Psychotropic drugs attenuate lipopolysaccharide-induced hypothermia by altering hypothalamic levels of inflammatory mediators in rats

Neuroscience Letters 626 (2016) 59–67

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Research paper

Psychotropic drugs attenuate lipopolysaccharide-induced hypothermia by altering hypothalamic levels of inflammatory mediators in rats Ahmad Nassar a , Yael Sharon-Granit a , Abed N. Azab a,b,∗ a b

Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel School for Community Health Professions – Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

h i g h l i g h t s • Inflammation may contribute to the pathophysiology of mental disorders and psychotropic drugs are known to exert various effects on brain inflammation.

• Systemic administration of lipopolysaccharide (LPS) to rats causes robust production of inflammatory mediators and pathological changes in body temperature.

• Four psychotropic drugs significantly attenuated LPS-induced hypothermia in rats. • Lithium, carbamazepine, haloperidol and imipramine differently affected levels of prostaglandin E2, tumor necrosis factor-␣ and phosphorylated p65 levels in plasma and hypothalamus of LPS-treated rats.

a r t i c l e

i n f o

Article history: Received 22 March 2016 Received in revised form 26 April 2016 Accepted 11 May 2016 Available online 12 May 2016 Keywords: Carbamazepine Haloperidol Imipramine Inflammation Lithium Pathophysiology

a b s t r a c t Recent evidence suggests that inflammation may contribute to the pathophysiology of mental disorders and that psychotropic drugs exert various effects on brain inflammation. The administration of bacterial endotoxin (lipopolysaccharide, LPS) to mammals is associated with robust production of inflammatory mediators and pathological changes in body temperature. The objective of the present study was to examine the effects of four different psychotropic drugs on LPS-induced hypothermia and production of prostaglandin (PG) E2 , tumor necrosis factor (TNF)-␣ and phosphorylated-p65 (P-p65) levels in hypothalamus of LPS-treated rats. Rats were treated once daily with lithium (100 mg/kg), carbamazepine (40 mg/kg), haloperidol (2 mg/kg), imipramine (20 mg/kg) or vehicle (NaCl 0.9%) for 29 days. On day 29, rats were injected with LPS (1 mg/kg) or saline. At 1.5 h post LPS injection body temperature was measured, rats were sacrificed, blood was collected and their hypothalami were excised, homogenized and centrifuged. PGE2 , TNF-␣ and nuclear P-p65 levels were determined by specific ELISA kits. We found that lithium, carbamazepine, haloperidol and imipramine significantly attenuated LPSinduced hypothermia, resembling the effect of classic anti-inflammatory drugs. Moreover, lithium, carbamazepine, haloperidol and imipramine differently but significantly affected the levels of PGE2 , TNF-␣ and P-p65 in plasma and hypothalamus of LPS-treated rats. The results suggest that psychotropic drugs attenuate LPS-induced hypothermia by reducing hypothalamic production of inflammatory constituents, particularly PGE2 . The effects of psychotropic drugs on brain inflammation may contribute to their therapeutic mechanism but also to their toxicological profile. © 2016 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: BT, body temperature; CBZ, carbamazepine; HPL, haloperidol; HT, hypothalamus; IL, interleukin; IMP, imipramine; LIT, lithium; LPS, lipopolysaccharide; NF␬B, nuclear factor ␬ B; P-p65, phosphorylated-p65; PGE2 , prostaglandin E2 ; TNF-␣, tumor necrosis factor-␣. ∗ Corresponding author at: Department of Nursing, School for Community Health Professions, Ben-Gurion University of the Negev, P.O.B 653, Beer-Sheva 84105, Israel. E-mail address: [email protected] (A.N. Azab). http://dx.doi.org/10.1016/j.neulet.2016.05.019 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction A large body of data has accumulated suggesting that inflammation may contribute to the pathophysiological mechanisms underlying mental illnesses such as depression [1,2] bipolar disorder [3,4] and schizophrenia [5,6]. Supporting evidence indicates that psychotropic drugs exert multiple anti-inflammatory effects [7–13]. These observations have laid down the foundation for the progressively acknowledged “inflammation hypothesis” of mental disorders. In support for this hypothesis, many studies demonstrated that regular anti-inflammatory drugs reduce the severity of symptoms among psychiatric patients [14–17]. For example, drugs that inhibit the enzyme cyclooxygenase (COX) and reduce prostaglandins (PGs) synthesis have been shown to exert beneficial effects as add-on therapy in patients with mental disorders [14–17]. Systemic inflammation occurs when infectious pathogens invade the body of mammals and circulate in the blood. For example, systemic administration of bacterial endotoxin (lipopolysaccharide, LPS) to mammals leads to a profound inflammatory response which includes robust production of inflammatory mediators, pathological changes in body temperature (BT), hypotension, among other pathological features [18–21]. In rats, LPS induces a biphasic change in BT—an initial decrease (hypothermia) followed by an elevation (fever) [20,21]. The mechanism of the hypothermic response to LPS is not fully understood. It seems that several inflammatory constituents contribute to this complex process [18,20,21], among which PGE2 is known to play a pivotal role [20,21]. Pathological changes in BT (both hypothermia and fever) are associated with increased levels of PGE2 in the hypothalamus. Consistently, anti-inflammatory drugs that decrease hypothalamic production of PGE2 reduce LPS-induced hypothermia [20,22]. Brain inflammation occurs in response to pathological processes such as invasion of infectious microorganisms, traumatic injury, ischemia and degeneration [23–25]. Glia cells play a central role in most types of brain inflammation as they secrete pro-inflammatory substances such as interleukin (IL) 1-␤, IL-6, PGE2 and tumor necrosis factor (TNF)-␣ [26,27]. On the other hand, glia cells can suppress the inflammatory response by producing anti-inflammatory elements such as IL-10 [26,27]. However, not all brain inflammatory processes are detrimental as they may reflect tissue homeostasis [28]. Thus, inhibition of inflammation does not always necessarily benefit the brain. Inflammatory mediators modulate a number of crucial processes in the brain. For example, PGE2 regulates synaptic transmission, hypothalamus-pituitary-adrenal axis function, neurotransmitter release, thermoregulation and appetite [29]. TNF-␣ regulates the expression of many genes that are important for neuron function and survival [30]. Nuclear factor ␬ B (NF-␬B) is another cellular pathway that is activated during brain inflammation. Mammalian NF-␬B family consists of several members such as p50 and p65 which are involved in healthy as well as pathologic processes [31]. At resting conditions, NF-␬B interacts with inhibitor of ␬B (I␬B) proteins which inhibit its activity by preventing its translocation to the nucleus [31]. Upon activation, phosphorylation of I␬B leads to its dissociation from NF-␬B and translocation of the latter to the nucleus for target gene transcription [31]. As mentioned above, psychotropic drugs exert various antiinflammatory effects [7–13], however, they have also been shown to have pro-inflammatory properties [6,11,32]. Lithium in particular exerts multiple effects on inflammation [12]. We have shown that acute treatment with lithium attenuated the hypothermic response to a high dose of LPS in rats, which was accompanied by a reduction in hypothalamic PGE2 levels [21].

