Role of oxygen and nitrogen species in experimental uveitis: anti-inflammatory activity of the synthetic antioxidant ebselen

Free Radical Biology & Medicine, Vol. 33, No. 5, pp. 669 – 675, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter

PII S0891-5849(02)00954-1

Original Contribution ROLE OF OXYGEN AND NITROGEN SPECIES IN EXPERIMENTAL UVEITIS: ANTI-INFLAMMATORY ACTIVITY OF THE SYNTHETIC ANTIOXIDANT EBSELEN FRANCISCO BOSCH-MORELL,* JOAQU´ıN ROMA´ ,† NURIA MAR´ıN,† BELE´ N ROMERO,† ANTONIO RODRIGUEZ-GALIETERO,‡ SIV JOHNSEN-SORIANO,* MANUEL Dı´AZ-LLOPIS,§㛳 and FRANCISCO J. ROMERO*㛳 *Department of Physiology, Pharmacology, and Toxicology, Universidad Cardenal Herrera-CEU, Valencia, Spain; †Department of Physiology, University of Valencia, Valencia, Spain; ‡Ophthalmology Unit, University Hospital La Fe, Valencia, Spain; § Ophthalmology Unit, General University Hospital, Valencia, Spain; and 㛳“Fundacio´n Oftalmolo´gica del Mediterra´neo”, Valencia, Spain (Received 12 April 2002; Accepted 16 May 2002)

Abstract—This study was aimed at examining the role of oxygen and nitrogen reactive species in a model of experimental uveitis upon intravitreal injection of bacterial endotoxin to albino New Zealand rabbits. The inflammatory response was evaluated in terms of: (i) the integrity of the blood aqueous barrier (protein and cell content in samples of aqueous humor), (ii) histopathological changes of the eyes, (iii) clinical evaluation (with a score index based on clinical symptoms), and (iv) the concentration of malondialdehyde (MDA), in aqueous humor, as a marker of oxidative stress. Betamethasone was used as reference treatment, superoxide dismutase as quencher of superoxide anion, L-NG-nitro-L-arginine-methyl-esther (L-NAME) and chlorpromazine as nitric oxide synthase inhibitors, and ebselen, a glutathione peroxidase mimic, as peroxynitrite reductant. All the substances were injected subconjunctivally to the rabbits immediately after the intravitreal endotoxin injection. Ebselen was the only treatment able to decrease MDA concentration to control values, exerting an effect similar to that elicited by L-NAME on the rest of the parameters tested. The data presented render ebselen a notable choice for the treatment of uveitis, with implications for clinical trials. © 2002 Elsevier Science Inc. Keywords—Anti-inflammatory agents, Antioxidants, Lipid peroxidation, Superoxide dismutase, Uveitis, Free radicals

INTRODUCTION

These cells are responsible for the generation of the reactive species that lead, among other phenomena, to ocular lipid peroxidation [6]. Using the uveitis model of endotoxin injection into the rat footpad, we have recently reported the efficacy of ebselen, an antioxidant with anti-inflammatory properties [7]. Ebselen is a selenoorganic compound (Fig. 1) that was originally described as a glutathione peroxidase (GPx) mimic [8,9]. In the last few years evidence has accummulated that provides a role for nitric oxide (NO) and peroxynitrite in ocular inflammation [6,10 –13], and for ebselen as peroxynitrite reductant [14 –17]. Following these evidences, this study was aimed at examining the specific contribution of superoxide anion, NO, and peroxynitrite to the inflammatory process. Accordingly, we studied the integrity of the blood-aqueous barrier (BAB) and markers of oxidative stress, in control rabbit eyes, eyes intravitreally

Reactive oxygen species and oxidative stress have been involved in the pathophysiology of ocular inflammation in the anterior segment of the eye and also the retina [1– 4]. In animal models, the basic mechanisms of the immune response are not yet clear, though a widespread mechanism for the endotoxin-induced uveitis (EIU) (after systemic administration of bacterial endotoxin) involves cytokine production by resident uveal macrophages as initiator of the inflammatory response, with subsequent neutrophil and mononuclear infiltration [5]. Address correspondence to: Prof. Dr. Francisco J. Romero, Department of Physiology, Pharmacology, and Toxicology, Universidad Cardenal Herrera-CEU, Edificio Seminario s/n, 46113-Moncada (Valencia), Spain; Tel: ⫹34 (96) 136-9000; Fax: ⫹34 (96) 139-5272; E-Mail: [email protected]. 669

