Identification of free radicals produced during phacoemulsification.

PURPOSE: To detect, identify, and quantitate free radicals produced during conditions similar to phacoemulsification cataract surgery. SETTING: Research laboratory at the Biotechnology Center, Utah State University, Logan, Utah, USA. METHODS: All experiments were performed using a Series Ten Thousand phacoemulsifier (Alcon Laboratories) modified to make a 10 mL continuous circulation loop (to increase sensitivity). The irrigating solution was passed through a 3 mL chamber in line with the circulation loop, and electron spin resonance spin trapping methods were used to detect, identify, and quantitate free radical production during phacoemulsification. As an additional indication of hydroxyl radical production, the hydroxylation of salicylate and thiocyanate was detected by high-performance liquid chromatography and spectrophotometry, respectively. RESULTS: The hydroxyl radical was formed when phacoemulsification was performed in the presence of solutions containing spin trap in double deionized water or balanced salt solution (BSS). Hydroxyl radical production was linear with respect to phacoemulsification time. Production of the hydroxyl radical was not observed when phacoemulsification was performed with anaerobic solutions, indicating a requirement for oxygen in radical production. The concentration of trapped hydroxyl radical was reduced in the presence of balanced salt solution with bicarbonate, dextrose, and glutathione (BSS Plus). Upon phacoemulsification, both salicylate and thiocyanate underwent hydroxylation when included in the irrigating solution, confirming the generation of the hydroxyl radical. Additional tests discounted the formation of superoxide or hydrogen peroxide during phacoemulsification. CONCLUSIONS: Hydroxyl radical was produced by phacoemulsification in the presence of aerobic solutions. Hydroxyl radical production was dependent on the presence of molecular oxygen and was not generated as a result of the homolytic cleavage of water. The amount of hydroxyl radical detected was directly proportional to phacoemulsification time and was reduced in the presence of BSS Plus. Other reactive oxygen species such as superoxide, hydrogen peroxide, and ozone were not detected during phacoemulsification under these conditions.

Quantitative method to determine the cohesion of viscoelastic agents by dynamic aspiration.

PURPOSE: To quantitatively determine the cohesion of ophthalmic viscoelastic agents using an in vitro method based on dynamic aspiration kinetics. SETTING: Alcon Laboratories, Inc., Fort Worth, Texas, USA. METHOD: Five viscoelastic agents were tested: Healon GV (sodium hyaluronate 1.4%); Provisc (sodium hyaluronate 1.0%); Healon (sodium hyaluronate 1.0%); Amvisc Plus (sodium hyaluronate 1.6%); Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%). Samples were placed into a tared polypropylene container using a positive displacement pipette. Calibrated vacuum was applied for 2 seconds to a polypropylene tip (inner diameter 0.5 mm) lowered into the viscoelastic sample. The quantity of viscoelastic agent remaining in the container after aspiration was determined gravimetrically. The procedure was repeated at various vacuum levels between 100 and 700 mm Hg. The percentage of viscoelastic agent aspirated was plotted against vacuum pressure. The slopes of these curves indicate the relative cohesion of the viscoelastic sample. RESULTS: The cohesion-dispersion indices (percentage viscoelastic agent aspirated/100 mm Hg) were Healon GV (72.3) > Provisc (46.0) > Healon (31.2) = Amvisc Plus (21.4) > Viscoat (3.4). CONCLUSION: The method provided a quantitative basis for the clinical classification of viscoelastic materials as cohesive or dispersive. The aspiration kinetics profile (curve shape), maximum rate of removal, and vacuum levels at which the bolus removal of the viscoelastic agent started (break point) were useful in characterizing the viscoelastic agents. Because the results agree with the clinical impression of cohesion/dispersion, this method may be used to predict the surgical performance of viscoelastic agents.

New method to measure the retention of viscoelastic agents on a rabbit corneal endothelial cell line after irrigation and aspiration.

