Retinomotor activity and the c-wave of the hypoxic trout retina.

The teleost retina exhibits retinomotor activity in response to changing light intensity. We have shown that hypoxia interfers with normal retinomotor activity in the dark-adapted rainbow trout, so that the retina assumes an essentially light-adapted configuration with the cones contracted, the rods extended toward the pigmented epithelium, and epithelial pigment expanded. These cell movements appear to be correlated with the marked increase in ERG c-wave amplitude which we consistently observe during hypoxia in trout. Since the a-wave is not immediately affected by hypoxia, this increase in c-wave amplitude may be related to the movement of the rods toward the pigmented epithelium, which would cause a greater than normal change in extracellular [K+] near the apical membrane in response to a light stimulus, leading to an increase in c-wave amplitude.

The rabbit lacrimal gland in vitamin A deficiency.

PURPOSE: To investigate the morphology, ultrastructure, and protein secretion of the vitamin A-deficient rabbit lacrimal gland. METHODS: The lacrimal glands of vitamin A-deficient rabbits and age-matched controls were fixed, processed by standard methods, and examined by light and transmission electron microscopy. Protein secreted by the lacrimal gland was analyzed using gel filtration chromatography and electrophoresis. RESULTS: By light microscopy, the glands of experimental and control rabbits were indistinguishable. Electron microscopy showed little effect of vitamin A deficiency on the lacrimal acini, although occasional pyknotic nuclei were observed. The intralobular ductal epithelium was unaffected. Protein concentration and composition were essentially unchanged in lacrimal gland fluid of vitamin A-deficient rabbits compared to controls. CONCLUSIONS: The rabbit lacrimal gland is minimally affected by vitamin A deficiency, suggesting species differences between rabbits and rats in the vitamin A requirements of the lacrimal gland. Normal lacrimal gland structure and function in vitamin A deficiency allow for the prompt secretion of retinol on the restoration of vitamin A to the diet.

Esterification of retinol in lacrimal gland. Evidence for acyl-CoA:retinol acyltransferase activity.

Vitamin A is stored in cells as long-chain fatty acyl esters of retinol. Esterification in many tissues is catalyzed in part by acyl-CoA:retinol acyltransferase (ARAT). Since the lacrimal gland contains stores of retinyl esters, it was the goal of this study to determine whether the lacrimal gland contains ARAT activity. Rabbit lacrimal gland microsomes incubated with 3H-retinol synthesized retinyl esters. The reaction rate was stimulated 30-fold in the presence of a fatty acyl-CoA generating system, producing a mixture of esters including retinyl laurate, retinyl linoleate, retinyl palmitate, and retinyl stearate as determined by reverse-phase HPLC. Retinyl palmitate was synthesized at 1944 pmole/mg protein/30 min, representing 50% of total ester synthesis, and this activity was directly proportional to microsomal protein concentration. In the presence of 180 microM 3H-retinol and 100 microM palmitoyl-CoA, retinyl palmitate was synthesized at 175-220 pmole/mg/min, and the reaction fit Michaelis-Menten kinetics as a function of retinal concentration (theoretical Vmax = 329.4 pmole/mg/min). Lauroyl CoA and stearoyl CoA, but not linoleoyl CoA, were as effective as palmitoyl CoA as substrates for the reaction. The enzyme activity was inhibited by p-chloromercuriphenyl sulfonic acid and Na-taurocholate. The data show that the lacrimal gland synthesizes retinyl esters and that the characteristics of synthesis are consistent with the presence of acyl-CoA:retinol acyltransferase in lacrimal gland.

Corneal endothelial healing rate and the effect of topical retinoic acid.

These studies were undertaken to evaluate wound healing rates of the corneal endothelium in vivo. After insertion of a 26-gauge needle into the anterior chamber of the rabbit eye through the limbus, a 5-0 nylon monofilament was introduced through the needle, and endothelial wounds were made by scratching the cells with the filament. The wounds were photographed with a wide-field specular microscope at various intervals. Montages of the wounds were made, and the areas of the wounds were determined by planimetry. Wound closure occurred rapidly in a linear manner during the first 6 hr after wounding, after which the rate of cell migration decreased. Healing rates (micron2/hr) during the first 6 hr were calculated by linear regression analysis. There was a direct linear correlation between the healing rate and initial wound area. The slope of this line for nine normal (untreated) corneas was 0.093 hr-1. Nine corneas were treated with 0.1% retinoic acid in petrolatum ointment, while eight control corneas received vehicle alone. The slope of healing rate versus initial wound area for treated corneas (0.11 hr-1) was significantly greater than control (0.097 hr-1). This was interpreted as a stimulation of corneal endothelial migration during healing by retinoic acid. As a result of this study, a method for analysis of corneal endothelial healing rate has been developed which can be used for comparison of healing rates among treatments when initial wound area cannot be standardized.

