The purpose of this study was to evaluate the usefulness of MEMRI in chronic pain research using whole brain analysis, as well as to measure changes in brain activities after drug administration in a chronic pain model. In this study, we demonstrated cortico-cortical neural excitation of several ROIs under chronic neuropathy using MEMRI, as well as changes in Mn-enhanced signals of the pain-related cortex area after direct infusion of mTOR inhibitors in the brain. Until now, the effects of mTOR inhibitor using animal MRI have not been studied. Several studies have used the MEMRI method to assess pain in the central nervous system or to observe changes in pain through analgesic use [3, 10, 24]. However, the MEMRI method has several difficulties in observing the effects of pain reduction or changes in neuronal activities. In order to abbreviate these problems, we tried to observe dynamic changes in the brain using ECA injection with MnCl2.
4.1 The meaning of Mn2+ enhancement in ROIs
MEMRI has advantages of a fast influx and enhancements of Mn2+ in the tissues.  In addition, the cumulative Mn2+ pattern is retained for several days, and it also provides a good opportunity to observe activated brain changes upon stimulation when viewed through T1-weighted MRI . Quantitative evaluation of contrast enhancement showed that MnCl2 injection via ECA may be the best way to show hind paw stimulation-induced pain in rats, with the best signal-to-noise ratio . The Mn-injection method through ECA could observe the signal changes only on the ipsilateral side, which is related to blood circulation in the brain . In order to observe whole brain activation with MEMRI, Mn-injection through subarachnoid or direct injection into the brain regions were also considered. Nevertheless, Mn-injection through the ECA for MEMRI was considered to better facilitate the observation of specific structures and activity, rather than mere signal enhancement by Mn-diffusion.
Reliable brain activities were observed using the MEMRI method in the control, Torin1-, and XL388-infused groups. Mn2+ enhancement of individual slices was measured and analyzed in the rostral and caudal regions. Integrated signal analysis of the IC showed higher activated signals in the rostral region than in the caudal region of control animals. These results were consistent with previous findings that the rostral part of the IC is more involved in pain processing [26, 27]. A previous anatomical study  revealed that the rostral part of the IC receives considerable input from the centrolateral and mediodorsal thalamus, which showed strong activity in response to noxious stimulus in MEMRI results. The functional characteristics of the caudal part of IC were clearly distinct from those of the rostral part of IC, which has received attention for its role in nociceptive processing . The rostral part of IC directly accepts the pain caused by stimulation, while the caudal part of IC may function pathologically with persistent maintenance of mechanical allodynia . In this study, rats infused with Torin1 or XL388 showed not only a reduction of signals in the rostral part of IC, but also a reduction of signals in the caudal part of IC. In addition to the IC, other cortical target regions, such as the S1HL and ACC, have been investigated to evaluate the pain inhibition effects after medication. In the S1HL, which directly reflects noxious stimulation, subcutaneous electrical stimulation in the hind paw could activate Aβ fibers from cutaneous receptors . Evoked pain sensation was reflected in the S1HL. In addition, the ACC was consistently activated in nociception in human and animal studies . Previous research has indicated that the neuronal plasticity in ACC is highly correlated with the development of chronic pain [32, 33]. Although the ACC has been shown to be associated with different aspects of pain, precise functional mapping of these aspects still remains largely unknown.
Interestingly, we observed enhanced signals in the M1/2 region upon noxious hind paw stimulation. Although M1/2 is not a part of the pain matrix, it is known that the M1/2 has wide connections to some of the sensory relay nuclei in the thalamus, as well as to efferent and afferent fibers in the spinal cord, that are responsible for the transmission of painful stimuli and modulation of the motor response to noxious contact. Previous investigators have indicated that the motor cortex in rats and mice partially overlaps the S1 to form a “sensorimotor amalgam” that largely involves representations of the hind limb [34-36]. Despite this knowledge, it is difficult to explain how noxious stimuli in the hind paw activate the M1/2. Further research is required to explain the role of the M1/2 in pain perception.
