Advance Publication
The Journal of Veterinary Medical Science Accepted Date: 7 Sep 2017 J-STAGE Advance Published Date: 21 Sep 2017
1
Physiology
2
Note
3 4 5 6
Antidepressant-Like Effect of Milk-derived Lactoferrin
7
in the Repeated Forced-swim Stress Mouse Model
8 9
Takashi TAKEUCHI*, Kana MATSUNAGA and Akihiko SUGIYAMA
10 11
Course of Veterinary Laboratory Medicine, Department of Veterinary Medicine,
12
Faculty of Agriculture, Tottori University, Tottori 680-0945, Japan
13 14 15
*Corresponding Address; Takashi TAKEUCHI, Course of Veterinary Laboratory
16
Medicine, Department of Veterinary Medicine, Faculty of Agriculture, Tottori
17
University, Tottori 680-8553, Japan
18
Phone & Fax: +81-857-31-5645
19
e-mail:
[email protected]
20 21
Running head; Antidepressant-like effect of lactoferrin
22 23
1
24
ABSTRACT
25
We investigated the antidepressant-like effect of lactoferrin (Lf) in a repeated
26
forced-swim test (FST) stress mouse model. FST was performed on days 1, 2, 7 and 14.
27
Bovine Lf (bLf) or bovine serum albumin (BSA) was supplemented at 1% to the
28
commercial diet after the first FST throughout the experimental period. The FST-control
29
and FST+BSA group showed a marked increase in immobility time on day 2, which
30
remained increased up to the 14th day, while the FST+bLf group showed a significant
31
lower immobility time. Brain-derived neurotrophic factor (BDNF) content in the
32
hippocampus significantly decreased in all of FST treated groups. These results suggest
33
that bLf may improve the depressive-like symptoms induced by repeated FST.
34 35
KEY
36
forced-swim test
WORDS:
antidepressant,
BDNF,
37
2
corticosterone,
lactoferrin,
repeated
38
Lactoferrin (Lf) is an iron-binding glycoprotein present in body fluids, such as
39
tears, saliva, milk, pancreatic juice and bile. Lf has many bioactive effects as an
40
antiviral [1], antibacterial [12], anti-tumor [17] and anti-inflammatory agent [19].
41
clarify the physiological effects of Lf, we reported the characteristic transporting system
42
for Lf [14]. Intraduodenally administered bovine Lf (bLf) is transported into the blood
43
circulation via thoracic duct lymph fluid in adult rats. This finding indicates that the bLf
44
transported to epithelial cells from the lumen reaches the blood circulation via the
45
lymphatic pathway, suggesting that it could be distributed to the whole body, if an
46
effective dose was administered. Kamemori et al. [6] have reported the transportation of
47
intravenously administered bLf into the cerebrospinal fluid (CSF). Administered bLf
48
was immunohistochemically detected in the vesicular membranes of endothelial cells in
49
cerebral blood vessels 10 min after the infusion.
50
vesicles were also detected in the ependymal cells of the choroid plexus. Moreover,
51
the bLf concentration in the CSF was significantly increased at 1-2 hr after the
52
intravenous infusion of bLf (10 or 30 mg/kg).
53
Lf is possibly transported into the brain matter even in adult animals.
To
Numerous immunoreactive small
These findings clearly demonstrate that
54
Depressive disorders are a major psychological burden in humans and are treated
55
with selective serotonin reuptake inhibitors (SSRI) and other anti-depressants.
56
Repeated forced-swim stress is an animal model of chronic depression [2]. It shows
57
increasing immobilized behavior over time and sensitizes to the effects of SSRI or
58
other anti-depressant drugs.
59
In our previous studies, we have reported that bLf suppresses the distress induced
60
by electric-shock [5] or psychological stress in rats [13]. Lf also has a suppressive
61
effect on the increment of plasma corticosterone in maternal separated rat pups [13].