Retinol secretion by the lacrimal gland.

In order to determine the source of the retinol which has been identified in the tear fluid, the lacrimal gland ducts of rabbits and rats were cannulated and the collected lacrimal gland fluid was analyzed by high performance liquid chromatography. Retinol was identified in the lacrimal gland fluid of rabbits and rats, and it is concluded that the lacrimal gland is the source of retinol in the tears. Dose-response studies show that intravenously administered pilocarpine and intra-arterial acetylcholine stimulate secretion of retinol by the lacrimal gland. Intravenous administration of vasoactive intestinal peptide (VIP) also stimulates retinol secretion in a dose-response manner. These observations are similar to the effects of cholinergic drugs and VIP on protein secretion by the lacrimal gland.

A larger corneal epithelial wound closes at a faster rate.

In order to evaluate relationships between healing rates and initial wound area, epithelial wounds were made on rabbit corneas by scraping the epithelium within a 4-, 6.5-, or 8-mm trephine mark. The wounds were stained with fluorescein and photographed during healing. The wounded areas were measured by planimetry. Although larger wounds closed later than smaller wounds, all of the healing curves appeared to be linear. The mean healing rate of the 8-mm diameter wounds (0.91 mm2/hr) was significantly greater than that of the 6.5-mm diameter wounds (0.80 mm2/hr). The 4-mm diameter wounds healed at a significantly slower rate (0.37 mm2/hr) when compared to the 6.5-mm diameter wounds. The authors found a strong positive correlation between the healing rates and the initial wound areas. By comparison, regardless of the initial wound area, the wound diameter decreased at a rate of approximately 0.1 mm/hr, which may explain the dependency of the healing rate on the initial wound area. The healing rate varied considerably between animals with the same diameter wounds, but both eyes of each animal showed a similar healing rate.

The relationship between secretion of retinol and protein by the lacrimal gland.

Secretion of retinol and protein by the rabbit lacrimal gland appear to be closely related, suggesting that they are secreted by the same mechanism. Pilocarpine and vasoactive intestinal peptide (VIP) both stimulate protein and retinol secretion rate in a dose-dependent manner, but the concentrations of retinol and protein in the lacrimal gland fluid are independent of fluid flow at flow rates in excess of 1 microliter/min. Under all conditions of stimulation with pilocarpine and VIP that were studied, the retinol:protein ratio in lacrimal gland fluid of normal rabbits remained constant at 3.3 ng retinol/mg protein. This correlation between retinol and protein secretion by the lacrimal gland suggests that retinol is protein-bound in lacrimal gland fluid. To identify this protein, vitamin A-deficient rabbits were treated orally with 3H-retinyl acetate. Lacrimal gland fluid was collected and analyzed for 3H-retinol and protein. The 3H-retinol in lacrimal gland fluid was identified by reverse-phase HPLC and analysis of protein by gel filtration chromatography showed that this 3H-retinol was associated with protein which eluted from the columns in the 20 kD range.

A comparison of healing of corneal epithelial wounds stained with fluorescein or Richardson's stain.

The effects of fluorescein and Richardson's stain on corneal epithelial wound healing were compared in eyes of rabbits whose corneas had the epithelium removed by scraping or by n-heptanol. One eye of each rabbit was stained with fluorescein and the other eye was stained with Richardson's stain at intervals throughout the healing process, and the wounds were photographed for planimetry and determination of re-epithelialization rate. Corneal thickness was also measured throughout the re-epithelialization. These studies showed that Richardson's stain, as compared with fluorescein, decreases re-epithelialization rate, delays wound closure, and slows the return of the edematous cornea to normal thickness. Therefore fluorescein rather than Richardson's stain should be used to stain epithelial defects in corneal wound healing studies and in the evaluation of the corneal toxicity of chemical agents.

Corneal endothelial morphology in the rat. Effects of aging, diabetes, and topical aldose reductase inhibitor treatment.