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Ko¨ ln, Germany) (15 mg/kg). Ebselen was previously encapsulated in lecithine liposomes (ebselen:lecithine, 1:4, w/w), in view of its low solubility in H2O, as previously described [21]. Injection of liposomes alone showed no effect on any of the parameters tested (data not shown). Unless otherwise stated, all chemicals were from Sigma Quı´mica SA. Integrity of the blood-aqueous barrier (BAB) Fig. 1. Chemical structure of ebselen (2-phenyl-1,2-benzisoselenazol3(2H)-one).

injected with endotoxin, and eyes subconjunctivally injected with a variety of compounds. The results presented here confirm the efficacy of ebselen as an antiinflammatory drug and strengthen the role of peroxynitrite in the pathophysiological mechanisms involved in this experimental model of endotoxin-induced uveitis. MATERIALS AND METHODS

Animal model of uveitis Albino New Zealand rabbits (1.2–1.5 kg) were used throughout the study, according to [18]. Animals were grouped for the different treatments randomly and 10 animals were assigned to each group. The right eye of each animal was used in all cases and the left eye was kept free of injections. Animals were anesthetized with ketamine (35 mg/kg intramuscular injection) and 1 ␮g endotoxin (lipopolysacharide from Salmonella typhimurium, Sigma Quı´mica SA, Alcobendas, Spain, as in [19]) in 0.1 ml saline was injected into the eye vitreous after extraction of the same AH volume, to avoid increases of intraocular pressure. This procedure minimizes systemic inflammation because: (i) endotoxin is injected locally, and (ii) any systemic inflammatory mediator will affect the left eye that is used as control [20]. Eyes were enucleated 24 h after the induction of uveitis. Animal care conformed the NIH and ARVO statements for the use of animals in research. Drug treatments All substances were injected subconjunctivally in a final volume of 0.5 ml of saline to assure the same penetration in all groups, immediately after intravitreal endotoxin injection. The doses used were: betamethasone (1.5 mg/kg), bovine liver SOD (Peroxinorm, Grunenthal GmbH, Aachen, Germany; 0.06 mg/kg), LNAME (75 mg/kg), chlorpromazine (1.85 mg/kg), and ebselen (Rhoˆ ne-Poulenc Rorer Arzneimittel GmbH,

Two parameters were assayed to assess the BAB integrity: cell count and protein concentration in AH samples 24 h after the induction of uveitis. One microliter of AH was adequately diluted and counted under microscope in a hemocytometer. Protein was assayed by the method of Lowry et al. [22]. Histopathology score Right eyes from each group were enucleated 24 h after the induction of uveitis, fixated with 10% formaldehyde for 7 d (the sclera was punctured to allow an adequate penetration of the formaldehyde), dissected in three portions (one central and two lateral), each portion embedded in paraffin. Sections (6 – 8 ␮m thick) were stained with hematoxyline and eosine and evaluated for six different structures (the ones in the vicinity of the injected endotoxin): anterior chamber angle, iris, cilliary body, choroid, cornea, and retina. In the first four structures the severity of vasodilation, interstitial edema, and tissue hemorrhage was evaluated applying the following scale: 0-none, 1-minimum, 2-discrete, 3-moderate, 4-severe, 5-intense. Corneal inflammation was evaluated considering the degree of interstitial edema (IE) and endothelial adhesion of leucocytes (ADH) as follows: 0-none (no IE, no ADH), 1-minimum (either minimal IE or ADH), 2-discrete (both minimal IE and ADH), 3-moderate (minimal IE and severe ADH), 4-severe (severe IE and minimal ADH), 5-intense (severe IE and ADH). Finally, retinal inflammation was considered upon the presence of infiltration of inflammatory cells (0 absence, 1 presence). Thus, the score for each eye could be 0 (absence of inflammation) to 26 (highest). Each specimen was evaluated by at least two different observers. Clinical score Six different parameters (the more classical ones in any routine ophthalmoscopic study) were considered when examining the rabbits’ eyes 24 h after the intravitreal injection of endotoxin or saline in the case of controls: degree of hyperemia, degree of iris vasodila-

