Neuroscience Letters 484 (2010) 4750

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Acupuncture is effective to attenuate stress and stimulate lymphocyte

proliferation in the elderly
Tiago S. Pavo b , Priscila Vianna c , Micheli M. Pillat b , Amanda B. Machado b , Moiss E. Bauer a,b,
a
b
c

Faculty of Biosciences, Pontical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre (RS), Brazil
Laboratory of Cellular and Molecular Immunology, Institute of Biomedical Research, Pontical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre (RS), Brazil
Department of Genetics, Federal University of Rio Grande do Sul, Porto Alegre (RS), Brazil

a r t i c l e

i n f o

Article history:
Received 24 June 2010
Received in revised form 27 July 2010
Accepted 6 August 2010
Keywords:
Acupuncture
Psychological stress
Proliferation
Glucocorticoids

a b s t r a c t
Acupuncture has increasingly been used to treat many conditions, including psychiatric disorders
and immunological-related disorders. However, the effects of acupuncture as stress management and
immune functions in the elderly are largely unclear. Here we investigated the effects of acupuncture on
stress-related psychological symptoms and cellular immunity in young adults and elderly subjects. The
acupuncture treatment consisted of six sessions and the procedures included the insertion of needles
at bilateral acupoints LI4, SP6 and ST36. Psychological variables (depression, anxiety and stress) were
investigated by means of self-assessment inventories. Peripheral blood mononuclear cells were isolated
and cultured in vitro to measure mitogen-induced T-cell proliferation as well as cellular sensitivity to
dexamethasone. All data were assessed before and after the intervention. Acupuncture was able to signicantly reduce depression (p < 0.001), anxiety (p < 0.001) and stress (p < 0.001) scores. The intervention
also increased T-cell proliferation, with greater intensity in the elderly group (p = 0.004). No changes in
cellular sensitivity to dexamethasone were observed following acupuncture. We conclude that acupuncture was efcient to attenuate the psychological distress as well as to increase an important feature of
cellular immunosenescence.
2010 Elsevier Ireland Ltd. All rights reserved.

Acupuncture is certainly the most popular intervention of traditional Chinese medicine in western countries. It has been used for
treating many diseases, including pain, asthma and major depression [13,20]. Both experimental and clinical studies indicated that
electrical or applied acupuncture to certain acupoints, including
the SP6, was effective to attenuate anxiety [8]. An important eld of
interest is thus the possibility to inuence the quality of life through
interventions with acupuncture.
Human aging has been associated with signicant psychological distress and related behavioral changes that certainly impact
the quality of life of elderly subjects. We have previously demonstrated that strictly healthy (SENIEUR) elders were signicantly
more distressed in parallel with increased daily cortisol levels
[4,17]. Changes in lymphocyte subsets [9], reduced T-cell proliferation and lower cellular sensitivity to steroids [3] have been also
observed and associated with psychoneuroendocrine changes. The
immunosenescence may be closely related to chronic stress and

Corresponding author at: Laboratory of Cellular and Molecular Immunology,

Institute of Biomedical Research, Hospital So Lucas - PUCRS, Av. Ipiranga 6690,
2 andar PO Box 1429, 90.610-000 Porto Alegre, RS, Brazil.
Tel.: +55 51 3320 3000x2725; fax: +55 51 33203312.
E-mail address: mebauer@pucrs.br (M.E. Bauer).
0304-3940/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2010.08.016

stress factors during healthy aging [4]. Therefore, interventions

aimed to increase cellular immune functions in the elderly are
required.
Acupuncture is associated with signicant immunological
changes. It has been shown that electro- or applied stimulation
on specic acupoints (e.g. LI4 or ST36) produced both enumerative and functional cellular immune changes. For instance, applied
acupuncture has been associated with either signicant increase
[22] or decrease [12] in major lymphocyte subsets in adults. Applied
stimulation on ST36, BL18, BL20 and BL23 acupoints has been
associated with higher intracellular levels of IL-4, IL-1 and IFN-
[22]. A recent study demonstrated that applied acupuncture significantly increased mitogen-induced lymphocyte proliferation and
natural killer (NK) cell activity in anxious women 1 month following the end of the intervention [2]. However, the effectiveness
of acupuncture in promoting relaxation and immunoregulation in
elderly individuals has not been addressed yet. Here, we investigated the effects of repeated applied acupuncture on psychological
variables and lymphocyte proliferation in healthy adults and
elders.
Twenty-four healthy subjects were recruited at the Center for
Physical Activities Terra Brasilis (Porto Alegre, Brazil) and subgrouped into 12 young adults (mean age 27.6 years, 2338 years; 6
females) and 12 healthy elders (mean age 65.6 years, 6081 years; 6

