A Na+/Ca2+ exchanger isoform, NCX1, is involved in retinal cell death after N-methyl-D-aspartate injection and ischemia-reperfusion.

We investigated the expression of Na(+)/Ca(2+) exchanger (NCX) and the functional role of NCX in retinal damage by using NCX1-heterozygous deficient mice (NCX1(+/-)) and SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline), a selective NCX inhibitor in vivo. We also examined the role of NCX in oxygen-glucose deprivation (OGD) stress with a retinal ganglion cell line (RGC-5) cell culture in vitro. The expression of NCX1 was confirmed and entirely localized in retina by immunoblotting and immunohistochemistry, respectively. NCX1(+/-) mice possessed significant protection against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA). SEA0400 at 3 and 10 mg/kg significantly reduced NMDA- or high intraocular pressure-induced retinal cell damage in mice. Furthermore, SEA0400 reduced the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells and the expression of phosphorylated mitogen-activated protein kinases (ERK1/2, JNK, p38) induced by NMDA injection. In RGC-5, SEA0400 at 0.3 and 1 microM significantly inhibited OGD-induced cell damage. OGD-induced cell damage was aggravated by ouabain (a Na(+),K(+)-ATPase inhibitor) at 100 microM, and this increased damage was significantly reduced by SEA0400 at 1 microM. In conclusion, these results suggest that NCX1 may play a role in retinal cell death induced by NMDA and ischemia-reperfusion.

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis.

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

Ets family transcription factor ESE-1 is expressed in corneal epithelial cells and is involved in their differentiation.

Involvement of an epithelium-specific transcription factor ESE-1/ESX/ELF3/jen (ESE-1) in corneal epithelial cell differentiation was investigated. ESE-1 was reported to be induced during terminal differentiation of the epidermis and primary keratinocytes and to transactivate target genes through ets binding sites. However, its expression and function in corneal epithelium have not been examined. We report here that ESE-1 is upregulated upon differentiation in mouse corneal epithelium and in immortalized human corneal epithelial cells (HCE). ESE-1 transactivates through the regulatory element of cornea-specific K12 keratin. Moreover, introduction of ESE-1 antisense RNA in HCE cells affect their differentiation. These data suggest the involvement of ESE-1 in differentiation of corneal epithelial cells.

Optic disc topographic parameters measured in the normal cynomolgus monkey by confocal scanning laser tomography.

AIM: To study optic disc topographic parameters in normal cynomolgus monkeys by Heidelberg retina tomograph (HRT). METHODS: 12 optic disc topographic parameters were investigated in 36 normal eyes in 18 male monkeys. Mean (SD) and interocular differences were obtained for each parameter from three independent measurements made during a 1 week period. Correlations among the topographic parameters were analysed, too. RESULTS: No significant differences between right and left eyes were detected for any topographic parameters. Disc area, rim area, and height variation contour showed smaller right-left differences than other parameters. The coefficients of variation for rim area, height variation contour, rim volume, mean cup depth, maximum cup depth, mean retinal nerve fibre layer (RNFL) thickness, and RNFL cross section area were less than 10% (for rim area, less than 5%). Rim area and height variation contour showed relatively weak interrelations and neither showed a correlation with disc area. CONCLUSION: For evaluating time related changes in the optic disc by HRT in monkeys, rim area and height variation contour might be useful parameters because coefficients of variation and right-left differences were lower than for other parameters and because these parameters showed weak interrelations and no correlation with disc area.

Time course of changes in aqueous protein concentration and flow rate after oral acetazolamide.

The coefficient of plasma protein entry into the aqueous humor, kin, was calculated in the human eyes from the aqueous protein concentration measured with a flare-cell meter and from the aqueous flow rate determined with fluorophotometry. The value of kin averaged 3.47 +/- 0.25 x 10(-5) min-1 (mean +/- SEM) in 12 eyes of six normal young volunteers. The time course of changes in aqueous protein concentration after oral administration of 500 mg acetazolamide was measured with a flare-cell meter in 24 eyes of 12 subjects. Aqueous protein concentration significantly increased from 2-10 hr postadministration with a maximum increase of 41 +/- 7% (mean +/- SEM) at 6 hr postadministration. Assuming that kin is not affected by the drug treatment, we calculated the time change of aqueous flow rate from that of aqueous protein concentration using the value of kin above. The calculated flow rate after the administration of acetazolamide decreased between 1.25 and 8 hr, with a maximum reduction of 40 +/- 11% at 1.75 hr postadministration. These measurements obtained with the flare-cell meter corresponded well to those obtained by fluorophotometry in a separate group of volunteers given the same treatment. It was shown that oral acetazolamide increases aqueous protein concentration, and that the time change of its effect on aqueous flow rate can be monitored by measuring aqueous protein concentration.

