Cytotoxicity evaluation of multipurpose contact lens solutions using an in vitro test battery.

PURPOSE: Many in vitro alternatives to eye irritation testing are not mechanism-specific and do not employ ocular cell lines. We have developed an effective and reliable test battery that reveals toxicity mechanisms of contact lens solutions on cell metabolism and proliferation. METHODS: Cytotoxicity endpoints were quantified using bovine corneal epithelial cultures in 96-well microplates. A kenacid blue assay provided information on total cell protein, while lactate production and alamarBlue assays served as indicators of aerobic/anaerobic metabolism and redox state of cells grown in serum-free Dulbecco's modified Eagle's/Ham's F12 medium (DMEM/F12). Concentrations (% v/v) causing 10-90% inhibition of the control assay responses were used for correlations with in vivo data. RESULTS: Cytotoxicities of contact lens solutions correlated better with irritant symptoms than with corneal staining, and were ranked as follows: Lens Plus << Opti-Free < or = ContaClair < or = ReNu. Lens Plus was not toxic to cell glycolysis, respiration, and proliferation for up to 20% v/v. However, the multi-purpose solutions inhibited these endpoints in a concentration-dependent manner. Opti-Free and ReNu, containing Dymed and Polyquad (ammonium surfactants), showed non-specific cell inhibition. The lactate production assay had a flatter log concentration-response curve than the other two assays. CONCLUSIONS: The proposed biochemically-based test battery using the target corneal epithelium has the potential to be a simple and effective method for screening and defining toxicity profiles of contact lens care solutions. The model can be applicable to small- or large-scale testing programs and research and development of new ocular products.

A technique for quantitation of protein deposits on rigid gas permeable contact lenses.

PURPOSE: The purpose of this study was to develop a method for quantitating protein on rigid gas permeable (RGP) lenses and apply it to worn lenses. METHODS: We built a video microscope and wrote software to measure light absorbance by contact lenses before and after protein staining with Coomassie brilliant blue. We corrected for the temporal stability and spatial uniformity of the system, and set the iris aperture so that both lens surfaces could be simultaneously focused. We examined four RGP lens types worn by 22 patients. Standard curves were prepared with plastic discs spiked with dialyzed Coomassie blue-stained bovine serum albumin. RESULTS: The method was linear (R2 = 0.99) from 14 to over 100 microg protein per image and independent of dioptric power from -6 to +14 diopters. Protein quantities on worn Equalens II, Advent, Quantum II, and Fluoroperm 92 lenses were not significantly different (123 +/- 36, 111 +/- 28, 110 +/- 23, and 83 +/- 15 microg/lens; means +/- SEMs, P > 0.7). Patients differed (P < 0.05) in protein deposition, independently of lens type, and fit a Poisson distribution. DISCUSSION: The method is adequate for quantitating protein on RGP lenses or for examining the efficacy of cleaning regimens or care systems. However, because of the non-Gaussian distribution of patient protein deposits, paired or cross-over experimental design and testing is recommended for studying protein deposition in clinical trials.

Limitations of in vitro contact lens dehydration/rehydration data in predicting on-eye dehydration.

PURPOSE: The purpose of this study was to use in vitro dehydration and rehydration data to model the predicted hydration changes that may occur as a soft contact lens loses and gains water on the eye between blinks. METHODS: Using a recent in vitro data set for four lens materials dehydrated and rehydrated in saline, we derived a mathematical model to describe dehydration and rehydration time courses. The model further combined the dehydration and rehydration data iteratively, as a function of blink frequency, and pre-lens break-up time. RESULTS: The model showed that reduced break-up times or decreased blink frequencies significantly affected dehydration rates and steady state dehydration for lenses of a variety of water contents. However, the model did not agree with the commonly accepted clinical belief that high water content lens materials dehydrate more than low water content materials. DISCUSSION: The discrepancy of the model with historical observations may be accounted for by one or more of the following factors, which were not accounted for in the present model: 1) temperature dependent dehydration (as a lens is taken from a room temperature vial and warmed when placed on the eye); 2) the colloid osmotic pressure, ionicity, pH, and chemical potential of tears (compared to saline); and 3) dehydration by other non-evaporative mechanisms.

New cryoprotectant for cryorefractive surgery.

