Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 373: 79–84 DOI 10.1007/s00210-006-0058-1
SH ORT COMMUNI CATIO N
Ewa Obuchowicz . Agnieszka Marcinowska . Łukasz Drzyzga . Jacek Wójcikowski . Władysława A. Daniel . Zbigniew S. Herman
Effect of chronic treatment with perazine on lipopolysaccharide-induced interleukin-1β levels in the rat brain Received: 14 November 2005 / Accepted: 2 March 2006 / Published online: 1 April 2006 # Springer-Verlag 2006
Abstract In the present study, we sought to determine whether chronic treatment with perazine alters lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β) levels in the following rat brain regions: the hypothalamus, frontal cortex, striatum and hippocampus. Male Wistar rats were administered perazine dimaleate (15 or 30 mg/kg/day) in drinking water for 21 days. On day 22, LPS was injected i. p. (125 μg/kg) 2 h before decapitation. Concentrations of perazine and its metabolites in plasma and brain was assessed by HPLC. The levels of IL-1β were determined using ELISA. Treatment with perazine (30 mg/kg/day) reduced LPS-stimulated IL-1β levels in the hypothalamus, and a tendency to its decrease in the striatum and frontal cortex was observed. This in vivo study suggests for the first time that long-term oral administration of perazine modulates reactivity of cells producing IL-1β. Keywords Perazine . Interleukin-1β . Lipopolysaccharide . Brain . Rat
Introduction In recent years, clinical studies have provided a number of pieces of evidence that schizophrenia may be accompanied by immune abnormalities, suggesting suppression of some and activation of other immune functions (Gaughran 2002). Several studies have shown alterations in cytokines, E. Obuchowicz (*) . A. Marcinowska . Ł. Drzyzga . Z. S. Herman Department of Clinical Pharmacology, Silesian University School of Medicine, Medykow 18 Street, 40-752 Katowice, Poland e-mail:
[email protected] Tel.: +48-32-2523902 Fax: +48-32-2523902 J. Wójcikowski . W. A. Daniel Department of Pharmacokinetics and Drug Metabolism, Polish Academy of Sciences, Institute of Pharmacology, 31-343 Kraków, Poland
which are important mediators of interactions between the central nervous system (CNS) and the endocrine and immune systems. In the brain, cytokines are produced by glial cells (mainly microglial cells) and, to a small extent, by neurons. Also cytokines synthesized in the periphery by the immunocompetent cells may gain access to the brain. In the CNS, cytokines are implicated in various regulatory mechanisms, including initiation of an inflammatory response, regulation of the hypothalamic-pituitary-adrenal (HPA) axis activity, modulation of synaptic transmission, and action on neuronal survival and apoptosis (Muller and Ackenheil 1998). These direct actions exerted by cytokines on the CNS processes triggered an interest in their potential role in the effect of antipsychotic drugs. Interleukin-1β (IL-1β), a pro-inflammatory cytokine, mediates psychological stress response and, therefore, is implicated in various psychiatric diseases. Administration of IL-1β into the rat brain elicits a number of behavioral effects which are similar to those observed in stressexposed animals (Dantzer 2004). Moreover, it has been shown that some types of stress enhanced IL-1β level in the hypothalamus (Deak et al. 2005). Stress is a wellknown factor that may induce or exacerbate schizophrenia in susceptible individuals (Horan et al. 2005; MyinGermeys et al. 2005). Some clinical studies suggest that impaired signaling or production of the IL-1 family is associated with schizophrenia (Katila et al. 1999; Kowalski et al. 2001; Theodoropoulou et al. 2001; Toyooka et al. 2003). Up until now, the influence of antipsychotic drugs on cytokine networks has been studied by evaluation of their effect on cytokine levels in peripheral blood or cerebrospinal fluid, and only a few studies demonstrated their effect on cytokine secretion in rat glial cell cultures (Pollmächer et al. 2000; Labuzek et al. 2005). However, there is no data concerning their influence on brain cytokine levels. In the present study, we examined whether chronic administration of perazine to rats affects lipopolysaccharide (LPS)induced IL-1β levels in various brain regions. Although perazine belongs to the group of phenothiazines with piperazine structure in a side chain, it is not a potent
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antagonist of dopaminergic D2 receptors. Moreover, it is a weak antagonist of serotonergic 5-HT2 and adrenergic α1 and α2 receptors, with a higher affinity for histaminergic H1 receptors (Leysen et al. 1993). Therefore, perazine rarely produces extrapyramidal side-effects and adverse effects in the autonomic nerve system (Jarema and Konieczynska 2000). As a drug with moderate potency, it is used in chronic therapy of schizophrenia and schizophrenia-like psychoses (Terminska and Mrowiec 1989). The effect of perazine on IL-1β levels was estimated in the hypothalamus, frontal cortex, striatum and hippocampus, because these structures mediate pharmacological action of antipsychotic drugs (Baldessarini and Tarazi 2006). On the other hand, it has been shown that LPS injected intraperitoneally (i.p.) dose-dependently increases IL-1β protein levels in the rat hypothalamus, hippocampus and cortex (Hansen et al. 2000).