4.2 How can mTOR inhibition reduce brain activities in response to chronic pain?
Modulation of chronic pain through mTOR inhibition is a newly studied target for chronic pain control [16, 37]. Previous studies have observed changes in chronic pain through the inhibition of mTOR signaling in the spinal cord [38-41], and recent studies have demonstrated effective modulation of chronic pain by direct mTOR signaling control in the brain [16, 32, 42], The activation of mTOR regulates protein synthesis by phosphorylating downstream effectors, which influence a wide range of physiological and pathological states, including neuropathic, inflammatory, and cancer-related pain [16-18]. The mTOR inhibitors Torin1 and XL388 used in this study are ATP-competitive inhibitors that suppress the phosphorylation of downstream effectors to regulate mRNA translation and protein synthesis . Our previous research revealed how Torin1/XL388 can change chronic pain after nerve injury [44, 45]. In these studies, Torin1 or XL388 was microinjected into the brain of nerve-injured animals and behavioral changes were assessed. The results indicated that the administration of Torin1 or XL388 significantly increased mechanical thresholds and reduced mechanical allodynia. These results strongly suggest that Torin1 and XL388 may attenuate neuropathic pain via inhibition of mTOR in the brain. Remarkably, the Torin1- and XL388-infused rats showed reduced Mn-enhanced signals in the cortical area, including the IC, S1HL, M1/2, and ACC. These results demonstrate that mTOR inhibition may have an effect on activation of other brain regions, as well as pain reduction through the inhibition of phosphorylation by mTOR control [13, 32, 38]. The maintenance of long-term potentiation (LTP) is dependent on protein synthesis, and mTOR signaling is involved in the local protein synthesis required for the maintenance of LTP. In the process of pain-related neural plasticity, mTOR kinase is one of the key factors related to protein synthesis. Also, increases in mTOR following nerve injury has been shown to increase phosphorylation, indicating that the protein synthesis involved in neural plasticity causing neuropathic pain is increased. Our another previous research has reported that the expressions of mTOR-related translation factors, such as 4E-BP1 and p70S6K, are increased under chronic pain conditions . In addition, several studies have confirmed activation of PKCα, which follows mTOR phosphorylation [46, 47]. PKCα is known to play a critical role in cytoskeleton rearrangement induced by mTOR. Moreover, down-regulation of the PKCα generates an abnormal cell shape or excessive actin cytoskeleton and alters neurons by rearranging their configuration, volume, or length. Thus, we assume that the reduced signal intensities observed in the Torin1- and XL388-treatment groups may be attributable to the relief of neuropathic pain due to inhibition of protein synthesis and cytoskeleton rearrangement associated with mTOR.
In our results, there were no differences in signal enhancement ratios between experimental groups in the V1/2 region. These results indicate that the inhibitory effect of mTOR signaling is not significantly different in response to similar visual cues. Recent studies have suggested that mTOR signaling may be involved in the initiation of pain perception [15, 19], which could effectively reduce chronic pain through inhibition of mTOR. However, the amount of research on how pain is controlled due to regulation of mTOR is still insufficient. A better understanding of these signaling pathways would provide great insights into the underlying mechanisms and lead to more refined therapeutic approaches.
In conclusion, the activity-dependent Mn-enhanced magnetic resonance signals after mTOR inhibition reported herein indicated that pharmaceutical inhibition of mTOR signaling in the brain could reduce abnormal cortex activities after chronic neuropathic pain. Furthermore, rostral-caudal analysis revealed that the rostral regions of IC and M1/2 are more related to pain perceptions than caudal regions. Nevertheless, MRI studies with manganese ions have many challenges, due to the restrictive pass capability of the blood-brain barrier, in addition to difficulties in controlling the accumulation of manganese in tissues and physiological changes in the body. The development of various studies using improved MR techniques, real-time optical imaging with voltage-sensitive dye, and brain slice recordings may help contribute to future studies of neural mechanisms and help to advance knowledge of cerebral activation in chronic pain.