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T.S. Pavo et al. / Neuroscience Letters 484 (2010) 4750

females). All participants gave their written informed consent and

the protocol was approved by the ethics committee of the Pontical
Catholic University of Rio Grande do Sul (PUCRS, #07-03877).
Applied acupuncture was performed bilaterally into acupoints
chosen according to previous studies [8,18,22]: SP6 (Sanyinjiao:
located on the inside of the ankle, three nger-breadths above
the ankle bone), LI4 (Hegu: located on the dorsum of the hand,
between the rst and second metacarpal bones) and ST36 (Zusanli:
located one nger breadth lateral to the tibias anterior crest). The
intervention consisted of six sessions of acupuncture performed
in each individual twice weekly during three consecutive weeks.
Briey, disposable acupuncture needles (30 mm, 160 m in diameter) were inserted into the acupoints and manipulated every 5 min
to induce the special needle sensation (DeQi) [12]. During the interventions, the subject was lying on a stretcher in supine position.
Data was collected before and 48 h following the last acupuncture
session.
The psychological status was assessed by clinical interviews performed by a trained investigator. The stress levels were assessed by
the Inventory of Stress Symptoms Lipp for adults (ISSL) [14]. This
inventory has been used to evaluate current or past stress symptoms in both young and elderly populations [9,11]. The scoring was
performed by means of three different stages related to the duration
(Q1 = last 24 h, Q2 = last week and Q3 = last month) and intensity
of stress symptoms. The State-Trait Anxiety Inventory (STAI) was
employed as self-report for generic measures of anxiety [5]. STAI
is a 20-item assessment device which includes separate measures
of state and trait anxiety. The beck depression inventory (BDI) was
employed as self-report scale for assessing depression levels [10].
BDI consists of 21 items to assess the intensity of depression in
clinical and normal patients. The KuderRichardson reliability coefcients for these scales were always higher than 0.90.

Ten millilitres of peripheral blood were collected by venepuncture in the morning and stored in heparinized tubes prior to
analyses. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and cell viability was
always higher than 95%. PBMCs were cultured (3 106 cells/mL)
in RPMI-1640 medium supplemented with 10% fetal calf serum for
96 h at 37 C in 5% CO2 atmosphere. PBMCs were stimulated in vitro
with phytohemagglutinin (PHA, Gibco, USA) to assess T-cell proliferation. PBMCs sensitivity to glucocorticoids was estimated by
functional assays developed to measure the ability of dexamethasone (DEX) to suppress T-cell proliferation. Data are presented as
percentage of basal proliferation (stimulated without steroids). The
proliferative responses were determined by MTT assays as previously described [16].
Psychological assessments were performed by two-way ANOVA
that included two between-subjects variables (Group Time) and
one within-subjects variable (stress, anxiety or depression scores).
Proliferation data were analyzed by repeated measures ANOVA that
included two between-subjects variables (Group Time) and one
within-subjects variable (mitogen or DEX concentration). Multiple
comparisons among levels were checked with Tukey post hoc test.
Differences were considered statistically signicant when p < 0.05.
Data are expressed as mean SE.
Mitogen-induced T-cell proliferation was evaluated as an index
of cell-mediated immunity (Table 1). The elderly group had a
signicantly lower proliferation than young adults at baseline,
F(1,22) = 6.89, p = 0.01. However, elderly and young adults had similar lymphocyte sensitivity to dexamethasone in vitro at baseline
(data not shown), F(1,19) = 0.40, p = 0.53.
Here, we demonstrate that acupuncture was highly effective
to reduce psychosocial distress in both young and elderly populations (Fig. 1). In particular, stress (Q2 and Q3, all p < 0.0001),

Fig. 1. Psychological assessments following six sessions of acupuncture. (A and B) Stress scores were assessed in the last 24 h (Q1), last week (Q2) and last month (Q3).
Depression (BDI) and anxiety (STAI) scores are shown in gures C and D. The data were measured before (Pre) and after 6 sessions of acupuncture (Post). BDI, beck depression
inventory; STAI, State-Trait Anxiety Inventory. Signicant differences are shown: *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Pre.

