- Email: [email protected]
Effects of anti-glaucoma drugs timolol and GLC756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, on endotoxin-induced-uveitis and -arthritis in rats
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 57 (2005) 127–134
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Effects of anti-glaucoma drugs timolol and GLC756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, on endotoxin-induced-uveitis and -arthritis in rats U.W. Laenglea,, M. Courta, R. Marksteinb, P.G. Germanna, V. Noguesa, D. Romana a
Department of Toxicology/Pathology, Novartis Pharma AG, MUT-2881.133, CH-4002 Basel, Switzerland Department of Ophthalmics, Novartis Pharma AG, CH-4002 Basel, Switzerland
b
Received 1 December 2004; accepted 24 February 2005
Abstract Anti-glaucoma drugs exhibiting anti-inflammatory properties are desirable for the long-term treatment of glaucoma since they may reduce the risk for treatment-related inflammatory processes in outer compartments of the eye. The purpose of this study was to evaluate potential anti-inflammatory effects of two topically and systemically applied antiglaucoma drugs i.e. GLC 756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, and timolol a b-adrenoceptor antagonist using endotoxin-induced uveitis (EIU) and arthritis in rats as an in vivo model. For EIU, 8week-old Lewis rats were intravenously injected at 160 mg lipopolysaccharide (LPS) from Salmonella typhimurium. GLC756, timolol, or betamethasone, as a positive control, were either topically (0.4%, 0.5%, and 0.1%, respectively, 16-times 20 mL eye drops during 48 h) or systemically (1 mg/kg subcutaneous for 5 days) administered. Cell infiltration in tissue of the eye and knee joint were assessed histopathologically and in special compartments of the eye by confocal microscopy 48 h after LPS-induction. Numerous infiltrating cells were detected in the eyes after LPS-induction and half of the animals showed arthritis. Topical and systemic pre-treatment with GLC756 and timolol resulted in reduced cell infiltration in the eye. In addition, GLC756 reduced, whereas timolol increased the incidence of arthritis. Betamethasone suppressed almost completely the cell infiltration in the eye and the incidence of arthritis. In conclusion, the observations that GLC756 reduced cell infiltration in the eye and the incidence of arthritis after LPS-induction is compatible with anti-inflammatory properties of this drug. By contrast, timolol produced no consistent anti-inflammatory effect since both inhibitory as well as stimulatory effects on inflammatory processes were seen. r 2005 Elsevier GmbH. All rights reserved. Keywords: Anti-glaucoma drugs; GLC756; Dopamine D2 agonist and D1 antagonist; Timolol; Betamethasone; EIU; Arthritis; Infiltrating cells; Rat
Introduction Corresponding author. Tel.: +41 61 324 1891; fax: +41 61 324 1561. E-mail address: [email protected] (U.W. Laengle).
0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.02.008
Glaucoma designates a group of chronic eye diseases which are characterized by progressive atrophy of the optic nerve head and visual field loss and, in most cases, by elevation of intraocular pressure (IOP). The etiology
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of glaucoma is still unknown but elevated IOP and impaired perfusion of the optic nerve has been suggested to be important risk factors. Current medical treatment of glaucoma is focused mainly on lowering IOP. Timolol belongs to the class of b-adrenoceptor blocking agents with IOP lowering properties (Schuman, 2002). Dopaminergic drugs are another class of compounds that lower IOP (Pruente and Flammer, 1995) and in addition can improve perfusion of the optic nerve (Pru¨nte et al., 1995) and may therefore be of particular value for the treatment of glaucoma (Chiou and Chen, 1992; Markstein et al., 1996; Prunte et al., 1996). Increased numbers of inflammatory cells in outer compartments of the eye were found in humans who have been treated with conventional anti-glaucoma drugs (Sherwood et al., 1989; Ariturk et al., 1997; Broadway et al., 1994). Mediators of inflammatory response might mediate proliferation of the fibroblasts in the anterior parts of the eye (Lark et al., 1999). Both, increased numbers of inflammatory cells and of fibroblasts may contribute to the failure of glaucoma filtering surgery (Sherwood et al., 1989; Maumenee, 1960; Flaxel and Swan, 1969; Miller et al., 1989; Addicks et al., 1983) which is necessary in some glaucoma patients who do not respond well to conventional anti-glaucoma drugs. Therefore, anti-glaucoma drugs with additional antiinflammatory properties may be of particular therapeutic value. Endotoxin-induced uveitis (EIU) in the Lewis rat is a model for severe uveitis in humans (Rosenbaum et al., 1980). Systemic injection of endotoxin, a lipopolysaccharide (LPS) from the outer membrane of gramnegative bacteria, induces an acute and self-limited bilateral uveitis (Rosenbaum et al., 1980; Hoekzema et al., 1992). The ocular inflammation is characterized by a cellular infiltration mainly with polymorphonuclear neutrophils (McMenamin and Crewe, 1995; Kogiso et al., 1992) into the outer compartments of the eye. It is also shown that the Lewis rat is arthritis susceptible after intravenous challenge with enteropathogenic bacteria (Hill and Yu, 1987). In this study, effects of systemic and topical medicated anti-glaucoma drugs GLC756, a novel dopamine D2 agonist and D1 antagonist, and timolol on EIU and arthritis were examined. The results were compared with that of betamethasone which served as a positive control.
Material and methods Animals and study design Eight-week-old Lewis rats, weighing 140–180 g, were obtained from Charles River Company, WIGA, Sulzfeld, Germany. EIU and arthritis were induced in
eight groups of five rats each, receiving an intravenous injection of LPS from Salmonella typhimurium (Sigma, Switzerland.) at 160 mg per rat in a volume of 160 mL. Two of these groups were kept as positive controls. The remaining groups were treated with GLC756, betamethasone, or timolol either topically (0.4%, 0.1%, and 0.5% respectively, 16-times 20 mL eye drops during 48 h, LPS intravenous about 1 h after the 1st eye drop) or systemically (1 mg/kg/day subcutaneously for 5 days; LPS intravenously on the 3rd day). An additional group of four animals was kept as control group treated topically with 0.9% NaCl (16-times 20 mL eye drops during 48 h into the right eye; left eye stayed untreated). Animals were sacrificed 48 h after LPS-induction.
