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Contents lists available at ScienceDirect
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Research report
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Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats
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Leila Arbabi a , Mohamad. Taufik Hidayat Baharuldin a,∗ , Mohamad Aris Mohamad Moklas a , Sharida Fakurazi a , Sani Ismaila Muhammad b a Human Anatomy Laboratory, Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia b Laboratory of Molecular Biomedicine, Institute of Bioscience, UniversitiPutra Malaysia, Serdang 43400, Selangor, Malaysia
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h i g h l i g h t s • • • •
The first evaluation of antidepressant effect of omega-3 on postpartum depression model of rat. The first measurement of pro-inflammatory cytokines in postpartum-induced rats. The first evaluation of impact of omega-3 on pro-inflammatory cytokines in postpartum-induced rats. The first evaluation of effect of omega-3 on corticosterone levels in postpartum-induced rats.
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Article history: Received 12 April 2014 Received in revised form 14 May 2014 Accepted 16 May 2014 Available online xxx
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Keywords: Postpartum depression Omega-3 fatty acid Hormone simulated pregnancy Forced swimming test Pro-inflammatory cytokine Corticosterone
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1. Introduction
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Postpartum depression (PPD) is a psychiatric disorder that occurs in 10–15% of childbearing women. It is hypothesized that omega-3 fatty acids, which are components of fish oil, may attenuate depression symptoms. In order to examine this hypothesis, the animal model of postpartum depression was established in the present study. Ovariectomized female rats underwent hormone-simulated pregnancy (HSP) regimen and received progesterone and estradiol benzoate or vehicle for 23 days, mimicking the actual rat’s pregnancy. The days after hormone termination were considered as the postpartum period. Forced feeding of menhaden fish oil, as a source of omega-3, with three doses of 1, 3, and 9 g/kg/d, fluoxetine 15 mg/kg/d, and distilled water 2 ml/d per rat started in five postpartum-induced and one vehicle group on postpartum day 1 and continued for 15 consecutive days. On postpartum day 15, all groups were tested in the forced swimming test (FST) and open field test (OFT), followed by a biochemical assay. Results showed that the postpartum-induced rats not treated with menhaden fish oil, exhibited an increase in immobility time seen in FST, hippocampal concentration of corticosterone and plasmatic level of corticosterone, and pro-inflammatory cytokines. These depression-related effects were attenuated by supplementation of menhaden fish oil with doses of 3 and 9 g/kg. Moreover, results of rats supplemented with menhaden fish oil were comparable to rats treated with the clinically effective antidepressant, fluoxetine. Taken together, these results suggest that menhaden fish oil, rich in omega-3, exerts beneficial effect on postpartum depression and decreases the biomarkers related to depression such as corticosterone and pro-inflammatory cytokines. © 2014 Published by Elsevier B.V.
Postpartum depression (PPD) is a psychiatric disorder, defined as a subtype of major depressive disorder (MDD). It has been reported that 10–15% women suffer from PPD following childbirth [1]. Although the underlying etiology of postpartum depression
∗ Corresponding author. Tel.: +60389472356; fax: +60389472356. E-mail address: taufi[email protected] (Mohamad.T.H. Baharuldin).
remains unknown, the abrupt changes in reproductive hormones that women undergo in post-delivery period may cause postpartum depression [2]. In addition to the role of estrogen and progesterone, some other biological factors such as hypothalamic–pituitary–adrenal (HPA) axis hormones, altered immune system and cytokines, and altered fatty acid have been proposed to play a role in causing postpartum depression [3]. Not diagnosing and treating postpartum depression has significant adverse effects on depressed individuals and their families [4]. Due to side effects of medical treatment on breastfed infants and the
http://dx.doi.org/10.1016/j.bbr.2014.05.036 0166-4328/© 2014 Published by Elsevier B.V.
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negative effects of untreated depression, mothers face a dilemma about how to deal with depression symptoms. Therefore, another alternative treatment should be considered to lessen depressive symptoms with lower side effects for both mother and baby. The relationship between omega-3 and depression has been reported in previous studies. There are many reasons indicating an inverse relationship between omega-3 fatty acids and depression. This link has been observed in both observational and experimental research [5,6]. Fish and fish oil, which are rich in omega-3 fatty acids, are the best dietary sources of omega-3 [7]. Although it is believed that omega-3 plays a vital role in the body, and in particular, in the nervous system and mood disorder, the underlying mechanism is poorly understood. While numerous studies have been carried out to evaluate the effects of omega-3 on depression and other mood disorders, the researches carried out to determine the effects of omega-3 on postpartum depression are few and the results are inconsistent. Due to the contradictions among the results of previous researches in this regard, the present study was performed to clarify these discrepancies by investigating the effects of menhaden fish oil (rich in omega-3) on postpartum-induced rats. In order to study the antidepressant-like effects of omega-3 on postpartum-induced rats, a hormone manipulation was used to establish the animal model of postpartum depression, and then these PPD-induced rats were supplemented with menhaden fish oil. The antidepressantlike effect of omega-3 was evaluated using a standard behavioral test (FST) and also the effect of omega-3 on HPA axis was assayed by measuring corticosterone levels in the plasma and hippocampus of PPD-induced rats. Moreover, to determine the relationship between omega-3 and immune system responses, the plasma levels of pro-inflammatory cytokines were also measured.
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2. Materials and method
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2.1. Subjects
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Forty-two female Sprague-Dawley rats (60–70 days old, weighing 180–200 g at the beginning of the experiment), obtained from Saintik Enterprise Company, were used in this study. Animals were kept in Animal House, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. They were housed 3 per cage under a normal 12-h/12-h light/dark schedule with the lights on at 07:00 am, in a temperature of 22 ± 1 ◦ C and with free access to water and standard rat food pellets. Animals were allowed 14 days to acclimatize to the laboratory conditions prior to the experiment. Experiments were conducted between 8:00 a.m. and 3:00 p.m. All procedures involving animals were conducted in accordance with ethical guidelines and with approval from the Animal Care and Use Committee (ACUC), Faculty of Medicines and Health Sciences, Universiti Putra Malaysia. In order to minimize animal suffering, the minimum number of animals and duration of observations were employed to gain reliable data.
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2.2. Surgery
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At the beginning of the experiment, rats were ovariectomized (OVX). Using an aseptic technique, the ovariectomy was carried out bilaterally under anesthesia with xylazine 10 mg/kg and ketamine 80 mg/kg i.m. Ovariectomized rats were allowed to recover for one week following surgery, while their surgery scars were closely observed daily [8].
