Neuroprotective Effect and Intraocular Penetration of Nipradilol, a β-Blocker with Nitric Oxide Donative Action

Ken Mizuno; Takashi Koide; Mitsuo Yoshimura; Makoto Araie

Abstract

purpose. To investigate the effect of nipradilol, an α₁,β-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, [¹⁴C]-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.

Nipradilol—(3,4-dihydro-8-(2-hydroxy-3-isopropylamino)propoxy-3-nitroxy-2H-1-benzopyran; molecular weight [MW]: 326.35)—is a novel antiglaucoma ophthalmic agent that has nonselective β-receptor and selectiveα ₁-receptor blocking properties¹ ² ³ with a nitric oxide (NO) donative action.⁴ Topical administration of 0.25% nipradilol lowered intraocular pressure (IOP) both in rabbits and humans by reducing aqueous production and by increasing uveoscleral outflow.⁵ ⁶ The ocular hypotensive effect was equipotent to that of 0.5% timolol in glaucoma patients with less systemicβ -blocking effects,⁷ and nipradilol has therefore been registered as an antiglaucoma ophthalmic solution in Japan. Further, unilateral topical application of nipradilol significantly increased the optic nerve head (ONH) blood velocity on the ipsilateral treated side in rabbits, possibly through the NO donative and/orα ₁-blocking action of nipradilol, which penetrated locally.⁵

Evidence has been accumulating that NO plays a crucial role in neural degeneration, including the loss of retinal ganglion cells.⁸ ⁹ ¹⁰ ¹¹ ¹² Vorwerk et al.¹⁰ have shown that the NO released from neuronal NO synthase (nNOS) is a prerequisite for the full expression of excitotoxicity in the retina by use of nNOS-deficient mice. Additionally, Neufeld et al.¹³ reported that the inhibition of inducible NOS (iNOS) prevented ganglion cell loss in an elevated IOP model. These results suggest that NO released from several isoforms of NOS participates in various degenerated states and tissues. On the contrary, NO plays important roles in physiologic maintenance such as basal tone in the retinal circulation and visual transduction,⁸ and reports have suggested that it has neuroprotective effects.¹⁴ ¹⁵ ¹⁶ ¹⁷ ¹⁸

To investigate these, we first investigated the neuroprotective effects of nipradilol on N-methyl-d-aspartate (NMDA)–induced retinal damage in comparison with those of the nonselective β-blocker timolol, anα ₁-selective blocker bunazosin,¹⁹ and a NO-donor sodium nitroprusside (SNP) in rats. Second, we studied by two methods in rabbits whether topically instilled nipradilol could penetrate the posterior retina and choroid. The first method was direct measurement of topically applied[ ¹⁴C]-nipradilol in retina–choroid, and the second was a look at whether topical nipradilol reaches the retinal inner limiting layer at a pharmacologically active level by use of the inhibitory action of nipradilol against endothelin (ET)-1–induced retinal artery contraction as an in vivo parameter. The results of the second study may also be applicable to other topical drugs.

Methods

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Effects on NMDA-Induced Retinal Damage

Male Sprague–Dawley rats (7 weeks old; Japan Laboratory Animals, Tokyo) were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL). The methods for determining the volume of intravitreal injection, doses, and histologic evaluations were essentially the same as described in previous reports.²⁰ ²¹ ²² ²³ Briefly, body temperature was kept at 37°C with a heating pad (KN-474-S; Natsume, Tokyo, Japan) throughout the experiment. A 33-gauge needle was inserted into the midvitreous of one eye chosen at random under a stereoscopic microscope with care to avoid lens injury. The other eye received the vehicle solution as a control. A single 5-μl injection of the drug into one eye was completed over 1 minute. The following drugs were administered with or without 4 × 10⁻ ² M NMDA (Sigma, St. Louis, MO): 2 × 10⁻ ³ M (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; Nacalai Tesque, Kyoto, Japan), 10⁻ ¹⁰ to 10⁻ ⁶ M nipradilol hydrochloride, 10⁻ ⁸ to 10⁻ ⁶ M timolol maleate (Sigma), 10⁻ ⁸ to 10⁻ ⁶ M bunazosin hydrochloride, and 10⁻ ⁸ to 10⁻ ⁶ M SNP (Sigma). Nipradilol (purity, more than 99.5% by high–performance liquid chromatography [HPLC]) and bunazosin (purity, more than 99.5% by HPLC) were synthesized in our laboratory. Nipradilol was dissolved in equimolar amounts of hydrogen chloride to obtain a 10⁻ ²-M solution and diluted by 0.1 M phosphate buffer (pH 7.0). Other drugs were dissolved in 0.1 M phosphate buffer (pH 7.0).

