Ultrastructural changes in rabbit ciliary body after extraocular mitomycin C.

PURPOSE: To study the ultrastructural changes in ciliary body epithelium of the rabbit eye after subconjunctival injections of mitomycin C. METHODS: One eye of six New Zealand white rabbits was given a subconjunctival injection at the 12-o'clock position with 0.005, 0.02, 0.08, 0.1, 0.12, or 0.16 mg mitomycin C. The fellow eye was given a subconjunctival injection of balanced salt solution. Two weeks after treatment, the eyes were enucleated, and the ciliary body was exposed and submerged in fresh 4% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 degrees C. Electron microscopy of the ciliary body was performed at two sites: the injection site (12-o'clock position) and 180 degrees away (6-o'clock position). RESULTS: At dosages of 0.1 mg and higher, ciliary body epithelial cells beneath the injection site were thinned. There were vacuoles and expansion of intracellular and intercellular spaces. Plasma membrane infoldings were disrupted, and the apical membrane was thinned. Mitochondria and nuclei were normal. Ciliary body epithelium at 6-o'clock position showed only mild architectural distortion of the plasma membrane infoldings. Eyes that received lower doses of mitomycin C (0.005 mg, 0.02 mg, and 0.08 mg) and balanced salt solution showed normal ciliary body epithelium at the injection site and 180 degrees away. CONCLUSIONS: Subconjunctival injection of mitomycin C in the rabbit produces dose-dependent localized ultrastructural changes of the ciliary body epithelium.

The cardiovascular, pulmonary, and ocular hypotensive effects of 0.2% brimonidine.

OBJECTIVE: To compare the cardiovascular, pulmonary, and ocular hypotensive effects of 0.2% brimonidine tartrate with those of 0.5% timolol maleate, 0.25% betaxolol suspension, and brimonidine vehicle. DESIGN AND PATIENTS: A single-center, double-masked, randomized, crossover study of 24 young, healthy men. INTERVENTIONS: Baseline heart rate, blood pressure, respiratory rate, and intraocular pressure were recorded at hour 0. At hour 2, heart rate, blood pressure, respiratory rate, and forced expiratory volume in 1 second were measured and a 15-minute treadmill test performed. Hour 0 measurements were repeated at hour 4. On four subsequent visits, we instilled one drop of a study medication into each eye after the baseline measurements at hour 0. RESULTS: Timolol reduced resting (-5.3 to -6.5 beats/min, P < or = .004) and exercise-induced heart rate (-4.3 to -13.6 beats/min; P < or = .022) compared with brimonidine, betaxolol suspension, and brimonidine vehicle. At hour 4, brimonidine reduced resting systolic blood pressure compared with all other study medications (-5.2 to -7.3 mm Hg; P < or = .024). Timolol reduced systolic blood pressure during exercise and brimonidine reduced systolic blood pressure during recovery more than betaxolol suspension and brimonidine vehicle (-5.1 to -7.7 mm Hg; P < or = .033; and -5.4 to -6.0 mm Hg; P < or = .002, respectively). Mean respiratory rate and forced expiratory volume in 1 second were not significantly altered by any study medication. At hour 4, brimonidine lowered intraocular pressure as well as timolol and better than betaxolol suspension (-1.9 mm Hg; P < .001) or brimonidine vehicle (-1.8 mm Hg; P < .001). CONCLUSIONS: The cardiopulmonary effects of 0.2% brimonidine were limited to a slight reduction in systolic blood pressure during recovery from exercise and at 4 hours after instillation. The ocular hypotensive effect of brimonidine was comparable to that of timolol and greater than that of betaxolol suspension in this patient population.

Anterior lamellar sclerectomy for nanophthalmos.

A 38-year-old woman with nanophthalmos suffered bilateral acute angle closure glaucoma. Peripheral iridotomies were unsuccessful in maintaining low intraocular pressure in either eye. The left eye had probable ciliary block glaucoma and was treated with subsequent pars plana vitrectomy and trabeculectomy. Further postoperative complications included a serous choroidal detachment treated by a lamellar sclerectomy in three quadrants with sclerotomies and drainage of suprachoroidal fluid. These procedures were successful in achieving a well-formed left globe with normal intraocular pressures. The right eye, however, continued to manifest elevated intraocular pressure despite peripheral iridoplasties. Before further intraocular surgery in the right eye, prophylactic lamellar sclerectomies in each quadrant, as well as sclerotomies in the inferior two quadrants, were performed in the hope that preplacement might reduce postoperative sequelae of anticipated filtering surgery and/ or vitrectomy. Surprisingly, the lamellar sclerectomies alone resulted in excellent intraocular pressure control. The pathogenesis of nanophthalmos is discussed in view of this finding.

