Effect of Acupuncture on Hypothalamic–Pituitary–Adrenal System in Maternal Separation Rats

Abstract

The maternal separation (MS) animal model has been widely used to study early life stress and several psychiatric conditions such as depression and anxiety. In this study, we investigated the effect of acupuncture on anxiety-related behaviors and hypothalamic–pituitary–adrenal (HPA) system in MS-induced early life stress of Sprague–Dawley rat pups (14–21 postnatal days). For determining anxiety-related behaviors, the elevated plus-maze test was performed. The effects of acupuncture on the activation of stress were measured by assessing plasma levels of corticosterone (CORT) and adrenocorticotropin hormone (ACTH). The hypothalamic immunoreactivity (IR) of arginine vasopressin (AVP) was also examined. Acupuncture was conducted at acupoint HT7, which is used to treat mental disorders in Oriental medicine, for seven consecutive days. Acupuncture significantly decreased the frequencies of open arm entries and the amount of time spent in the open arms in MS rats. In addition, acupuncture reduced CORT and ACTH levels in plasma of MS rats, and AVP-IR in the hypothalamic paraventricular nucleus of MS rats. In conclusion, acupuncture reduced anxiety-related behaviors and modulated the HPA system. These findings suggest that acupuncture at HT7 may be useful as a therapeutic treatment in MS-induced early life stress.

Introduction

Individuals with a history of early adverse experiences such as child abuse, social isolation, and parental loss often reveal antisocial personality symptoms with serious psychiatric problems like depression and anxiety disorders (Agid et al. 1999; Barnow et al. 2001).

Maternal separation (MS) in rats during the early postnatal period has been established as an animal model of early adverse experiences (Park et al. 2005; Veenema et al. 2006, 2007). MS rats are characterized as increase of anxiety- and depression-related behaviors (Kalinichev et al. 2002; Wigger and Neumann 1999) and dysfunction of the hypothalamic–pituitary–adrenal (HPA) system such as elevated plasma levels of corticosteron (CORT) and adrenocorticotropin hormone (ACTH) (Levine et al. 1991; Plotsky and Meaney 1993). In addition, MS has been shown to alter the activity of neuropeptide systems including arginine vasopressin (AVP) (Veenema et al. 2006, 2007).

Acupuncture therapy has long been practiced in East-Asian countries for treating various medical conditions, and has recently been considered a new alternative method of medicine in some Western countries (Cherkin et al. 2003; Kaptchuk 2002). We previously reported the effects of acupuncture at acupoint HT7 (Shenmen), which has been used to treat mental disorders in Oriental medicine (Lin 1995), on early life stress using the MS animal models. In our previous study, acupuncture at HT7 reduced anxiety-related behaviors, enhanced neuropeptide Y expression in the amygdala and hippocampus, and increased cell proliferation in the dentate gyrus of MS rats (Lim et al. 2003; Park et al. 2002, 2005). In this study, we further investigated the ability of acupuncture at acupoint HT7 on early life stress by analyzing anxiety-related behaviors, and the activation of the HPA system using the MS animal model.

Materials and Methods

Timed-pregnant Sprague–Dawley rats were provided on gestation day 17 from Orient Bio (Seoul, South Korea), housed under a 12-h light/dark cycle at a standard temperature (22 ± 3°C) with food and water freely available. On postnatal day (pnd) 14 (Lim et al. 2003; Park et al. 2002), pups were randomly assigned to one of four groups (n = 8 per group): control, acupuncture, MS, and acupuncture-treated MS groups. Pups of control and acupuncture groups were housed with their mothers under standard conditions; those of MS and acupuncture-treated MS groups were maintained individually for 7 days (pnd 14–21). All subsequent experiments were performed on pnd 21. Experimental procedures were carried out in accordance with the animal care guidelines of the National Institute for Health (NIH) Guide and the Korean Academy of Medical Sciences.

For the HT7 acupuncture stimulation (Lim et al. 2003; Park et al. 2002, 2005), stainless needles (0.2 mm diameter) were inserted about 3 mm into the left and right side at the selected acupuncture points. The needles were twisted twice a second for 30 s and then removed. The stimulated areas corresponded to acupuncture points in humans. HT7 is located at the end of the transverse crease of the ulnar wrist of the forepaw. The MS and the control groups were both slightly immobilized for 30 s. All treatments were repeated every day (pnd 14–21).