Seven days after the injection, both eyes were enucleated, and three sequential meridian sections (3 μm thick) were made through the optic disc. Sections were stained with hematoxylin and eosin, and the number of cells in the ganglion cell layer (GCL), and the thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were measured as previously reported.²⁰ ²¹ ²² Morphologically distinguishable glial cells and vascular endothelial cells were excluded from the cell count, as described by Lam et al.²⁴ Data from three sequential sections were averaged for each eye. The values for the treated eye of each animal were normalized to those for the contralateral vehicle-treated control and were shown as a percentage. All histologic measurements were performed by an investigator masked to the treatment.

Posterior Penetration of Topically Instilled Nipradilol

[3-¹⁴C]-nipradilol (Code CFQ11032, radiochemical purity 98%, specific radioactivity 1.55 MBq (43μ Ci)/mg) was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Four male Japanese White (JW) rabbits (10 weeks old, Japan Laboratory Animals) were used. Rabbits were placed in a restraining cage and [¹⁴C]-nipradilol (1%, 100μ l, 1.5 MBq [41 μCi]/dose) was instilled twice daily (10 AM and 6 PM) for 7 days into the lower cul-de-sac of the right eye. Thirty minutes after the final instillation, rabbits were bled from the marginal ear artery and deeply anesthetized with pentobarbital. To minimize contamination of the intraocular tissues, enucleation and dissection of the eye were performed by the following procedure. A cut was made along the edge of the orbit, and both eyelids were clamped by surgical clip. Grasping the clamped eyelids enabled enucleation of the globe together with the eyelids and surrounding tissues, as a pouch. This pouch was immersed immediately in a mixture of hexane and solid carbon dioxide for 2 minutes and then stored at −15°C. The next day, posterior surrounding connective tissues were removed, and the frozen globe was cut circle-wise at a radius of 5 mm with the optic nerve for its center for division into anterior and posterior cups. Vitreous and then retina–choroid were exfoliated from the vitreous side of the posterior cup. The lens, iris, aqueous humor, and cornea were isolated from the vitreous side of the anterior cup. Isolation of the tissue was performed under semifrozen conditions. All tissues were dissolved, and radioactivity was measured by a liquid scintillation counter (Tri-Carb Liquid Scintillation Analyzer 2700TR; Packard Instruments, Meriden, CT).

Thirteen male JW rabbits (10 weeks old; Japan Laboratory Animals) were used in the intravitreal endothelin-1 (ET-1 human; Sigma) injection study. To serve as a control for the later experiments, physiological saline (50 μl) was instilled twice daily (10 AM and 6 PM) for 7 days into the lower cul-de-sac of both eyes. Thirty minutes after the final instillation, 20 μl of 5 × 10⁻⁸ M ET-1 was injected into the vitreous of both eyes. Care was taken to inject accurately between the lens and posterior fundus. Photographs of the ocular fundus were obtained with a fundus camera (RC-XV3; Kowa, Tokyo, Japan) at 5 minutes before and 15, 30, 45, and 60 minutes after the ET-1 injection and were captured in an image filing system (VK-2; Kowa). The diameter of the two major retinal arteries just at the rim of the ONH and the diameter of the ONH were measured. The average diameter of the arteries was normalized by the ONH diameter and was expressed as a percentage of the diameter 5 minutes before the ET-1 injection. IOP was measured 5 minutes before the final instillation of nipradilol or timolol and 5 minutes before the ET-1 injection. Nipradilol (0.25%, 50 μl) or timolol (0.5%, 50 μl) was instilled in one randomly selected eye for 7 days according to the schedule described earlier. Vehicle or saline was instilled in the other eye. Fundus photographs were obtained 5 minutes before and 60 minutes after the ET-1 injection at the base of the control experiment. All procedures were performed by an investigator masked to the treatment.