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2.3. Hormone simulating pregnancy (HSP)
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After one-week’s rest following the ovariectomy procedure, hormone regimens were started. Ovariectomized rats were
Table 1 Hormone-simulated pregnancy (HSP) regimen. Hormones
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Estradiol benzoate Progesterone
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administered hormones (estradiol and progesterone), dissolved in 0.1 ml sesame oil, for 23 days to establish hormone simulated pregnancy (HSP) as shown in Table 1. The duration and doses of the HSP regimen mimicked of the rat’s normal gestation and were chosen based on previous studies reporting that this amount of hormones can efficiently produce maternal behavior in nulliparous female rats [8–12]. This study included five HSP groups, one vehicle group, and one normal control group. The vehicle group was subcutaneously injected with sesame oil (vehicle) after ovariectomy, with the same volume and duration of hormones. Rats in the normal control group were virgin female rats, which had no experience of ovariectomy and injection. In order to determine whether the HSPtreatment produced maternal behavior (and whether the vehicle treatment did not), all rats were placed individually in cages (one rat/cage) after injection on day 23 of HSP regimen where they were provided with paper towels as nesting materials. After 24 h, each cage was observed for the presence or absence of nesting behavior. Maternal behavior was scored positive when rats made paper towels shredded into small pieces and arranged them into a circular nest in one corner of the cage. The absence of nesting behavior was considered as negative maternal behavior [8]. 2.4. Supplementation protocols Based on the fact that the amount of food consumed varies for each rat, oral administration of fish oil through gavage was chosen to ensure sufficient amount of omega-3 received by each rat. After termination of hormones and vehicle injection, rats which were fed on the normal diet, were given different forced feed regimens as described below for 15 consecutive days (Fig. 1). The amount of menhaden fish oil (Sigma F8020) administered was calculated daily after weighing rats, using the formula [13] as below: for dose of 1 g/kg: (body weight (g) × 0.001)/density (g/ml) for dose of 3 g/kg: (body weight (g) × 0.003)/density (g/ml) for dose of 9 g/kg: (body weight (g) × 0.009)/density (g/ml) According to the rats’ weights, which were in the range of 200–230 g, and the menhaden fish oil’s density that was 0.93 g/ml, the maximum amount of oil administered was not more than 2.2 ml/gavage. This amount of fish oil was high enough to show effects without causing diarrhea or stomach regurgitation. Menhaden fish oil, which includes approximately 30% omega-3 fatty acids, has been previously used as the source of omega-3 supplementation in animal [14] and human [15] studies. 2.5. Behavioral tests 2.5.1. Forced swim test (FST) In order to determine whether this animal model of postpartum induces depression-like behavior and whether omega-3 may attenuate this depression, an FST was conducted on postpartum day 15 followed by open field test (OFT). In order to minimize the number of rats used, rats exposed to the open field test were also tested in the forced swimming test (approximately 1 h between tests). Basically, the apparatus of the FST is a rectangular tank (25 cm × 25 cm w × 60 cm h) containing water at temperature of 27 ◦ C (±2 ◦ C) and 30 cm deep [16]. On postpartum day 14, pretest of FST was conducted. Animals were placed in the swim tank individually for 15 min to induce a state of helplessness. On postpartum day 15, rats were placed again in the swim tank for a 5-min
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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Fig. 1. Timeline of hormone and treatment regimen, OVX = ovariectomy, PP = postpartum, MFO = menhaden fish oil, FLX = fluoxetine.
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test session [17]. The rat’s behavior, including immobility time was videotaped during test session. After each FST trial, the rat was dried with a towel, placed under a heat lamp to avoid hypothermia, and then returned to its cage [16]. The water was changed after every test session to avoid any influence on the next rat. The videotapes were observed precisely by an observer blind to the rats’ treatment design to measure total duration of immobility (seconds) based on what is described by Johnson et al. [16].
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2.5.2. Open field test (OFT) Spontaneous locomotor activities of experimental rats were evaluated in the open field paradigm over a 5-min period. The apparatus consisted of a square box 75 × 75 cm wide and 42 cm high. The floor of the open field was divided by black lines into 25 squares of equal dimension of 15 cm2 . On the testing day, animals were transferred from the animal house to the behavioral test room at least 1 h before the test for habituation. The trial was recorded by a camera installed above the arena. The videotapes would be scored later by an observer (ignorant of the treatment design) based on what is described by Galea et al. [10].
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2.6. Biochemical analysis
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2.6.1. Blood and brain samples collection Animals were sacrificed by decapitation on postpartum day 15 following exposure to behavioral tests. Since anesthetic drugs affect the levels of corticosterone [18,19], decapitation was done without anesthesia. The procedure was carried out by an expert and trained researcher. Trunk blood was collected from the site where the animal was decapitated in EDTA tubes. Tubes were then centrifuged at 1000 × g at 4 ◦ C for 15 min. The plasma was aspirated and stored in −80 ◦ C for further analysis. The whole brain was quickly removed and dissected on an ice-cold plate to harvest the hippocampus. The hippocampus was immediately rinsed with cold 1× PBS, weighed, and homogenized in cold 1× PBS (every 100 mg tissue in 1 ml of 1× PBS). The homogenates were then centrifuged at 5000 × g, 4 ◦ C for 5 min and the supernatants were pipetted and stored at −20 ◦ C until assayed.
2.6.2. Corticosterone assays The levels of plasma and hippocampus corticosterone were run in duplicate by using the appropriate enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, Wuhan, China) according to the manufacturer’s instruction. 2.6.3. Pro-inflammatory cytokines assay The concentrations of plasma pro-inflammatory cytokines, including IL-1, TNF-␣ and INF-␥ were measured using a rat Procarta® Immunoassay Kit according to the instructions of the manufacturer (Affymetrix, USA). 2.7. Statistics All data was analyzed using GraphPad Prism 6 software. The results are expressed as mean ± SEM. Significant differences between groups were evaluated using one-way analysis of variance (ANOVA), followed by the post hoc Tukey’s multiple comparison test when appropriate p < 0.05 was considered significant.