Increases in corneal endothelial cell polymegathism and pleomorphism are characteristic of diabetic human and dog corneas. This study investigated the rat as a model for age- and diabetes-related changes in endothelial cell morphology. As in most mammals, aging of the normal rat results in a progressive decrease in cell density as well as reduced numbers of hexagonal endothelial cells and increased coefficient of variation of cell size after age 34 weeks. Streptozotocin-induced diabetes produced an early progressive increase in the coefficient of variation of cell size and decrease in percentage of hexagonal cells so that diabetic rats were significantly different from age-matched normal rats by 24 weeks of age. Topical treatment with the aldose reductase inhibitor, AL 1576, begun immediately after diabetes induction, prevents endothelial cell changes and cataract formation. Topical aldose reductase inhibition also reverses endothelial cell changes when treatment is begun 8 weeks after streptozotocin injection. These results indicate that the rat is a good model for studying diabetes-induced corneal endothelial changes and that topical aldose reductase inhibitors may be effective in preventing or reversing diabetic corneal endothelial cell changes.

Retinoid permeability and uptake in corneas of normal and vitamin A-deficient rabbits.

In vitro perfusion of corneas of normal and vitamin A-deficient rabbits provided a model in which to study the pharmacokinetics of corneal permeability and uptake of retinoic acid and retinol. The permeability coefficients of retinoic acid and retinol were 1.49 x 10(-5) and 0.61 x 10(-5) cm/s, respectively. Removal of the corneal epithelium did not affect the permeability of these lipid-soluble retinoids; however, diffusion through xerophthalmic, vitamin A-deficient corneas was significantly reduced. The corneal uptake of retinoic acid and retinol was reduced by 50% on removal of the epithelium, was nonspecific, and was not affected by xerophthalmia. High-performance liquid chromatography indicated that these retinoids were not metabolized during diffusion through the cornea. These results show that topical application of retinoids is a rational approach to the treatment of such corneal diseases as xerophthalmia and epithelial defects.

Isotretinoin in lacrimal gland fluid and tears.

Isotretinoin (13-cis-retinoic acid) is used in the treatment of severe cystic acne. Adverse ocular reactions, including blepharoconjunctivitis and dry eye symptoms, are frequent side effects of this drug. Our previous observation that retinol is present in tears and lacrimal gland fluid suggests that isotretinoin may also be secreted by the lacrimal gland. Rabbits were treated with isotretinoin, and lacrimal gland fluid was collected from the cannulated lacrimal gland duct. Tears were collected from patients who were being treated with isotretinoin. Lacrimal gland fluid and tears were analyzed by reverse-phase high-pressure liquid chromatography and a peak eluted from each sample, which was identified as isotretinoin. We conclude that the lacrimal gland is able to secrete isotretinoin in addition to retinol and that, in animals and patients treated systemically with isotretinoin, the ocular surface is exposed to the drug via the tear film.

Corneal epithelial wound healing in normal and diabetic rabbits treated with tretinoin.

The rate of corneal epithelial wound healing has been shown to be faster in diabetic than in normal rabbits when the epithelial cells are removed by scraping or freezing; both methods of epithelial removal, however, damage the basement membrane in corneas of diabetic but not normal rabbits. In this study, we compared the rate of wound healing and the increase in corneal thickness in normal and diabetic rabbits in which the epithelial cells were removed with heptanol, a method that does not damage the basement membrane in either group. In addition, the effect of tretinoin on the rate of wound healing was compared in both groups. There was no statistical difference between the rate of epithelial healing in the untreated control and in untreated diabetic eyes. Treatment with tretinoin resulted in a significant increase in the rate of healing in control but not in diabetic eyes. Corneal thickness increased in all groups after epithelial removal, but the increase was significantly less in the corneas of diabetic rabbits at 24 hours. These results indicate that tretinoin may be more effective in promoting epithelial healing in eyes of normal patients than in diabetic patients.

Effects of preservative-free artificial tear solutions on corneal epithelial structure and function.

