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Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like effect of fluoxetine in an animal model of depression
Accepted Manuscript
Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like effect of fluoxetine in an animal model of depression Silvia Alboni , Cristina Benatti , Giacomo Capone , Fabio Tascedda , Nicoletta Brunello PII: DOI: Reference:
S0165-0327(18)30052-1 10.1016/j.jad.2018.04.063 JAD 9718
To appear in:
Journal of Affective Disorders
Received date: Revised date: Accepted date:
11 January 2018 14 March 2018 5 April 2018
Please cite this article as: Silvia Alboni , Cristina Benatti , Giacomo Capone , Fabio Tascedda , Nicoletta Brunello , Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like effect of fluoxetine in an animal model of depression, Journal of Affective Disorders (2018), doi: 10.1016/j.jad.2018.04.063
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ACCEPTED MANUSCRIPT Highlights ASA accelerates the effect of fluoxetine in the CED model after 7 days Fluoxetine plus ASA effects in restoring escape response depends on ASA dose Flurbiprofen or celecoxib are not as effective as ASA associated with fluoxetine The chronic escape deficit model as tool to study NSAIDs effects in depression
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ACCEPTED MANUSCRIPT Title page To be submitted to JOURNAL OF AFFECTIVE DISORDERS Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like
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effect of fluoxetine in an animal model of depression
Silvia Albonia*, Cristina Benattia,b*, Giacomo Caponec, Fabio Tasceddaa,b, Nicoletta Brunelloa,b
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Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena
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(Italy) b
Center for Neuroscience and Neurotechnology University of Modena and Reggio Emilia, Modena (Italy)
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AIFA - Agenzia Italiana del Farmaco - Via del Tritone, 181 - 00187 Roma, Italy
Corresponding author: Dr. Silvia Alboni PhD
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* These authors equally contributed as first author
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Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125, Modena, Italy; E-mail address: [email protected]
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phone: +39 059 2055383 fax: + 39 059 2055376
ABSTRACT Introduction:
Non-steroidal anti-inflammatory drugs (NSAIDs) have been studied as possible adjunctive
therapy in the treatment of depression. However, administering NSAIDs to increase the effectiveness of anti-depressant has yielded inconsistent results.
ACCEPTED MANUSCRIPT Methods: We evaluated the effect of the co-administration of fluoxetine (5 mg/kg) and flurbiprofen (5 mg/kg) or fluoxetine (5 mg/kg) and celecoxib (5 mg/kg) in the chronic escape deficit (CED) model of depression after 7 days of treatment. The co-administration fluoxetine plus acetylsalicylic acid (ASA, 45mg/kg i.p.) was used as a positive control. Moreover, we tested the behavioural effect of different doses (45, 22.5, and 11.25 mg/Kg i.p.) of ASA as potentiating agent of the effect of fluoxetine in the same
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paradigm. Results: Our study showed that only the co-administration of ASA with fluoxetine was able to revert the stress-induced condition of escape deficit after 7 days of treatment, and that the amplitude of the antidepressant-like effect of ASA was dose dependent. In the same experimental conditions, celecoxib with
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fluoxetine only partially resolved the stress-induced impaired behaviour while flurbiprofen/fluoxetine cotreatment was ineffective.
Limitations: Our study is still exploratory, more doses, longer treatment regimens, and different behavioural outcomes must be investigated to draw a clear conclusion.
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Conclusion: Our results further stress the importance of the type and dose when NSAIDs are associated
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with antidepressants to ameliorate a clinical response.
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Keywords: animal model of depression, behaviour, NSAIDs, acetylsalicylic acid, fluoxetine
ACCEPTED MANUSCRIPT 1. Introduction Inflammation and pathogenesis of depression appear to be clinically and mechanistically intertwined (Benatti et al., 2016; Wang et al., 2011a), thus non-steroidal anti-inflammatory drugs (NSAIDs) have been studied as possible adjunctive therapy in the treatment of depression (Baune, 2017; Eyre et al., 2015).
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Results from clinical and preclinical research are mixed: while some support their effectiveness as add-on therapy, others report negative findings or suggest that only a sub-population of depressed patients with elevated inflammation may benefit from treatment with NSAIDs (Köhler et al., 2014; Warner-Schmidt et al., 2011). Lack of a clear conclusion may depend on the methodological heterogeneity, i.e. method of
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depression measure, severity of depression, and presence of underlying medical conditions. Moreover, these studies employed different NSAIDs that are a very heterogeneous group of drug; NSAIDs can have many different effects on peripheral or central inflammation but also off-target effects, depending on the dose (Raison, 2017; Schrör, 2016). The need to find a simple model to study the interaction between
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antidepressants and NSAIDs, and improve their manageability in the clinic is pressing.
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To dig into the effectiveness of anti-inflammatory drugs in modulating antidepressants activity, we choose to investigate the effects of different NSAIDs in the same experimental paradigm in which we
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previously demonstrated that acetylsalicylic acid (ASA) is effective in shortening and potentiating the effect
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al., 2006).
