Optimal Concentration of hEGF for Epithelial Healing in Experimental Corneal Alkali Wounds

PURPOSE: An optimal concentration of hEGF to enhance epithelial healing was determined in the rabbit eyes after alkali wounds. METHODS: Alkali-injured corneas (=5.5 mm, 1N NaOH, 60s) were treated with 0 (control), 2, 5, 10, and 50microgram/ml hEGF 3 times per day. The measurement of epithelial healing rate, transmission electron microscopy and immunohistochemical observation were performed after 7 days treatment. RESULTS: No significant difference was observed in the rates of epithelial healing between control and each hEGF-treated group. Among the tested concentrations, 5microgram/ml hEGF induced the most active proliferation of basal cells and 50microgram/ml hEGF remarkably produced a vascular ingrowth to the central wound area. The number of epithelial layers was increased by hEGF in a concentration-dependent manner. CONCLUSIONS: A low concentration of hEGF may selectively enhance epithelial healing without inducing corneal neovascularization. The optimal concentration of hEGF for the stimulation of basal cell proliferation appears to be 5 microgram/ml in rabbit corneal alkali wounds.

Effect of Topically Applied Human Epidermal Growth Factor on Stromal Keratocyte Activity in Experimental Corneal Alkali Wounds

PURPOSE: The Influence of topically applied human epidermal growth factor(hEGF) on stromal keratocyte was evaluated in the experimental corneal alkali wounds. METHODS: Corneal alkali wounds were induced by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the center of rabbit cornea for 60 seconds. The eyes were treated topically with 5 micrometer/ml hEGF(DaeWoong Pharma. Co. Ltd. Seoul, Korea) or PBS, 3 times per day. After 3- and 28-days treatment, the corneas were processed for transmission electron microscopy(TEM). RESULTS: After 3-days treatment, the basement membrane integrity was maintained and the stromal keratocyte remained damaged in both groups. However, after 28-days treatment, the basement membrane was disrupted and the keratocytes were activated in both groups. CONCLUSIONS: The findings of present study indicated that the optimal concentration of hEGF which is known to stimulate the epithelial healing was failed to affect the keratocyte activities as long as the basement membrane integrity was maintained.

Epidemiology of Infectious Keratitis(II): A Multi-center Study

To identify risk factors and causative organisms, and to evaluate clinical manifestations, methods and results of treatment in infectious keratitis, an epidemiological study was performed prospectively under the identical protocol from April 1995 to March 2000.Logistic regression analysis was used to evaluate possible risk factors. The 1474 cases of infectious keratitis reported from 22 hospitals were studied. Five hundred forty-four organisms(442 bacteria, 82 fungi, 20 A c a n t h a m o e b a)were detected in 1320 eyes with infectious keratitis excluding 154 herpetic keratitis. The Pseudomonas aeruginosa was the most common organism in bacterial keratitis, and Fusariumspp. was the major isolate in fungal keratitis. Contact lens wear and occupation(industry, forester, miner, fisherman)were the risk factors for bacterial keratitis. Risk factors in fungal keratitis were fifth decade of age, farmer, and systemic diseases(diabetes mellitus etc.). Risk factors in herpetic keratitis were male and occupation(office worker, service, student, housewife). Risk factors in Acanthamoeba keratitis was contact lens wear.

Effects of Topically Applied Na-Hyaluronan on Epithelial Healing and Aqueous Composition in Experimental Corneal Alkali Wounds

The effects of topical sodium-hyaluronan (Na-HA)on the epithelial healing and aqueous compositions were examined in the repair process of experimen-tal corneal alkali wounds.Corneal alkali wounds were produced in one eye of each rabbit by applying a 5.5 mmround filter paper soaked in 1N NaOH onto the central cornea for 60 seconds.Then the eyes were treated topically with either 1%Na-HA (the treatment group)or a phosphate buffered saline (PBS) (the control group)4 times per day.The epithelial healing was assessed using a quantitative method and the changes of the concentration of ascorbic acid and glucose in aqueous were evaluated.The initial epithelial healing rate was similar between 1%Na-HA treated and control groups and the later healing was enhanced in the treated group but statistically insignificant.The concentrations of ascorbic acid and glucose in aqueous humor are not statistically different in the early phase, but ascorbic acid concentration in later healing phase was higher in the treated group when compared with the control group.The present findings in dicate that topical-ly applied 1%Na-HA affects late epithelial healing and ascorbic acid concentration in aqueous humor during the repair process after corneal alkali wounds.