Iontophoresis of 5-fluorouracil into the conjunctiva and sclera.

It was investigated in the rabbit eye whether the method of iontophoresis could introduce a sufficient amount of 5-fluorouracil (5-FU) to inhibit fibroblast proliferation into the conjunctiva and sclera, thus considerably reducing the total amount of 5-FU which must be given to the eye. Five-FU was introduced with a simple apparatus which was filled with 5% 5-FU solution and connected to the negative pole of a current source. When the apparatus was placed over the cornea, the 5-FU penetration into the cornea showed a correlation with strength of current (0-0.75 mA). When the apparatus was placed over the bulbar conjunctiva, a sufficient amount of 5-FU to inhibit fibroblast proliferation was introduced into the conjunctiva and sclera with a current of 0.5 mA passed for 30 seconds. Immediately after iontophoresis, mean 5-FU concentrations in the conjunctiva and sclera at the iontophoresis site were 480 and 168 micrograms/g, respectively. They decreased to 0.6 and 1.2 micrograms/g by 10 hr, but were still above the reported ID50 levels for the cultured conjunctival fibroblast. On the other hand, the total amount of 5-FU introduced into the eye averaged only 3.7 micrograms and the current used was much lower than that formerly applied to the patient's cornea.

Movement of fluorescein and its glucuronide across retinal pigment epithelium-choroid.

PURPOSE: To characterize movement of fluorescein and its glucuronide across the blood-retinal barrier. METHODS: Retinal pigment epithelium (RPE)-choroid preparations from New Zealand albino rabbit were sealed in an Ussing-type chamber in a stabilized condition for 3 hr, where movement of fluorescein and fluorescein glucuronide across the RPE-choroid was studied under a short circuit condition. RESULTS: The outward (vitreous-choroid) permeability to fluorescein determined at a concentration of 15 mumol/l was about 4 times greater than the inward (choroid-vitreous) permeability (P < 0.01). The outward permeability was significantly decreased by 50-65% by metabolic or competitive inhibitors (1 mumol/l ouabain, 10 mumol/l 2,4-dinitrophenol, 100 mumol/l probenecid, 30 mmol/l hippurate, or 5 mmol/l iodipamide), whereas the inward permeability was not affected by any of the above competitive inhibitors. As the fluorescein concentration was increased from 15 to 150 mumol/l, the net fluorescein movement across the tissue indicated saturation, and a Lineweaver-Burk plot gave an apparent Km of 26 mumol/l and Vmax of 1.56 nmol/hr/cm2. The outward permeability to fluorescein glucuronide determined at 15 mumol/l was about double the inward permeability (P < 0.01) and about 1/3 of the outward permeability to fluorescein (P < 0.01). The outward permeability to fluorescein glucuronide was significantly decreased by about 50% by 1 mumol/l ouabain, 10 mumol/l 2,4-dinitrophenol, or 100 mumol/l probenecid, whereas the inward permeability was not affected by 100 mumol/l probenecid. CONCLUSION: These results suggest that the majority of the outward fluorescein movement across the tissue and part of that of fluorescein glucuronide depends on an active transport mechanism, whereas the inward movement of both fluorescein and fluorescein glucuronide occurs by a passive mechanism.

Effect of oxidized glutathione on the barrier function of the corneal endothelium.

