Neurobiology of Pain

BackgroundPain is one of the primary symptoms of endometriosis, a chronic inflammatory condition characterised by the presence of endometrial tissue outside the uterus. Endometriosis-associated pain is commonly considered as nociceptive in nature but its clinical presentation suggests that it might have neuropathic-like properties in a subgroup of patients. MethodsThis is a cross sectional study using an online survey. The survey was distributed by patient support websites. The survey was composed of validated questionnaires assessing pain symptoms, psychological measures and questions about number of surgeries. Main results and the role of chanceWe had 1417 responses which met the inclusion criteria. Using standard painDETECT cut-off scores, we found that pain was classified as neuropathic in 40% of patients and as mixed neuropathic/nociceptive in a further 35%. In line with observations in other neuropathic conditions, the neuropathic subgroup reported higher pain intensities, greater psychological distress and cognitive impairment. Neuropathic pain was also more likely in those with more surgeries to the abdomen and a longer history of pain. As revealed by a cluster analysis, those with a neuropathic pain component could further be divided into two subgroups based on their sensory profile. ConclusionsThe data presented here indicate that endometriosis-associated pain includes a neuropathic-like component in a substantial proportion of women. Although further investigation is required, our finding challenges the current conceptualisation of endometriosis-associated pain as nociceptive and advocates for a new perspective on this type of pain, which is so debilitating to a large number of women.

In human dorsal root ganglia (DRG), and trigeminal (TG) neurons, the various voltage-gated sodium channel (Nav) isoforms play critical roles in the firing of action potentials, which drive electrical impulses that encode somatosensations including, itch, and pain. The SCN11A gene encodes the tetrodotoxin (TTX)-resistant voltage-gated sodium channel Nav1.9, characterized by unique gating properties. Unlike other isoforms, the Nav1.9 channel activates and inactivates slowly and has a hyperpolarized voltage-dependence of activation and depolarized voltage-dependence of inactivation. This leads to a large window current that has been suggested to function as a regulator of the resting membrane potential of neurons. Mutations in Nav1.9 channels lead to congenital insensitivity to pain (gain-of-function) or familial episodic pain syndrome (loss-of-function) suggesting the channel is a critical mediator of pain. Despite its relevance in pain pathophysiology, most existing data relies on rodent models or heterologous expression systems, leaving the specific pharmacology and biophysical behavior of these channels in human primary neurons largely unknown. In this study, we pharmacologically isolated and characterized native Nav1.9 channel currents in human DRG and TG neurons to compare their biophysical profiles. Our findings reveal significant kinetic and voltage-dependent differences between the two populations. Specifically, Nav1.9 channels in TG neurons exhibit a right-shifted steady-state inactivation curve, a larger window current, and faster activation kinetics compared to those in DRG neurons. In addition, conditions that simulate inflammatory states in-vivo greatly potentiates the Nav1.9 currents consistent with similar observations in rodent models. By detailing these distinct biophysical properties, this research offers crucial insights into Nav1.9 channel function relevant for drug discovery efforts aimed at developing analgesics for both acute and chronic pain.

Understanding the complex network that regulates pain is fundamental to develop strategies to combat its growing prevalence and increase useful therapeutics. Although extensive literature identifies the importance of cannabinoid receptors and endocannabinoids in controlling pain, their efficacy and loci of action remain debated. To directly test the actions of peripherally restricted cannabinoids and elucidate the minimal circuitry capable of producing cannabinoid-mediated analgesia, we utilized a novel genetic approach that allows for cell-specific reactivation of cannabinoid receptor 1 (CB1R) selectively in peripheral sensory neurons using newly developed CB1R floxed-stop-floxed mice (CB1RLOXTB) crossed with Nav1.8-cre mice (Nav1.8+/-:CB1RLOXTB). Ex vivo and in vivo experiments confirmed successful knockout and reactivation of CB1R. Wildtype littermate controls, but neither Nav1.8+/-:CB1RLOXTB nor CB1RLOXTB animals, exhibited robust analgesia after systemic WIN55,212-2 (WIN) treatment in the tail flick assay. Furthermore, the presence of CB1R on Nav1.8 neurons was not associated with either a difference in the development of inflammatory pain or the response to WIN. However, after neuropathic injury, CB1RLOXTB animals displayed an earlier onset of both mechanical and thermal hypersensitivity than their Nav1.8+/-:CB1RLOXTB or wildtype counterparts, suggesting a dual role for CB1R in inflammatory and neuropathic pain. These studies represent an important approach to further improve our mechanistic understanding of cannabinoid modulation of pain in the nervous system and begins to settle long-standing controversies in cannabinoid literature. Table of ContentsPeripherally restricted cannabinoids show strong preclinical analgesic efficacy but have not translated clinically. Using a genetic model restricting CB1R to Nav1.8-expressing sensory neurons, we show peripheral neuronal endocannabinoid signaling is required for chronic, but not acute pain modulation. This dissociation suggests clinical failures may reflect testing peripheral cannabinoids in acute rather than chronic pain paradigms, informing future translational strategies.