All data are presented as the mean ± SEM. Data were analyzed using the paired t-test, Wilcoxon signed-rank test, or Dunnett or Bonferroni multiple comparison test, as appropriate. P < 0.05 was considered statistically significant.

Results

Effects on NMDA-Induced Retinal Damage

Intravitreous injection of NMDA decreased the number of cells in the GCL by 50.4% ± 2.6% and the IPL thickness to 47.8% ± 4.9% (n = 8) of the respective control value, whereas the INL and ONL thicknesses were unchanged (Fig. 1) . The coadministration of MK-801 with NMDA abolished the cell loss in the GCL and the reduction of IPL thickness (n = 8, Fig. 1 ).

Intravitreous injection of nipradilol alone had no effect on the number of cells in the GCL or the thickness of each layer (n = 8–10, Fig. 2A ). The cell loss in the GCL and the reduction of IPL thickness by NMDA were partially suppressed by the coadministration of nipradilol, which had a dose-dependent effect that was significant at doses of 10⁻ ⁸ M and higher and 10⁻ ⁷ M or higher, respectively (n = 8–10). Figure 3 shows typical light microscopic photographs of the results.

Intravitreous injection of timolol and bunazosin produced no histologic alterations in the retina. The coadministration of timolol or bunazosin with NMDA had neither an adverse nor a protective effect on NMDA-induced retinal damage (n = 6, Fig. 4 ).

Intravitreous injections of SNP had no effect on the retina (n= 7, Fig. 5A ). The cell loss in GCL and the reduction of IPL thickness by NMDA were partially prevented by the coadministration of SNP at doses of 10⁻ ⁷ M and more and at 10⁻ ⁶ M, respectively (n = 7, Fig. 5B ).

Posterior Penetration of Topically Instilled Nipradilol

Table 1 shows the tissue and blood concentrations of nipradilol in the ocular region 30 minutes after the final instillation (n = 4). Nipradilol concentrations in all the tissues of the treated eye were significantly higher than those in the nontreated control eye. In the nontreated eye, the concentrations of nipradilol were approximately the same as or lower than those in blood.

Table 2 shows the effects of saline, nipradilol or timolol on ET-1–induced retinal artery contraction. The retinal artery contracted significantly and time dependently after the intravitreous injection of ET-1. The contraction gradually increased to a maximum at 30 minutes after the injection (n = 4, Table 2 , upper lane). In the control group, the difference in diameter between each eye and the variance of the data were minimal at 60 minutes after the injection. Therefore, we evaluated the effect of nipradilol or timolol at this time point. There was no significant difference in diameter at 5 minutes before the ET-1 injection among three groups. No significant difference was seen between the timolol-treated and control eyes (n = 4, Table 2 , lower lane). In the nipradilol-treated eyes, the retinal artery contracted significantly less than in the vehicle-treated eyes (n= 5, P = 0.02) and timolol-treated eyes (n= 4–5, P = 0.006). The bilateral difference in diameter between the nipradilol- and vehicle-treated eyes was significantly greater than in the saline-treated control group (n= 4–5, P = 0.048). Both nipradilol and timolol significantly lowered the IOP to a similar extent (n = 4–5, Table 3 ).