3
62
Increased hypothalamus-pituitary-adrenal (HPA) axis activity has been reported to
63
highly relate with depressaive disorder [3]. We have also reported an anti-nociceptive
64
effect of Lf in a mice tail-flick model [16], suggesting Lf is effective in the central
65
nervous system in both rats and mice. Within these contexts, we hypothesized that Lf
66
has an anti-depressant-like effect. The major purpose of this study was to assess the
67
antidepressant-like effect of Lf using the repeated forced-swim stress mouse model.
68
We also investigated glucocorticoid release and brain-derived neurotrophic factor
69
(BDNF) content in the hippocampus in relation to the antidepressant-like effect of Lf.
70
Male ICR strain mice aged 5 weeks were purchased from the Tokyo Experimental
71
Animal Co., Ltd. (Tokyo, Japan). The mice were acclimated for 1 week, and then used
72
for experiments at 6 weeks of age. The animals were housed in a temperature-controlled
73
(22 ± 1 oC) room under a constant day/night cycle (lights on 07:00-19:00). Mice were
74
housed 3 per cage for all experiments. Food (standard chow, CE-2, Nihon-Crea, Tokyo,
75
Japan) and water were available ad libitum. The experimental procedure used in the
76
present study and care of animals were approved by the Experimental Animal
77
Committee of Tottori University.
78
Bovine Lf was purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan), and
79
bovine serum albumin (BSA) was purchased from Sigma Aldrich Japan (Tokyo,
80
Japan).
81
The forced-swim test (FST) was conducted using a plexiglass cylinder with a 15
82
cm diameter and 25 cm height, filled with warm water (25oC) to a depth of 10 cm.
83
Mice were divided into 6 groups: groups 1 and 4 received a normal diet (control),
84
groups 2 and 5 received a 1% BSA-supplemented diet, and groups 3 and 6 received a
85
1% bLf-supplemented diet. FST was performed only in groups 4 (FST-control), 5
4
86
(FST+BSA) and 6 (FST+bLf). Repeated FST was performed according to a previous
87
report [8] with slight modifications. Before the FST, the mice were left quiet for at least
88
1 hr to adapt to the experimental room. Each mouse was placed individually in the
89
cylinder for 15 min on day 1 and for 5 min on days 2, 7 and 14. All experiments were
90
carried out between 13:00 and 17:00. The behavior of each mouse was recorded during
91
the FST using a video camera (Handycam, Sony, Tokyo, Japan), and immobility time
92
was measured using SMART 3.0 software (Panlab Harvard Apparatus, Barcelona,
93
Spain). Each mouse was judged to be immobile when it ceased struggling and remained
94
floating motionless in the water.
95
cm2/sec from preliminary experiments (data are not shown) and judged every 0.5 sec by
96
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
97
Blood samples (0.1 ml) were collected at 30 min after the FST on days 2, 7 and 14
98
from the tail vein. Blood samples were also collected at15:00-16:00 on day 15 to
99
measure basal plasma corticosterone levels. The blood was transferred into an
100
EDTA-containing tube and centrifuged (5,000×g, 5 min, 4oC). All samples were stored
101
at -80oC until measurement for levels of corticosterone. Plasma corticosterone was
102
determined by an ELISA kit (Assaypro, St. Charles, MO, USA).
103
After all behavioral experiments and blood collections were completed, mice were
104
sacrificed by decapitation. The hippocampus was rapidly removed and stored at -80oC
105
until assays were performed. For the measurement of BDNF content, protein samples
106
were obtained from the hippocampus. Protein concentration was determined by a
107
protein assay reagent (Bio-Rad, Hercules, CA, USA). BDNF content was determined by
108
an ELISA kit (Promega, Madison, WI, USA). Normalized BDNF concentration was
109
shown as BDNF/protein concentration.
5
110
All data are expressed as mean ± SD. Differences between the groups were
111
statistically investigated using two-way repeated analysis of variance (ANOVA) for the
112
immobility time in the FST and plasma corticosterone levels immediately after the FST.
113
We also used two-way ANOVA for the basal plasma corticosterone level and
114
hippocampal BDNF content. If there is significant difference by the factor, we used
115
Bonferroni’s post hoc test. In all cases, a probability (P) value of
