Advance Publication
The Journal of Veterinary Medical Science Accepted Date: 7 Sep 2017 J-STAGE Advance Published Date: 21 Sep 2017
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Physiology
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Note
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Antidepressant-Like Effect of Milk-derived Lactoferrin
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in the Repeated Forced-swim Stress Mouse Model
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Takashi TAKEUCHI*, Kana MATSUNAGA and Akihiko SUGIYAMA
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Course of Veterinary Laboratory Medicine, Department of Veterinary Medicine,
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Faculty of Agriculture, Tottori University, Tottori 680-0945, Japan
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*Corresponding Address; Takashi TAKEUCHI, Course of Veterinary Laboratory
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Medicine, Department of Veterinary Medicine, Faculty of Agriculture, Tottori
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University, Tottori 680-8553, Japan
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Phone & Fax: +81-857-31-5645
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e-mail:
[email protected]
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Running head; Antidepressant-like effect of lactoferrin
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ABSTRACT
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We investigated the antidepressant-like effect of lactoferrin (Lf) in a repeated
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forced-swim test (FST) stress mouse model. FST was performed on days 1, 2, 7 and 14.
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Bovine Lf (bLf) or bovine serum albumin (BSA) was supplemented at 1% to the
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commercial diet after the first FST throughout the experimental period. The FST-control
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and FST+BSA group showed a marked increase in immobility time on day 2, which
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remained increased up to the 14th day, while the FST+bLf group showed a significant
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lower immobility time. Brain-derived neurotrophic factor (BDNF) content in the
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hippocampus significantly decreased in all of FST treated groups. These results suggest
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that bLf may improve the depressive-like symptoms induced by repeated FST.
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KEY
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forced-swim test
WORDS:
antidepressant,
BDNF,
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corticosterone,
lactoferrin,
repeated
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Lactoferrin (Lf) is an iron-binding glycoprotein present in body fluids, such as
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tears, saliva, milk, pancreatic juice and bile. Lf has many bioactive effects as an
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antiviral [1], antibacterial [12], anti-tumor [17] and anti-inflammatory agent [19].
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clarify the physiological effects of Lf, we reported the characteristic transporting system
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for Lf [14]. Intraduodenally administered bovine Lf (bLf) is transported into the blood
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circulation via thoracic duct lymph fluid in adult rats. This finding indicates that the bLf
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transported to epithelial cells from the lumen reaches the blood circulation via the
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lymphatic pathway, suggesting that it could be distributed to the whole body, if an
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effective dose was administered. Kamemori et al. [6] have reported the transportation of
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intravenously administered bLf into the cerebrospinal fluid (CSF). Administered bLf
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was immunohistochemically detected in the vesicular membranes of endothelial cells in
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cerebral blood vessels 10 min after the infusion.
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vesicles were also detected in the ependymal cells of the choroid plexus. Moreover,
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the bLf concentration in the CSF was significantly increased at 1-2 hr after the
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intravenous infusion of bLf (10 or 30 mg/kg).
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Lf is possibly transported into the brain matter even in adult animals.
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Numerous immunoreactive small
These findings clearly demonstrate that
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Depressive disorders are a major psychological burden in humans and are treated
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with selective serotonin reuptake inhibitors (SSRI) and other anti-depressants.
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Repeated forced-swim stress is an animal model of chronic depression [2]. It shows
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increasing immobilized behavior over time and sensitizes to the effects of SSRI or
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other anti-depressant drugs.
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In our previous studies, we have reported that bLf suppresses the distress induced
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by electric-shock [5] or psychological stress in rats [13]. Lf also has a suppressive
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effect on the increment of plasma corticosterone in maternal separated rat pups [13].
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Increased hypothalamus-pituitary-adrenal (HPA) axis activity has been reported to
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highly relate with depressaive disorder [3]. We have also reported an anti-nociceptive
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effect of Lf in a mice tail-flick model [16], suggesting Lf is effective in the central
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nervous system in both rats and mice. Within these contexts, we hypothesized that Lf
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has an anti-depressant-like effect. The major purpose of this study was to assess the
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antidepressant-like effect of Lf using the repeated forced-swim stress mouse model.
