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Nucleotides are naturally occurring substances present in tear film that can stimulate tear secretion in animals and humans. We investigated whether certain nucleotides can affect the rate of wound healing in the cornea of white rabbits. In the absence of any added compound, the rate of healing was 72.4 +/- 2.2 microm h(-1). Of all the tested nucleotides, UTP and Ap(4)A were the most active ones, maximally increasing the rate of healing to 121.6 +/- 3.7 and 93.7 +/- 3.2 microm h(-1), respectively. Responses to UTP were dose dependent. UTP had a pD(2) value of 8.9 +/- 0.1 (EC(50): 1.25 nM). P2 purinoceptor antagonists such as suramin and reactive blue-2, inhibited the effect of UTP indicating the involvement of P2Y receptors. Mitogen-activated protein kinase (MAPK) cascade inhibitors also abolished the effects of UTP, suggesting that P2Y receptors are coupled to the MAPK cascade, and that this is involved in controlling the rate of epithelial cell migration.

The extracellular matrix (ECM) plays a crucial role in cell adhesion, differentiation and wound-healing. Its stability is tightly controlled by enzymes that regulate the metabolism of its components (e.g collagen, fibronectin, laminin). We have found that in the cornea, a potent lipid inflammatory mediator platelet-activating factor (PAF) activates the expression of two metalloproteinases (MMP-1 and MMP-9) as well as urokinase-plasminogen activator (uPA). uPA is of particular interest because, as a serine protease, it is at the top of the protease cascade. PAF may contribute to the destruction of the ECM and the formation of epithelial defects and corneal ulcers by activating uPA and then proteases. We also investigated how several PAF antagonists with different binding affinities can block the expression of the uPA gene. Our results suggest that PAF antagonists with affinities for intracellular binding sites and/or specific structures derived from triazolobenzodiazepine could be of therapeutic use to limit the breakdown of the ECM and the development of ulcer formation.

Purpose: To determine whether there is an association between epidermal growth factor (EGF)-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase) and stimulation of wound closure in rabbit corneal epithelial cells., Methods: Immortalized rabbit corneal epithelial cells were cultured in 24-well plates until they became confluent. Circular wounds were created in confluent cultures by cell denudation and then incubated in the absence and presence of EGF for varying intervals. Wound closure was monitored by staining the cells with Giemsa and quantifying the wound area with SigmaS can computer program. Cell proliferation during wound repair was estimated by measuring the incorporation of [3H]thymidine into nuclear DNA. Changes in PI 3-kinase activity were assessed by measuring the production of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] in 32P-labeled cells as well as by immunoprecipitating and assaying PI 3-kinase activity with phosphatidylinositol 4,5-bisphosphate and [gamma-32P]ATP as substrates. The enzyme product, PIP3, was analyzed by a combination of thin-layer and high-pressure liquid chromatography., Results: Addition of 10 ng/ml EGF to the wounded corneal epithelial cells stimulated wound closure in a time-dependent manner, and the wound closed completely within 48 hours. The effect of EGF was dose dependent, and maximal wound closure occurred at 10 ng/ml EGF. As the epithelial cells were undergoing EGF-stimulated wound closure, there was a time-dependent increase in PI 3-kinase activity. The enzyme activity increased maximally at 24 hours and then decreased gradually as the incubation was continued to 48 hours. When the cells were treated with wortmannin, a PI 3-kinase inhibitor, the EGF-stimulated PIP3 formation as well as the wound closure were inhibited significantly. Treatment of the cells with genistein or tyrphostin B42 also decreased both EGF-stimulated PIP3 formation and wound closure in a dose-dependent manner. Concomitant with stimulation of wound repair, the growth factor increased [3H]thymidine incorporation into nuclear DNA, and this effect was inhibited by pretreatment of the cell with wortmannin., Conclusions: The data suggest a close correlation between EGF-stimulated wound closure and activation of PI 3-kinase in corneal epithelial cells. It can be concluded that PI 3-kinase might be an important component in signal transduction cascade initiated by EGF-receptor interaction, which leads to mitosis and cell proliferation during wound closure in corneal epithelial cells.

