Melatonin reverses neurochemical alterations induced by 6-OHDA in rat striatum

Life Sciences 70 (2002) 1041–1051

Melatonin reverses neurochemical alterations induced by 6-OHDA in rat striatum L.M.V. Aguiar, S.M.M. Vasconcelos, F.C.F. Sousa, G.S.B. Viana* Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Rua Cel. Nunes de Melo 1127, Fortaleza, 60431-970, Ceará, Brazil Received 19 April 2001; accepted 24 August 2001

Abstract The present work showed that the intrastriatal injection of 6-OHDA significantly decreases DA, DOPAC and HVA levels in that rat brain structure. Although there is also a decrease in 5-HT levels no changes were observed in 5-HIAA levels as compared to controls. On the other hand, melatonin (2, 5, 10 and 25 mg/kg, i.p., daily for 7 days) treatment starting 1 h after 6-OHDA lesions, partially reverses the decreases caused by 6-OHDA lesions on these neurotransmitter levels, and contents were brought to approximately 50% of that observed in the contralateral sides of controls or of melatonin treated group. Melatonin was more efficient at the doses of 5 and 10 mg/kg, i.p., and effects were similar between the lowest and highest doses characteristic of a bell-shaped type of response. The apomorphineinduced rotational behavior (3 mg/kg, i.p.) was blocked by 60, 89, 78 and 47% after the doses of 2, 5, 10 and 25 mg/kg, i.p., respectively. Similarly, in this case the doses of 5 and 10 mg/kg were also more efficient. Melatonin (5mg/kg) produced an upregulation of D1 receptors associated with a decrease in Kd value. While no change was observed in maximum density of D2 receptors, the Kd value was also decreased. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Melatonin; 6-OHDA; Dopamine receptors; Rotational behavior; Monoamine levels; Neuroprotection; Parkinson’s disease

Introduction Molecular damage by oxygen radicals has been potentially linked to a large number of diseases and is believed to contribute to neuronal loss during aging and aging-related dementia. While the brain is by no means the only organ exposed to free radicals, it consumes large quantities of oxygen, and has paucity of oxidative defense mechanisms what place this organ in risk for damage mediated by reactive oxygen species. There is extensive evidence that free

* Corresponding author. Fax: 55-85-288-8333. E-mail address: [email protected] (G.S.B. Viana) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 8 0 -1

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radical damage is particularly prevalent in the brain and it is an important factor in a number of age-related neurodegenerative diseases, including Parkinson’s disease [1]. Parkinson’s disease (PD) is characterized by a gradual degeneration of the nigrostriatal dopaminergic system, that results in an associated reduction in dopamine-containing fibers in the striatum, and is accompanied by a variety of sensory and motor impairments including tremor, rigidity and akinesia. While there may be other neuronal systems involved, a primary feature of PD is the loss of the dopaminergic neurons [1,2]. Although the cause of PD remains obscure, it is known that the excessive generation of free radicals mediated by oxidative stress is the hypothesis that has received a great increase in experimental support [3–5]. A similar degeneration of nigral dopamine neurons can be produced in animal models of PD based upon the unilateral stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA) [6,7]. The oxidative stress-mediated neurotoxicity of 6-OHDA plays a pivotal role in the degeneration of nigrostriatal dopaminergic system [6,8], suggesting protective effects of antioxidants in the prevention of 6-OHDA neurotoxicity. Melatonin, a neurohormone mainly secreted by the pineal gland, was recently found to possess free radical scavenging and antioxidative properties [9]. Experiments in vivo and in vitro have demonstrated that melatonin can protect cells, tissues, and organs against oxidative damage induced by a variety of free radical generating agents and processes [2,10,11,12]. The mechanism of this activity appears to be related to direct scavenging of free radicals and/ or to the increase of both the mRNA [13,14] and the activity of several antioxidative enzymes [12]. The purpose of this work was to study the neuroprotective properties of systemically administered melatonin, using an animal model of PD induced by the stereotaxic injection of 6-OHDA into the rat striatum, in order to evaluate behavioral and biochemical changes produced by this neurohormone. Materials and methods Animals Male Wistar rats (200–250 g) from the Animal House of the Federal University of Ceará, maintained in a 12 h light/dark cycle were used. All experiments were performed according to the Guide for the care and use of laboratory animals from the US Department of Health and Human Services[15]. Drugs Melatonin, 6-OHDA, ascorbic acid, apomorphin, sodium octanesulfonic acid, acetonitrile, tetrahydrofuran, mianserine, dopamine, butaclamol were purchased from Sigma Chemical Co. USA. [3H]-SCH23390 and [3H]-spiroperidol were purchased from Amersham, USA. All other drugs were from analytical grade. Experimental protocol Animals were anesthetized with thiopental (30 mg/kg, i.p.) and chloral hydrate (200 mg/ kg, i.p.) and received a unilateral stereotaxic injection of 6-OHDA (two injections of 12 mg per one ml of saline containing 0.2% ascorbic acid) into the right striatum (AP 0.9/1.4, ML