Trigeminal neuralgia (TN) is a severe and disabling facial pain condition and is characterized by intermittent, severe, electric shock-like pain in one (or more) trigeminal subdivisions. This pain can be triggered by an innocuous stimulus or can be spontaneous. Presently available therapies for TN include both surgical and pharmacological management; however, the lack of a known etiology for TN contributes to the unpredictable response to treatment and the variability in long-term clinical outcomes. Given this, a range of peripheral and central mechanisms underlying TN pain remain to be understood. We acquired functional magnetic resonance imaging (fMRI) data from TN patients who (1) rested comfortably in the scanner during a resting state session and (2) rated their pain levels in real time using a calibrated tracking ball-controlled scale in a pain tracking session. Following data acquisition, the data was analyzed using the conventional correlation analysis and two artificial intelligence (AI)-inspired deep learning methods: convolutional neural network (CNN) and graph convolutional neural network (GCNN). Each of the three methods yielded a set of brain regions related to the generation and perception of pain in TN. There were six regions that were identified by all three methods, including the superior temporal cortex, the insula, the fusiform, the precentral gyrus, the superior frontal gyrus, and the supramarginal gyrus. Additionally, 17 regions, including dorsal anterior cingulate cortex(dACC) and the thalamus, were identified by at least two of the three methods. Collectively, these 23 regions represent signature centers of TN pain and provide target areas for future studies relating to central mechanisms of TN.

IntroductionThe role of pain as a warning system necessitates a rapid transmission of information from the periphery for the execution of appropriate motor responses. The nociceptive withdrawal reflex (NWR) is a physiological response to protect the limb from a painful stimulus and is often considered an objective measure of spinal nociceptive excitability. The NWR is commonly defined by its latency in the presumed Ad-fiber range consistent with the canonical view that "fast pain" is signaled by Ad nociceptors. We recently demonstrated that human skin is equipped with ultrafast (A{beta} range) nociceptors. Here, we investigated the short-latency component of the reflex and explored the relationship between reflex latency and pain perception. MethodsWe revisited our earlier work on NWR measurements in which, following convention, only those reflex responses were selected that were in the presumed Ad range (taken to be latencies [≥]90 ms in that study). In our current analysis, we expanded the time window to search for shorter latency responses and compared those with pain ratings. ResultsIn both cohorts, we found an abundance of recordings with short-latency reflex responses. In nearly 90% of successful recordings, only single reflex responses (not dual) were seen which allowed us to compare pain ratings to reflex latencies. We found that shorter latency reflexes were just as painful as those in the conventional latency range. DiscussionWe found a preponderance of short-latency painful reflex responses. Based on this finding, we suggest that short-latency responses must be considered in future studies. We predict these might be signaled by the ultrafast nociceptors, warranting further investigation.

