Effects of calcium channel blockers on rabbit corneal endothelial function.

The effects of calcium channel antagonists and agents that alter intracellular Ca2+ mobilization on corneal endothelial function have been examined. All experiments, except where specifically designated, were performed in the continuous presence of extracellular Ca2+. Verapamil (at 50 microM) increased the swelling rate of corneas bathed in normal Ringer solution whereas nifedipine and diltiazem (both up to 100 microM) were without effect. The nifedipine analog nisoldipine caused corneal swelling at 10 microM and 50 microM but nimodipine was without effect. When briefly exposed to a Ca(2+)-free solution corneal swelling was enhanced after subsequent exposure to 50 microM verapamil in normal Ringer but not after 50 microM diltiazem in normal Ringer, indicating that Ca2+ entry from the bathing solution into the cell was important and was apparently impeded by verapamil. Cadmium (0.6 and 1 mM) but not nickel (up to 250 microM) caused swelling of corneas bathed in normal Ringer. A Ca2+ channel agonist, BAY-K-8644, alone did not influence corneal thickness but when presented to the endothelium with 50 microM verapamil the swelling rate was much reduced compared to verapamil alone. The agonist, therefore, presumably maintained some Ca2+ channels open in face of the Ca2+ channel blocker. An agent that inhibited the release of intracellular Ca2+ stores (TMB-8) caused an initial corneal swelling over the first 1.5 hr of perfusion but thereafter had no effect on corneal thickness. In the presence of continued extracellular Ca2+ one explanation for the results is that modulation of intracellular Ca2+ by agents that alter plasma membrane transfer of Ca2+ influences apical junction permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood-ocular barrier permeability and electroretinogram after intravitreal silicone oils of varying composition.

Silicone oils, used as long-term retinal tamponades, cause retinal toxicity that may be related to certain ingredients. Specific additives, proven to increase corneal endothelial permeability, were added to a purified oil, and placed into the vitreous of rabbits to assess their effects on the retina. Oils were exchanged for vitreous at constant intraocular pressure to 1 ml oil volume. Blood-ocular barrier permeability was measured with fluorophotometry after intravenous dye, and retinal function was measured using dark-adapted electroretinography (ERG). Each parameter was determined at eight week intervals: this periodicity was chosen to allow any toxicity to develop based upon prior data in the literature. The fluorescein concentration in different ocular compartments indicated an increased aqueous humor fluorescein concentration after pure oil (a non-statistically significant 50% increase) or oil plus long chain additive (a significant 240% increase). After oil plus a linear series of compounds both aqueous humor (2000%) and anterior vitreous fluorescence (8000%) was statistically significantly increased, indicating a breakdown of the blood-aqueous barrier. The height of the b-wave of the ERG was unaffected by any oil in the presence or absence of additive. Overt changes were minimal with oil alone, increased with oil containing linear chain additive, and were extensive with oil with long chain additive.

Perfluorocarbon effects on rabbit blood-retinal barrier permeability.

Studies have been made of the effects of intravitreal perfluoro-n-octane on the permeability to fluorescein of the blood-retinal barrier in rabbits. At day 1 after injection, there is increased aqueous humor fluorescence that reflects the physical disturbance to the eye following injection. From that time through 7 weeks, there is no evidence of any overt toxicity to the blood-retinal barrier. The retention in the vitreous of a small volume of perfluoro-n-octane following its intraoperative use would not be expected to induce a toxic response. Some effect on an already compromised retina cannot, however, be excluded.

Drug interaction with intraocular lenses of different materials.