PURPOSE: To quantify the surface retention of several ophthalmic viscoelastic agents following irrigation and aspiration (I/A) using a new in vitro method. SETTING: Alcon Laboratories, Inc., Fort Worth, Texas, USA. METHODS: A rabbit corneal endothelial cell line was cultured to confluency in 24-well plates, and the cells were labeled quantitatively with internalized neutral red dye. Five ophthalmic viscoelastic agents were applied to cover the monolayer of cells: sodium hyaluronate (Healon, Provisc, and Amvisc Plus), Formulation A (a dispersive, nonproteinaceous, synthetic polymer), and sodium chondroitin sulfate, sodium hyaluronate (Viscoat). Irrigation and aspiration (with fluid turbulence similar to that encountered in phacoemulsification surgery) were performed on each well for 3 minutes, using 120 mL of balanced salt solution with bicarbonate. dextrose, and glutathione (BSS Plus). The cells were treated with an acidified ethanol solution to extract the dye from the cells left without a viscoelastic cover. The extracted dye was measured by spectrophotometry and compared with the total dye recovered from control cells. RESULTS: The retention value, which represented the percentage of cells with viscoelastic retained on the surface, was calculated as follows: Healon, 7; Provisc, 16; Amvisc Plus, 17; Formulation A, 55; Viscoat, 90. On a nonadsorptive, non-cell surface, the retention values of the five viscoelastics were significantly less than those on cells. CONCLUSION: The results of this experimental model suggest that cohesive viscoelastics are readily removed from the cells, while dispersive viscoelastics are highly retained. In addition, mutual surface interaction (electrical charge and other properties) plays a significant role in determining the retention of viscoelastics on the corneal endothelial cell surface following I/A.

G protein-mediated activation of a nonspecific cation current in cultured rat retinal pigment epithelial cells.

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mM KCl intracellular and 130 mM NaCl extracellular solutions, rat RPE cells possessed both inward and outward K+ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5'-O-(3-thiophosphate) (GTPgammaS, 0.1 mM), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs+-aspartate in the pipette, and was not affected by alterations in the extracellular Ca2+ or Cl- concentration. The GTPgammaS-activated current was found to be permeable to several monovalent cations (K+, Na+, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPgammaS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca2+ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K+ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K+.

Endothelin subtypes: effect on isolated rhesus monkey ciliary muscle.

The effects of endothelin (ET)-1, ET-2, ET-3 and sarafotoxin S6C on the contractile response in the longitudinal and coronal vectors of the isolated rhesus monkey ciliary muscle were studied. Fresh ciliary muscle strips from young and middleaged rhesus monkeys were mounted in an apparatus capable of monitoring contractile force simultaneously in the two vectors. The responses to the ET compounds were measured and compared to those produced with 1 microM carbachol. ET-1 produced contractions of up to 15% of the near-maximum response to carbachol in both vectors of 66% of ciliary muscle strips studied. In responsive strips, the maximal ET-1 induced contraction was approximately equal in both vectors, but the longitudinal vector was approximately 5-fold more sensitive than the circular. All ciliary muscle strips responded reproducibly to carbachol. None of the other ET compounds tested had any effect, suggesting that the ETA receptor may predominate in rhesus monkey ciliary muscle. Because ET-1 induces only weak and interindividually variable contraction in isolated rhesus monkey ciliary muscle strips, ET-1's enhancement of outflow facility with only minimal induction of accommodation in the living monkey may be due to effects directly on the trabecular meshwork. However, the 5-fold greater potency of ET-1 in the longitudinal compared to the circular contractile vector may indicate that selective contraction of the longitudinal portion of the ciliary muscle with consequent deformation of the trabecular meshwork but not the lens is also involved.

Prostaglandin F2 alpha effects on isolated rhesus monkey ciliary muscle.

PURPOSE: The effects of prostaglandin (PG)F2 alpha on the contractile response in the longitudinal and coronal vectors of the isolated rhesus monkey ciliary muscle were studied. METHODS: Fresh rhesus monkey ciliary muscle strips from young and middle-aged animals were mounted in an apparatus capable of monitoring contractile force simultaneously in the two vectors. The responses to PGF2 alpha were measured and compared to those produced with 1 microM carbachol. RESULTS: PGF2 alpha consistently relaxed the carbachol precontracted ciliary muscle in a dose-dependent manner with equal potency and efficacy in both vectors, but it did not alter the tone of muscle strips at resting tension. CONCLUSIONS: The relaxation of precontracted ciliary muscle by PGF2 alpha may help explain how a single topical dose of this prostanoid increases uveoscleral outflow and antagonizes resting myopia in the living monkey.

The effect of muscarinic agonists and selective receptor subtype antagonists on the contractile response of the isolated rhesus monkey ciliary muscle.