Effects of Topically Applied Na-Hya luronan on Stromal and Endothelial Healing in Experimental Corneal Alkali Wounds

The effects of topical sodium-hyaluronan on the stromal and endothelial healing were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were produced in one eye of each rabbit by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the central cornea for 60 seconds. Then the eyes were treated topically with either 1% Na-HA(the treatment group)or a phosphate buffered saline(PBS)(the con-trol group)4 times per day for 3 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The stromal healing was assessed by counting polymorphonuclear leukocytes(PMNs)and keratocytes in the central and marginal wounds areas. The stroma treated with Na-HA had less PMNs than that of the control group during the early healing period. The defect area of the endothelium was significantly smaller in the Na-HA treated group than in the control corneas. The present findings indicate that topically applied 1%Na-HA affects stromal and endothelial healing during the early repair process after corneal alkali wounds.

Effects of Topically Applied 0.1%Dexamethasone on Endothelial Healing and Aqueous Composition Following Experimental Corneal Alkali Wounds

The effects of topical dexamethasone on the endothelial healing and the change of aqueous composition were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were induced, then the eyes were treated topically with either 0.1%dexamethasone or abalanced salt solution[BSS]4 times per day for 8 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The concentrations of ascorbic acid, glucose, and the ions, Na, K , Ca2 and Mg2 , were measured in the aqueous humor. Endothelial healing in control corneas showed a biphasic pattern of healing:an initial short-term healing for the first week and then a late long-term healing following a secondary endothelial breakdown. Topical administration of 0.1%dexamethasone deterred endothelial healing during the early period and prevented secondary endothelial breakdown. Total repair process of endothelium was accelerated by the dexamethasone treatment. Among the various components of the aqueous humor examined, ascorbic acid seemed most sensitive to change caused by the alkali injury and dexametha-sone treatment. The present data indicate that dexamethasone may have a therapeutic potential in the management of endothelial healing after corneal alkali injury.

Optimal Concentrations of hEGF for Epithelial and Endothelial Healing in Corneal Alkali Wounds: An Organ Cultured Study

Optimal concentrations of hEGF for epithelial and endothelial healing after alkali-injury were determined in an organ cultured system. By using a 5.5 mmround filter paper, corneal alkali wounds (1N NaOH, 60sec.) were induced in the rabbit eyes and then the whole cornea with 1 mmscleral rim were excised.The excised corneas were immersed in the Eagle`s minimum essential medium supplemented with 5% Dextran, 1%antibiotics and different concentrations of hEGF (DaeWoong Pharma.Co.Ltd. Seoul, Korea), i.e., O (control), 0.01, 0.1, 1.0, 10, 100ng/ml. After tryphan blue staining, morphometric analysis was performed at 1, 2, 3, 4 and 6 days time periods. The healing rates were calculated by using an image analysis system. After 7 days, the corneas were processed for an immunohistochemical observation of proliferating cell nuclear antigen (PCNA) positive cells in the re-surfaced epithelium. Epithelial re-surfacing was significantly enhanced by the hEGF treatment at a concentration of 1.0ng/ml. The thickness of regenerated epithelium appeared to be increased in a dose-dependent man-ner. The rate of endothelial healing was increased at the concentration of 100ng/ml. The results indicate that epithelial healing was stimulated at about 100 times lower concentration of hEGF than endothelial healing.

Complains of Injection in the Left Eye

No abstract available.