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3. Results
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Twenty four hours after termination of HSP regimen, rats were observed for the presence or absence of nesting behavior using paper towels that were provided a day earlier. In the present study, all HSP-treated rats exhibited positive nesting behavior. Meanwhile none of the rats in the vehicle group exhibited positive nesting behavior. Our results are consistent with previous studies reporting that this animal model of rat’s pregnancy produced maternal behavior [8,9,11,12]. 3.2. Effects of menhaden fish oil on immobility time in FST Following 15 consecutive days of treatment, with three different doses of menhaden fish oil, the immobility time of animals seen in FST was reduced as compared to that of the negative control group (PP + water). The effect was found to be dose dependent where menhaden fish oil at doses of 3 g and 9 g/kg/d has shown significant
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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antidepressant fluoxetine at 15 mg/kg/d, which significantly reduced the duration of immobility time of rats in FST, also produced no significant difference in number of crossing activity. 3.4. Effects of menhaden fish oil on pro-inflammatory cytokines The results showed that the plasma levels of IL-1 and INF-␥ in postpartum depression-induced rats treated with menhaden fish oil at doses of 3 and 9 g/kg/d, and the plasma levels of TNF-␣ in group treated with 9 g/kg/d were decreased significantly (p < 0.05) compared to the negative control group (PP + water). The levels of IL-1 and INF-␥ in groups treated with 3 and 9 g/kg/d and the levels of TNF-␣ in group treated with 9 g/kg/d were comparable to the level seen in rats treated with fluoxetine. Rats in the PP + water group exhibited the elevated levels of IL-1, INF-␥ and TNF-␣, as compared to the normal control group (Fig. 4). 3.5. Effects of menhaden fish oil on plasma level of corticosterone
Fig. 2. Effects of menhaden fish oil (1 g/kg, 3 g/kg and 9 g/kg) on immobility time in the forced swim test in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of immobility time of animals in each group (N = 6). abcd Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. * Values with different superscripts are significantly different at p < 0.01 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
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reduction in immobility time (p < 0.05). The results were found to show a similar pattern of reduction with the positive control group, which received fluoxetine (Fig. 2).
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3.3. Effects of menhaden fish oil on locomotor activity in OFT
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The OFT results showed that the number of squares crossed by rats did not differ between groups (p > 0.05) (Fig. 3). Treatment with menhaden fish oil, which significantly reduced duration of immobility time in FST, produced no significant difference in number of crossing activity of rats in OFT. The clinically effective
As shown in Fig. 5, the plasma levels of corticosterone in postpartum depression-induced rats, treated with menhaden fish oil at doses of 3 and 9 g/kg/d, were significantly decreased (p < 0.05) as compared to the negative control group (PP + water). Meanwhile, rats in PP + water group had higher level of corticosterone as compared to the normal control group (p < 0.05). The antidepressant, fluoxetine 15 mg/kg/d, as a positive control also decreased plasma corticosterone concentrations significantly (p < 0.01) as compared to the negative control group (PP + water). 3.6. Effects of menhaden fish oil on hippocampus level of corticosterone The results showed that the hippocampus level of corticosterone in postpartum depression-induced rats treated with menhaden fish oil at a dose of 9 g/kg/d decreased significantly (p < 0.05) compared to the negative control group (PP + water). The level of corticosterone in the group treated with 9 g/kg/d was comparable to the level seen in rats treated with fluoxetine. The PP + water group maintained the elevated level of corticosterone as compared to the normal control group (Fig. 6). 4. Discussion
Fig. 3. Effects of menhaden fish oil (1 g/kg, 3 g/kg and 9 g/kg) and fluoxetine (15 mg/kg) on number of crossings in open field test (OFT) in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of number of squares crossed by animals in each group (N = 6). A one-way ANOVA followed by post hoc Tukey’s multiple comparison test, p > 0.05. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
In order to study the antidepressant effects of omega-3 on postpartum-induced rats, the animal model of postpartum depression was established as described above. In the present study, all HSP-treated rats and none of the vehicle rats exhibited positive maternal behavior at the end of hormone regimen, indicating this model of rat’s pregnancy was effective to produce maternal behavior. The days after hormone termination were considered as the postpartum period. Menhaden fish oil was then supplemented to these PPD-induced rats as a source of omega-3 for 15 consecutive days during the postpartum period. To evaluate antidepressant-like behavior of experimental rats, FST was conducted on postpartum day 15. FST is one of the behavioral tests widely employed to screen antidepressant efficacy and depressive-like behavior in rodents [20]. In this test, increasing immobility time was the indicator for depressive-like symptomatology [17]. The findings of this study showed that 15 days forced feeding with three different doses of menhaden fish oil (MFO) decreased immobility time in FST compared to the negative control group (PP + water). The effect was found to be dose dependent where menhaden fish oil 3 g and 9 g/kg/d have shown significant reduction in immobility time. Generally, compounds that are able to increase locomotor activity can decrease the immobility time in FST and produce a false
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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Fig. 5. Effects of menhaden fish oil (1 g/kg/d, 3 g/kg/d and 9 g/kg/d) on plasma levels of corticosterone in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of plasma corticosterone concentrations (ng/ml) of six animals in each group (N = 6). ab Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. * Values with different superscripts are significantly different at p < 0.01 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartuminduced, FLX = fluoxetine, MFO = menhaden fish oil.
positive result in FST. Some agents such as stimulants, convulsants, and anticholinergic cause increased locomotor activity [21]. Thus, we used OFT to rule out any probable effect of menhaden fish oil on locomotor activity that can contribute to bias on FST results. The results of OFT in the present study showed that there were no significant differences among the groups in terms of the number of squares crossed by rats. These findings indicated that menhaden fish oil supplementation, which was seen to reduce immobility time in FST, did not have a significant effect on locomotor activity.