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We also investigated glucocorticoid release and brain-derived neurotrophic factor
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(BDNF) content in the hippocampus in relation to the antidepressant-like effect of Lf.
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Male ICR strain mice aged 5 weeks were purchased from the Tokyo Experimental
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Animal Co., Ltd. (Tokyo, Japan). The mice were acclimated for 1 week, and then used
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for experiments at 6 weeks of age. The animals were housed in a temperature-controlled
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(22 ± 1 oC) room under a constant day/night cycle (lights on 07:00-19:00). Mice were
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housed 3 per cage for all experiments. Food (standard chow, CE-2, Nihon-Crea, Tokyo,
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Japan) and water were available ad libitum. The experimental procedure used in the
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present study and care of animals were approved by the Experimental Animal
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Committee of Tottori University.
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Bovine Lf was purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan), and
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bovine serum albumin (BSA) was purchased from Sigma Aldrich Japan (Tokyo,
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Japan).
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The forced-swim test (FST) was conducted using a plexiglass cylinder with a 15
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cm diameter and 25 cm height, filled with warm water (25oC) to a depth of 10 cm.
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Mice were divided into 6 groups: groups 1 and 4 received a normal diet (control),
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groups 2 and 5 received a 1% BSA-supplemented diet, and groups 3 and 6 received a
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1% bLf-supplemented diet. FST was performed only in groups 4 (FST-control), 5
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(FST+BSA) and 6 (FST+bLf). Repeated FST was performed according to a previous
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report [8] with slight modifications. Before the FST, the mice were left quiet for at least
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1 hr to adapt to the experimental room. Each mouse was placed individually in the
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cylinder for 15 min on day 1 and for 5 min on days 2, 7 and 14. All experiments were
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carried out between 13:00 and 17:00. The behavior of each mouse was recorded during
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the FST using a video camera (Handycam, Sony, Tokyo, Japan), and immobility time
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was measured using SMART 3.0 software (Panlab Harvard Apparatus, Barcelona,
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Spain). Each mouse was judged to be immobile when it ceased struggling and remained
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floating motionless in the water.
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cm2/sec from preliminary experiments (data are not shown) and judged every 0.5 sec by
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the software. Following the first FST trial, we started to feed BSA or bLf to the animal.
Immobile criteria were determined as less than 0.6
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Blood samples (0.1 ml) were collected at 30 min after the FST on days 2, 7 and 14
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from the tail vein. Blood samples were also collected at15:00-16:00 on day 15 to
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measure basal plasma corticosterone levels. The blood was transferred into an
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EDTA-containing tube and centrifuged (5,000×g, 5 min, 4oC). All samples were stored
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at -80oC until measurement for levels of corticosterone. Plasma corticosterone was
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determined by an ELISA kit (Assaypro, St. Charles, MO, USA).
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After all behavioral experiments and blood collections were completed, mice were
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sacrificed by decapitation. The hippocampus was rapidly removed and stored at -80oC
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until assays were performed. For the measurement of BDNF content, protein samples
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were obtained from the hippocampus. Protein concentration was determined by a
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protein assay reagent (Bio-Rad, Hercules, CA, USA). BDNF content was determined by
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an ELISA kit (Promega, Madison, WI, USA). Normalized BDNF concentration was
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shown as BDNF/protein concentration.
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All data are expressed as mean ± SD. Differences between the groups were
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statistically investigated using two-way repeated analysis of variance (ANOVA) for the
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immobility time in the FST and plasma corticosterone levels immediately after the FST.
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We also used two-way ANOVA for the basal plasma corticosterone level and
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hippocampal BDNF content. If there is significant difference by the factor, we used
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Bonferroni’s post hoc test. In all cases, a probability (P) value of