Pathophysiology of postpartum depression (PPD) has been associated with many factors, such as neuroendocrine, neuroinflammation and neurotransmitter changes. Fish oil (FO) improves PPD both in humans and animals. However, little is known with regards to its pharmacology on a PPD-like rat model. Hence, the current study aimed at investigating the effects of FO on a PPD-like rat model. Female rats were induced with PPD-like symptoms and then randomly divided into six groups (n = 6) for two experimental protocols. Protocol 1 consisted of PPD-like rats (2 mL distilled water), PPD-like + FO (9 g/kg/d) and PPD-like + Fluoxetine (FLX) (15 mg/kg/d) groups of rats, whereas Protocol 2 consisted of PPD-like rats (2 mL distilled water) + PCPA (p-chlorophenylalanine) 150 mg/kg, PPD-like + FO (9 g/kg/d) + PCPA 150 mg/kg and PPD-like + FLX (15 mg/d) + PCPA 150 mg/kg groups of rats, respectively. All treatments were administered orally for 10 days postpartum, except PCPA, which was given intraperitoneally. Prior to euthanasia, the antidepressant-like effect of the FO was evaluated using the forced swimming test (FST) and open field test (OFT) on day 10 postpartum. Biochemical analysis of serotonin, serotonin metabolite and serotonin turnover from their prefrontal cortex and hippocampus were also measured. The results showed that FO decreased immobility time and increased swimming time significantly, but not climbing time in FST. Further, it also decreased serotonin metabolite and turnover significantly in the hippocampus of the PPD-like rats. In contrast, administration with PCPA reversed all the outcomes. The antidepressant-like effects of FO were found to be similar with that of FLX. Thus, it can be concluded that FO exerts its antidepressant-like effects in PPD-like rats through modulation of serotonergic system.
Chemicals used in this study include hormones, estradiol (E8515, Sigma Chemicals, St Louis, MO, USA) and progesterone (P0130, Sigma Chemicals, St Louis, MO, USA) dissolved in 0.1 mL sesame oil (S3547, Sigma Chemicals, St Louis, MO, USA). The specifications of the sesame oil used in this study, as provided by Sigma Chemicals, were as follows: appearance (turbidity): clear; appearance (color): yellow to yellow-green; appearance (form): liquid; identity: conforms; specific gravity: 0.916–0.921; solidification range: 20–25 °C; free fatty acids: (0.02 N NaOH) <2.0 mL; iodine value: 103–116; saponification value: 188–195; unsaponifiable matter: <1.5%; heavy metal: < 0.001%; and cottonseed oil absent: confirmed. Fish oil (F8020, Sigma Chemicals, St Louis, MO, USA) and fluoxetine (FLX) (F132, Sigma Chemicals, St Louis, MO, USA) were also used; other chemicals used for the study include p-chlorophenylalanine methyl ester (PCPA, Sigma Chemicals, St Louis, MO, USA), an inhibitor of the rate limiting enzyme, tryptophan hydroxylase, which is responsible for 5HT production (Vines et al., 2012). Further, ELISA kits for the measurements of 5HT (ST/5HT, E-EL-0033, Elabscience, Houston, TX, USA) and its metabolites 5-Hydroxyindolacetic acid (5HIAA, E-EL-0075, Elabscience, Houston, TX, USA) were also used. Female albino Wistar rats, aged 60–70 days (180–200 g), were purchased at the beginning of the experiment from Saintik Enterprise Company, Malaysia, and housed at the Animal House, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. The Animal Experimental Unit (Animal House) lies on a latitude of 2.978705 and a longitude of 101.717662, while the altitude of Serdang, where the animal house is located, is about 0–500 m above sea level. All rats were housed two per cage under 12 hrs/12 hrs light/dark cycles at 22 ± 1 °C with free access to water and standard rat chow (Gold Coin, Malaysia, Table 1). Prior to the experiment, all the rats were acclimatized for 14 days under laboratory conditions. The experiments were conducted from 8 am to 3 pm every day. All experimental procedures were conducted in accordance with the animal ethics guidelines, which were approved by the Institutional Unit of Animal Care and Use Committee (IACUC), Universiti Putra Malaysia, with the reference number UPM/IACUC/AUP-R097/2015. Composition of rat chow (Gold coin Malaysia) used for the feeding of the rats [21]. The induction of PPD in rats involves ovariectomy, a hormone-simulated pregnancy (HSP) regime followed by the withdrawal of ovarian hormones; then, confirmation through maternal nesting behavior, as previously described by Arbabi and Shukkoor [13,17], is conducted. Ovariectomy was carried out by a veterinarian; this lowered the risk of the death of rats in the experiment. Both ovaries were removed under anesthesia, ketamine (80 mg/kg intramuscular (i.m)) and xylazine (10 mg/kg i.m). In the maternal nesting behavior test, a paper towel was placed for each rat in its cage to use as a nesting material. The presence of a circular-shaped nest-like structure made from the paper towel observed in the rat’s cage was an indicator of success for the induction of HSP, while absence of a nest-like structure in the cages indicates failure for the induction of HSP and these rats were excluded from the studies. This study recorded a 95% success rate for the induction of HSP, as confirmed by the nesting behavior exhibited by the rats. The supplementation with FO was based on an established protocol [13]. The dose of the FO (Menhaden fish oil, Sigma F8020) was calculated daily using the formula (body weight (g) × 0.009)/density (g/mL) for dose 9 g/kg. Its composition includes 30% omega-3 fatty acid with 10%–15% eicosapentaenoic acid and 8%–15% docosahexaenoic acid, which has been proven to be safe in rats. FO and FLX were administered orally