Chronic pain, a complex multidimensional disorder, remains a major health care issue and a therapeutic challenge. Neuropathic pain is a chronic pain condition that results from damage or dysfunction in the nervous system. While mechanisms of neuropathic pain at the peripheral and spinal cord level have been extensively studied, pain mechanisms in the brain remain underexplored. The amygdala, a limbic brain region, has emerged as a critical brain area for the emotional-affective dimension of pain and pain modulation. Amygdala neuroplasticity has been associated with pain states, but exact molecular and cellular mechanisms underlying these states and the transition from acute to chronic pain are not well understood. Here, we used the spinal nerve ligation (SNL) model of neuropathic pain in male rats to investigate changes in gene expression in the amygdala at the chronic pain stage using RNA sequencing (RNA-Seq). Two amygdala nuclei, basolateral (BLA) and central (CeA), were investigated in a hemisphere-dependent manner. We used an integrative approach that focuses on functional significance and cell type specificity of differentially expressed genes (DEGs) to nominate mechanistic targets for central regulation of chronic pain. Our integrative transcriptomic and bioinformatic analyses identified individual genes (e.g., Cxcl10, Cxcl12, Mbp, Plp1, Mag, Mog, Slc17a6, Gad1, Sst), molecular pathways (e.g., cytokine-mediated signaling pathway), biological processes (e.g., myelination, synaptic transmission), and specific cell types (e.g., oligodendrocytes, glutamatergic and GABA-ergic neurons) affected by chronic pain. Our results also provide evidence for the emerging concept of hemispheric lateralization of pain processing in the amygdala. Overall, our study proposes oligodendrocyte dysfunction in the amygdala, neuroimmune signaling in CeA, and glutamatergic neurotransmission in BLA as mechanistic determinants of and potential therapeutic targets for the management of chronic neuropathic pain.

Nerve growth factor (NGF) is a key mediator of pain through activation of the high-affinity tropomyosin receptor kinase A (TrkA) and the low-affinity neurotrophin receptor (p75NTR). Although neuronal TrkA signaling is well established, the contribution of non-neuronal cells to NGF- dependent pain remains unclear. Here, we show that NGF and its precursor proNGF engage distinct cellular mechanisms. Intraplantar NGF induced acute nociception, heat hyperalgesia, mechanical allodynia, and cold hypersensitivity, whereas cleavage-resistant proNGF selectively evoked mechanical allodynia and cold hypersensitivity. Pharmacological and cell-specific genetic approaches demonstrated that acute nociception and heat hyperalgesia require neuronal TrkA, whereas mechanical and cold hypersensitivity depend on p75NTR activation in Schwann cells. In Schwann cells, NGF and proNGF induced p75NTR-dependent calcium release, followed by TRPA1 activation, mitochondrial ROS production, and NOX1-dependent oxidative amplification. Inhibition of ROS or TRPA1, or Schwann cell-specific Trpa1 deletion, markedly reduced mechanical allodynia and cold hypersensitivity without affecting acute nociception or heat hyperalgesia. These findings identify a Schwann cell p75NTR-ROS-TRPA1 pathway sustaining persistent pain and highlight non-neuronal p75NTR signaling as a potential therapeutic target.

Neuropathic pain is a leading cause of high impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the DRG is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human DRGs from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain associated DRGs. We sequenced 70 human DRGs, including over 50 having mRNA libraries with neuronal mRNA. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14, and OSM in male and including CCL1, CCL21, PENK and TLR3 in female DRGs associated with neuropathic pain. Co-expression modules revealed enrichment in members of JUN-FOS signaling in males, and centromere protein coding genes in females. Neuro-immune signaling pathways revealed distinct cytokine signaling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization. Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.