The present study was undertaken to examine the effects of chronic treatment with lithium (LIT), carbamazepine (CBZ), haloperidol (HPL) and imipramine (IMP) on LPS-induced hypothermia and hypothalamic production of inflammatory mediators in rats. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 200–220 g at the beginning of the experiments were used throughout the studies. Animals were housed 3 per cage and maintained under controlled environmental conditions (ambient temperature 22 ± 1 ◦ C, humidity 55–60%, photoperiod cycle 12 h light: 12 h dark), fed Purina Lab Chow and water ad libitum. Only animals with no overt pathology have been included in the studies. The procedures of the study were in accordance with the guidelines of the Committee for the Use and Care of Laboratory Animals in Ben-Gurion University of the Negev (authorization # IL-61-11-2010). 2.2. Drug treatment LIT, CBZ, HPL and IMP were administered for 4 weeks through a single daily intraperitoneal (ip) injection. Control rats were injected with vehicle. The drugs were given at the following doses: LIT 100 mg/kg; CBZ 40 mg/kg; HPL 2 mg/kg; and, IMP 20 mg/kg, similar to previous studies which used similar protocols [33–36]. 2.3. Measurement of body temperature (BT) BT was measured with a thermocouple probe (HL 600 Thermometer, Anristu Meter Co., Japan) inserted into the rectum. Rats were acclimated to this procedure during 3 days before experiments were initiated. Chronic treatment with LIT, CBZ, HPL and IMP did not significantly alter BT in the very most of measurement points during the initial 4 weeks of drug treatment (data not shown). It is important to mention that rats’ BT is highly affected by various environmental factors such as ambient temperature, light, and surrounding noise. Short-term changes in BT do not necessarily reflect a pathologic process but may be a response to an environmental stimulus. In the present study, rats with persistent alterations in BT (in 2 successive measurements) were excluded from the studies. 2.4. Induction of inflammation by LPS LPS from Escherichia coli was dissolved in sterile NaCl 0.9%. On day 29 of the treatment protocol, LPS 1 mg/kg was given ip at 2 h after drug/saline injection. Control rats were injected ip with sterile NaCl 0.9%. The dose of LPS (1 mg/kg) was chosen in order to induce an inflammatory response of a mild to moderate magnitude [10], as we wished to test the drugs under condition that resemble a possibly-occurring chronic low-grade inflammation that takes place in the brain of mentally affected patients. A high dose of LPS would have led to a severe inflammatory response [10] which would be difficult to influence by pharmacological interventions that are not potent anti-inflammatory drugs. 2.5. Blood collection and preparation of hypothalamic samples BT was measured before and at ∼1.5 h after LPS injection. Then, rats were briefly anesthetized with a mixture of 4% isoflurane in 100% oxygen and immediately sacrificed by decapitation. Blood was collected (in heparin-containing tubes) for plasma separation