OBJECTIVES: To test the efficacy of a bicarbonate-containing artificial physiologic tear solution (solution PT) in providing an environment in which the damaged corneal epithelium can recover its normal barrier function and to compare this solution with other available artificial tears. Also, to investigate the effects on the corneal mucin layer and epithelial ultrastructure. METHODS: The corneal epithelial permeability of anesthetized rabbits was increased by exposure to 0.1% benzalkonium chloride. The corneas were then exposed to solution PT, with or without bicarbonate, or one of four commercially available artificial tear solutions for 1.5 hours, followed by a 5-minute exposure to 5(6)-carboxyfluorescein. Frozen sections of the corneas were examined by fluorescence microscopy. The fluorescence intensity (FI) of the epithelium was measured by image analysis. Undamaged corneas exposed to tear solutions were examined by transmission electron microscopy after fixation of the mucin layer with cetylpyridinium chloride. RESULTS: The FI of corneas damaged by benzalkonium chloride was increased threefold above those of undamaged controls. Damaged corneas treated with either of two commercial isotonic tear solutions partially recovered their barrier function, but the FI did not reach control levels. Corneas treated with hypotonic solutions containing ethylenediaminetetraacetic acid (EDTA) did not recover. In contrast, the FI of corneas treated with solution PT returned to control levels. This effect was lost in the absence of bicarbonate. Solution PT and the two isotonic solutions maintained normal corneal ultrastructure and mucin layer. Lack of bicarbonate in solution PT resulted in focal damage to superficial epithelial cells, whereas the EDTA-containing solutions destroyed the first two cell layers and reduced the mucin thickness. CONCLUSIONS: Bicarbonate-containing solution PT is superior to the other tear solutions tested in promoting recovery of the damaged corneal epithelial barrier and maintaining normal ultrastructure. The presence of bicarbonate appears to be essential to this process.

Healing of experimental corneal wounds treated with topically applied retinoids.

We treated experimental corneal epithelial wounds in rabbits with topical retinoids. Treatment with 0.1% all-trans-retinoic acid three times per day resulted in a 21% increase in the healing rate compared to the control eyes. Treatment five times a day resulted in a 35% increase in healing rate. Treatment with topical retinoic acid also promoted corneal deturgescence. Retinyl palmitate, retinyl acetate, retinol, and 13-cis-retinoic acid had no effect on corneal wound healing. These data suggested that topically applied all-trans-retinoic acid may be effective in promoting corneal healing after surgery and in the treatment of persistent and recurring corneal epithelial defects.

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

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 rabbit's 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.

Artificial tear composition and promotion of recovery of the damaged corneal epithelium.

In severe dry eye syndromes the corneal epithelium is compromised with development of punctate erosions and increased permeability. In the present study the ability of artificial tear solutions to promote recovery of the corneal epithelial barrier was determined by measurement of corneal uptake of 5,6 carboxyfluorescein (CF). Corneas of anesthetized rabbits were exposed to 0.01% benzalkonium for 5 min to increase epithelial permeability. The cornea was then exposed to an artificial tear solution for 1.5 h followed by measurement of CF uptake. During exposure to three commercial isotonic, nonpreserved solutions and a solution preserved with polyquaternium-1, CF uptake decreased significantly but did not return to control. No recovery of the epithelial barrier occurred during exposure of corneas to nonpreserved hypotonic solutions. During exposure to an experimental tear solution with an electrolyte composition similar to human tears, buffered with bicarbonate, CF uptake returned to control levels. Bicarbonate is an essential component of this solution because the same formula buffered with borate or without buffer was ineffective in promoting recovery of the damaged corneal epithelium.

The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium.

PURPOSE: The eicosanoid, 15-(S)-hydroxyeicosa-5Z, 8Z-11Z, 13E-tetraenoic acid (15-(S)-HETE), is known to stimulate production of mucin glycoprotein by airway epithelium. This study investigated the effect of 15-(S)-HETE on the mucin glycoprotein secretion by the corneal epithelium. METHODS: To determine the effect of dose, corneas of anesthetized New Zealand White rabbits were treated with 50, 500, or 5,000 nM 15-(S)-HETE in artificial tears for 120 minutes. To determine the time to onset of the response, corneas were treated with 500 or 1,000 nM 15-(S)-HETE in balanced salt solution for periods ranging from 5 to 120 minutes. Corneas were fixed for electron microscopy in fixative containing 0.5% cetylpyridinium chloride (CPC) to stabilize the layer of mucin-like glycoprotein on the corneal surface. The mucin layer thickness was measured by image analysis of electron micrographs. RESULTS: The layer of CPC-fixed mucin-like glycoprotein on the surface of control corneas was 0.46 +/- 0.04 microm thick. After treatment with 15-(S)-HETE, the thickness of the mucin layer increased to 0.64 +/- 0.1 microm at 50 or 5,000 nM HETE and as much as 1.02 +/- 0.2 microm in response to 500 nM HETE. Mucin thickness reached a statistical maximum of 0.59 +/- 0.1 microm after only 5 minutes of exposure to 500 or 1,000 nM HETE. CONCLUSIONS: Exposure of the cornea to 15-(S)-HETE causes a rapid-onset increase in the thickness of a layer of mucin-like glycoprotein on the surface of the corneal epithelium. This supports previous reports that corneal epithelial cells produce mucin and suggests that treatment with topical 15-(S)-HETE may be effective in treating ocular surface mucin deficiency in dry eye syndrome.