The elevated plus-maze (EPM) test was performed as described previously (Park et al. 2005). After habituated to the testing room for 20 min, each rat was placed in the center of a cross maze facing an open arm. Time spent and the numbers of entries into the open or closed arms were recorded during a 5-min test session. An entry was defined as two forepaws being on the arm. As parameters of anxiety-related behavior, the percentage of time spent in the open arms and the percentage of open arm entries were measured.

Plasma levels of CORT and ACTH were measured using Coat-A-count Rat CORT and ACTH kit (Siemens, Los Angeles, CA, USA), respectively. Blood samples were collected into ice-cold heparinized tubes, and centrifuged at 2,600×g for 10 min at 4°C. Plasma was stored at −20°C until assayed. For assessing CORT level, after adding ¹²⁵I-labeled rat CORT and plasma in CORT Ab-coated tube, the tube was incubated for 2 h at room temperature, and was then washed out. For measuring ACTH level, ¹²⁵I-labeled rat ACTH and ACTH Ab-coated tube were used. The level of ¹²⁵I-labeled CORT and ¹²⁵I-labeled ACTH was measured using a Cobra II gamma counter (Packard BioScience, Dreieich, Germany).

Rats were transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4), and then with chilled 4% paraformaldehyde. After post-fixation, the brains were cryoprotected in 30% sucrose, sectioned coronally (40 μm) on a freezing microtome and collected in cryoprotectant. Free-floating tissue sections for the hypothalamic paraventricular nucleus (PVN) were blocked with 10% normal goat serum for 1 h, and were incubated overnight with rabbit AVP antibody (Chemicon, Temecula, CA, USA). The sections were incubated with biotinylate-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), and then with Vectastatin Elite ABC kit (Vector Laboratories). The sections were treated with a DAB substrate kit (Zymed Laboratories; San Francisco, CA).

To assess co-localization of AVP and FOS in the PVN, the hypothalamic sections were first stained by mouse FOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using above mentioned procedure. Then, the sections were incubated with rabbit AVP antibody, and detection of AVP immunostaining was performed using nickel-DAB substrate kit (Zymed Laboratories).

For analysis of immunostaining, Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Bethesda, MD, USA) was used. The levels of AVP-IR were measured quantitatively in terms of optical density on the PVN region. To assess co-localization of FOS-IR and AVP-IR, the numbers of FOS- and AVP-positive cells in the same sections were counted manually under microscopy (Olympus, Japan), and the percentage of FOS-positive cells of AVP-positive cells were calculated. All data were analyzed by ANOVA with Tukey’s HSD post-hoc test using SPSS (Windows version 12.0; SPSS Inc., Chicago, IL, USA). P values less than 0.05 were deemed to indicate statistical significance. All values are expressed as the mean ± SEM.

Results

The results of the EPM test are illustrated in Fig. 1. The MS rats had a significantly lower percentage of open arm entries than the control rats. In comparison, the percentage in the acupuncture-treated MS rats increased compared to that in the MS rats (Fig. 1a). Furthermore, the MS rats spent less time in the open arms compared to the control rats, whereas the acupuncture-treated MS rats spent more time than the MS rats (Fig. 1b).

Fig. 1.

Effect of acupuncture on anxiety-related behavior of MS rats using the elevated plus-maze. Total open and closed arm entries in the EPM during a 5-min test were measured. Anxiety-related behavior is indicated by the percentage of time spent in the open arms (a) and the percentage of open arm entries (b). The vertical bars on the graph indicate the SEM; *P < 0.05, the MS group compared to the control group at each time point; ^† P < 0.05, the acupuncture-treated MS group compared to the MS group

The plasma levels of CORT and ACTH were examined in MS rats with or without acupuncture treatment (Fig. 2). CORT plasma level was elevated in MS rats (332.4 ± 20.3 ng/ml) compared to the control rats (147.0 ± 10.1 ng/ml), whereas acupuncture (131.7 ± 14.1 ng/ml) suppressed the elevation of CORT level by MS. MS also increased ACTH plasma level (246.2 ± 36.3 ng/ml in MS rats, 97.0 ± 10.1 ng/ml in control rats), and acupuncture (66.3 ± 18.0 ng/ml) reduced the increase of ACTH by MS.

Fig. 2.