Neonatal pain is a significant clinical issue but the mechanisms by which pain is produced early in life are poorly understood. Our recent work has linked the transcription factor serum response factor downstream of local growth hormone (GH) signaling to incision-related hypersensitivity in neonates. However, it remains unclear if similar mechanisms contribute to inflammatory pain in neonates. We found that local GH treatment inhibited neonatal inflammatory myalgia but appeared to do so through a unique signal transducer and activator of transcription (STAT) dependent pathway within sensory neurons. The STAT1 transcription factor appeared to regulate peripheral inflammation itself by modulation of monocyte chemoattractant protein 1 (MCP1) release from sensory neurons. Data suggests that STAT1 upregulation, downstream of GH signaling, contributes to neonatal nociception during muscle inflammation through a novel neuroimmune loop involving cytokine release from primary afferents. Results could uncover new ways to treat muscle pain and inflammation in neonates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/605393v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@18b0bdorg.highwire.dtl.DTLVardef@fb5ee9org.highwire.dtl.DTLVardef@114179aorg.highwire.dtl.DTLVardef@19979b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Patients with schwannomatosis (SWN) develop multiple tumors along major peripheral nerves, with most experiencing significant pain, though each patients symptoms are unique. Neuropathic, nociceptive, and inflammatory pain types have been reported, but many patients describe severe pain when a schwannoma is palpated or even lightly touched. Currently, the only effective treatment for pain relief is surgical removal. We are investigating the root causes of tumor-induced pain. In some cases, tumor growth increases pressure on nearby nerves, resulting in pain. Additionally, schwannoma cells in culture secrete proinflammatory cytokines into the surrounding medium. This conditioned medium (CM) sensitizes sensory neurons to painful stimuli both in vitro and in vivo. When injected into the glabrous skin of a mouse hindpaw, CM from painful schwannomas increases neuron sensitivity to light touch, as demonstrated by a fourfold reduction in paw withdrawal threshold (measured using the Von Frey assay) one hour post-injection (p = 0.006), with effects persisting for 24 hours (p = 0.002).We hypothesize that this increase in sensitivity is linked to mechanosensitive ion channels (MSCs), which detect pressure and stretch. These channels can be blocked by the peptide GsMTx-4. This peptide penetrates deeper into cell membranes under mechanical pressure to block MSCs from opening without affecting other ion channels. When co-injected with CM into the mouse hindpaw, 10 {micro}M GsMTx-4 prevents heightened sensitivity to light touch. Moreover, GsMTx-4 can reverse hyperalgesia, restoring withdrawal thresholds to baseline levels. Thus, local injection of GsMTx-4 near painful tumors presents a promising, minimally invasive therapeutic approach for SWN patients. SignificancePain is a confounding comorbidity in the multiple tumor syndrome schwannomatosis. Patients harbor benign peripheral nerve sheath tumors that rarely become malignant or cause neurological deficits. Yet, patients undergo numerous surgeries for the removal of painful tumors. A non-invasive treatment for tumor-related pain is in dire need. We are examining the small peptide GsMTx-4, a blocker of mechanosensitive ion channels, as a potential therapy for painful tumors in the context of schwannomatosis.

IntroductionOffset analgesia (OA) is defined as a disproportionate reduction in pain perception following a small decrease in noxious stimulation. However, the mechanisms underlying this phenomenon remain unclear, with ongoing debate on peripheral versus central contributions. ObjectivesThis experimental study aimed to differentiate first and second pain perception during the OA paradigm, thereby assessing fiber-specific influences on OA. MethodsThirty-two healthy participants were asked to distinguish a double pain sensation (first and second pain), to assess pain quality descriptors related to A-{delta} and C-fibers, and to indicate response times to brief noxious heat stimuli. This procedure was repeated while implementing heat pulses in an OA paradigm. ResultsNo significant differences were found between offset and constant trials in the reported double pain sensation or the fiber specific pain descriptors (p > 0.05). Nevertheless, significant differences in response times were observed depending on the type of trial and the timing of the stimulus. Response time to noxious stimuli was delayed after prolonged stimulation in both offset and constant trials (p < 0.05). ConclusionsThe findings suggest that A-{delta} and C-fiber response characteristics were unaffected during the OA paradigm; however, higher stimulation intensities or prolonged pain induce a notable response delay. This indicates a negligible role of specific peripheral nerve fibers in OA, emphasizing the predominance of central mechanisms, particularly those related to attention and cognitive resources, which merit further investigation.

BackgroundThe experience of pain has been dissociated into two interwoven aspects: a sensory-discriminative aspect and an affective-motivational aspect. We aimed to explore which of the pain descriptors is more deeply rooted in the human brain. FindingsParticipants were asked to evaluate applied cold pain. The majority of the trials showed distinct ratings: some were rated higher for unpleasantness and others for intensity. We compared the relationship between functional data recorded from 7 tesla MRI with unpleasantness and intensity ratings and revealed a stronger relationship between cortical data and unpleasantness ratings. ConclusionsThe present study underlines the importance of the emotional-affective aspects of pain-related cortical processes in the brain. The findings corroborate previous studies showing a higher sensitivity to pain unpleasantness compared to ratings of pain intensity. For the processing of pain in healthy subjects, this effect may reflect the more direct and intuitive evaluation of emotional aspects of the pain system, which is to prevent harm and to preserve the physical integrity of the body.