Fig. 4. Effects of menhaden fish oil (1 g/kg/d, 3 g/kg/d and 9 g/kg/d) on plasma levels of IL-1 (panel A), TNF-␣ (panel B) and INF-␥ (panel C) in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of plasma cytokines concentration (pg/ml) of animals in each group (N = 6). ab Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. * Values with different superscripts are significantly different at p < 0.01 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
Fig. 6. Effects of menhaden fish oil (1 g/kg/d, 3 g/kg/d and 9 g/kg/d) on hippocampus corticosterone levels in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of hippocampus corticosterone concentration (ng/g) of six animals in each group (N = 6). ab Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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The group treated with fluoxetine, an established antidepressant, which were found to demonstrate significantly decreased immobility time in FST, did not have a significant effect on ambulation activity shown by similar numbers of squares crossed by the rats as compared to other groups. This result is consistent with previous studies reporting omega-3 enriched diet produced no significant effects on locomotor activity in experimental animals [22,23]. Thereby, in the present study, the immobility time reduction seen in FST following menhaden fish oil treatment was specifically due to its antidepressant mechanism and was not attributed to locomotion effects. The FST results of this study are consistent with previous animal studies reported that rats supplemented with menhaden fish oil [23] and dietary omega-3 [22] exhibited reduction in immobility time. In addition, an omega-3 deficient diet in rats increased immobility time by about 30% in FST relative to controls [24]. Earlier studies, with few exceptions, stated that there is a relationship between omega-3 deficiency and depression [5,6]. Some nutritional studies have stated that supplementation with omega3 is inversely related to the prevalence and severity of depression [25,26]. As fish are the main source of omega-3 [27], it is not surprising that there is a relationship between fish consumption and the prevalence of unipolar and postpartum depression [28,29]. Lower concentration of omega-3 was also reported in women with postpartum depression [30]. The relation between depression and immune system has been reported in previous studies. Cytokines (including proinflammatory and anti-inflammatory cytokines) are signaling molecules, responsible for regulation of immune responses. Proinflammatory cytokines such as TNF-␣, IFN-␥, IL-1, IL-6 are involved in inflammatory processes, while anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 reduce the immune responses [31]. The results of the present study show that rats in the PP + water group, which had a higher immobility time in FST, exhibit significant increases (p < 0.05) in plasma levels of TNF-␣, IFN-␥ and IL-1 as compared to normal control group. These findings indicate that the postpartum model of rats, which produce depression-like behavior, is associated with high concentration of pro-inflammatory cytokines. These findings are similar to earlier studies that reported the increased release of pro-inflammatory cytokines such as IL-1, TNF-␣ and INF-␥ in depression [32,33]. Thus, pro-inflammatory cytokines, which are released due to inflammatory process, may be involved in the pathophysiology of depression. Although the precise mechanism by which cytokines may influence mood and behavior are unknown, there are a number of suggested theories regarding this issue. One of these theories states that cytokines have been found to affect glucocorticoid receptors (GR). GR have a critical role in regulating immune responses and inflammation. Disruption of GR functioning by cytokines causes glucocorticoid resistance and leads to dysfunction in negative feedback of inflammatory responses [34]. Furthermore, cytokines increase the release of corticotropin releasing hormone (CRH) and activity of the HPA axis [35]. All of these effects of cytokines, including dysfunction of GR and HPA axis, have been found in depressed patients and support the relation between depression and proinflammatory cytokines [36]. The activated hypothalamus–pituitary–adrenal (HPA) axis releases glucocorticoids (cortisol in human and corticosterone in the rodents) into the blood. Glucocorticoid crosses the blood brain barrier and goes to the brain where it regulates its own secretion through a negative feedback. This negative feedback, in healthy organisms, limits the continuous release of HPA to control the glucocorticoids (GC) levels while disturbance of this feedback results in a feed-forward cascade of increasing glucocorticoids [37]. Apart from hypothalamus, the other parts of the brain such as the
hippocampus and the prefrontal cortex are involved in this negative feedback. High levels of corticosterone can damage the structure and function of hippocampus and prefrontal cortex [38]. The results of the present study showed that rats in the negative control group (PP + water) exhibited a significant elevation (p < 0.05) in the plasma and hippocampus levels of corticosterone as compared to the normal control group (Figs. 5 and 6). These findings are consistent with previous studies which reported the high glucocorticoids level in both humans [39] and rodents [40] in the postpartum period. In addition, it has been stated that high levels of corticosterone during the postpartum period decreases body weight, maternal care, and hippocampal cell proliferation, and increases depressive-like behavior in female rats [41]. In another study, Craft et al. [9] compared plasma levels of corticosterone in postpartum period in animal model and rat’s normal pregnancy versus vehicle and virgin control rats, respectively. His findings indicated that the plasma levels of corticosterone in both animal model and the actual postpartum period were significantly increased compared to control groups. The results of FST in the present study showed that the immobility time of PP + water group was significantly higher than the immobility time of normal control group; moreover, the PP + water had higher levels of corticosterone. This finding is similar to the findings of previous studies which reported that high corticosterone during the postpartum period results in more immobility in the forced swim test [42]. Animal models studies have shown that elevated blood corticosterone concentrations and HPA axis hyperactivity following exposure to chronic stress cause depression-like behavior [43]. The results of the present study revealed that 15 days’ supplementation with MFO decreased the plasma and hippocampal levels of corticosterone and plasma levels of pro-inflammatory cytokines as compared to the negative control group (PP + water). This reduction was found to be significant following treatment with doses of 3 and 9 g/kg/d of MFO for plasma levels of IL-1, INF-␥ and corticosterone and with dose of 9 g/kg/d of MFO for plasma level of TNF-␣ and hippocampal level of corticosterone compared to PP + water. Fluoxetine 15 mg/kg/d, an established antidepressant, as a positive control drug also significantly reduced plasma level of cytokines and corticosterone compared to negative control group (PP + water). These findings are consistent with earlier studies, which reported that omega-3 fatty acids reduce the levels of pro-inflammatory cytokines [44] and corticosterone in rats exposed to stress [45]. Polyunsaturated fatty acids (PUFA), which include omega-3 and omega-6, have a major influence over inflammatory pathways. While omega-3 has anti-inflammatory effects, omega-6 produces inflammatory responses. Omega-3 fatty acids have been proposed to attenuate the depressive and anxiolytic effects of pro-inflammatory cytokines. Pro-inflammatory cytokines appear to play an important role in depression and increase corticosterone. IL-1, which is a pro-inflammatory cytokine, affects brain functions by mediating a pathway-involved prostaglandin (PG) E2 . In this pathway, phospholipase A2 (PLA2) is responsible for triggering the release of arachidonic acid (AA), an omega-6 fatty acid and a precursor for eicosanoids synthesis [46]. Following this, eicosanoids produce PGE2 and pro-inflammatory cytokines. PGE2 activates paraventricular hypothalamic nuclei (PVN) to release corticotrophin-releasing factor (CRF) [47]. CRF, subsequently, triggers the release of glucocorticoids via HPA axis. An increase of n-6 fatty acid arachidonic acid (AA) has been reported in animal models of depression [48]. In contrast, n-3 fatty acids, found richly in fish oil, limit the action of AA in terms of eicosanoids production. An omega3-rich diet also decreases the availability of AA for eicosanoids synthesis. Therefore, omega-3 fatty acids can decrease the level of PGE2 and pro-inflammatory cytokines [49,50] and subsequently decrease CRF expression and CORT concentration [51]. Conversely,
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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an omega-3 fatty acids-deficient diet enhanced serum corticosterone concentrations in restraint stress-induced and increases immobility time in forced swimming test [52].
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In conclusion, the results of the present study clearly reveal that supplementation of omega-3 is able to produce an antidepressantlike effect in postpartum-induced rats, which is not attributed to its locomotor activity effect. Findings also show that omega-3 reduces the levels of corticosterone and pro-inflammatory cytokines, which were observed to be increased in PPD-induced rats. These findings suggest that the antidepressant effects of omega-3 might be due to interaction with neuroendocrine HPA axis systems and the neuroimmune system.
