We present an automated MATLAB-based GUI for the analysis of microfluorimetry data inisolated neuronal cultures. The pipeline integrates signal extraction, quality control,responder classification, and stimulus-evoked quantification into a single workflowcompatible with both single-wavelength and ratiometric dyes. Applied to thermal and drugstimulations, the automated analysis reproduces the classification and quantitative outputsobtained using manual approaches, while substantially reducing analysis time. The GUI wedeveloped provides a practical and reproducible solution for higher throughput [Ca²⁺]ᵢimaging analysis across neuroscience, cell biology, and related fields.

SARS-CoV-2 infection is frequently associated with persistent pain, yet the underlying mechanisms remain poorly understood. Given that viral structural proteins can act as pathogen-associated molecular patterns, we investigated whether the Spike protein is sufficient to induce long-lasting nociceptive alterations and the immune pathways involved. Intraplantar administration of recombinant Spike protein in mice elicited robust and persistent mechanical and thermal hypersensitivity, accompanied by transient local inflammation, sustained systemic cytokine alterations, and prolonged spinal neuroinflammation. Spike-induced paw inflammation and persistent mechanical hypersensitivity were dependent on Toll-like receptor 4 (TLR4), as both pharmacological inhibition and genetic deficiency of TLR4 abolished these responses. Mechanistically, depletion of mononuclear phagocytes or blockade of T-cell co-stimulation prevented the development of persistent mechanical hypersensitivity, supporting a role for coordinated innate and adaptive immune mechanisms in the establishment of long-lasting nociceptive sensitization. In parallel, Spike induced increases in lymphocyte-associated cytokines, including IFN-γ, IL-4, and IL-17, with modest and transient changes in peripheral compartments but pronounced and sustained elevations in the spinal cord. Among these, IFN-γ emerged as a critical contributor to these responses, as its genetic deletion abolished both inflammation and persistent hypersensitivity. Collectively, these findings demonstrate that the SARS-CoV-2 Spike protein is sufficient to trigger persistent pain-like states through a TLR4-driven neuroimmune cascade involving phagocytes, T cells, and IFN-γ signaling. This study provides mechanistic insight into neuroimmune pathways that may contribute to persistent pain following SARS-CoV-2 infection and establishes a tractable mechanistic experimental model for investigating Spike-induced neuroimmune mechanisms of chronic pain.

Fibromyalgia (FM) is a chronic pain disorder with a severe impact on a person's health-related quality of life. In addition to the characteristic widespread pain and fatigue, people with FM regularly experience sensory abnormalities and report lingering, often painful sensations after mechanical probing of the skin. The neurobiological changes which underlie the varied symptoms in FM are incompletely understood, but many symptoms and signs can be transferred from patients to mice by administration of patient IgG. The present study aimed to explore whether sensory afferents, including nociceptors, in rodents injected with FM patient IgG displayed ongoing firing after mechanical stimulation. Skin saphenous nerve recordings were retrospectively analyzed to assess the presence and quality of ongoing discharges following mechanical stimulation of sensory afferents in mouse skin after passive-transfer of FM patient IgG. Our analysis revealed that C-mechano and Aδ-mechano afferents innervating the skin in FM-IgG treated animals fired significantly more action potentials after, but not during, mechanical force steps compared to healthy control-IgG treated afferents. This sustained mechanically evoked activity mirrors the nociceptor hyperexcitability observed in people with FM, suggesting that autoreactive IgG might underly this phenomenon in patients. This adds to the growing body of evidence demonstrating that FM symptoms arise from changes in the peripheral nervous system generated by circulating autoreactive IgG. KEY POINTS: Passive transfer of fibromyalgia (FM)-IgG causes sensitization of mechano-sensitive afferents in the skin of mice. Aδ-mechano and C-mechano sensitive afferents in skin from mice injected with FM-IgG display more after-discharge following mechanical stimulation than mice injected with healthy control-IgG. Changes in the peripheral nervous system caused by circulating autoreactive IgG play a role in FM signs and symptoms.