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3.8, DV 3.3, from Bregma), according to the Atlas of Paxinos and Watson [16] using a 5 ml Hamilton syringe. The syringe was left in place for 2 min before slowly retracting it. Shamoperated animals were used for controls. Melatonin suspended in 8% Tween 80 was administered usually around 4 p.m. (2, 5, 10 and 25 mg/kg, i.p.) 1h after 6-OHDA lesion and, thereafter, daily for 7 days. Four weeks after 6-OHDA injection, rotational behavior was assessed by monitoring body rotations induced by apomorphine (3 mg/kg, i.p.) and the number of net rotations was recorded for 60 min. Animals were decapitated 24 h after and immediately their brains were dissected on ice. The striatum was used to prepare 10% homogenates. For the measurement of monoamine levels, the brain tissue was sonicated in 0.1 M HClO4, for 30 s, centrifuged at 4 8C for 15 min at 15.000 rpm, and the supernatant was filtered (0.2 mm, Millipore). A 20 ml sample was then injected in the high-performance liquid chromatograph (HPLC) column. The mobile phase was 0.163 M citric acid, pH 3.0, containing 0.02 mM EDTA, with 0.69 mM sodium octanesulfonic acid (SOS), as ion pairing reagent, 4% v/v acetonitrile and 1.7 % v/v tetrahyrofuran. DA, 5HT and their metabolites were electrochemically detected using an amperometric detector (Shimadzu, Japan), by oxidation on a glassy carbon electrode at 0.85 V relative to the Ag-AgCl reference electrode. The amount of monoamine was determined by comparison with standards injected into the HPLC column at the day of experiment. Levels were expressed as ng/mg tissue. Binding studies: For binding assays, the methods of Meltzer et al. [17] and Kessler et al. [18] were used for the D1- and D2-like receptors respectively. Animals (5–6 per group) were decapitated, and the striatum was dissected under ice for the preparation of homogenates. The homogenate containing 80–160 mg of protein was incubated in Tris-HCl buffer (50 mM, pH 7.4), in the presence of 10 mM mianserine for blocking of serotonergic receptors, in the case of D2-like receptors. Several ligand concentrations of [3H]-SCH 23390 (87 Ci/mmol) ranging from 0.135 to 10.8 nM or [3H]-spiroperidol (114 Ci/mmol) ranging from 0.04 to 2.29 nM, were used for D1 and D2-like receptors respectively, in a final volume of 0.2ml. The reaction media was incubated at 37 8C for 60 min, and the reaction was terminated by filtration in vacuum through Whatman GF/B filter paper on a cell harvester apparatus from Brandel, Maryland, USA. Filters were washed 5 times with cold saline, dried out in the oven for 2 h at 60 8C, and placed in vials containing 3 ml of a toluene-based scintillation cocktail. Radioactivity was measured in a Beckman LS 100 counter with a 68% efficiency. The specific binding was calculated by subtracting the total minus nonspecific binding done in the presence of 5 mM dopamine or 10 mM butaclamol, in the case of D1 and D2-like receptors respectively. Data were expressed as femtomoles/per mg of protein. Protein was determined by the method of Lowry et al. [19] using bovine serum albumin as standard. Statistical analyses All results were presented as means 6 S.E.M. For the Scatchard analyses of [3H]-spiroperidol and [3H]-SCH23390 binding data were compared with unpaired Student’s t-test. Results were considered significant at p,0.05. Results Results from Table 1 show that 6-OHDA decreased from 77 to 85% the contents of monoamines and their metabolites in the ipsilateral as compared to the contralateral side in