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of fluoxetine: a seven-day treatment in the chronic escape deficit (CED) model of depression (Brunello et
2. Methods:
2.1 Animals:
Male Sprague-Dawley rats (Charles River, Italy) weighing 200-250 g, were group housed in an environment maintained at constant temperature, controlled humidity, ad libitum access to food and water and 12 h inverted light-dark cycle. Experiments were carried out under a red light. Animals were handled and weighed daily. The procedures used were in accordance with European legislation on the use and care
ACCEPTED MANUSCRIPT of laboratory animals (EU directive 2010/63/EU) and were approved by the Ministry of Health and of the local Ethical Committee. All efforts were made to minimize the number of animals used and their suffering. 2.2 Behavioural procedures & pharmacological treatments: The escape deficit was induced as already described and was maintained by repeated exposure to minor unavoidable stress on alternate days (Benatti et al., 2014; Brunello et al., 2006). Two independent
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behavioural experiments were conducted. Animals not exposed to the unavoidable stress (naive), with intact performance on the escape test, were tested as well in both experiments on day 1.
Experiment 1: From day 1, rats developing an escape deficit were randomly divided into groups [F(3;20)=0.382; p=0.767; Fig. 1B] and subjected to the following pharmacological treatments for 7 days (Fig.
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1A): 1) Saline or methocel 0.5% on alternate days (Stress; n=7) 2) Fluoxetine + Flurbiprofen (n=5)
4) Fluoxetine + ASA (n=5)
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3) Fluoxetine + Celecoxib (n=7)
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On day 8, rats from these groups, together with naive animals (n=10), were exposed to the escape test again to assess their reactivity toward an avoidable aversive stimulus.
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Experiment 2: From day 1, rats developing an escape deficit were randomly divided into groups
2A):
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[F(3;50)=1.681; p=0.183; Fig. 2B] and subjected to the following pharmacological treatments for 7 days (Fig.
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1) Saline (Stress; n=24) 2) Fluoxetine + ASA 45 (n=10) 3) Fluoxetine + ASA 22.5 (n=10) 4) Fluoxetine + ASA 11.25 (n=10) On day 8, rats from these groups, together with naive animals (n=19), were exposed to the escape test again to assess their reactivity toward an avoidable aversive stimulus. 2.3 Drugs:
ACCEPTED MANUSCRIPT Fluoxetine HCl (Polichimica s.r.l., Italy), 5 mg/kg, was dissolved in sterile distilled water, and acetylsalicylate of lysine (Sanofi-Synthelabo s.p.a., Italy) in sterile saline at a dose equivalent to 11.25, 22.5 or 45 mg/kg of ASA. Fluoxetine, ASA, and saline were injected via an intraperitoneal route (i.p.; 1 ml/kg). Flurbiprofen (Sigma Aldrich), 5mg/kg, and celecoxib, 5mg/kg, were dissolved in methocel (Fluka) 0.5%, sonicated 30 seconds and administered via intra gastric (i.g.) route (5ml/kg). We also tested higher doses of
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flurbiprofen (50 mg/kg) and celecoxib (20 mg/kg), but this procedure was associated with high mortality rate and severe adverse effects. In particular, rats receiving the higher doses of celecoxib and flurbiprofen showed signs of gastric damage, lethargy, anorexia, hunched posture, dark stools, and in flurbiprofen/fluoxetine receiving animals nose bleeding was also present given that flurbiprofen decreases
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platelet count, this could be due to an antiaggregant effect. All sick animals that did not improve with supportive care and all moribund animals were euthanized for ethical reasons and not tested for escape ability. We cannot exclude that associating fluoxetine with higher doses of either celecoxib or flurbiprofen in our conditions resulted in unwanted, possibly lethal, side effects as already reported in rats (Takeuchi et
2.4 Statistical analyses:
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al., 2011).
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Behavioural results were analysed with an analysis of variance (ANOVA) followed by Tukey post-hoc
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test (with p<0.05 significance level) using SPSS for Windows® v.23.
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3. Results:
3.1 Experiment 1: only ASA, but not flurbiprofen or celecoxib when co-administered with fluoxetine induces an antidepressant-like effect in the CED model after a 7-day treatment
One-way ANOVA revealed a main effect of the pharmacological treatments on the stress-induced escape deficit [F(4; 29)=6.887; p<0.001; Fig. 1C]. Only the co-administration of fluoxetine plus aspirin (45 mg/kg) was able to completely restore the escape competence of rats in the CED model of depression, the mean of escapes of the fluoxetine plus ASA group was not different from naive animals (p>0.05) while being significantly higher than the stress group (p<0.05). On the other hand, the stress-induced behaviour
ACCEPTED MANUSCRIPT was not reverted in the group receiving fluoxetine plus flurbiprofen, which scored significantly less than naive animals (p<0.05) while not being statistically different from their saline-receiving stressed counterparts. The mean of escapes of animals receiving celecoxib plus fluoxetine was not different from other groups (p>0.05; Fig. 1C). We tested higher doses of flurbiprofen (50 mg/kg) and celecoxib (20 mg/kg), but this procedure was associated with high mortality rate (80% and 25% respectively). We cannot exclude
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that associating fluoxetine with higher doses of either celecoxib or flurbiprofen in our conditions resulted in unwanted, possibly lethal, side effects as already reported in rats (Takeuchi et al., 2011) .