Pain management has been challenging and a major obstacle lies in the limited translational success between preclinical studies, often based on rodent models and evoked nociception behavioral assays, whose validity is often questioned. The dorsal root ganglia (DRG) contains diverse nociceptor subtypes that serve as the primary afferent pathways for detecting painful stimuli and analgesics often target proteins expressed in nociceptors. This makes the distinct protein repertoires and molecular interactors within nociceptor subtypes a key focus for understanding which molecular players drive pain processing and how they may be therapeutically targeted. The confirmation of cross-species conservation of pain-related signaling pathways, mediated by nociceptors, could help to elucidate the molecular mechanisms by which the drugs act across species. In this context, we constructed and compared experimentally-validated protein-protein interaction (PPI) networks based on drug targets and their direct binding partners for nociceptor subtypes supported by single-nuclei transcriptome data from mouse and human DRGs. We found that overall gene expression is more conserved across mice than in human nociceptor subtypes, indicating a higher degree of molecular specialization of human nociceptors. Overall signaling network analyses revealed subtype- and species-specific conservation related to pain signaling, with some particularities, in which key drug targets mediate broader cellular processes beyond pain signaling and neuronal depolarization. Altogether, this resource may help to further understand the molecular mechanisms of specific drug targeting, and the proposed workflow can be used to identify and prioritize pain-related pathways in the DRG, advancing target identification and translational medicine.

Chemotherapy-induced peripheral neuropathy (CIPN) is the de facto clinical side effect that limits the administration of anti-cancer treatments. Recently, we reported that intrathecally injected bone marrow stromal cells (BMSCs) reduced nerve trauma-induced neuropathic pain in male mice via TGF-{beta}1 signaling. In this study, we examined sex-dependent pain relief mediated by intrathecally delivered BMSCs and TGF-{beta}1 in paclitaxel (PTX)-induced CIPN. BMSCs were prepared from primary cultures of male or female mice separately. A single intrathecal injection of BMSCs, prepared from male donors, completely prevented the development of PTX-evoked mechanical allodynia in male mice. However, female mice showed no analgesic response to either male or female BMSCs. Additionally, male mice did not demonstrate an analgesic response to BMSCs from female donors. Intrathecal injection of TGF-{beta}1 neutralizing antibody reversed the analgesic action of BMSCs. Interestingly, spinal administration of TGF-{beta}1 reduced mechanical allodynia in male mice but not in female mice. Ex vivo patch-clamp recordings in spinal cord slices revealed that TGF-{beta}1 inhibited PTX-induced synaptic plasticity, i.e. increase in spontaneous excitatory synaptic currents (sEPsCs), in spinal cord neurons from male mice only. Intrathecal TGF-{beta}1 increased the paw withdrawal threshold in von Frey testing in naive mice of males but not females, and the antinociceptive effect of TGF-{beta}1 in males was blocked by orchiectomy-induced androgen deficiency. Together, these findings reveal sex dimorphism in BMSC control of mechanical pain through spinal TGF-{beta}1 signaling.

There is an urgent need for new pain-relieving therapies. We have previously shown using mouse models of persistent pain that a single intrathecal injection of substance P conjugated to the light chain of botulinum toxin (SP-BOT) silenced neurons in the dorsal horn of the spinal cord and alleviated mechanical hypersensitivity. The SP-BOT construct selectively silenced neurokinin 1 receptor positive (NK1R+) neurons in the superficial dorsal horn of the spinal cord. A subset of these NK1R+ neurons are nociceptive projection neurons and convey injury-related information to the brainstem, initiating and maintaining programmes of escape and recovery essential for healing. Previously, we observed a reduction in mechanical hypersensitivity in a spared nerve injury (SNI) model of neuropathic pain state after intrathecal injection of SP-BOT over the lumbar spinal cord and lasting for up to 40 days. In this latest study, we have extended these observations and now show that thermal and affective measures of pain behaviour were also alleviated by a single intrathecal injection of SP-BOT. By introducing SNI 30 days, 60 days, 90 days or 120 days after injection of SP-BOT we have established that NK1R+ spinal neurons in the superficial lamina of the dorsal horn were silenced for up to 120 days following a single intrathecal injection of the botulinum construct. We also show that behavioural alleviation of neuropathic pain symptoms could be reinstated by a second injection of SP-BOT at 120 days. Taken together this research demonstrates that this recently developed botulinum toxin conjugate provides a powerful new way of providing long term pain relief without toxicity following a single injection and also has a therapeutic potential for repeated dosing when pain begins to return.