Cryorefractive surgeries, keratomileusis, keratophakia, and epikeratophakia cause destruction of keratocytes, which may result in postoperative corneal haze. We examined the effects of two cryoprotectants on keratocyte survival following freeze injury. We compared the ability of CPTES and the standard cryoprotectant KM-26 to prevent keratocyte death by altering the length of time corneal tissue was exposed to the cryoprotectant. When corneal stroma was immersed in CPTES for five minutes prior to freezing, 66.5% of the keratocytes survived; when tissue was immersed in KM-26 for the same length of time, 27.5% survived (P less than .01). Immersion for one to 30 minutes in CPTES prior to freezing produced keratocyte viabilities that were 40% to 80% of those of fresh, unfrozen tissue; immersion in KM-26 produced keratocyte viabilities of 20% to 60%. We compared the ability of these cryoprotectants to reduce corneal haze following freeze injury using our rabbit model of lamellar keratoplasty. The postoperative data were comparable to those in the cell culture experiments. Based on our findings in rabbit corneas, a cryoprotective medium such as CPTES may promote cell survival and thereby speed recovery from cryorefractive procedures in humans.

Effects of temperature and conditioning on contact lens wetting angles.

Because wettability is not always examined under standard conditions, we investigated the temperature dependence of saline wettability on unconditioned and conditioned polymethylmethacrylate (PMMA), cellulose acetate butyrate (CAB), and three silicone acrylate lens materials. Sessile drop contact angles were measured in a humidity chamber at 23 degrees C and 34 degrees C using laser-assisted contact angle goniometry. In separate experiments, saline-stored and preconditioned lenses were examined either with or without rinsing. Sessile drop contact angles at 34 degrees C were within 2 degrees to 5 degrees of the room temperature values for both conditioned and unconditioned lenses, demonstrating a negligible temperature dependence. At both temperatures, the conditioned PMMA, CAB, silafocon A, and pasifocon C lenses wet slightly better, by 1 degree to 12 degrees, than unconditioned lenses. However, this increase was only significant with PMMA and silafocon A (P less than 0.05) and reversed when the preconditioned lenses were rinsed repeatedly in saline and reexamined. The results suggest that for these materials: 1) in vitro saline contact angles do not approach those seen on the eye, and this discrepancy can not be explained by temperature or conditioning; and 2) conditioning does not increase material wettability but merely forms a temporary hydrophilic interface that is more wettable than the lens material.

Studies on initiation of silicone elastomer lens adhesion in vitro: binding before the indentation ring.

We studied silicone elastomer lens binding in vitro to determine what factors may influence its development on the cornea or corneosclera. Lens binding to corneas was not influenced by corneal toricity (0-20 D), corneal fitting relationship (2 D steep to 4 D flat), mucin (2 or 5%) in the tear-bath, or transcorneal pressure (11-22 mm/Hg). In isolated corneas or in whole eyes, transient intraocular pressure changes did not influence keratometry readings, ruling these out as potential mechanisms for corneal binding during sleep. Corneoscleral preparations were also examined to simulate a decentered lens. Corneoscleral binding occurred with a significantly (P less than 0.001) greater frequency than corneal binding and was not influenced by corneal toricity, corneal fitting relationship (up to .5 mm steeper than K), or mucin concentration. Unlike the final stages of clinical lens adhesion, the binding we observed permitted lateral lens movement and occurred without leaving an indentation ring. These findings may suggest that the system models the initiation of corneoscleral binding, involving decentration and suction onto the corneoscleral junction. Corneal binding, however, cannot be explained by a chemical attraction between the silicone elastomer lens surface and cornea, with or without mucin interaction, and must be accounted for by other factors found in vivo.

Contact lens-induced edema in vitro. Pharmacology and metabolic considerations.

The effects of physiologic and pharmacologic manipulations on contact lens-induced edema were studied. In isolated superfused rabbit corneas bathed in Ringer's solution and covered with large-diameter polymethylmethacrylate (PMMA) lenses, corneal swelling rates of 17-26 microns/hr (versus -5-5 microns/hr in paired controls) were observed. Neither the calcium antagonist diltiazem (10(-4) M), the glucocorticoid dexamethasone (10(-7) M), the glucose substitute fructose (20 mM), nor 0.5 mM adenosine and 0.3 mM reduced glutathione mitigated the edema. Lens-induced edema was 25 microns/hr in corneas bathed at pH 8.2 and decreased to 9 microns/hr at pH 7.0. In corneas without lenses, however, decreasing the pH from 7.4-7.0 caused significant swelling (P less than 0.05). The pyruvate dehydrogenase stimulant sodium dichloroacetate (3.2 mM) on the tears side ameliorated the edema, and its congener, 3.2 mM 2-chloropropionate, was less effective. These latter agents are known to relieve lactic acidosis systemically and had no significant effect on corneas without lenses. In tissues bathed with 20 mM lactate Ringer's, normal thickness was maintained in both control and PMMA-treated corneas throughout the 3-hr period. These findings suggest that the contact lens-induced edema does not involve the acute cytotoxic mechanisms seen in severe tissue ischemia or hypoxia. The edema appears to result in part from acidosis but mainly from stromal lactate accumulation.

