J Life Sci Res 2013; 11: 1-4Ⓒ 2013 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Rapid Communication

Protective Activity of Antioxidants in the Hypothalamic Paraventricular Nucleus of
Chronic Restraint-Stressed Mice
Hideo SHINAGAWA†１,２,３, Mariko YAMANO２, Tatsuyoshi SAIJO１,４, and Makoto MURATSUGU２
１

Research Center for Behavioral Economics, The Institute of Social and Economic Research (ISER), Osaka University, 6-1 Mihogaoka,
Ibaraki-shi, Osaka 565-0047, Japan; ２College of Health and Human Sciences, Osaka Prefecture University, 3-7-30 Habikino, Habikinoshi, Osaka 583-8555, Japan; ３Junior College, Higashi Osaka University, 3-1-1 Nishitsutsumigakuen-machi, Higashiosaka-shi, Osaka
577-8567, Japan; ４Department of Management, Kochi University of Technology, 185 Tosayamada, Kami-shi, Kochi 782-8502, Japan.
Received 5 November 2013; accepted 2 December 2013
Key words : antioxidants; hypothalamic paraventricular nucleus; chronic stress

1 Introduction
Antioxidants are a group of phytochemicals, vitamins,
and other nutrients that protect cells from free radical-induced damage.1 They are believed to play a role in preventing the development of such chronic diseases as cancer,
heart disease, hypertension, ataxia, and cataracts.2-6 Cells
that use oxygen naturally produce free radicals, and thesecan cause severe damage. Antioxidants act as "free radical
scavengers" and prevent or repair free-radical-induced damage.
Astaxanthin (ASX) and proanthocyanidin (PAC) areboth classified as antioxidants. ASX is a carotenoid that iscommonly found in crustaceans such as shrimp and crab,
as well as marine organisms including salmon, salmon roe,krill, and algae. On the other hand, PAC, a flavonoid, ismainly extracted from maritime pine bark. A number of invitro and in vivo studies recently demonstrated the antioxidant and neuroprotective effects of ASX and PAC.7-10 However, their neuroprotective action against chronic stress inthe brain, especially in the hypothalamic paraventricular nucleus, has yet to be investigated.
Stress can be positive, causing the release of adrenaline in humans, which can enhance decision-making andproblem-solving abilities. However, chronic stress, whichis constant and persists over an extended period of time, canbe debilitating and overwhelming. Chronic stress can affectboth the physical and psychological well-being by causing
various problems including anxiety, insomnia, muscle pain,high blood pressure, and a weakened immune system. Previous studies demonstrated that stress contributed to the development of mental disorders such as depression,†

Corresponding author, Email: sinagawa@iser.osaka-u.ac.jp

agoraphobia, and anxiety.11-14 Therefore, although the consequences of chronic stress can be very serious, many people under prolonged stress do not make the necessary
lifestyle changes to reduce stress and ultimately prevent
health problems. In addition, the neural substrates underlying stress resilience remain unknown. The expression of cFos has generally been used as a marker to detect
stress-induced neural activation in the brain.15,16 Particularstressors have been shown to increase the immunoreactivityof c-Fos in a regionally-specific manner. For example, limbic areas are activated in response to neurogenic, but not
systemic stressors, while the hypothalamic paraventricular
nucleus is activated regardless of the stress type. BDNF isalso an important mediator of activity-dependent functionsin the nervous system and its overexpression induces learning and memory impairments.17 Blood corticosterone levels
have been used as a peripheral stress marker.18 Previous
studies reported that the expression of c-Fos and BDNF in
the hypothalamic paraventricular nucleus and blood corticosterone levels in restraint-stressed mice were significantly
higher than those in non-restraint control mice.15,19 Therefore, the purpose of the present study was to clarify immunohistologically the protective effects of antioxidants
against chronic stress in the hypothalamic paraventricular
nucleus of mice.
2 Materials and Methods
2.1 Animals
Six male ICR mice (over 8 weeks old) were purchasedfrom SLC Inc. (Hamamatsu, Japan). They were individuallyhoused under standard conditions (23−25°C and 50±5% humidity), with a 12-h light/dark cycle (lights on at 0800 and
off at 2000). Food and water were available ad libitum. An-