Epidemiology of infectious KeratitisI A Multi-center Study

Infectious keratitis is the most common serious ocular infection, and may be caused by various bacteria, fungi, viruses and parasites. The authors performed prospectively an epidemiological study to identify risk factors and causative organisms, and to evaluate clinical manifestations, methods and results of treatment in infectious keratitis under the identical protocol from April 1995 to September 1997. Logistic regression analysis [univariate analysis and multivariate analysis] was used to evaluate possible risk factors. Six hundred sixty cases of infectious keratitis reported from 19 hospitals were studied. Two hundred eighty-three organisms[247 bacteria, 32 fungi, 4 acanthamoeba] were detected in 626 eyes with infectious keratitis excluding 34 pherpetic keratitis. The Pseudomonas aeruginosa, coagulase negative staphylococcus, Staphylococcus aureus and Serratia marcescens were the major orgnisms in bacterial keratitis. Aspergillus, Fusarium and Candida were the major isolates in fungal keratitis. Contact lens wear was a risk factor for bacterial keratitis. Female, age[less than 40 years] and occupation[student, house-wife, office worker, servise] were associated with bacterial keratitis. Risk factors in herpetic keratitis were age[between 40 and 59 years] and ocular adnexal diseases. Male was associated factor with herpetic keratitis.

The Effect of 0/1% Dexamethasone on Repair Process of Eithelial Basement Membrane after Corneal Alkali Wounds: Electron Microscoic Examination

An intact basement membrane which provides a firm attachment of basal epithelial cells to the maintain the corneal integrity in alkali wound healing. Despite many therapeutic trials tested, the fate of present investigation, the repair process of basal epithelium and its basement membrane was evaluated morphologically by utilizing a transmission electron microscope. New Zealand white rabbits, weighing 2.5kg, total of 16(16 eyes), were used in this experiment. Initial epithelial wounds were induced by applying a 5.5mm round filter paper, soaked in 1N NaOH, put on the central cornea for 60 seconds. Animals were divided into two groups and treated with 0.1% dexamethasone in the treated group and with balanced salt solution(BSS) in the control eyes q,i,d. for 8 weeks;. In control eyes, the basement membrane started focal disruption at 2 days, became the worst state at 4 weeks, and regained the normal integrity at 8 weeks after initial alkai damage, In dexamethansone-treated eyes. the basement membrance structure was fairly well visible up to 4 weeks and was almost completely disappeared at 8 weeks after intial alkali wounds.

Clinical Effect of Levocabastine Eye Drops on Allergic Conjunctivitis

Levocabastine, a selective H1-receptor antagonist, has been evaluated in the management of allergic conjunctivitis and its efficacy was compared between the skin test positive and negative groups. A total of 20 patients who was clinically diagnosed as allergic conjunctivitis were processed for the skin test. According to the results of skin test, patients were divided into two groups; skin test positive (15) and negative (5). Livostin(levocabastine 0.5mg/ml) eye drops were topically instilled 2 times per day for 1 week. Both subjective and objective findings were assessed by a 4-point scase (0=none, 1=mild, 2=moderate, 3=severe). The parameters used for the examination of drug effectiveness were itching, tearing, chemosis, lid swelling and injection. 85.5% (17 put of 20) of the patients showed effects on the management of their symptoms and signs. No significant difference was observed in its effectiveness between skin test positive and negative groups. The results of present clinical trials suggest that Livostin eye drops may be an attractive therapeutic option for the management of allergic conjunctivitis.

Morphological Studies on Fuchs`Dystrophy

Light andelectron microscopic findings of Fuchs`dystrophy were examined in a corneal button obtained following a penetrating keratoplasty. In light microscopic study, an avascular connective tissue was observed between the epithelial basement membrane and Bowman`s layer. Fibroblast-like cells scattered on the denuded surface of the Descemet`s membrane were found in a flat-mounted specimen examined after alizarine red and tryphan blue staining. A slightly thickened Descemet`s membrane covered with the disorganized endothelium was observed in a electron microscopy. Swollen organelles and vacuoles in the cytoplasm were found as an internal structure of the endothelium.

The Therapeutic Effect of Topically Applied 1% Sodium Hyaluronic Acid(Na-HA) on Corneal Alkali Burn

The effects of topically applied 1% Na-HA on corneal epithelial healing and visual outcomes were evaluated in 15 eyes of 12 corneal alkali-burned patients. 1 % Na-HA (Healon, Pharmacia, Sweden) together with antibiotics(Tarivid eye oint, Japan) in the treatment group and antibiotics ointment alone in the control group were given four times a day for the first one week and then antibiotics were used in both groups. For the assessment of the initial epitheilal healing, the eyes were examined at about 8 to 12 hours intervals during the first week. Individual epithelial healing rates were calculated by a linear regresion analysis from the epithelial defect areas. The best corrected visual acuity was compared between two groups. Initial epithelial healing rates were 0.81+/-0.53(mm2/hour) in the 1% Na-HA treated group and 0.40+/-0.21(mm2/hour) in the control group, respectively. A progressive improvement of visual acuity was observed in the 1% Na-HA treated group, while no change of vision was found in the control group. This clinical study suggests that topical application of 1 % Na-HA in combination with topical antibiotics could be considered for the management of corneal alkali wound.