Drug preparation GLC756 was received as a powder and as 0.4% eye drops from Novartis Pharma AG, Switzerland. The GLC756 powder was reported to be 100% pure by certificate of analysis. The powder was dissolved in a 2.5% glycerol solution. Timolol was available commercially as a powder (100% pure) from Sigma, Switzerland, and as 0.5% eye drops from Ursapharm, Germany. The powder was dissolved in a 2.5% glycerol solution. Betamethasone was available commercially as a powder (99.7% pure) from Sigma, Switzerland and as 0.1% eye drops from Dr. Winzer Pharma GmbH, Germany. The powder was dissolved in a 40% polyethylenglycol 300 solution.
Tissue sampling After sampling of the aqueous humor for special examinations (Laengle et al., 2005) the right eye and a knee joint of all treated animals was removed for histopathological processing and the remaining eye for confocal laser scanning microscopy (CLSM). In the control group, the left eye of three animals and the right eye of one animal were used for histopathology and the remaining eyes for CLSM.
Histopathological processing The eyes with harderian glands were fixed in Davidson’s solution (96% alcohol, 40% formaldehyde, acetic acid, distilled water, 1% methylene blue) and the knee joints in phosphate-buffered 10% formalin. The bone was demineralized with 10% formic acid. The tissues were embedded in Paraplasts. A section of 4 mm was stained with hematoxylin and eosin. Severity of inflammatory reaction in iris, ciliary/vitreous body, retina, and choroidea 48 h after LPS-induction, and systemic or topical pre-treatment with GLC756, betamethasone, or timolol was based on a sum-up of a
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Fig. 1. Histological section through an eye 48 h after LPS-induction showing infiltrating cells, polymorphonuclear neutrophils (PMNs), monocytes/macrophages (Mon). Main picture 300, small picture 750. (A) Inflammatory cell infiltration into iris, (B) inflammatory cell infiltration into the ciliary body, (C) inflammatory cell infiltration into the vitreous body, and (D) inflammatory cell infiltration into the vitreous body and with remarkably lower grade into the optic nerve head.
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grading, minimal ¼ 1, slight ¼ 2, moderate ¼ 3, and severe ¼ 4.
Confocal laser scanning microscopy The double immunofluorescence labeled slides were examined using a Leica TCS-NT (Leica Microsystems Ltd.), argon/krypton CLSM coupled to a Leica DM IRBE inverted microscope. The Argon/krypton laser was filtered using a double dichromic mirror so that dual excitation wavelengths of 488 and 568 nm were used. The emission of fluorescence was detected using different barrier filter sets. Antibody coupled to Alexa was detected using a band pass filter set at 530 nm and the propidium iodide was detached using a long pass filter set at 590 nm. Using a 10 air and 63 water immersion objectives the upper and lower limits of the regions of interest were selected and at the same time adjustments were made to the gain (photomultiplier detector voltage) and offset (image black levels). Between 10 and 50 consecutive z-series, optical sections were taken of various thicknesses between 0.2 and 1.0 mm, to capture the entire field of interest. Each optical section was cleaned using a 4 -averaging filter to improve the signal/noise ratio. Collected z-series optical sections were saved as the raw image data, which were in the format of multi-image TIFF files. Collected optical sections were also projected on to one another to give a single 2D reconstruction of the data.
Processing for CLSM After sampling the tissue for normal histopathology the animals were perfused with 4% paraformaldehyde, for 2–3 min, via the left ventricle. Thereafter, the remaining eyes were removed, fixed in fresh fixative for a further 1.5 h, and kept in cold phosphate buffered saline (PBS, 4 1C) for 1 h until dissection under a binocular microscope into the segment, iris (McMenamin, 2000). The segment was then quartered and placed in a well containing PBS. The eye segments of the systemically treated animals were then incubated 12 h at 4 1C with the mouse IgM anti-rat HIS48 (Serotec, Switzerland) at 1/100, diluted in PBS, 1% BSA, 0.005% Tween 20 for the staining of granulocytes. The tissue were then washed twice in PBS and further incubated with the anti-mouse IgM coupled to Alexa 488 at 1/200 in PBS, 1% BSA, 0.005% Tween 20, for 30 min at room temperature. Finally the tissues were washed twice in PBS and counterstained in propidium iodide (for the detection of the nuclei) at 1 mg/ml in PBS for 15 min at room temperature. Erythroid cells are also stained by the anti-HIS48 antibody (De Vos et al., 2000).
Topical Treatment LPS 160 µg
3.50
GLC756 0.4%
Betamethasone 0.1%
Timolol 0.5%
3.00
Grading
2.50
2.00
** 1.50
** 1.00
**
**
0.50
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
0.00
Fig. 2. Inflammatory reaction in iris, ciliary/vitreous body, retina, and choroidea 48 h after LPS-induction, and topical pretreatment with GLC756, betamethasone, or timolol. Severity was based on a sum-up of a grading, minimal ¼ 1, slight ¼ 2, moderate ¼ 3, severe ¼ 4. Each column is a mean of five values and the bars indicate SD. Means were considered significantly different from those of the LPS-group at Po0:005.
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of the eye, in particular the anterior/posterior chamber, iris, ciliary and vitreous body, retina, choroidea, and optic nerve head (Fig. 1). The infiltration was most severe in the vitreous body, consisting mainly of neutrophils and a few monocytes and macrophages, as well as in the ciliary body and retina (Figs. 2 and 3), here predominantly of neutrophils. GLC756 produced a statistically significant reduction in inflammatory cell infiltration in the retina after topical and in the ciliary/vitreous body, and retina after systemic pre-treatment (Figs. 2 and 3). Topical and systemic pre-treatment with betamethasone, reduced statistically significant the cellular infiltration in all compartments of the eye (Figs. 2 and 3). Timolol caused a statistically significant reduction in inflammatory cell infiltration in the vitreous body only after systemic treatment but was inactive after topical administration (Figs. 2 and 3). Coincident with the histopathological evaluation, CLSM of immunostained granulocytes illustrates the systemic pre-treatment-related effects in samples from the iris (Fig. 4). In LPS-treated animals HIS48-labeled cells were scattered throughout the iris (Fig. 4A). Mainly granulocytes are labeled (Fig. 4A0 ). In comparison, a decrease in HIS48-labeled cells was apparent after pretreatment with GLC756, betamethasone or timolol (Figs. 4B–D, B0 –D0 ).