BackgroundChronic pain is present in about 20% of the population and is a major burden to the health care system. About 30-40% of these patients report neuropathic pain. Neuropathic pain is defined as pain caused by injury or disease of the somatosensory nervous system. Current available treatments for neuropathic pain have limited efficacy and substantial side effects. MethodsTo address the need for more effective and safer treatments for neuropathic pain, this study aimed to test whether boldine, a naturally occurring alkaloid, could attenuate neuropathic pain in a murine model of spared nerve injury (SNI). Von Frey filament test, hot/cold plate test and dynamic weight bearing test were used to assess pain phenotypes following SNI. ResultsWe found that boldine inhibited the lipopolysaccharide-induced overexpression of inflammatory markers in BV-2 microglial cells. Oral administration of boldine at 50 mg/kg body weight/day resulted in significant reduction of SNI-induced mechanical and thermal hypersensitivity. Boldine also corrected SNI-induced weight bearing deficits, which are an indication of spontaneous pain. Boldine significantly inhibited SNI-induced peripheral inflammation as indicated by reduced levels of inflammatory cytokines/chemokines in the serum. Immunofluorescence studies revealed that boldine reduced the number of reactive astrocytes and inhibited microglia activation in lumbar spinal cord. ConclusionOur findings suggest that boldine may be a promising therapeutic candidate for the treatment of neuropathic pain, possibly through inhibition of glia activation and neuroinflammation.

Pathological pain is the most common neurological disorder in people living with HIV-1/AIDS (PLWHA), and rationale-based effective treatment is not available. Multiple neuropathologies develop in the pain transmission pathways in of HIV patients, consistent with their nociceptive dysfunction1,2. One of the prominent neuropathologies associating with the manifestation of pain in HIV patients is astrogliosis (a.k.a. reactive astrocytes) in the spinal dorsal horn (SDH)1, the spinal center for the transmission of pain signals from peripheral organs to the brain. However, the pathogenic role and the activation mechanism of astrogliosis are unclear. Here, we show that the astrogliosis is crucial for the pain pathogenesis induced by HIV-1 gp120, a key etiologically relevant protein2, and that a neuron-to-astrocyte Wnt5a signal controls the astrogliosis. We found that ablation of astrogliosis blocked the development of gp120-induced mechanical hyperalgesia, and concomitantly the expression of neural circuit polarization (NCP) in the SDH. In addition, we demonstrated that conditional knockout (CKO) of either Wnt5a in neurons or its receptor ROR2 in astrocytes abolished not only gp120-induced astrogliosis but also the hyperalgesia and the NCP. Furthermore, we found that the astrogliosis promoted expression of the NCP and the hyperalgesia via IL-1{beta} regulated by a Wnt5a-ROR2-MMP2 axis. Our results elucidate an important role and a novel mechanism of astrogliosis in the pathogenesis of HIV-associated pain. Targeting reactive astrocytes by manipulating the mechanistic processes identified here may lead to the development of effective therapy to treat the pain syndrome in HIV patients.

