**3.3 Microglia and stress-induced pain**

It has been established that microglia in the dorsal horn of the spinal cord play major roles in the mechanism of chronic neuropathic pain. Activated microglia

highly express P2X4 and P2X7 receptors, which enhance ATP/P2 receptors signaling and increase IL-1β, TNFα, BDNF release, leading to increased glutamatergic receptor function and decreased GABA receptor function in dorsal horn neurons of the spinal cord. This is the mechanism of chronic neuropathic pain by microglial activation [46].

As a mechanism by which pain is enhanced by stress, changes at the respective levels of the spinal cord and the central nervous system can be considered. Several animal studies have investigated the relationship between stress-induced pain and changes in microglia. Sawacki loaded social defeat stress for 6 days to mice and evaluated pain behavior, gene expressions of inflammatory mediators in the spinal cord. Social defeat stress enhanced mechanical allodynia and increased the number of microglia and expressions of IL-1, TNF, TLR4, CC chemokine ligand2. Selective removal of microglia by CSF-1 inhibitor attenuated these changes [47]. Another series of studies using SPS reported that SPS also enhances mechanical allodynia, increases the number of microglia and expressions of inflammatory mediators in the spinal cord, and induces microglial priming there. Administration of respectively, angiotensin II type 1 receptor antagonist, alpha-7 nicotinic acetylcholine receptor agonist,


#### **Table 3.**

*List of papers regarding microglia in stress induced pain, which include animal studies, PET with TSPO, and an study with iMG cells.*

and glucocorticoid receptor antagonist were shown to attenuate rat pain sensitivity and inflammatory changes in the spinal cord [48–50]. Thus, these receptors may be involved in the mechanism of stress-induced pain.

Activation of microglia in the central nervous system as well as in the spinal cord may also be involved in stress-induced pain. Intrahippocampal injection of minocycline was shown to normalize SPS-induced hippocampal microglial inflammatory changes and mechanical allodynia [51]. The rostral ventromedial medulla (RVM) is considered to be one of the brain regions that directly project to the dorsal horn of the spinal cord to regulate pain. In pathological pain, serotonin neurons in the RVM are excited to increase serotonin release in the spinal cord. Wei et al. induced postoperative chronic pain in rats by skin/muscle incision and retraction. It caused inflammatory changes in microglia and elevated serotonin levels in the RVM. Inhibiting microglial inflammatory changes in the RVM reduced rat pain sensitivity and serotonin levels in the spinal cord [52]. Given that it has been reported that chronic restraint stress increases serotonin levels in the RVM and increases pain [53], inflammatory changes in microglia in the RVM may be relevant to the stress-induced pain.

Fibromyalgia is characterized by a wide range of pain for a long period of 3 months or more, strong stiffness, and various symptoms such as severe fatigue, insomnia, headache, and depressed mood. Psychosocial stress is related to its onset and chronicity. A PET study targeting TSPO in patients with fibromyalgia was reported in 2019. The expression level of TSPO in the anterior and posterior middle cingulate cortex increased in the patient group and was also correlated with clinical symptoms [54].

In our laboratory, we generated iMG cells from patients with fibromyalgia and investigated the details of their activation at the cellular level. We found that patientderived iMG cells have an increased ability to release TNF-α and it correlates with the degree of pain [55]. We summarize animal, PET, and iMG studies indicating microglial involvement in stress-induced pain and fibromyalgia in **Table 3**.