Effects of glutathione on the corneal endothelium were reexamined. Four kinds of solutions were made: oxidized glutathione (GSSG) was added to a basic solution which does not contain glutathione (GSSG-0) at a concentration of 0.03 mM, 0.3 mM or 3 mM to make GSSG-0.03, GSSG-0.3 or GSSG-3, respectively. Paired rabbit corneas were perfused separately, and the endothelial permeability (Pac) to carboxyfluorescein was determined. Between the paired corneas perfused with GSSG-0 and GSSG-0 or GSSG-0 and GSSG-0.03, there was no significant difference in the Pac. A significant difference in this factor was seen between the paired corneas perfused with GSSG-0 and GSSG-0.3 or GSSG-0 and GSSG-3 (P less than 0.01). The ratio of GSSG-0 to GSSG-0.3 for Pac, 1.18 +/- 0.16, and that of GSSG-0 to GSSG-3, 1.14 +/- 0.07, were significantly greater than the left-right ratio for Pac obtained when the paired corneas were perfused with GSSG-0, 1.01 +/- 0.10 (mean +/- SD, n = 8) (P less than 0.025). The corneal swelling rate (micron/hr) was 7.9 +/- 4.9 for the corneas perfused with GSSG-0 and 8.4 +/- 5.4 (mean +/- SD, n = 6) for those perfused with GSSG-0.3; difference was not significant. Addition of GSSG at a concentration of 0.3 mM or more to the irrigating solution was further beneficial to the corneal endothelial barrier function and a solution containing GSSG may be safer for patients with vulnerable corneas.

Human corneal endothelial permeability to fluorescein and fluorescein glucuronide.

The corneal endothelial permeability coefficient (Pac) for fluorescein and fluorescein glucuronide was determined in ten normal young volunteers. After oral administration of fluorescein, the apparent concentrations of both dyes in the corneal stroma and the anterior chamber were measured by differential fluorometry. The apparent dye levels calculated directly from the in vivo fluorometric measurements were converted to the true ones, based on the result of a normalization experiment performed in rabbit eyes. The value of Pac averaged 5.44 +/- 1.77 X 10(-4) cm/min for fluorescein and 3.77 +/- 1.10 X 10(-4) cm/min for fluorescein glucuronide (mean +/- SD, N = 20); the former was significantly greater than the latter (paired t-test, P less than 0.001). The aqueous-cornea distribution ratio was 0.50 +/- 0.14 for fluorescein and 0.66 +/- 0.16 for fluorescein glucuronide; the latter was significantly greater than the former (paired t-test, P less than 0.001). It was suggested that the previously reported values of Pac for fluorescein in the human eye were underestimates.

Effects of pilocarpine and tropicamide on blood-aqueous barrier permeability in man.

The time courses of changes in the effects of topical pilocarpine and tropicamide on the index of the blood-aqueous barrier permeability to plasma protein (Pin) were determined in normal volunteers. Before and after drug instillation in one eye, protein concentration in the anterior chamber (Ca) was determined from aqueous flare intensity with a laser flare-cell meter and from aqueous flow by fluorophotometry. The Pin was calculated from the Ca, plasma protein concentration, and aqueous flow. One percent pilocarpine produced a maximum increase of 21 +/- 10% in the Ca (mean +/- SEM, n = 10), no significant change in the aqueous flow (n = 5), and a maximum increase of 29 +/- 10% in the Pin (n = 10). Three percent pilocarpine produced a maximum increase of 55 +/- 11% in the Ca (n = 8), a maximum increase of 34 +/- 13% in the aqueous flow (n = 5), and a maximum increase of 74 +/- 18% in the Pin (n = 8). Tropicamide (0.4%) produced a maximum decrease of 17 +/- 7% in the Ca (n = 8), a maximum decrease of 15 +/- 11% in the aqueous flow (n = 8), and a maximum decrease of 24 +/- 13% in the Pin (n = 8). The results indicated that pilocarpine increased the blood-aqueous barrier permeability to plasma protein in a dose-dependent manner and that tropicamide reduced it.

In vivo measurement of iridial circulation using laser speckle phenomenon.