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3.2 Experiment 2: the amplitude of the augmenting effect of ASA on fluoxetine is dose dependent in the CED model
One-way ANOVA revealed a main effect of the doses of ASA, ranging from 45 to 11.25 mg/kg, in affecting depressive-like behaviour [F(4; 68)=33.076; p<0.0001; Fig. 2C]. As already demonstrated, a week-
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long co-administration of ASA at the highest dose tested (45 mg/kg) with fluoxetine completely restored
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the escape competence of stressed animals: the number of escapes of the fluoxetine plus ASA 45mg/kg group was not different from naive animals (p>0.05) and was significantly higher than the stress group
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(p<0.0001) (Brunello et al., 2006). When decreasing the dose of ASA (22.5 and 11.25 mg/kg) the number of escapes decreased as well: administering for 7 days fluoxetine with lower doses of ASA resulted in a
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performance in the escape test significantly different from both naive (p<0.01) and stress groups (p<0.05).
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The escape competence of the fluoxetine plus ASA 11.25 mg/kg group was significantly lower than their counterparts receiving fluoxetine with 45mg/kg of ASA (p<0.01) (Fig. 2C). The percentage of the antidepressant response was about 90%, 60% and 40% for animals receiving fluoxetine plus ASA at the dose of 45, 22.5 or 11.25 mg/kg, respectively. We previously demonstrated that ASA at the highest dose tested here and fluoxetine (5 mg/kg) do not possess antidepressant properties in the CED model after one week of treatment when administered alone (Brunello et al., 2006).
4. Discussion:
ACCEPTED MANUSCRIPT In this study we combined fluoxetine with three NSAIDs possessing different selectivity for cyclooxygenase (COX): aspirin is an irreversible COX inhibitor, more potent in inhibiting COX-1 than COX-2, flurbiprofen is a non-selective reversible COX inhibitor, while celecoxib is a selective COX-2 inhibitor (Warner et al., 1999). We demonstrated that neither celecoxib nor flurbiprofen were as effective as ASA in potentiating the effect of fluoxetine in the CED animal model of depression. However, whereas a seven-day
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treatment with celecoxib plus fluoxetine partially restored the ability to avoid a noxious stimulus, the combined treatment fluoxetine plus flurbiprofen did not. Moreover, the potentiating effect of ASA on fluoxetine in the CED model was dose dependent.
Although no randomized double-blind placebo-controlled study exists on ibuprofen as add-on
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treatment, in a longitudinal population based cohort study the concomitant use of ibuprofen and SSRI reduced the risk of psychiatric contact in secondary health care system (Köhler et al., 2015). In contrast, in mice Warner-Schmidt and co-workers demonstrated that a 14-day co-treatment with ibuprofen plus citalopram blocked the behavioural and molecular effects elicited by citalopram alone (Warner-Schmidt et
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al., 2011). Here we demonstrated that also in the CED model flurbiprofen, a more potent arylpropionic acid
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derivative than ibuprofen (Schrör, 2016), combined with fluoxetine was not able to revert the stressinduced behaviour.
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Celecoxib may hasten the onset and increase response in depressive disorders: clinical trials in depressed patients suggest a benefit for short-term celecoxib in monotherapy or in combination with
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antidepressants (Müller et al., 2006), while in a population-based cohort study of SSRI-users COX-2
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selective inhibitors showed an increased mortality risk and no adjunctive antidepressant effects (Köhler et al., 2014). In our model, treatment with celecoxib plus fluoxetine for 7 days was able to partially revert the stress-induced escape deficit, thus, celecoxib may be partially effective as adjunctive agent, or possibly longer treatments or different drug dosage are necessary to fully appreciate its antidepressant-like effect (Guo et al., 2009; Santiago et al., 2014). Clinical studies exploring the effect of ASA as an adjunctive drug in depression are few and with a small sample size, limiting the generalization of any conclusion. Indeed, in an open-label study, we demonstrated that a 28-day treatment with SSRIs plus ASA at low dose in non-responder patients (n= 26) is
ACCEPTED MANUSCRIPT associated with a response rate of 52.4% (Mendlewicz et al., 2006). Unlike us, in another open-label study on depressed inpatients (n=40), Zdanowicz and colleagues found that the co-administration of ASA with a noradrenergic agent is more effective than a SSRI alone (escitalopram), whereas ASA plus escitalopram is not more effective (Zdanowicz et al., 2017). The lack of effect on depressive state between SSRI (fluoxetine) treated group and fluoxetine plus ASA treated group was also confirmed by Galecki after 3 months of
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treatment in patients (n=50) with the first depressive episode (Gałecki et al., 2009). Finally, it has also been suggested that co-administration of ASA with citalopram (n= 10) may be associated with the occurrence of important side effects (Baune, 2017). This later finding is in disagreement with the conclusion of a study population (n= 123,351; SSRIs treated subjects) that fails to demonstrate a negative impact on safety while
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highlighting reduced risk of psychiatric contact (Köhler et al., 2015).
