John L. Ubels

Quantitative evaluation of the corneal epithelial barrier : effect of artificial tears and preservatives

The effect of artificial tear solutions on the corneal epithelial barrier was evaluated by measuring corneal uptake of 5, 6 carboxyfluorescein (CF) after exposure of rabbit corneas to various formulations in a conjunctival cup. Four tear solutions containing 0.01% benzalkonium chloride (BAC), a formulation containing 0.001% Polyquad, a contact lens re-wetting solution containing 0.004% thimerosal, and each of the above preservatives in balanced salt solution (BSS) were evaluated. Four non-preserved solutions were also tested. After treatment with the solutions corneas were exposed to the CF, removed from the eyes and dialyzed in balanced salt solution. The CF concentration in the dialysate was measured by fluorometry. Solutions containing 0.01% BAC caused a 9.24 to 99.28 fold increase in CF uptake as compared to control. Solutions preserved with Polyquad or thimerosal caused only a 0 to 4 fold increase in CF uptake while non-preserved solutions caused no change compared to control. Corneas prepared for transmission electron microscopy using fixative containing ruthenium red exhibited damage which correlated well with CF uptake; the ruthenium red penetrated the epithelium to the basal cell layer after corneal exposure to solutions containing BAC while only superficial cell layers were stained after exposure to the other test solutions. The method used in this study allows statistical comparison of artificial tear formulations. The data show that patients with severe dry-eye who use artificial tears frequently should avoid tear solutions containing BAC and that non-preserved solutions are preferable for treatment of these patients.

Vitamin A is present as retinol in the tears of humans and rabbits

Vitamin A is required for the normal growth maintenance and maturation of the corneal epithelium and is effective in the treatment of xerophthalmia and experimental corneal epithelial wounds when applied topically as retinoic acid. The normal route of delivery of vitamin A to the cornea has remained undefined. We collected tears from normal and vitamin A deficient rabbits and from humans and analyzed them by high pressure liquid chromatography. A peak corresponding to a retinol standard was eluted from normal rabbit and human tears but was absent from the vitamin A deficient rabbit tears. The retinol concentration in rabbit tears was 69 ng/ml (0.2 X 10(-6)M) and in human tears was 16 ng/ml (0.5 X 10(-7)M). This demonstration that vitamin A is present in the tears as retinol establishes the rationale for treatment of corneal disease with topical vitamin A.

The efficacy of retinoic acid ointment for treatment of xerophthalmia and corneal epithelial wounds

In previous studies of topical application of retinoic acid to the eye, retinoic acid in an oil vehicle has been used. An all-trans-retinoic acid (Tretinoin, USP) formulation in a petrolatum-based ophthalmic ointment vehicle has now been developed which is chemically stable for at least one year. The ointment is effective at retinoic acid concentrations of 0.01-0.1% in reversing corneal keratinization in vitamin A-deficient, xerophthalmic rabbits. At 0.1% it also stimulates the healing rate of corneal epithelial wounds in rabbits and monkeys. Retinoic acid ointment may prove to be valuable clinically in the treatment of xerophthalmia, corneal epithelial erosions, and related ocular surface abnormalities.

Drug uptake and release by a hydrogel intraocular lens and the human crystalline lens

ABSTRACT Implantation of a hydrogel (IOGEL) intraocular lens in humans has been reported. The polyhydroxyethyl methacrylate (poly HEMA) matrix of this hydrogel is permeable to water soluble drugs and may adsorb agents used intracamerally during cataract extraction or topically during the postoperative period. This study compared the in vitro uptake and release of chloramphenicol, dexamethasone, epinephrine, pilocarpine, and bovine serum albumin by polymethylmethacrylate and hydrogel intraocular lenses with that of the intact crystalline lens of humans and rabbits. An in vivo study compared the uptake and release of chloramphenicol and dexamethasone by hydrogel lenses implanted in the anterior chamber of rabbit eyes with that of the rabbits crystalline lens. The in vitro uptake and washout of epinephrine and pilocarpine by the hydrogel lens was comparable to the human lens. Uptake of chloramphenicol and dexamethasone by the hydrogel lens exceeded that of the human lens and, following a two‐hour washout period, the dexamethasone content of the hydrogel lens remained significantly greater than the human lens. The uptake and washout of bovine serum albumin by the hydrogel lens was half that of the human lens. In vivo, the hydrogel lens efficiently eluted both chloramphenicol and dexamethasone. These studies show that a hydrogel lens will not act as a significant depot for drugs in the eye.

The Effect of Retinoic Acid on Thymidine Incorporation and Morphology of Corneal Stromal Fibroblasts

The effect of retinoic acid on DNA synthesis and cell morphology was studied using corneal stromal fibroblasts in culture. All-trans retinoic acid induces an increase in DNA synthesis after 24 hours of exposure. Autoradiographic studies of 3H-thymidine incorporation into corneal stromal cells exposed to 10(-6) M retinoic acid for 24 hours showed an increase in labeling which ranged from 19.2% to 67.6% over control cultures. Scintillation analysis of labeled cultures also showed an increase in incorporation of 3H-thymidine into cells treated with 10(-6) M retinoic acid, with increases ranging from 21.8% to 114.7% above control cultures. Exposure of cultured corneal stromal cells to 10(-6) M retinoic acid resulted in a dramatic change in cell morphology such that they changed from spindle-shaped to round, flattened cells which were epithelioid in appearance. These data demonstrate that biological activity of retinoic acid in stromal fibroblasts and imply a role for vitamin A in maintenance of stroma structure and function.