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Table 1 Effects of melatonin treatment on levels of dopamine (DA), serotonin (5HT) and their metabolites (ng/mg tissue) after 6-OHDA-induced lesions in rat striatum Group

DA

DOPAC

HVA

5HT

5HIAA

Cont C Cont I

4728.7 6 339.4 703.0 6 52.3a

2018.1 6 92.5 365.5 6 37.1a

498.3 6 49.3 84.5 6 6.8a

697.8 6 62.6 162.1 6 12.2a

569.5 6 43.4 481.6 6 32.7

Mel 2 C Mel 2 I

4652.5 6 265.1 1911.3 6 186.0b

1673.8 6 125.7 1012.6 6 67.7b

453.3 6 18.6 243.0 6 18.5b

693.0 6 46.2 491.1 6 53.7b,c

482.0 6 38.2 328.0 6 27.7

Mel 5 C Mel 5 I

5019.4 6 492.5 2646.4 6 189.6b

1674.0 6 122.1 658.2 6 74.7b

311.7 6 54.9 185.9 6 23.3

815.7 6 60.1 683.1 6 41.1b

367.7 6 16.7 358.9 6 29.2

Mel 10 C Mel 10 I

4163.2 6 315.2 2179.1 6 186.4b

1729.7 6 212.1 1010.2 6 123.1b

461.7 6 25.8 332.7 6 49.8b

560.7 6 62.3 410.0 6 43.9b,c

566.5 6 31.8 469.8 6 39.5

Mel 25 C Mel 25 I

4112.1 6 153.7 1585.3 6 163.5b,c,d

1694.6 6 108.4 937.5 6 89.2b

535.9 6 27.5 270.4 6 20.1b

622.5 6 45.7 697.5 6 79.2b,d

590.2 6 26.3 492.3 6 30.0

Melatonin (Mel) was administered (2, 5, 10 and 25 mg/kg, i.p.) daily for 7 days, 1h after 6-OHDA lesion. Four weeks after 6-OHDA injection into the striatum, animals were decapitated for monoamine measurements. Data are means 6 SEM from 5 – 15 experiments. For statistical analyses, ANOVA and the Student-Newman-Keuls test were used. a vs Cont C, b, vs. controls (Cont, 6-OHDA); c, vs. Mel 5; d, vs. Mel 10 at p , 0.05. I 5 ipsilateral; C 5 contralateral side. DA, 3,4 dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were electrochemically detected using HPLC.