In a preclinical study ASA, at an approximate dose of 210 mg/kg, for 5-7 days blocked the acute effect of citalopram in the TST (Warner-Schmidt et al., 2011), while a three-week treatment with aspirin 20 mg/kg was able to reverse the stress-induced behavioural alterations in rats exposed to a CUMS resistant to
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a 4-week exposure to fluoxetine (Wang et al., 2011b). The pharmacological effects of ASA are highly dose
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dependent: a dose of 200 mg/kg is more analgesic/anti-inflammatory, while the doses employed by us and Wang are more on the low-medium range (anti-platelet effect) (Schrör, 2016).
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ASA targets a wide plethora of biological systems. For example, like other NSAIDs, it can modulate the production of pro-inflammatory cytokines and oxidative and nitrosative stress pathway, but, at low-to-
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medium concentrations, can also transacetylate numerous macromolecules, including enzymes, DNA,
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plasmatic protein, and transcription factors (Berk et al., 2013; Schrör, 2016). Moreover, Di Matteo and colleagues demonstrated that ASA, but not the COX-2 specific inhibitor meloxicam, is able to prevent neurotoxin-induced degeneration of striatal dopaminergic neurons likely through COX-2- independent mechanisms (Di Matteo et al., 2006). It has been demonstrated that stress-induced behavioural impairments in the CED model are associated to reduced dopaminergic tone and blunted dopaminergic response to positive stimuli in the nucleus accumbens shell (Scheggi et al., 2016). Pharmacological treatments that stabilize or regulate the dopaminergic system in specific brain area have been effective in reinstating stress- impaired behaviour (Scheggi et al., 2018). Here we confirmed that simply by adding a
ACCEPTED MANUSCRIPT low dose of ASA to fluoxetine treatment we completely restored the depressive-like behaviour, suggesting that off-target effects may underlie the antidepressant-like effect. Limitations Herein we operate a 7-day treatment with three different NSAIDs in the CED to achieve our aim, however to provide further useful information translatable to the clinic, more doses and longer treatment regimens
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must be evaluated, and the molecular correlate subtending the behavioural outcome must be investigated. Conclusion
These data, albeit preliminary, confirmed that neither all NSAIDs, nor all doses, may be equally useful in the treatment of depression. The dose and the class of the inflammatory medications employed
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are key factors that can strongly impact the molecular mechanisms underlying the effects of the combination of NSAIDs with antidepressants. Whether anti-inflammatory drugs may potentiate antidepressant efficacy or not, and at which dose and time may depend on COX-dependent antiinflammatory effects as well as on off-target effects (Raison, 2017; Tegeder et al., 2001).
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Our model may represent a useful tool to dissect the role played by these molecular mechanisms in
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the interplay between anti-inflammatory drugs and antidepressants (Benatti et al., 2017). Improvement of evidence based knowledge of the use of NSAIDs as adjunctive treatment in depression, increase of
Contributions:
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therapeutic efficacy and avoidance of uncritical practice remain a pressing clinical challenge.
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AS, BC, and CG performed behavioural experiments. BC analysed the behavioural data. AS, and BC drafted
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the article. CG, TF and BN provided feedback on the manuscript. All authors have reviewed the manuscript and approved the final version submitted for publication.
Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest: none.
ACCEPTED MANUSCRIPT Acknowledgements: none
Disclaimer The views expressed in this work by Dr Giacomo Capone are personal and may not be understood or
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quoted as being made on behalf of or reflecting the position of AIFA.
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Figure legends:
1. Experiment 1: a 7-day treatment with fluoxetine induces an antidepressant-like effect in the CED model only when co-administered with acetylsalicylic acid (ASA), but not with flurbiprofen or celecoxib
ACCEPTED MANUSCRIPT (A) Timeline of the chronic escape deficit (CED) protocol and pharmacological treatments. (B) Induction of the escape deficit at day 1. (C) Results of the escape test at day 8. Scores are expressed as the mean number of escapes ± S.E.M. out of 30 trials. Comparisons were made by one way ANOVA (### p<0.0001; ##p<0.01, # p<0.05) followed by Tukey post-hoc test ***p<0.0001, **p<0.01, *p<0.05 vs naive; §p<0.05 vs
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stress.
2. Experiment 2: the accelerating action of acetylsalicylic acid (ASA) on the antidepressant-like effects of fluoxetine in the CED model is dose dependent
(A) Timeline of the chronic escape deficit (CED) protocol and pharmacological treatments. (B) Induction of the escape deficit at day 1. (C) Results of the escape test at day 8. Scores are expressed as the mean number of escapes ± S.E.M. out of 30 trials. Comparisons were made by one way ANOVA (### p<0.0001; #
ACCEPTED MANUSCRIPT p<0.05) followed by Tukey post-hoc test ***p<0.0001, **p<0.01, *p<0.05 vs naive; §§§p<0.0001, §p<0.05
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vs stress; °°p<0.01 vs fluoxetine/ASA 45mg/kg.
