Quantification of stromal destruction in the inflamed cornea.

An intrastromal injection of endotoxin lipopolysaccharide (LPS) in one eye of New Zealand albino rabbits induced a prominent keratitis characterized clinically and microscopically by edema and infiltration. Polymorphonuclear leukocytes (PMNs) constituted the primary invading leukocytic element. Collagen synthesis was measured by pulsing the corneas with 3H-proline before inducing inflammation. The invasion of the cornea by leukocytes did not alter the conversion of proline to hydroxyproline significantly in the stroma during the 14-day observation period, signifying that there were only negligible changes in the rate of collagen synthesis. However, the percentage of total stromal protein represented by collagen (ie, collagen/total protein) was only 50% of that in comparable corneas receiving an injection of phosphate-buffered saline. Some animals were rendered leukopenic by intravenous nitrogen mustard before intrastromal LPS injection caused a less severe corneal inflammatory response, characterized microscopically by fewer infiltrating leukocytes. Similarly, in nonleukopenic rabbits, topical therapy with 1% prednisolone acetate markedly reduced the corneal inflammatory response which also was characterized by fewer invading leukocytes. In neither instance was there extreme collagen loss, suggesting that the loss of stromal collagen is related to PMN infiltration.

Effect of concurrent topical corticosteroid and NSAID therapy of experimental keratitis.

The ability of suprofen, a nonsteroidal anti-inflammatory drug, and prednisolone acetate, a corticosteroid, to suppress polymorphonuclear leukocyte invasion of the rabbit cornea during an experimental keratitis was evaluated following topical ophthalmic administration of either drug alone or both drugs concurrently. Suprofen therapy initiated immediately after induction of inflammation was ineffective. However, if suprofen therapy was begun 48 hr prior to the induction of inflammation, the drug was effective. In contrast, prednisolone acetate therapy begun after the induction of inflammation was effective; 48 hr of pretreatment with the corticosteroid produced a marked increase in its therapeutic effect. When administered according to the same regimen, concurrent therapy with suprofen and prednisolone acetate was significantly more effective than treatment with either drug alone. This result was obtained irrespective of whether concurrent therapy was initiated prior to or after the inflammatory event.

Bioavailability and corneal anti-inflammatory effect of topical suprofen.

The bioavailability in rabbit cornea and aqueous humor of an ophthalmic formulation of suprofen, a nonsteroidal anti-inflammatory drug, was evaluated following topical administration of a single dose to the eye. The drug penetrated rapidly into the uninflamed cornea with intact epithelium; highest levels occurred during the first 30 to 45 min after instillation and decreased thereafter. The bioavailability of suprofen in cornea and aqueous humor following administration of a 1.0% concentration was twice that produced by a 0.5% concentration of the drug. Topical application of multiple doses of suprofen failed to suppress polymorphonuclear leukocyte invasion of the cornea if treatment was started after the induction of inflammation. Suprofen therapy initiated prior to the induction of corneal inflammation and maintained into the post-inflammation period did produce a significant (P less than 0.01) decrease in the numbers of PMNs that invaded the inflamed cornea. There was no significant difference (P greater than 0.05) in the corneal anti-inflammatory effect achieved by the 0.5% and 1.0% concentrations of suprofen when administered according to this regimen.

In vitro evaluation of fibroplasia in a porous polymer.

We evaluated a polybutylene/polypropylene blend in a blown microfiber configuration in vitro for potential use as the peripheral area of a keratoprosthetic device. Material properties such as ultimate tensile strength and ultimate elongation were measured. Stromal fibroblasts were seeded onto the material in vitro, and cell proliferation between uncoated and Type I collagen coated discs did not differ significantly. Fibroblasts could be seen migrating along the fibers and also traversing the fibers. The synthesis of connective proteins was examined. Laminin, fibronectin, and Type I collagen were detected by day 8. The experiments demonstrate that stromal fibroblasts can adhere onto the fibers, proliferate, and synthesize connective tissue proteins. Experiments are now being conducted to further evaluate the material in vivo.

In vivo fibroplasia of a porous polymer in the cornea.