Effects of Corneal Epithelial Abrasion on Corneal Transparency, Aqueous Humor Composition, and Lens of Fish

Abstract Transporting largemouth bass (Micropterus salmoides) in tanks at high density can result in a high incidence of corneal cloudiness. The structural integrity of the corneal epithelium is essential for maintenance of corneal transparency. In this report we show that removal of the corneal epithelium in rainbow trout (Salmo gairdneri), alewife (Alosa pseudoharengus), and longhorn sculpin (Myoxocephalus octodecemspinosus) causes corneal edema; cataracts form but clear as the epithelium heals. Removal of the corneal epithelium from teleosts, spiny dogfish (Squalus acanthias), and little skates (Raja erinacea) causes changes in aqueous humor osmolality and sodium concentration that reflect the osmolality and salinity of the environment. An intact corneal epithelium is, therefore, essential for maintenance of normal ocular function in fish. We suggest that factors such as repeated contact of the eye with spines and scales of other fish, nets, and tank walls can cause corneal epithelial abrasion during t...

Vitamin A is stored as fatty acyl esters of retinol in the lacrimal gland

Many tissues which require vitamin A store the vitamin as long-chain fatty acyl esters of retinol. As part of a study designed to characterize vitamin A metabolism in the lacrimal gland, which transports retinol from blood to lacrimal gland fluid, extracts from lacrimal glands of rabbits and rats were analyzed by non-aqueous high performance liquid chromatography. Retinyl linoleate, retinyl palmitate, and retinyl stearate were identified in these extracts by their co-elution with standards, their retention time relative to retinyl palmitate, and their susceptibility to hydrolysis by saponification. Retinyl palmitate was present in rabbit lacrimal gland at 51.0 +/- 10.1 ng/g tissue. After treatment of vitamin A-deficient rabbits with orally administered [11,12-3H] retinyl acetate, the radiolabeled esters retinyl linoleate, palmitate, and stearate were extracted from the lacrimal glands. These data show that the lacrimal gland stores vitamin A as fatty acyl esters of retinol.

Ocular Toxicity of Diethyltoluamide (Deet)

AbstractThe ocular toxicity following topical application (10, 30, 100 μl) of 100% DEET, 100% ethanol, and 80% ethanol-20% DEET was evaluated by measuring corneal thickness, area of corneal epithelial damage by fluorescein staining, and corneal reepithelialization. These measurements were compared to the standard toxicological techniques presently used to evaluate eye irritancy. These studies have indicated that corneal thickness, fluorescein staining, and corneal reepithelialization are objective predictors of ocular damage. The 10-μl volume of test substance appeared to be the best volume to predict toxicity, and the corneal reepithelialization studies provided the most accurate method to rank relative toxicity. The current investigation has shown that 100% ethanol is the least toxic followed by 100% DEET and 80% ethanol-20% DEET. Although all test substances showed ocular toxicity, reversibility was obtained by 10 days.

Retinyl ester hydrolysis in the rabbit lacrimal gland

The lacrimal gland stores retinyl esters which are synthesized by the enzyme acyl CoA:retinyl acyl transferase. Retinol is released from retinyl ester reserves by retinyl ester hydrolase (REH). Since the lacrimal gland secretes retinol, this gland should also contain this enzyme. To identify bile salt-dependent REH activity, rabbit lacrimal glands were homogenized in 0.05 M Trismaleate buffer, and enzyme activity was determined in the tissue homogenate, in the membrane fraction and in the cytosolic fraction by measurement of production of retinol from retinyl palmitate (nmol retinol produced/mg protein/h). In the lacrimal gland, production of retinol was optimal in the presence of 200 mM CHAPS at pH 7. The REH activity in the presence of 1000 microM retinyl palmitate was 2.38 +/- 0.18 nmol/mg/h in the homogenate, 1.13 +/- 0.16/nmol/mg/h in membranes and 3.25 +/- 0.26 nmol/mg/h in cytosol. By comparison, REH activity in rabbit liver was 6.58 +/- 0.75 nmol/mg/h. The REH activity in lacrimal gland was not affected by vitamin A deficiency. These data are consistent with the presence of retinyl ester hydrolase activity in the lacrimal gland and provide further evidence that this gland is adapted for metabolism and secretion of retinol.

EVALUATION OF OCULAR IRRITANCY OF HAIR CARE PRODUCTS

AbstractFour hair care products—dandruff shampoo, baby shampoo, and experimental formulations designated I and II—were evaluated for ocular irritancy by Draize testing, biomicroscopic examination, measurement of corneal thickness, and analysis of effects on cell migration rate during healing following topical application. The goal of the study was to determine the utility of a new statistical method for analysis of Draize data, known as time-weighted averaging, and to determine whether increasing the degree of ethoxylation of the surfactant ammonium lauryl ether sulfate (ALES) from 1 mole (formulation I) to 3 moles (formulation II) reduces ocular irritancy. The results of analyses by each method were in agreement, with dandruff and baby shampoos rated as severe and nonirritants, respectively. The experimental formulations were intermediate to these extremes and, based on time-weighted average Draize scores and cell migration rate (40 µm/hr formulation I, 67 µm/hr formulation II, 72 µm/hr control), it is c...