Peripheral neuropathy is a frequent complication of type 2 diabetes mellitus (T2DM). We investigated whether human islet amyloid polypeptide (hIAPP), which forms pathogenic aggregates that damage pancreatic islet β-cells in T2DM, is involved in T2DM-associated peripheral neuropathy. In vitro, hIAPP incubation with sensory neurons reduced neurite outgrowth and increased levels of mitochondrial reactive oxygen species. Transgenic hIAPP mice that have elevated plasma hIAPP levels without hyperglycemia developed peripheral neuropathy as evidenced by pain-associated behavior and reduced intra-epidermal nerve fiber (IENF) density. Similarly, hIAPP Ob/Ob mice that have hyperglycaemia in combination with elevated plasma hIAPP levels had signs of neuropathy, although more aggravated.In wild-type mice, intraplantar and intravenous hIAPP injections induced long-lasting allodynia and decreased IENF density. Non-aggregating murine IAPP, mutated hIAPP (Pramlintide), or hIAPP with pharmacologically inhibited aggregation did not induce these effects. T2DM patients had reduced IENF density and more hIAPP oligomers in the skin compared to non-T2DM controls. Thus, we provide evidence that hIAPP aggregation is neurotoxic and mediates peripheral neuropathy in mice. The increased abundance of hIAPP aggregates in the skin of T2DM patients supports the notion that hIAPP is a potential contributor to T2DM neuropathy in humans.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as CGRP and substance P, inducing neurogenic inflammation which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked if inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 and TRPA1 lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid (LPA) or 4-hydroxy-2-nonenal (4HNE), finding each induces cold pain that is dependent on the cold-gated channel TRPM8. Inhibition of either CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Lastly, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GFRα3. These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 and the neurotrophin receptor GFRα3 that leads to cold pain, providing select targets for potential therapies for this pain modality.

Inflammation causes pain by shifting the balance of ionic currents in nociceptors toward depolarization, leading to hyperexcitability. The ensemble of ion channels within the plasma membrane is regulated by processes including biogenesis, transport, and degradation. Thus, alterations in ion channel trafficking may influence excitability. Sodium channel Na1.7 and potassium channel K7.2 promote and oppose excitability in nociceptors, respectively. We used live-cell imaging to investigate mechanisms by which inflammatory mediators (IM) modulate the abundance of these channels at axonal surfaces through transcription, vesicular loading, axonal transport, exocytosis, and endocytosis. Inflammatory mediators induced a Na1.7-dependent increase in activity in distal axons. Further, inflammation increased the abundance of Na1.7, but not of K7.2, at axonal surfaces by selectively increasing channel loading into anterograde transport vesicles and insertion at the membrane, without affecting retrograde transport. These results uncover a cell biological mechanism for inflammatory pain and suggest Na1.7 trafficking as a potential therapeutic target.

Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive. Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses.

The impact of the gut microbiota on glial cell growth and maturation via the gut-brain axis is highlighted herein. Considering that glial activation is crucial for onset and maintenance of neuropathic pain, we assessed the putative involvement of gut microbiota in the pathogenesis of neuropathic pain. Depletion of mouse gut microbiota with chronic antibiotics cocktail treatment prevented nerve injury-induced mechanical allodynia and thermal hyperalgesia both in male and female mice. Furthermore, post-injury treatment with antibiotics cocktail relieved ongoing pain in neuropathic pain-established mice. Upon recolonization of the gut microbiota after cessation of antibiotics, nerve injury-induced mechanical allodynia relapsed. Depletion of gut microbiota accompanied a decrease in nerve injury-induced TNF- α expression in the spinal cord. Notably, nerve injury changed the diversity and composition of the gut microbiome, which was measured by 16s rRNA sequencing. We then tested if probiotic administration ameliorating dysbiosis affected the development of neuropathic pain after nerve injury. Probiotic treatment for three weeks prior to nerve injury inhibited nerve injury-induced TNF- α expression in the spinal cord and pain sensitization. Our data reveal an unexpected link between the gut microbiota and development and maintenance of nerve injury-induced neuropathic pain, and we propose a novel strategy to relieve neuropathic pain through the gut-brain axis.

By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.

Chronic pain is a significant problem that afflicts individuals and society, and for which the current clinical treatment is inadequate. In addition, the neural circuit and molecular mechanisms subserving chronic pain remain largely uncharacterized. Herein we identified enhanced activity of a glutamatergic neuronal circuit that encompasses projections from the ventral posterolateral nucleus (VPL) to the glutamatergic neurons of the hindlimb primary somatosensory cortex (S1HL), driving allodynia in mouse models of chronic pain. Optogenetic inhibition of this VPL→S1HL circuit reversed allodynia, whereas the enhancement of its activity provoked hyperalgesia in control mice. In addition, we found that the expression and function of the HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel 2) were increased in VPL neurons under conditions of chronic pain. Using calcium imaging, we demonstrated that downregulation of HCN2 channels in the VPL neurons abrogated the rise in S1HL neuronal activity while alleviating allodynia in mice with chronic pain. With these data, we propose that dysfunction in HCN2 channels in the VPL→S1HL thalamocortical circuit and their upregulation occupy essential roles in the development of chronic pain.