Oxygen and nitrogen species in uveitis Table 1. Effect of Different Substances on the Integrity of the Blood-aqueous Barrier in Rabbit Eyes with Endotoxin-induced Uveitis Cell count

Protein concentration

Group

(10 cells/ml)

% of uveitis group

Uveitis Betamethasone SOD L-NAME Chlorpromazine Ebselen

7.28 ⫾ 0.98 0.94 ⫾ 1.37* 5.84 ⫾ 1.20 1.53 ⫾ 0.32* 0.86 ⫾ 0.24* 1.23 ⫾ 0.31*

100 13 80 21 12 17

3

(mg/ml)

% of uveitis group

31.25 ⫾ 2.78 5.72 ⫾ 0.96* 20.35 ⫾ 2.68* 22.10 ⫾ 1.47* 22.84 ⫾ 3.02* 21.53 ⫾ 2.08*

100 18 65 71 73 69

Results are means ⫾ standard deviations, of at least five different eyes. *p ⬍ .005 vs. uveitis (Student’s t-test). In control animals, cells were not detectable, and protein concentration was 0.54 ⫾ 0.23 mg/ml in aqueous humor samples.

tion, Tyndall effect in the anterior chamber, presence of exudates at the pupil rim, and degree of myosis; all these parameters were scored 0 –3 (3 highest). Finally, the absence or presence of a pupil membrane was scored 0 or 1, respectively. Thus, the highest score possible may be expected to be 16. Each eye was examined by at least two different observers. Malondialdehyde assay Malondialdehyde (MDA) concentration was measured in AH samples 24 h after the intravitreal injection of endotoxin or, in the case of controls, saline, according to a modification of the high pressure liquid chromatography method of Richard et al. [23], as previously reported [24]. Briefly: 0.1 ml of sample (or standard solutions prepared daily from 1,1,3,3-tetraethoxypropane) and 0.75 ml of working solution (thiobarbituric acid 0.37% and perchloric acid 6.4%, 2:1 v/v) were mixed and heated to 95°C for 1 h. After cooling (10 min in ice-water bath), the flocculent precipitate was removed Table 2. Histopathological and Clinical Scores of Rabbit Eyes with Endotoxin-induced Uveitis Treated with Different Substances % of uveitis group Group

Histopathology score

Uveitis Betamethasone SOD L-NAME Chlorpromazine Ebselen

23.10 ⫾ 0.91 5.20 ⫾ 1.22* 21.40 ⫾ 1.67 11.70 ⫾ 1.14* 11.10 ⫾ 2.35* 9.70 ⫾ 1.52*

100 23 93 51 48 42

Clinical score 14.50 ⫾ 1.78 5.20 ⫾ 1.55† 14.00 ⫾ 1.33 7.50 ⫾ 2.12† 7.30 ⫾ 2.21‡ 8.70 ⫾ 1.83†

100 36 97 51 50 60

Results are means ⫾ standard deviations, of at least five different eyes. *p ⬍ .05, †p ⬍ .001 vs. uveitis (Mann-Withney U test). In control animals both scores were zero.

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by centrifugation at 3200 ⫻ g for 10 min. The supernatant was neutralized and filtered (0.22 ␮m) prior to injection on a ODS 5 ␮m column (250 ⫻ 4.6 mm). The mobile phase consisted of 50 mM phosphate buffer (pH 6.0): methanol (6:4, v/v). Isocratic separation was performed with 1.0 ml/min flow and detection at 532 nm. Calibration curves were run daily.

Statistical analysis Data are reported as mean ⫾ standard deviation. Analysis of variance established the statistical differences and the statistical significance between the different groups was established by means of the unpaired Student’s t-test. RESULTS