The uptake and washout kinetics of four drugs representing different classes of ophthalmic medications were measured in intraocular lenses of different materials. The materials ranged from hydrogel lenses to poly(methyl methacrylate) (PMMA), Acrysof (acrylic/methacrylic), and two types of silicone lenses (Chiroflex and AMO SI-18NB). Uptake was determined after seven days of immersion in a large volume of Ringer's solution containing drug concentrations that equaled those found in aqueous humor 30 minutes to one hour after topical administration. Washout was determined after placing lenses in 1 ml of 0.9% saline for 24 hours. Only hydrogel lenses could be digested in acid to measure lens uptake directly. The PMMA, Acrysof, and silicone lenses behaved similarly toward gentamicin and dexamethasone--low uptake (less than 3.5 ng/mg lens) and low washout (less than 4.0%). Their uptake of norepinephrine was lower (less than 0.7 ng/mg lens) but the washout varied from 29% (AMO silicone) to 100% (PMMA and Acrysof). The pilocarpine uptake was the lowest of drugs tested (less than 0.03 ng/mg lens) and the washout varied from 1.5% (acrylic) to 100% (PMMA and Chiroflex silicone). Hydrogel lenses took up the most drug in the following order: dexamethasone greater than pilocarpine greater than gentamicin greater than norepinephrine. Washout was high, ranging from 83% to 98%. Despite the greater uptake and washout, the maximum drug uptake would only provide one-tenth of the greatest aqueous humor concentration that occurs after topical drug administration. Intraocular lenses of the materials tested did not interfere with the intraocular drug pharmacokinetics, nor did the data indicate that presoaking intraocular lenses of these materials in drugs would enhance post-surgical intraocular drug concentrations.

Influence of vehicle and anterior chamber protein concentration on cyclosporine penetration through the isolated rabbit cornea.

The transcorneal penetration of cyclosporine A has been determined from each of three vehicles across isolated cornea into simulated aqueous humor containing either 50 mg % protein (0.5 mg/ml; as found in a normal eye) or 5000 mg % protein (50 mg/ml; as found in an inflamed eye). Cyclosporine entered the corneal epithelium and stroma/endothelium as well as passed through the cornea from an alpha cyclodextrin vehicle. Entry into the epithelium and stroma/endothelium occurred from an ointment vehicle with limited detectable anterior chamber penetration using 50 mg % protein solution in the anterior chamber. From corn oil vehicle, cyclosporine penetrated across the cornea with a permeability equal to that of alpha cyclodextrin vehicle. The concentration of cyclosporine in both corn oil and ointment vehicles is 8 times greater than that in alpha cyclodextrin vehicle resulting in a flux from corn oil vehicle about 7 or 8 times greater than that seen after alpha cyclodextrin vehicle. The amounts retained in the cornea, however, were relatively low after corn oil compared to cyclodextrin. The penetration of cyclosporine from either the cyclodextrin vehicle or ointment was at least doubled in the presence of 5000 mg % protein in the simulated aqueous humor relative to that seen in 50 mg % protein. This data indicates that the (presumed) absorption and binding of drug by the excess protein in the simulated aqueous humor may have removed free cyclosporine from the solution and sustained a high concentration gradient of free solute across the cornea.(ABSTRACT TRUNCATED AT 250 WORDS)

Corneal endothelial toxicity of dapiprazole hydrochloride.

A new adrenergic antagonist designed for topical use to induce pupillary miosis has been tested for direct toxicity on isolated rabbit corneal endothelium. Dapiprazole hydrochloride was perfused across endothelia in the specular microscope at concentrations from 1.25 micrograms/ml to 1000 micrograms/ml. No toxicity was observed, as determined by corneal thickness determinations over a 3 hour perfusion period, until concentrations greater than 125 micrograms/ml were reached. At 250 micrograms/ml a swelling rate of 17.8 microns/hour occurred, and at 500 micrograms/ml the swelling rate was 17.1 microns/hour; with 1000 micrograms/ml inducing a swelling rate of 23.3 microns/hour. It is evident that the drug concentration that reaches the endothelium after topical application has no toxic effect on the cornea, and that the drug should only be used as directed and not used as an anterior chamber perfusate.

Ocular toxicity of subconjunctival gentamicin.