2imals were handled in accordance with the guidelines established by the Institutional Animal Care and Ethical Committee at Osaka Prefecture University.
2.2 Food and restraint stress procedure
Mice were randomly divided into three experimentalgroups (i.e., control, ASX, and PAC). Each group was maintained on their individual diets for 4 weeks before the restraint stress procedure. Standard laboratory food (Oriental
Yeast Co., Ltd., Tokyo, Japan) was provided to the control
group. Standard laboratory food containing 1% astaxanthin([ASX] AstaREAL, Fuji Chemical Industry Co., Ltd.,
Toyama, Japan) and 1% proanthocyanidin ([PAC] Pycnogenol, Horphag Research, Geneva, Switzerland), respectively, was provided to the ASX and PAC groups. The dosesof these antioxidants were considered appropriate and adequate amounts based on the findings of a previous study.18
Chronic restraint stress was applied for 10 consecutive days.
A stainless mesh was used to allow for a close fit to the mice
(4 h/day between 0800 and 2000).20
2.3 Immunohistochemistry
After stress, mice were deeply anesthetized with diethyl ether, and transcardially perfused with ice-cold saline
followed by 4% paraformaldehyde in 0.1 M phosphatebuffered saline (PBS, pH 7.4). The brain was post-fixed in
the same fixative and cryoprotected in 0.1 M PBS (30% sucrose). The brain was embedded in Tissue-Tek O.C.T. compound (Sakura Finetech Japan Co., Ltd., Tokyo, Japan).
Coronal sections (20 μm) were made by a cryostat(HM550E, MICROM, Walldorf, Germany), and all sections
between stereotaxic coordinates 2.2 and 3.0 anterior to thebregma, according to the brain atlas of Paxinos and Franklin(2004), were collected. Standard avidin–biotin immunohistochemistry (ABC Elite System Vector Laboratories;
Burlingame, CA, USA) was used to reveal c-Fos, a protooncogene, (1:1500; Oncogene, Cambridge, MA, USA) or
BDNF, a brain-derived neurotrophic factor, (1:1500; Santa
Cruz Biotechnology, Santa Cruz, CA, USA). Free-floating
sections were incubated in 0.1% hydrogen peroxide in 0.1
M PBS for 30 min, rinsed 5 times, and then blocked for 1
hr in 0.1M PBS containing 10% normal donkey serum and
1% albumin. Sections were then incubated overnight at 4°C
in 0.1 M PBS containing the primary rabbit polyclonal cFos or BDNF antibody. After rinses in PBS, sections wereincubated for 3 hr in biotinylated goat anti-rabbit secondary
IgG. They were then rinsed 5 times, and incubatedovernight in the avidin-biotin complex. Sections were thenprocessed in 0.02% diaminobenzidine tetrahydrochloride
and 0.005% hydrogen peroxide to yield a black product in
c-Fos- or BDNF-containing structures. Immunostained cells
were observed using a light microscope.
2.4 Measurement of corticosterone levels

