In this paper, we employed functional connectivity analysis methods with a new NRER-fMRI design to investigate the sustained effects of acupuncture. In most previous fMRI studies, the model-dependent analysis methods using a block design require prior knowledge of event timing from which an anticipated hemodynamic response can be modeled. However, this type of analysis methods cannot be used without a predictable hemodynamic response reflecting the actual BOLD signal changes induced by acupuncture. In the current study, we used a model-independent approach, i.e. the new NRER-fMRI design, to explore systematic changes of the BOLD signal without a decrease in the statistical power and the bias on the results.
Our work presents the first fMRI connectivity study of acupuncture using a modular approach in connectivity analysis , followed by a more recent connectivity study on the defaut mode network . We have defined a resting-state brain network that is associated with an amygdalar region activated during acupuncture stimulation (Fig. 4). Our results further showed that the amygdalar-specific network consisting of brain regions overlapped with the pain-matrix to some extent. These regions include extensive areas in the SI and SII, insula, ACC and PFC as well as the hypothalamus and PAG (Table 1). While these brain regions were activated by sham or acupuncture stimulation, the corresponding network associated with sham could be dissociated from acupuncture modulation effects by differentiating functional connectivity patterns among these regions (Fig. 4).
As the amygdala plays a dual role of facilitating as well inhibiting in the modulation of pain behavior and nociceptive processing at different levels of the pain matrix, the amygdala-associated network during the resting state may be crucial in both pain and analgesia systems implicated in the effects of acupuncture stimulation [2, 20, 34]. Many previous studies have found a basic network derived from the important concept of "default mode" in the resting brain [10, 35]. Within this default mode, the temporal correlation of the fMRI signal during a resting-state provides complementary information about the intrinsic interaction between different brain regions. With such a baseline, the verum acupuncture- or sham-induced changes in the temporal correlation may represent the modulation of region-to-region interactions. It should be noted that the low-frequency temporal correlations in the resting state are also related to uncontrolled brain activities . Continuous stimulation during ACUP or SHAM may result in a higher degree of low frequency correlation than the "resting" uncontrolled stimulus state in these regions, which may provide an explanation for the increase in connectivity in both sham and acupuncture conditions (Figs. 4, 5).
The change in the region-to-region connectivity is an indication of functional changes of the network, and may provide complementary information for exploring modulating effects of acupuncture or sham on the network of interest (Figs. 4, 5). Based on the pain-related network in the resting brain, we can expect that either verum or sham acupuncture (using pain-related stimulation) may modulate the functional connectivity in some specific brain regions implicated in this network. The connectivity network during the post-acupuncture condition was similar to the sham except for more extensive and stronger connectivity in the limbic system. Furthermore, since the procedure during SHAM intervention, as a whole, is the similar to that of ACUP and all of subjects are naïve to acupuncture, we expect that SHAM is believed to be the same procedure as verum acupuncture to our subjects in terms of potential placebo effects of pain relief. Therefore, the observed differences between these two conditions may constitute a specific physiological effect. Comparing ACUP and SHAM, we found connectivity increases specifically in the PAG and INS (Fig. 4). The PAG has abundant opiate receptors and participates in both opioid analgesia and acupuncture analgesia [5, 8]. In addition, a recent PET study involving patients in pain, has clearly identified a hyperactivation of the ipsilateral insula, suggesting a specific neural structure underpinning the effect of acupuncture for the treatment of chronic musculoskeletal pain. The insula is a key modulator of the visceromotor system. The increases in the connectivity of the amygdala with both the insula as well PAG indicate that the uncrossed visceroceptive autonomic pathways may be engaged, which seems to be crucial for acupuncture analgesia effects reflecting its specific action on the central nervous system.