There are ultrastructural and histochemical differences between the longitudinal (putatively more relevant to outflow facility) and circular (putatively more relevant to accommodation) portions of the primate ciliary muscle. Oxotremorine, a muscarinic agonist putatively somewhat selective for the M2 receptor subtype, binds preferentially to the longitudinal rather than the circular portion. Aceclidine, a putatively non-subtype selective muscarinic agonist, can dissociate accommodative and outflow facility responses in monkeys and humans. We used the muscarinic receptor subtype antagonists 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), 11-([2-(diethylamino)methyl]-1-piperidinyl)acetyl]- 5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116), and pirenzepine to inhibit contractile responses to the muscarinic agonists carbachol, aceclidine and oxotremorine in the longitudinal and circular vectors of the rhesus monkey ciliary muscle in vitro. Oxotremorine generated dose-response curves that were similar in both the circular and longitudinal vectors and intermediate to those previously reported for carbachol and aceclidine. 4-DAMP (M3 selective) was the most potent inhibitor of contractile responses to all three agonists, with IC50 values ranging from 33 to 68 nM for the circular and from 27 to 63 nM for the longitudinal vector, depending on the agonist used to elicit contraction. Pirenzepine (M1 selective) was > or = 25-fold less potent and AF-DX 116 (M2 selective) was > or = 108-fold less potent at inhibiting contractile responses to all three agonists in either vector, indicating that M3 is the predominant receptor subtype mediating ciliary muscle contraction in both vectors.(ABSTRACT TRUNCATED AT 250 WORDS)

Age does not affect contractile responses of the isolated rhesus monkey ciliary muscle to muscarinic agonists.

In primates, ciliary muscle contraction causes accommodation and facilitates aqueous outflow. In living rhesus monkeys, accommodative, outflow facility, and ciliary muscle movement responses to cholinergic agonists all decline with age. We developed an apparatus to determine in vitro whether the latter is related to intra- or extra-ciliary muscle factors, and whether ciliary muscle contraction in the coronal (putatively more accommodation-relevant) and longitudinal (putatively more facility-relevant) vectors can be dissociated pharmacologically. In fresh ciliary muscle strips, carbachol and aceclidine each induced dose-dependent contraction in the longitudinal and coronal vectors. With neither drug was there any apparent dissociation of the responses in the two vectors. Atropine pretreatment completely prevented a supramaximal dose of carbachol from inducing ciliary muscle contraction in either vector. Ciliary muscle strips responded to several cholinergic agonists as well on day 2 (24-32 hours post-enucleation) as on day 1 (1-9 hours post-enucleation) when kept in a cell culture medium at 4 degrees C. By light microscopy, the general architecture of the ciliary muscle, the muscle bundles, and the single muscle cells appeared normal; however, cellular and nuclear swelling were apparent following the 32-hour culturing period. Contractile responses to near-maximal doses of carbachol and aceclidine did not vary markedly with age in either vector, suggesting that the age-related decrease in ciliary muscle mobility in vivo is due to extra-muscular restrictive factors rather than diminished muscular contractility.

The effect of topical PGF2 alpha on uveoscleral outflow and outflow facility in the rabbit eye.

Prostaglandin F2 alpha (PGF2 alpha) is a powerful ocular hypotensive agent in rabbit, cat, dog, monkey and human. In cynomolgus monkeys, the intraocular pressure (IOP) lowering is due to increased uveoscleral outflow (Fu). Because the anatomy of the rabbit outflow apparatus differs significantly from that of the primate, we sought to determine whether the mechanism of the PGF2 alpha-induced IOP fall was the same. PGF2 alpha tromethamine salt (PGF2 alpha-TS) (50 micrograms) applied to one eye of 14 conscious rabbits produced a significant IOP fall of 7.4 +/- 0.9 mmHg (P less than 0.001). In untreated control eyes, Fu determined from the quantity of intracamerally perfused [125I]albumin found in the ocular and periocular tissues accounted for 5-8% of total aqueous outflow. In 15 unilaterally PGF2 alpha-treated rabbits, after 4-6 hr dosing Fu was 49 +/- 14% higher in the treated than in the contralateral control eyes. Total outflow facility of outflow from the anterior chamber to the general circulation were measured concurrently in 11 rabbits using a two-level constant pressure perfusion and isotope accumulation technique. Both facilities tended to be higher in the treated eyes than in the controls, with a strong correlation between drug-induced changes in total facility and changes in facility of flow to blood (r = 0.85, P less than 0.001). In eight rabbits treated unilaterally with 50 micrograms PGF2 alpha-TS, the fluorophotometrically determined aqueous formation rate was probably not decreased relative to control eyes. Protein levels in the aqueous humor were approximately eight-fold higher in PG-treated vs. control eyes, suggesting a drug-induced compromise of the blood-aqueous barrier.(ABSTRACT TRUNCATED AT 250 WORDS)