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Acknowledgment
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This study was supported by Fundamental Research Grants Q2 Scheme (Vote No. 9305100), Universiti Putra Malaysia, UPM, Ser452 dang, Selangor, Malaysia. 453 451
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Research report
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Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats
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Leila Arbabi a , Mohamad. Taufik Hidayat Baharuldin a,∗ , Mohamad Aris Mohamad Moklas a , Sharida Fakurazi a , Sani Ismaila Muhammad b a Human Anatomy Laboratory, Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia b Laboratory of Molecular Biomedicine, Institute of Bioscience, UniversitiPutra Malaysia, Serdang 43400, Selangor, Malaysia
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h i g h l i g h t s • • • •
The first evaluation of antidepressant effect of omega-3 on postpartum depression model of rat. The first measurement of pro-inflammatory cytokines in postpartum-induced rats. The first evaluation of impact of omega-3 on pro-inflammatory cytokines in postpartum-induced rats. The first evaluation of effect of omega-3 on corticosterone levels in postpartum-induced rats.
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Article history: Received 12 April 2014 Received in revised form 14 May 2014 Accepted 16 May 2014 Available online xxx
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Keywords: Postpartum depression Omega-3 fatty acid Hormone simulated pregnancy Forced swimming test Pro-inflammatory cytokine Corticosterone
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1. Introduction
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Postpartum depression (PPD) is a psychiatric disorder that occurs in 10–15% of childbearing women. It is hypothesized that omega-3 fatty acids, which are components of fish oil, may attenuate depression symptoms. In order to examine this hypothesis, the animal model of postpartum depression was established in the present study. Ovariectomized female rats underwent hormone-simulated pregnancy (HSP) regimen and received progesterone and estradiol benzoate or vehicle for 23 days, mimicking the actual rat’s pregnancy. The days after hormone termination were considered as the postpartum period. Forced feeding of menhaden fish oil, as a source of omega-3, with three doses of 1, 3, and 9 g/kg/d, fluoxetine 15 mg/kg/d, and distilled water 2 ml/d per rat started in five postpartum-induced and one vehicle group on postpartum day 1 and continued for 15 consecutive days. On postpartum day 15, all groups were tested in the forced swimming test (FST) and open field test (OFT), followed by a biochemical assay. Results showed that the postpartum-induced rats not treated with menhaden fish oil, exhibited an increase in immobility time seen in FST, hippocampal concentration of corticosterone and plasmatic level of corticosterone, and pro-inflammatory cytokines. These depression-related effects were attenuated by supplementation of menhaden fish oil with doses of 3 and 9 g/kg. Moreover, results of rats supplemented with menhaden fish oil were comparable to rats treated with the clinically effective antidepressant, fluoxetine. Taken together, these results suggest that menhaden fish oil, rich in omega-3, exerts beneficial effect on postpartum depression and decreases the biomarkers related to depression such as corticosterone and pro-inflammatory cytokines. © 2014 Published by Elsevier B.V.
Postpartum depression (PPD) is a psychiatric disorder, defined as a subtype of major depressive disorder (MDD). It has been reported that 10–15% women suffer from PPD following childbirth [1]. Although the underlying etiology of postpartum depression
∗ Corresponding author. Tel.: +60389472356; fax: +60389472356. E-mail address: taufi[email protected] (Mohamad.T.H. Baharuldin).
remains unknown, the abrupt changes in reproductive hormones that women undergo in post-delivery period may cause postpartum depression [2]. In addition to the role of estrogen and progesterone, some other biological factors such as hypothalamic–pituitary–adrenal (HPA) axis hormones, altered immune system and cytokines, and altered fatty acid have been proposed to play a role in causing postpartum depression [3]. Not diagnosing and treating postpartum depression has significant adverse effects on depressed individuals and their families [4]. Due to side effects of medical treatment on breastfed infants and the
http://dx.doi.org/10.1016/j.bbr.2014.05.036 0166-4328/© 2014 Published by Elsevier B.V.
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negative effects of untreated depression, mothers face a dilemma about how to deal with depression symptoms. Therefore, another alternative treatment should be considered to lessen depressive symptoms with lower side effects for both mother and baby. The relationship between omega-3 and depression has been reported in previous studies. There are many reasons indicating an inverse relationship between omega-3 fatty acids and depression. This link has been observed in both observational and experimental research [5,6]. Fish and fish oil, which are rich in omega-3 fatty acids, are the best dietary sources of omega-3 [7]. Although it is believed that omega-3 plays a vital role in the body, and in particular, in the nervous system and mood disorder, the underlying mechanism is poorly understood. While numerous studies have been carried out to evaluate the effects of omega-3 on depression and other mood disorders, the researches carried out to determine the effects of omega-3 on postpartum depression are few and the results are inconsistent. Due to the contradictions among the results of previous researches in this regard, the present study was performed to clarify these discrepancies by investigating the effects of menhaden fish oil (rich in omega-3) on postpartum-induced rats. In order to study the antidepressant-like effects of omega-3 on postpartum-induced rats, a hormone manipulation was used to establish the animal model of postpartum depression, and then these PPD-induced rats were supplemented with menhaden fish oil. The antidepressantlike effect of omega-3 was evaluated using a standard behavioral test (FST) and also the effect of omega-3 on HPA axis was assayed by measuring corticosterone levels in the plasma and hippocampus of PPD-induced rats. Moreover, to determine the relationship between omega-3 and immune system responses, the plasma levels of pro-inflammatory cytokines were also measured.
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2. Materials and method
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2.1. Subjects
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Forty-two female Sprague-Dawley rats (60–70 days old, weighing 180–200 g at the beginning of the experiment), obtained from Saintik Enterprise Company, were used in this study. Animals were kept in Animal House, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. They were housed 3 per cage under a normal 12-h/12-h light/dark schedule with the lights on at 07:00 am, in a temperature of 22 ± 1 ◦ C and with free access to water and standard rat food pellets. Animals were allowed 14 days to acclimatize to the laboratory conditions prior to the experiment. Experiments were conducted between 8:00 a.m. and 3:00 p.m. All procedures involving animals were conducted in accordance with ethical guidelines and with approval from the Animal Care and Use Committee (ACUC), Faculty of Medicines and Health Sciences, Universiti Putra Malaysia. In order to minimize animal suffering, the minimum number of animals and duration of observations were employed to gain reliable data.
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2.2. Surgery
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At the beginning of the experiment, rats were ovariectomized (OVX). Using an aseptic technique, the ovariectomy was carried out bilaterally under anesthesia with xylazine 10 mg/kg and ketamine 80 mg/kg i.m. Ovariectomized rats were allowed to recover for one week following surgery, while their surgery scars were closely observed daily [8].