We have developed a melt-blown fibrous construction of polybutylene/polypropylene in which we previously demonstrated keratocyte ingrowth and collagen synthesis in vitro. In the present studies, we evaluated this material in vivo in interlamellar corneal pockets for periods of up to six months. By day 42, the porous interstices of the disc were heavily populated with keratocytes. Extracellular matrix deposition occurred and there was a 5000-fold increase in total protein and a 1000-fold increase in total collagen over background. The cells within the disc continued to be synthetically active for the six months of our study. Discs remained in corneas for periods of up to one year without any extrusion. This material has great promise as a porous peripheral component of a keratoprosthetic device.

Quantitation of herpes simplex virus in rabbit corneal epithelium.

The authors have developed an objective method for quantitation of herpes simplex virus in the corneal epithelium of rabbits. At appropriate times postinfection, full-thickness rabbit corneas were removed by trephination and subjected to one cycle of freezing and thawing. The corneal epithelium was then disrupted by sonication. The amount of infectious virus recovered from sonicated specimens was determined by an in vitro plaque assay, providing a measure of the quantity of virus present during the acute stage of herpetic keratitis. Using this technique, the authors found that the mean virus titer was reduced from 1.5 X 10(5) plaque forming units (pfu) per cornea in control rabbits to less than 200 pfu per cornea in rabbits treated topically for 2 days with 1% trifluridine. In contrast, instillation of 1% prednisolone acetate resulted in the persistence of higher levels of virus (275 pfu) than those observed in control rabbits (3 pfu) 4 days after the cessation of therapy.

Topical antibiotic therapy of Pseudomonas aeruginosa keratitis.

The in vivo antibacterial effectiveness in the rabbit cornea of several commercially available ophthalmic antibiotic preparations was determined against a single strain of Pseudomonas aeruginosa isolated from a human corneal ulcer. Each antibiotic was instilled topically at hourly intervals, and the number of residual viable organisms in the cornea subsequently was ascertained. In vivo measurements correlated well with in vitro data and with generally held clinical impressions. Three antibiotics, gentamicin sulfate, polymyxin B sulfate, and colistin sulfate, suppressed corneal growth of P aeruginosa in commercially available concentrations. Gentamicin was slightly more effective than polymyxin B; both drugs were substantially more effective than colistin. Formulations of gentamicin and polymyxin B containing approximately four times the quantity of drug found in commercial preparations eliminated this P aeruginosa strain from the cornea much more rapidly than did the commercial preparations.

Topically administered corticosteroids: effect on antibiotic-treated bacterial keratitis.

The effect of a topically administered corticosteroid, 1.0% prednisolone acetate, on bacterial replication in rabbit cornea receiving adequate antibiotic therapy was determined. Staphylococcus aureus keratitis was treated either with neomycin sulfate or gentamicin sulfate, while Pseudomonas aeruginosa keratitis was treated either with gentamicin or polymyxin B sulfate. Each antibiotic was administered topically at hourly intervals in both the commercially available concentration and as a formulation containing four times the quantity of drug found in the commercial preparations. In each instance, the antibiotic regimen sharply reduced the number of viable organisms in the cornea, although the concentrated preparations did so more rapidly and effectively. The addition of 1.0% prednisolone acetate had no measurable effect on outcome. In no instance was there a statistically significant difference between number of residual viable organisms in antibiotic-treated corneas and antibiotic/corticosteroid-treated corneas.

Topical antibiotic therapy of staphylococcal keratitis.

The in vivo antibacterial effectiveness in the rabbit cornea of a number of commercially available ophthalmic antibiotic preparations was determined against a single strain of penicillinase-producing Staphylococcus aureus isolated from a human corneal ulcer. Each antibiotic was instilled topically at hourly intervals, and the number of residual viable organisms in the cornea subsequently was ascertained. In vivo measurements demonstrated that five antibiotics--neomycin sulfate, gentamicin sulfate, erythromycin, tetracycline hydrochloride, and chlortetracycline hydrochloride--were equally effective in suppressing growth of the strain of S aureus studied. Therapeutic results were the same whether the corneal epithelium was present of absent for each of the drugs studied. With one exception (chloramphenicol), there was excellent correlation between in vivo and in vitro findings.

Optimal frequency of topical prednisolone administration.

