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(Investigative Ophthalmology and Visual Science. 2002;43:1142-1145.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Effects of Topical Antiglaucoma Eye Drops on Prostaglandin E2–Induced Aqueous Flare Elevation in Pigmented Rabbits

Yoriko Hayasaka, Seiji Hayasaka, Xue-Yun Zhang and Yasunori Nagaki

From the Department of Ophthalmology, Toyama Medical and Pharmaceutical University, Toyama, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To evaluate the role of topical instillation of some antiglaucoma agents on experimental elevation of aqueous flare induced by prostaglandin E2 (PGE2) in pigmented rabbits.

METHODS. Transcorneal diffusion of PGE2 (25 µg/mL or 7.09 x 10-2 mM) with the use of a glass cylinder was achieved to produce aqueous flare elevation in pigmented rabbits. An antiglaucoma agent was topically administered before application of PGE2. Aqueous flare was measured with a laser flare cell meter.

RESULTS. A single instillation of apraclonidine 1.15%, two instillations of epinephrine 1.25%, two instillations of dipivefrin 0.1%, and two instillations and one instillation of dipivefrin 0.04% eye drops inhibited 98%, 96%, 87%, 73%, and 47% of PGE2-induced aqueous flare elevation, respectively. Timolol 0.5%, nipradilol 0.25%, dorzolamide 1%, and pilocarpine 2% eye drops had no effects on the increase of PGE2-induced flare.

CONCLUSIONS. Apraclonidine, epinephrine, and dipivefrin eye drops inhibit PGE2-induced elevation of aqueous flare in pigmented rabbits.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Studies from our laboratory have shown that transcorneal diffusion of prostaglandin E2 (PGE2) with use of a glass cylinder induces elevation of aqueous flare in pigmented rabbits and that the elevation is reproducible when PGE2 is reapplied more than 1 week later.1 2 A glass cylinder (11 mm in diameter) was attached to the cornea to avoid transconjunctival diffusion.1

Aqueous flare elevation decreased after repeated applications of PGE2 within a short time (hourly or daily). However, weekly applications of PGE2 did not change the aqueous flare reaction in pigmented rabbits.2 We have also reported that a single instillation (30 minutes before PGE2) of 0.25% clonidine and two instillations (60 and 30 minutes before PGE2) of 0.5% betaxolol inhibits 89% and 32% of PGE2-induced aqueous flare elevation, respectively.3 4 In the present study, we evaluated the effect of topical instillation of antiglaucoma agents on aqueous flare elevation induced by PGE2 in pigmented rabbits.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Fifty-two pigmented male rabbits (Japanese mongrel) weighing 2.5 to 3.5 kg were used. The animals were housed and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Institutional Animal Care and Utilization Committee, Toyama Medical and Pharmaceutical University, Toyama, Japan. One eye of each animal was used to determine the effect of each drug. The eyes received two transcorneal applications of PGE2 at 1- or 2-week intervals. Three months later, the other eye of the animal was used to determine the effect of another drug.

Chemicals
Dipivefrin hydrochloride (Pivalephrine, a ß2-agonist) and pilocarpine (Sanpilo, a cholinergic agent) ophthalmic solutions were obtained from Santen Pharmaceutical Company (Osaka, Japan). Timolol maleate (Timoptol, a ß-antagonist) and dorzolamide (Trusopt, a carbonic anhydrase inhibitor) were from Banyu Pharmaceutical Company (Tokyo, Japan). Apraclonidine (Iopidine, an {alpha}2-agonist), epinephrine (Epista, a ß2-agonist), and nipradilol (Nipranol, a ß-antagonist with an {alpha}1-antagonist) ophthalmic solutions were purchased from Alcon Laboratories (Fort Worth, TX), Senju (Osaka, Japan), and Teika (Toyama, Japan) pharmaceutical companies, respectively.

PGE2 was obtained from Funakoshi Chemicals (Tokyo, Japan). PGE2 was dissolved in 100% ethanol and stored at -70°C. PGE2 solution was diluted to 5% ethanol with 0.9% NaCl just before use. Epinephrine ophthalmic solution was diluted with 0.9% NaCl.

Topical Instillation of Antiglaucoma Agent or Placebo
In one eye, 50 µL 0.1% antiglaucoma agent or placebo (0.9% NaCl) was topically instilled. Instillation took place twice (60 and 30 minutes before PGE2). We also performed single instillations to examine dose and time dependency. The bottles were masked, and the person who administered the eye drops had no preliminary knowledge of the contents.