Long-term storage of frozen lenticules for cryorefractive surgery.

We studied freezing rates, cryoprotectants, and storage times on keratocyte viability, using rabbit corneal buttons incubated in either KM-26, CPTES, K-Sol, or TC 199 for 30 minutes at 4 degrees C. Using a controlled freezing rate (2 degrees/min to -40 degrees C), viabilities were 69 +/- 7% for KM-26, 113 +/- 21% for CPTES, 0.1 +/- 0.08% for K-Sol, and 0 +/- 0% for TC 199. The KM-26 and CPTES were further studied in corneas stored one to 30 days at -80 or -196 degrees C; CPTES had a better cryoprotective efficacy over one, three, and seven days of -80 degrees storage, and at liquid nitrogen storage temperature (-196 degrees) over one, 14, and 30 days storage. The findings demonstrate the superiority of CPTES. It provides better viability than KM-26 under similar conditions, and may enable long-term frozen storage of lenticules for later use in cryorefractive surgeries, with minimal loss of keratocyte viability.

Proton NMR relaxation in hydrogel contact lenses: correlation with in vivo lens dehydration data.

A study of hydrogel contact lenses was undertaken to determine whether NMR relaxation data can be used as a predictor for on-eye lens dehydration. Proton NMR relaxation times (T1 and T2), were determined for a series of contact lenses for which on-eye dehydration data were also available. NMR relaxation times were found to depend upon lens water content, but the dependence was not monotonic. T1 values varied between 100 and 800 msec, and T2 values varied between 6 and 85 msec for the lenses studied. In this study, the NMR signal and corresponding relaxation times are average values, derived both from lens water protons as well as from exchangeable polymer protons. A simple analysis of the data indicates that the mobility of these protons varies by more than a factor of 10 for the lenses studied. A test for linear correlation between NMR relaxation rate, 1/T1 and relative change in lens water mass, % delta mw gave r = -0.830 for all data, and r = 0.904 if one lens was excluded.

Contact lens-induced edema in vitro. Ion transport and metabolic considerations.

The relationship of contact lens-induced edema to epithelial and endothelial function was determined in isolated superfused rabbit corneas. Placement of a polymethyl methacrylate (PMMA) contact lens on the cornea caused swelling rates of 15-28 microns/hr compared to 0-6 microns/hr in paired control corneas. The edema increased with temperature (P less than 0.01). PMMA-induced swelling was significant in: 1) bicarbonate-free Ringer's solution; 2) chloride-free Ringer's; 3) 0.3 mM furosemide-treated corneas; and 4) deepithelialized corneas. The swelling did not occur in corneas with silicone oil replacing the endothelium to block fluid uptake. The effluent aqueous bathing fluid from edematous corneas did not induce edema in normoxic corneas. These studies demonstrate that contact lens-induced edema depends on metabolism, involves a significant stromal contribution, and requires fluid absorption across the endothelial layer, but is not a direct result of epithelial and endothelial ion transport inhibition.

Effects of sodium lactate on isolated rabbit corneas.

Corneal stromal lactate accumulation may result from epithelial hypoxia and contact lens wear, but the possible corneal toxicity of lactate has not been reported. Isolated superfused whole rabbit corneas were examined for thickness changes during exposure to neutral sodium lactate (NaL) or excess sodium chloride (NaCl) in Krebs-bicarbonate Ringer's solution for a 3-hr period. Placed in the tears side bath, 5 mM NaL significantly thinned corneas (swelling rates of 1 +/- 1 micron/hr in Ringer's controls vs -11 +/- 1 micron/hr in lactate-treated corneas; mean +/- SD). Excesses of 5 mM NaCl had essentially identical effects (0 +/- 1 micron/hr in controls vs -13 +/- 3 micron/hr in experimentals). When placed on the aqueous side of normal-thickness corneas, neither 20 mM NaL nor 20 mM excess NaCl affected corneal thickness, but both solutions stimulated endothelium-mediated deswelling in preswollen deepithelialized corneas. When "loaded" into the stroma of deepithelialized corneas, Ringer containing 20 mM lactate caused more swelling than Ringer's alone (491 +/- 18 microns in controls vs 558 +/- 20 microns in loaded corneas; mean +/- SEM). A similar swelling occurred when 20 mM excess NaCl was loaded into the stroma (483 +/- 15 vs 565 +/- 20 microns in controls and loaded corneas, respectively), due to fluid uptake into the hypertonic stroma across the endothelium from the aqueous side (Ringer's) bath. Corneas both loaded and superfused with either NaL or excess NaCl swelled and subsequently deswelled similar to controls swollen and superfused in Ringer's.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 6. Trans-6-[2-(substituted-1-naphthyl)ethyl(or ethenyl)]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-ones.