PURPOSE: To evaluate the use of the laser speckle phenomenon for noninvasive in vivo consecutive measurement of the iridial circulation. METHODS: A pigmented rabbit iris was illuminated using a diode laser, and the normalized blur of the resulting laser speckle pattern, NBiris, was determined as a quantitative index of blood velocity in the iridial tissue. The authors compared data on positional variation, reproducibility, and correlation to iridial blood velocity derived with this technique with the blood flow rate simultaneously determined by the microsphere technique. They also evaluated the effects on iridial circulation of ocular perfusion pressure (OPP) change, rectus muscle excisions, and instillation of topical timolol or betaxolol. RESULTS: The NBiris increased gradually from the pupil margin to the periphery; the coefficient of variation of NBiris was lowest at the center of this area. The coefficient of reproducibility of two NBiris measurements at 5-minute intervals was 8.8%; at 24-hour intervals, it was 14.1%. The NBiris correlated well with the microsphere technique measurements of blood flow rate at several intraocular pressures (IOP) (r = 0.61, P = 0.0002, n = 40) and with the comparison of preinstillation and postinstillation unoprostone (r = 0.93, P = 0.0068, n = 8). The NBiris decreased with OPP reduction, decreased temporarily after excision of the superior or inferior rectus, and showed no significant change after excision of the medial or lateral rectus. Instillation of timolol caused a significant decrease in IOP but did not significantly change the NBiris. Topically applied betaxolol decreased IOP and increased NBiris at 2.5 hours after instillation in an ipsilateral eye. CONCLUSIONS: The laser speckle method permits noninvasive, semiquantitative, consecutive measurement of the iridial circulation, with reasonable reproducibility.

Effects of topical nipradilol, a beta-blocking agent with alpha-blocking and nitroglycerin-like activities, on aqueous humor dynamics and fundus circulation.

PURPOSE: To study the effects of nipradilol, a nonselective beta-blocker with alpha 1-blocking activity and nitroglycerin-like activity, on aqueous humor dynamics and optic nerve head (ONH) circulation in albino rabbits. METHODS: Experiments were carried out during the dark phase, in conscious rabbits conditioned to a schedule of alternating 12-hour periods of light and dark. The blood-aqueous barrier permeability and the aqueous flow rate were determined fluorophotometrically. The effect on outflow to general blood circulation and uveoscleral outflow were determined by using the fluorophotometric Diamox technique, and the effect on the uveoscleral outflow was further assessed by using the anterior chamber perfusion method. The ONH circulation was estimated by using the laser speckle method. RESULTS: Unilateral topical administration of 0.25% nipradilol solution lowered intraocular pressure (IOP) with relatively weak contralateral effects in a dose-dependent manner with a maximum reduction of 6 mm Hg and an effect duration of 6 hours. Twice-daily instillation for 14 days showed no attenuation of the effects. Single instillation of 0.25% nipradilol showed no significant effect on blood-aqueous barrier permeability and decreased aqueous flow rate in the treated eye (17%; P < 0.01) and in the contralateral eye (9%, P < 0.05). Nipradilol produced no significant effect on outflow facility to general blood circulation, whereas it substantially increased uveoscleral outflow. Twice-daily 0.25% nipradilol instillation increased ONH tissue blood velocity by 13% (P < 0.01), which was probably attributable to locally penetrating drug. CONCLUSIONS: Because of its ability to lower IOP and to increase uveoscleral outflow and optic nerve head circulation in rabbits, further studies are warranted to determine whether nipradilol has potential as an antiglaucoma agent in humans.

Effect of calcium ion concentration on the permeability of the corneal endothelium.

The effect of free Ca2+ ion concentration on the integrity of the barrier function of the corneal endothelium was studied using the endothelial permeability to carboxyfluorescein (Pac) as a quantitative index according to the method of Araie. Paired rabbit corneas were isolated and mounted in a chamber. To serve as a control, one eye of each pair was perfused with a glucose-glutathione-bicarbonate solution at a Ca2+ concentration of 1.1 mEq, the other with a solution at various Ca2+ concentrations ranging from 0.23-1.1 mEq. The Pac ratio of a solution with a Ca2+ concentration of 0.38 mEq or higher to the control solution was close to unity, and the Pac ratio of a solution with a Ca2+ concentration of 0.33 mEq or lower to the control solution was significantly greater than unity. In a separate experiment, it was found that only slight swelling was seen when a solution with a Ca2+ concentration of 0.23 mEq or higher was used; significant swelling was seen with a solution of a Ca2+ concentration of 0.17 mEq or lower. The lowest free Ca2+ concentration needed for maintaining the barrier function of the corneal endothelium, 0.38 mEq, was found to be higher than that needed for maintaining the corneal thickness, 0.23 mEq.

