lr

C

mtt

Music Exposure Induced Prolongation of Cardiac Allograft Survivaland Generated Regulatory CD4� Cells in Mice

M. Uchiyama, X. Jin, Q. Zhang, A. Amano, T. Watanabe, and M. Niimi

ABSTRACT

In clinical practice, music has been used to decrease stress, heart rate, and blood pressure andto provide a distraction from disease symptoms. We investigated sound effects on alloimmuneresponses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA,H2K) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to 1 of3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6different single sound frequencies for 7 days. An adoptive transfer study was performed todetermine whether regulatory cells were generated in allograft recipients. Cell-proliferation,cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graftexposed to opera and classical music had significantly prolonged allograft survival (mediansurvival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single soundfrequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreatedand eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively).Adoptive transfer of whole splenocytes, CD4� cells, and CD4�CD25� cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naivesecondary recipients (MSTs, 36, 68, and �50 days, respectively). Cell-proliferation, interleukin(IL)-2 and interferon-� were suppressed in opera-exposed mice, whereas IL-4 and IL-10 fromopera-exposed recipients were up-regulated. Flow cytometry studies showed an increasedCD4�CD25�Foxp3� cell population in splenocytes from opera-exposed mice. In conclusion,exposure to some types of music may induce prolonged survival of fully allogeneic cardiac

allografts and generate CD4�CD25�Foxp3� regulatory cells.

MUSIC can improve performance of reasoning tasks,1

reduce stress, enhance feelings of comfort and re-axation, provide a distraction from pain, and improve theesults of clinical therapy.2 In the transplantation field,

however, the use of music therapy remains controversial,and little is known about mechanisms by which such treat-ment might modulate alloimmune responses. The currentstudy investigated sound effects on alloimmune responses ina murine model of cardiac allograft transplantation.

METHODSHeart and Skin Transplantation, Perforation of theTympanic Membrane, and In Vitro Study

Hearts from male C57BL/6 (B6, H2b) were transplanted into maleBA/N (CBA, H2K) recipients. Heart and skin transplantation and

in vitro studies were conducted as described previously.3 Tympanicembrane perforation was conducted by means of tweezers, and

he loss of tympanic membrane was confirmed by visual observa-

ion.

0041-1345/12/$–see front matterdoi:10.1016/j.transproceed.2012.02.008

1076

Exposure to Music

CBA recipients of cardiac allografts were randomly assigned to 1 of5 groups, which were either not exposed to music (no treatmentgroup) or were exposed to opera, classical music, New Age music,or 6 different sound frequencies from the day of transplantationuntil 6 days afterward. All experiments were conducted in anenvironment in which a cycle of 12 hours of light and 12 hours ofdarkness and a room temperature of 24°C were maintained and thelevel of ambient noise (such as that produced by the air condi-tioner) was about 40 dB (the sound level in the no treatment

From the Department of Cardiovascular Surgery (M.U., A.A.),Juntendo University Hospital, Tokyo, Japan; Department ofSurgery (M.U., X.J., Q.Z., T.W., M.N.), Teikyo University, Tokyo,Japan; and Department of Cardiovascular and Thoracic Surgery(X.J.), The 4th Affiliated Hospital to Harbin Medical University,Harbin, China.

Address reprint requests to Masanori Niimi, MD, PhD, Depart-ment of Surgery, Teikyo University, 2-11-1 Kaga, Itabashi-ku,

Tokyo 173-8605, Japan. E-mail: mniimi@med.ikyo-u.ac.jp

© 2012 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 44, 1076–1079 (2012)

arIpI

(ti

agdnmd

ep(.tnsCdcefit

C

MUSIC EXPOSURE 1077

group). The source of the sound in mice exposed to either thesound frequencies or music was about 1 m from the cages housingthe animals, and the sound level was about 60 dB. In each of the 3music-exposed groups, the musical selection was played repeatedlyon a CD player with an amplifier for 24 h/d.

Adoptive Transfer Studies

Adoptive transfer studies were conducted to determine whetherregulatory cells were generated in mice exposed to opera music.Thus, 10 days after transplantation of B6 hearts into primary CBArecipients exposed to opera music for 7 days after grafting,splenocytes (5.0 � 107) from primary recipients with functioningllografts were adoptively transferred into naïve secondary CBAecipients by means of intravenous injection into the penile vein.mmediately afterward, the secondary recipients underwent trans-lantation of a B6 (donor-specific) or BALB/c (third-party) heart.n some experiments, CD4� cells were purified from the spleens of

primary transplant recipients by positive selection using a magnet-ically activated cell sorter (MACS) and CD4 microbeads (MiltenyiBiotec, Auburn, Calif, United States; purity �98%), and CD4�

cells (2.0 � 107) were adoptively transferred into naïve secondaryrecipients, which then immediately underwent transplantation of aB6 heart. In other experiments, CD4�CD25� cells were purifiedfrom the spleens of primary recipients exposed to opera using aMACS and a mouse CD4�CD25� regulatory T-cell isolation kitMiltenyi Biotec). CD4�CD25� cells (2.0 � 106) were then adop-ively transferred into naïve secondary recipients, which thenmmediately underwent transplantation of a B6 heart.