T.S. Pavo et al. / Neuroscience Letters 484 (2010) 4750

Table 1
T-cell proliferation at baseline.
Young adults (n = 12)

Unstimulated (0)
PHA (0.5%)
PHA (1%)
PHA (2%)

Elderly (n = 12)

Mean

SE

Mean

SE

0.09
0.25
0.23
0.18

0.01
0.03
0.03
0.02

0.07
0.17
0.14
0.11

0.01
0.01
0.01
0.01

NS
<0.01
<0.01
<0.01

Note: BDI, beck depression inventory; Q1, stress last 24 h; Q2, stress last week; Q3,
stress last month; STAI, State-Trait Anxiety Inventory. PHA = phytohemagglutinin.

anxiety (STAI-Trait or STAI-State, all p < 0.0001) or depression (BDI,

p < 0.0001) scores were found similarly reduced in both young and
elderly groups following the intervention. The magnitude of stress
management ranged from 15% to 47% reductions in all analyzed
scores. There was a signicant Group Time interaction (p < 0.05)
in Q1 variable, indicating that acupuncture was more effective to
reduce stress in the last 24 h among the young adults (47%) as
compared to elderly subjects (28%).
We next assessed the effects of acupuncture on lymphocyte
proliferation, a pivotal function of adaptive immunity. Although
acupuncture was unable to change the mitogen-induced T-cell proliferation in the young group (Fig. 2A; F(1,22) = 1.07, p = 0.31), we
observed higher proliferative responses in the elderly group following the intervention (Fig. 2B), F(1,22) = 10.48, p = 0.004. Notably, the
cell proliferation of the elderly group reached similar levels of those
found in the young adults. The magnitude of acupuncture-induced
increments in cell proliferation varied from 42% to 52%. Young and
elderly subjects had similar cellular sensitivity to DEX following the
intervention (Fig. 3), p = 0.81 and p = 0.25, respectively.

49

Previous studies have demonstrated that applied or electroacupuncture can attenuate psychological distress in healthy or
depressed adults [1,7,15,21]. Our results indicate for the rst
time that repeated applied acupuncture can efciently reduce
stress, anxiety and depressed symptoms in healthy elders as
well. The magnitude of acupuncture-induced global relaxation
was the same in both cohorts analyzed, with the exception of
Q1 scores that were found further reduced in young adults.
We could speculate that young adults had a more important
attenuation of this parameter due to the higher psychological
morbidity found in the pre-intervention time than the elderly
group. The acupoints chosen in this study are known to attenuate anxiety/pain (SP6) [8] or to produce immunological changes
(LI4 or ST36) [12,18,22] in healthy adults. The mechanisms
underlying the inuence of acupuncture to attenuate psychological distress have, as yet, to be claried. Acupuncture may
exert its effects by inuencing neurotransmitter and hormonal
pathways underlying emotional states. Indeed, acupuncture stimulation leads to secretion of serotonin and norepinephrine [6],
two monoamine neurotransmitters implicated in the etiology of
depression. Furthermore, acupuncture may also stimulate sensory
nerves, induce the release of endogenous opioids and modulate
the autonomic nervous system [2] and, perhaps, other hormone
functions inuencing mood. Future studies should investigate
whether repeated applied acupuncture can also reduce peripheral stress factors. A pilot study demonstrated that cortisol and
norepinephrine plasma levels were found unchanged following
applied acupuncture in adults [12]. We next assessed whether
acupuncture could also modulate cellular immune functions in the
elderly.