Image analysis Selected z-series optical sections were transferred to a Silicon Graphics (SGI) workstation and further analyzed using Imaris software (Bitplane). Images were opened into the ‘sections’ module of the Imaris program. Single optical sections of the confocal images and both x and y plane profiles were analyzed to localize and identify inflammatory cells deeper in the tissue. No filters or image correction were applied to the images.
Statistical analysis For all groups mean7SD were obtained. These values were subjected to an ANOVA 1-way Dunnett’s test. Values were considered significantly different from corresponding control at Po0:05.
Results Histopathological evaluation showed no obvious cellular infiltration in tissues of normal or 0.9% NaCl treated control eyes. In animals treated with LPS, cellular infiltration could be observed 48 h post-dose in almost all compartments
Systemic Treatment 2.50
LPS 160 µg
GLC756 0.4%
Betamethasone 0.1%
Timolol 0.5%
2.00
** Grading
1.50
* *
1.00
**
**
0.50
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
** ** vitreous body
ciliary body
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
iris
** **
0.00
Fig. 3. Inflammatory reaction in iris, ciliary/vitreous body, retina, and choroidea 48 h after LPS-induction, and systemic pretreatment with GLC756, betamethasone, or timolol. Severity was based on a sum-up of a grading, minimal ¼ 1, slight ¼ 2, moderate ¼ 3, severe ¼ 4. Each column is a mean of five values and the bars indicate SD. Means were considered significantly different from those of the LPS-group at Po0:05 and Po0:005.
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Histopathological evaluation of a knee joint (Table 1) revealed that control animals were devoid of any inflammatory cell infiltration. After LPS-induction, five out of ten animals showed minimal to slight inflamma-
tory cell infiltration, mainly neutrophils. Topical and systemic pre-treatment with GLC756, like the positive control betamethasone reduced the incidence of articular inflammation. In the case of timolol, the incidence of
Fig. 4. Confocal photomicrographs showing HIS48 staining in the iris 48 h after LPS-induction (A), and systemic pre-treatment with GLC756 (B), betamethaosone (C), or timolol (D). Mainly granulocytes are labeled A0 –D0 . Bars in main picture ¼ 100 mm; Bars in small picture ¼ 20 mm.
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Table 1. timolol
133
Incidence of arthritis 48 h after LPS-induction and topical- or systemic pre-treatment with GLC756, betamethasone, or
Examined animals (n) Affected animals (n)
Controls
LPS
GLC756
Betamethasone
Timolol
10 0
10 5
10 2
10 1
10 7
articular inflammation was even higher than in the LPSgroup.
Discussion Increased numbers of inflammatory cells in outer compartments of the eye were found in humans who have been treated with anti-glaucoma drugs (Sherwood et al., 1989; Ariturk et al., 1997; Broadway et al., 1994). Mediators of inflammatory response might mediate proliferation of the fibroblasts in the anterior parts of the eye (Lark et al., 1999). Both effects may contribute to the failure of glaucoma filtering surgery (Sherwood et al., 1989; Maumenee, 1960; Flaxel and Swan, 1969; Miller et al., 1989; Addicks et al., 1983). Therefore, one can assume that anti-glaucoma drugs with additional anti-inflammatory properties may prevent these adverse effects. To evaluate potential anti-inflammatory properties of anti-glaucoma drugs, an in vivo study using an EIU model in Lewis rats was performed. In this study, GLC756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, and timolol as well as betamethasone as a positive control, were assessed for their effects on cellular infiltration in the eye and knee joint 48 h after LPS-administration. Systemic administration of endotoxin, LPS, a component of gram-negative bacterial outer membranes, was first reported to induce an acute bilateral anterior uveitis in the Lewis rat, affecting only the anterior chamber, iris, ciliary body, and conjunctiva (Rosenbaum et al., 1980; Herbort et al., 1988; Cousins et al., 1984). However, further investigations showed that inflammatory cells also infiltrate posterior eye segments, including the retina and choroidea (Ruiz-Moreno et al., 1992). Furthermore, inflammatory infiltration of most of the polymorphonuclear and a few mononuclear cells was also reported in the vitreous body of an EIU susceptible mouse strain (Kogiso et al., 1992). Besides induction of EIU, it is also shown that the Lewis rat is arthritis susceptible after intravenous challenge with enteropathogenic bacteria (Hill and Yu, 1987). In the present study, systemic administration of LPS induced inflammatory cell infiltration in almost all compartments of the eye, in particular the anterior/ posterior chamber, iris, ciliary and vitreous body, retina,
choroidea, and optic nerve head 48 h post-administration. The highest inflammatory cell reactions were graded for the vitreous body, followed by the ciliary body and retina. A notably lower rating resulted for the iris and choroidea. Inflammatory cell reaction was also noted in the optic nerve head. The inflammatory cell infiltration was composed mainly of neutrophils and a few monocytes and macrophages. GLC756 produced a statistically significant reduction in inflammatory cell infiltration in the retina after topical and in the ciliary/vitreous body and retina after systemic pre-treatment. This anti-inflammatory property of GLC756 might be related to its significant TNF-a reducing effect which was measured in the serum (Laengle et al., in submission). TNF-a as being one of the first cytokines released in the cascade of inflammation (Fong and Lowry, 1990) is an important mediator of inflammation (Durum and Oppenheim, 1989). TNF-a can be produced mainly by ocular resident cells (i.e., macrophages, endothelial, and epithelial cells) as well as by infiltrating cells (i.e., polymorphonuclear cells and monocytes) (De Vos et al., 1994). The anti-inflammatory effect of GLC756 was also confirmed by reducing the incidence of arthritis in comparison to that of the LPS-group. The effect was similar than that of the positive control with betamethasone. Betamethasone was, however, much more efficient in the reduction of inflammatory cell infiltration in the eye than GLC756. The difference is very likely related to the highly immunosuppressive property of betamathasone which indicates also that immune mechanisms contribute to the inflammation in EIU (Rosenbaum and Boney, 1993). For timolol, a statistically significant reduction in inflammatory cell infiltration was seen in the vitreous body after systemic pre-treatment. This seemingly antiinflammatory effect of timolol in the eye was in contrast to the finding in the knee joint where no effect on reduced incidence in arthritis was noted. In summary, our in vivo study in Lewis rats demonstrated that GLC756, a novel dopaminergic compound, showed slight inhibitory effects on EIU and arthritis in the Lewis rat. Therefore it is suggested that dopaminergic compounds like GLC756 can prevent or counteract slight inflammatory processes in outer compartments of the eye which represents an additional benefit in the treatment of glaucoma.