controls. No significant differences were seen among the values measured in the contralateral side from the melatonin treated, not treated (controls) and sham-operated groups. Melatonin 2, 5, 10, 25 mg/kg (ipsilateral side) decreased in 52, 44, 54 and 67% the DA contents as related to the contralateral side of controls, and in 48, 67, 50, and 54% those of DOPAC, while HVA contents decreased to 51, 63, 33 and 46% also compared to the contralateral side of controls. In the melatonin treated groups, DA contents were brought to values of 3.2; 3.8; 3.1 and 2.3 times those of 6-OHDA, and DOPAC contents to values of 2.9; 2.2; 3.9 and 3.2 times. A significant difference was also seen among DA levels in melatonin 2, 5, 10, and 25 mg/kg treated groups. In this condition, the protection offered by melatonin in 6-OHDA lesioned rats was inversely proportional to its dose. Fig. 1 shows a 83% decrease in DA content in the 6-OHDA lesioned rats (controls) in the ipsilateral side as related to the contralateral side. All values were obtained from the same animals. On the other hand, in the melatonin treated group at the lower doses of 2 and 5 mg/kg, this decrease was only 48 and 38% respectively. However, the effect of melatonin was inversely proportional to its dose, and the percentage decreases were 51.5 and 63.5% with the doses of 10 and 25 mg/kg, i.p., respectively. A decrease of 72% was observed in 5HT levels in the 6-OHDA lesioned group as compared to the sham-operated group. In the melatonin treated group at the doses of 2, 5 and 10 mg/kg, decreases were 39, 27 and 31%, respectively, while at the higher dose (25 mg/kg), the decrease was 51%. Except for a 34% decrease observed in 5-HIAA levels with melatonin at the dose of 2 mg/ kg, i.p., no other effect was observed in this parameter. No difference was seen between the sham-operated (contralateral and ipsilateral sides) and the contralateral side of the 6-OHDAlesioned rats (controls) (Fig.2).

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Fig. 1. Effect of the melatonin treatment (2, 5, 10 and 25 mg/kg, i.p. daily for 7 days, 1h after 6-OHDA lesion) on levels of DA, DOPAC and HVA in rat striatum (ipsilateral/contralateral side, %) (ANOVA and Student-NewmanKeuls test were used) a, vs. sham-operated; b, vs. controls (6-OHDA); c, vs. Mel 2; d, vs. Mel 5; e, vs Mel 10 at p , 0.05. Experimental conditions were the same as those described in Table 1.

Rats with striatal lesions presented a characteristic rotational behavior after the administration of apomorphine (controls: 152.8616.3 turns/hour). This stereotyped behavior was significantly decreased in 60, 89, 78 and 47%, by melatonin (2, 5, 10 and 25 mg/kg, i.p.) treatment as related to controls, respectively. Maximum effect was observed with 5 and 10 mg/kg, and was somewhat smaller with the highest melatonin dose (25 mg/kg) (Fig.3). Data from dopaminergic binding show that melatonin (5 mg/kg, i.p.) significantly increased in 43% [3H]-SCH 23390 binding indicative of an upregulation of D1 receptor. This

Fig. 2. Effect of the melatonin treatment (2, 5, 10 and 25 mg/kg, i.p. daily for 7 days, 1h after 6-OHDA lesion) on levels of 5-HT and 5-HIAA in rat striatum (ipsilateral/contralateral side, %) (ANOVA and Student-NewmanKeuls test were used) a, vs. sham-operated; b, vs. controls (6-OHDA); c, vs. Mel 2; d, vs. Mel 5; e, vs. Mel 10 at p , 0.05. Experimental conditions were the same as those described in Table 1.

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Fig. 3. Effect of the melatonin treatment (2, 5, 10 and 25 mg/kg, i.p. daily for 7 days, 1h after 6-OHDA lesion) on apomorphine-induced (3 mg/kg, i.p.) rotational behavior in 6-OHDA lesioned rats. Four weeks after 6-OHDA striatal injection, the number of contralateral rotation was counted for 60 min. Data are reported as means 6 SEM from 5 – 15 experiments. (ANOVA and Student-Newman-Keuls test were used) a,b vs. sham-operated and controls (6-OHDA-lesioned without melatonin), respectively, at p , 0.05).

effect was opposite to the one occurring in Kd values which showed a 48% decrease instead, as compared to controls. No effect was observed in D2-like receptor number. Although not significant, a 25% decrease was also observed in Kd values from D2 receptors (Table 2). Discussion The neurotoxin 6-hydroxydopamine (6-OHDA) is used experimentally to induce Parkinson’s disease, which is associated with degeneration of the nigrostriatal system and a consequent reduction of dopamine content in the striatum and substantia nigra. This compound destroys Table 2 Effects of melatonin on D1 and D2 dopaminergic receptor subtypes in 6 OH-DA-induced lesion in rat striatum [3H]-SCH23390 binding

