Alternatively spliced fibronectin molecules in the wounded cornea: analysis by PCR.

PURPOSE: To determine whether certain fibronectin isoforms participate in corneal epithelial wound healing, the authors used the polymerase chain reaction to detect different splicing patterns of the EIIIA segment of fibronectin mRNA in epithelial scrape-wounded cornea of rats. METHODS: Specific fibronectin cDNA sequences synthesized from rat cornea with total RNA were amplified with various sets of synthetic oligonucleotide primers. RESULTS: The authors detected both the EIIIA+ and EIIIA- fibronectin mRNA isoforms during corneal wound healing. The kinetics of corneal expression of both total fibronectin mRNA and the EIIIA- fibronectin mRNA isoform was polyphasic; an initial decrease was followed by an increase at 45 minutes, a second increase at 2 hours, and a third increase at 4 days after wounding. EIIIA+ fibronectin mRNA, not found in normal cornea, also was detected during healing. CONCLUSIONS: The expression of total fibronectin mRNA and both the EIIIA+ and EIIIA- fibronectin mRNA is upregulated during corneal epithelial wound healing. The expression of EIIIA+ fibronectin mRNA during wound healing, a fibronectin isoform that was highly expressed in embryonic tissue, suggests that this fibronectin isoform is involved functionally in corneal wound healing.

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.

Immunopathology of scleritis.

Conjunctival and scleral biopsies from 25 patients with necrotizing scleritis and 5 patients with recurrent nonnecrotizing scleritis were studied by histopathologic, immunofluorescence, and immunoperoxidase techniques. Vasculitis with fibrinoid necrosis and neutrophil invasion of the vessel wall was present in 75% of the scleral and 52% of the conjunctival specimens. Vascular immunodeposits were found in 93% of the scleral and 79% of the conjunctival tissue tested by immunofluorescence techniques. A dramatic increase in the number of inflammatory cells over normal controls was detected in both tissues by immunoperoxidase techniques. In the conjunctival epithelium, there were significantly more T-helpers, macrophages, and B cells. In the conjunctival substantia propria, there were significantly more T cells of all types, macrophages, and B cells. Likewise, scleral specimens showed an increase over controls of T cells of all types and macrophages. HLA-DR expression was dramatically increased in both tissues. Immune-complex-mediated vasculitis plays a pivotal role in the pathogenesis of necrotizing scleritis and recurrent nonnecrotizing scleritis. Induced HLA-DR expression on ocular nonimmune cells and T cell controlled responses also may participate.

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 topically administered epidermal growth factor on corneal wound strength.

A 9-mm perforating corneal wound was created in one eye of New Zealand albino rabbits, sutured, and treated with mouse-derived epidermal growth factor (1 mg/L), human-derived epidermal growth factor (1.0 mg/L to 100 mg/L), or buffered saline, instilled once, twice, or four times daily. Both mouse-derived epidermal growth factor and human-derived epidermal growth factor significantly increased the tensile strength of full-thickness corneal wounds after 9 days of topical therapy. For human-derived epidermal growth factor, a concentration of 10 mg/L administered twice daily produced the maximal effect. An increase in either the concentration of epidermal growth factor or its frequency of administration failed to induce a further increase in wound strength. Indeed, at a concentration of 100 mg/L, human-derived epidermal growth factor appeared to lose its ability to accelerate healing of full-thickness corneal wounds.

Suppression of chemotaxis to corneal inflammation by nitrous oxide.

Immune competency is depressed in the perioperative period. The role of anesthetic agents in immune reactivity remains unclear. The chemotactic migration of polymorphonuclear leukocytes (PMNs) to the cornea of rabbits injured by clove oil was studied. PMNs were previously radiolabeled with tritiated (3H) thymidine. Immediately following injury, the rabbits entered isolation chambers and breathed either air or air containing 10%, 20% or 40% nitrous oxide (N2O) for 24 hours. After sacrifice, the radioactivity of a 10 mm corneal button, removed by trephination, was determined by scintillation counting technique. Peripheral blood was obtained for hemoglobin, white cell and platelet count. The N2O dosage affected on the migration of PMNs to the cornea. 3H was decreased 15.4% by 20% N2O and 38.8% for 40% N2O-exposed rabbits. Peripheral blood values did not differ. N2O can suppress chemotaxis of PMNs in the rabbit, thereby adversely affecting the inflammatory component of immune defense.

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.

The effect of alcohol intoxication on inflammation of the cornea.

New Zealand albino rabbits received daily intraperitoneal injections of alcohol (ethyl alcohol), 1.6 g/kg and the effect of short-term (three days) and long-term (six weeks) administration on corneal inflammation was studied. Both regimens produced an average peak serum concentration of more than 0.200 g/dL, a level consistent with gross intoxication in the majority of humans. Clinical signs of intoxication were present in all animals, manifested by a gross disturbance of equilibrium and gait. Neither regimen produced measurable liver damage. Nonetheless, following both regimens of alcohol administration, significantly fewer polymorphonuclear leukocytes invaded the corneas of animals receiving alcohol than invaded the corneas of simultaneously run controls receiving intraperitoneal saline. These data provide a mechanism to explain why an alcoholic individual might not cope with a corneal infection as well as a nonalcoholic person, an observation long thought to be true clinically.

Effect of suprofen on corneal wound healing.