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Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like effect of fluoxetine in an animal model of depression Silvia Alboni , Cristina Benatti , Giacomo Capone , Fabio Tascedda , Nicoletta Brunello PII: DOI: Reference:
S0165-0327(18)30052-1 10.1016/j.jad.2018.04.063 JAD 9718
To appear in:
Journal of Affective Disorders
Received date: Revised date: Accepted date:
11 January 2018 14 March 2018 5 April 2018
Please cite this article as: Silvia Alboni , Cristina Benatti , Giacomo Capone , Fabio Tascedda , Nicoletta Brunello , Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like effect of fluoxetine in an animal model of depression, Journal of Affective Disorders (2018), doi: 10.1016/j.jad.2018.04.063
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ACCEPTED MANUSCRIPT Highlights ASA accelerates the effect of fluoxetine in the CED model after 7 days Fluoxetine plus ASA effects in restoring escape response depends on ASA dose Flurbiprofen or celecoxib are not as effective as ASA associated with fluoxetine The chronic escape deficit model as tool to study NSAIDs effects in depression
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ACCEPTED MANUSCRIPT Title page To be submitted to JOURNAL OF AFFECTIVE DISORDERS Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like
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effect of fluoxetine in an animal model of depression
Silvia Albonia*, Cristina Benattia,b*, Giacomo Caponec, Fabio Tasceddaa,b, Nicoletta Brunelloa,b
a
Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena
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(Italy) b
Center for Neuroscience and Neurotechnology University of Modena and Reggio Emilia, Modena (Italy)
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AIFA - Agenzia Italiana del Farmaco - Via del Tritone, 181 - 00187 Roma, Italy
Corresponding author: Dr. Silvia Alboni PhD
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* These authors equally contributed as first author
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Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125, Modena, Italy; E-mail address: [email protected]
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phone: +39 059 2055383 fax: + 39 059 2055376
ABSTRACT Introduction:
Non-steroidal anti-inflammatory drugs (NSAIDs) have been studied as possible adjunctive
therapy in the treatment of depression. However, administering NSAIDs to increase the effectiveness of anti-depressant has yielded inconsistent results.
ACCEPTED MANUSCRIPT Methods: We evaluated the effect of the co-administration of fluoxetine (5 mg/kg) and flurbiprofen (5 mg/kg) or fluoxetine (5 mg/kg) and celecoxib (5 mg/kg) in the chronic escape deficit (CED) model of depression after 7 days of treatment. The co-administration fluoxetine plus acetylsalicylic acid (ASA, 45mg/kg i.p.) was used as a positive control. Moreover, we tested the behavioural effect of different doses (45, 22.5, and 11.25 mg/Kg i.p.) of ASA as potentiating agent of the effect of fluoxetine in the same
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paradigm. Results: Our study showed that only the co-administration of ASA with fluoxetine was able to revert the stress-induced condition of escape deficit after 7 days of treatment, and that the amplitude of the antidepressant-like effect of ASA was dose dependent. In the same experimental conditions, celecoxib with
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fluoxetine only partially resolved the stress-induced impaired behaviour while flurbiprofen/fluoxetine cotreatment was ineffective.
Limitations: Our study is still exploratory, more doses, longer treatment regimens, and different behavioural outcomes must be investigated to draw a clear conclusion.
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Conclusion: Our results further stress the importance of the type and dose when NSAIDs are associated
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with antidepressants to ameliorate a clinical response.
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Keywords: animal model of depression, behaviour, NSAIDs, acetylsalicylic acid, fluoxetine
ACCEPTED MANUSCRIPT 1. Introduction Inflammation and pathogenesis of depression appear to be clinically and mechanistically intertwined (Benatti et al., 2016; Wang et al., 2011a), thus non-steroidal anti-inflammatory drugs (NSAIDs) have been studied as possible adjunctive therapy in the treatment of depression (Baune, 2017; Eyre et al., 2015).
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Results from clinical and preclinical research are mixed: while some support their effectiveness as add-on therapy, others report negative findings or suggest that only a sub-population of depressed patients with elevated inflammation may benefit from treatment with NSAIDs (Köhler et al., 2014; Warner-Schmidt et al., 2011). Lack of a clear conclusion may depend on the methodological heterogeneity, i.e. method of
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depression measure, severity of depression, and presence of underlying medical conditions. Moreover, these studies employed different NSAIDs that are a very heterogeneous group of drug; NSAIDs can have many different effects on peripheral or central inflammation but also off-target effects, depending on the dose (Raison, 2017; Schrör, 2016). The need to find a simple model to study the interaction between
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antidepressants and NSAIDs, and improve their manageability in the clinic is pressing.
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To dig into the effectiveness of anti-inflammatory drugs in modulating antidepressants activity, we choose to investigate the effects of different NSAIDs in the same experimental paradigm in which we
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previously demonstrated that acetylsalicylic acid (ASA) is effective in shortening and potentiating the effect
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al., 2006).