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2.3. Hormone simulating pregnancy (HSP)
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After one-week’s rest following the ovariectomy procedure, hormone regimens were started. Ovariectomized rats were
Table 1 Hormone-simulated pregnancy (HSP) regimen. Hormones
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Estradiol benzoate Progesterone
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50 g/rat –
administered hormones (estradiol and progesterone), dissolved in 0.1 ml sesame oil, for 23 days to establish hormone simulated pregnancy (HSP) as shown in Table 1. The duration and doses of the HSP regimen mimicked of the rat’s normal gestation and were chosen based on previous studies reporting that this amount of hormones can efficiently produce maternal behavior in nulliparous female rats [8–12]. This study included five HSP groups, one vehicle group, and one normal control group. The vehicle group was subcutaneously injected with sesame oil (vehicle) after ovariectomy, with the same volume and duration of hormones. Rats in the normal control group were virgin female rats, which had no experience of ovariectomy and injection. In order to determine whether the HSPtreatment produced maternal behavior (and whether the vehicle treatment did not), all rats were placed individually in cages (one rat/cage) after injection on day 23 of HSP regimen where they were provided with paper towels as nesting materials. After 24 h, each cage was observed for the presence or absence of nesting behavior. Maternal behavior was scored positive when rats made paper towels shredded into small pieces and arranged them into a circular nest in one corner of the cage. The absence of nesting behavior was considered as negative maternal behavior [8]. 2.4. Supplementation protocols Based on the fact that the amount of food consumed varies for each rat, oral administration of fish oil through gavage was chosen to ensure sufficient amount of omega-3 received by each rat. After termination of hormones and vehicle injection, rats which were fed on the normal diet, were given different forced feed regimens as described below for 15 consecutive days (Fig. 1). The amount of menhaden fish oil (Sigma F8020) administered was calculated daily after weighing rats, using the formula [13] as below: for dose of 1 g/kg: (body weight (g) × 0.001)/density (g/ml) for dose of 3 g/kg: (body weight (g) × 0.003)/density (g/ml) for dose of 9 g/kg: (body weight (g) × 0.009)/density (g/ml) According to the rats’ weights, which were in the range of 200–230 g, and the menhaden fish oil’s density that was 0.93 g/ml, the maximum amount of oil administered was not more than 2.2 ml/gavage. This amount of fish oil was high enough to show effects without causing diarrhea or stomach regurgitation. Menhaden fish oil, which includes approximately 30% omega-3 fatty acids, has been previously used as the source of omega-3 supplementation in animal [14] and human [15] studies. 2.5. Behavioral tests 2.5.1. Forced swim test (FST) In order to determine whether this animal model of postpartum induces depression-like behavior and whether omega-3 may attenuate this depression, an FST was conducted on postpartum day 15 followed by open field test (OFT). In order to minimize the number of rats used, rats exposed to the open field test were also tested in the forced swimming test (approximately 1 h between tests). Basically, the apparatus of the FST is a rectangular tank (25 cm × 25 cm w × 60 cm h) containing water at temperature of 27 ◦ C (±2 ◦ C) and 30 cm deep [16]. On postpartum day 14, pretest of FST was conducted. Animals were placed in the swim tank individually for 15 min to induce a state of helplessness. On postpartum day 15, rats were placed again in the swim tank for a 5-min
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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Fig. 1. Timeline of hormone and treatment regimen, OVX = ovariectomy, PP = postpartum, MFO = menhaden fish oil, FLX = fluoxetine.
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test session [17]. The rat’s behavior, including immobility time was videotaped during test session. After each FST trial, the rat was dried with a towel, placed under a heat lamp to avoid hypothermia, and then returned to its cage [16]. The water was changed after every test session to avoid any influence on the next rat. The videotapes were observed precisely by an observer blind to the rats’ treatment design to measure total duration of immobility (seconds) based on what is described by Johnson et al. [16].
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2.5.2. Open field test (OFT) Spontaneous locomotor activities of experimental rats were evaluated in the open field paradigm over a 5-min period. The apparatus consisted of a square box 75 × 75 cm wide and 42 cm high. The floor of the open field was divided by black lines into 25 squares of equal dimension of 15 cm2 . On the testing day, animals were transferred from the animal house to the behavioral test room at least 1 h before the test for habituation. The trial was recorded by a camera installed above the arena. The videotapes would be scored later by an observer (ignorant of the treatment design) based on what is described by Galea et al. [10].
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2.6. Biochemical analysis
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2.6.1. Blood and brain samples collection Animals were sacrificed by decapitation on postpartum day 15 following exposure to behavioral tests. Since anesthetic drugs affect the levels of corticosterone [18,19], decapitation was done without anesthesia. The procedure was carried out by an expert and trained researcher. Trunk blood was collected from the site where the animal was decapitated in EDTA tubes. Tubes were then centrifuged at 1000 × g at 4 ◦ C for 15 min. The plasma was aspirated and stored in −80 ◦ C for further analysis. The whole brain was quickly removed and dissected on an ice-cold plate to harvest the hippocampus. The hippocampus was immediately rinsed with cold 1× PBS, weighed, and homogenized in cold 1× PBS (every 100 mg tissue in 1 ml of 1× PBS). The homogenates were then centrifuged at 5000 × g, 4 ◦ C for 5 min and the supernatants were pipetted and stored at −20 ◦ C until assayed.
2.6.2. Corticosterone assays The levels of plasma and hippocampus corticosterone were run in duplicate by using the appropriate enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, Wuhan, China) according to the manufacturer’s instruction. 2.6.3. Pro-inflammatory cytokines assay The concentrations of plasma pro-inflammatory cytokines, including IL-1, TNF-␣ and INF-␥ were measured using a rat Procarta® Immunoassay Kit according to the instructions of the manufacturer (Affymetrix, USA). 2.7. Statistics All data was analyzed using GraphPad Prism 6 software. The results are expressed as mean ± SEM. Significant differences between groups were evaluated using one-way analysis of variance (ANOVA), followed by the post hoc Tukey’s multiple comparison test when appropriate p < 0.05 was considered significant.
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Twenty four hours after termination of HSP regimen, rats were observed for the presence or absence of nesting behavior using paper towels that were provided a day earlier. In the present study, all HSP-treated rats exhibited positive nesting behavior. Meanwhile none of the rats in the vehicle group exhibited positive nesting behavior. Our results are consistent with previous studies reporting that this animal model of rat’s pregnancy produced maternal behavior [8,9,11,12]. 3.2. Effects of menhaden fish oil on immobility time in FST Following 15 consecutive days of treatment, with three different doses of menhaden fish oil, the immobility time of animals seen in FST was reduced as compared to that of the negative control group (PP + water). The effect was found to be dose dependent where menhaden fish oil at doses of 3 g and 9 g/kg/d has shown significant
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antidepressant fluoxetine at 15 mg/kg/d, which significantly reduced the duration of immobility time of rats in FST, also produced no significant difference in number of crossing activity. 3.4. Effects of menhaden fish oil on pro-inflammatory cytokines The results showed that the plasma levels of IL-1 and INF-␥ in postpartum depression-induced rats treated with menhaden fish oil at doses of 3 and 9 g/kg/d, and the plasma levels of TNF-␣ in group treated with 9 g/kg/d were decreased significantly (p < 0.05) compared to the negative control group (PP + water). The levels of IL-1 and INF-␥ in groups treated with 3 and 9 g/kg/d and the levels of TNF-␣ in group treated with 9 g/kg/d were comparable to the level seen in rats treated with fluoxetine. Rats in the PP + water group exhibited the elevated levels of IL-1, INF-␥ and TNF-␣, as compared to the normal control group (Fig. 4). 3.5. Effects of menhaden fish oil on plasma level of corticosterone
Fig. 2. Effects of menhaden fish oil (1 g/kg, 3 g/kg and 9 g/kg) on immobility time in the forced swim test in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of immobility time of animals in each group (N = 6). abcd Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. * Values with different superscripts are significantly different at p < 0.01 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
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reduction in immobility time (p < 0.05). The results were found to show a similar pattern of reduction with the positive control group, which received fluoxetine (Fig. 2).