Neuroprotective effect and intraocular penetration of nipradilol, a beta-blocker with nitric oxide donative action.

PURPOSE: To investigate the effect of nipradilol, an alpha(1),beta-blocker with a nitric oxide donative action, on N:-methyl-D-aspartate (NMDA)-induced retinal damage in rats and to determine whether topically instilled nipradilol penetrates the ipsilateral posterior retina-choroid at pharmacologically active concentrations in rabbits. METHODS: To determine effects on NMDA-induced damage, drugs were injected alone or with NMDA into the vitreous of one eye, and cell loss in the ganglion cell layer (GCL) and thinning of the retinal neural cell layers were histologically evaluated. To evaluate posterior penetration, first, [(14)C]-nipradilol was instilled, and its tissue concentration was measured. Second, nipradilol or timolol was instilled, and their effects on intravitreal injection of endothelin-1-induced retinal artery contraction were compared, to evaluate whether a pharmacologically active level of nipradilol penetrates the inner limiting layer by topical application. RESULTS: Intravitreous injection of NMDA reduced cell numbers in the GCL and the thickness of the inner plexiform layer (IPL) to 50.4% +/- 2.6% and 47.8% +/- 4.9% (n = 8) of control, respectively. Nipradilol alone had no effect. Coadministration of nipradilol with NMDA reduced cell numbers in the GCL and IPL thickness to 67.8% +/- 2.2% and 74.4% +/- 5.2% of control, respectively (P: < 0.05-0.01). Sodium nitroprusside, but not timolol or bunazosin, also significantly prevented the NMDA-induced reduction of cell numbers in the GCL and IPL thickness. Radioactivity of nipradilol was found in the ipsilateral posterior retina-choroid at 318.6 +/- 42.9 ng/g (n = 4), which was significantly higher than in the contralateral control (107.4 +/- 21.8 ng/g). Topical application of nipradilol, but not timolol, significantly suppressed the endothelin-1-induced contraction of the retinal artery (83.95% +/- 8.15% and 35.24% +/- 5.62% of baseline vessel diameter for nipradilol and timolol, respectively). CONCLUSIONS: Nipradilol suppressed the NMDA-induced retinal damage in rats for which nitric oxide released from nipradilol may be responsible. Posterior penetration studies suggested that an effective concentration of nipradilol reached the posterior retina after topical application.

Cholestanol induces apoptosis of corneal endothelial and lens epithelial cells.

PURPOSE: To determine whether cholestanol induces cornea endothelial and lens epithelial cell death in vitro. METHODS: Cornea endothelial and lens epithelial cells were cultured in minimum essential media with 10% fetal bovine serum containing 10 microg/ml cholesterol in ethanol, 10 microg/ml cholestanol in ethanol, or 1% ethanol. These cells, stained using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) method, were analyzed by laser cytometer. The activities of ICE and CPP32 proteases in cells were also measured. RESULTS: Both cornea endothelial and lens epithelial cells cultured with 10 microg/ml cholestanol showed a significant loss of viability. The nuclei of these cells cultured with 10 microg/ml cholestanol were more frequently stained than those exposed to 10 microg/ml cholesterol or 1% ethanol. Quantitative analysis of apoptotic DNA fragmentation confirmed that the cholestanol induced apoptosis of these cells in a time-dependent manner. The activities of interleukin-1beta-converting enzyme (ICE) and CPP32 proteases for cells cultured with 10 microg/ml cholestanol were significantly higher than those observed in control cells. CONCLUSIONS: In vitro, cholestanol was taken up by corneal endothelial cells and lens epithelial cells, an event that led to apoptosis of these cells.

Noncontact, two-dimensional measurement of retinal microcirculation using laser speckle phenomenon.