We studied the effect of suprofen, a new ophthalmic nonsteroidal anti-inflammatory agent, on corneal wound healing. Nine-millimeter, central, perforating corneal wounds were made in albino rabbits and sutured with 10-0 nylon. The animals were randomly treated with balanced salt solution, suprofen vehicle, 1% suprofen, or 0.1% dexamethasone sodium phosphate administered topically for six days. On the seventh postoperative day, the sutures were removed and, in situ, the intraocular pressure was increased in a controlled manner until the wound burst. Dexamethasone applied four times a day significantly inhibited corneal wound healing, whereas suprofen given as often as hourly did not. Pretreatment with hourly administered suprofen for two days prior to surgery, in addition to the same postoperative hourly therapy, also did not significantly decrease stromal wound strength.

Lipid angiogenic factor from omentum.

Placing the omentum on the brain surface by surgical transposition or transplantation will result in the development of numerous neovascular connections between these two structures. This phenomenon occurs even in the absence of cerebral ischemia, which raised the question as to whether an angiogenic factor was causing the response. A lipid material obtained from the omentum contains a potent angiogenic factor extractable in a chloroform-methanol solvent mixture. Angiogenesis created by this material was observed in the rabbit cornea after only a single injection of the substance. The angiogenic material obtained from the omentum is abundant in supply. This important characteristic offers promise for the purification and identification of its structure, which should allow for extensive animal and clinical studies dealing with the development or inhibition of angiogenesis.

Fluorometholone acetate. A new ophthalmic derivative of fluorometholone.

Hourly topical administration of 0.1% fluorometholone acetate ophthalmic suspension produced, on the average, a 47% reduction in the polymorphonuclear leukocytes invading the cornea during an experimentally induced inflammatory keratitis. This is a significantly greater anti-inflammatory effect than we have previously reported for the alcohol derivative of fluorometholone and is not significantly different from the therapeutic effect of 1.0% prednisolone acetate ophthalmic suspension, the most effective corneal anti-inflammatory agent that we have studied to date. Fluorometholone acetate (0.1%) formulated as a high-viscosity carbomer gel and applied at three-hour intervals reduced invading leukocytes in the cornea an average of 48%, an effect not significantly different from hourly administration of the suspension.

Prolongation of anti-inflammatory effect of prednisolone acetate. Influence of formulation in high-viscosity gel.

Prednisolone acetate was formulated in a high-viscosity carboxypolymethylene gel at concentrations of 0.125% and 1.0%. The ability of these gel preparations to suppress inflammation in the cornea was assessed and compared with the antiinflammatory capabilities of conventional commercially available prednisolone acetate ophthalmic suspensions. When administered hourly, the gel formulations produced no greater anti-inflammatory effect than the conventional suspensions. However, the gel formulations were equal in effect to the suspensions and maintained their effects considerably longer than did the suspensions. When applied at intervals up to and including four hours, there was no falloff in effect; the gel demonstrated a level of anti-inflammatory effectiveness that could not be distinguished from hourly administration of the suspension.

Route of antibiotic administration in bacterial keratitis.

The in vivo antibacterial effectiveness in the rabbit cornea of several antibiotics delivered by topical application, by periocular injection, and by intravenous (IV) inoculation was determined against Staphylococcus aureus and Pseudomonas aeruginosa. Topical instillation of antibiotic was highly effective in eliminating these organisms from the cornea. In contrast, despite a considerable increase in the quantity of antibiotic administered, we could demonstrate no statistically significant reduction in the number of viable staphylococcal or Pseudomonas organisms in the cornea when the antibiotic was given by periocular or by IV injection.

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.

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.

Antiinflammatory medications.

Corticosteroids are by far the most frequently used agents to treat ocular inflammation. When administered topically to the eye, different derivatives of the same corticosteroid base are not equivalent in their antiinflammatory properties. A change in the derivative of a corticosteroid base alters its behavior as an antiinflammatory agent. To date, the acetate derivative of each corticosteroid base studied has been the most effective, and among commercially available ophthalmic formulations, 1.0 percent prednisolone acetate is the drug of choice for maximal antiinflammatory effect. Hourly instillation produces a greater, more rapid reduction of corneal inflammation than does instillation of the drug every 4 hours, while instillation at 15-minute intervals results in an even greater therapeutic effect. Topical delivery of five doses of 1.0 percent prednisolone acetate at 1-minute intervals each hour results in an antiinflammatory effect comparable to that achieved by instillation every 15 minutes. Topical instillation of a corticosteroid produces a greater reduction in inflammatory cells invading the cornea than does periocular injection of a steroid. Administration of corticosteroids concurrently by topical and subconjunctival routes produces an additive antiinflammatory effect. Addition of a topically applied corticosteroid to an effective topical antibiotic regimen containing a bactericidal agent does not enhance bacterial replication in the cornea if the corticosteroid is not instilled more frequently than the antibiotic. Corticosteroids enhance viral proliferation and are contraindicated in active epithelial herpetic keratitis. Many instances of stromal herpetic keratitis appear to be a toxic or immune response to incomplete, nonreplicating viral particles rather than alteration of tissue by multiplying live virus, and the judicious use of corticosteroids is advocated along with an antiviral antimetabolite. Because control of replicating fungal organisms by specific antifungal agents is often difficult to achieve, corticosteroids should not be used in the treatment of mycotic keratitis.

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.

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.