[3H]-spiroperidol binding

Group

Bmax

Kd

Bmax

Kd

Control Mel 5

194.8619.0(6) 277.8625.8(7)*

2.960.38(6) 1.560.10(7)**

209.3616.64(6) 203.2613.00(7)

2.760.25(6) 2.160.23(7)#

Melatonin was administered (2, 5, 10 and 25 mg/kg, i.p.) daily for 7 days and controls received only saline 0.9%, 1h after 6-OHDA lesion. Four weeks after 6-OHDA injection into the right (6-OHDA lesioned) striatum, animals were decapitated for binding assays. Numbers are means 6 SEM of the number of experiments shown in parenthesis (student t-test). [3H]-SCH 23390 and [3H]-spiroperidol were used as ligands for D1 and D2 receptors binding assays respectively. * t(11) 5 2.514; p 5 0.0144; ** t(11) 5 3.602; p 5 0.0021; # t(11)5 1.802; p50.0495.

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catecholaminergic neurons by producing oxygen species through non-enzymatic oxidation reaction [20]. There is increasing evidence that the neurotoxicity of 6-OHDA is mediated by oxidative stress, leading to excessive generation of free radicals and cell death [21–23], thus oxidative damage caused by 6-OHDA can be prevented by a variety of antioxidants [24]. In the rat model of PD, the nigrostriatal fibers are destroyed unilaterally with 6-OHDA, and animals respond to systemic dopaminergic agonists, including apomorphine, with contralateral turning [25,26]. On the other hand, the degeneration of dopaminergic neurons in the substantia nigra and the resulting loss of their nerve terminals in the striatum are responsible for most of the motor disturbances seen in Parkinson’s disease. Melatonin (N-acetyl-methoxy-tryptamine), the pineal hormone, acts as a free radical scavenger and antioxidant, and crosses easily the blood-brain barrier entering cells and subcellular compartments [5,27]. Additionally, melatonin may stimulate some important antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and glutathione reductase and also stabilizes cell membranes [1]. Besides, in experimental models used to study neurodegenerative diseases, including Parkinson’s disease, melatonin was found to be effective in reducing neuronal damage. Studies have shown the antioxidative actions of melatonin in vivo and in vitro [28–30]. Melatonin can reduce lipid peroxidation [31–37], protein damage [38] and DNA damage [39–42]. Kim et al. [2] showed that melatonin treatment rescues nigrostriatal dopaminergic neurons from cell death in 6-OHDA lesioned rats suggesting that melatonin has a potent antioxidative action. In the present work, we showed that 6-OHDA induced significant decreases in DA, DOPAC and HVA levels when measured from the ipsilateral side compared with the contralateral side from the same animal. We also demonstrated that 5-HT levels decreased but levels of its metabolite, 5HIAA, were unchanged. These results are consistent with older studies describing biochemical changes in the denervated striatum following intracerebral 6-OHDA injection [43,44], which decreases dopaminergic function and deplete dopamine levels. Although several works [45,46,47] in the literature reported significant increases in rat striatal 5-HT and 5-HIAA levels after 6-OHDA lesions, most of them used 3 day old and not adult rats as in the present paper, others [48], showed an increase and no change in striatal 5-HT and 5-HIAA contents working with neonatal and adult rats respectively. In addition, Zhou et al., 1991 [49] showed a significant increase in 5-HT and 5-HIAA levels in adult rat striatum. However, in their case the decrease in DA level in the striatum exceeded 90%, while in ours it was around 80%. However, our data agrees with that by Karstaedt et al 1994 [50], who showed that 6 weeks after 6-OHDA lesion, levels of 5-HT and 5-HIAA were significantly decrease in rat striatum. According to these authors the loss of DA innervation in the striatum triggers an increase in 5-HT turnover and a net depletion of 5-HT in the striatum. Our results showed that melatonin partially restores contents of monoamines and their metabolites in 6-OHDA lesioned rats when administered 1h after the lesion. Similarly, Kim et al. [2] showed that melatonin treatment rescues nigrostriatal dopaminergic neurons from cell death in 6-OHDA lesioned rats suggesting that this beneficial effect was a consequence of the potent antioxidative action of melatonin. In the present work, although the percentage decreases (comparing ipsilateral against contralateral sides) were similar with the three