Fig. 2. Effects of acupuncture on T-cell proliferation. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated in vitro with medium (0) or phytohemagglutinin
(PHA) for 96 h. Cell proliferation/viability was estimated by MTT assays before (Pre) and following the acupuncture (Post). The optical density (OD) was determined at a
wavelength of 570 and 630 nm. Signicant differences are shown: *p < 0.05, **p < 0.001 vs. Pre.

Fig. 3. Effects of acupuncture on cellular sensitivity to dexamethasone (DEX). The cellular sensitivity to DEX is presented as percentage of basal proliferation (0 = PHA 1%
without steroids). Cell proliferation/viability was estimated by MTT assays before (Pre) and following the acupuncture (Post). The optical density (OD) was determined at a
wavelength of 570 and 630 nm.

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T.S. Pavo et al. / Neuroscience Letters 484 (2010) 4750

Lymphocyte proliferation is a pivotal feature of adaptive cellular

immune responses and it was mimicked here by mitogen-induced
proliferation assays. Here, we found that healthy elderly subjects
had a blunted T-cell proliferation when compared to young adults
at baseline, corroborating previous studies [3,16]. This is a hallmark
of mammal immunosenescence and directly related to impaired
immunity observed in elderly populations. We also showed that
acupuncture can modulate this important age-related feature. At
the end of six sessions of acupuncture we observed a signicant
increase in the T-cell proliferation in the elderly, without reciprocal changes in the young adults. Notably, the cell proliferation
of the elderly group reached similar levels of those found in the
young adults. These data are in accordance to a previous study
showing increased PHA-induced proliferation in anxious women
following applied acupuncture [2]. Future studies should explore
the underlying mechanisms of this important positive change.
It is tempting to speculate that acupuncture may increase IL-2
secretion, a crucial cytokine for T-cell proliferation. This change
could be also associated with increased counts of peripheral T cells
observed following the acupuncture [22]. Taken together, these
data suggest that acupuncture may positively change the process
of immunosenescence.
Here we did not observe signicant changes in the cellular sensitivity to a synthetic glucocorticoid (DEX). We initially hypothesized
that reduced stress symptoms could be associated with a parallel
increase in cellular sensitivity to glucocorticoids. This hypothesis
was not conrmed here. However, corroborating these ndings, we
have recently shown that relaxation and visualization therapy was
effective in alleviating the psychological distress in breast cancer
patients without changing salivary cortisol levels or cellular sensitivity to glucocorticoids [19]. As cellular responses to steroids in
vitro are inversely correlated to peripheral cortisol levels [3,19], it
is tempting to speculate that acupuncture is not capable to change
glucocorticoids [12].
In conclusion, we demonstrated that repeated applied acupuncture was effective to attenuate stress and stimulate lymphocyte
proliferation in both young and elderly populations. However, it
should be taken into account that acupuncture session itself may
inuence the subjective state of the individual and would theoretically confound the ndings. To demonstrate denitive usefulness of
acupuncture stimulation, double-blind, placebo-controlled studies
with a large number of subjects should be required in the future.
Future studies should examine whether these changes are longlasting, to investigate the underlying mechanisms and their clinical
relevance. A recent study demonstrated that several immunological changes are still observed 1 month following the end of repeated
applied acupuncture [2].
Acknowledgements
This work was supported by grants from CNPq and CAPES.