Discussion

The toxicity of glutamate to the retina has been well documented.²⁵ ²⁶ ²⁷ ²⁸ ²⁹ ³⁰ ³¹ Degeneration of the inner part of the retina was observed after intravitreal injection of glutamate,³² and many studies have suggested that the predominant form of excitotoxicity in retinal ganglion cells is mediated by overstimulation of glutamate receptors.³³ ³⁴ ³⁵ In this study, NMDA injected into the rat vitreous caused cell loss in the GCL and reduction of IPL thickness that were consistent with previous findings.²⁰ NMDA receptors are mainly expressed in ganglion cells, displaced amacrine cells in the GCL, and subsets of amacrine cells in the INL,³⁶ which seems to be consistent with the area damaged by NMDA in the present study. The NMDA-induced retinal damage was abolished by coadministration of MK-801, an NMDA-receptor–specific antagonist, which also suggests that the observed retinal damage induced by NMDA was mediated by NMDA receptor.

NMDA-induced cell loss in the GCL and reduction of IPL thickness were significantly suppressed by the coadministration of nipradilol. Assuming that the volume of the rat vitreous is approximately 50μ l,³⁷ the effective concentration at the retinal surface would be one tenth of that obtained when nipradilol diffused uniformly, or somewhat higher. Because the negative logarithm of dissociation constant for competitive antagonist (pA₂) values of nipradilol were 8.9 for nonselective β-blocking and 6.5 for selectiveα ₁-blocking actions, respectively,¹ ² ³ it is conceivable that the effective concentration of nipradilol elicits both receptor blocking actions at the retina. We also examined the effects of a nonselective β-blocker, timolol,³⁸ and a selectiveα ₁-blocker, bunazosin,¹⁹ on the NMDA-induced retinal damage. But neither blocker had any effect at concentrations sufficient to cause β- orα ₁-receptor blockade.

In this study, we found a protective effect of nipradilol, as well as of SNP, on NMDA-induced damage. Nipradilol was not histologically toxic to normal retina up to 10⁻ ⁴ M (data not shown). The underlying mechanism by which nipradilol or the related NO donor SNP protects neural cells against NMDA-induced retinal damage is not clear. There is evidence that NMDA-induced retinal ganglion cell damage can be modulated by the β₁-selective blocker, betaxolol, the mechanism for which is reported at least as a Ca²⁺ channel-blocking action.³⁹ ⁴⁰ This does not apply to nipradilol, however, because nipradilol does not have Ca²⁺ channel-blocking activity. One explanation for the protective effect is that it causes vasodilation. A previous report⁴ and Table 2 in this article suggest that nipradilol has vasodilating activity in the retinal artery. Moreover, the result obtained with SNP, which also has vasodilating activity, suggests that vasodilating agents can have neuroprotective effects. Another possibility is that the NMDA receptor has a redox modulatory site that consists of thiol groups, where disulfide bonds form on the surface of the receptor, and redox agents modulate the receptor activity through changes at this site.¹⁴ ⁴¹ ⁴² It is reported that reducing agents upregulate receptor activity and oxidizing agents downregulate it by forming disulfide bonds. NO acting as an oxidant may cause S-nitrosylation of NMDA-receptor thiol to downregulate the receptor activity.¹⁴ ⁴¹ ⁴² Thus, it may be possible that nipradilol and SNP downregulate the NMDA receptor activity and produce the neuroprotective effect through NO-donating action, but this mechanism of action should be studied in an in vitro system without blood supply.

The current results indicate that nipradilol has neuroprotective properties in NMDA-induced retinal damage, but it was not known whether topically applied nipradilol could reach the retina at a pharmacologically active concentration. To investigate this issue, the ocular penetration of topically instilled nipradilol was studied in rabbits.