A variety of trans-6-[2-(substituted-1-naphthyl)ethyl(or ethenyl)]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-ones were prepared and, upon conversion to their 3,5-dihydroxy carboxylates, were found to have good inhibitory activity against the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-determining enzyme in cholesterogenesis. The most active compounds are 2,4,6- and 2,4,7-trichloro derivatives and would be expected to display about the same potency as the standard compactin upon resolution.

Evaluation of variation in oxygen transmission in rigid contact lens extended wear.

Variation in thickness is known to affect the Dk/L of rigid gas permeable contact lenses. Our study was designed to evaluate the variability of central and average thickness of Oxyflow EW lenses. In addition, the relationship between overnight swelling of the central cornea and Dk/L was examined to determine whether Dk/L calculated with central lens thickness correlated better with lens-induced edema than Dk/L calculated with average lens thickness. The results showed marked variation among lens center thicknesses for all powers (-6 D, -2 D, +2 D, +6 D) examined, and, as expected, more variation for centrally calculated Dk/L with high minus lenses. Average Dk/L did not vary significantly with power. Average and central Dk/L values had a near 1:1 relationship for -2 D lenses for the base curve (7.8 mm) ordered; but only when power was kept constant was a good correlation seen between central and average Dk/L. A one-patient study evaluating the overnight central swelling response to three +6 D and three -6 D (high, medium, and low center thickness) lenses showed that central Dk/L correlated better with lens-induced edema than average Dk/L. Thus, specifying central thickness on lens orders appears to be a primary variable influencing extended wear edema at night. Peripheral Dk/L, however, may influence corneal physiological variables other than central edema and warrants further study.

Parameter and environmental influences on rigid contact lens wettability.

The present investigation was designed to determine the effect of lens parameters and lens environment on measurements of contact angle. The sessile drop contact angle of saline on four rigid [polymethyl methacrylate (PMMA) and silicone/acrylate] contact lens materials was examined with a Ramé-Hart goniometer to determine how front surface radius, drop size, time after drop placement, humidity, and desiccation affect measurements of lens wettability in vitro. Contact angles of Silafocon A and PMMA were relatively uninfluenced by front surface radii between 7.7 and 8.85 and 7.3 to 8.8 mm, respectively. Contact angles of Pasifocon C and modified PMMA were slightly but significantly influenced by front surface radii between 6.4 and 7.5 mm. For drop volumes from 2 to 20 microliter, all materials yielded contact angles, which were unaffected by drop size. The contact angle of lenses stored in the hydrated or dehydrated state was not affected by chamber humidity between 31 and 76%. In the ranges tested, drop size, humidity, and hydration had no significant effect on the contact angle within 1 to 6 min after drop placement. In addition, surface scratches had no effect on lens wettability. The results suggest that goniometry on contact lens surfaces, for the most part, is uninfluenced by lens parameters and environmental conditions.

Laser-assisted contact angle measurements.

A laser beam, aimed at the solid-liquid-air interface of a sessile drop on a contact lens, produces two lines of diffraction which are theoretically normal to the profile of the fluid and solid surfaces at the point of contact. In the present experiments, the beam and the diffraction patterns were projected on a flat screen perpendicular to the beam, and the angle was measured as a contact angle. The angle was measured on curved surfaces (contact lenses) of 6.5 to 8.9 mm radius and for each material studied, the angle: (1) was independent of the front surface radius, (2) was independent of droplet volume at 2 and 10 microliter, and (3) decreased with time after drop placement. The laser-derived measurements correlated well with contact angles measured by goniometry on contact lenses and a variety of other materials (r = 0.86; p less than 0.0001). Because the diffraction lines are straight and are generated at the point of contact, the angle formed by them is measured easily with a protractor, yielding a convenient and reproducible contact angle measurement. An application of the method was demonstrated by measuring contact angles for saline-containing 0 to 2% bovine serum albumin or bovine submaxillary mucin on Silafocon-A (Polycon II), Pasifocon C (Paragon EW), and polymethyl methacrylate (generic PMMA and Paragon 18) lenses. On each material, physiological concentrations of mucin, but not of albumin, significantly (p greater than 0.01) decreased contact angles by 8 to 15 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Lovastatin, an inhibitor of cholesterol synthesis, induces hydroxymethylglutaryl-coenzyme A reductase directly on membranes of expanded smooth endoplasmic reticulum in rat hepatocytes.