Purpose: The differential effects of transforming growth factor (TGF) alpha, beta 1 and beta 2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing., Methods: Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGF alpha, TGF beta 1 or TGF beta 2. After 24 or 48 hours in culture, cryostat sections of explants were processed for immunofluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1., Results: A comparison of antibody labeling patterns and relative fluorescence intensity of antibody labeling to controls suggested that TGF alpha inhibits the spatial polarization of proteins into the reforming adhesion complex during early stages of wound healing. Both TGF beta 1 and beta 2 enhanced the linear localization of the three proteins to the site of the reforming adhesion complex. However, in our model TGF beta isoforms did not have identical functions. TGF beta 2 accelerated the temporal localization of collagen type VII to the adhesion complex, an effect which was not observed with TGF beta 1., Conclusions: TGF beta, but not TGF alpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

To test the hypothesis that a mucin ophthalmic solution could accelerate the healing of corneal epithelium, we conducted a study in rabbits using an ophthalmic solution containing mucin extracted from bovine submaxillary gland. The corneas of rabbits were abraded with a razor blade in the central portion over an area demarcated with a 7-mm trephine. Mucin-containing eye drops were instilled into the right eye 4 times a day for 3 days, and the vehicle was instilled into the left eye. After enucleation, the eyes were observed by scanning and transmission electron microscopy as well as light microscopy. Mucin-containing eye drops were shown to reduce the epithelial defect area more rapidly than the vehicle. Thus, the eye drops accelerated corneal epithelial wound healing.

The effect of corneal epithelial wound creation on epidermal growth factor (EGF) concentration in tears was evaluated in order to better understand the effects of EGF on the wound healing process. The tears of New Zealand white rabbits were sampled by micropipette one day prior to wounding, immediately prior to the creation of a 7.5 mm diameter anterior keratectomy wound, immediately following wound creation, and at 1, 2, 3, 7, and 14 days following wounding. A volume of 50 microL was taken at each sampling time, and all tear samples were assayed for EGF by an enzyme linked immunosorbent assay (ELISA). The results demonstrated that the concentration of EGF in the tear layer rises dramatically immediately following wound creation. The basal measured EGF concentration was approximately 600 pg/mL; immediately following wound creation, this rose to approximately 1600 pg/mL. By 1 day following creation of the wound, the concentration of EGF in the tears had returned to the basal level. A second, marginally significant increase in the tear concentration was noted at 3 days post wounding. The EGF concentration in the tears were not significantly different at any other time. The measured dramatic rise in EGF concentration in the tears in response to the creation of a corneal epithelial wound provides further evidence of the importance of tear EGF in the wound healing process. The concentrations in all cases were on the order of ng/mL, suggesting that the intercellular concentrations in this range result in optimal cell stimulation.

Purpose: To establish a method for evaluation of less irritating contact lens materials by dynamic contact with cornea, we examined epithelial and endothelial cell injury to the porcine cornea caused by rotatory rubbing with four kinds of hard contact lenses (HCL)., Methods: The HCLs used were (1) polymethylmethacrylate (PMMA) HCL, (2) gas-permeable HCL composed of a graft co-polymer of dextran derivative and methylmethacrylate (MMA) (Suncon Mild II(TM), 12 Dk), (3) gas-permeable HCL composed of a graft co-polymer of dextran derivative, a monomer containing silicone, a monomer containing fluorine and MMA (New Dx HCL-136, 32 Dk), and (4) gas-permeable HCL composed of a monomer containing silicone, a monomer containing fluorine and MMA(RGPL-A, 216 Dk). Using a specially designed apparatus, we produced a standardized injury to the epithelium or endothelium of porcine corneas by holding the HCL against the corneal surface while rotating the lens rapidly., Results: After rotatory rubbing of the HCL on the epithelium, the degree of rose bengal staining of the epithelial cells, indicating degeneration of the cells and mucin detachment, were significantly different among these HCLs (New Dx HCL-136 < Suncon Mild II(TM), PPMA < RGPL-A), and the cell injury rates of the endothelium were also significantly different among them (Suncon Mild II(TM) < New Dx HCL-136, PMMA < RGPL-A). The water-wettability of HCLs was not directly correlated with cell injury rates on epithelial and endothelial cells., Conclusions: Both the New Dx HCL-136 and Suncon Mild II(TM), which have a common composition of graft co-polymer of dextran derivative, are less irritating to the epithelium and to the endothelium. Also a very few patients complaints regarding Suncon Mild II(TM) wearing in individuals with dry eyes have been reported. Therefore, we would expect that the New Dx HCL-136 should be satisfactory for wear by individuals with dry eyes.