It is difficult to enucleate an eyeball free of contamination when enucleation scissors are inserted through the conjunctiva, which has a very high concentration of radioactivity. To minimize such contamination, we enucleated the eye through the orbital skin, bisected the frozen globe into anterior and posterior cups, and isolated the tissue from the vitreous side on which the radioactivity was thought to be the lowest. Additionally, it was confirmed that no radioactivity was present in the hexane that was used to freeze the eyeballs. Each tissue in the treated eye had a higher concentration of nipradilol than that in the nontreated contralateral control, where nipradilol concentrations were approximately the same as or lower than those in blood. Nipradilol concentrations measured in the cornea, aqueous humor or lens were comparable to those found in the previous study for nipradilol⁴³ and for betaxolol.⁴⁰ The nipradilol concentration in the posterior retina–choroid was 0.98 and 0.33 μM in the treated eye and nontreated contralateral control, respectively. The former value was significantly higher than the latter. The difference was not considered attributable to contamination during the dissecting procedure, because the concentration in the posterior vitreous isolated first was much lower, and the concentration in the retina–choroid in the control eye and was similar to that in the blood.

Rather, we believe this difference indicates that topically applied nipradilol reaches the posterior retina–choroid at an effective concentration, not through the vitreous but through an as yet unidentified route, at least in the rabbit eye. One possibility may be the periocular route. Sponsel et al.⁴⁴ reported that Tenon capsules accumulate betaxolol or timolol at much higher concentrations than can be obtained intraocularly after long-term topical therapy and suggested that periocular accumulation provides more immediate access to the posterior segment and proximal ocular vasculature for topical drugs. Additionally, Geroski and Edelbauser⁴⁵ documented that the sclera is quite permeable to a wide range (molecular weight: 285–70,000) of solutes, and the permeability constant for transscleral solutes was inversely related to solute molecular weight. The molecular weight of nipradilol is 326.35, and there is therefore a possibility that nipradilol reaches the retina–choroid through the periocular transscleral route.

In this study, we used 1% nipradilol (100 μl) to evaluate the tissue concentration of drug. The clinical dose and volume of nipradilol are 0.25% and 50 μl, respectively. The minimal estimated concentration of nipradilol in the posterior retina–choroid after application of a 0.25% solution and 50-μl drop would be 0.12 and 0.04 μM in the treated eye and contralateral control, respectively, and the concentration in blood, 0.03 μM. At these concentrations, nipradilol is considered to show pharmacologic activity,¹ ² ³ ⁴ including neuroprotective action as seen in the current rat experiment. But there is a possibility that most of the drug is bound to tissue(s) and not pharmacologically active, and it was difficult to determine the concentration in the retina in this way because of difficulty in isolating the retinal layer from the choroid in a frozen state.

To determine whether pharmacologically active nipradilol could reach the retinal layer after topical application, we designed the ET-1 intravitreal injection study. Nipradilol exhibits NO-related vasodilating activity by activating the soluble guanylate cyclase and increasing the production of cyclic guanosine monophosphate in the vascular cells.⁴ We focused on the retinal artery that is present between the vitreous and the retinal inner limiting layer and examined the inhibitory effect of topical nipradilol on ET-1–induced retinal artery contraction. Topical nipradilol significantly suppressed the ET-1–induced contraction of the retinal artery.

The vascular contraction in the vehicle-treated side in the nipradilol group tended to be less than that in the saline- or timolol-treated group. This may be because of the effect of systemically absorbed nipradilol. According to the result of the first rabbit experiment, the nipradilol concentration in blood in the second rabbit experiment would be 0.03 μM or higher. The effective vasodilating concentration of nipradilol was 0.01 μM and higher.⁴ The suppressive effect against ET-1–induced contraction, however, was significantly stronger in the nipradilol-treated than the vehicle-treated eye. On the contrary, timolol had an equipotent ocular hypotensive effect with nipradilol but no effect on ET-1–induced contraction of the retinal artery, which suggests that the effect of nipradilol was not mediated by the ocular hypotensive effect.

These results suggest that topically applied nipradilol reached the ipsilateral retina at a pharmacologically active concentration in the normal rabbit eye, not through the systemic circulation, but through local penetration. A route through the vitreous seems unlikely, because the concentration in the vitreous was very low. The active nipradilol concentration in the ipsilateral retina is not known, but it should be higher than the lowest vasodilating concentration (0.01μ M).⁴ This concentration range is not far from the estimated vitreous concentration of nipradilol that showed neuroprotective effect against NMDA-induced retinal damage.