Effects of acupuncture on plasma levels of CORT and ACTH in MS rats. The levels of CORT (a) and ACTH (b) were measured in plasma of MS rats with or without acupuncture treatment. The vertical bars on the graph indicate the SEM; *P < 0.05, the MS group compared to the control group at each time point; ^† P < 0.05, the acupuncture-treated MS group compared to the MS group. Independent experiments were performed twice using four or five mice per group. Con control, Acu acupuncture treatment only, MS maternal separation, MS + Acu acupuncture-treated MS

The effects of acupuncture on AVP-IR were examined in the hypothalamic PVN of MS rats (Fig. 3a, b). AVP-IR was higher in the PVN of MS rats than in that of control rats. In comparison, acupuncture reduced the AVP-IR in the PVN of MS rats.

Fig. 3.

Effects of acupuncture on AVP-IR in MS rats. The expression of AVP was detected in the hypothalamic PVN of MS rats with or without acupuncture treatment (a), and the optical density of PVN region stained by AVP was measured (b). c To characterize AVP-positive cells in the PVN, FOS, and AVP was also double-labeled. d The number of FOS-positive cells (nuclear immunoreaction) of the AVP-containing cells (cytoplasmic immunoreaction) in the PVN was counted, and was presented as the percentage. The vertical bars on the graph indicate the SEM; *P < 0.05, the MS group compared to the control group at each time point; ^† P < 0.05, the acupuncture-treated MS group compared to the MS group. Scale bars indicate 50 μm. Con control, Acu acupuncture treatment only, MS maternal separation, MS + Acu acupuncture-treated MS

FOS-IR has been extensively used as a marker of neuronal activation (Sagar et al. 1998). We double-labeled FOS and AVP in the PVN, and detected the FOS activity in the AVP-expressing cells. As shown in Fig. 3c, d, MS increased the number of the FOS-positive cells of AVP-containing cells, whereas acupuncture decreased the number in MS rats.

Discussion

In this study, acupuncture (HT7) reduced anxiety-related behaviors in MS rats, decreased CORT and ACTH levels in plasma of MS rats, and inhibited AVP-IR in the hypothalamic PVN of MS rats.

Previous studies showed that MS increased anxiety-related behaviors reducing the frequencies of open arm entries and the amount of time spent in the open arms in EPM test (Kalinichev et al. 2002; Wigger and Neumann 1999). Similar results were also observed in our MS rat. In comparison, acupuncture-treated MS rats showed less avoidance of the aversive open arms than MS rats. These results indicated that acupuncture decreased anxiety-related behaviors in MS rats.

Anxiety-related symptoms have been associated with HPA axis abnormalities (Haller et al. 2004; de Kloet 2003). Indeed, in MS rats, plasma CORT and ACTH levels were elevated being proportional to the duration of separation (Levine et al. 1991) with increased anxiety-related behaviors (Kalinichev et al. 2002; Wigger and Neumann 1999). In addition, previous studies have indicated an important role for AVP regulating neuroendocrine and anxiety-related behavior (Veenema et al. 2006; Wigger et al. 2004). At the level of the hypothalamic PVN and supraoptic nucleus, locally released AVP regulated ACTH release from the pituitary (Antoni 1993; Wotjak et al. 2002). Infusion of an AVP V1-receptor antagonist into the PVN of rats bred for high trait anxiety resulted in a decrease of anxiety behaviors (Wigger et al. 2004). Also, MS induced the increase of the hypothalamic AVP expression with an increase of anxiety-related behavior (Veenema et al. 2006, 2007). In our study, MS increased CORT and ACTH plasma levels, AVP-IR in the PVN, and FOS activity in the AVP-containing cells, whereas acupuncture reduced these increase. These results indicated that acupuncture could prevent MS-induced activation of the HPA system.

In this study, we also examined the effect of acupuncture at acupoint ST36 (Zusanli), which has been used to regulate gastrointestinal function, relieve pain, and modulate nicotine addiction (Lin 1995), in MS rats for observing the specificity of the effect of acupoint HT7. In our previous studies (Lim et al. 2003; Park et al. 2002, 2005), the acupuncture at ST36 did not show any significant effect on MS rat unlike the HT7 acupuncture. Also, in this study, the ST36 acupuncture showed no significant changes as compared to the MS group (data not shown). These results indicate that each acupuncture point exhibits different effects.

In conclusion, acupuncture at HT7 protected MS-induced anxiety-related behaviors and activation of the HPA system. These results suggest that acupuncture at acupoint HT7 may be useful as a therapy for early life stress.