References
[1] J. Allen, R. Schnyer, A. Chambers, S. Hitt, F. Moreno, R. Mamber, Acupuncture for depression: a randomized controlled trial, J. Clin. Psychiatry 67 (2006)
16651667.
[2] L. Arranz, N. Guayerbas, L. Siboni, M. De la Fuente, Effect of acupuncture treatment on the immune function impairment found in anxious women, Am. J.
Chin. Med. 35 (2007) 3551.
[3] M.E. Bauer, Stress, glucocorticoids and ageing of the immune system, Stress 8
(2005) 6983.
[4] M.E. Bauer, C.M. Jeckel, C. Luz, The role of stress factors during aging of the
immune system, Ann. N. Y. Acad. Sci. 1153 (2009) 139152.
[5] A. Biaggio, Manual: IDATE, inventrio de ansiedade Traco Estado, CEPA, Rio
de Janeiro, 2003.
[6] S. Ceniceros, G.R. Brown, Acupuncture: a review of its history, theories, and
indications, South Med. J. 91 (1998) 11211125.
[7] B.H. Chang, U. Boehmer, Y. Zhao, E. Sommers, The combined effect of relaxation
response and acupuncture on quality of life in patients with HIV: a pilot study,
J. Altern. Complement. Med. 13 (2007) 807815.
[8] H.-M. Chen, C.-H. Chen, Effects of acupressure at the Sanyinjiao point on primary dysmenorrhoea, J. Adv. Nurs. 48 (2004) 380387.
[9] D. Collaziol, C. Luz, F. Dornelles, I. Cruz, M. Bauer, Psychoneuroendocrine correlates of lymphocyte subsets during healthy ageing, Mech. Ageing Dev. 125
(2004) 219227.
[10] C. Gorenstein, L. Andrade, Validation of a Portuguese version of the beck depression inventory and the state-trait anxiety inventory in Brazilian subjects, Braz.
J. Med. Biol. Res. 29 (1996) 453457.
[11] C.M. Jeckel, R.P. Lopes, M.C. Berleze, C. Luz, L. Feix, I.I. Argimon, L.M.
Stein, M.E. Bauer, Neuroendocrine and immunological correlates of chronic
stress in strictly healthy populations, Neuroimmunomodulation 17 (2009)
918.
[12] W. Kou, J.D. Bell, I. Gareus, G. Pacheco-Lopez, M.U. Goebel, G. Spahn, M.
Stratmann, O.E. Janssen, M. Schedlowski, G.J. Dobos, Repeated acupuncture
treatment affects leukocyte circulation in healthy young male subjects: a randomized single-blind two-period crossover study, Brain Behav. Immun. 19
(2005) 318324.
[13] R.J. Leo, J.S. Ligot Jr., A systematic review of randomized controlled trials
of acupuncture in the treatment of depression, J. Affect. Disord. 97 (2007)
1322.
[14] M. Lipp, Manual do Inventrio de Sintomas de Stress para Adultos, Casa do
Psiclogo, So Paulo, 2000.
[15] H. Luo, F. Meng, Y. Jia, X. Zhao, Clinical research on the therapeutic effect of
the electo-acupuncture treatment in patients with depression, Psychiatry Clin.
Neurosci. 52 (1998) 338340.
[16] C. Luz, D. Collaziol, T. Preissler, I.M. da Cruz, L. Glock, M.E. Bauer, Healthy aging
is associated with unaltered production of immunoreactive growth hormone
but impaired neuroimmunomodulation, Neuroimmunomodulation 13 (2006)
9099.
[17] C. Luz, F. Dornelles, T. Preissler, D. Collaziol, I.M. da Cruz, M.E. Bauer, Impact of
psychological and endocrine factors on cytokine production of healthy elderly
people, Mech. Ageing Dev. 124 (2003) 887895.
[18] H. Mori, K. Nishijo, H. Kawamura, T. Abo, Unique immunomodulation by
electro-acupuncture in humans possibly via stimulation of the autonomic nervous system, Neurosci. Lett. 320 (2002) 2124.
[19] D.F. Nunes, A.L. Rodriguez, F. da Silva Hoffmann, C. Luz, A.P. Braga Filho,
M.C. Muller, M.E. Bauer, Relaxation and guided imagery program in patients
with breast cancer undergoing radiotherapy is not associated with neuroimmunomodulatory effects, J. Psychosom. Res. 63 (2007) 647655.
[20] P.H. Richardson, C.A. Vincent, Acupuncture for the treatment of pain: a review
of evaluative research, Pain 24 (1986) 1540.
[21] B.M. Shuaib, M.F. Haq, Electro-acupuncture treatment in psychiatry, Am. J. Chin.
Med. 5 (1977) 8590.
[22] N. Yamaguchi, T. Takahashi, M. Sakuma, T. Sugita, K. Uchikawa, S. Sakaihara, T.
Kanda, M. Arai, K. Kawakita, Acupuncture Regulates Leukocyte Subpopulations
in Human Peripheral Blood, Evid. Based Complement. Altern. Med. 4 (2007)
447453.