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and hypothalamus (HT) of each rat was gently extracted and immediately transferred to −80 ◦ C. Thereafter, HT samples were weighed and manually homogenized for 10 s in 500 ␮l of a homogenizing buffer (cold phosphate-buffered saline solution containing a cocktail of protease inhibitors purchased from Sigma) and centrifuged at 10,000g, 4 ◦ C for 10 min. Supernatants and pellets were separated, collected and immediately transferred to −80 ◦ C for further determination. 2.6. Determination of PGE2 and TNF-˛ levels Levels of PGE2 and TNF-␣ in plasma and hypothalamic samples (supernatants of homogenates) were measured using rat DuoSet ELISA kits according to manufacturer’s protocols (R&D Systems; Minneapolis, USA). The detection limits of the assays were as follows: 39–2500 pg/ml for PGE2 and 62.5–4000 pg/ml for TNF-␣. 2.7. Measurement of phosphorylated p65 (P-p65) levels in nuclear extracts of hypothalamic samples Preparation of nuclear extracts was performed based on a previous protocol [37], with some modifications. Pellets of hypothalamic

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homogenates (Section 2.5) were re-suspended in 110 ␮l per sample of (lysis) buffer A [25 mM HEPES, 0.3 M NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.1% Triton x-100, 20 mM ␤-glycerophosphate, 1 mM sodium orthovandate, 1 mM PMSF, 1 mM DTT, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, completed to 10 ml with dd-H2 O] and aggressively vortexed for 10 s. Then, samples were incubated on ice for 15 min and subjected to rapid passage through a syringe needle (28G) 8 times and centrifuged at 13,000g for 1 min at 4 ◦ C. Pellets were collected and re-suspended in 220 ␮l of buffer B [10 mM HEPES, 0.3 M KCl, 0.1 mM EDTA, 0.1 mM EGTA, 20 mM ␤-glycerophosphate, 0.1 mM sodium orthovandate, 1 mM PMSF, 1 mM DTT, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, completed to 10 ml with dd-H2 O] with aggressive vortex. Samples were incubated on ice for 15 min and centrifuged at 13,000g for 5 min at 4 ◦ C. Pellets were collected and re-suspended in 110 ␮l of buffer C [10 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM ␤glycerophosphate, 1 mM sodium orthovandate, 1 mM PMSF, 1 mM DTT, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, completed to 10 ml with dd-H2 O] with aggressive vortex for 20 s, and incubated on ice for 15 min. Then, samples were centrifuged at 13,000g for 5 min at 4 ◦ C and supernatants were collected and stored at −80 ◦ C. Those supernatants were regarded as containing the nuclear fraction.

Fig. 1. Effects of psychotropic drugs on LPS-induced hypothermia in rats. Rats were injected once daily (ip) with LIT (A), CBZ (B), HPL (C), IMP (D) for 28 days. On day 29, rats were treated with those drugs at 2 h before LPS (1 mg/kg, ip) injection. BT was measured before and at 1.5 h post LPS injection. The graphs show the difference (delta) in BT after and before LPS injection. Values are mean ± SEM of 12 rats. * P < 0.05 vs. Control; ˆP < 0.05 vs. LPS.

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Fig. 2. Effects of psychotropic drugs on plasma TNF-␣ levels in LPS-treated rats. Rats were injected once daily (ip) with LIT (A), CBZ (B), HPL (C), or IMP (D) for 28 days. On day 29, rats were treated with those drugs at 2 h before LPS (1 mg/kg, ip) injection. Rats were sacrificed at ∼2 h post LPS injection and blood was collected for plasma separation. Plasma TNF-␣ levels were determined by ELISA and are presented relative to their level in the LPS group, which is expressed as 100%. Values are means ± SEM of 12 rats. UD denotes undetectable. * P < 0.05 vs. Control; Pˆ < 0.05 vs. LPS.