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Acknowledgements We thank Lothar Kruppa, Novartis Pharma AG, for his engagement in performing the in-life phase and Merdol Ibrahim for doing the confocal microscopy of this study.
References Addicks EM, Quigley HA, Greene WR, Robin AL. Histologic characteristics of filtering blebs in glaucomatous eyes. Arch Ophthalmol 1983;101:795–8. Ariturk N, Oge I, Baris S, Erkan D, Sullu Y, Koc F. The effects of antiglaucomatous agents on conjunctiva used for various durations. Int Ophthalmol 1997;20:57–62. Broadway DC, Grierson I, O’Brien C, Hitchings RA. Adverse effects of topical antiglaucoma medication. I. The conjunctival cell profile. Arch Ophthalmol 1994;112:1437–45. Chiou GC, Chen YJ. Effects of dopamine agonist, bromocriptine, and some dopamine antagonists on ocular blood flow. J Ocul Pharmacol 1992;8:285–94. Cousins SW, Guss RB, Howes Jr EL, Rosenbaum JT. Endotoxin-induced uveitis in the rat: observations on altered vascular permeability, clinical findings, and histology. Exp Eye Res 1984;39(5):665–76. De Vos AF, van Haren MA, Verhagen C, Hoekzema R, Kijlstra A. Kinetics of intraocular tumor necrosis factor and interleukin-6 in endotoxin-induced uveitis in the rat. Invest Ophthalmol Vis Sci 1994;35:1100–6. De Vos AF, Dick AD, Klooster J, Broersma L, McMenamin PG, Kijlstra A. Analysis of the cellular infiltrate in the iris during experimental autoimmune encephalomyelitis. Invest Ophthalmol Vis Sci 2000;41:3001–10. Durum SL, Oppenheim JJ. In: Paul WE, editor. Fundamental immunology. New York: Ravens Press; 1989. p. 639–61. Flaxel JT, Swan KC. Limbal wound healing after cataract extraction. Arch Ophthalmol 1969;81:653–9. Fong Y, Lowry SF. Tumor necrosis factor in the pathophysiology of infection and sepsis. Clin Immunol Immuopathol 1990;55:157–70. Herbort CP, Okumura A, Mochizuki M. Endotoxin-induced uveitis in the rat. A study of the role of inflammation mediators. Graefes Arch Clin Exp Ophthalmol 1988;226:553–8. Hill JL, Yu DT. Development of an experimental animal model for reactive arthritis induced by Yersinia enterocolitica infection. Infect Immun 1987;55:721–6. Hoekzema R, Verhagen C, van Haren M, Kijlstra A. Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6. Invest Ophthalmol Vis Sci 1992;33:532–9. Kogiso M, Tanouchi Y, Mimura Y, Nagasawa H, Himeno K. Endotoxin-induced uveitis in mice. 1. Induction of uveitis and role of T lymphocytes. Jpn J Ophthalmol 1992;36:281–90. Laengle UW, Markstein R, Schneider V, Greiner B, Roman D. Effects of antiglaucoma drugs timolol and GLC756, a
novel dopamine D2 agonist and D1 antagonist, on edotoxin-induced-uveitis in rats. Exp Eye Res 2005;80: 847–52. Laengle UW, Markstein R, Schneider V, Roman D. Effects of antiglaucoma drugs timolol and GLC756, a novel dopamine D2 agonist and D1 antagonist, on endotoxin-induced TNF-alpha release in serum of rats. Journal of Glaucoma, submitted. Lark KK, Pasha AS, Yan X, Edward DP. The effect of latanoprost and brimonidine on rabbit subconjunctival fibroblasts. J Glaucoma 1999;8:72–6. Markstein R, Gull P, Rudeberg C, Urwyler S, Jaton AL, Kalkman HO, Dixon AK, Hoyer D. SDZ GLC 756, a novel octahydrobenzo[g]quinoline derivative exerts opposing effects on dopamine D1 and D2 receptors. J Neural Transm 1996;103:17–30. Maumenee AE. External filtering operations for glaucoma: the mechanism of function and failure. Trans Am Ophthalmol Soc 1960;58:319–28. McMenamin PG. Optimal methods for preparation and immunostaining of iris, ciliary body, and choroidal wholemounts. Invest Ophthalmol Vis Sci 2000;41:3043–8. McMenamin PG, Crewe J. Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body. Invest Ophthalmol Vis Sci 1995;36:1949–59. Miller MH, Grierson I, Unger WI, Hitchings RA. Wound healing in an animal model of glaucoma fistulizing surgery in the rabbit. Ophthalmic Surg 1989;20:350–7. Pruente C, Flammer J. The novel dopamine D1 antagonist and D2 agonist, SDZ GLC 756, lowers intraocular pressure in healthy human volunteers and in patients with glaucoma. Ophthalmology 1995;102:1291–7. Pru¨nte C, Flammer J, Markstein R, Rudin M. Quantification of optic nerve blood flow using MR-imaging. Invest Ophthalmol Vis Sci 1995;36:247–51. Prunte C, Nuttli I, Markstein R. Topical SDZ GLC-756, a novel dopamine D-1 antagonist and D-2 agonist, lowers intraocular pressure in conscious rabbits. Jpn J Ophthalmol 1996;40:167–73. Rosenbaum JT, Boney RS. Failure to inhibit endotoxininduced uveitis with antibodies that neutralize tumor necrosis factor. Reg Immunol 1993;5:299–303. Rosenbaum JT, McDevitt HO, Guss RB, Egbert PR. Endotoxin-induced uveitis in rats as a model for human disease. Nature 1980;286:611–3. Ruiz-Moreno JM, Thillaye B, de Kozak Y. Retino-choroidal changes in endotoxin-induced uveitis in the rat. Ophthalmic Res 1992;24:162–8. Schuman JS. Short- and long-term safety of glaucoma drugs. Expert Opin Drug Saf 2002;1:181–94. Sherwood MB, Grierson I, Millar L, Hitchings RA. Longterm morphologic effects of antiglaucoma drugs on the conjunctiva and Tenon’s capsule in glaucomatous patients. Ophthalmology 1989;96:327–35.