A redesigned corneal holder for the bovine cornea opacity and permeability assay that maintains normal corneal morphology.

The bovine cornea opacity and permeability assay (BCOP) has been in use for nearly 10 years but has not been submitted for regulatory approval. In previous reports we have presented corneal hydration and endothelial damage as additional endpoints in this assay and have suggested that the design of the BCOP's corneal holder should be modified. The standard holder used in the BCOP assay induces physical damage to the cornea because it contacts clear cornea causing edge damage to the epithelial, stromal and endothelial layers. Second, by forcing a curved, oval-shaped bovine cornea into a flat, circular opening, corneal wrinkling occurs which can alter the cornea's optical characteristics and, most importantly, induces endothelial damage. We now report on a redesigned BCOP corneal holder that clamps onto the sclera, maintains normal corneal shape and does not cause damage to the endothelium. This ensures that irritancy tests are conducted using healthy, anatomically normal tissue. Tests of this holder using acetone, trichloroacetic acid, isopropanol and benzalkonium chloride show that it is now possible to evaluate effects of chemical substances on the endothelium. The effects of these compounds on corneal opacity and hydration in the new holder are similar to their effects on the cornea in the standard holder.

Corneal permeability in a redesigned corneal holder for the bovine cornea opacity and permeability assay.

The bovine cornea opacity and permeability assay (BCOP) is a proposed alternative to the Draize rabbit test for potential eye irritants. In the standard BCOP, bovine corneas are mounted in a holder on a flat surface between two identical chambers. The flat configuration of the standard holder does not conform to the normal curved shape of the bovine cornea and it comes into direct contact with the cornea tissue. Mounting corneas in this holder causes extensive damage to both epithelial and endothelial corneal cell layers. Our laboratory has designed a new holder that allows the cornea to maintain its natural curvature and does not damage the cornea. Previous tests, using both the new and standard holders, and comparing corneal opacity, hydration and endothelial morphology, have shown that the new holder is a significant improvement over the standard holder. The present study extends the comparisons of the new and standard holders to measurement of corneal fluorescein permeability. The permeability (ng/cm(2)/min) of intact corneas, corneas with no epithelium, and corneas treated with 1% NaOH, isopropanol, acetone, 30% trichloroacetic acid or 30% sodium dodecysulfate for either 1 or 10 min was determined by measuring fluorescence of samples taken from the endothelial chamber after 90 min epithelial exposure to 0.04% sodium fluorescein. In all trials, the redesigned holders yielded not only lower permeability measurements but also decreased measurement variability. The data provide further evidence that the new holder is an improvement over the standard holder and should be incorporated into a new protocol for the BCOP.

Corneal opacity, hydration and endothelial morphology in the bovine cornea opacity and permeability assay using reduced treatment times.

The purpose of this study was to determine whether the standard bovine cornea opacity and permeability (BCOP) assay exposure time of 10 minutes overestimates the ocular irritancy of chemical substances. Corneas were subjected to BCOP protocol following 30-second and 1-minute exposures to irritants. Corneal opacity and hydration (mg H(2)O/mg cornea) were then measured and compared to data obtained after 10 minute irritant treatments. For most test substances corneal opacity and hydration were lower following reduced exposure times. It is suggested that using shorter exposure times in BCOP protocol may be more predictive of human response to ocular irritants, since irritants are usually in brief contact with the ocular surface during accidental exposure. A second purpose of this study was to examine effects of irritants on the corneal endothelium. Corneas were treated according to BCOP protocol following exposure to irritants for 1 or 10 minutes. The endothelium was stained with Alizarin Red and trypan blue, and examined using light microscopy. Severe irritants, such as NaOH and trichloroacetic acid, cause endothelial cell death. It was also determined that simply mounting the cornea in the BCOP assay holders caused damage to 20% of the endothelial cells. Because the endothelium is essential for normal corneal transparency and hydration, it is suggested that examination of the endothelium be added to the BCOP assay and that optimization of the assay will require modification of the cornea holders.