Transcorneal Diffusion of PGE2
For transcorneal diffusion, a glass cylinder (11 mm in diameter) was attached to the cornea, as described by Hirata et al.1 Next, 600 µL of PGE2 solution (25 µg/mL or 7.09 x 10-2 mM) was delivered into the cylinder and pipetted out 4 minutes later. The cylinder was removed, and the corneal surface and conjunctival sac were rinsed with 20 mL 0.9% NaCl. The eyes received a second transcorneal application of PGE2, 1 or 2 weeks later (Fig. 1) . PGE2-induced flare elevation was measured in the eye pretreated with antiglaucoma agent or placebo (0.9% NaCl) and again after the second PGE2 application.



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  		Figure 1. Two applications of PGE2. The eyes received two transcorneal diffusions of PGE2 at 1- or 2-week intervals.

 
Aqueous Flare Measurement
Aqueous flare was measured with a laser flare cell meter (model FC 1000; Kowa, Tokyo, Japan), according to the method described by Sawa et al.5 A laser flare-cell meter measured intracameral proteins. Five measurements were taken at each time point to obtain a mean value. The measurement was taken in the midportion of the anterior chamber. The sampling area was 0.075 mm2.

Aqueous flare elevation was expressed as the area under the curve (AUC) for each eye. Inhibition was estimated from the AUCs in the same eye by the following equation: inhibition (%) = 1 - [(AUC with treatment)/(AUC without treatment)] x100. The measurer had no preliminary knowledge of the treatment.

Statistics
Statistical analysis was performed using the Dunn multiple comparisons procedure. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After topical instillation of epinephrine 1.25%, the iris became slightly pale. Other eye drops induced no change in iris color. Two instillations (60 and 30 minutes before PGE2) of apraclonidine, epinephrine, dipivefrin, timolol, nipradilol, dorzolamide, and pilocarpine did not induce aqueous flare elevation.

No marked changes in the systemic condition, including body weight and behavior, were noted after the transcorneal diffusion of PGE2 (7.09 x 10-2 mM).

After PGE2 was administered, aqueous flare increased, reached its maximum (470 ± 37 photon counts/ms) at 60 to 90 minutes, and then gradually decreased and returned to baseline level after 7 to 8 hours (Fig. 2) . When apraclonidine 1.15% was topically instilled 60 minutes before PGE2, aqueous flare did not increase. Single instillation of apraclonidine 1.15%, two instillations of epinephrine 1.25%, two instillations of dipivefrin 0.1%, and two and one instillations of dipivefrin 0.04% eye drops inhibited 98%, 96%, 87%, 73%, and 47% of PGE2-induced increase in aqueous flare, respectively (Table 1) .



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  		Figure 2. Changes in flare intensity after transcorneal diffusion of PGE2, with and without topical instillation of apraclonidine. (•) Transcorneal application of PGE2 (7.09 x 10–2 mM) for 4 minutes (solid arrow). ({circ}) Apraclonidine (1.15%) topically instilled 60 minutes before PGE2 application (open arrow). Data are the mean (n = 6).

 

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  		Table 1. Effects of Eye Drops on PGE2-Induced Aqueous Flare Elevation in Pigmented Rabbits

 
Timolol 0.5%, nipradilol 0.25%, dorzolamide 1%, and pilocarpine 2% eye drops had no effect on the increase in PGE2-induced flare. The effect of a single instillation of epinephrine on the increase in PGE2-induced aqueous flare in pigmented rabbits is shown in Table 2 . Topical instillation of epinephrine inhibited flare elevation in a dose-dependent manner (0.2%–1.25%). Instillation of 1.25% epinephrine 30 minutes before PGE2 application inhibited 90% of PGE2-induced aqueous flare elevation.


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  		Table 2. Effects of a Single Instillation of Epinephrine on PGE2-Induced Aqueous Flare Elevation in Pigmented Rabbits

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have reported that topical clonidine inhibits the increase in PGE2-induced aqueous flare.3 Topical apraclonidine (p-aminoclonidine ) reduces the increase in intraocular pressure and aqueous humor protein after YAG and argon laser irradiation of the rabbit iris.6 7 In the present study, topical apraclonidine also inhibited the PGE2-induced elevation in aqueous flare in rabbits.