The results obtained from the posterior penetration experiment for nipradilol may be also applicable to other topically applied drugs with a similar molecular weight and lipophilicity. For example, betaxolol has a molecular weight similar to that of nipradilol but is much more lipophilic.⁴⁶ It is possible that topically instilled betaxolol would reach the ipsilateral retina at a higher concentration than presently observed for nipradilol in normal rabbits.⁴⁰ The results of the posterior penetration studies should not be directly extrapolated to the human eye, but the possibility that a topically applied drug can reach the posterior parts of the human eye at a pharmacologic concentration is not to be excluded.

In summary, nipradilol had a dose-dependent and significant protective effect on NMDA-induced retinal damage in the rat eye. SNP, but not theβ - and α₁-blockers, showed a comparable effect on the NMDA-induced retinal damage. Moreover, it was indicated that nipradilol could reach the posterior retina after topical application at pharmacologically active concentrations by local penetration in the normal rabbit eye. These properties may be advantageous in an antiglaucoma eye drop.

Submitted for publication July 14, 2000; revised November 6, 2000; accepted November 30, 2000.

Commercial relationships policy: E (KM, TK, MY); N (MA).

Corresponding author: Ken Mizuno, Department of Pharmacology, Tokyo Research Laboratories, Kowa Co., Ltd., 2-17-43 Noguchicho, Higashimurayama, Tokyo 189-0022, Japan. [email protected]

Figure 1.

The effects of vehicle (0.1 M phosphate buffer, □), 4 ×
10− 2 M of NMDA ( ) and 2 ×
10− 3 M of MK-801 (▪) on cell number in GCL
and thickness of IPL, INL, and ONL. Data are the mean ± SEM;** P < 0.01 versus vehicle (Dunnett multiple
comparison test); n = 8.

View Original Download Slide

The effects of vehicle (0.1 M phosphate buffer, □), 4 × 10⁻ ² M of NMDA ( Image not available

) and 2 × 10⁻ ³ M of MK-801 (▪) on cell number in GCL and thickness of IPL, INL, and ONL. Data are the mean ± SEM;** P < 0.01 versus vehicle (Dunnett multiple comparison test); n = 8.

Figure 2.

View Original Download Slide

Effect of nipradilol on intact retina (A) and nipradilol induced suppression of NMDA-induced retinal damage (B). Data are the mean ± SEM; *P < 0.05,** P < 0.01 versus NMDA in GCL (Dunnett), and†† P < 0.01 versus NMDA in IPL (Dunnett). NP, nipradilol; (□), cell number in GCL; (▪), IPL thickness; n= 8 to 10.

Figure 3.

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Typical light micrographs illustrating the effect of NMDA on reducing cell number in the GCL, thickness of the IPL, and protection by nipradilol. NP, nipradilol. Vehicle-treated control retina (A) and retinas treated with 4 × 10⁻ ² M NMDA (B), + 10⁻ ¹⁰ M (C), + 10⁻ ⁹ M (D), + 10⁻ ⁸ M (E), + 10⁻ ⁷ M (F), + 10⁻ ⁶ M (G) nipradilol. Magnifi-cation, ×66; scale bar, 50μ m.

Figure 4.

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Effect of timolol (A) and bunazosin (B) on NMDA-induced retinal damage. Data are the mean ± SEM; TM, timolol; Bu, Bunazosin; (□), cell number in the GCL; (▪), IPL thickness; n = 6.

Figure 5.

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Effect of SNP on intact retina (A) and NMDA-induced retinal damage (B). Data are the mean ± SEM;* P < 0.05, **P < 0.01 versus NMDA in GCL (Dunnett), and ††P < 0.01 versus NMDA in IPL (Dunnett). (□), cell number in the GCL; (▪), IPL thickness; n= 7.

Table 1.

View Table

Intraocular Concentration of Nipradilol at 30 Minutes after Topical Application

Table 1.