The relationship between the frequency with which 0.125% and 1.0% prednisolone acetate ophthalmic suspensions are instilled and the anti-inflammatory effect they achieve in the cornea was studied. Within the time limits of the experimental protocol, application of the drug at four-hour intervals failed to produce an effect while hourly administration of both concentrations of the corticosteroid produced a substantial anti-inflammatory effect. Instillation at 15-minute intervals resulted in a significantly (P less than .05) greater reduction of the polymorphonuclear leukocytes invading the cornea than did administration of the medication every hour. If five doses of prednisolone acetate were applied topically at one-minute intervals each hour, both concentrations of this corticosteroid produced a therapeutic effect in the cornea equal to that achieved by administration of the drug every 15 minutes.

Betaxolol. A new beta-adrenergic blocking agent for treatment of glaucoma.

The intraocular pressure response to topically instilled 0.25% betaxolol hydrochloride was evaluated in 12 patients with chronic open angle glaucoma or ocular hypertension. The drug produced a significant lowering of IOP in all 12 of the patients under study. A 30% to 35% decrease below baseline IOP was observed and was maintained during the one-year observation period. Visual acuity was stable in all subjects throughout the study and corneal anesthesia was not encountered. Tear secretion was not adversely effected by betaxolol, nor did topical administration of the drug produce any systemic cardiovascular response of consequence. Blood pressure and pulse rate remained stable throughout the year. These data suggest that an ophthalmic formulation of betaxolol may have substantial clinical potential for the treatment of glaucoma.

Drug interaction in the eye. Concurrent corticosteroid-antibiotic therapy for inflammatory keratitis.

Concurrent instillation of individual preparations of a corticosteroid and an antibiotic resulted in significantly (P less than .05) lower peak corneal and aqueous humor steroid levels than those achieved by the steroid alone. Both the interval elapsing between instillation of the two drugs and the sequence in which they were administered influenced subsequent steroid bioavailability. Corticosteroid levels in the cornea after administration of a combination steroid-antibiotic preparation were not significantly different (P less than .05) from those detected after instillation of the same steroid alone, suggesting that, for the treatment of corneal disorders, use of a combination preparation may offer a method to circumvent certain drug interactions. The decrease in ocular steroid bioavailability could not be directly equated with differences in antinflammatory effectiveness, so that the therapeutic relevance of the demonstrated drug interaction is not known.

Penetration of fluorometholone into the cornea and aqueous humor.

In each of the experimental conditions studied, fluorometholone penetrated into the cornea and aqueous humor following topical administration of a standard drop. The amount of drug measured in each location was less than that previously documented for dexamethasone and prednisolone preparations. In contrast to these more conventional steroids, the ocular penetration of fluorometholone appeared to be unaffected by the presence or absence of the corneal epithelium or of intraocular inflammation.

Therapeutic effectiveness of fluorometholone in inflammatory keratitis.

During an experimentally-induced inflammatory keratitis, we measured the ability of 0.1% fluorometholone ophthalmic suspension to reduce the numbers of polymorphonuclear leukocytes that invaded the cornea. The data demonstrate that topically administered fluorometholone is an effective therapeutic agent and that it compares favorably in anti-inflammatory activity with dexamethasone and prednisolone preparations. Comparison of our results with comparable studies of dexamethasone and prednisolone formulations indicates that 1.0% prednisolone acetate ophthalmic suspension is still the most effective corneal anti-inflammatory agent that we have investigated to date. However, the decreased potential of fluorometholone to produce secondary elevation of the intraocular pressure would appear to make it the drug of choice in situations in which maximum pharmacologic suppression of inflammation is not required and in chronic inflammatory conditions that require prolonged treatment.

Quantitation of bacterial infection and antibiotic effect in the cornea.

We report an experimental model that allows objective quantitation of bacterial keratitis. The model permits direct measurement of the number of viable organisms in the cornea after varying periods of in vivo growth. The size of the inoculum used to produce the corneal infection is critical, and the experimental organism must be standardized for its growth characteristics in the cornea. The end point is an objective one, productive of numerical data that can be subjected to statistical analysis. The findings are highly reproducible and the system is sufficiently sensitive to indicate the ability of a topically administered antibiotic to reduce the number of viable organisms in the cornea of an outbred rabbit population.