Flow Cytometry Analysis of CD4, CD25, andFoxp3 Expression

Splenocytes were obtained from naïve CBA mice and from opera-exposed and untreated cardiac allograft recipients 1, 2, and 4 weeksafter transplantation. The cells were stained with fluorochrome-conjugated anti-CD4 or anti-CD25 monoclonal antibody (mAb)(RM4–5 and PC61, respectively; BD Biosciences, San Jose, Calif)or anti-mouse Foxp3 mAb (FJK-16s; eBioscience, San Diego, Calif,United States), as well as their isotype controls (eBioscience). Thestained cells were analyzed using a fluorescence-activated cellsorting (FACS) Canto2 system (BD Biosciences). The percentageof CD4�CD25�Foxp3� in CD4� cells was determined.

Statistical Analysis

Cardiac allograft survival in the 2 experimental groups was com-pared by using log-rank test. In the cell proliferation, cytokine, andflow cytometry studies, the difference between the 2 groups wasassessed using unpaired Student t tests. P � .05 was consideredsignificant.

RESULTSSurvival of Fully Mismatched Cardiac Allografts and ofSkin Grafts in Mice Exposed to Opera Music

CBA recipients of B6 cardiac allografts that were giveneither no treatment or were exposed to 1 of 6 single soundfrequencies rejected their grafts acutely (Table 1), as didmice with tympanic membrane perforation exposed toopera (median survival time [MST], 8 days; Fig 1). Incontrast, CBA allograft recipients exposed to opera or

classical music from the day of transplantation until 6 days

fterward had significantly prolonged survival of their B6rafts (MSTs, 26.5 and 20 days, respectively; P � .001 forifference between either music groups compared with theo treatment group; Fig. 1). Mice exposed to New Ageusic had little prolongation of allograft survival (MST, 11

ays; P � .01 compared with the no treatment group; Fig 1).These results indicate that exposure to opera music mayinduce hyporesponsiveness to cardiac allografts. Moreover,B6 (donor-specific) skin grafts survived significantly longerthan BALB/c (third-party) skin grafts (MSTs, 17 and 12days, respectively; P � .01).

Generation of Regulatory Cells in Mice Exposed toOpera Music

We previously found that some anti-inflammatory or immuno-modulatory agents induce hyporesponsiveness to fully allo-geneic grafts by means of generation of regulatory cells.4–6

In the current investigation, adoptive transfer studies wereconducted to determine whether generation of regulatorycells was involved in the induction of hyporesponsiveness inthe opera-exposed mice. We found that naïve secondaryCBA allograft recipients given adoptive transfer of spleno-cytes, CD4� cells, or CD4�CD25� cells from opera-xposed primary CBA recipients 10 days after heart trans-lantation had significantly prolonged survival of B6 heartsMSTs, 36, 68, and �100 days, respectively; P � .01, P �001, and P � .005, respectively, compared with transfer ofhe 3 cell types from naïve CBA mice; Table 2). In contrast,aïve secondary CBA recipients given adoptive transfer ofplenocytes, CD4� cells, or CD4�CD25� cells from naï,BA mice rejected B6 hearts acutely (MSTs, 10, 8, and 8ays, respectively; Table 2). Moreover, when whole spleno-ytes from opera-exposed primary CBA transplant recipi-nts with functioning B6 allografts were adoptively trans-erred into naïve secondary CBA recipients that thenmmediately underwent transplantation of a BALB/c heart,he BALB/c allografts were rejected acutely (MST, 7 days).

Flow cytometry studies showed that the population ofD4�CD25�Foxp3� cells in the CD4� cells was increased

in the spleens of opera-exposed recipients compared with

Table 1. Cardiac Allograft Survival in Mice Exposed to a SingleSound Frequency

Group N Individual STs (d) MST (d)

No exposure 5 6, 7, 7, 7, 8 7Frequency

100 Hz 5 7, 7, 7, 8, 12 7500 Hz 5 7, 7, 8, 8, 10 81000 Hz 5 7, 8, 9, 9, 10 95000 Hz 5 7, 7, 8, 8, 11 810,000 Hz 5 7, 7, 8, 9, 18 820,000 Hz 5 7, 8, 8, 9, 17 8

Note: All exposures were at 60 dB; there were no significant differencesbetween the no-exposure group and any of the single-frequency groups onlog-rank testing.