Detection of P-p65 in nuclear extracts was done using a special ELISA kit according to manufacturer’s protocol (eBioscience; San Diego, USA). 2.8. Statistical analysis Statistical evaluations were carried out using two-tailed Student’s t-test. Results are presented as mean ± SEM for the sample size as indicated in each figure. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Effects of psychotropic drugs on LPS-induced hypothermia Consistent with previous studies [20,21], treatment with LPS led to a significant decrease in BT at 1.5 h post injection. Pretreatment with LIT, CBZ, HPL and IMP significantly attenuated LPS-induced hypothermia (Fig. 1).

3.2. Effects of psychotropic drugs on plasma levels of TNF-˛ and PGE2 The administration of LPS led to a significant increase in plasma levels of TNF-␣ in rats (Fig. 2). Pretreatment with LIT and IMP significantly reduced LPS-induced elevation in TNF-␣ levels, whereas CBZ and HPL did not have a significant effect (Fig. 2). Moreover, LPS led to a significant increase in plasma PGE2 levels in some of the experiments performed (Fig. 3). Mostly, the drugs did not significantly alter PGE2 levels in LPS-treated rats (Fig. 3). 3.3. Effects of psychotropic drugs on hypothalamic levels of TNF-˛ and PGE2 The administration of LPS did not significantly alter TNF-␣ levels in HT (Fig. 4). Pretreatment with LIT, CBZ and IMP did not significantly alter HT TNF-␣ levels in control and LPS-treated rats. On the other hand, HPL significantly reduced HT TNF-␣ levels both in vehicle- and LPS-treated rats (Fig. 4). Furthermore, LPS significantly increased PGE2 levels in rats’ hypothalamus (Fig. 5). Pretreatment

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Fig. 3. Effects of psychotropic drugs on plasma PGE2 levels in LPS-treated rats. Rats were injected once daily (ip) with LIT (A), CBZ (B), HPL (C), or IMP (D) for 28 days. On day 29, rats were treated with those drugs at 2 h before LPS (1 mg/kg, ip) injection. Rats were sacrificed at ∼2 h post LPS injection and blood was collected for plasma separation. Plasma PGE2 levels were determined by ELISA and are presented relative to their level in the LPS group, which is expressed as 100%. Values are means ± SEM of 12 rats. * P < 0.05 vs. Control; Pˆ < 0.05 vs. LPS.

with LIT, CBZ and IMP significantly reduced HT PGE2 levels in LPS-treated rats (CBZ and IMP also decreased HT PGE2 levels in vehicle-treated rats). In contrast, HPL did not significantly alter HT PGE2 levels in LPS-treated rats (Fig. 5). 3.4. Effects of psychotropic drugs on hypothalamic levels of nuclear P-p65 As seen in Fig. 6, the administration of LPS led to a significant increase in HT P-p65 levels. Pretreatment with HPL led to a significant decrease in P-p65 levels while LIT led to a nearly significant decrease. Contrastingly, CBZ led to a significant increase in HT P-p65 levels while IMP did not have a significant effect (Fig. 6). 4. Discussion The major finding of the present study was that chronic treatment with LIT, CBZ, HPL and IMP attenuated LPS-induced hypothermia in rats. Moreover, similar to previous studies, this study demonstrated that LIT, CBZ, HPL and IMP exerted both antiand pro-inflammatory effects.

It is known that PGE2 plays a pivotal role in regulating BT under normal and pathological conditions [20,38]. Particularly, pathological changes in BT are usually associated with alterations in PGE2 levels in the hypothalamus [20,21]. We found that LIT, CBZ, HPL and IMP – all are not typical anti-inflammatory or BT-modulating drugs – significantly attenuated LPS-induced hypothermia in rats (Fig. 1). LIT [39], CBZ [34] and HPL [40] have been shown to decrease PGE2 levels in rat brain. Contrastingly, IMP was previously found to increase PGE2 levels in rat brain [36]. It is tempting to speculate that the anti-hypothermic effect of the drugs in the present study is due to a reduction in PGE2 levels in HT. This assumption is supported by: i) our findings that LIT, CBZ and IMP significantly decreased PGE2 levels in HT (Fig. 5); ii) the established ability of COX inhibitors to reduce PGE2 in HT and attenuate changes in BT [20,22,29], and, iii) our previous observation that LIT significantly attenuated hypothermia and HT PGE2 levels in rats treated with a high dose of LPS [21]. However, this assumption is complicated by the fact that HPL (which significantly attenuated LPS-induced hypothermia; Fig. 1) did not significantly reduce HT PGE2 levels (Fig. 5). It is possible that the attenuating effect of HPL on LPS-induced hypothermia is due to other BT-modulating effects. In this regard,