PURPOSE: To report a new apparatus for noncontact, two-dimensional measurement of retinal microcirculation using the laser speckle phenomenon and to demonstrate that this apparatus can document known or expected changes in retinal blood flow. METHODS: The rabbit fundus was illuminated by an argon (blue) laser spot (0.62 x 0.62 mm), and its image speckle was detected with an image sensor. The difference between the average of the speckle intensity (Imean) and the speckle intensity for successive scannings was calculated, and the ratio of Imean to this difference was defined as normalized blur (NB), a quantitative index of blood velocity in the retinal microcirculation. The results were displayed on a color monitor showing the two-dimensional variation of the NB level in the measurement area. Using this apparatus in the rabbit, the NB in the retinal field free of visible surface vessels was determined and compared with the retinal blood flow rate measured using the microsphere technique in the same eye simultaneously. In addition, the effect of the ocular perfusion pressure (OPP) on NB was studied. In the above experiments, a stepwise reduction in OPP was introduced by elevating the intraocular pressure manometrically. RESULTS: The relative decrease in the average NB (NBav) over the field measured, with the reduction in OPP, showed significant correlation with the relative change in the blood flow rate determined using the microsphere technique (r = 0.59, P < 0.001). Although NBav in the retina was little affected by OPP change when OPP was greater than 50 mm Hg, NB decreased along with OPP at levels less than 50 mm Hg. CONCLUSIONS: The NBav showed significant correlation with the retinal blood flow rate determined with microsphere technique. Retinal microcirculation under various conditions can be studied two dimensionally and noninvasively in the living eye with the present apparatus.

Effects of nilvadipine, a calcium antagonist, on rabbit ocular circulation and optic nerve head circulation in NTG subjects.

PURPOSE: To study the effects of nilvadipine, a Ca2+ antagonist, on tissue circulation in the optic nerve head (ONH), choroid, and retina in rabbits and on the ONH circulation in normal tension glaucoma (NTG) patients. METHODS: Nilvadipine (3.2 microg/kg) or vehicle solution was injected intravenously into urethane-anesthetized rabbits, and the normalized blur value (NB), a quantitative index of in vivo tissue blood velocity, was measured in the choroid and in an area of the ONH and retina free of visible surface vessels before and for 90 minutes after injection, using the laser speckle method. The effects of nilvadipine on the ONH circulation was also studied using the H2 gas clearance method in separate groups of rabbits. Oral nilvadipine (4 mg/d) or placebo was administered to NTG patients in a double-masked manner, and NB in an area of the ONH rim free of visible surface vessels was measured by the same method before and 2, 4, 8, and 12 weeks after administration. RESULTS: The NB obtained from the ONH, choroid, or retina during the experimental period was increased by approximately 10% to 25% in the nilvadipine group compared with the NB in the control group (P < 0.0001, ANOVA), although systemic condition parameters and intraocular pressure (IOP) showed no significant intergroup difference except for a transient decrease in blood pressure in the nilvadipine groups. Blood flow rate in the ONH determined by the H2 gas clearance method also showed an approximately 25% increase in the nilvadipine group. The NB in the ONH of the oral nilvadipine-treated patients was significantly increased, by approximately 20% compared with the placebo-treated patients throughout the follow-up period. No significant intergroup difference was seen in blood pressure, pulse rate, or IOP. CONCLUSIONS: Nilvadipine increased blood velocity and, probably, blood flow in the ONH, choroid, and retina of rabbits. It also increased blood velocity in the ONH of NTG patients.

Effects of retinal glial cells on isolated rat retinal ganglion cells.

PURPOSE: The effect of retinal glial cells on retinal ganglion cell (RGC) survival was investigated in cocultures of pure, isolated retinal glial cells with pure, isolated RGCs. METHODS: RGCs from 2-day-old rats were cocultured for 48 hours, avoiding direct contact between cell types, with either nonconfluent retinal glial cells from 3-day-old rats or confluent retinal glial cells from 3-day-old, 12-day-old, or 1-year-old rats. Survival of RGCs was evaluated by flow cytometry. Amino acids were determined in culture medium. The effects of glutamate antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione and MK801, a nitric oxide (NO) scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), and an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), were examined. RESULTS: Nonconfluent retinal glial cells significantly reduced the survival of small and large RGCs, but confluent retinal glial cells reduced the survival of only small RGCs, regardless of the rat's age at the time of retinal glial cell harvesting. Profiles of some amino acids significantly varied, depending on the culture condition. Cocultures of RGCs with nonconfluent retinal glial cells released significantly more glutamate into the medium than cocultures of RGCs with confluent retinal glial cells or RGCs in pure culture. The glutamate antagonists improved the survival of RGCs cocultured with nonconfluent retinal glial cells, especially when the two were administered in combination, and in the case of large RGCs. c-PTIO and L-NAME, also improved the survival of RGCs cocultured with nonconfluent retinal glial cells. CONCLUSIONS: Adverse effects of retinal glial cells on the survival of RGCs varied by size of the RGCs and retinal glial cell confluence. Glutamate and NO may be involved in retinal glial cell-related antisurvival effects.