Abbreviations: STs, allograft survival times; MST, median allograft survivaltime.

those of no treatment CBA mice after 4 weeks of trans-

T

(of0

amcpn5u

abb

C

1078 UCHIYAMA, JIN, ZHANG ET AL

plantation (12.15% � 0.27 vs 6.43% � 0.14; P � .001).hese data suggest that the CD4� regulatory cells con-

tained a population that was CD4�CD25�Foxp3�.

Cell Proliferation and Cytokine Production in Mice Exposedto Opera Music

Proliferation of splenocytes from CBA transplant recipientsexposed to opera was markedly suppressed compared withthat of splenocytes from untreated recipients (mean 0.017optical density (OD) � standard error of the mean (SEM)0.01 vs 0.033 OD � 0.01; P � .001). Levels of interleukinIL)-4 and IL-10 in splenocytes from CBA mice exposed topera were significantly higher than those in splenocytesrom untreated mice (12.27 ng/mL � 6.12 vs 0.59 ng/mL �.14 and 36.13 ng/mL � 21.32 vs 8.55 ng/mL � 6.03; P � .01

Fig 1. Cardiac graft survivalin CBA recipients of a B6heart that was exposed to op-era, classical, New Age music,or a single sound frequency(100 Hz) from the day of trans-plantation to 6 days afterward,untreated allograft recipients,or allograft recipients withtympanic membrane perfora-tion exposed to opera fromthe day of transplantation to 6days afterward. #P � .01,##P � .005, and ###P � .001for differences between the 2groups.

Table 2. Cardiac Allograft Survival After Adoptive Transfer ofWhole Splenocytes, CD4� Cells, or CD4�CD25� Cells

Group N Individual STs (d) MST (d)

Whole splenocytes fromprimary recipients

8 8, 10, 34, 34, 38, �100,�100, �100

36

CD4� cells from primaryrecipients

7 26, 26, 33, 68, �100,�100, �100

68

CD4�CD25� fromprimary recipients

5 32, 37, �100, �100,�100

�100

ontrolsWhole splenocytes from

naïve CBA6 8, 8, 8, 12, 14, 14 10

CD4� from naïve CBA 5 7, 7, 8, 8, 9 8CD4�CD25� from 5 6, 7, 8, 9, 10 8

naïve CBA

nd P � .05, respectively, compared with untreated CBAice). However, levels of IL-2 and interferon (IFN)-� were

onsiderably decreased in opera-exposed recipients com-ared with untreated recipients (2.14 ng/mL � 0.41 vs 7.07g/mL � 3.90 and 318.51 ng/mL � 9.09 vs 385.90 mg/mL �0.84; P � .05 and P � .05, respectively, compared withntreated CBA mice).

DISCUSSION

An increasing number of scientific studies using animalmodels to assess the physiological effects of music andsound are being conducted, mainly in the field of neurosci-ence. In the current study, CBA mice with perforatedtympanic membranes did not have prolonged allograftsurvival after exposure to opera, unlike mice with normalauditory function. On the basis of these findings, we hy-pothesize that the interaction between brain function stim-ulated by music and the autoimmune response includes thegeneration of regulatory cells. The results also indicate thatexposure to music can influence brain function, probablythrough the modulation of neurotransmitters or otherneuronal mediators. In support of this hypothesis are datashowing that music composed by Mozart may modulatedopamine release in the striatum in spontaneously hyper-tensive rats7 and that loud music potentiates methamphet-mine toxicity in mice.8 Moreover, exposure to music haseen reported to improve performance in maze learning inoth rats9 and mice,10 increase neurogenesis in the hip-

pocampus of the developing rat brain,11,12 and produce an

anxiolytic effect in mice.13,14 The possible effect of music

svreIier

eserC

J

rb

i1

i

oR

mN

nh

e2

m1

12

MUSIC EXPOSURE 1079

and sound on the immune response in murine heart trans-plantation has not previously been explored.

In the current study, 1 week of exposure to opera musicapparently induced significantly prolonged survival of fullymismatched cardiac allografts in a murine model. In con-trast, in eardrum-ruptured CBA recipients of B6 allograftsexposed to opera and CBA recipients exposed to 1 of 6single sound frequencies, the allografts were rejectedacutely. Exposure to opera also generated CD4�CD25�

regulatory cells in allograft recipients, and these cells hadsuppressive activity in mixed leukocyte cultures. Further-more, splenocytes from opera-exposed mice had increasedlevels of IL-4 and IL-10 and down-regulation of IL-2 andIFN-� in MLCs.