Intraocular Concentration of Nipradilol at 30 Minutes after Topical Application

	Treated	Control
Anterior
Cornea	104038.9 ± 18967.4 (318.80 ± 58.12)^*	81.0 ± 10.7 (0.25 ± 0.03)
Aqueous humor	2681.7 ± 337.1 (8.22 ± 1.03)^*	5.3 ± 1.1 (0.02 ± 0.00)
Iris	10507.3 ± 2174.5 (32.20 ± 6.63)^*	82.7 ± 2.9 (0.25 ± 0.01)
Lens	346.8 ± 15.4 (1.06 ± 0.05)^*	15.4 ± 1.1 (0.05 ± 0.00)
Posterior
Vitreous	4.3 ± 0.4 (0.01 ± 0.00)^{, †}	2.1 ± 0.3 (0.01 ± 0.00)
Retina–choroid	318.6 ± 42.9 (0.98 ± 0.13)^*	107.4 ± 21.8 (0.33 ± 0.01)
Blood	84.0± 9.1 (0.26± 0.03)

Data are the mean nanograms equivalent per gram or milliliter (micromolar) ± SEM of four preparations.

* P < 0.01 versus control (paired t-test).

† P < 0.05 versus control (paired t-test).

Table 2.

View Table

Effect of Nipradilol and Timolol on ET-1–Induced Contraction in Retinal Artery in Rabbits

Table 2.

Effect of Nipradilol and Timolol on ET-1–Induced Contraction in Retinal Artery in Rabbits

	Time (min)
		15	30	45	60
Saline (right)	84.09 ± 9.01	68.57 ± 8.53^*	60.93 ± 12.48^*	47.58 ± 5.573^{, †} ^{, ‡}
Saline (left)	92.19 ± 8.91	54.74 ± 10.63^*	48.67 ± 7.58^{, †} ^{, ‡}	49.64 ± 6.89^{, †} ^{, ‡}
Difference	8.10 ± 16.76	13.83 ± 12.29	12.26 ± 11.03	2.06 ± 4.08
Nipradilol	—	—	—	83.95 ± 8.15^{, †} ^{, §} ^{, ∥}
Vehicle	—	—	—	61.54 ± 4.12^{, †}
Difference	—	—	—	22.42 ± 10.45^{, ¶}
Timolol	—	—	—	35.24 ± 5.62^{, †}
Saline	—	—	—	50.32 ± 13.32^{, †}
Difference	—	—	—	15.08 ± 8.91

Data are the mean percentage vessel diameter compared with baseline ± SEM of four to five preparations. Twenty microliter of 5 × 10⁻⁸ M ET-1 was injected into the vitreous of all eyes 30 minutes after the final instillation of saline or drugs.

* P < 0.05 versus pretreatment.

† P < 0.01 versus pretreatment.

‡ P < 0.05 versus 15 minutes (Bonferroni).

§ P < 0.05 versus contralateral vehicle-treated side (Wilcoxon signed-rank test).

∥ P < 0.01 versus timolol-treated side (Wilcoxon rank-sum test).

¶ P < 0.05 versus saline-treated group (Wilcoxon signed-rank test).

Table 3.

View Table

Effect of Nipradilol and Timolol on IOP in Rabbits

Table 3.

Effect of Nipradilol and Timolol on IOP in Rabbits

	IOP
		Pretreatment	Posttreatment
Nipradilol	20.25 ± 2.73	16.58 ± 2.00^**
Vehicle	18.83 ± 2.42	18.50 ± 2.39
Timolol	20.60 ± 0.19	17.00 ± 0.88^**
Saline	20.50 ± 1.16	19.90 ± 0.56

Data are the mean millimeters of mercury ± SEM of five animals.

** P < 0.01 versus vehicle or saline-treated side (paired t-test).

The authors thank Hideki Fujino, Yoshihiko Tsunenari, and Junko Mori for assistance and Kiyoshi Ishii for technical advice.

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