It is now believed that sham does have a physiological effect, as well as many of the central neural substrates may be involved in the sham-related pain sensation. Compared to the ACUP, an increased connectivity was shown among the MFC, PCG (and the SII) and PCC during the SHAM. The MFC has been shown to be involved in the modulation of pain by regulating attention and affective emotion [8, 9]. The MFC and PCC may also transform the memory or sensory information to assign meaning to pain, and subserve planning and execution of coping strategies. Previous imaging studies have shown that the SII and PCC have connections to memory-related temporal-lobe structures and the motor system, suggesting that these cortices may contribute to learning and memory of pain, as well as to pain-motor integration [37–40]. In addition, the PCC near the 'unpleasantness region' is associated with response selection, conflict monitoring and attention, which is considered to be more reliably activated by pain [37–39]. Therefore, the increased connections with the amygdala shown in the SII and PCC during SHAM may be due to more intense sensations commonly induced by sham stimulation. As a result, we speculate that the action of sham may involve non-specific effects supporting both sensory and affective pain perception. Although no significant statistical differences between acupuncture and sham scores on subjects' perceptions of sensations (P < 0.05), the sharp pain levels showed an elevated tendency during the sham stimulation (paired t-test, P < 0.07), primarily due to the low scores (typically less than 1) measured during the acupuncture stimulation.
The involvement of the ACC in both sham and acupuncture is an interesting finding in our study. The functional connectivity results are consistent with the previous neuroanatomical and electrophysiological findings that the efferents of the amygdala have bidirectional relations with the ACC [41–43]. Although a considerable volume of literature documented the role of the ACC in autonomic regulation and emotion , some studies pointed out that a pain response as shown in the ACC might be associated with the "suffering" component of pain  or with the opioid pathway [46, 47]. Placebo effects following conditioning with surreptitious variation of heat pain can induce decreased activity within the ACC. We hypothesized that placebo analgesia may arise from changes in the expectation of pain within higher cognitive centers such as the ACC. The ACC contains a high concentration of opioid receptors , and has been regarded as a functional region in opioid analgesia and in other forms of pain modulation [49, 50], which may suggest a similar involvement of higher order control of opioid-dependent placebo analgesia. We therefore suggest that the ACC activation found in both ACUP and SHAM is linked to the expectation of therapeutic benefit and exerts a top-down effect on the midbrain which is at the same site of the PAG activation reported by Petrovic et al . Previous studies also showed that acupuncture might stimulate both pain modulation and analgesia systems by releasing endogenous opioids . However, there was no significant difference in the amygdala-ACC connectivity between SHAM and ACUP (Fig. 4.), suggesting that the ACC may not mediate the effects specific to the sustained effects of acupuncture, but participate in non-specific components such as expectation and pain-related affective processes.
The activation of the insula has been reported in previous acupuncture studies [9, 53, 54]. The increased connectivity of the insula in our study is consistent with electrophysiological studies and clinical investigations , which showed the insula's involvement in emotional processing (fear, uneasiness, etc) and ascending visceral symptoms . The involvement of the insula in the post-acupuncture functional network is also consistent with TCM's viewpoint that the known healing effects of Zusanli (ST36) acupuncture on gastroenteric disorders such as gastroenteritis and gastroenteric spasm may be mediated through the insular visceral feedback pathway. Furthermore, the unaltered connectivity of the ACC may therefore suggest that the expectation during the treatment may have a physiological effect on the brain, which mediates a potentially powerful non-specific response to acupuncture. On the other hand, our results showed that the SII and cerebellum were more associated with SHAM, suggesting that the post-sham effects may mostly be represented in modulating responses in sensory processes . More compelling evidence supporting central effects specific to acupuncture was from direct comparisons between the ACUP and SHAM connectivity maps (Fig. 6 and Table 4). Our ROI-based temporal analyses in Figure 7 indicated a dynamic relationship between the amygdala and the other four regions implicated in the pain-related network. When the region-to-region connectivity patterns were further explored, our results showed differential modulatory effects of acupuncture and sham stimulations on the corresponding networks, in which the modulatory effects were mediated in a time-dependent way. Therefore, these findings suggest that the difference in functional connectivity is region-specific, which provides indirect evidence in support of the discrepancy between verum and sham acupuncture.
It has been recently suggested that acupuncture may be effective in pain relief regardless of acupoint locations, although there are differences in their efficacies . However, as shown in our results using a new fMRI approach, the verum acupuncture and sham stimulation (at a non-acupoint) induced the activations of differential brain networks. The specific pattern of correlation during the post-acupuncture condition provides a reasonable explanation for the actual analgesia effect of acupuncture as well as direct evidence supporting that an acupuncture point may have its own functional specificity.