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of fluoxetine: a seven-day treatment in the chronic escape deficit (CED) model of depression (Brunello et
2. Methods:
2.1 Animals:
Male Sprague-Dawley rats (Charles River, Italy) weighing 200-250 g, were group housed in an environment maintained at constant temperature, controlled humidity, ad libitum access to food and water and 12 h inverted light-dark cycle. Experiments were carried out under a red light. Animals were handled and weighed daily. The procedures used were in accordance with European legislation on the use and care
ACCEPTED MANUSCRIPT of laboratory animals (EU directive 2010/63/EU) and were approved by the Ministry of Health and of the local Ethical Committee. All efforts were made to minimize the number of animals used and their suffering. 2.2 Behavioural procedures & pharmacological treatments: The escape deficit was induced as already described and was maintained by repeated exposure to minor unavoidable stress on alternate days (Benatti et al., 2014; Brunello et al., 2006). Two independent
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behavioural experiments were conducted. Animals not exposed to the unavoidable stress (naive), with intact performance on the escape test, were tested as well in both experiments on day 1.
Experiment 1: From day 1, rats developing an escape deficit were randomly divided into groups [F(3;20)=0.382; p=0.767; Fig. 1B] and subjected to the following pharmacological treatments for 7 days (Fig.
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1A): 1) Saline or methocel 0.5% on alternate days (Stress; n=7) 2) Fluoxetine + Flurbiprofen (n=5)
4) Fluoxetine + ASA (n=5)
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3) Fluoxetine + Celecoxib (n=7)
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On day 8, rats from these groups, together with naive animals (n=10), were exposed to the escape test again to assess their reactivity toward an avoidable aversive stimulus.
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Experiment 2: From day 1, rats developing an escape deficit were randomly divided into groups
2A):
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[F(3;50)=1.681; p=0.183; Fig. 2B] and subjected to the following pharmacological treatments for 7 days (Fig.
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1) Saline (Stress; n=24) 2) Fluoxetine + ASA 45 (n=10) 3) Fluoxetine + ASA 22.5 (n=10) 4) Fluoxetine + ASA 11.25 (n=10) On day 8, rats from these groups, together with naive animals (n=19), were exposed to the escape test again to assess their reactivity toward an avoidable aversive stimulus. 2.3 Drugs:
ACCEPTED MANUSCRIPT Fluoxetine HCl (Polichimica s.r.l., Italy), 5 mg/kg, was dissolved in sterile distilled water, and acetylsalicylate of lysine (Sanofi-Synthelabo s.p.a., Italy) in sterile saline at a dose equivalent to 11.25, 22.5 or 45 mg/kg of ASA. Fluoxetine, ASA, and saline were injected via an intraperitoneal route (i.p.; 1 ml/kg). Flurbiprofen (Sigma Aldrich), 5mg/kg, and celecoxib, 5mg/kg, were dissolved in methocel (Fluka) 0.5%, sonicated 30 seconds and administered via intra gastric (i.g.) route (5ml/kg). We also tested higher doses of
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flurbiprofen (50 mg/kg) and celecoxib (20 mg/kg), but this procedure was associated with high mortality rate and severe adverse effects. In particular, rats receiving the higher doses of celecoxib and flurbiprofen showed signs of gastric damage, lethargy, anorexia, hunched posture, dark stools, and in flurbiprofen/fluoxetine receiving animals nose bleeding was also present given that flurbiprofen decreases
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platelet count, this could be due to an antiaggregant effect. All sick animals that did not improve with supportive care and all moribund animals were euthanized for ethical reasons and not tested for escape ability. We cannot exclude that associating fluoxetine with higher doses of either celecoxib or flurbiprofen in our conditions resulted in unwanted, possibly lethal, side effects as already reported in rats (Takeuchi et
2.4 Statistical analyses:
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al., 2011).
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Behavioural results were analysed with an analysis of variance (ANOVA) followed by Tukey post-hoc
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test (with p<0.05 significance level) using SPSS for Windows® v.23.
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3. Results:
3.1 Experiment 1: only ASA, but not flurbiprofen or celecoxib when co-administered with fluoxetine induces an antidepressant-like effect in the CED model after a 7-day treatment
One-way ANOVA revealed a main effect of the pharmacological treatments on the stress-induced escape deficit [F(4; 29)=6.887; p<0.001; Fig. 1C]. Only the co-administration of fluoxetine plus aspirin (45 mg/kg) was able to completely restore the escape competence of rats in the CED model of depression, the mean of escapes of the fluoxetine plus ASA group was not different from naive animals (p>0.05) while being significantly higher than the stress group (p<0.05). On the other hand, the stress-induced behaviour
ACCEPTED MANUSCRIPT was not reverted in the group receiving fluoxetine plus flurbiprofen, which scored significantly less than naive animals (p<0.05) while not being statistically different from their saline-receiving stressed counterparts. The mean of escapes of animals receiving celecoxib plus fluoxetine was not different from other groups (p>0.05; Fig. 1C). We tested higher doses of flurbiprofen (50 mg/kg) and celecoxib (20 mg/kg), but this procedure was associated with high mortality rate (80% and 25% respectively). We cannot exclude
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that associating fluoxetine with higher doses of either celecoxib or flurbiprofen in our conditions resulted in unwanted, possibly lethal, side effects as already reported in rats (Takeuchi et al., 2011) .