Corticosterone levels were measured with an AssayMax Corticosterone ELISA kit (Assaypro, St. Charles, MO,
USA) according to the manufacturer's manual, with minor
modifications. Briefly, mice were sacrificed and 1 mL ofblood was collected with 30 μL of 100 mM EDTA immediately after the final restraint stress. Samples were centrifuged (5 min, 4000 rpm, 4°C). Each supernatant wasadded to a well in a 96-well plate at a dilution of 1:100 in
duplicate and subjected to the immunoassay. The opticaldensity of the enzyme products was read at 420 nm (Model
680XR Microplate Reader, Bio-Rad Labs. Inc., Hercules,
CA, USA). A 4-parameter logistic equation was used forthe quantitative analysis of corticosterone levels.
3 Results
The expression of c-Fos and BDNF in the hypothalamic paraventricular nucleus of mice is shown in Figure 1.
The expression of both c-Fos and BDNF was lower in both
the ASX and PAC groups than in the control group. Especially in the ASX group, the expressions were lower thanthose in the control group.
Blood corticosterone levels after chronic restraint
stress were markedly lower in the ASX group than in boththe control and PAC groups. The levels of corticosterone
level in the ASX group were approximately one-tenth thatin the control group, and less than that in the PAC group(Data not shown).

Fig. 1 The expression of c-Fos (A) and BDNF (B) in the hypothalamic paraventricular nucleus (circled with white dotlines) of each group.

4 Discussion
The relationship between corticosteroids (endogenousand exogenous) and stress is well known.21-23 The hypothalamic-pituitary-adrenal (HPA) axis exhibits a circadianrhythm, and is activated by stress and inhibited by corticosteroids. The stress-induced facilitation of HPA axis activity
may be mediated by a parallel stress-induced (corticotropinreleasing hormone [CRH]-dependent) increase in the ca-

3pacity of brain noradrenergic cell groups to respond to
stress.24-26 In addition, free radicals are known to be a naturally occurring byproduct of normal metabolic processes
and are normally regulated by the body's immune system.
However, a marked increase in the level of free radicals has
been observed with chronic stress in humans, and this overwhelms the body's natural defenses, resulting in severe
damage to the body. Increases in corticosterone levels andthe expression of c-Fos and BDNF have also been reported
in the blood and the brain, respectively, under this condition.
We focused on the hypothalamic paraventricular nucleus in the brain in the present study. Two antioxidants (i.e.,
ASX and PAC) were used to assess the effects of antioxidants on mental and physical stress. Both of these antioxidants reduced the expression of c-Fos and BDNF, whichindicated that they may protect the hypothalamic paraventricular nucleus from chronic stress-induced damage. The
expression of c-Fos and BDNF was significantly lower inthe ASX group than in the PAC group, which indicates that
ASX is more effective against stress than PAC. The notabledifference between these antioxidants is that ASX is lipid
soluble, whereas the majority of ASX ingested is also
egested. Even though low levels of ASX are absorbed, it
may still penetrate into the cell membrane and prevent celldegeneration due to oxidative damage. On the other hand,water-soluble PAC does not cross the cell membrane easily
and is not very effective in the hypothalamic paraventricular
nucleus.
The levels of corticosterone observed in the blood
were consistent with the results obtained for immunohistological expression. These results showed that the restraint
stress used in the present study caused appropriate damage
both centrally and peripherally. In other words, mechanicalchronic stress activated the HPA axis and changed bloodcorticosterone levels. Therefore, the blood corticosterone
levels were highest in the control group and lower in the
ASX and PAC groups. This result suggests that these antioxidants also reduced the effects of chronic stress peripherally. Thus, antioxidants such as ASX and PAC may bebeneficial for both the nervous system and vasculature andprotect the brain and body from stress-induced damage.
However, whether antioxidants initially act to produce aneffect centrally or peripherally has yet to be clarified. Further studies are needed to address this issue.
5 Conclusion
When ASX and PAC were systematically administeredby ingestion before and during chronic restraint stress, theymarkedly reduced the expression of c-Fos and BDNF in the
hypothalamic paraventricular nucleus, which should havebeen increased by the chronic restraint stress protocol used.
These results suggest that antioxidants such as ASX andPAC may protect the brain against chronic stress.
Acknowledgements
This study was supported by the Japan Society of thePromotion of Science (JSPS); Grant-in-Aid for Research
Activity Start-up (21890132), for Young Scientists(24792353), and for Scientific Research on Priority Areas(19046003).
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