The purpose of this study was to determine the role of programmed cell death (apoptosis) in the disappearance of keratocytes beneath an epithelial debridement wound in the cornea and to investigate a potential role of interleukin-1 (IL-1) in induction of apoptosis in stromal fibroblasts in vitro and keratocytes in vivo. Keratocyte and stromal fibroblast cell morphology was examined in wounded and unwounded mouse corneas using transmission electron microscopy. Nuclear DNA fragmentation was detected with the TUNEL assay for 3'-hydroxyl DNA ends. The effect of IL-1 on keratocytes in vivo was determined by microinjection of IL-1 alpha into the central corneal stroma via a limbal entry site. The in vitro effects of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were determined with primary cultures of human corneal stromal and dermal fibroblasts. Cell shrinkage, blebbing with formation of membrane bound bodies, condensation and fragmentation of the chromatin, and DNA fragmentation, consistent with apoptosis were detected in anterior stromal keratocytes after epithelial scrape wounds. Thus, disappearance of keratocytes from the underlying stroma following epithelial debridement is mediated by apoptosis. Microinjection of IL-1 alpha into the central stroma of the mouse cornea caused a redistribution of keratocytes in the stroma via apoptosis and, possibly, negative chemotaxis. IL-1 alpha and IL-1 beta induced apoptosis in corneal stromal and dermal fibroblasts in vitro. The epithelial/endothelial-stromal IL-1 system may mediate corneal tissue organization and responses to mechanical- and pathogen-induced injury through induction of keratocyte apoptosis. Keratocyte apoptosis is likely an initiating event in wound healing following corneal surgery. We hypothesize that derangement's in this system may have a role in the pathogenesis of keratoconus and other diseases of the cornea.

Background: Post-traumatic recurrent corneal erosion syndrome is a common clinical problem in which a definitive diagnosis is not always possible based solely upon biomicroscopic corneal signs. Indeed, in some cases archetypical symptoms or a preceding history of epithelial trauma cannot always be elucidated., Methods: Four groups of patients underwent corneal analysis using computer videokeratography. Group 1 (n = 11), subjects who presented with simple corneal epithelial trauma approximately three months before the study. Group 2 (n = 11), prospective analysis of subjects presenting with simple corneal epithelial trauma. Group 3 (n = 12), prospective analysis subjects with an established diagnosis of post-traumatic recurrent erosion syndrome. Group 4 (n = 135), prospective analysis of healthy control subjects., Results: Small, well delineated areas (1.0 to 1.5 mm in diameter) of markedly reduced corneal dioptric power (greater than 2 dioptres), which we have termed 'corneal topographic lagoons', were identified more commonly in eyes with recurrent corneal erosion syndrome and in eyes that had recently sustained corneal epithelial trauma; being found in 18.2% of Group 1, 9.1% of Group 2, 58.3% of Group 3 but only 1.5% of Group 4 (control)., Conclusions: The identification of areas of focal abnormality in the corneas of patients with post-traumatic recurrent corneal erosion syndrome remains a significant clinical problem. Corneal topographic analysis has demonstrated a focal abnormality, the 'corneal topographic lagoon', which is much more prevalent in cases of recent corneal trauma and recurrent corneal erosion syndrome than in the 'normal' population. Computerised videokeratography therefore adds a new tool to the armamentarium of the clinician seeking to identify and locate abnormalities in these difficult cases.