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3.2 Experiment 2: the amplitude of the augmenting effect of ASA on fluoxetine is dose dependent in the CED model
One-way ANOVA revealed a main effect of the doses of ASA, ranging from 45 to 11.25 mg/kg, in affecting depressive-like behaviour [F(4; 68)=33.076; p<0.0001; Fig. 2C]. As already demonstrated, a week-
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long co-administration of ASA at the highest dose tested (45 mg/kg) with fluoxetine completely restored
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the escape competence of stressed animals: the number of escapes of the fluoxetine plus ASA 45mg/kg group was not different from naive animals (p>0.05) and was significantly higher than the stress group
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(p<0.0001) (Brunello et al., 2006). When decreasing the dose of ASA (22.5 and 11.25 mg/kg) the number of escapes decreased as well: administering for 7 days fluoxetine with lower doses of ASA resulted in a
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performance in the escape test significantly different from both naive (p<0.01) and stress groups (p<0.05).
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The escape competence of the fluoxetine plus ASA 11.25 mg/kg group was significantly lower than their counterparts receiving fluoxetine with 45mg/kg of ASA (p<0.01) (Fig. 2C). The percentage of the antidepressant response was about 90%, 60% and 40% for animals receiving fluoxetine plus ASA at the dose of 45, 22.5 or 11.25 mg/kg, respectively. We previously demonstrated that ASA at the highest dose tested here and fluoxetine (5 mg/kg) do not possess antidepressant properties in the CED model after one week of treatment when administered alone (Brunello et al., 2006).
4. Discussion:
ACCEPTED MANUSCRIPT In this study we combined fluoxetine with three NSAIDs possessing different selectivity for cyclooxygenase (COX): aspirin is an irreversible COX inhibitor, more potent in inhibiting COX-1 than COX-2, flurbiprofen is a non-selective reversible COX inhibitor, while celecoxib is a selective COX-2 inhibitor (Warner et al., 1999). We demonstrated that neither celecoxib nor flurbiprofen were as effective as ASA in potentiating the effect of fluoxetine in the CED animal model of depression. However, whereas a seven-day
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treatment with celecoxib plus fluoxetine partially restored the ability to avoid a noxious stimulus, the combined treatment fluoxetine plus flurbiprofen did not. Moreover, the potentiating effect of ASA on fluoxetine in the CED model was dose dependent.
Although no randomized double-blind placebo-controlled study exists on ibuprofen as add-on
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treatment, in a longitudinal population based cohort study the concomitant use of ibuprofen and SSRI reduced the risk of psychiatric contact in secondary health care system (Köhler et al., 2015). In contrast, in mice Warner-Schmidt and co-workers demonstrated that a 14-day co-treatment with ibuprofen plus citalopram blocked the behavioural and molecular effects elicited by citalopram alone (Warner-Schmidt et
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al., 2011). Here we demonstrated that also in the CED model flurbiprofen, a more potent arylpropionic acid
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derivative than ibuprofen (Schrör, 2016), combined with fluoxetine was not able to revert the stressinduced behaviour.
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Celecoxib may hasten the onset and increase response in depressive disorders: clinical trials in depressed patients suggest a benefit for short-term celecoxib in monotherapy or in combination with
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antidepressants (Müller et al., 2006), while in a population-based cohort study of SSRI-users COX-2
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selective inhibitors showed an increased mortality risk and no adjunctive antidepressant effects (Köhler et al., 2014). In our model, treatment with celecoxib plus fluoxetine for 7 days was able to partially revert the stress-induced escape deficit, thus, celecoxib may be partially effective as adjunctive agent, or possibly longer treatments or different drug dosage are necessary to fully appreciate its antidepressant-like effect (Guo et al., 2009; Santiago et al., 2014). Clinical studies exploring the effect of ASA as an adjunctive drug in depression are few and with a small sample size, limiting the generalization of any conclusion. Indeed, in an open-label study, we demonstrated that a 28-day treatment with SSRIs plus ASA at low dose in non-responder patients (n= 26) is
ACCEPTED MANUSCRIPT associated with a response rate of 52.4% (Mendlewicz et al., 2006). Unlike us, in another open-label study on depressed inpatients (n=40), Zdanowicz and colleagues found that the co-administration of ASA with a noradrenergic agent is more effective than a SSRI alone (escitalopram), whereas ASA plus escitalopram is not more effective (Zdanowicz et al., 2017). The lack of effect on depressive state between SSRI (fluoxetine) treated group and fluoxetine plus ASA treated group was also confirmed by Galecki after 3 months of
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treatment in patients (n=50) with the first depressive episode (Gałecki et al., 2009). Finally, it has also been suggested that co-administration of ASA with citalopram (n= 10) may be associated with the occurrence of important side effects (Baune, 2017). This later finding is in disagreement with the conclusion of a study population (n= 123,351; SSRIs treated subjects) that fails to demonstrate a negative impact on safety while
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highlighting reduced risk of psychiatric contact (Köhler et al., 2015).