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Fig. 4. Effects of psychotropic drugs on TNF-␣ levels in hypothalamus of LPS-treated rats. Rats were injected once daily (ip) with LIT (A), CBZ (B), HPL (C), or IMP (D) for 28 days. On day 29, rats were treated with those drugs at 2 h before LPS (1 mg/kg, ip) injection. Rats were sacrificed at ∼2 h post LPS injection immediately after which hypothalamus (HT) was removed and further processed as described in Section 2. TNF-␣ levels in HT were determined by ELISA and are presented relative to their level in the LPS group, which is expressed as 100%. Values are means ± SEM of 12 rats. * P < 0.05 vs. Control; Pˆ < 0.05 vs. LPS.

other inflammatory mediators than PGE2 (e.g., leukotrienes) have also been shown to regulate LPS-induced hypothermia in rodents [18]. Among the four tested drugs only HPL significantly reduced TNF-␣ and P-p65 levels in HT of LPS-treated rats (Figs. 4 and 6, respectively). Moreover, we found that HPL significantly decreased PGE2 levels and totally abolished TNF-␣ in hippocampus of LPStreated rats (data not shown). A linkage between inflammatory mediators and brain function has been reported in several studies. For example, TNF-␣ was found to increase the activity of a serotonin transporter in mice [41]. TNF-␣ also increased plasma concentrations of corticosterone, altered central norepinephrine, dopamine and serotonin turnover, and induced sickness behavior in mice [42]. Moreover, patients suffering from psychiatric illnesses have been reported to have altered plasma concentrations of inflammatory mediators [1–6]. Thus, alterations in inflammatory mediators’ levels in plasma and brain regions by LIT, CBZ, HPL and IMP as seen in the present study may be associated with their therapeutic and/or toxic effects. The transcription factor NF-␬B plays a critical role in immune homeostasis, cell growth, and survival [31]. Previous studies have

shown that systemic administration of LPS stimulates NF-␬B activation in whole brain but particularly in HT [43]. Consistently, we found that nuclear P-p65 levels were increased in HT of LPS-treated rats (Fig. 6). P-p65 levels in HT were prominently reduced by LIT and HPL, whereas CBZ significantly increased P-p65 levels and IMP had no effect (Fig. 6). Emerging evidence attest for an inhibitory effect for psychotropic drugs on the NF-␬B pathway [13]. Thus, it would be of high scientific value to examine the efficacy of specific NF-␬B inhibitors in animal models of depression and mania. A possible limitation of our study is that rats were chronically administered with psychotropic drug before induction with LPS. In this regard, emerging evidence suggests that brain inflammation may precede the onset of psychiatric symptoms and the administration of pharmacological interventions [44]. Thus, it is reasonable to speculate why in the present study LPS was given to rats after the administration of psychotropic drugs? Two reasons accounted for the choice of our experimental design: 1) the inflammatory effects of acute LPS administration in vivo are known to resolve very rapidly, particularly when it is given at a low dose. Therefore it was not rational to administer LPS a month before the extraction of brain

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Fig. 5. Effects of psychotropic drugs on PGE2 levels in hypothalamus of LPS-treated rats. Rats were injected once daily (ip) with LIT (A), CBZ (B), HPL (C), or IMP (D) for 28 days. On day 29, rats were treated with those drugs at 2 h before LPS (1 mg/kg, ip) injection. Rats were sacrificed at ∼2 h post LPS injection immediately after which hypothalamus (HT) was removed and further processed as described in Section 2. PGE2 levels in HT were determined by ELISA and are presented relative to their level in the LPS group, which is expressed as 100%. Values are means ± SEM of 12 rats. * P < 0.05 vs. Control; Pˆ < 0.05 vs. LPS.

samples. 2) Repetitive exposure to LPS on the other hand is associated with tolerance to and gradual reduction in the magnitude of its inflammatory effects [45], hindering the possibility of chronic LPS administration. The experimental design was set to examine the effects of the drugs on LPS-induced inflammation under a unified model system and following a chronic treatment protocol in order to mimic a therapeutically-relevant regimen. Previous studies have used similar experimental designs [46]. In summary, the present study demonstrated that LIT, CBZ, HPL and IMP significantly attenuated LPS-induced hypothermia but differently affected the production of inflammatory mediators in plasma and hypothalamus of LPS-treated rats. The differences in the drugs’ influence on inflammatory mediators do not enable drawing an unequivocal conclusion regarding a possible common mechanism that may contribute to their therapeutic efficacy. Future studies may benefit from utilizing mediator-specific pharmacological interventions which will shed more light on the involvement of inflammatory mediators in the pathophysiology and treatment of mental disorders.

Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this paper.

Funding This study was supported by a grant from the Israel Science Foundation (Grant # 198/12). Authors’ Contribution A.N. conducted most of the in-vivo experiments in rats; contributed a major part to the biochemical analysis of the samples; participated in the analysis of the data and writing of the manuscript. Y.S.G. participated in the in-vivo experiments in rats; contributed to the biochemical analysis of the samples; participated in the analysis of the data and writing of the manuscript. A.N.A. designed the study protocols; coordinated all study procedures including the in-vivo experiments and biochemical analysis of the samples, analysis of the data and writing the manuscript.

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Fig. 6. Effects of psychotropic drugs on nuclear P-p65 levels in hypothalamus of LPS-treated rats. Rats were injected once daily (ip) with LIT (A), CBZ (B), HPL (C), or IMP (D) for 28 days. On day 29, rats were treated with those drugs at 2 h before LPS (1 mg/kg, ip) injection. Rats were sacrificed at ∼2 h post LPS injection immediately after which hypothalamus (HT) was removed and further processed as described in Section 2. Nuclear P-p65 levels in HT were determined by ELISA and are presented relative to their level in the control group, which is expressed as 1. Values are means ± SEM of 8 rats. * P < 0.05 vs. Control; Pˆ < 0.05 vs. LPS.

Acknowledgement The authors would like to acknowledge Mrs. Caroline FaourNmarne for initiating the in-vivo experiments with haloperidol. References [1] J. Licinio, M.L. Wong, The role of inflammatory mediators in the biology of major depression: central nervous system cytokines modulate the biological substrate of depressive symptoms, regulate stress-responsive systems, and contribute to neurotoxicity and neuroprotection, Mol. Psychiatry 4 (1999) 317–327. [2] A.H. Miller, V. Maletic, C.L. Raison, Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression, Biol. Psychiatry 65 (2009) 732–741. [3] J.S. Rao, G.J. Harry, S.I. Rapoport, H.W. Kim, Increased excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from bipolar disorder patients, Mol. Psychiatry 15 (2010) 384–392. [4] A. Modabbernia, S. Taslimi, E. Brietzke, M. Ashrafi, Cytokine alterations in bipolar disorder: a meta-analysis of 30 studies, Biol. Psychiatry 74 (2013) 15–25. [5] S. Potvin, E. Stip, A.A. Sepehry, A. Gendron, R. Bah, E. Kouassi, Inflammatory cytokine alterations in schizophrenia: a systematic quantitative review, Biol. Psychiatry 63 (2008) 801–808.

[6] J.M. Meyer, J.P. McEvoy, V.G. Davis, D.C. Goff, H.A. Nasrallah, S.M. Davis, J.K. Hsiao, M.S. Swartz, T.S. Stroup, J.A. Lieberman, Inflammatory markers in schizophrenia: comparing antipsychotic effects in phase 1 of the clinical antipsychotic trials of intervention effectiveness study, Biol. Psychiatry 66 (2009) 1013–1022. [7] N. Castanon, B.E. Leonard, P.J. Neveu, R. Yirmiya, Effects of antidepressants on cytokine production and actions, Brain Behav. Immun. 16 (2002) 569–574. [8] Y.K. Kim, I.B. Suh, H. Kim, C.S. Han, C.S. Lim, S.H. Choi, J. Licinio, The plasma levels of interleukin-12 in schizophrenia, major depression, and bipolar mania: effects of psychotropic drugs, Mol. Psychiatry 7 (2002) 1107–1114. [9] S.I. Rapoport, M. Basselin, H.W. Kim, J.S. Rao, Bipolar disorder and mechanisms of action of mood stabilizers, Brain Res. Rev. 61 (2009) 185–209. [10] K.S. MacDowell, B. García-Bueno, J.L. Madrigal, M. Parellada, C. Arango, J.A. Micó, J.C. Leza, Risperidone normalizes increased inflammatory parameters and restores anti-inflammatory pathways in a model of neuroinflammation, Int. J. Neuropsychopharmacol. 16 (2013) 121–135. [11] V. Tourjman, É. Kouassi, M.È. Koué, M. Rocchetti, S. Fortin-Fournier, P. Fusar-Poli, S. Potvin, Antipsychotics’ effects on blood levels of cytokines in schizophrenia: a meta-analysis, Schizophr. Res. 151 (2013) 43–47. [12] A. Nassar, A.N. Azab, Effects of lithium on inflammation, ACS. Chem. Neurosci. 5 (2014) 451–458. [13] A. Troib, A.N. Azab, Effects of psychotropic drugs on nuclear factor kappa B, Eur. Rev. Med. Pharmacol. Sci. 19 (2015) 1198–1208. [14] N. Müller, M. Riedel, C. Scheppach, B. Brandstätter, S. Sokullu, K. Krampe, M. Ulmschneider, R.R. Engel, H.J. Möller, M.J. Schwarz, Beneficial antipsychotic