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3.3. Effects of menhaden fish oil on locomotor activity in OFT
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The OFT results showed that the number of squares crossed by rats did not differ between groups (p > 0.05) (Fig. 3). Treatment with menhaden fish oil, which significantly reduced duration of immobility time in FST, produced no significant difference in number of crossing activity of rats in OFT. The clinically effective
As shown in Fig. 5, the plasma levels of corticosterone in postpartum depression-induced rats, treated with menhaden fish oil at doses of 3 and 9 g/kg/d, were significantly decreased (p < 0.05) as compared to the negative control group (PP + water). Meanwhile, rats in PP + water group had higher level of corticosterone as compared to the normal control group (p < 0.05). The antidepressant, fluoxetine 15 mg/kg/d, as a positive control also decreased plasma corticosterone concentrations significantly (p < 0.01) as compared to the negative control group (PP + water). 3.6. Effects of menhaden fish oil on hippocampus level of corticosterone The results showed that the hippocampus level of corticosterone in postpartum depression-induced rats treated with menhaden fish oil at a dose of 9 g/kg/d decreased significantly (p < 0.05) compared to the negative control group (PP + water). The level of corticosterone in the group treated with 9 g/kg/d was comparable to the level seen in rats treated with fluoxetine. The PP + water group maintained the elevated level of corticosterone as compared to the normal control group (Fig. 6). 4. Discussion
Fig. 3. Effects of menhaden fish oil (1 g/kg, 3 g/kg and 9 g/kg) and fluoxetine (15 mg/kg) on number of crossings in open field test (OFT) in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of number of squares crossed by animals in each group (N = 6). A one-way ANOVA followed by post hoc Tukey’s multiple comparison test, p > 0.05. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
In order to study the antidepressant effects of omega-3 on postpartum-induced rats, the animal model of postpartum depression was established as described above. In the present study, all HSP-treated rats and none of the vehicle rats exhibited positive maternal behavior at the end of hormone regimen, indicating this model of rat’s pregnancy was effective to produce maternal behavior. The days after hormone termination were considered as the postpartum period. Menhaden fish oil was then supplemented to these PPD-induced rats as a source of omega-3 for 15 consecutive days during the postpartum period. To evaluate antidepressant-like behavior of experimental rats, FST was conducted on postpartum day 15. FST is one of the behavioral tests widely employed to screen antidepressant efficacy and depressive-like behavior in rodents [20]. In this test, increasing immobility time was the indicator for depressive-like symptomatology [17]. The findings of this study showed that 15 days forced feeding with three different doses of menhaden fish oil (MFO) decreased immobility time in FST compared to the negative control group (PP + water). The effect was found to be dose dependent where menhaden fish oil 3 g and 9 g/kg/d have shown significant reduction in immobility time. Generally, compounds that are able to increase locomotor activity can decrease the immobility time in FST and produce a false
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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Fig. 5. Effects of menhaden fish oil (1 g/kg/d, 3 g/kg/d and 9 g/kg/d) on plasma levels of corticosterone in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of plasma corticosterone concentrations (ng/ml) of six animals in each group (N = 6). ab Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. * Values with different superscripts are significantly different at p < 0.01 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartuminduced, FLX = fluoxetine, MFO = menhaden fish oil.
positive result in FST. Some agents such as stimulants, convulsants, and anticholinergic cause increased locomotor activity [21]. Thus, we used OFT to rule out any probable effect of menhaden fish oil on locomotor activity that can contribute to bias on FST results. The results of OFT in the present study showed that there were no significant differences among the groups in terms of the number of squares crossed by rats. These findings indicated that menhaden fish oil supplementation, which was seen to reduce immobility time in FST, did not have a significant effect on locomotor activity.
Fig. 4. Effects of menhaden fish oil (1 g/kg/d, 3 g/kg/d and 9 g/kg/d) on plasma levels of IL-1 (panel A), TNF-␣ (panel B) and INF-␥ (panel C) in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of plasma cytokines concentration (pg/ml) of animals in each group (N = 6). ab Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. * Values with different superscripts are significantly different at p < 0.01 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
Fig. 6. Effects of menhaden fish oil (1 g/kg/d, 3 g/kg/d and 9 g/kg/d) on hippocampus corticosterone levels in the postpartum depression-induced rats. Each column represents the mean ± S.E.M of hippocampus corticosterone concentration (ng/g) of six animals in each group (N = 6). ab Values with different superscripts are significantly different at p < 0.05 amongst groups after Tukey’s post-hoc multiple comparison tests. PP = postpartum-induced, FLX = fluoxetine, MFO = menhaden fish oil.