Lovastatin is a potent competitive inhibitor of the rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [HMG-CoA reductase; (S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. We determined the subcellular distribution of HMG-CoA reductase at high resolution by means of immunoelectron microscopy on ultrathin frozen liver sections of rats treated with lovastatin and cholestyramine. High concentrations of reductase were located on the outer (cytoplasmic) surfaces of smooth endoplasmic reticulum (SER) membranes induced in hepatocytes by acute drug administration. The enzyme was specifically localized over the whorled SER membranes and was absent from nonwhorled SER, rough endoplasmic reticulum, and peroxisomes. Intense HMG-CoA reductase labeling was only observed in hepatocytes containing high levels of HMG-CoA reductase activity; no staining was detected in untreated livers. These observations show that HMG-CoA reductase is induced as an integral component of the SER membranes that form in rat hepatocytes subsequent to lovastatin treatment and suggest that the formation of SER whorls in rat hepatocytes is due to mechanism-based effects of lovastatin.

Hydroxymethylglutaryl-coenzyme A reductase exhibits graded distribution in normal and mevinolin-treated ileum.

Because the small bowel is a site of significant cholesterol synthesis, we determined the ileal distribution of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of the cholesterol biosynthetic pathway. Immunofluorescence microscopy on unfixed snap-frozen sections of ileum and jejunum from untreated rats or dogs showed HMG-CoA reductase in the absorptive villus epithelial cells and this appeared to be strikingly localized in their apical cytoplasm. This pattern of HMG-CoA reductase staining approximated a gradient along the villus-crypt axis with the distal villi labeling most intensely. Treatment of rats with mevinolin and/or cholestyramine for 12 days induced a 5- to 11-fold increase in ileal HMG-CoA reductase activity, and yielded a higher intensity of immunostaining without altering the pattern of enzyme distribution observed in control intestines. Also, rats with maximal induction of ileal HMG-CoA reductase exhibited a twofold increase in the number of epithelial villus cells containing prominent stacks of smooth-surfaced membranes in their apical cytoplasm as seen with electron microscopy. These observations suggest that the distal villus absorptive epithelial cells of the ileum contain high concentrations of HMG-CoA reductase, and therefore might be capable of contributing significant quantities of cholesterol to the circulation.

Contact lens-induced edema in vitro--amelioration by lactate dehydrogenase inhibitors.

Isolated rabbit corneas bathed in Krebs-bicarbonate Ringer solution were observed for thickness changes after a 90 minute equilibration period. Control corneas swelled an average of 0.5 micron/hr, and placement of a polymethylmechacrylate (PMMA) contact lens on the epithelial surface caused the corneas to swell 24.5 microns/hr, an effect similar to 0.5 mM epithelial cyanide exposure. The pronounced swelling induced by PMMA lens placement was much less however, in the epithelial presence of 3.2 mM sodium oxalate (3.22 microns/hr) or 3.2 mM sodium oxamate (5.38 microns/hr). An equiosmotic excess of 4.8 mM NaCl was least active (15.89 microns/hr). On normal isolated corneas (without contact lenses), the Ringer containing an excess of 4.8 mM NaCl significantly deswelled the corneas (-13.44 microns/hr), which contrasted with oxalate and oxamate containing Ringer solutions (1.17 and 1.33 micron/hr respectively). The present study supports the notion that contact lens-induced edema results from stromal lactate accumulation, and suggests a potential alternative to osmotic therapy for its amelioration. These LDH inhibitors, in the concentrations used, have no acute osmotic or toxic effect on normal corneas in vitro.

Contact lens surface changes after exposure to surfactant and abrasive cleaning procedures.

Proper lens maintenance is required if contact lens wear is to be successful. Poor compliance or inadequate cleaning may lead to contact lens failures and potentially damage to the eye. With phase contrast microscopy, we addressed the effect of cleaning systems on the physical integrity of a surface-modified lens--the Silcon contact lens. Several cleaners were evaluated for their ability to clean Silcon lenses with minimal damage to the lens surface. The data demonstrated that: all cleaning techniques alter the surface appearance; scratches develop more readily on lenses received with surface irregularities; wettability does not correlate with the extent of surface scratching; recommended cleaning procedures do not directly alter the wettability of the contact lens material; and phase contrast microscopy may be a useful addition to laboratory quality control.