Experimental and Toxicologic Pathology 57 (2005) 127–134
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Effects of anti-glaucoma drugs timolol and GLC756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, on endotoxin-induced-uveitis and -arthritis in rats U.W. Laenglea,, M. Courta, R. Marksteinb, P.G. Germanna, V. Noguesa, D. Romana a
Department of Toxicology/Pathology, Novartis Pharma AG, MUT-2881.133, CH-4002 Basel, Switzerland Department of Ophthalmics, Novartis Pharma AG, CH-4002 Basel, Switzerland
b
Received 1 December 2004; accepted 24 February 2005
Abstract Anti-glaucoma drugs exhibiting anti-inflammatory properties are desirable for the long-term treatment of glaucoma since they may reduce the risk for treatment-related inflammatory processes in outer compartments of the eye. The purpose of this study was to evaluate potential anti-inflammatory effects of two topically and systemically applied antiglaucoma drugs i.e. GLC 756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, and timolol a b-adrenoceptor antagonist using endotoxin-induced uveitis (EIU) and arthritis in rats as an in vivo model. For EIU, 8week-old Lewis rats were intravenously injected at 160 mg lipopolysaccharide (LPS) from Salmonella typhimurium. GLC756, timolol, or betamethasone, as a positive control, were either topically (0.4%, 0.5%, and 0.1%, respectively, 16-times 20 mL eye drops during 48 h) or systemically (1 mg/kg subcutaneous for 5 days) administered. Cell infiltration in tissue of the eye and knee joint were assessed histopathologically and in special compartments of the eye by confocal microscopy 48 h after LPS-induction. Numerous infiltrating cells were detected in the eyes after LPS-induction and half of the animals showed arthritis. Topical and systemic pre-treatment with GLC756 and timolol resulted in reduced cell infiltration in the eye. In addition, GLC756 reduced, whereas timolol increased the incidence of arthritis. Betamethasone suppressed almost completely the cell infiltration in the eye and the incidence of arthritis. In conclusion, the observations that GLC756 reduced cell infiltration in the eye and the incidence of arthritis after LPS-induction is compatible with anti-inflammatory properties of this drug. By contrast, timolol produced no consistent anti-inflammatory effect since both inhibitory as well as stimulatory effects on inflammatory processes were seen. r 2005 Elsevier GmbH. All rights reserved. Keywords: Anti-glaucoma drugs; GLC756; Dopamine D2 agonist and D1 antagonist; Timolol; Betamethasone; EIU; Arthritis; Infiltrating cells; Rat
Introduction Corresponding author. Tel.: +41 61 324 1891; fax: +41 61 324 1561. E-mail address: [email protected] (U.W. Laengle).
0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.02.008
Glaucoma designates a group of chronic eye diseases which are characterized by progressive atrophy of the optic nerve head and visual field loss and, in most cases, by elevation of intraocular pressure (IOP). The etiology
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of glaucoma is still unknown but elevated IOP and impaired perfusion of the optic nerve has been suggested to be important risk factors. Current medical treatment of glaucoma is focused mainly on lowering IOP. Timolol belongs to the class of b-adrenoceptor blocking agents with IOP lowering properties (Schuman, 2002). Dopaminergic drugs are another class of compounds that lower IOP (Pruente and Flammer, 1995) and in addition can improve perfusion of the optic nerve (Pru¨nte et al., 1995) and may therefore be of particular value for the treatment of glaucoma (Chiou and Chen, 1992; Markstein et al., 1996; Prunte et al., 1996). Increased numbers of inflammatory cells in outer compartments of the eye were found in humans who have been treated with conventional anti-glaucoma drugs (Sherwood et al., 1989; Ariturk et al., 1997; Broadway et al., 1994). Mediators of inflammatory response might mediate proliferation of the fibroblasts in the anterior parts of the eye (Lark et al., 1999). Both, increased numbers of inflammatory cells and of fibroblasts may contribute to the failure of glaucoma filtering surgery (Sherwood et al., 1989; Maumenee, 1960; Flaxel and Swan, 1969; Miller et al., 1989; Addicks et al., 1983) which is necessary in some glaucoma patients who do not respond well to conventional anti-glaucoma drugs. Therefore, anti-glaucoma drugs with additional antiinflammatory properties may be of particular therapeutic value. Endotoxin-induced uveitis (EIU) in the Lewis rat is a model for severe uveitis in humans (Rosenbaum et al., 1980). Systemic injection of endotoxin, a lipopolysaccharide (LPS) from the outer membrane of gramnegative bacteria, induces an acute and self-limited bilateral uveitis (Rosenbaum et al., 1980; Hoekzema et al., 1992). The ocular inflammation is characterized by a cellular infiltration mainly with polymorphonuclear neutrophils (McMenamin and Crewe, 1995; Kogiso et al., 1992) into the outer compartments of the eye. It is also shown that the Lewis rat is arthritis susceptible after intravenous challenge with enteropathogenic bacteria (Hill and Yu, 1987). In this study, effects of systemic and topical medicated anti-glaucoma drugs GLC756, a novel dopamine D2 agonist and D1 antagonist, and timolol on EIU and arthritis were examined. The results were compared with that of betamethasone which served as a positive control.
Material and methods Animals and study design Eight-week-old Lewis rats, weighing 140–180 g, were obtained from Charles River Company, WIGA, Sulzfeld, Germany. EIU and arthritis were induced in
eight groups of five rats each, receiving an intravenous injection of LPS from Salmonella typhimurium (Sigma, Switzerland.) at 160 mg per rat in a volume of 160 mL. Two of these groups were kept as positive controls. The remaining groups were treated with GLC756, betamethasone, or timolol either topically (0.4%, 0.1%, and 0.5% respectively, 16-times 20 mL eye drops during 48 h, LPS intravenous about 1 h after the 1st eye drop) or systemically (1 mg/kg/day subcutaneously for 5 days; LPS intravenously on the 3rd day). An additional group of four animals was kept as control group treated topically with 0.9% NaCl (16-times 20 mL eye drops during 48 h into the right eye; left eye stayed untreated). Animals were sacrificed 48 h after LPS-induction.