Several researchers have reported a relationship between epinephrine and aqueous humor: Townsend and Brubaker8 postulated that epinephrine increases the rate of the uveoscleral outflow pathway in humans. Camras et al.9 reported on the inhibition of epinephrine-induced reduction of intraocular pressure by systemic indomethacin in humans. Miyake et al.10 11 reported that epinephrine induces disruption of the blood-aqueous barrier several months after drug administration in rabbits and humans. Anderson and Wilson12 described inhibition by indomethacin of the increased facility of outflow induced by adrenaline in rabbits. Mori et al.13 reported that a single instillation of epinephrine affects neither the protein concentration in the anterior chamber nor the aqueous flow rate in humans. In our present study, topical instillation of epinephrine and dipivefrin (dipivalyl epinephrine) inhibited the PGE2-induced elevation of aqueous flare in rabbits. Miyake et al.14 further reported synthesis of PGE2 in rabbit eyes with topically applied epinephrine.

However, our results showed that a single instillation of 1.25% epinephrine 30 minutes before PGE2 inhibited 90% of the increase in PGE2-induced flare. The difference between our results and the findings reported by Miyake et al.10 14 may be due to the varied instillation times. Okada and Shimada15 reported that intravenous epinephrine and intramuscular steroid inhibit the increase of permeability of the blood–aqueous barrier induced by reverse passive Arthus reactions in rabbits. Our results were similar to those described by Okada and Shimada.15 The iris in our animals became slightly pale after topical instillation of 1.25% epinephrine. Vasoconstriction induced by epinephrine may play a role in inhibition of the PGE2-induced elevation in aqueous flare. Topical epinephrine or dipivefrin effects on the elevation of aqueous flare after argon laser iridotomy should be examined in humans.

We have reported that betaxolol inhibits the PGE2-induced elevation in aqueous flare in rabbits and suggest that the calcium-channel blocking activity of betaxolol may be involved in the inhibition.4 Miichi and Nagataki16 reported that timolol does not alter the function of the blood–aqueous barrier in the cynomolgus monkey. Kanno et al.17 reported that a single instillation of nipradilol shows no significant effect on blood–aqueous barrier permeability in rabbits.

In our present study, timolol and nipradilol did not inhibit elevation of aqueous flare. It is unlikely that the ß-blocking activity of the drugs is involved in the inhibition of the elevation of flare in rabbits. Mori and Araie18 reported that timolol induces elevation of protein concentration in humans. The discrepancy between our results and the findings reported by Mori and Araie18 may be due to the difference in species.19

In the present study, topical dorzolamide and pilocarpine did not alter the PGE2-induced elevation of aqueous flare. Miichi and Nagataki16 reported that the blood–aqueous barrier was not altered by pilocarpine in the cynomolgus monkey. Our findings support this.16

PGE2-like activity was detected in the aqueous humor after paracentesis in rabbits,20 and it may be involved in traumatic iridocyclitis in rabbits. The blood–aqueous barrier in rabbits has a unique sensitivity to PGs.19 Therefore, the findings in the present study are not representative of the effects seen in humans. The mechanisms of inhibition by epinephrine and dipivefrin of the PGE2-induced elevation of aqueous flare in rabbits should be investigated.


   Footnotes
 
Submitted for publication June 22, 2001; revised November 16, 2001; accepted November 30, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Yoriko Hayasaka, Department of Ophthalmology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan; ophthal{at}ms.toyama-mpu.ac.jp