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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The group treated with fluoxetine, an established antidepressant, which were found to demonstrate significantly decreased immobility time in FST, did not have a significant effect on ambulation activity shown by similar numbers of squares crossed by the rats as compared to other groups. This result is consistent with previous studies reporting omega-3 enriched diet produced no significant effects on locomotor activity in experimental animals [22,23]. Thereby, in the present study, the immobility time reduction seen in FST following menhaden fish oil treatment was specifically due to its antidepressant mechanism and was not attributed to locomotion effects. The FST results of this study are consistent with previous animal studies reported that rats supplemented with menhaden fish oil [23] and dietary omega-3 [22] exhibited reduction in immobility time. In addition, an omega-3 deficient diet in rats increased immobility time by about 30% in FST relative to controls [24]. Earlier studies, with few exceptions, stated that there is a relationship between omega-3 deficiency and depression [5,6]. Some nutritional studies have stated that supplementation with omega3 is inversely related to the prevalence and severity of depression [25,26]. As fish are the main source of omega-3 [27], it is not surprising that there is a relationship between fish consumption and the prevalence of unipolar and postpartum depression [28,29]. Lower concentration of omega-3 was also reported in women with postpartum depression [30]. The relation between depression and immune system has been reported in previous studies. Cytokines (including proinflammatory and anti-inflammatory cytokines) are signaling molecules, responsible for regulation of immune responses. Proinflammatory cytokines such as TNF-␣, IFN-␥, IL-1, IL-6 are involved in inflammatory processes, while anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 reduce the immune responses [31]. The results of the present study show that rats in the PP + water group, which had a higher immobility time in FST, exhibit significant increases (p < 0.05) in plasma levels of TNF-␣, IFN-␥ and IL-1 as compared to normal control group. These findings indicate that the postpartum model of rats, which produce depression-like behavior, is associated with high concentration of pro-inflammatory cytokines. These findings are similar to earlier studies that reported the increased release of pro-inflammatory cytokines such as IL-1, TNF-␣ and INF-␥ in depression [32,33]. Thus, pro-inflammatory cytokines, which are released due to inflammatory process, may be involved in the pathophysiology of depression. Although the precise mechanism by which cytokines may influence mood and behavior are unknown, there are a number of suggested theories regarding this issue. One of these theories states that cytokines have been found to affect glucocorticoid receptors (GR). GR have a critical role in regulating immune responses and inflammation. Disruption of GR functioning by cytokines causes glucocorticoid resistance and leads to dysfunction in negative feedback of inflammatory responses [34]. Furthermore, cytokines increase the release of corticotropin releasing hormone (CRH) and activity of the HPA axis [35]. All of these effects of cytokines, including dysfunction of GR and HPA axis, have been found in depressed patients and support the relation between depression and proinflammatory cytokines [36]. The activated hypothalamus–pituitary–adrenal (HPA) axis releases glucocorticoids (cortisol in human and corticosterone in the rodents) into the blood. Glucocorticoid crosses the blood brain barrier and goes to the brain where it regulates its own secretion through a negative feedback. This negative feedback, in healthy organisms, limits the continuous release of HPA to control the glucocorticoids (GC) levels while disturbance of this feedback results in a feed-forward cascade of increasing glucocorticoids [37]. Apart from hypothalamus, the other parts of the brain such as the
hippocampus and the prefrontal cortex are involved in this negative feedback. High levels of corticosterone can damage the structure and function of hippocampus and prefrontal cortex [38]. The results of the present study showed that rats in the negative control group (PP + water) exhibited a significant elevation (p < 0.05) in the plasma and hippocampus levels of corticosterone as compared to the normal control group (Figs. 5 and 6). These findings are consistent with previous studies which reported the high glucocorticoids level in both humans [39] and rodents [40] in the postpartum period. In addition, it has been stated that high levels of corticosterone during the postpartum period decreases body weight, maternal care, and hippocampal cell proliferation, and increases depressive-like behavior in female rats [41]. In another study, Craft et al. [9] compared plasma levels of corticosterone in postpartum period in animal model and rat’s normal pregnancy versus vehicle and virgin control rats, respectively. His findings indicated that the plasma levels of corticosterone in both animal model and the actual postpartum period were significantly increased compared to control groups. The results of FST in the present study showed that the immobility time of PP + water group was significantly higher than the immobility time of normal control group; moreover, the PP + water had higher levels of corticosterone. This finding is similar to the findings of previous studies which reported that high corticosterone during the postpartum period results in more immobility in the forced swim test [42]. Animal models studies have shown that elevated blood corticosterone concentrations and HPA axis hyperactivity following exposure to chronic stress cause depression-like behavior [43]. The results of the present study revealed that 15 days’ supplementation with MFO decreased the plasma and hippocampal levels of corticosterone and plasma levels of pro-inflammatory cytokines as compared to the negative control group (PP + water). This reduction was found to be significant following treatment with doses of 3 and 9 g/kg/d of MFO for plasma levels of IL-1, INF-␥ and corticosterone and with dose of 9 g/kg/d of MFO for plasma level of TNF-␣ and hippocampal level of corticosterone compared to PP + water. Fluoxetine 15 mg/kg/d, an established antidepressant, as a positive control drug also significantly reduced plasma level of cytokines and corticosterone compared to negative control group (PP + water). These findings are consistent with earlier studies, which reported that omega-3 fatty acids reduce the levels of pro-inflammatory cytokines [44] and corticosterone in rats exposed to stress [45]. Polyunsaturated fatty acids (PUFA), which include omega-3 and omega-6, have a major influence over inflammatory pathways. While omega-3 has anti-inflammatory effects, omega-6 produces inflammatory responses. Omega-3 fatty acids have been proposed to attenuate the depressive and anxiolytic effects of pro-inflammatory cytokines. Pro-inflammatory cytokines appear to play an important role in depression and increase corticosterone. IL-1, which is a pro-inflammatory cytokine, affects brain functions by mediating a pathway-involved prostaglandin (PG) E2 . In this pathway, phospholipase A2 (PLA2) is responsible for triggering the release of arachidonic acid (AA), an omega-6 fatty acid and a precursor for eicosanoids synthesis [46]. Following this, eicosanoids produce PGE2 and pro-inflammatory cytokines. PGE2 activates paraventricular hypothalamic nuclei (PVN) to release corticotrophin-releasing factor (CRF) [47]. CRF, subsequently, triggers the release of glucocorticoids via HPA axis. An increase of n-6 fatty acid arachidonic acid (AA) has been reported in animal models of depression [48]. In contrast, n-3 fatty acids, found richly in fish oil, limit the action of AA in terms of eicosanoids production. An omega3-rich diet also decreases the availability of AA for eicosanoids synthesis. Therefore, omega-3 fatty acids can decrease the level of PGE2 and pro-inflammatory cytokines [49,50] and subsequently decrease CRF expression and CORT concentration [51]. Conversely,
Please cite this article in press as: Arbabi L, et al. Antidepressant-like effects of omega-3 fatty acids in postpartum model of depression in rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.036
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an omega-3 fatty acids-deficient diet enhanced serum corticosterone concentrations in restraint stress-induced and increases immobility time in forced swimming test [52].
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In conclusion, the results of the present study clearly reveal that supplementation of omega-3 is able to produce an antidepressantlike effect in postpartum-induced rats, which is not attributed to its locomotor activity effect. Findings also show that omega-3 reduces the levels of corticosterone and pro-inflammatory cytokines, which were observed to be increased in PPD-induced rats. These findings suggest that the antidepressant effects of omega-3 might be due to interaction with neuroendocrine HPA axis systems and the neuroimmune system.
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Acknowledgment
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This study was supported by Fundamental Research Grants Q2 Scheme (Vote No. 9305100), Universiti Putra Malaysia, UPM, Ser452 dang, Selangor, Malaysia. 453 451
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