Shigeyoshi Hiraki

Effects of Kakkon-to and Sairei-to on experimental elevation of aqueous flare in pigmented rabbits.

PURPOSE To evaluate the possible inhibitory effects of Kakkon-to and Sairei-to, traditional Sino-Japanese herbal medicines, on experimental aqueous flare elevation in pigmented rabbits. METHODS Anterior uveitis was induced either by an application of prostaglandin E2 (PGE2), 10 microg/mL, to the cornea, or an intravenous injection of lipopolysaccharides (LPS), 0.5 microg/kg, in an ear vein. Dose dependency of experimental uveitis induced by LPS (0.1, 0.25, 0.5, or 1.0 microg/kg) was also determined. For pretreatment, about 150 g/day of food containing Kakkon-to (1% w/w) or Sairei-to (0.6% or 2% w/w) was given to two groups of animals for 5 days before experimental uveitis was induced. A third group of animals underwent pretreatment with betamethasone, 130 microg/kg, injection into an ear vein 4 hours before experimental uveitis was induced. A fourth group of rabbits with no herbal medicine or betamethasone pretreatment served as controls. Aqueous flare was measured using a laser flare-cell meter. Aqueous flare intensity was expressed as the area under the curve (AUC) in arbitrary units. RESULTS The increase in aqueous flare induced by LPS was dose-dependent. The AUC of PGE2 (10 microg/mL) and LPS (0.5 microg/mL) induced aqueous flare elevations were 1,119 and 4,950 arbitrary units, respectively. Kakkon-to (AUC, 1,055) and Sairei-to (AUC, 965) did not inhibit the aqueous flare elevation induced by PGE2. Beta-methasone did inhibit the elevation (AUC, 271). Kakkon-to (AUC, 4,495) did not suppress the aqueous flare elevation induced by LPS. Both 0.6% and 2% Sairei-to (AUC, 2,478, and 978) and beta-methasone (AUC, 443) did suppress the aqueous flare elevation induced by LPS significantly (P < .05). CONCLUSION Sairei-to could have an inhibitory effect on experimental anterior uveitis induced by LPS.

Decreased Aqueous-Flare Reaction to Repeated Applications of Prostaglandin E2 to the Cornea in Pigmented Rabbits

To evaluate the effect of single or repeated topical applications of prostaglandin E2 (PGE2) to the cornea on the blood-aqueous barrier, we have measured the extent of flare in the anterior chamber of rabbit eyes at various time intervals. PGE2 was applied to the cornea for 4 min with the use of a glass cylinder in pigmented rabbits. Aqueous flare was measured by a laser flare cell meter. The flare intensity following the second application of 25 or 50 micrograms/ml of PGE2 was smaller than that following the first application. When 50 micrograms/ml of PGE2 was applied every day for 6 days, the flare intensity decreased significantly day by day. After consecutive applications of 10 micrograms/ml of PGE2 at hourly intervals, flare intensity increased up to 3 h, remained elevated from 3 to 5 h, and decreased thereafter. Repeated applications of 10 micrograms/ml of PGE2, every hour for 14 h every Monday, significantly decreased flare intensity week by week. Weekly applications of 50 micrograms/ml of PGE2 did not change flare intensity. This study indicates that the extent of the breakdown of the blood-aqueous barrier in the rabbit eye, as measured by the development of flare in the anterior chamber, is reduced with repeated PGE2 application.

Nilvadipine Inhibits Acute Rise of Aqueous Flare and Intraocular Pressure Induced by Prostaglandin E2 in Pigmented Rabbits

To evaluate the possible role of calcium channel blocker on changes in aqueous flare and intraocular pressure induced by prostaglandin E2 (PGE2) in pigmented rabbits, we examined the effects of nilvadipine and nicardipine (calcium channel blockers). PGE2, 25 µg/ml, was administered using a glass cylinder. Nilvadipine or nicardipine was injected intravenously. Aqueous flare was measured by a laser flare cell meter. Intraocular pressure was measured by a noncontact tonometer. After administration of PGE2, aqueous flare and intraocular pressure increased and then decreased. Increased flare and elevated intraocular pressure following PGE2 administration were inhibited by nilvadipine in a dose-dependent manner (5–500 µg/kg body weight). These responses were inhibited only slightly by nicardipine at the same concentration. Nilvadipine injected 30 min before PGE2 application inhibited the increase maximally. The inhibitory effect was found on days 1, 3, 5, and 7 by daily administration of nilvadipine. A good correlation between the inhibition of intraocular pressure and the inhibition of increased flare by nilvadipine was found. We believe that Ca2+, or calcium channel blocker-related substances may be involved in the mechanism of PGE2-induced elevation of aqueous flare and intraocular pressure in the pigmented rabbit.