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smaller doses of melatonin, they were much lower than that detected in controls (6-OHDAlesioned rats without melatonin treatment). However, the percentage decrease was larger with the highest melatonin dose and similar to that observed with the smallest one, giving as a result a bell-shaped dose-response curve. The 6-OHDA-injection caused a loss of dopamine cell bodies in the substantia nigra and their terminals in the striatum [51], resulting in a characteristic motor dysfunction. That loss was reduced by the treatment with dopamine receptors agonists, such as apomorphine which induced rotational behavior in a direction contralateral to the lesion (contralateral side) [52]. Our results showed that intrastriatal administration of 6-OHDA produced a number of apomorphine-induced rotations that were significantly decreased by melatonin treatment, suggesting increases in DA levels in the denervated striatum and reduction of receptor supersensitivity. These findings were supported by mensurements of DA levels which were partially recovered in the ipsilateral side of the melatonin treated group. Melatonin dose-response curve was bell-shaped. A similar feature was also observed elsewhere [53]. The loss of dopamine neurons in humans is associated with diseases of the aged, notably PD. There is strong evidence indicating that the destruction of DA neurons in PD involves the accumulation of oxygen free radicals. Because of these actions, the present results support the hypothesis that melatonin may have beneficial effects on therapeutic approaches for the treatment of oxidative stress-induced neurodegenerative diseases such as PD. We showed that melatonin was able to rescue DA neurons from death in the model of 6-OHDA-induced striatal lesion associated with oxidative stress. We also demonstrated that melatonin significantly increased D1 receptor number indicative of an upregulation type of phenomenon, and increased at a similar magnitude D1-like receptor affinity. It is largely documented that melatonin effects may involve modulation of dopaminergic system [54,55]. In addition, melatonin has been shown to be clinically effective in PD and neuroleptic-induced PD suggesting an interaction with the DA system. The D2 subtype of DA receptors have been suggested to play an important role in a number of neurological disorders such as Alzheimer’s and Parkinson’s diseases, and to mediate psychomotor DA behaviors including rotation and locomotion [56]. Recent work [57] showed that the affinity (Kd) but not the number (Bmax) of D2- DA receptors in rat striatum was altered by melatonin. The author postulated that melatonin increases D2- DA receptors affinity through conformational changes of the receptor binding site. This finding may represent a mechanism through which melatonin exerts a modulatory influence on DA system. Data from Escames et al., 1996, reported that iontophoresis of melatonin attenuated the excitatory response in striatal neurons while that of a D2 antagonist produced an increase in the excitatory response. These results showed that the same striatal neurons may be driven by melatonin and D2 receptors [58]. However, according to these authors, the effects of the two compounds are mediated by different receptor intracellular messengers. Other data [59] indicated that D1- DA receptor binding was affected by pinealectomy in the suprachiasmatic nuclei of fetal and neonatal rats as well as in that of their mothers. In conclusion, we demonstrated that melatonin reverted the decreases in striatal levels of DA, DOPAC, HVA, 5-HT after 6-OHDA lesions. These effects occurred predominantly on the dopaminergic system suggesting a somewhat stronger interaction between melatonin and

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DA. Another finding in favor of this interaction was the melatonin blockade of stereotyped behavior (rotational behavior) in striatal 6-OHDA lesioned rats. In addition, dopaminergic receptors mainly of the D2 subtype seem to play an important role in the expression of apomorphine-induced rotational behavior, and although the maximum density of D2 receptors was unchanged after melatonin treatment, it altered receptors affinity. Finally, in the present work a receptor upregulation phenomenon was observed only with the D1 receptor subtype after melatonin treatment, which also altered the affinity of the D1 subtype of dopaminergic receptor.

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