The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.

Intranasal formulations can provide treatment options for people with migraine in whom oral drugs are ineffective, slow-acting, or intolerable because of nausea and vomiting. Zavegepant, an intranasally administered small molecule calcitonin gene-related peptide (CGRP) receptor antagonist, was previously assessed in a phase 2/3 trial. This phase 3 trial aimed to compare the efficacy, tolerability, safety, and timecourse of response for zavegepant nasal spray with placebo in the acute treatment of migraine.

Numerous studies have investigated the impacts of common types of chronic pain (CP) on patients’ cognitive function and observed that CP was associated with later dementia. More recently, there is a growing recognition that CP conditions frequently coexist at multiple body sites and may bring more burdens on patients’ overall health. However, whether and how multisite CP (MCP) contributes to an increased risk of dementia, compared to single-site CP (SCP) and pain-free (PF), is largely unclear. In the current study, utilizing the UK Biobank cohort, we first investigated dementia risk in individuals (n = 354,943) with different numbers of coexisting CP sites using Cox proportional hazards regression models. We then applied generalized additive models to investigate whether MCP leads to excessive deterioration of participants’ (n = 19,116) cognition and brain structure. We found that individuals with MCP were associated with significantly higher dementia risk, broader and faster cognitive impairment, and greater hippocampal atrophy than both PF individuals and those with SCP. Moreover, the detrimental effects of MCP on dementia risk and hippocampal volume aggravated along with the number of coexisting CP sites. Mediation analyses further revealed that the decline of fluid intelligence in MCP individuals was partially mediated by hippocampal atrophy. Our results suggested that cognitive decline and hippocampal atrophy interact biologically and may underlie the increased risk of dementia associated with MCP.

Spreading depolarization (SD), the underlying mechanism of migraine aura, may trigger the opening of the Pannexin-1 (Panx1) pore to sustain the cortical neuroinflammatory cascades involved in the genesis of headache. Yet, the mechanism underlying SD-evoked neuroinflammation and trigeminovascular activation remains incompletely understood. We characterized the identity of inflammasome activated following SD-evoked Panx1 opening. Pharmacological inhibitors targeting Panx1 or NLRP3 as well as genetic ablation of Nlrp3 and Il1b were applied to investigate the molecular mechanism of the downstream neuroinflammatory cascades. In addition, we examined whether SDs-triggered microglial activation facilitates neuronal NLRP3-mediated inflammatory cascades. Pharmacological inhibition of toll-like receptors TLR2/4, the potential receptors of the damage-associated molecular pattern HMGB1, was further employed to interrogate the neuron-microglia interplay in SD-induced neuroinflammation. We found that NLRP3 but not NLRP1 or NLRP2 inflammasome was activated following Panx1 opening after single or multiple SDs evoked by either KCl topical application or noninvasively with optogenetics. The SD-evoked NLRP3 inflammasome activation was observed exclusively in neurons but not microglia or astrocytes. Proximity ligation assay demonstrated that the assembly of NLRP3 inflammasome was as early as 15 mins after SD. Genetic ablation of Nlrp3 or Il1b or pharmacological inhibition of Panx1 or NLRP3 ameliorated SD-induced neuronal inflammation, middle meningeal artery dilatation, calcitonin gene-related peptide expression in trigeminal ganglion, and c-Fos expression in trigeminal nucleus caudalis. Moreover, multiple SDs induced microglial activation subsequent to neuronal NLRP3 inflammasome activation, which in turn orchestrated with neurons to mediate cortical neuroinflammation, as demonstrated by decreased neuronal inflammation after pharmacological inhibition of microglia activation or blockade of the TLR2/4 receptors. To conclude, single or multiple SDs evoked activation of neuronal NLRP3 inflammasomes and its downstream inflammatory cascades to mediate cortical neuroinflammation and trigeminovascular activation. In the context of multiple SDs, the cortical inflammatory processes could be facilitated by SDs-evoked microglia activation. These findings may implicate the potential role of innate immunity in migraine pathogenesis.