Effects of a nitric oxide synthase inhibitor on prostaglandin-induced aqueous flare elevation in pigmented rabbits.

To evaluate the possible role of nitric oxide in the pathogenesis of exogenous prostaglandin (PG) induced aqueous flare elevation, we examined the effect of NG-nitro-L-arginine methyl ester (L-NAME), an L-arginine analogue acting as a specific inhibitor of nitric oxide synthase, on the elevation of aqueous flare in pigmented rabbits. L-NAME was injected into the ear vein of the animals. PGE1, PGE2, and PGF2 alpha were administered topically to the cornea using a glass cylinder. Aqueous flare was measured using a laser flare cell meter. PGE1, PGE2, and PGF2 alpha increased the aqueous flare dose dependently (5-100 micrograms/ml). L-NAME inhibited PGE1-, PGE2-, or PGF2 alpha-induced aqueous flare elevation. L-NAME, injected 30-90 min before PG application, inhibited the aqueous flare elevation maximally. We believe that nitric oxide may be involved in the pathogenesis of PG-induced aqueous flare elevation in rabbits.

Nicardipine inhibits acute rise of aqueous flare and intraocular pressure induced by argon laser photocoagulation.

The purpose of this study was to examine the anti-inflammatory effect of the calcium channel blocker nicardipine. Intraocular inflammation was induced by argon laser photocoagulation of the iris of pigmented rabbits and was assessed by measuring aqueous flare and intraocular pressure. This resulted in a marked increase in the aqueous flare that peaked at approximately one hour following coagulation and returning to the original values after six hours. Intraocular pressure increased within 15 minutes following laser treatment and returned to baseline levels at 60 minutes. Pre-treatment of the rabbits with an intravenous injection of 2 mg/kg of nicardipine completely abolished both the increase in aqueous flare and intraocular pressure induced by laser photocoagulation as compared to the control experiment. Earlier work from the authors group has shown that nicardipine can also block aqueous flare and intraocular pressure increases induced by topical administration of prostaglandin E2. From these combined experiments they would like to draw the conclusion that the inflammatory reaction induced by photocoagulation of the iris is partly mediated by prostaglandins and that blockade of calcium channels by nicardipine can inhibit the effects induced by prostaglandin E2.

Effects of Nilvadipine, Nicardipine and Verapamil on Acute Rise of Aqueous Flare Induced by Iris Photocoagulation or Intravenous Lipopolysaccharides in Pigmented Rabbits

The effects of nilvadipine, nicardipine and verapamil on the acute rise of aqueous flare induced by argon laser photocoagulation of the iris or by intravenous injection of lipopolysaccharides (LPS, 0.5 µg/kg) were investigated in pigmented rabbits. Nilvadipine, nicardipine and verapamil were injected intravenously. Aqueous flare was measured with a laser flare cell meter. Following photocoagulation, aqueous flare increased, reached its maximum at 45–75 min and then decreased. After administration of LPS, aqueous flare increased, reached its maximum at 4 h and then returned to baseline levels at about 24 h. Flare reactions were inhibited by nilvadipine in a dose-dependent manner. The elevations were maximally inhibited by nilvadipine 30 min before photocoagulation or intravenous LPS. Two hundred micrograms per kilogram of nilvadipine inhibited 81% of photocoagulation-induced flare elevation, while the same dose of nicardipine and verapamil inhibited 19 and 9% of the elevation, respectively. The same dose of nilvadipine inhibited 51% of LPS-induced flare elevation, while the same dose of nicardipine and verapamil inhibited 6 and 4% of the elevation, respectively. In conclusion, nilvadipine inhibited the experimental elevation of aqueous flare more effectively than did nicardipine and verapamil.

