Objectives Fibromyalgia is commonly considered a stress-related chronic pain disorder, and daily stressors are known triggers. However, the relation between stress and pain development remains poorly defined by clinical approaches. Also, the aetiology remains largely unknown. Methods We used a newly developed mouse model and lipidomic approaches to probe the causation and explore the biological plausibility for how perceived stress translates into chronic non-inflammatory pain. Clinical and lipidomic investigations of fibromyalgia were conducted for human validation. Results Using non-painful sound stimuli as psychological stressors, we demonstrated that mice developed long-lasting non-inflammatory hyperalgesia after repeated and intermittent sound stress exposure. Elevated serum malondialdehyde level in stressed mice indicated excessive oxidative stress and lipid oxidative damage. Lipidomics revealed upregulation of lysophosphatidylcholine 16:0 (LPC16:0), a product of lipid oxidisation, in stressed mice. Intramuscular LPC16:0 injection triggered nociceptive responses and a hyperalgesic priming-like effect that caused long-lasting hypersensitivity. Pharmacological or genetic inhibition of acid-sensing ion channel 3 impeded the development of LPC16:0-induced chronic hyperalgesia. Darapladib and antioxidants could effectively alleviate the stress-induced hyperalgesia by inhibiting LPC16:0 synthesis. Clinical investigations showed that excessive oxidative stress and LPC16:0 expression also exist in patients with fibromyalgia. Moreover, LPC16:0 expression was correlated with pain symptoms in patients with high oxidative stress and disease severity. Conclusions Our study provides experimental evidence for the causal effect of psychological stressors on chronic pain development. The findings identify a possible pathophysiological mechanism of stress-induced chronic non-inflammatory pain at molecular, behavioural and clinical levels that might indicate a new therapeutic approach for fibromyalgia.

Significance: An estimated 20 million people in the United States have chronic neuropathic pain, but current analgesics are nonspecific or insufficiently effective. Here we show that advillin, a sensory neuron-specific protein, modulates axonal regeneration of a specific subset of pain-sensing afferent neurons (nociceptors) that binds with isolectin B4 and neuropathic pain. In addition, we identify the cell behavior of advillin shed-off from the growth cone in the context of axonal regeneration and thus detected advillin protein in the cerebrospinal fluid in mice with painful peripheral neuropathy. Advillin is a potential biosignature to diagnose the lesion cause of neuropathic pain associated with isolectin B4 ⁺ nociceptors.

Accumulating evidence has shown transcranial low-intensity ultrasound can be potentially a non-invasive neural modulation tool to treat brain diseases. However, the underlying mechanism remains elusive and the majority of studies on animal models applying rather high-intensity ultrasound that cannot be safely used in humans. Here, we showed low-intensity ultrasound was able to activate neurons in the mouse brain and repeated ultrasound stimulation resulted in adult neurogenesis in specific brain regions. In vitro calcium imaging studies showed that a specific ultrasound stimulation mode, which combined with both ultrasound-induced pressure and acoustic streaming mechanotransduction, is required to activate cultured cortical neurons. ASIC1a and cytoskeletal proteins were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents. In contrast, the inhibition of mechanical-sensitive channels involved in bilayer-model mechanotransduction like Piezo or TRP proteins did not repress the ultrasound-mediated neuronal activation as efficiently. The ASIC1a-mediated ultrasound effects in mouse brain such as immediate response of ERK phosphorylation and DCX marked neurogenesis were statistically significantly compromised by ASIC1a gene deletion. Collated data suggest that ASIC1a is the molecular determinant involved in the mechano-signaling of low-intensity ultrasound that modulates neural activation in mouse brain.

Background: Tissue acidosis is effective in causing chronic muscle pain. However, how muscle nociceptors contribute to the transition from acute to chronic pain is largely unknown. Results: Here we showed that a single intramuscular acid injection induced a priming effect on muscle nociceptors of mice. The primed muscle nociceptors were plastic and permitted the development of long-lasting chronic hyperalgesia induced by a second acid insult. The plastic changes of muscle nociceptors were modality-specific and required the activation of acid-sensing ion channel 3 (***ASIC3) or transient receptor potential cation channel V1 (TRPV1). Activation of ASIC3 was associated with increased activity of tetrodotoxin (TTX)-sensitive voltage-gated sodium channels but not protein kinase Cε (PKCε) in isolectin B4 (IB4)-negative muscle nociceptors. In contrast, increased activity of TTX-resistant voltage-gated sodium channels with ASIC3 or TRPV1 activation in Na_V1.8-positive muscle nociceptors was required for the development of chronic hyperalgesia. Accordingly, compared to wild type mice, Na_V1.8-null mice showed briefer acid-induced hyperalgesia (5 days vs. >27 days). Conclusion: ASIC3 activation may manifest a new type of nociceptor priming in IB4-negative muscle nociceptors. The activation of ASIC3 and TRPV1 as well as enhanced Na_V1.8 activity are essential for the development of long-lasting hyperalgesia in acid-induced, chronic, widespread muscle pain.

Significance: Marine cone snails provide one of the highest toxin diversities among venomous animals, the conotoxins, which often target ion channels. In addition, cone snail venoms contain short conopeptides, the molecular targets of which are mostly unknown. We identified a new conopeptide from the venom of the cone snail Conus textile that targets the proton-gated ion channel ASIC3, which detects painful acidosis. This peptide belongs to the subgroup of cono-RFamides and potentiates ASIC3 currents, thereby increasing the excitability of nociceptors. Injection of the cono-RFamide in mice muscle enhanced acid-induced muscle pain. Our results thus identify ASIC3 as the first molecular target of cono-RFamides. These findings underline the role of ASIC3 in pain perception and the importance of nontoxic venom components.