The integrity of the BAB is disrupted in EIU and cell and protein contents increase in the anterior chamber of the eye. Table 1 shows the effect of the different substances employed to recover BAB integrity. SOD did not show any statistical significant effect on cell count; maximal effects were observed for the following substances: chlorpromazine ⬎ betamethasone ⬎ ebselen ⬎ L-NAME. When considering the ability of the drugs examined to maintain BAB integrity for proteins, all of them, except betamethasone, failed to show effects higher than 50% (considering uveitis group as 100%). Table 2 shows the results of the effect of ebselen and the different substances examined on the histopathological and clinical parameters. A score index was used (as described in Materials and Methods section) in the evaluation of histopathological and clinical modifications in the different groups. The highest protective effect was exerted by betamethasone and the lowest by SOD. In between these effects, the other compounds examined elicited approximately 50% effect on both indexes. Figures 2 and 3 show different histopathological aspects of rabbit eyes with EIU compared to control eyes and eyes treated with betamethasone or ebselen. Leucocyte adhesion to corneal endothelium, as well as stromal edema, are seen in EIU eyes (compare Fig. 2A control vs. 2B uveitis), along with inflammatory infiltration of the anterior chamber angle (compare Fig. 3A control vs. 3B uveitis), both signs were greatly reduced by betamethasone and ebselen (cf. Figs. 2C, 2D, and 3C, 3D). Other signs observed in EIU eyes are iridian and coroidean vasodilation, together with some scattered foci of coroiditis (data not shown). Interestingly, Fig. 4 shows a strong effect of ebselen on MDA concentration. Of note, this was the only treatment able to return MDA concentrations to control values. Betamethasone and SOD showed

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Fig. 2. Micrographs of hematoxylin and eosine stained sections of control (A), endotoxin-treated (B) rabbit eyes, and endotoxin eyes treated with bethamethasone (C), or ebselen (D), representative out of at least four different preparations. Endotoxin promotes adhesion of inflammatory cells to corneal endothelium (arrow heads, B), not present in controls (A), or scarcely in (C) or (D). Magnification ⫻300.

a statistically significant difference with the uveitis group, whereas L-NAME and chlorpromazine had no major effect.

DISCUSSION

Intravitreal injection of endotoxin is an adequate model to study uveitis, as has been repeatedly reported [19,20,25,26]. Evidence has accumulated that reactive nitrogen species, mainly NO, play a role in inflammation [27]. Moreover, it has been described recently how changes in the generation or actions of NO in the eye could contribute to pathological states such as inflammation and neurodegeneration or degenerative diseases [28]. The localization of lipid peroxidation products in the corneal endothelium and trabecular meshwork [4] are in agreement with the data shown in Fig. 2. The fact that peroxynitrite plaques accumulate in the vicinity of the infiltrating phagocytes [6] suggests the diffusion of NO, superoxide anion, and/or its reaction product peroxynitrite to loci where lipid peroxidation can be easily induced (this is especially true for the photoreceptors in

view of their high concentration of docosahexaenoic acid [29]). Considering this important role of oxidative stress, therapeutic strategies and experimental approaches based on the intervention of reactive oxygen [3] and nitrogen [10 –13] species have been designed separately, showing partially positive results. It is herein reported that inhibition of NO synthase or quenching of superoxide anion by SOD led to an amelioration of inflammatory signs that do not tally the maximal effects of betamethasone described (Tables 1 and 2). These findings are consistent with previous reports showing that (i) NO synthase inhibitors can abort the lipopolysacharide-induced inflammatory response if administered at early stages [30], and (ii) L-NAME partially decreases corneal edema [11]. The inhibitory effect of chlorpromazine on either the constitutive NO synthase or the induction of other NO synthase forms [31] is confirmed herein in view of the comparable results obtained with this substance and LNAME (Tables 1 and 2 and Fig. 4). Administration of antioxidants such as allopurinol, oxypurinol [32], SOD, catalase, or sodium benzoate [33] led to a decrease in ocular tissue inflammation and tissue damage. Moreover,

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Fig. 3. Micrographs of hematoxylin and eosine stained sections of control (A), endotoxin-treated (B) rabbit eyes, and endotoxin eyes treated with bethamethasone (C) or ebselen (D), representative out of at least four different preparations. Anterior chamber angle appears empty in control eyes (arrow heads, A) and in eyes treated with bethamethasone (C) or ebselen (D), whereas intense inflammatory cell infiltration appears after endotoxin treatment (arrow heads, B). Magnification ⫻150.

other reports show that cell-permeable lecithinized SOD associated with ebselen were able to prevent lipid peroxidation and oxidative DNA damage in a model of

acute renal ischemia [34]. In endotoxin-mediated inflammatory models, myeloperoxidase (MPO) is increased, and it has been proposed that the increase in free radical

Fig. 4. Histogram of malondialdehyde concentrations in aqueous humor of rabbit eyes, treated as in Materials and Methods. Results are means ⫾ standard deviations, of at least five different eyes. *p ⬍ .001 vs. Group 1; **p ⬍ .001 vs. Group 1 and not significantly different from group 0; #p ⬍ .05 vs. Group 0.