Drug preparation GLC756 was received as a powder and as 0.4% eye drops from Novartis Pharma AG, Switzerland. The GLC756 powder was reported to be 100% pure by certificate of analysis. The powder was dissolved in a 2.5% glycerol solution. Timolol was available commercially as a powder (100% pure) from Sigma, Switzerland, and as 0.5% eye drops from Ursapharm, Germany. The powder was dissolved in a 2.5% glycerol solution. Betamethasone was available commercially as a powder (99.7% pure) from Sigma, Switzerland and as 0.1% eye drops from Dr. Winzer Pharma GmbH, Germany. The powder was dissolved in a 40% polyethylenglycol 300 solution.
Tissue sampling After sampling of the aqueous humor for special examinations (Laengle et al., 2005) the right eye and a knee joint of all treated animals was removed for histopathological processing and the remaining eye for confocal laser scanning microscopy (CLSM). In the control group, the left eye of three animals and the right eye of one animal were used for histopathology and the remaining eyes for CLSM.
Histopathological processing The eyes with harderian glands were fixed in Davidson’s solution (96% alcohol, 40% formaldehyde, acetic acid, distilled water, 1% methylene blue) and the knee joints in phosphate-buffered 10% formalin. The bone was demineralized with 10% formic acid. The tissues were embedded in Paraplasts. A section of 4 mm was stained with hematoxylin and eosin. Severity of inflammatory reaction in iris, ciliary/vitreous body, retina, and choroidea 48 h after LPS-induction, and systemic or topical pre-treatment with GLC756, betamethasone, or timolol was based on a sum-up of a
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Fig. 1. Histological section through an eye 48 h after LPS-induction showing infiltrating cells, polymorphonuclear neutrophils (PMNs), monocytes/macrophages (Mon). Main picture 300, small picture 750. (A) Inflammatory cell infiltration into iris, (B) inflammatory cell infiltration into the ciliary body, (C) inflammatory cell infiltration into the vitreous body, and (D) inflammatory cell infiltration into the vitreous body and with remarkably lower grade into the optic nerve head.
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grading, minimal ¼ 1, slight ¼ 2, moderate ¼ 3, and severe ¼ 4.
Confocal laser scanning microscopy The double immunofluorescence labeled slides were examined using a Leica TCS-NT (Leica Microsystems Ltd.), argon/krypton CLSM coupled to a Leica DM IRBE inverted microscope. The Argon/krypton laser was filtered using a double dichromic mirror so that dual excitation wavelengths of 488 and 568 nm were used. The emission of fluorescence was detected using different barrier filter sets. Antibody coupled to Alexa was detected using a band pass filter set at 530 nm and the propidium iodide was detached using a long pass filter set at 590 nm. Using a 10 air and 63 water immersion objectives the upper and lower limits of the regions of interest were selected and at the same time adjustments were made to the gain (photomultiplier detector voltage) and offset (image black levels). Between 10 and 50 consecutive z-series, optical sections were taken of various thicknesses between 0.2 and 1.0 mm, to capture the entire field of interest. Each optical section was cleaned using a 4 -averaging filter to improve the signal/noise ratio. Collected z-series optical sections were saved as the raw image data, which were in the format of multi-image TIFF files. Collected optical sections were also projected on to one another to give a single 2D reconstruction of the data.
Processing for CLSM After sampling the tissue for normal histopathology the animals were perfused with 4% paraformaldehyde, for 2–3 min, via the left ventricle. Thereafter, the remaining eyes were removed, fixed in fresh fixative for a further 1.5 h, and kept in cold phosphate buffered saline (PBS, 4 1C) for 1 h until dissection under a binocular microscope into the segment, iris (McMenamin, 2000). The segment was then quartered and placed in a well containing PBS. The eye segments of the systemically treated animals were then incubated 12 h at 4 1C with the mouse IgM anti-rat HIS48 (Serotec, Switzerland) at 1/100, diluted in PBS, 1% BSA, 0.005% Tween 20 for the staining of granulocytes. The tissue were then washed twice in PBS and further incubated with the anti-mouse IgM coupled to Alexa 488 at 1/200 in PBS, 1% BSA, 0.005% Tween 20, for 30 min at room temperature. Finally the tissues were washed twice in PBS and counterstained in propidium iodide (for the detection of the nuclei) at 1 mg/ml in PBS for 15 min at room temperature. Erythroid cells are also stained by the anti-HIS48 antibody (De Vos et al., 2000).
Topical Treatment LPS 160 µg
3.50
GLC756 0.4%
Betamethasone 0.1%
Timolol 0.5%
3.00
Grading
2.50
2.00
** 1.50
** 1.00
**
**
0.50
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
0.00
Fig. 2. Inflammatory reaction in iris, ciliary/vitreous body, retina, and choroidea 48 h after LPS-induction, and topical pretreatment with GLC756, betamethasone, or timolol. Severity was based on a sum-up of a grading, minimal ¼ 1, slight ¼ 2, moderate ¼ 3, severe ¼ 4. Each column is a mean of five values and the bars indicate SD. Means were considered significantly different from those of the LPS-group at Po0:005.
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of the eye, in particular the anterior/posterior chamber, iris, ciliary and vitreous body, retina, choroidea, and optic nerve head (Fig. 1). The infiltration was most severe in the vitreous body, consisting mainly of neutrophils and a few monocytes and macrophages, as well as in the ciliary body and retina (Figs. 2 and 3), here predominantly of neutrophils. GLC756 produced a statistically significant reduction in inflammatory cell infiltration in the retina after topical and in the ciliary/vitreous body, and retina after systemic pre-treatment (Figs. 2 and 3). Topical and systemic pre-treatment with betamethasone, reduced statistically significant the cellular infiltration in all compartments of the eye (Figs. 2 and 3). Timolol caused a statistically significant reduction in inflammatory cell infiltration in the vitreous body only after systemic treatment but was inactive after topical administration (Figs. 2 and 3). Coincident with the histopathological evaluation, CLSM of immunostained granulocytes illustrates the systemic pre-treatment-related effects in samples from the iris (Fig. 4). In LPS-treated animals HIS48-labeled cells were scattered throughout the iris (Fig. 4A). Mainly granulocytes are labeled (Fig. 4A0 ). In comparison, a decrease in HIS48-labeled cells was apparent after pretreatment with GLC756, betamethasone or timolol (Figs. 4B–D, B0 –D0 ).
