Animal Studies

Mu-opioid receptors (MORs) are crucial for analgesia by both exogenous and endogenous opioids. However, the distinct mechanisms underlying these two types of opioid analgesia remains largely unknown. Here, we demonstrate that analgesic effects of exogenous and endogenous opioids on inflammatory pain are mediated by MORs expressed in distinct subpopulations of neurons in mouse. We found that the exogenous opioid-induced analgesia of inflammatory pain is mediated by MORs in Vglut2 glutamatergic but not GABAergic neurons. In contrast, analgesia by endogenous opioids is mediated by MORs in GABAergic rather than Vglut2 glutamatergic neurons. Furthermore, MORs expressed at the spinal level is mainly involved in the analgesic effect of morphine in acute pain, but not in endogenous opioid analgesia during chronic inflammatory pain. Thus, our study revealed distinct mechanisms underlying analgesia by exogenous and endogenous opioids, and laid the foundation for further dissecting the circuit mechanism underlying opioid analgesia.

Phosphorylation of the 5' cap-binding protein eIF4E by MAPK interacting kinases MNK1/2 is important for nociceptor sensitization and the development of chronic pain. IL-6 induced DRG nociceptor excitability is attenuated in mice lacking eIF4E phosphorylation, in MNK1/2 mice and by the nonselective MNK1/2 inhibitor cercosporamide. Here, we sought to better understand the neurophysiological mechanisms underlying how IL-6 causes nociceptor excitability via MNK-eIF4E signaling using the highly selective MNK inhibitor eFT508 . Dorsal root ganglion (DRG) neurons were cultured from male and female ICR mice, 4-7 weeks old. DRG cultures were treated with vehicle, IL-6, eFT508 (pretreat) followed by IL-6 or eFT508 alone. Whole-cell patch clamp recordings were done on small diameter neurons (20-30 pF) to measure membrane excitability in response to ramp depolarization. One hr IL-6 treatment resulted in increased action potential firing compared to vehicle at all ramp intensities, an effect that was blocked by pretreatment with eFT508. Basic membrane properties, including resting membrane potential, input resistance and rheobase, were similar across groups. Latency to the first action potential in the ramp protocol was lower in the IL-6 group, and rescued by eFT508 pretreatment. We also found that the amplitudes of T-type voltage-gated calcium channels (VGCCs) were increased in the DRG following IL-6 treatment, but not in the eFT508 co-treatment group. Our findings are consistent with a model wherein MNK-eIF4E signaling controls the translation of signaling factors that regulate T-type VGCCs in response to IL-6 treatment. Inhibition of MNK with eFT508 disrupts these events, thereby preventing nociceptor hyperexcitability.

Chronic pelvic pain syndrome (CPPS), is a multi-symptom syndrome with unknown etiology. The experimental autoimmune prostatitis (EAP) mouse model of CPPS is associated with immune cell infiltration into the prostate, expression of C-C Chemokine ligand 2 (CCL2) and neuroinflammation in the spinal cord. Here, we studied CCL2 expression in tissues along the nociceptive pathway and its association with neuroimmune cells during pain development. Examination of prostate tissues at days 14 and 28 after EAP induction revealed CCL2 expression was increased in epithelial cells and was associated with increased numbers of macrophages lying in close apposition to PGP9.5-positive afferent neuronal fibers. CCL2 immunoreactivity was elevated to a similar degree in the DRG at day 14 and day 28. D14 of EAP was associated with elevated IBA1 cells in the DRG that were not evident at D28. Adoptive transfer of GFP+ leukocytes into EAP mice demonstrated monocytes are capable of infiltrating the spinal cord from peripheral blood with what appeared to be a proinflammatory phenotype. In the lower dorsal spinal cord, CCL2 expression localized to NeuN expressing neurons and GFAP-expressing astrocytes. Myeloid derived cell infiltration into the spinal cord in EAP was observed in the L6-S2 dorsal horn. Myeloid derived CD45+ IBA1+ cells were localized with IBA1+ TMEM199+ microglia in the dorsal horn of the spinal cord in EAP, with intimate association of the two cell types suggesting cell-cell interactions. Lastly, intrathecal administration of liposomal clodronate ameliorated pelvic pain symptoms, suggesting a mechanistic role for macrophages and microglia in chronic pelvic pain.

Mlxipl regulates glucose metabolism, lipogenesis and tumorigenesis and has a wide-ranging impact on human health and disease. However, the role of Mlxipl in neuropathic pain remains unknown. In this study, we found that Mlxipl was increased in the ipsilateral L4-L6 spinal dorsal horn after Spared Nerve Injury surgery. Knockdown of Mlxipl in the ipsilateral L4-L6 spinal dorsal horn by intraspinal microinjection aggravated Spared Nerve Injury-induced mechanical allodynia and inflammation in the spinal dorsal horn, on the contrary, overexpression of Mlxipl inhibited mechanical allodynia and inflammation. Subsequently, the rat Mlxipl promoter was analyzed using bioinformatics methods to predict the upstream transcription factor cJun. Luciferase assays and ChIP-qPCR confirmed that cJun bound to the promoter of Mlxipl and enhanced its expression. Finally, we demonstrated that Mlxipl inhibited the inflammatory responses of lipopolysaccharide-induced microglia and that Mlxipl was regulated by the transcription factor cJun. These findings suggested that cJun-induced Mlxipl upregulation in the spinal dorsal horn after peripheral nerve injury provided a protective mechanism for the development and progression of neuropathic pain by inhibiting microglial-derived neuroinflammation. Targeting Mlxipl in the spinal dorsal horn might represent an effective strategy for the treatment of neuropathic pain.