OBJECTIVE:Osteoarthritis (OA) is a degenerative joint disease characterized by chronic pain. We investigated whether the ion channel transient receptor potential melastatin 3 (TRPM3), expressed in sensory neurons, mediates OA pain. DESIGN:We used genetically modified mice, pharmacological tools, and behavioural assessments to evaluate the role of TRPM3 in OA pain induced by monosodium iodoacetate (MIA) or partial medial meniscectomy (PMM). Mice with global Trpm3 knockout (Trpm3-/-) and conditional deletion in sensory neurons (Advillin-Cre/Trpm3fl/fl) were compared with control mice. Selective TRPM3 antagonists (ononetin and isosakuranetin) were tested for their ability to reverse established pain. Histological analyses were performed to assess cartilage damage. RESULTS:Global deletion of Trpm3 prevented the development of pain behaviours in both MIA (mean difference [MD] = -7.8, 95% CI: -13.6 to -2.1) and PMM (MD = -13.6, 95% CI: -22.3 to -4.9) models without inhibiting structural cartilage damage. Sensory neuron-specific Trpm3 deletion replicated this effect in PMM mice (MD = -9.0, 95% CI: -15.0 to -3.1), demonstrating a neuronal contribution. Furthermore, pharmacological inhibition of TRPM3 with ononetin (MIA: MD = -2.8, 95% CI: -4.4 to -1.4; PMM: MD = -1.5, 95% CI: -2.2 to -0.7) or isosakuranetin (MIA: MD = -3.0, 95% CI: -4.4 to -1.6; PMM: MD = -1.5, 95% CI: -2.2 to -0.8) reversed established mechanical hypersensitivity in OA mice. CONCLUSIONS:TRPM3 expressed in sensory neurons is a critical mediator of OA pain in mice. Selective TRPM3 antagonism effectively alleviates established pain, supporting this channel as a potential therapeutic target for chronic pain associated with OA.

The endogenous peroxisome proliferator-activated receptors (PPARγ) agonist 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) stimulates sensory neurons by activating transient receptor potential A1 (TRPA1). Synthetic thiazolidinedione PPARγ agonists have been used as antidiabetic agents but have also been explored as experimental analgesics. Here, we have used intracellular Ca2+-measurements and voltage-clamp recordings to examine the effects of several PPARγ ligands on TRPA1 in sensory neurons and cell lines and examined nociception produced by local intraplantar administration of troglitazone. Troglitazone, rosiglitazone, nTZDpa, and the PPARγ antagonist GW9662 evoked concentration-dependent Ca2+-influx responses in TRPA1 expressing, but not untransfected Chinese hamster ovary (CHO)cells. Furthermore, troglitazone, nTZDpa, and GW9662 evoked [Ca2+]i-responses in mouse DRG neurons expressing TRPA1. Responses were abolished by the TRPA1 antagonist A967079 and were absent in DRG neurons from Trpa1-/- mice. The TRPA1 agonist activity of troglitazone, nTZDpa and GW9662 were unaffected by incubation with an excess of cysteine-methyl ester, indicating that these ligands do not act by covalent modification of cysteine residues, but rather through a non-covalent interaction with TRPA1. The cysteine reducing agent DTT did not reverse the effects of Troglitazone, nTZDpa and GW9662, which suggests that the observed agonist effects were independent of cysteine oxidation. Intraplantar injections of troglitazone evoked pain-responses in wild-type mice, but not in Trpa1-/- mice. Our molecular docking studies indicate that nTZDpa and troglitazone bind to overlapping sites in a hydrophobic pocket in the pre-S1 helix These observations demonstrate that multiple PPARγ ligands stimulate TRPA1 and that nTZDpa may be a useful tool for investigations of TRPA1.