Massive intra-articular infiltration of proinflammatory macrophages is a prominent feature of rheumatoid arthritis (RA) lesions, which are thought to underlie articular immune dysfunction, severe synovitis and ultimately joint erosion. Here we report an efferocytosis-informed nanoimitator (EINI) for in situ targeted reprogramming of synovial inflammatory macrophages (SIMs) that thwarts their autoimmune attack and reestablishes articular immune homeostasis, which mitigates RA. The EINI consists of a drug-based core with an oxidative stress-responsive phosphatidylserine (PtdSer) corona and a shell composed of a P-selectin-blocking motif, low molecular weight heparin (LMWH). When systemically administered, the LMWH on the EINI first binds to P-selectin overexpressed on the endothelium in subsynovial capillaries, which functions as an antagonist, disrupting neutrophil synovial trafficking. Due to the strong dysregulation of the synovial microvasculature, the EINI is subsequently enriched in the joint synovium where the shell is disassembled upon the reactive oxygen species stimulation, and PtdSer corona is then exposed. In an efferocytosis-like manner, the PtdSer-coroneted core is in turn phagocytosed by SIMs, which synergistically terminate SIM-initiated pathological cascades and serially reestablish intra-articular immune homeostasis, conferring a chondroprotective effect. These findings demonstrate that SIMs can be precisely remodeled via the efferocytosis-mimetic strategy, which holds potential for RA treatment.

Chemotherapy-induced peripheral neuropathy (CIPN) is a primary dose-limiting side effect caused by antineoplastic agents, such as paclitaxel. A primary symptom of this neuropathy is pain. Currently, there are no effective treatments for CIPN, which can lead to long-term morbidity in cancer patients and survivors. Neuro-immune interactions occur in CIPN pain and have been implicated both in the development and progression of pain in CIPN and the resolution of pain in CIPN. We investigated the potential role of inducible co-stimulatory molecule (ICOS) in the resolution of CIPN pain-like behaviors in mice. ICOS is an immune checkpoint molecule that is expressed on the surface of activated T cells and promotes proliferation and differentiation of T cells. We found that intrathecal administration of ICOS agonist antibody (ICOSaa) alleviates mechanical hypersensitivity caused by paclitaxel and facilitates the resolution of mechanical hypersensitivity in female mice. Administration of ICOSaa reduced astrogliosis in the spinal cord and satellite cell gliosis in the DRG of mice previously treated with paclitaxel. Mechanistically, ICOSaa intrathecal treatment promoted mechanical hypersensitivity resolution by increasing interleukin 10 (IL-10) expression in the dorsal root ganglion. In line with these observations, blocking IL-10 receptor (IL-10R) activity occluded the effects of ICOSaa treatment on mechanical hypersensitivity in female mice. Suggesting a broader activity in neuropathic pain, ICOSaa also partially resolved mechanical hypersensitivity in the spared nerve injury (SNI) model. Our findings support a model wherein ICOSaa administration induces IL-10 expression to facilitate neuropathic pain relief in female mice. ICOSaa treatment is in clinical development for solid tumors and given our observation of T cells in the human DRG, ICOSaa therapy could be developed for combination chemotherapy-CIPN clinical trials.

Increased low frequency cortical oscillations are observed in people with neuropathic pain, but the cause of such elevated cortical oscillations and their impact on pain development remain unclear. By imaging neuronal activity in a spared nerve injury (SNI) mouse model of neuropathic pain, we show that neurons in dorsal root ganglia (DRG) and somatosensory cortex (S1) exhibit synchronized activity after peripheral nerve injury. Notably, synchronized activity of DRG neurons occurs within hours after injury and 1-2 days before increased cortical oscillations. This DRG synchrony is initiated by axotomized neurons and mediated by local purinergic signaling at the site of nerve injury. We further show that synchronized DRG activity after SNI is responsible for increasing low frequency cortical oscillations and synaptic remodeling in S1, as well as for inducing animals’ pain-like behaviors. In naive mice, enhancing the synchrony, not the level, of DRG neuronal activity causes synaptic changes in S1 and pain-like behaviors similar to SNI mice. Taken together, these results reveal the critical role of synchronized DRG neuronal activity in increasing cortical plasticity and oscillations in a neuropathic pain model. These findings also suggest the potential importance of detection and suppression of elevated cortical oscillations in neuropathic pain states.