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production by polymorphonuclear leukocytes (PMNs) is apparently mediated by NO and MPO, because NOS inhibitors inhibit arachidonic acid-induced free radical generation from endotoxin-treated PMNs [35]. In other inflammatory models such as necrotizing pancreatitis, use of SOD extracellularly does not decrease MPO levels, whereas these levels decrease with an anti-ICAM-1 antibody [36]. This suggests, as a hypothesis, that, in our model, SOD extracellularly may not, in fact, be able to modulate ICAM-1 expression, explaining the results in Table 1 (practically no reduction of cell count) and, furthermore, the lack of effect of SOD on the histopathology and clinical score (Table 2). On the other hand, SOD is able to reduce to approximately 50% the concentration of MDA in aqueous humor (Fig. 4), most probably depending only on the induced reduction of superoxide anion steady-state concentration. The treatment of uveitis, a disease with potentially blinding sequelae, lacks specificity [5,37] and is part of a systemic treatment entailing either steroids, which, of course, elicit severe side effects [38], or cyclosporin, which is associated with a wide and varied patient response [37]. The results presented confirm the partial failure of the approaches tried so far for this inflammatory disorder in experimental models, i.e. the partial efficacy of NO or superoxide anion quenching independently. Ebselen shows similar partial effects, ameliorating the BAB breakdown or clinical or histopathological scores (Table 2), however, it was the only one able to restore control values of aqueous MDA concentration (Fig. 4). This may be explained by its double effect as glutathione peroxidase mimic [8] and peroxynitrite reductant [17], which would lead to a very effective reduction of peroxynitrite concentration, assuming, again as a hypothesis, that peroxynitrite could be the final common mediator of reactive oxygen and nitrogen species’ action. On the one hand, ebselen would increase GPx activity via maintenance of native GPx levels and its own GPxlike activity [7]. Indeed, it has been repeatedly demonstrated that GPx activity decreases in neural tissues, including neuroretina, during free radical-mediated processes, such as uveitis [7], alcoholic peripheral neuropathy [39], and experimental diabetic neuropathy [40,41]. These decreases in GPx activity can be explained by either its direct free radical-mediated degradation [42, 43], or the inhibitory effects of the lipid peroxidation products generated during these processes [44]. On the other hand, peroxynitrite is effectively and directly reduced by ebselen [14,15] and GPx [16,17]. As a consequence of the ebselen-induced increase in GPx activity, the steady-state concentration of hydrogen peroxide is expected to decrease, as is that of peroxynitrite. If so, the demonstrated peroxynitrite-dependent nitration observed during uveitis [6] may also be reduced; current

experiments are trying to confirm this hypothesis. Thus, this lower steady-state level of peroxynitrite might account for decreased levels of lipid peroxidation. The ebselen-dependent inhibition of lipid peroxidation has a direct preserving effect on the integrity of cell membranes of photoreceptors and other neural cells of the retina in the vicinity of the inflammatory loci. This is a desirable effect, in view of the fact that most of the eicosanoids and docosanoids (both inflammatory mediators, the latter group of the utmost importance in the retina, in view of the high content of docosahexaenoic acid of these membranes) are generated via a peroxidation process and their decrease would lead to a long-term anti-inflammatory effect. The ability of ebselen to maintain the concentration of lipid peroxidation products to control values in this experimental model (Fig. 4), and to protect, in vivo, tissue antioxidant systems such as glutathione peroxidase [7], together with its ability to cross the blood-aqueous barrier [7] and its extremely low toxicity in animals and humans [45], makes this compound a valuable option to be considered for the treatment of uveitis in clinical trials. Acknowledgements — Supported by grants PM99-0177 from Direccio´ n General de Ensen˜ anza Superior (Madrid, Spain), 99/0568 from Fondo de Investigacio´ n Sanitaria (Madrid, Spain) and a grant from Fundacio´ n Oftalmolo´ gica del Mediterra´ neo with funds from BANCAIXA, to F.J.R. The authors wish to thank Prof. Enrique Cadenas for his helpful criticisms and for carefully revising the manuscript.

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