The immunolocalization of 70kD stress-protein (SP 70) was investigated in white rabbit corneas with alkali burns. Corneal alkali burn injuries were induced with 6 mm circular filter paper discs soaked in 1N NaOH. Corneas of 1, 3, 5, and 7 days after alkali injury were immunostained with mouse monoclonal anti-SP 70 antibody. In the control rabbit cornea, immunostaining for SP 70 was observed in the nuclei of the basal cells and of the wing cells. On day 1 after alkali injury, total epithelial defect was observed. Immunostaining for SP 70 was not seen in corneas with total epithelial defect. Three days after injury, epithelial resurfacing with corneal edema was observed. Basal cells showed immunostaining for SP 70 in their nuclei and cytoplasm. Five days after injury, irregular basal cells and a few wing cells with cytoplasmic processes were recognized. Both cells showed diffuse cytoplasmic and nuclear immunostaining for SP 70. Seven days after injury, the corneal wound was healed, and SP 70 was observed in the nuclei of the epithelial cells again. It appears that SP 70 has a role in corneal epithelial healing after alkali burn injury.

We performed quantitative analyses of glycosaminoglycans in the tear fluids in a rabbit wound healing model. We ablated rabbit corneal epithelium with trephine and spatula, and sampled tear fluids before the epithelial ablation, and at 3, 24, 48, and 72 hours after it. After an instillation of 200 microliters saline solution in the conjunctival sac, as much tear fluid as possible was collected from the lower cul-del-sac. The glycosaminoglycans in the tears were then treated with chondoroitinase ABC to make fractions of disaccharides. The quantities of disaccharides were determined with high-performance liquid chromatography as weight per unit protein in the tears. The concentrations of delta Di-HA in the tear fluids at 3 and 24 hours were significantly higher than those before the treatment and returned to the initial value at 72 hours after making the epithelial wound. Among the disaccharides of chondroitin sulfate, delta Di-0S and delta Di-6S showed a significant increase at 3 hours after the treatment but delta Di-4S did not show any significant variation. The results suggest that the glycosaminoglycans in the rabbit tear fluids may play an important role in the corneal epithelial wound healing process.

Purpose: The polymerase chain reaction was used to examine fibronectin (FN) expression during corneal scrape wounding with specific attention to the presence, absence, or gross changes of alternatively spliced FN as differentially expressed in the corneal stroma versus the epithelium in normal and wounded tissue., Methods: Specific FN cDNA sequences were synthesized from rat cornea with total RNA and were amplified using various sets of synthetic oligonucleotide primers., Results: The authors observed the presence and sustained the expression of total FN, EIIIA, EIIIB, and V-region FN mRNA in normal and injured corneal stroma for up to 3 weeks after scrape wounding. In contrast, complementary overlying epithelial samples were virtually devoid of FN message., Conclusions: These data suggest that functionally different, alternatively spliced FN isoforms may be involved both in the maintenance of the normal cornea and in wound healing, and that their synthesis occurs in situ principally by the stroma rather than by the epithelium.

Two patients were splashed in the eye accidentally with an aliquot of Preeglox-L, a herbicide containing paraquat, diquat and surfactants. Despite immediate flushing of the eye with copious amounts of water, in both cases the corneal epithelium deteriorated one to two weeks after the incident. Thereafter, the lesions in each eye healed gradually.

To study the efficacy, safety and comfort of porcine collagen shields in the treatment of traumatic corneal abrasion, patients with corneal abrasions that had occurred within 24 hours before examination were treated with either a collagen shield (18 patients) or tight patching (12 patients). There was no significant difference in the rate of healing or the proportion of corneas healed between the two groups. The collagen shield was significantly more comfortable than the patch (p < 0.05). After 1 hour of wear the collagen shield allowed useful vision (mean 20/100) in nine healthy volunteer subjects. Collagen shields may be of benefit in carefully selected cases of traumatic corneal abrasion.