measured every day, so that the correct doses were maintained. The average doses of perazine actually ingested by rats were 14.88±0.48 or 29.41±0.88 mg/kg/ day (mean ± SEM), respectively. There was no difference in body weight gain between the treatment groups and the control rats which received tap water. Throughout the study, water intake at the higher concentration of drug solution was about 10% lower than water intake in the control group. LPS (Escherichia coli serotype 0111 : B4 ; Sigma Chemical Co., St. Louis, MO, USA) was dissolved in sterilized, pyrogen-free 0.9% saline. The applied dose of LPS and the time point of tissue collection was chosen on the basis of previous research (Hansen et al. 2000). Moreover, in our pilot study no stronger effect of the 4-fold higher dose of LPS had been found. Tissue collection and measurement of IL-1β levels
Materials and methods Animals Male Wistar rats, of initial body weight between 200 – 255 g, from the Animal Farm of the Silesian University School of Medicine, were allowed 1 week to adapt to the new conditions before the study began. They were housed 2 per cage (55×32×18 cm) under a controlled environment (12 h/12 h light-dark cycle, lights on from 7 a.m. to 7 p.m.; 22±2°C ambient temperature) with free access to chow and water. This study was approved by the Bioethical Committee of the Silesian University School of Medicine. Experimental procedure Four groups of rats were used in the experiments: 1. The rats were given tap water for 21 days and were injected i.p. with sterilized, pyrogen-free saline on day 22 2. The rats were given tap water for 21 days and LPS (125 μg/kg) was administered i.p. on day 22 3. The rats were given perazine (15 mg/kg/day) in drinking water for 21 days and were injected i.p. with LPS (125 μg/kg) on day 22 4. The rats were given perazine (30 mg/kg/day) in drinking water for 21 days and were injected i.p. with LPS (125 μg/kg) on day 22 The rats (n=6 per group) were sacrificed by decapitation (between 10 a.m. and 12 a.m.) with guillotine 2 h after i.p. injection of either LPS or saline on day 22 of the experiment. Perazine dimaleate (Labor, Wrocław, Poland) was dissolved in water every day before use, and the drug solution was given in light-proof drinking bottles. The rats were weighed every 3–4 days, and the concentration of perazine in water was adjusted according to the weight of the animals. The amount of liquid consumed was also
Following decapitation, brains were rapidly removed and placed on a Petri dish filled with ice. The hypothalamus, frontal cortex, striatum and hippocampus were dissected out after localization according to the atlas by Paxinos and Watson (1986). The brain structures were immediately frozen on dry ice, weighed and stored at −70°C until assayed for IL-1β. The samples were prepared as described by Deak et al. (2005). The dissected structures were sonicated in 7 vol of ice cold buffer for 10–12 s using an ultrasonic cell disruptor (Misonix Inc., Farmingdale, NY, USA) at a setting 5. The buffer (pH 7.2, 4°C) consisted of 50 mM Tris, 1mM EDTA, 6 mM MgCl2, 3 mM benzamidine hydrochloride, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml of leupeptin, 1 μg/ml of aprotinin, 1 μg/ ml of antipain, and 1 μg/ml of soybean trypsin inhibitor. All components were purchased from Sigma Chemical Co., St. Louis, MO, USA. Sonicated samples were then centrifuged (32,000×g, 15 min, 4°C ). Afterwards, supernatants were collected and centrifuged once more (32,000×g, 10 min, 4°C). The IL-1β levels were measured using rat IL-1β ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The optical density was measured using a UV microplate reader (Dynex Technologies Inc., Chantilly, VA, USA). The intra-assay precision coefficient of variation was 8.7% and the detection limit of the assay was 5 pg/ml. All results are expressed as pg of IL-1 β/ml of supernatant. Measurement of perazine and its metabolites by HPLC The rats (n=10 per group) were sacrificed by decapitation with guillotine between 8 a.m. and 9 a.m. The rats’ trunk blood was collected in tubes moistened with a 30% solution of sodium citrate, and their brains were rapidly removed and frozen on dry ice. Plasma aliquots for determination of concentration of perazine and its metabolites were kept frozen at −70°C until assay. Concentrations of perazine and its main metabolites (5sulfoxide and N-desmethylperazine) were assessed by the
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HPLC method based on Daniel et al. (1998). The brains were homogenized in distilled water (1:3 w/v). The homogenates were alkalized with 3 M NaOH to achieve pH=12 (approximately 300 μl of NaOH). Perazine and its metabolites were extracted with dichloromethane and hexane (1:1; 6 ml) for 45 min. After cooling overnight at −20°C, the samples were centrifuged at 2,000×g for 15 min. The organic phase containing the neuroleptic and its metabolites was transferred to clean tubes and evaporated under nitrogen at 40°C. Plasma samples were processed in the following way: after precipitation of proteins (1.5 ml of plasma + 600 μl of methanol), the plasma was mixed with 3 ml of distilled water. After alkalization to pH=12 (80 μl of 3 M NaOH), the extraction and evaporation of the organic phase was performed as above. During the whole procedure the samples were protected from light. The residue obtained after evaporation of the plasma or brain extracts was dissolved in 100 μl of the mobile phase described below. An aliquot of 20 μl was injected into the HPLC system LaChrom (Merck-Hitachi), equipped with a UV detector, L-7100 pump and D-7000 System Manager. The analytical column (Econosphere C18, 5 μm, 4.6×250 mm) was purchased from Alltech (Carnforth, England). The mobile phase consisted of acetate buffer, pH=3.4 (100 mmol of ammonium acetate, 20 mmol of citric acid and 1 ml of triethylamine in 1,000 ml of the buffer adjusted to pH=3.4 with an 85% phosphoric acid), and acetonitrile at a proportion of 1:1 (v/v). Elution proceeded at an ambient temperature at a flow rate of 1.2 ml/min. The absorbance of perazine and its metabolites was measured at a wavelength of 265 nm. The compounds were eluted in the following order: perazine 5-sulfoxide (6.08 min), N-desmethylperazine (7.09 min), perazine (11.97 min). All the reagents used in the whole procedure Fig. 1 Effect of perazine on IL-1β levels induced by lipopolysaccharide (LPS) in the rat hypothalamus, frontal cortex, striatum and hippocampus. Perazine dimaleate (15 or 30 mg/kg/day) (PER 15 or PER 30) was administered in drinking water for 21 days. On day 22, LPS (125 μg/kg) was injected i.p. 2 h before decapitation. Control rats were given tap water for 21 days and were injected i.p. with pyrogen-free saline or LPS (125 μg/kg) on day 22. Data are expressed as pg of IL-1β/ml of supernatant; means ± SEM; n=6; *** P