Pain is one of the most severe complications affecting the quality of life of cancer patients. Although substantial progress has been made in the diagnosis and treatment of cancer, the neurobiological mechanism of cancer pain is still unclear. In the present study, we identified the critical role of CXC chemokine 2 (CXCL2), released by Schwann cells after being activated by cancer cells, in maintaining cancer-induced macrophage infiltration and the resulting mechanical hypersensitivity and persistent spontaneous nociception. In vitro, Schwann cells cocultured with breast cancer cells exhibited a significant increase in CXCL2 expression; in addition, conditioned medium from Schwann cells activated by breast cancer cells had a similar effect to recombinant CXCL2 in terms of inducing macrophage migration. Targeting CXCL2 signaling by both CXC chemokine receptor 2 (CXCR2) antagonist pharmacological blockade and anti-CXCL2 mAb immunological blockade robustly prevented conditioned medium-induced macrophage migration. In vivo, both application of recombinant CXCL2 and perineural breast cancer cell implantation resulted in mechanical hypersensitivity and persistent spontaneous nociception in mice, along with increased macrophage infiltration into the sciatic nerves. Similar to the in vitro results, inhibition of CXCL2/CXCR2 signaling or conditional knockdown of CXCL2 in sciatic nerve Schwann cells effectively attenuated breast cancer cell-induced mechanical hypersensitivity, persistent spontaneous nociception, and macrophage recruitment in the sciatic nerve. Mechanistically, we found that redox effector factor-1 (Ref-1) secreted by breast cancer cells activated hypoxia inducible factor-1α (HIF-1α) expression and inhibited reactive oxygen species (ROS) production in Schwann cells, ultimately inducing CXCL2 expression in Schwann cells. In brief, the present study expands new insights into cancer pain mechanisms from promising animal models to provide new strategies for the control of cancer pain.

Opioid analgesic tolerance, a root cause of opioid overdose and misuse, can develop through an associative learning. Despite intensive research, the locus and central pathway subserving the associative opioid analgesic tolerance (AOAT) remains unclear. Using a combination of chemo/optogenetic manipulation with calcium imaging and slice physiology, here we identify neuronal ensembles in a hierarchically organized pathway essential for AOAT. The association of morphine-induced analgesia with an environmental condition drives glutamatergic signaling from ventral hippocampus (vHPC) to dorsomedial prefrontal cortex (dmPFC) cholecystokininergic (CCKergic) neurons. Excitation of CCKergic neurons, which project and release CCK to basolateral amygdala (BLA) glutamatergic neurons, relays AOAT signal through inhibition of BLA μ-opioid receptor function, thereby leading to further loss of morphine analgesic efficacy. This work provides evidence for a circuit across different brain regions distinct for opioid analgesic tolerance. The components of this pathway are potential targets to treat opioid overdose and abuse.

Cross-talk between peripheral neurons and immune cells is important in pain sensation. We identified Snx25 as a pain-modulating gene in a transgenic mouse line with reduced pain sensitivity. Conditional deletion of Snx25 in monocytes and macrophages, but not in peripheral sensory neurons, in mice (Snx25 mice) reduced pain responses in both normal and neuropathic conditions. Bone marrow transplantation using Snx25 and wild-type mice indicated that macrophages modulated pain sensitivity. Expression of sorting nexin (SNX)25 in dermal macrophages enhanced expression of the neurotrophic factor NGF through the inhibition of ubiquitin-mediated degradation of Nrf2, a transcription factor that activates transcription of Ngf. As such, dermal macrophages set the threshold for pain sensitivity through the production and secretion of NGF into the dermis, and they may cooperate with dorsal root ganglion macrophages in pain perception.

Neuropathic pain is a debilitating condition resulting from damage to the nervous system. Imbalance of spinal excitation and inhibition has been proposed to contribute to neuropathic pain. However, the structural basis of this imbalance remains unknown. Using a preclinical model of neuropathic pain, we show that microglia selectively engulf spinal synapses that are formed by central neurons and spare those of peripheral sensory neurons. Furthermore, we reveal that removal of inhibitory and excitatory synapses exhibits distinct temporal patterns, in which microglia-mediated inhibitory synapse removal precedes excitatory synapse removal. We also find selective and gradual increase in complement depositions on dorsal horn synapses that corresponds to the temporal pattern of microglial synapse pruning activity and type-specific synapse loss. Together, these results define a specific role for microglia in the progression of neuropathic pain pathogenesis and implicate these immune cells in structural remodeling of dorsal horn circuitry.

Endometriosis is a common condition in women that causes chronic pain and infertility and is associated with an elevated risk of ovarian cancer. We profiled transcriptomes of >370,000 individual cells from endometriomas (n = 8), endometriosis (n = 28), eutopic endometrium (n = 10), unaffected ovary (n = 4) and endometriosis-free peritoneum (n = 4), generating a cellular atlas of endometrial-type epithelial cells, stromal cells and microenvironmental cell populations across tissue sites. Cellular and molecular signatures of endometrial-type epithelium and stroma differed across tissue types, suggesting a role for cellular restructuring and transcriptional reprogramming in the disease. Epithelium, stroma and proximal mesothelial cells of endometriomas showed dysregulation of pro-inflammatory pathways and upregulation of complement proteins. Somatic ARID1A mutation in epithelial cells was associated with upregulation of pro-angiogenic and pro-lymphangiogenic factors and remodeling of the endothelial cell compartment, with enrichment of lymphatic endothelial cells. Finally, signatures of ciliated epithelial cells were enriched in ovarian cancers, reinforcing epidemiologic associations between these two diseases.