using oral gavage. In this study, the experimental procedure consisted of two protocols: the first protocol, evaluating the effect of FO on PPD-like rats, and the second protocol, evaluating the effect of FO on PPD-like rats after the administration of PCPA, a 5HT blocker. Protocol 1 (Figure 1) was conducted to evaluate the antidepressant-like effects of 10 days of FO supplementation on a PPD-like rat model through FST and OFT, followed by biochemical analysis of serotonin, serotonin metabolite and serotonin turnover. The effects of FO were compared with FLX, which is a common antidepressant readily available in the market. The rats were randomly divided into three groups (n = 6). Experimental design for the protocol 1, evaluation of FO on PPD-like rat model. PPD—postpartum depression, FO—fish oil, FLX—fluoxetine, PCPA—p-chlorophenylalanine methyl ester, FST—forced swimming test, OFT—open field test, OVX—ovariectomy, H2O—water, X–euthanasia. Protocol 2 (Figure 2) was carried out to evaluate the antidepressant-like effects of 10 days of supplementation of FO on a PPD rat model after administration of PCPA on PPD days 8, 9 and 10 through FST and OFT, followed by biochemical analysis of 5HT, serotonin metabolite and 5HT turn over. The effects of FO were compared with FLX. The rats were randomly divided into three groups (n = 6). Experimental design for protocol 2, evaluation of FO on PPD rat model after PCPA administration. PPD—postpartum depression, FO—fish oil, FLX—fluoxetine, PCPA—p-chlorophenylalanine methyl ester, FST—forces swim test, OFT—open field test, OVX—ovariectomy, H2O—water, X—euthanasia. OFT was carried out to evaluate spontaneous locomotor activities of the experimental rats. The locomotor activities of the rats were evaluated by counting the number of small squares (25 squares of equal dimension of 15 cm square each) crossed by the rats at the bottom of an open field arena, a square box measuring 75 × 75 cm length and width, respectively, and 42 cm high. At the beginning of the test, rats were left for 1 h in the animal behavior room for habituation. The lux of the animal testing behavior room ranged from 25 to 100, depending on the number of lights on, but during the testing it was about 25 lux. Then, the rats were placed at the center of the open field arena and were observed for 5 min for their locomotor activities (number of lines crossed) and all changes were recorded using a video camera for later analysis, as described by Chiroma [22]. A rat is considered to have crossed a line when all of its four paws are within one square. The data obtained from the test were rated blindly by two trained independent observers. The average of the data reported by the two observers was used for statistical analysis. FST was carried out 1 h after OFT was completed. Three behavioral patterns were assessed through FST; the immobility behavior (a floating-like behavior without any necessary movement, unless for maintaining the rat’s nose above the water level), swimming behavior (an active behavior with horizontal movement of the forelimb or hind limb in a paddling fashion) and climbing behavior (an active behavior with vertical movement of the rats’ front paw that breaks the surface of the water up to the wall). In FST, the time spent on each behavioral pattern by rats after the given treatment was observed in a plastic cylinder (25 cm in diameter and 50 cm height) which contained water 30 cm deep at a temperature of 27 °C. FST was conducted in two sessions; the first session was a pre-test, whereby rats were forced to swim for 15 min to induce the state of helplessness, and the second session was conducted after 24 h, with a 5 min period of swimming. All behavioral changes were recorded using a video camera for subsequent analysis [23,24]. The video obtained was analyzed by two independent raters who were blinded to the rat’s groupings in order to avoid bias. Inter-rater differences were avoided by using the averages of the two raters as the final data for analysis to ascertain the depressive-like behaviors of the rats. On day 10 postpartum, the rats were euthanized through decapitation; the whole brain was harvested and placed on a cold plate, followed by rapid isolation of their hippocampus and prefrontal cortex (PFC). Then, the rats were rinsed in cold phosphate buffer saline (PBS) and weighed. The tissues were then homogenized in cold PBS with every 100 mg tissue minced in 1 mL of 1× PBS. The homogenates were then centrifuged for 5 min at 5000× g 4 °C. The supernatants were aliquot into small volume and stored at −80 °C for later use. ELISA kits were used to measure the concentration of monoamines neurotransmitter, serotonin (ST/5HT, E-EL-0033, Elabscience, Houston, TX, USA) and its metabolites 5-Hydroxyindolacetic Acid (5HIAA, E-EL-0075, Elabscience, Houston, TX, USA) in brain PFC and hippocampus. Both of the ELISA kits were rat-specific commercialized ELISA kits [25]. The assays were prepared based on the manufacturer’s instruction and the measurements were read using photospectrometer at an optical density 450 nm. Serotonin turnover ratio (5HIAA/5HT) was then calculated using the measurement of 5HT and 5HIAA for both the PFC and hippocampus. The data obtained were statistically analyzed using SPSS version 23 and GraphPad prism version 6 (ISI, San Diego, CA, USA) software. All the results were expressed as mean ± standard error of mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s post hoc tests, where appropriate, for multiple comparison. A significant difference was accepted when the p value was less than 0.05 (p < 0.05).
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