Effect of Topical Na-Hyaluronan on Epithelial Cell Morphogenesis and Superficial Stromal Healing in Corneal Wound

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

Effects of 0.1% Dexamethasone on Experimental corneal Epithelial Healing Following Alkali Wounds

The effect of topical steroid on corneal alkali wound has been controversial due to a complexity of its mechanism and a risk of ulceration and perforation. In this study, the effect of topical 0,1% dexamethasone on corneal epithelial wound healing following alkali burn was evaluated quantitatively. 22 eyes wounded by alkali burn out of 22 white rabbits were divided into 3 groups ; A group treated with topical 0.1% dexamethasone for eight weeks, B grouptreated with topical 0.1% dexamethasone for the first one week followed by another seven weeks without treatment, C group with no treatment served as a control. The area of corneal epithelial defect was examined at 6, 18, 30, 42 hours following corneal alkali wound for initial wound healing and examined every other day from 4 days to 48 days following corneal alkali wound for late wound healing. In this study, it was found that topical 0.1% dexamethasone delayed corneal epithelial healing following corneal alkali burn in both stage of initial and late wound healing.

The Effect of Topically Applied Sodium-hyaluronic acid(Na-HA) on Corneal Epithelial Healing

To evaluate the effect of Na-hyaluronic acid (Na-HA) on corneal epithelial wound healing, twenty admitted patients (twenty-four eyes) with corneal epithelial abrasion were randomly divided into 2 groups. 1% Na-HA was given 4 times daily to 12 treated eyes while 12 control cases received antibiotic ointment therapy. Epithelial defect area was photodocumented by 6-12 hours interval and individual healing rates were calculated by linear regression analysis. The healing rate of 1% Na-HA treated cornea was 1.02 +/- 0.26mm2/hour and that of the control eyes was 0.67 +/- 0.19 mm/hour(p<0.05). The result suggests that topically applied Na-HA enhances epithelial re~surfacing when compared to the antibiotics ointment.

Effect of topically applied Na-hyaluronan on experimental corneal alkali wound healing

The effect of topically applied 1% sodium hyaluronate (Na-HA) on the healing of a standardized corneal alkali wound was studied. The healing of the epithelium, stroma, and endothelium was evaluated separately, using quantitative methods. Central corneal alkali wound was produced in one eye of the rabbits by applying a 5.5-mm round filter paper, soaked in 1 N NaOH, for 60 seconds. 1% Na-HA in the treatment group and phosphate buffered saline (PBS) in the control group were given topically 4 times per day for 2 days, 1- and 3-weeks. Epithelial and endothelial healing was assessed morphometrically from standardized photographs and micrographs, respectively. Stromal healing was determined by counting polymorphonuclear leukocytes (PMN) and keratocytes in the central and marginal wound areas. A positive healing influence was observed in the epithelium. In stromal healing, 1% Na-HA treated corneas showed less PMNs and more keratocytes than the control group, suggesting that topically applied 1% Na-HA may suppress the stromal PMN infiltration and enhance the keratocyte repopulation during corneal alkali wound healing. However, no significant difference was found in morphometric evaluation of endothelial healing between the two groups.

Comparison of Central Corneal Epithelial Healing Rates Between Different Wound Conditions

The role of limbal epithelial cells on the initial re-surfacing of the central epithelial defect was evaluated. Central or peripheral corneal wounds were produced by using n-heptanol in New Zealand white rabbit, and the epithelial healing rates were evaluated. Central corneal wounds made by applying a filter paper soaked in 0.2 N NaOH with vs without limbal damage were produced for the paralleled set of experiment. A 5.5 mm round filter paper for central wound and a 2 mm filter paper strip for limbal damage were used for the initial wounding. Epithelial defect areas were photodocumented after fluorescein stainmg at 0.6, 18, 30, 42, 48 hours following initial damage. Epithelial healing rates were measured by linear regression analysis, The epithelial healing rates of central and peripheral heptatlol wounds were 0.71 +/- 0.14 mm2/h, and 0.79 +/- 0.10 mm2/h(p>0.05), respectively, NaOH central wound without limbal damage and with limbal damage were 0.77 +/- 0.11 mm2/h, and 0.76 +/- 0.11 mm2/h, respectively(p>0.05). These data suggest that the limbal epithelium has no influence on the initial re-surfacing of the central epithelial defect.