Though it is well-known that a high-salt diet (HSD) is associated with many chronic diseases, the effects of long-term high-salt intake on physiological functions and homeostasis remain elusive. In this study, we investigated whether and how an HSD affects mouse nociceptive thresholds, and myeloid cell trafficking and activation.

The medial prefrontal cortex (mPFC) modulates a range of behaviors, including responses to noxious stimuli. While various pain modalities alter mPFC function, our understanding of changes to specific cell types underlying pain-induced mPFC dysfunction remains incomplete. Proper activity of cortical GABAergic interneurons is essential for normal circuit function. We find that nerve injury increases excitability of layer 5 parvalbumin-expressing neurons in the prelimbic (PL) region of the mPFC from male, but not female, mice. Conversely, nerve injury dampens excitability in somatostatin-expressing neurons in layer 2/3 of the PL region; however, effects are differential between males and females. Nerve injury slightly increases the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) in layer 5 parvalbumin-expressing neurons in males but reduces frequency of sEPSCs in layer 2/3 somatostatin-expressing neurons in females. Our findings provide key insight into how nerve injury drives maladaptive and sex-specific alterations to GABAergic circuits in cortical regions implicated in chronic pain.

Spinal cord injury (SCI) causes loss of locomotor function and chronic neuropathic pain (NeP). Hematogenous macrophages and activated microglia are key monocytic lineage cell types in the response to SCI, and each has M1- and M2-phenotypes. To understand the roles of these cells in neuronal regeneration and chronic NeP after SCI, differences in distribution and phenotypes of activated microglia and infiltrated macrophages after SCI were examined at the injured site and the lumbar enlargement, as a remote region. Chimeric mice were used for differentiating activated microglia from hematogenous macrophages. The prevalences of activated microglia and infiltrating macrophages increased at day 14 after SCI, at the time of most severe pain hypersensitivity, with mainly M1-type hematogenous macrophages at the injured site and M2-type activated microglia at the lumbar enlargement. Peak expression of TNF-α, an M1-induced cytokine, occurred on day 4 post-SCI at the injured site, but not until day 14 at the lumbar enlargement. Expression of IL-4, a M2-induced cytokine, peaked at 4 days after SCI at both sites. These results suggest different roles of activated microglia and hematogenous macrophages, including both phenotypes of each cell, in neuronal regeneration and chronic NeP after SCI at the injured site and lumbar enlargement. The prevalence of the M1 over the M2 phenotype at the injured site until the subacute phase after SCI may be partially responsible for the lack of functional recovery and chronic NeP after SCI. Activation of M2-type microglia at the lumbar enlargement in response to inflammatory cytokines from the injured site might be important in chronic below-level pain. These findings are useful for establishment of a therapeutic target for prevention of motor deterioration and NeP in the time-dependent response to SCI.

Effective analgesic treatment for neuropathic pain remains an unmet need, so previous evidence that epidermal growth factor receptor inhibitors (EGFRIs) provide unexpected rapid pain relief in a clinical setting points to a novel therapeutic opportunity. The present study utilises rodent models to address the cellular and molecular basis for the findings, focusing on primary sensory neurons because clinical pain relief is provided not only by small molecule EGFRIs, but also by the anti-EGFR antibodies cetuximab and panitumumab, which are unlikely to access the central nervous system in therapeutic concentrations. We report robust, rapid and dose-dependent analgesic effects of EGFRIs in two neuropathic pain models, matched by evidence with highly selective antibodies that expression of the EGFR (ErbB1 protein) is limited to small nociceptive afferent neurons. As other ErbB family members can heterodimerise with ErbB1, we investigated their distribution, showing consistent co-expression of ErbB2 but not ErbB3 or ErbB4, with ErbB1 in cell bodies of nociceptors, as well as providing evidence for direct molecular interaction of ErbB1 with ErbB2 in situ. Co-administration of selective ErbB1 and ErbB2 inhibitors produced clear evidence of greater-than-additive, synergistic analgesia; highlighting the prospect of a unique new combination therapy in which enhanced efficacy could be accompanied by minimisation of side-effects. Peripheral (intraplantar) administration of EGF elicited hypersensitivity only following nerve injury and this was reversed by local co-administration of selective inhibitors of either ErbB1 or ErbB2. Investigating how ErbB1 is activated in neuropathic pain, we found evidence for a role of Src tyrosine kinase, which can be activated by signals from inflammatory mediators, chemokines and cytokines during neuroinflammation. Considering downstream consequences of ErbB1 activation in neuropathic pain, we found direct recruitment to ErbB1 of an adapter for PI 3-kinase and Akt signalling together with clear Akt activation and robust analgesia from selective Akt inhibitors. The known Akt target and regulator of vesicular trafficking, AS160 was strongly phosphorylated at a perinuclear location during neuropathic pain in an ErbB1-, ErbB2- and Akt-dependent manner, corresponding to clustering and translocation of an AS160-partner, the vesicular chaperone, LRP1. Exploring whether neuronal ion channels that could contribute to hyperexcitability might be transported by this vesicular trafficking pathway we were able to identify Na1.9, (Na1.8) and Ca1.2 moving towards the plasma membrane or into proximal axonal locations – a process prevented by ErbB1 or Akt inhibitors. Overall these findings newly reveal both upstream and downstream signals to explain how ErbB1 can act as a signalling hub in neuropathic pain models and identify the trafficking of key ion channels to neuronal subcellular locations likely to contribute to hyperexcitability. The new concept of combined treatment with ErbB1 plus ErbB2 blockers is mechanistically validated as a promising strategy for the relief of neuropathic pain.