epithelium of the conjunctival phenotype, verified by the immunofluorescent staining positive to AM-3 monoclonal antibody but negative to AE-5 monoclonal antibody. Thus, a deficiency of limbal stem cells contributes to the triad of conjunctival epithelial ingrowth, corneal vascularization, and delayed healing with recurrent erosion. In PLD, corneal epithelium is still compromised, particularly when a large epithelial cell mass is removed.     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

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

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

Polymorphonuclear neutrophils delay corneal epithelial wound healing in vitro

Using an in vitro system for measuring epithelial wound healing, we studied the effect of polymorphonuclear neutrophils (PMNs) and PMN lysate on rat corneal epithelial wound healing. After 22 hr of organ culture, epithelial defects that were originally 3 mm in diameter (7.06 mm2) measured 0.41 mm2 (range, 0.17-0.72 mm2) in control rat corneas, 2.11 mm2 (range, 0.81-3.91 mm2) in corneas incubated in medium containing 5 X 10(6) PMN/ml, and 2.49 mm2 (range, 2.21-2.76 mm2) in corneas incubated with lysate obtained from an equivalent number of PMNs. Scanning and transmission electron microscopy showed similar morphology in the three groups. PMNs selectively adhered to the leading edge of the wound in corneas incubated with PMNs. These data indicate that PMNs and PMN lysate significantly slow corneal epithelial wound healing in vitro.     

Flow cytometry measurements of the DNA content of corneal epithelial cells during wound healing

This study presents the first application of flow cytometry (FCM) techniques to the assessment of cell cycle dynamics in the corneal epithelium after experimental wounding. Anterior keratectomies 6 mm in diameter were created in the central corneas of albino rabbits. The authors sampled the epithelial tissue obtained outside the wound at 12-hr intervals until wound closure at 72 hr. Regenerated epithelium from the surface of the wounded area was collected at 78 hr. The percentages of nuclei in the G0/G1 (growth), S (DNA synthesis), and G2/M (tetraploid/mitosis) phases were determined by FCM. An increase in the percentage of nuclei in the G2/M phase at 36 hr was seen, compared with cell populations in samples from unwounded control corneas. The authors found an increase in mitotic activity in the corneal epithelium during the period of cell migration before wound closure.     

Distribution of fibronectin in human and rabbit corneas

In order to study the possible role of fibronectin (FN) in corneal wound healing and the relationship between FN and sensory innervation, FN was demonstrated immunohistochemically in both normal and sensorily denervated rabbit corneas and in normal or tissue-cultured human corneas. The distribution of FN was the same in the groups examined: a thin subepithelial band of FN-like immunoreactivity was seen at the level of epithelial basement membrane and at the stromal side of Descemet's membrane. Epithelial abrasions were also performed in both normal and denervated rabbit corneas. The results were compared with those obtained from organ-cultured human corneas. Following abrasion of the corneal epithelium, FN was detected in the anterior margin of the denuded stroma 18 hr after the operation in the areas where the epithelium had not healed, but not 49 hr after. Sensory denervation did not affect the distribution of FN in normal, denervated or healing rabbit cornea. It is concluded that FN is probably not controlled by sensory innervation.     

Effects of substance P and IGF-1 in corneal epithelial barrier function and wound healing in a rat model of neurotrophic keratopathy

PURPOSE: To establish a rat model of neurotrophic keratopathy and to examine the effects of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and wound healing in this model. METHODS: Corneal denervation was achieved by thermocoagulation of the ophthalmic branch of the trigeminal nerve. A modified Schirmer test was performed without topical anesthesia. Corneal epithelial barrier function was assessed by measurement of fluorescein permeability with an anterior fluorophotometer. Epithelial wound healing was evaluated by measurement of the area of the defect at various times after removal of the entire epithelium. Eye drops containing both 1 mM SP and IGF-1 (1 micro g/mL) were administered six times daily. RESULTS: The Schirmer test result in eyes subjected to trigeminal denervation was lower than that in control eyes. The fluorescein permeability of the corneal epithelium of denervated eyes was increased relative to that of control eyes. Furthermore, trigeminal denervation induced a delay in corneal epithelial wound healing. Application of eye drops containing SP and IGF-1 to denervated corneas restored the fluorescein permeability of the corneal epithelium to control levels and abolished the delay in epithelial wound healing. CONCLUSIONS: A rat model of neurotrophic keratopathy, characterized by reduced tear secretion, loss of corneal sensation, impaired epithelial barrier function, and delayed epithelial wound healing, was established by trigeminal denervation. Treatment with both SP and IGF-1 improved corneal epithelial barrier function and stimulated corneal epithelial wound healing in this model.     

A new in vitro corneal preparation to study epithelial wound healing.

Corneal epithelial wound healing is an important process necessary for maintenance of visual integrity. Corneal epithelial wound healing occurs by cellular migration and proliferation. However, the molecular basis of reepithelialization is not known. To investigate individual molecular contributions to the wound healing process, an in vitro corneal preparation comparable to the in vivo condition is needed. This investigation developed a new whole mount in vitro rabbit cornea preparation and studied epithelial wound healing rates for epithelial and subepithelial wounds. The wound closure rates obtained in this study for epithelial and subepithelial wound healing (52 +/- 14 microns/hr and 38 +/- 7 microns/hr, respectively) are comparable to in vivo rates of wound healing determined by other laboratories for rabbits. This preparation, achieved by functionally separating the epithelial and endothelial sides of the cornea, allows application of agents to the cornea in a manner that approximates the in vivo condition. This in vitro system is promising for future studies designed to investigate corneal wound healing while reducing potential ocular discomfort associated with in vivo corneal wounding.     

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis,mitosis, and myofibroblast transformation

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.     

Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy.

PURPOSE: To investigate the effects of mitomycin C (MMC) on rabbit cornea wound healing after photorefractive keratectomy (PRK). MATERIALS AND METHODS: Rabbit corneas were stained with dichlorotriazinyl aminofluorescein immediately after PRK. MMC was applied to the right eye and phosphate-buffered salt solution (PBS) to the left. Corneal epithelial wound healing rate and corneal haze were examined. Ultrasound pachymetry was performed. Stromal collagen regeneration was evaluated by fluorescent microscopy. We used terminal deoxyribonucleotidyl transferase-mediated D-uridine 5'-triphosphated-digoxigenin nick-end labeling (TUNEL) assay and transmission electron microscopy (TEM) to evaluate keratocyte apoptosis. RESULTS: In eyes treated with MMC, there was no delay to the healing rate of corneal epithelial wound, and less haze 4 weeks after PRK. Ultrasound pachymetry showed thinner corneal thickness in MMC-treated eyes at week 4. Corneal stromal thickness regression was less in MMC-treated eyes observed by fluorescent microscope at week 4. Keratocyte apoptosis was noted in both MMC- and PBS-treated eyes by TUNEL assay and TEM observation. This study discovered the phenomenon that MMC prolongs keratocyte apoptosis. CONCLUSIONS: Applying MMC after PRK is an effective method to decrease haze formation and corneal stromal thickness regression in rabbit corneas. The effect may be related to MMC prolonging keratocyte apoptosis.     

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.