Effects of α2-Adrenergic agonists on lipopolysaccharide-induced aqueous flare elevation in pigmented rabbits

PURPOSE To evaluate the effects of the alpha(2)-adrenergic agonists (clonidine, apraclonidine, and guanfacine) on lipopolysaccharide (LPS)-induced aqueous flare elevation in pigmented rabbits. METHODS Anterior uveitis was induced with an intravenous injection of LPS (0.5 microg/kg) in an ear vein. The reproducibility of experimental uveitis induced by LPS (0.5 microg/kg) was also determined. Clonidine (0.01, 0.05, 0.25, or 1%), apraclonidine (1%), or guanfacine (1%) was topically instilled in the right eye 30 and 5 minutes before and 30 minutes after LPS application (N = 6 animals, respectively). Clonidine (0.25%) was topically administered three times at 30-minute intervals from 240 or 120 minutes before, or 120 or 240 minutes after LPS application (N = 6 animals, respectively). Then 1 mg/kg of yohimbine was injected into an ear vein 30 minutes before each topical three-time instillation of clonidine 1%, apraclonidine 1% or guanfacine 1% (N = 6 animals, respectively). Aqueous flare was measured with a laser flare-cell meter. Aqueous flare elevation was expressed as the area under the curve (AUC) in arbitrary units. Rabbits received the first LPS intravenous injection, and the control values of the AUC were obtained. Three months later, the alpha(2)-agonist and the second LPS administration were given to the same animals. RESULTS The AUCs (5,184 +/- 1,255 units) after the first application of LPS were similar to those (5,033 +/- 1,290) after the second application 3 months after the first administration. Topical instillation of clonidine inhibited LPS-induced aqueous flare elevation in a dose-dependent manner (0.01-0.25%). Topical instillation of clonidine 1%, apraclonidine 1% or guanfacine 1% inhibited LPS-induced aqueous flare elevation by 98 +/- 2.0% (mean +/- SD), 86 +/- 14% and 94 +/- 5.7%, respectively. Pretreatment with intravenous yohimbine prevented the inhibitory effect on flare elevation induced by each agent. CONCLUSION The present findings suggested that topical instillation of some alpha(2)-agonists may have an inhibitory effect on ocular inflammation, which is mediated in part by alpha(2)-receptors.

Sites of Disruption of the Blood-Aqueous Barrier after Application of Prostaglandin E2 in Pigmented Rabbits

We examined the disruption of the blood-aqueous barrier following prostaglandin (PG)-E2 application in rabbits. Vehicle or PGE2 in 10, 50 or 250 microg/ml concentration was applied to the cornea of pigmented rabbit with the use of a glass cylinder. After PGE2 administration, horseradish peroxidase (HRP) was injected intravenously. Then the eyes were enucleated, and distribution of HRP in the anterior segments was examined by electron microscopy. In control eyes, diffusion of HRP was blocked by vascular endothelial cells in the iris and by nonpigmented epithelial cells in the iridial and ciliary processes. In the iridial and ciliary processes of the eyes treated with 10 microg/ml PGE2, no HRP reaction product was seen in intercellular spaces of the nonpigmented epithelial cells, but it was found in pinocytotic vesicles. In the eyes treated with 50 microg/ml PGE2 HRP reaction product was found in intercellular spaces of the nonpigmented cells in the iridial processes. In the eyes treated with 250 microg/ml PGE2 HRP reaction product was further distributed in the iris stroma. The present study demonstrated that the sites of breakdown of the blood-aqueous barrier depended upon the doses of exogenous PGE2.

Intracameral Ascorbic Acid, Glutathione and Protein Levels in Albino and Pigmented Rabbits

We performed a comparative investigation into differences between albino and pigmented rabbits in respect of intracameral concentrations of ascorbic acid, glutathione and total protein with high-performance liquid chromatography. The intracameral concentrations of reduced ascorbic acid, total ascorbic acid, reduced glutathione, total glutathione and protein in albino rabbits were similar (p > 0.05) to those in pigmented rabbits.

Effects of Topical Clonidine on Prostaglandin-E2-Induced Aqueous Flare Elevation in Pigmented Rabbits

We evaluated the role of topical clonidine on experimental ocular inflammation. Transcorneal diffusion of prostaglandin (PG) E2, 7.09 × 10–2 mmol/l, with the use of a glass cylinder was employed to produce aqueous flare elevation in pigmented rabbits. Clonidine was topically administered and yohimbine was injected intravenously. Aqueous flare was measured with a laser flare cell meter. Topical instillation of 0.25% clonidine inhibited 89% of PGE2-induced aqueous flare elevation. Instillation of clonidine at 60 or 30 min before and 10 min after PGE2 inhibited flare significantly. Pretreatment with intravenous yohimbine decreased the clonidine-induced inhibition of the flare elevation in a dose-dependent manner. It is possible that the anti-inflammatory action of topical clonidine may be mediated partly by α2-receptors.