Clearance of Amphotericin B in Rabbit Vitreous

The object of this paper is to determine the clearance of amphotericin B from the rabbit vitreous. In vitro susceptability of Candida guilliermondi to amphotericin B in antibiotic medium 20(Difco) provided the lowest values of the minimal inhibitory concentration(MIC) among several Candida strains and showed the same MIC afteir incubation at either 30 degrees C or 35 degrees C for 48 hours. A volume of 0.05 ml of solution containing 5 microgram of amphotericin B was injected into the rabbit vitreous, and animals were killed at various intervals. Both eyes were immediately enucleated and frozen to prevent further diffusion of the amphotericin B out of the vitreous. The vitreous was removed intact while it was still frozen. After the vitreous had thawed, its volume was measured. Sterile saline was added and total volume was adjust to 2 ml. The mixture was homogenized and then centrifuged at 2,000 rpm for 20 minutes. One ml of supernatant was removed from the sediment, and the concentration of amphotericin B was determined by broth dilution technique with Candida guilliermondi. The clearance studies indicated that 5 microgram of amphotericin B injected intravititreally, maintained an antibiotic level of greater than 1 microgram/ml for longer than 96 hours, with a half-life of 12 hours. If all factors are considered equal, presumed clearance data for intravitreal injection of 10 microgram of amphotericin B in humans may be calculated from the result in rabbit vitreous. Therapeutic levels of amphotericin B(1 microgram/ml) may be maintained at least 48 hours after the initial intravitreal injection of antibiotic in human eyes.

Fluorescein Fundus Angiographic Findings in Diabetics

At least 50% of the diabetics are affected by retinopathy in the western world since the 1950s and in Japan since the 1960s. In Korea, the incidence seems to be about 30%. The lower incidence of diabetic retinopathy in Korea may not be related with racial differences but rather with social and economic problems. This study covered 80 Korean diabetics (120 eyes) who were examined by means of the fluorescein fundus angiography and the results were compared with ordinary ophthalmoscopic findings. Diabetic retinopathy was found in 71% of the total studied cases by means of the fluorescein fundus angiography, but only 33% of the cases were recognized having diabetic retinopathy by ophthalmoscopic examination. It is very important to detect vascular changes in their early stage of diabetic retinopathy from view point of treatment. The earliest fluorescein fundus angiographic findings in diabetic retinopathy are microaneurysm, microvascular leakage, and capillary nonperfusion. On the fluorescein fundus angiographic findings, microaneurysm was found in 75 out of 120 eyes studied but only 30 eyes were found to have microaneurysms on the ophthalmoscopic examination. Among the retinal lesions referred to duration of diabetes mellitus, microaneurysms were observed in 13 out of 34 eyes in less than one year duration, and in 31 of 52 eyes during the period from one to five years. Leakage from microaneurysm was found in 18 out of 86 eyes within five years duration. Capillary non perfusion area was found in 10 among 34 eyes in less than one year duration, and 27 out of 52 eyes in one to five years duration. The longer the patient suffered from diabetes, the greater number of eyes showed microaneurysms and capillary non perfusion areas. Of 120 eyes examined by the fluorescein fundus angiography, 53 eyes were found having retinal edema and of 53 eyes, 6(4 patients) showed macular edema but through the ophthalmoscopy, only 10 eyes were found having retinal edema. Retinal neovasculation was found in 7 eyes (4 patients) through the fluorescein fundus angiography but only 5 eyes (3 patieuts) were found by ophthalmoscopic examination. The rate of having macular edema and neovascularization which were revealed through the fluorescein fundus angiographic findings clearly indicates the fact that at least 10% out of toral diabetic retinopathy studied so far are definitely indicative of photocoagulation therapy.