Pain and fatigue are common but poorly understood features of post-COVID Syndrome (PCS). To probe the mechanistic basis of these symptoms, we investigated sensory functions in patients with widespread pain attributed to PCS. Quantitative sensory testing revealed increased mechanical pain sensitivity and altered thermal sensitivities and microneurography demonstrated that patients with pain displayed abnormal spontaneous C-fibre activity. Administration of IgG from PCS patients with pain and fatigue (PCS-PF) to mice, conferred mechanical and cold hypersensitivities and reduced intra-epidermal nerve fibre density (IENFd). IgG from patients with fatigue but without pain (PCS-F) did not induce hypersensitivities but similarly reduced IENFd. In line with behavioural responses, sensory nerves from PCS-PF IgG treated mice showed increased responsiveness to mechanical and cold stimulation. PCS-PF IgG bound to isolated sensory neurons with staining intensities that correlated with the level of pain experienced by patients with PCS. These results indicate a causal role for autoantibodies in the pathogenesis of pain and sensory disturbances associated with PCS.

Fibromyalgia syndrome (FM) is characterized by widespread pain and fatigue. People living with FM also experience tactile allodynia, cold-evoked pain, paraesthesia and dysaesthesia. There is evidence of small fibre neuropathy and hyperexcitability of nociceptors in FM; however, the presence of other sensory abnormalities suggests involvement of large diameter sensory fibres. The passive transfer of FM IgG to mice causes cold and mechanical hyperalgesia associated with changes in A- and C-nociceptor function. However, whether FM IgG also confers sensitivity to light touch and whether large diameter sensory fibres contribute to symptoms evoked by cold is unknown. Here we demonstrate that the presence of sensory abnormalities such as tingling, correlate with the impact of FM, and that people with FM describe the sensation of cutaneous cooling with neuropathic descriptors such as tingling/pins and needles. We find a causal link between circulating FM IgG and the sensitization of large diameter, Aβ low threshold mechanoreceptors (Aβ-LTMRs) to mechanical and cold stimuli in mice ex vivo and in vivo. In keeping with our experimental observations, a larger proportion of Aβ-LTMRs respond to cold stimulation in people with FM, but in contrast to our results ex vivo, the same fibres display reduced responses to mechanical stimuli. These results expand the pathophysiological role of IgG in FM and will inform future studies of sensory symptoms and pain in people with FM.

The platinum chemotherapeutic oxaliplatin produces dose-limiting pain, dysesthesia, and cold hypersensitivity in most patients immediately after infusion. An improved understanding of the me-chanisms underlying these symptoms is urgently required to facilitate the development of symptomatic or preventative therapies. In this study, we have used skin-saphenous nerve recordings in vitro and behavioral experiments in mice to characterize the direct effects of oxaliplatin on different types of sensory afferent fibers. Our results confirmed that mice injected with oxaliplatin rapidly develop mechanical and cold hypersensitivities. We further noted profound changes to A fiber activity after the application of oxaliplatin to the receptive fields in the skin. Most oxaliplatin-treated A delta-and rapidly adapting A beta-units lost mechanical sensitivity, but units that retained responsiveness additionally displayed a novel, aberrant cold sensitivity. Slowly adapting A beta-units did not display mechanical tachyphylaxis, and a subset of these fibers was sensitized to mechanical and cold stimulation after oxaliplatin treatment. C fiber afferents were less affected by acute applications of oxaliplatin, but a subset gained cold sensitivity. Taken together, our findings suggest that direct effects on peripheral A fibers play a dominant role in the development of acute oxaliplatin-induced cold hypersensitivity, numbness, and dysesthesia.Perspective: The chemotherapeutic drug oxaliplatin rapidly gives rise to dose-limiting cold pain and dysesthesia. Here, we have used behavioral and electrophysiological studies of mice to characterize the responsible neurons. We show that oxaliplatin directly confers aberrant cold responsiveness to subsets of A-fibers while silencing other fibers of the same type.(R) 2023 The Author(s). Published by Elsevier Inc. on behalf of United States Association for the Study of Pain, Inc This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/ 4.0/).