Trigeminal neuralgia is a prevalent chronic pain disorder characterized by recurring episodes of intense facial pain, which significantly impairs patients quality of life. MRI-based biomarkers have consistently demonstrated their ability to predict pain intensity and treatment outcomes. However, most studies have primarily focused on the trigeminal system, paying less attention to the extensive neural reorganization that occurs throughout the brain in response to chronic pain. In this study, we aimed to examine the thalamus, a key brain structure involved in information processing, and provide a detailed perspective on thalamic remodeling in response to chronic pain at the level of individual thalamic nuclei. We analyzed a sample of 62 patients with primary trigeminal neuralgia undergoing surgical treatment, along with 28 healthy participants. Our results revealed significant gray matter volume changes in thalamic nuclei among patients with trigeminal neuralgia. Notably, the intralaminar nuclei (centromedian/parafascicular) and nuclei associated with visual and auditory signal processing (lateral and medial geniculate bodies) exhibited significant alterations, contrasting with the ventral group nuclei involved in nociceptive processing. Additionally, we found no substantial volume increase in any of the studied nuclei following successful surgical intervention 6 months later. The volumes of thalamic nuclei were negatively correlated with pain intensity and disease duration. The findings obtained in this study, albeit preliminary, have promising clinical implications as they unveil previously unknown facets of chronic pain development. PerspectiveThe study examined alterations in gray matter volume within the thalamus of patients diagnosed with trigeminal neuralgia at the level of specific nuclei. The most significant changes were observed in the lateral and medial geniculate bodies, along with the pulvinar nuclei. HighlightsO_LIA detailed investigation of the thalamic nuclei in patients suffering from primary trigeminal neuralgia (TN) has been carried out for the first time. C_LIO_LIPatients with TN showed a significant gray matter volume decrease in intralaminar, sensory and associative nuclei. C_LIO_LIFollowing successful surgery, there was no observed increase in the volume of the investigated nuclei. C_LI

Spinal cord stimulation (SCS) is an established therapy for neuropathic pain, typically delivered at either low (60 Hz) or high (1 kHz) frequencies, with analgesic effects largely dependent on active stimulation. Here, we investigated whether combined-frequency SCS produces sustained analgesia beyond stimulation periods and explored the underlying mechanisms. Using a spared nerve injury (SNI) model in both rats and mice, we applied dual-frequency SCS (60 Hz + 1 kHz). This paradigm produced robust reversal of mechanical allodynia during stimulation and, notably, a progressive and long-lasting analgesic effect that persisted for days to weeks after stimulation cessation. RNA sequencing revealed pronounced immune-related transcriptional changes in the spinal cord, including upregulation of innate immune, pro-resolution, and neutrophil-associated pathways. Functional studies demonstrated that neutrophil depletion attenuated SCS-induced analgesia, whereas intrathecal S100A8 treatment mimicked therapeutic effects via CD69/SOCS3 signaling. These findings identify dual-frequency SCS as a promising strategy to prolong analgesia and highlight a critical role for neuroimmune modulation in sustained pain relief. HighlightsO_LICombined-frequency, not single-frequency SCS, sustains analgesia during washout C_LIO_LICombination SCS induces robust immune activation in spinal cord and DRG C_LIO_LICombination SCS increases spinal perfusion and promotes neutrophil recruitment C_LIO_LINeutrophil signaling contributes to sustained SCS analgesia C_LI

Pain in elderly persons is often not adequately treated, and current treatments may lead to poor outcomes. Therefore, new treatment strategies need to be developed based on a better understanding of the mechanisms underlying the development of chronic pain. Recent studies have shown that Tac1-/- mice display a significant decrease in nociceptive pain responses to moderate or intense stimuli but present no phenotypic changes following light or nonpainful stimuli. Moreover, the deletion of the Tac1 gene led to a deficit of opioid peptides, which are essential to endogenous pain control mechanisms. Thus, we investigated whether Tac1-/- mice show defective pain modulatory pathways by specifically profiling protein kinases in mice spinal cord using phosphoproteomics and bioinformatics. Protein phosphorylation is a key feature of the cellular regulatory mechanism, and phosphorylation status is related to the regulation and modulation of protein-protein binding. Bioinformatics analysis revealed that MAPK, tyrosine kinase, senescence, interleukin signaling, and TCR signaling are modulated in Tac1-/- mice. Interestingly, these processes are intimately linked with inflammatory responses leading to the release of cytokines and chemokines implicated in the interactions and communications between cells. They are key players involved in the initiation and persistence of pathologic pain. The absence of the Tac1 gene products may trigger a much wider cell response to compensate for the lack of important components of the nociceptive pain transmission system.