The subconjunctival injection of commercial gentamicin, given either as a single dose or as repeated injections, caused a hyperemic conjunctival response. Simultaneously there was a toxic myopathy induced in the extraocular muscles. The overall response, followed with light microscopy, occurred as a focal infiltrate of polymorphonuclear leukocytes with progression over a few days to a mononuclear cell infiltrate that accompanied fiber degeneration. Injections of each ingredient of commercial gentamicin individually showed that only gentamicin caused the muscle response; methylparaben, EDTA, and sodium bisulfite were without effect. The pH of the solution was eliminated as a contributing factor as was osmolarity of the injection. Increasing recognition is being given to post-operative complications caused by the prophylactic subconjunctival injection of gentamicin following cataract, and other intraocular surgery. The present data offer a mechanistic explanation for many of these toxic findings.

Subconjunctival gentamicin induction of extraocular toxic muscle myopathy.

Subconjunctival injection of commercially available gentamicin, delivered in one of several protocols, caused an acute toxic reaction myopathy of the extraocular muscles. The response began as a small focal infiltrate of polymorphonuclear leukocytes, and as the lesion progressed it assumed a mononuclear cell predominance accompanied by muscle fiber degeneration. The injection of individual components of the commercial preparation (gentamicin, methylparaben, ethylenediaminetetraacetic acid (EDTA) and sodium bisulfite), each at the same concentration as in the parent solution, gave no response except for gentamicin alone. Gentamicin caused acute toxic myopathy when injected away from the muscles and when given as an isotonic solution in saline compared to the hypotonic commercial solution. Solutions at acidic pH not containing gentamicin did not initiate myopathy. Several factors that have been inferred as participating in the toxic response to gentamicin have been eliminated. Gentamicin alone is responsible for the induction of the extraocular muscle myopathy.

Role of toxic ingredients in silicone oils in the induction of increased corneal endothelial permeability.

Silicone oils may induce pathological changes in corneas or retinas by unknown mechanisms but the effects are probably related to certain specific components. Low molecular weight compounds have been implicated in the induction of toxic tissue reactions. Several of these components, that occur as contaminants or by-products in crude silicone oils, were tested for their ability to alter corneal endothelial permeability. In vitro inulin/dextran permeability was measured after one week of in vivo exposure to a non-toxic oil to which various low molecular weight components were added. At least 75% of the anterior chamber volume was replaced with oil +/- additives. A long-chain silanol-terminated polydimethylsiloxane (1000 cps) at 2 mg/ml, tetramethyl-ammonium siloxanolate (a catalyst) at 1 mg/ml and a mixture of a series of linear compounds (MM through MD10M) each at 10 mg/ml all caused a large corneal endothelial permeability increase. A mixture of two short-chain silanol-terminated compounds was less damaging, as was a mixture of a cyclic series. Evidently certain compounds can induce toxic effects on the corneal endothelium whereas other compounds are much less toxic. The linear series and the catalyst, that induce corneal endothelial changes, have been shown to occur in silicone oils.

The effects of intraocular gases on rabbit blood-retinal barrier permeability.

The effects of intravitreal expansile gases, sulfur hexafluoride and octafluoropropane, as well as air and needle insertion alone were assessed by iris fluorescein angiography and blood-retinal barrier permeability determination. Iris angiography, at 3 days after injection, indicated no differences between experimental and paired control eyes. Vitreous fluorophotometry on day 1, day 4 and day 7 after gas or sham injection also showed no differences between paired control and experimental eyes regardless of which experimental condition was employed. The results indicate that these gases are non-toxic to the blood-retinal barrier over a 7 day time course.

Optimal pupil diameter for Scheimpflug slit image photography of the lens in man.

Densitometric analysis of Scheimpflug photography of the lens has been examined as a function of pupil diameter in normal volunteers. On the basis of both the length of the lens waveform and the integral area under the curve of the lens waveform, there is a critical pupil diameter above which the waveform is unchanged. Below this critical diameter the waveform becomes distorted by shortening and a change in shape. The critical pupil diameter is between 6 and 6.5 mm. This result is of importance either in longitudinal studies, where the pupil diameter may not be reproducible, or in studies where other drugs are used which may prevent full pupil dilatation.

Tear potassium contributes to maintenance of corneal thickness.