Image analysis Selected z-series optical sections were transferred to a Silicon Graphics (SGI) workstation and further analyzed using Imaris software (Bitplane). Images were opened into the ‘sections’ module of the Imaris program. Single optical sections of the confocal images and both x and y plane profiles were analyzed to localize and identify inflammatory cells deeper in the tissue. No filters or image correction were applied to the images.
Statistical analysis For all groups mean7SD were obtained. These values were subjected to an ANOVA 1-way Dunnett’s test. Values were considered significantly different from corresponding control at Po0:05.
Results Histopathological evaluation showed no obvious cellular infiltration in tissues of normal or 0.9% NaCl treated control eyes. In animals treated with LPS, cellular infiltration could be observed 48 h post-dose in almost all compartments
Systemic Treatment 2.50
LPS 160 µg
GLC756 0.4%
Betamethasone 0.1%
Timolol 0.5%
2.00
** Grading
1.50
* *
1.00
**
**
0.50
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
** ** vitreous body
ciliary body
choroida
retina
vitreous body
ciliary body
iris
choroida
retina
vitreous body
ciliary body
iris
iris
** **
0.00
Fig. 3. Inflammatory reaction in iris, ciliary/vitreous body, retina, and choroidea 48 h after LPS-induction, and systemic pretreatment with GLC756, betamethasone, or timolol. Severity was based on a sum-up of a grading, minimal ¼ 1, slight ¼ 2, moderate ¼ 3, severe ¼ 4. Each column is a mean of five values and the bars indicate SD. Means were considered significantly different from those of the LPS-group at Po0:05 and Po0:005.
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Histopathological evaluation of a knee joint (Table 1) revealed that control animals were devoid of any inflammatory cell infiltration. After LPS-induction, five out of ten animals showed minimal to slight inflamma-
tory cell infiltration, mainly neutrophils. Topical and systemic pre-treatment with GLC756, like the positive control betamethasone reduced the incidence of articular inflammation. In the case of timolol, the incidence of
Fig. 4. Confocal photomicrographs showing HIS48 staining in the iris 48 h after LPS-induction (A), and systemic pre-treatment with GLC756 (B), betamethaosone (C), or timolol (D). Mainly granulocytes are labeled A0 –D0 . Bars in main picture ¼ 100 mm; Bars in small picture ¼ 20 mm.
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Table 1. timolol
133
Incidence of arthritis 48 h after LPS-induction and topical- or systemic pre-treatment with GLC756, betamethasone, or
Examined animals (n) Affected animals (n)
Controls
LPS
GLC756
Betamethasone
Timolol
10 0
10 5
10 2
10 1
10 7
articular inflammation was even higher than in the LPSgroup.
Discussion Increased numbers of inflammatory cells in outer compartments of the eye were found in humans who have been treated with anti-glaucoma drugs (Sherwood et al., 1989; Ariturk et al., 1997; Broadway et al., 1994). Mediators of inflammatory response might mediate proliferation of the fibroblasts in the anterior parts of the eye (Lark et al., 1999). Both effects may contribute to the failure of glaucoma filtering surgery (Sherwood et al., 1989; Maumenee, 1960; Flaxel and Swan, 1969; Miller et al., 1989; Addicks et al., 1983). Therefore, one can assume that anti-glaucoma drugs with additional anti-inflammatory properties may prevent these adverse effects. To evaluate potential anti-inflammatory properties of anti-glaucoma drugs, an in vivo study using an EIU model in Lewis rats was performed. In this study, GLC756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, and timolol as well as betamethasone as a positive control, were assessed for their effects on cellular infiltration in the eye and knee joint 48 h after LPS-administration. Systemic administration of endotoxin, LPS, a component of gram-negative bacterial outer membranes, was first reported to induce an acute bilateral anterior uveitis in the Lewis rat, affecting only the anterior chamber, iris, ciliary body, and conjunctiva (Rosenbaum et al., 1980; Herbort et al., 1988; Cousins et al., 1984). However, further investigations showed that inflammatory cells also infiltrate posterior eye segments, including the retina and choroidea (Ruiz-Moreno et al., 1992). Furthermore, inflammatory infiltration of most of the polymorphonuclear and a few mononuclear cells was also reported in the vitreous body of an EIU susceptible mouse strain (Kogiso et al., 1992). Besides induction of EIU, it is also shown that the Lewis rat is arthritis susceptible after intravenous challenge with enteropathogenic bacteria (Hill and Yu, 1987). In the present study, systemic administration of LPS induced inflammatory cell infiltration in almost all compartments of the eye, in particular the anterior/ posterior chamber, iris, ciliary and vitreous body, retina,
choroidea, and optic nerve head 48 h post-administration. The highest inflammatory cell reactions were graded for the vitreous body, followed by the ciliary body and retina. A notably lower rating resulted for the iris and choroidea. Inflammatory cell reaction was also noted in the optic nerve head. The inflammatory cell infiltration was composed mainly of neutrophils and a few monocytes and macrophages. GLC756 produced a statistically significant reduction in inflammatory cell infiltration in the retina after topical and in the ciliary/vitreous body and retina after systemic pre-treatment. This anti-inflammatory property of GLC756 might be related to its significant TNF-a reducing effect which was measured in the serum (Laengle et al., in submission). TNF-a as being one of the first cytokines released in the cascade of inflammation (Fong and Lowry, 1990) is an important mediator of inflammation (Durum and Oppenheim, 1989). TNF-a can be produced mainly by ocular resident cells (i.e., macrophages, endothelial, and epithelial cells) as well as by infiltrating cells (i.e., polymorphonuclear cells and monocytes) (De Vos et al., 1994). The anti-inflammatory effect of GLC756 was also confirmed by reducing the incidence of arthritis in comparison to that of the LPS-group. The effect was similar than that of the positive control with betamethasone. Betamethasone was, however, much more efficient in the reduction of inflammatory cell infiltration in the eye than GLC756. The difference is very likely related to the highly immunosuppressive property of betamathasone which indicates also that immune mechanisms contribute to the inflammation in EIU (Rosenbaum and Boney, 1993). For timolol, a statistically significant reduction in inflammatory cell infiltration was seen in the vitreous body after systemic pre-treatment. This seemingly antiinflammatory effect of timolol in the eye was in contrast to the finding in the knee joint where no effect on reduced incidence in arthritis was noted. In summary, our in vivo study in Lewis rats demonstrated that GLC756, a novel dopaminergic compound, showed slight inhibitory effects on EIU and arthritis in the Lewis rat. Therefore it is suggested that dopaminergic compounds like GLC756 can prevent or counteract slight inflammatory processes in outer compartments of the eye which represents an additional benefit in the treatment of glaucoma.