Periocular injection of corticosteroids: an experimental evaluation of its role in the treatment of corneal inflammation.

The present experiments demonstrate that subconjunctivally injected corticosteroids are less effective in suppressing corneal inflammation than are topically instilled corticosteroids. Topical administration of 6.5 mg of prednisolone acetate over a 30-hour period reduced corneal inflammatory activity by 52%. Subconjunctival injection of 50 mg of the same steroid, also given over a 30-hour period, yielded a 15% reduction in corneal inflammation. A fourfold increase (200 mg) in the quantity of prednisolone acetate injected subconjunctivally resulted in a nonsignificant (P less than .05) increment (24%) in anti-inflammatory effect. Dexamethasone sodium phosphate achieved a 30% reduction in corneal inflammation, the maximum effect observed after subconjunctival administration. The data suggest that different modes of corneal penetration are involved after drug delivery via the two routes. Concurrent administration of corticosteroids by the topical and subconjunctival routes seemingly produced an additive anti-inflammatory effect.

Kinetics of topically administered prednisolone acetate. Optimal concentration for treatment of inflammatory keratitis.

Two types of quantitative measurements were made in rabbit corneas. First, the level that varying concentrations of topically administered prednisolone acetate attained in the cornea and aqueous humor was determined. Then, the ability of varying concentrations of this corticosteroid to suppress corneal inflammation was ascertained. The maximum dose-response curve for anti-inflammatory effect in the cornea was achieved by the 1.0% concentration, the highest concentration commercially available. Higher concentrations permitted greater quantities of the drug to gain access to the cornea and aqueous humor but produced no measurable increment in anti-inflammatory effect. These experimental observations suggest that concentrations of prednisolone acetate higher than 1.0% have an increased potential for toxicity without offering additional therapeutic benefit.

Comparative anti-inflammatory efficacy of topical corticosteroids with low glaucoma-inducing potential.

Fluorometholone and clobetasone butyrate have been developed as ophthalmic corticosteroids because of their lesser potential to elevate intraocular pressure. Nevertheless, their primary use is the inhibition of an inflammatory response. Quantification of their anti-inflammatory effect in the rabbit cornea indicates that 0.1% fluorometholone and 0.1% clobetasone butyrate are effective, but weak, anti-inflammatory agents. An increase in concentration of fluorometholone to 0.25% failed to enhance its anti-inflammatory effectiveness significantly, while an increase in concentration of clobetasone butyrate to 0.5% did significantly increase its anti-inflammatory effect. As with all other corticosteroid bases studied to date, formulation of fluorometholone as an acetate derivative significantly increased its effectiveness, rendering it as effective as 1.0% prednisolone acetate, the most effective of commercially available ophthalmic corticosteroids.

Specular microscopic appearance of the corneal endothelium in iridocorneal endothelial syndrome.

The corneal endothelium of a 71-year-old woman with iridocorneal endothelial syndrome had two types of cell structure and density demonstrated by specular microscopy. In parts of the central cornea, characterized by normal endothelium on examination with the slitlamp biomicroscope, a substantial increase in cell density over that in the opposite normal eye was observed. In other focal areas of the central cornea, characterized by a typical beaten-silver appearance on slit-lamp biomicroscopy, considerable cell loss and cellular pleomorphism were observed in comparison with the surrounding normal-appearing endothelium. Nevertheless, the endothelial cell density in these areas was not significantly greater than that of the opposite uninvolved eye. This finding supports the theory that endothelial proliferation in this disease occurs not only on the iris but also in the endothelial cell layer of the cornea.

Quantitation of anterior chamber inflammation and its response to therapy.

Anterior uveitis was produced in the rabbit eye by introducing a standardized clove oil globule into the anterior chamber. The response was characterized by an increase in the vascular permeability of the anterior uveal tract, resulting in the exudation of protein and the migration of leukocytes into the anterior chamber. Using radiolabeled agents and couting techniques, protein and cells in the aqueous humor were measured, providing an objective, quantitative assessment of the severity of inflammation in the anterior chamber. Frequent topical administration of 1.0% prednisolone acetate during the first 100 hours of the experimental anterior uveitis produced a significant decrease both in protein and in the number of leukocytes in the anterior chamber.