Expression regulation of hyaluronan synthase in corneal endothelial cells.

PURPOSE: Our previous study showed that hyaluronan synthase (HAS), the enzyme protein of hyaluronan (HA) biosynthesis, is expressed in ocular tissues including the corneal endothelium. In the current study, the mechanism that regulates HAS expression in bovine corneal endothelial cells (BCECs) was investigated. METHODS: Cultured BCECs were used. HAS expression in BCECs at the mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. The effects of transforming growth factor (TGF)-beta and platelet-derived growth factor (PDGF)-BB on HAS expression were examined by quantitative RT-PCR. The involvement of the Smad family (intracellular signal transducer of TGF-beta) was also investigated. The expression of HAS in BCECs at the protein level was confirmed by immunocytochemistry and Western blot analysis. RESULTS: Three HAS isoforms in BCECs were expressed at the mRNA level. The transcriptional sizes of each HAS in BCECs were 4.9 kb for HAS1, 2.8 kb for HAS2, and 1.6 kb for HAS3. The expression of HAS2 at the mRNA level was stimulated by TGF-beta1 and/or PDGF-BB treatment. In contrast, HAS1 and HAS3 expression was not affected by these growth factors. The additive effects of TGF-beta1 and PDGF-BB were observed in the stimulation of the expression levels of HAS2. HAS2 upregulation by these growth factors was also detected by Western blot analysis. The stimulation of the expression of HAS2 at the mRNA level by TGF-beta was accelerated by the overexpression of Smad2, Smad3, and Smad4 and inhibited by that of Smad7, all of which were confirmed to be involved in the signal transduction from TGF-beta through HAS expression. CONCLUSIONS: Although three HAS isoforms were expressed in the corneal endothelial cells, the expression of HAS2 was upregulated by TGF-beta1 and/or PDGF-BB. HAS2 expression was regulated by TGF-beta through Smad family members.

Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells.

PURPOSE: Myocilin gene (MYOC) was identified as one of the disease-causing genes of primary open-angle glaucoma. This study was conducted to establish a system for the investigation of the biological role of MYOC in vitro by using bovine eyes, which are easy to obtain and have been widely used to examine the aqueous outflow system. The cDNA sequence of the bovine MYOC was determined and its expression in bovine eyes was examined with a quantitative polymerase chain reaction (PCR) assay. METHODS: Bovine MYOC cDNA was obtained from cultured bovine trabecular meshwork cells, and part of its sequence was determined using a primer pair designed based on the known sequence of the human MYOC gene. The 3' and 5' ends of this sequence were determined using the method of 3' and 5' rapid amplification of cDNA ends. The induction of the MYOC gene in cultured bovine trabecular meshwork cells after exposure to dexamethasone was quantitatively examined with real-time quantitative PCR using a probe designed according to the sequence of the determined bovine MYOC gene. RESULTS: Bovine MYOC protein was composed of 490 amino acids, which was 81.6% identical with that of human MYOC protein. Most of the amino acid residues of which mutation was reported to cause glaucoma were conserved in the bovine MYOC protein. After 2 weeks of treatment with 500 nM dexamethasone, expression of bovine MYOC mRNA was amplified 14-fold (14.1+/-5.1-fold, mean +/- SEM) measured by real-time quantitative PCR. CONCLUSIONS: The cDNA sequence of the bovine MYOC gene had a high degree of similarity to that of the human MYOC gene. Investigation of the function of bovine MYOC may contribute to identifying the role of MYOC protein in the aqueous outflow system.