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[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

effects of celecoxib add-on therapy compared to risperidone alone in schizophrenia, Am. J. Psychiatry 159 (2002) 1029–1034. N. Müller, M.J. Schwarz, S. Dehning, A. Douhe, A. Cerovecki, B. Goldstein-Müller, I. Spellmann, G. Hetzel, K. Maino, N. Kleindienst, H.J. Möller, V. Arolt, M. Riedel, The cyclooxygenase-2 inhibitor celecoxib has therapeutic effects in major depression: results of a double-blind randomized, placebo controlled, add-on pilot study to reboxetine, Mol. Psychiatry 11 (2006) 680–684. F.G. Nery, E.S. Monkul, J.P. Hatch, M. Fonseca, G.B. Zunta-Soares, B.N. Frey, C.L. Bowden, J.C. Soares, Celecoxib as an adjunct in the treatment of depressive or mixed episodes of bipolar disorder: a double-blind randomized, placebo-controlled study, Hum. Psychopharmacol. 23 (2008) 87–94. W. Laan, D.E. Grobbee, J.P. Selten, C.J. Heijnen, R.S. Kahn, H. Burger, Adjuvant aspirin therapy reduces symptoms of schizophrenia spectrum disorders: results from a randomized double-blind, placebo-controlled trial, J. Clin. Psychiatry 71 (2010) 520–527. L. Paul, V. Fraifeld, J. Kaplanski, Evidence supporting involvement of leukotrienes in LPS-induced hypothermia in mice, Am. J. Physiol. 276 (1999) R52–58. K.P. Hoeflich, J. Luo, E.A. Rubie, M.S. Tsao, O. Jin, J.R. Woodgett, Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation, Nature 406 (2000) 86–90. A.N. Azab, S. Kobal, M. Rubin, J. Kaplanski, Inhibition of prostaglandins does not reduce the cardiovascular changes during endoxemia in rats, prostaglandins, Leukot. Essen. Fatty Acids 74 (2006) 135–142. J. Kaplanski, A. Nassar, Y. Sharon-Granit, A. Jabareen, S.L. Kobal, A.N. Azab, Lithium attenuates lipopolysaccharide-induced hypothermia in rats, Eur. Rev. Med. Pharmacol. Sci. 18 (2014) 1829–1837. A.N. Azab, S. Kobal, M. Rubin, J. Kaplanski, Effects of nimesulide a selective cyclooxygenase-2 inhibitor, on cardiovascular alterations in endotoxemia, Cardiology 103 (2005) 92–100. S.M. Lucas, N.J. Rothwell, R.M. Gibson, The role of inflammation in CNS injury and disease, Br. J. Pharmacol. 147 (2006) S232–S240. C. Iadecola, J. Anrather, The immunology of stroke: from mechanisms to translation, Nature Med. 17 (2011) 796–808. F.L. Heppner, R.M. Ransohoff, B. Becher, Immune attack: the role of inflammation in Alzheimer disease, Nat. Rev. Neurosci. 16 (2015) 358–372. A. Volterra, J. Meldolesi, Astrocytes, from brain glue to communication elements: the revolution continues, Nat. Rev. Neurosci. 6 (2005) 626–640. N.J. Allen, B.A. Barres, Glia—more than just brain glue, Nature 457 (2009) 675–677. A. Rolls, R. Shechter, M. Schwartz, The bright side of the glial scar in CNS repair, Nat. Rev. Neurosci. 10 (2009) 235–241. W.E. Kaufmann, K.I. Andreasson, P.C. Isakson, P.F. Worley, Cyclooxygenases and the central nervous system, Prostaglandins 54 (1997) 601–624. B. Beutler, E.T. Rietschel, Innate immune sensing and its roots: the story of endotoxin, Nat. Rev. Immunol. 3 (2003) 169–176. A. Oeckinghaus, M.S. Hayden, S. Ghosh, Crosstalk in NF-␬B signaling pathways, Nat. Immunol. 12 (2011) 695–708. A. Isgren, J. Jakobsson, E. Pålsson, C.J. Ekman, A.G. Johansson, C. Sellgren, K. Blennow, H. Zetterberg, M. Landén, Increased cerebrospinal fluid interleukin-8 in bipolar disorder patients associated with lithium and antipsychotic treatment, Brain Behav. Immun. 43 (2015) 198–204.