The possibility of epithelial injury of BALB/C mice cornea exposed to a cathode ray tube (CRT) of a commercially available television was investigated. Epithelial injury of the cornea was confirmed by fluorescein-positive staining after 24 hours of CRT exposure approximately 3-9 cm from the surface of the CRT. The corneal epithelial injury was shown to be mainly limited to the most superficial layer by transmission electron microscope finding showing slight deterioration of the basal cell layer. The corneal epithelial defect was further measured by a scanning electron microscope in relation to the time of exposure to the CRT. The impaired area increased in dose-dependent manner to the some extent. The epithelial defect increased at 5 hours of CRT exposure and reached a maximum, that is 46.6% of the entire area of the cornea, by 12 hours of exposure, thereafter maintaining an equilibrium. The mechanism of corneal epithelial injury by CRT was discussed.

A randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted in five centers to assess safety, ocular tolerance, and efficacy of an ophthalmic solution of epidermal growth factor (EGF) for the treatment of traumatic corneal epithelial defects. One hundred four patients completed the study: 47 received EGF and 57 placebo (the drug vehicle). Mean epithelial healing time was significantly enhanced in the EGF-treated group (44.17 h) compared with the placebo-treated treated group (61.05 h) (p < 0.01). The number of epithelial defects completely healed at 24, 48, and 72 h after the onset of treatment was significantly greater in the EGF-treated group. Local tolerance was adequate in both groups. These results indicated that topical EGF is well tolerated and may be a significant addition to the ophthalmologist's armamentarium for treating corneal epithelial defects.

A 20-year-old female developed periodically recurrent corneal erosion in a 10mm diameter area of the central cornea. Since the episodes of corneal erosion occurred suddenly in the early morning every 6-7 days and since medical treatments could not prevent recurrence, we suspected that the corneal erosion was due to self-inflicted injury such as mechanical damage and/or topical application of toxic drugs. Psychological tests also indicated that the patient had a tendency towards hysteria. The corneal erosion has not recurred since we completely protected both her eyes at night. These findings strongly suggested that the cause of this corneal erosion was due to self-inflicted injury.

Plasminogen activator (uPA) activities were determined in the tears of rabbits following mechanical (scraping) or chemical (n-heptanol) debridement and alkali burn of the central part of the corneal epithelium. All three types of injury enhanced the plasminogen activator activities in the tears. The increase in uPA activity was highest in alkali burn, lowest for n-heptanol debridement. Scraping yielded an intermediate increase in uPA activity. The maximum value in activator activity was reached at 5 hours for mechanical injury and at 24 hours for chemical injuries. uPA activity values returned to the normal range by the time of re-epithelialization for mechanical scraping and 1-3 days following re-epithelialization for heptanol debridement and alkali burn. A trend was observed between uPA activity level and the size of the wound but the correlation was not pronounced (R = 0.538).

The investigations were carried out on 3 groups of rabbits in which the corneal epithelium was removed and into the conjunctival sac were instilled drugs inhibiting the activity of prostaglandins such as indomethacin solution or a 0.5 p.c. solution of ibuprofen. It was demonstrated that these drugs lead in the period of the first 8 hours to reduction of the number of leucocytes in tears from 2 to 20 times in comparison with the control group. The presented observation show that prostaglandin inhibitors may be a successful mean leading to decrease of number of leucocytes in tears after corneal injury.