Isolated rabbit corneas were bathed on their endothelial surfaces with normal Krebs bicarbonate Ringer solution, while the epithelial surfaces were bathed in a basic tear solution containing sodium and potassium. When bathed in basic tear solution alone, corneal swelling occurred at an average of 12 microns/h over a 3-hour period. Corneal swelling occurred at a rate of about 21 microns/h when the epithelial solution was switched from normal basic tear solution to an iso-osmotic K(+)-free basic tear solution. Corneal swelling then slowed, and in the final hour of a 3-hour exposure to K(+)-free tear solution, the corneas deswelled at about 10 microns/h. The data indicate that potassium is a necessary solute for the maintenance of normal corneal thickness. The results suggest that a lacrimal dysfunction that would cause a decrease in the potassium content of tears may influence corneal thickness and also suggest that the inclusion of potassium in artificial tears is important.

Intraocular gas effects on corneal endothelial permeability.

We examined the effects of intraocular gases on the permeability of the rabbit corneal endothelium to inulin and dextran. Volumes of air (0.16 ml), sulfur hexafluoride (SF6) (0.08 ml), and octafluoropentane (C3F8) (0.04 ml) were infused into the anterior chamber at constant intraocular pressure so that all volumes were equal after expansion. The inulin/dextran permeability was statistically decreased by infusion with Ringer, while air caused an 8.4% increase in dextran permeability but no effect on inulin flux. These small effects were of no biological significance. SF6 caused a 16% and 13% increase in inulin and dextran permeability, respectively, while C3F8 caused an 18% increase in both inulin and dextran permeability. Longevity of gas in the anterior chamber appears important in delineating the deleterious effects. The gases per se do not appear toxic but rather disrupt normal physiologic function through physical process.

Perfluorocarbon liquid effects on corneal endothelial permeability.

We investigated the effects of perfluoro-n-octane liquid on the rabbit corneal endothelial permeability to inulin and dextran. Permeability measurements were made either after 10 minute in vitro exposure of the endothelium to 50 microliters of the test liquid or after one week exposure in vivo following injection of 50 microliters of the test fluid into the anterior chamber. Retention of the perfluorocarbon on the in vitro cornea and in the anterior chamber during the appropriate exposure time was visually verified. The corneal endothelial permeability was unchanged after either short or sub-chronic treatment with perfluoro-n-octane indicating an absence of toxicity for this substance should it reach the cornea either intraoperatively or postoperatively.

Intraocular silicone oil effects on rabbit blood-retinal barrier permeability.

Both short- (1 week) and longer-term (7 week) studies have been made on a series of silicone oils to determine their effects on blood-retinal barrier permeability. About 25% of the vitreous humor was replaced with the test oil, and fluorophotometric determinations made after intravenous fluorescein at intervals after oil injection. The short-term studies revealed a uniform disturbance of the eye at 24 hours after oil injection that probably represents the physical disturbance of the eye during injection. For most oils the readings on day 4 and day 7 after oil injection revealed no difference between the experimental and the paired control eye. Adatomed 5000 cps oil and a Japanese 1000 cps oil caused more sustained aqueous humor fluorescein values over the first week. Longer-term studies, with two oils shown in prior studies on corneal endothelium to increase permeability and one oil that had no influence on endothelial permeability, revealed no effects on fluorescein penetration into any ocular compartment. It is apparent that the corneal endothelium offers a more sensitive paradigm for examining the toxicity of oils for predictive behavior of long-term exposure of the retina.

Marijuana-derived material-induced changes in monkey ciliary processes differ from those in rabbit ciliary processes.

The morphologic changes in ciliary processes and the associated intraocular pressure (IOP) were observed in owl and squirrel monkeys after intravitreal (IVT) and intravenous (IV) injections of water soluble marijuana-derived material (MDM). The response in monkeys differed from that reported in rabbits wherein IV injection induced severe ciliary swelling and a significant decrease in IOP. Only moderate swelling occurs in monkey processes after IV injection of relatively high dose of MDM, and this change, which includes disruption of the basal lamina of the pigment epithelium, is not associated with a change in IOP. Severe swelling occurs in the crests of monkey ciliary processes after IVT injection, which is accompanied by a fall in IOP. The difference in the response in monkey versus rabbit ciliary processes after IV injection of MDM may be due to a more compact stroma in the monkey processes.