In a preclinical study ASA, at an approximate dose of 210 mg/kg, for 5-7 days blocked the acute effect of citalopram in the TST (Warner-Schmidt et al., 2011), while a three-week treatment with aspirin 20 mg/kg was able to reverse the stress-induced behavioural alterations in rats exposed to a CUMS resistant to
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a 4-week exposure to fluoxetine (Wang et al., 2011b). The pharmacological effects of ASA are highly dose
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dependent: a dose of 200 mg/kg is more analgesic/anti-inflammatory, while the doses employed by us and Wang are more on the low-medium range (anti-platelet effect) (Schrör, 2016).
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ASA targets a wide plethora of biological systems. For example, like other NSAIDs, it can modulate the production of pro-inflammatory cytokines and oxidative and nitrosative stress pathway, but, at low-to-
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medium concentrations, can also transacetylate numerous macromolecules, including enzymes, DNA,
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plasmatic protein, and transcription factors (Berk et al., 2013; Schrör, 2016). Moreover, Di Matteo and colleagues demonstrated that ASA, but not the COX-2 specific inhibitor meloxicam, is able to prevent neurotoxin-induced degeneration of striatal dopaminergic neurons likely through COX-2- independent mechanisms (Di Matteo et al., 2006). It has been demonstrated that stress-induced behavioural impairments in the CED model are associated to reduced dopaminergic tone and blunted dopaminergic response to positive stimuli in the nucleus accumbens shell (Scheggi et al., 2016). Pharmacological treatments that stabilize or regulate the dopaminergic system in specific brain area have been effective in reinstating stress- impaired behaviour (Scheggi et al., 2018). Here we confirmed that simply by adding a
ACCEPTED MANUSCRIPT low dose of ASA to fluoxetine treatment we completely restored the depressive-like behaviour, suggesting that off-target effects may underlie the antidepressant-like effect. Limitations Herein we operate a 7-day treatment with three different NSAIDs in the CED to achieve our aim, however to provide further useful information translatable to the clinic, more doses and longer treatment regimens
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must be evaluated, and the molecular correlate subtending the behavioural outcome must be investigated. Conclusion
These data, albeit preliminary, confirmed that neither all NSAIDs, nor all doses, may be equally useful in the treatment of depression. The dose and the class of the inflammatory medications employed
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are key factors that can strongly impact the molecular mechanisms underlying the effects of the combination of NSAIDs with antidepressants. Whether anti-inflammatory drugs may potentiate antidepressant efficacy or not, and at which dose and time may depend on COX-dependent antiinflammatory effects as well as on off-target effects (Raison, 2017; Tegeder et al., 2001).
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Our model may represent a useful tool to dissect the role played by these molecular mechanisms in
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the interplay between anti-inflammatory drugs and antidepressants (Benatti et al., 2017). Improvement of evidence based knowledge of the use of NSAIDs as adjunctive treatment in depression, increase of
Contributions:
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therapeutic efficacy and avoidance of uncritical practice remain a pressing clinical challenge.
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AS, BC, and CG performed behavioural experiments. BC analysed the behavioural data. AS, and BC drafted
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the article. CG, TF and BN provided feedback on the manuscript. All authors have reviewed the manuscript and approved the final version submitted for publication.
Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest: none.
ACCEPTED MANUSCRIPT Acknowledgements: none
Disclaimer The views expressed in this work by Dr Giacomo Capone are personal and may not be understood or
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quoted as being made on behalf of or reflecting the position of AIFA.
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Figure legends:
1. Experiment 1: a 7-day treatment with fluoxetine induces an antidepressant-like effect in the CED model only when co-administered with acetylsalicylic acid (ASA), but not with flurbiprofen or celecoxib
ACCEPTED MANUSCRIPT (A) Timeline of the chronic escape deficit (CED) protocol and pharmacological treatments. (B) Induction of the escape deficit at day 1. (C) Results of the escape test at day 8. Scores are expressed as the mean number of escapes ± S.E.M. out of 30 trials. Comparisons were made by one way ANOVA (### p<0.0001; ##p<0.01, # p<0.05) followed by Tukey post-hoc test ***p<0.0001, **p<0.01, *p<0.05 vs naive; §p<0.05 vs
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stress.
2. Experiment 2: the accelerating action of acetylsalicylic acid (ASA) on the antidepressant-like effects of fluoxetine in the CED model is dose dependent
(A) Timeline of the chronic escape deficit (CED) protocol and pharmacological treatments. (B) Induction of the escape deficit at day 1. (C) Results of the escape test at day 8. Scores are expressed as the mean number of escapes ± S.E.M. out of 30 trials. Comparisons were made by one way ANOVA (### p<0.0001; #
ACCEPTED MANUSCRIPT p<0.05) followed by Tukey post-hoc test ***p<0.0001, **p<0.01, *p<0.05 vs naive; §§§p<0.0001, §p<0.05
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vs stress; °°p<0.01 vs fluoxetine/ASA 45mg/kg.
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