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Acknowledgements We thank Lothar Kruppa, Novartis Pharma AG, for his engagement in performing the in-life phase and Merdol Ibrahim for doing the confocal microscopy of this study.
References Addicks EM, Quigley HA, Greene WR, Robin AL. Histologic characteristics of filtering blebs in glaucomatous eyes. Arch Ophthalmol 1983;101:795–8. Ariturk N, Oge I, Baris S, Erkan D, Sullu Y, Koc F. The effects of antiglaucomatous agents on conjunctiva used for various durations. Int Ophthalmol 1997;20:57–62. Broadway DC, Grierson I, O’Brien C, Hitchings RA. Adverse effects of topical antiglaucoma medication. I. The conjunctival cell profile. Arch Ophthalmol 1994;112:1437–45. Chiou GC, Chen YJ. Effects of dopamine agonist, bromocriptine, and some dopamine antagonists on ocular blood flow. J Ocul Pharmacol 1992;8:285–94. Cousins SW, Guss RB, Howes Jr EL, Rosenbaum JT. Endotoxin-induced uveitis in the rat: observations on altered vascular permeability, clinical findings, and histology. Exp Eye Res 1984;39(5):665–76. De Vos AF, van Haren MA, Verhagen C, Hoekzema R, Kijlstra A. Kinetics of intraocular tumor necrosis factor and interleukin-6 in endotoxin-induced uveitis in the rat. Invest Ophthalmol Vis Sci 1994;35:1100–6. De Vos AF, Dick AD, Klooster J, Broersma L, McMenamin PG, Kijlstra A. Analysis of the cellular infiltrate in the iris during experimental autoimmune encephalomyelitis. Invest Ophthalmol Vis Sci 2000;41:3001–10. Durum SL, Oppenheim JJ. In: Paul WE, editor. Fundamental immunology. New York: Ravens Press; 1989. p. 639–61. Flaxel JT, Swan KC. Limbal wound healing after cataract extraction. Arch Ophthalmol 1969;81:653–9. Fong Y, Lowry SF. Tumor necrosis factor in the pathophysiology of infection and sepsis. Clin Immunol Immuopathol 1990;55:157–70. Herbort CP, Okumura A, Mochizuki M. Endotoxin-induced uveitis in the rat. A study of the role of inflammation mediators. Graefes Arch Clin Exp Ophthalmol 1988;226:553–8. Hill JL, Yu DT. Development of an experimental animal model for reactive arthritis induced by Yersinia enterocolitica infection. Infect Immun 1987;55:721–6. Hoekzema R, Verhagen C, van Haren M, Kijlstra A. Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6. Invest Ophthalmol Vis Sci 1992;33:532–9. Kogiso M, Tanouchi Y, Mimura Y, Nagasawa H, Himeno K. Endotoxin-induced uveitis in mice. 1. Induction of uveitis and role of T lymphocytes. Jpn J Ophthalmol 1992;36:281–90. Laengle UW, Markstein R, Schneider V, Greiner B, Roman D. Effects of antiglaucoma drugs timolol and GLC756, a
novel dopamine D2 agonist and D1 antagonist, on edotoxin-induced-uveitis in rats. Exp Eye Res 2005;80: 847–52. Laengle UW, Markstein R, Schneider V, Roman D. Effects of antiglaucoma drugs timolol and GLC756, a novel dopamine D2 agonist and D1 antagonist, on endotoxin-induced TNF-alpha release in serum of rats. Journal of Glaucoma, submitted. Lark KK, Pasha AS, Yan X, Edward DP. The effect of latanoprost and brimonidine on rabbit subconjunctival fibroblasts. J Glaucoma 1999;8:72–6. Markstein R, Gull P, Rudeberg C, Urwyler S, Jaton AL, Kalkman HO, Dixon AK, Hoyer D. SDZ GLC 756, a novel octahydrobenzo[g]quinoline derivative exerts opposing effects on dopamine D1 and D2 receptors. J Neural Transm 1996;103:17–30. Maumenee AE. External filtering operations for glaucoma: the mechanism of function and failure. Trans Am Ophthalmol Soc 1960;58:319–28. McMenamin PG. Optimal methods for preparation and immunostaining of iris, ciliary body, and choroidal wholemounts. Invest Ophthalmol Vis Sci 2000;41:3043–8. McMenamin PG, Crewe J. Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body. Invest Ophthalmol Vis Sci 1995;36:1949–59. Miller MH, Grierson I, Unger WI, Hitchings RA. Wound healing in an animal model of glaucoma fistulizing surgery in the rabbit. Ophthalmic Surg 1989;20:350–7. Pruente C, Flammer J. The novel dopamine D1 antagonist and D2 agonist, SDZ GLC 756, lowers intraocular pressure in healthy human volunteers and in patients with glaucoma. Ophthalmology 1995;102:1291–7. Pru¨nte C, Flammer J, Markstein R, Rudin M. Quantification of optic nerve blood flow using MR-imaging. Invest Ophthalmol Vis Sci 1995;36:247–51. Prunte C, Nuttli I, Markstein R. Topical SDZ GLC-756, a novel dopamine D-1 antagonist and D-2 agonist, lowers intraocular pressure in conscious rabbits. Jpn J Ophthalmol 1996;40:167–73. Rosenbaum JT, Boney RS. Failure to inhibit endotoxininduced uveitis with antibodies that neutralize tumor necrosis factor. Reg Immunol 1993;5:299–303. Rosenbaum JT, McDevitt HO, Guss RB, Egbert PR. Endotoxin-induced uveitis in rats as a model for human disease. Nature 1980;286:611–3. Ruiz-Moreno JM, Thillaye B, de Kozak Y. Retino-choroidal changes in endotoxin-induced uveitis in the rat. Ophthalmic Res 1992;24:162–8. Schuman JS. Short- and long-term safety of glaucoma drugs. Expert Opin Drug Saf 2002;1:181–94. Sherwood MB, Grierson I, Millar L, Hitchings RA. Longterm morphologic effects of antiglaucoma drugs on the conjunctiva and Tenon’s capsule in glaucomatous patients. Ophthalmology 1989;96:327–35.