Absence of drug effects on rabbit blood-aqueous barrier permeability to non-electrolytes.

The effects of either topical epinephrine, norepinephrine or pilocarpine upon the entry of non-transported radioactive solutes into the anterior or posterior chamber of pigmented rabbit eyes was examined after intravenous solute perfusion. Solutes employed were urea, thiourea, sucrose and inulin. They were infused at a rate that maintained constant plasma levels from 1 minute through 5 hours of intravenous perfusion. Anterior and posterior chamber samples were taken at either 5 minutes or 5 hours after infusion of solutes, but in either case at 1 hour after drug administration. Differences between the 5 minute and 5 hour data showed more solute entry with the passage of time, and differences were found between the rate of solute entry at both times based upon molecular size. However, while occasional statistically significant differences were found between certain drug treatments, no drug effects were noted on solute entry into the treated eye either when compared to the contralateral untreated eye or to a separate untreated control group.

Effects of thymoxamine on corneal endothelium.

We have investigated the effects of 0.02 and 0.2% thymoxamine hydrochloride on the isolated rabbit corneal endothelium. The corneal swelling rate, measured by specular microscopy, indicated that 0.02% thymoxamine caused a swelling rate equal to controls while a 0.2% concentration caused a significantly increased swelling rate (34.1 vs 10.3 microns/h; P less than 0.05). The data suggests that the maximum recommended intracameral concentration of thymoxamine be 0.02% in order to allow a 10-fold safety factor for the corneal endothelium.

Hydrogen peroxide effects on ionic and non-ionic permeability of the rabbit corneal endothelium.

Perfusion of the isolated rabbit corneal endothelium with 0.3 mM hydrogen peroxide (H2O2) caused an increased passive permeability to bicarbonate relative to control tissues. This was accompanied by a reduction in the active flux that resulted in a reduced net bicarbonate flux. Perfusion with 0.3 mM H2O2 resulted in a marked increase in the active and net flux of sodium beginning at two hours. By four hours the net sodium flux had increased by nine-fold over control values. Perfusion with 0.3 mM H2O2 resulted in a 16% and 30% increase in endothelial permeability to inulin and dextran, respectively. Suppression of catalase activity by in vivo pretreatment with intravenous 3-aminotriazole (3AT) did not result in an increased sensitivity of the corneal endothelium to 0.2 mM H2O2: both bicarbonate and sodium fluxes were normal. Inhibition of glutathione synthesis with intravitreal buthionine sulfoximine (BSO) increased the sensitivity of the corneal endothelium to 0.2 mM H2O2 only in the case of sodium flux, with a 4.8-fold increase in net sodium flux at 3 hours after initiation of perfusion. Bicarbonate fluxes were unaffected after BSO pretreatment. The data show that ionic and non-ionic fluxes are altered by H2O2, that pretreatment with 3AT has a minimal effect on ion fluxes while BSO markedly alters sodium flux without changing bicarbonate fluxes, and that sodium and bicarbonate movement are not locked in a symport.

Corneal endothelial permeability after anterior chamber silicone oil.

One of six silicone oils, differing in both viscosity and manufacture, was infused into the anterior chambers of rabbit eyes. Polydimethylsiloxane oil, 5000 cps, caused an increased corneal endothelial permeability to inulin and dextran at 24, 96, and 168 hours after placement into the eye. Intraocular pressures were slightly elevated in the experimental eyes, compared with contralateral controls, at 24 and 144 hours after infusion. The effects of five other oils on corneal endothelial permeability were examined 168 hours after infusion. All oils increased permeability and caused thinning of endothelial cells, together with the appearance of a retrocorneal membrane, except Dow Corning Medical Fluid 360. The results indicated that contact of most silicone oils with corneal endothelium rapidly induces physiologic and morphologic changes.