Effect of cytochrome b5 on the transbilayer distribution of phospholipids in model membranes.

The transbilayer distribution of phosphatidylethanolamine was assessed in phosphatidylcholine-phosphatidylethanolamine vesicles that contained various amounts of cytochrome b5. The small vesicles, made by sonication, and the large vesicles, made by ethanol injection, were fractionated by centrifugation before cytochrome b5 was asymmetrically incorporated into the bilayer. The mole ratio of phospholipid to protein ranged from 280 to 560 in the small vesicles and from 100 to 500 in the large vesicles. The phosphatidylethanolamine distribution, determined by chemical labeling with trinitrobenzenesulfonic acid, was assessed in vesicles the contained intact cytochrome b5 molecules and in vesicles where only the hydrophobic tail remained associated with the bilayer. At every phospholipid to protein ratio examined, the transbilayer distribution of phosphatidylethanolamine in either the small or large unilamellar vesicles was not significantly different from the distribution in control vesicles that contained no protein. Ethanol was added to some cytochrome b5-vesicle preparations (20% v/v) in an attempt to facilitate rearrangement of the phospholipids. No differences in the transbilayer distribution were observed. These results are discussed in terms of transbilayer equilibrium and the perturbation induced by the protein.

Transbilayer distribution in small unilamellar phosphatidylglycerol-phosphatidylcholine vesicles.

The transbilayer distribution of the phospholipids in small unilamellar vesicles comprised of egg phosphatidylglycerol (PG) and egg phosphatidylcholine (PC) was ascertained by 31P NMR. These vesicles, containing 10-75 mol % PG, were formed by sonication (pH 7.6) and fractionated by centrifugation. Data from spectra accumulated in the presence and absence of a paramagnetic shift reagent, Mn2+, indicated that the phospholipids are randomly arranged across the bilayer. The absence of compositional asymmetry, which contradicts earlier reports, is also exhibited by small unilamellar vesicles (50 mol % PG) prepared by the rapid ethanol injection method. Control experiments showed that Mn2+ did not induce fusion, permeate the vesicles, or cause the phospholipids to migrate across the bilayer. It has been proposed that the transbilayer distribution of charged phospholipids in membranes is a consequence of the different surface charge densities on the opposing sides of the membrane. Our results suggest that it is the difference in the effective polar headgroup volumes of the components rather than the net charge of one component that determines the packing constraints for mixtures of phospholipids with the same acyl chains, at least in highly curved bilayers.

Transbilayer distribution of phosphatidylethanolamine in large and small unilamellar vesicles.

There is much evidence which strongly suggests that most constituents of biological membranes display a transbilayer compositional asymmetry. The tendency of binary mixtures of phospholipids to form compositionally asymmetric bilayers spontaneously has been studied extensively. In small unilamellar vesicles, most mixtures of phospholipids with different head groups have been reported to be nonrandomly arranged across the bilayer. In this study, the influence of the radius of curvature on the transbilayer phospholipid distribution has been investigated. The distribution of egg phosphatidylethanolamine in large unilamellar vesicles comprised of egg phosphatidylethanolamine and egg was determined by 2,4,6-trinitrobenzenesulfonic acid labeling. These large vesicles were obtained by modifying the ethanol injection procedure originally described by Batzri & Korn (1973) [Batzri, S., & Korn, E. D. (1973) Biochim. Biophys. Acta 298, 1015] by using a slow injection rate. After injection, the ethanol was removed by molecular sieve chromatography and the vesicle dispersion centrifuged. This results in a population of large, homogeneous, and unilamellar vesicles as determined by molecular sieve chromatography, 32P NMR, and electron microscopy. The phosphatidylethanolamine component in unilamellar vesicles of this type is equally distributed between the two monolayers. In contrast, phosphatidylethanolamine in small unilamellar vesicles is known to be preferentially localized in the outer monolayer at low phosphatidylethanolamine concentrations and in the inner monolayer at high phosphatidylethanolamine concentrations. These results suggest that while phospholipids may form asymmetric bilayers spontaneously in highly curved regions of biological membranes, other factors must be responsible for the generalized phospholipid asymmetry seen in these systems.