The records of 59 patients (66 eyes) with contact thermal burns of the cornea (unilateral in 52 cases and bilateral in 7) were reviewed to determine the causes, clinical presentation, management and complications of such injuries. Most of the injuries (90%) occurred at home. The commonest cause was splashing of boiling fluids (42% of cases), followed by contact with red-hot firecracker particles (18%) and lit match heads (17%). In 59 eyes (89%) the burn was limited to the corneal epithelium, in 5 (8%) the superficial stroma was also involved, and in 2 (3%) there was associated corneal perforation caused by mechanical impact of foreign bodies. Débridement of the coagulated epithelium with patching was the most successful treatment, healing 48 (73%) of the eyes. Postburn complications were rare, the most common being development of inferior symblepharon, in three eyes (4%).

aFGF expression was studied in normal and regenerating cornea of adult rats. aFGF mRNA and proteins were expressed mainly in corneal epithelium but not in stroma. After burning of the epithelium by iodine vapours, the intact epithelial cells migrated to cover the wounded area during the first 4 days and then divided to reconstitute a normal multilayered epithelium 6 days after injury. aFGF mRNA localized by in situ hybridization on regenerating epithelium showed a peak between 6 hr and 2 days after denudation, decreasing to basal levels 6 days later. This induction of aFGF mRNA preceded the increased amount of aFGF peptides, as assessed by indirect immunofluorescence staining. Thus aFGF overexpression is clearly correlated with active migration in epithelial wound healing.

Previous in vivo studies evaluating the effects of growth factors on epithelial regeneration have used the scrape injury model in rabbit eyes. Since growth factors act principally on the mitotic activity of regenerating cells, the rapid wound closure rates following scrape injury may not adequately access the effects of these agents on epithelial repair. In this study, we evaluated the rates of wound healing following scrape (8.6 mm) and lamellar keratectomy (8.6 mm) injury in 25 albino rabbits. Eyes were left untreated or received daily application (two to three times) of (a) Tears Naturale II, (b) 50 mM Tris/NaCl, and (c) commercial vehicle for EGF. Eyes were evaluated daily by fluorescein staining with Ophthalmic Fluoro-Strips followed by clinical photography. The area of staining was quantitated by computer-assisted planimetry and rates calculated by linear regression analysis. Eyes receiving scrape injuries epithelialized by 3 days following surgery. Rates of wound closure in two separate groups (six eyes each) were 25.83 mm2/day (r = 0.96) and 29.56 mm2/day 9r = 0.97). Lamellar keratectomy injuries epithelialized by 7 to 8 days, which is substantially longer than that observed for scrape injuries. Rates of wound healing in two separate untreated groups (8 and 10 eyes) were 10.88 mm2/day (r = 0.95) and 9.00 mm2/day (r = 0.93), respectively, which were not significantly different. Analysis of variance comparing rates of wound closure indicated that lamellar keratectomy injuries heal at a significantly slower rate when compared to scrape injury (p less than 0.0005).(ABSTRACT TRUNCATED AT 250 WORDS)

The corneal epithelium of Triturus cristatus was studied by light and electron microscopy. It consists of three cellular layers; the superficial cells, which show numerous short microvilli, are held together by typical junctional complexes. The basal cells adhere to the basal lamina by hemidesmosomes. Furthermore all cells form with the neighboring ones interdigitations and establish with them desmosomal contacts. Secretion granules and vesicles are present throughout the epithelium, mainly in the upper layer; they are filled with an electron dense material, containing glycoproteins as suggested by the histochemical analysis. Mitoses are very frequent in the basal layer. Occasionally exfoliating cells showing signs of degeneration were observed in the superficial layer. The morphological changes occurring during epithelization of wound linearly incised, were studied by light microscopy at several time intervals and the detachement and migration of the cells was particularly investigated. Very early after wounding (5') necrotic cells and cell debris are present at the wound edges. Soon after, thin protrusions originate from surrounding undamaged cells and push through the cellular fragments sliding on the underlining connective tissue. After 2 h, the wound is completely closed by such flattened protrusions. The newly formed epithelium becomes gradually thicker (3-7 h) and the mitotic activity till now suppressed, is resumed (24 h). Studies are in progress on the modifications of the junctional complexes and on the enzymatic mechanism possibly involved in cell mobilization.