There are several possible mechanisms by which expo-ure to opera might have induced increased allograft sur-ival in our model. The first possible mechanism of ouresults is the anti-inflammatory effect of music. Operaxposure of transplant recipients induced up-regulation ofL-10 in their splenocytes in cultures. IL-10 has anti-nflammatory and suppressive effects on most hematopoi-tic cells and is thus involved in the control of immuneesponses.15 In these findings, it seems possible that opera

music may inhibit immune responses against allografts.A second mechanism is production of immuno-modulatory

effects by the specific harmony and/or other features of themusic itself. In our model, none of 6 single sound frequen-cies were associated with prolongation of allograft survivaland also eardrum-ruptured CBA mice rejected B6 cardiacgrafts acutely. Previous studies have indicated that fre-quency is represented explicitly and predominantly in indi-vidual neurons in the human auditory cortex.16 However,little is known about possible mechanisms by which a singlesound frequency might modulate alloimmune responses.

A third mechanism is that the exposure resulted ingeneration of regulatory cells. In our adoptive transferstudies, most naïve secondary CBA recipients given wholesplenocytes, CD4� cells, and CD4�CD25� cells from opera-xposed primary CBA recipients had significantly prolongedurvival of their B6 cardiac grafts. These data suggest thatxposure to opera generated regulatory cells in the primaryecipients and that the regulatory population contained

D4�CD25� cells.

fc

In conclusion, exposure to some types of music mayinduce prolonged survival of fully allogeneic cardiac allo-grafts and generate CD4�CD25�Foxp3� regulatory cells.

REFERENCES

1. Rauscher FH, Shaw GL, Ky KN: Music and spatial taskperformance. Nature 365:611, 1993

2. Kemper KJ, Danhauer SC: Music as therapy. South Med J98:282, 2005

3. Iwami D, Zhang Q, Aramaki O, et al: Purified eicosapenta-enoic acid induces prolonged survival of cardiac allografts andgenerates regulatory T cells. Am J Transplant 9:1294, 2009

4. Inoue F, Zhang Q, Akiyoshi T, et al: Prolongation of survivalof fully allogeneic cardiac grafts and generation of regulatory cellsby a histamine receptor 2 antagonist. Transplantation 84:1288,2007

5. Aramaki O, Takayama T, Yokoyama T, et al: High dose ofantithrombin III induces indefinite survival of fully allogeneiccardiac grafts and generates regulatory cells. Transplantation 75:217, 2003

6. Yokoyama T, Aramaki O, Takayama T, et al: Selectivecyclooxygenase 2 inhibitor induces indefinite survival of fullyallogeneic cardiac grafts and generates CD4� regulatory cells.

Thorac Cardiovasc Surg 130:1167, 20057. Sutoo D, Akiyama K: Music improves dopaminergic neu-

otransmission: demonstration based on the effect of music onlood pressure regulation. Brain Res 1016:255, 20048. Morton AJ, Hickey MA, Dean LC: Methamphetamine toxic-

ty in mice is potentiated by exposure to loud music. Neuroreport2:3277, 20019. Rauscher FH, Robinson KD, Jens JJ: Improved maze learn-

ng through early music exposure in rats. Neurol Res 20:427, 199810. Aoun P, Jones T, Shaw GL, et al: Long-term enhancement

f maze learning in mice via a generalized Mozart effect. Neuroles 27:791, 200511. Amagdei A, Baltes FR, Avram J, et al: Perinatal exposure tousic protects spatial memory against callosal lesions. Int J Deveurosci 28:105, 201012. Kim H, Lee MH, Chang HK, et al: Influence of prenatal

oise and music on the spatial memory and neurogenesis in theippocampus of developing rats. Brain Dev 28:109, 200613. Li W, Yu H, Yang J, et al: Anxiolytic effect of music

xposure on BDNFMet/Met transgenic mice. Brain Res 1347:71,01014. Chikahisa S, Sano A, Kitaoka K, et al: Anxiolytic effect ofusic depends on ovarian steroid in female mice. Behav Brain Res

79:50, 200715. Moore KW, de Waal Malefyt R, Coffman RL, et al: Interleukin-

0 and the interleukin-10 receptor. Annu Rev Immunol 19:683,00116. Bitterman Y, Mukamel R, Malach R, et al: Ultra-fine

requency tuning revealed in single neurons of human auditoryortex. Nature 451:197, 2008