In the current study, one week of exposure to opera and Mozart music apparently induced much more significantly prolonged survival of fully MHC-mismatched cardiac allografts in a murine model compared with New Age music Enya. In contrast, CBA recipients exposed to one of six single sound frequencies or pre-treatment rejected acutely. According to above data, opera music itself may not induce Treg and also music genre may be unrelated to the graft results. However, CBA pre-treated with tympanic membrane perforation rejected acutely, suggesting that auditory brain function may play an important role of Treg induction and graft prolongation.

There are several possible mechanisms by which exposure to opera might have induced increased allograft survival in our model. One is that the exposure resulted in generation of regulatory cells. Acquisition of hyporesponsiveness to an allograft is a dynamic, multistep process involving many mechanisms, including immune regulation, deletion, anergy, and ignorance [22]. Among these, immune regulation, the control of alloimmune responses by regulatory cells, is considered as one of the most important. Active suppression by regulatory cells is involved in the induction and maintenance of self-tolerance [23] and unresponsiveness to allografts [24]. In our adoptive transfer studies, most naïve secondary CBA transplant recipients given splenocytes from opera-exposed primary CBA recipients with functioning B6 cardiac allografts had significantly prolonged survival of their allografts (MST, 36 days). Furthermore, adoptive transfer of CD4+ or CD4+CD25+ cells from opera-exposed primary transplant recipients resulted in longer or indefinite prolongation of allograft survival in secondary recipients (MSTs, 68 and > 100 days, respectively). These data suggest that exposure to opera generated regulatory cells in the primary recipients and that the regulatory population contained CD4+CD25+ cells. In addition, flow cytometry studies showed that the number of CD4+CD25+Foxp3+ cells and the percentage of CD4+CD25+Foxp3+ cells in CD4+ cells were increased in the primary allograft recipients.

Moreover, our MLC finding of up-regulation of IL-10 production by splenocytes in opera-exposed allograft recipients suggests that IL-10 contributed to the generation of regulatory cells. IL-10 has anti-inflammatory and suppressive effects on most hematopoietic cells, and it plays a crucial role not only in the function of regulatory cells but also in their generation [25]. We previously demonstrated the importance of IL-10 in generating regulatory cells in our murine cardiac transplantation model [26]. Thus, it is probable that in the current study, it was through the up-regulation of IL-10 that exposure to opera resulted in induction of CD4+CD25+ regulatory cells. Also, an anti-inflammatory effect may be induced through regulatory cells. Our histologic studies of allografts obtained from opera-exposed mice showed only minimal leukocyte infiltration. Furthermore, we observed that opera exposure in transplant recipients induced suppression of IL-2 and IFN-γ production and up-regulation of IL-4 and IL-10 in their splenocytes. IL-10 has anti-inflammatory and suppressive effects on most hematopoietic cells and is thus involved in the control of immune responses [27]. In the light of these findings, it appears possible that opera-induced regulatory cells may inhibit immune responses against allografts.

A second possible mechanism for the opera-induced hyporesponsiveness is the effects on brain function produced by the specific harmony and/or other features of the music itself. In our model, exposure to none of six single sound frequencies was associated with prolongation of allograft survival. Moreover, CBA recipients pre-treated with tympanic membrane perforation exposed to opera music rejected their graft acutely. Previous studies have indicated that frequency discrimination in humans is correlated with several cognitive skills, including facility with language [28], working memory [29], and ability to learn [30]. Moreover, frequency is represented explicitly and predominantly in individual neurons in the human auditory cortex [31]. However, little is known about possible mechanisms by which a single sound frequency might modulate alloimmune responses, and it has not been well determined that whether hearing loss induced by some immunosuppressants such as tacrolimus affects the graft survival [32]. In a previous study, exposure to music significantly enhanced levels of brain-derived neurotrophic factor and decreased levels of nerve growth factor in the hypothalamus of mice [33]. These findings suggest that music can influence brain function and development and that auditory stimulation from music, including specific harmony, may affect allograft survival.

A decrease in postoperative stress brought about by exposure to music is a third possible mechanism for the opera-induced hyporesponsiveness observed in our study. Stress has an important role in the development of symptoms and disease [34, 35]. Zhuang et al. [36] found that endogenous stress caused by heterotopic heart transplantation contributed to postoperative cardiac injury and allograft vasculopathy in rats. In experiments in hypertensive rats, exposure to music composed by Mozart significantly decreased heart rate and had no effect on blood pressure, thereby producing a small decrease in cardiac output, whereas music composed by Ligeti significantly increased blood pressure but reduced heart rate. The effects of music could not be attributed to a stress reaction because stress caused by switching cages increased both heart rate and blood pressure in the animals [37].

A fourth possible mechanism for the opera-induced hyporesponsiveness in our model is that exposure to music increased, rather than decreased, stress. Numerous studies have found that stress can suppress the immune response and thereby be detrimental to health [38]. In a study in mice, Wistar and Hildemann [39] found that chronic avoidance-learning stress depressed the immune reaction responsible for rejection of skin homografts to a moderate but significant degree. In another murine model, Nùñez et al. [40] observed that a chronic auditory stressor induced a significant reduction in both natural killer cell activity and in vivo and in vitro generation of cytotoxic T lymphocytes. In a rat model, however, neutrophils and macrophages from animals exposed to noise for a short period secreted significantly less superoxide anion and IL-1 than cells from control rats, but lymphocyte function remained unchanged [41]. In our model, the presence of music-induced stress was unlikely because the mice exposed to opera did not lose hair or weight and the mean weight of their adrenal glands at sacrifice was not different from that of mice in the no-treatment group (data not shown).