67

[33] T. Fukumoto, S. Morinobu, Y. Okamoto, A. Kagaya, S. Yamawaki, Chronic lithium treatment increases the expression of brain-derived neurotrophic factor in the rat brain, Psychopharmacology (Berl) 158 (2001) 100–106. [34] H. Alimohamad, L. Sutton, J. Mouyal, N. Rajakumar, W.J. Rushlow, The effects of antipsychotics on beta-catenin, glycogen synthase kinase-3 and dishevelled in the ventral midbrain of rats, J. Neurochem. 95 (2005) 513–525. [35] R.P. Bazinet, J.S. Rao, L. Chang, S.I. Rapoport, H.J. Lee, Chronic carbamazepine decreases the incorporation rate and turnover of arachidonic acid but not docosahexaenoic acid in brain phospholipids of the unanesthetized rat: relevance to bipolar disorder, Biol. Psychiatry 59 (2006) 401–407. [36] H.J. Lee, J.S. Rao, L. Chang, S.I. Rapoport, H.W. Kim, Chronic imipramine but not bupropion increases arachidonic acid signaling in rat brain: is this related to ‘switching’ in bipolar disorder? Mol. Psychiatry 15 (2010) 602–614. [37] H. Sakurai, S. Suzuki, N. Kawasaki, H. Nakano, T. Okazaki, A. Chino, T. Doi, I. Saiki, Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2 TRAF5, and TAK1 signaling pathway, J. Biol. Chem. 278 (2003) 36916–36923. [38] W.L. Smith, D.L. DeWitt, R.M. Garavito, Cyclooxygenases: structural, cellular, and molecular biology, Annu. Rev. Biochem. 69 (2000) 145–182. [39] M. Basselin, H.W. Kim, M. Chen, K. Ma, S.I. Rapoport, R.C. Murphy, S.E. Farias, Lithium modifies brain arachidonic and docosahexaenoic metabolism in rat lipopolysaccharide model of neuroinflammation, J. Lipid. Res. 51 (2010) 1049–1056. [40] C. Millia, F. Nicoletti, A.A. Grasso, F. Patti, D.F. Condorelli, E. Rapisarda, L. Rampello, G. Costa, U. Scapagnini, Effects of dopaminergic drugs on cerebellar prostaglandin concentrations, J. Neurochem. 41 (1983) 1190–1191. [41] C.B. Zhu, R.D. Blakely, W.A. Hewlett, The proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha activate serotonin transporters, Neuropsychopharmacology 31 (2006) 2121–2131. [42] S. Hayley, K. Brebner, S. Lacosta, Z. Merali, H. Anisman, Sensitization to the effects of tumor necrosis factor-alpha: neuroendocrine, central monoamine, and behavioral variations, J. Neurosci. 19 (1999) 5654–5665. [43] C.D. Munhoz, L.B. Lepsch, E.M. Kawamoto, M.B. Malta, S. Lima Lde, M.C. Avellar, R.M. Sapolsky, C. Scavone, Chronic unpredictable stress exacerbates lipopolysaccharide-induced activation of nuclear factor-kappaB in the frontal cortex and hippocampus via glucocorticoid secretion, J. Neurosci. 26 (2006) 3813–3820. [44] P.S. Bloomfield, S. Selvaraj, M. Veronese, G. Rizzo, A. Bertoldo, D.R. Owen, M.A. Bloomfield, I. Bonoldi, N. Kalk, F. Turkheimer, P. McGuire, V. de Paola, O.D. Howes, Microglial activity in people at ultra high risk of psychosis and in schizophrenia: an [(11)C]PBR28 PET brain imaging study, Am. J. Psychiatry 173 (2016) 44–52. [45] E. López-Collazo, C. del Fresno, Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences, Crit. Care 17 (2013) 242. [46] R. Molteni, F. Macchi, C. Zecchillo, M. Dell’agli, E. Colombo, F. Calabrese, G. Guidotti, G. Racagni, M.A. Riva, Modulation of the inflammatory response in rats chronically treated with the antidepressant agomelatine, Eur. Neuropsychopharmacol. 23 (2013) 1645–1655.