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hirata, H, Hiraki, S, Kaji, Y, Takeda, N, Fukuo, Y, Tachinami, K. (1994) The effects of trans-corneal administration of prostaglandin E2 on rabbit eyes J Jpn Ophthalmol Soc 98,927-934
  2. Watanabe, K, Hirata, H, Hiraki, S, Hayasaka, S. (1996) Decreased aqueous-flare reaction to repeated applications of prostaglandin E2 to the cornea in pigmented rabbits Ophthalmic Res 28,147-152[Medline][Order article via Infotrieve]
  3. Watanabe, K, Hayasaka, S, Hiraki, S, et al (2000) Effect of topical clonidine on prostaglandin E2-induced aqueous flare elevation in pigmented rabbits Ophthalmic Res 32,210-214[Medline][Order article via Infotrieve]
  4. Yanagisawa, S, Hayasaka, S, Zhang, XY, Hayasaka, Y, Nagaki, Y, Kitagawa, K. (2001) Effects of topical betaxolol on acute rise of aqueous flare induced by prostaglandin E2 in pigmented rabbits Jpn J Ophthalmol 45,669-671[Medline][Order article via Infotrieve]
  5. Sawa, M, Tsurimaki, Y, Tsuru, T, Shimizu, H. (1988) New quantitative method to determine protein concentration and cell number in aqueous in vivo Jpn J Ophthalmol 32,132-142[Medline][Order article via Infotrieve]
  6. Sugiyama, K, Kitazawa, Y, Kawai, K. (1990) Apraclonidine effects on ocular responses to YAG laser irradiation to the rabbit iris Invest Ophthalmol Vis Sci 31,708-714[Abstract/Free Full Text]
  7. Bonomi, L, Bellucci, R, Pagliarusco, A, Stefani, L. (1995) Apraclonidine protection of the blood-aqueous barrier from traumatic break-down J Ocul Pharmacol 11,25-35
  8. Townsend, DJ, Brubaker, RF. (1980) Immediate effect of epinephrine on aqueous formation in the normal human eye as measured by fluorophotometry Invest Ophthalmol Vis Sci 19,256-266[Abstract/Free Full Text]
  9. Camras, CB, Feldman, SG, Podos, SM, Christensen, RE, Gardner, SK, Fazio, DT. (1985) Inhibition of the epinephrine-induced reduction of intraocular pressure by systemic indomethacin in humans Am J Ophthalmol 100,169-175[Medline][Order article via Infotrieve]
  10. Miyake, K, Kayazawa, F, Manabe, R, Miyake, Y. (1987) Indomethacin and the epinephrine-induced breakdown of the blood-ocular barrier in rabbits Invest Ophthalmol Vis Sci 28,482-486[Abstract/Free Full Text]
  11. Miyake, K, Miyake, Y, Kuratomi, R. (1987) Long-term effects of topically applied epinephrine on the blood-ocular barrier in humans Arch Ophthalmol 105,1360-1363[Abstract]
  12. Anderson, L, Wilson, WS. (1990) Inhibition by indomethacine of the increased facility of outflow induced by adrenaline Exp Eye Res 50,119-126[Medline][Order article via Infotrieve]
  13. Mori, M, Sakurai, M, Araie, M. (1992) Effect of topical epinephrine on permeability of blood-aqueous barrier in human eyes Jpn J Ophthalmol 36,342-347[Medline][Order article via Infotrieve]
  14. Miyake, K, Shirasawa, E, Hikita, M, Miyake, Y, Kuratomi, R. (1988) Synthesis of prostaglandin E in rabbit eyes with topically applied epinephrine Invest Ophthalmol Vis Sci 29,332-334[Abstract/Free Full Text]
  15. Okada, M, Shimada, K. (1980) Effects of various pharmacologic agents on allergic inflammation of the eye Invest Ophthalmol Vis Sci 19,176-181[Abstract/Free Full Text]
  16. Miichi, H, Nagataki, S. (1983) Effects of pilocarpine, salbutamol, and timolol on aqueous humor formation in cynomolgus monkeys Invest Ophthalmol Vis Sci 24,1269-1275[Abstract/Free Full Text]
  17. Kanno, M, Araie, M, Tomita, K, Sawanobori, K. (1998) Effects of topical nipradilol, a ß-blocking agent with {alpha}-blocking and nitroglycerin-like activities, on aqueous humor dynamics and fundus circulation Invest Ophthalmol Vis Sci 39,736-743[Abstract/Free Full Text]
  18. Mori, M, Araie, M. (1991) A simple method of determining the time course of timolol’s effects on aqueous flow in humans Arch Ophthalmol 109,1099-1103[Abstract]
  19. Toris, CB, Camras, CB, Yablonski, ME, Brubaker, RF. (1997) Effects of exogenous prostaglandins on aqueous humor dynamics and blood-aqueous barrier function Surv Ophthalmol 41(suppl 2),S69-S75
  20. Miller, JD, Eakins, KE, Atwal, M. (1973) The release of PGE2-like activity into aqueous humor after paracentesis and its prevention by aspirin Invest Ophthalmol 12,939-942[Free Full Text]



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Y. Hayasaka, S. Hayasaka, X.-Y. Zhang, and Y. Nagaki
Effects of Topical Anti-inflammatory and Antiallergic Eyedrops on Prostaglandin E2-Induced Aqueous Flare Elevation in Pigmented Rabbits
Arch Ophthalmol, July 1, 2002; 120(7): 950 - 953.
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