Serumprogesterone sulfates were evaluated in the etiology of gestational diabetesmellitus (GDM). Serumprogesterone sulfates were measured using ultra-performance liquidchromatography-tandem mass spectrometry in four patient cohorts: 1) theHyperglycemia and Adverse Pregnancy Outcomes study; 2) London-based women ofmixed ancestry and 3) UK-based European-ancestry women with or without GDM; 4)11-13 week pregnant women with BMI≤25 or BMI≥35 with subsequent uncomplicatedpregnancies or GDM.Glucose-stimulatedinsulin secretion (GSIS) was evaluated in response to progesterone sulfates in mouseislets and human islets. Calcium fluorescence was measured in HEK293 cellsexpressing TRPM3. Computer modelling using Molecular Operating Environment (MOE)generated 3D structures of TRPM3. Epiallopregnanolonesulfate (PM5S) concentrations were reduced: in GDM (p<0.05); in womenwith higher fasting plasma glucose (p<0.010); and in early pregnancy samplesfrom women who subsequently developed GDM with BMI≥35 (p<0.05). In islets,50µM PM5S increased GSIS by at least 2-fold (P<0.001); isosakuranetin (TRPM3-inhibitor)abolished this effect. PM5S increased calcium influx in TRPM3-expressingHEK293 cells. Computer modelling and docking showed identical positioning ofPM5S to the natural ligand in TRPM3. PM5S increases GSISand is reduced in GDM serum. The activation of GSIS by PM5S is mediated by TRPM3in both mouse and human islets.

ABSTRACT TRPA1 expressed in peripheral sensory neurons is important for nociception. Pharmacological inhibition or genetic ablation of TRPA1 profoundly reduces normal behavioural sensitivity to noxious cold and mechanical stimulation, as well as sensory neuron responses to mechanical stimulation. TRPA1 inhibition also reverses cold and mechanical hypersensitivities in chronic pain models in vivo . Here we demonstrate that these striking effects of TRPA1 inactivation result from an increased constitutive activity of kappa opioid receptors (KOR) co-expressed with TRPA1 in sensory neurons. Inhibition of KOR in Trpa1 -/- mice restores nociception and neuronal activity to the levels observed in wild-type mice and reverses the analgesic effects of TRPA1 antagonism in naïve mice and in neuropathic and inflammatory pain conditions. TRPA1 regulation of KOR activity in sensory neurons provides a novel mechanism to produce peripherally mediated analgesia. Our findings suggest that TRP channel regulation of constitutive GPCR activity, may be a process of general physiological importance.

Background Fibromyalgia syndrome (FMS) is the most common chronic widespread pain condition in rheumatology. Until recently, no clear pathophysiological mechanism for fibromyalgia had been established, resulting in management challenges. Recent research has indicated that serum immunoglobulin Gs (IgGs) may play a role in FMS. We undertook a research prioritisation exercise to identify the most pertinent research approaches that may lead to clinically implementable outputs. Methods Research priority setting was conducted in five phases: situation analysis; design; expert group consultation; interim recommendations; consultation and revision. A dialogue model was used, and an international multi-stakeholder expert group was invited. Clinical, patient, industry, funder, and scientific expertise was represented throughout. Recommendation-consensus was determined via a voluntary closed eSurvey. Reporting guideline for priority setting of health research were employed to support implementation and maximise impact. Results Arising from the expert group consultation (n = 29 participants), 39 interim recommendations were defined. A response rate of 81.5% was achieved in the consensus survey. Six recommendations were identified as high priority- and 15 as medium level priority. The recommendations range from aspects of fibromyalgia features that should be considered in future autoantibody research, to specific immunological investigations, suggestions for trial design in FMS, and therapeutic interventions that should be assessed in trials. Conclusions By applying the principles of strategic priority setting we directed research towards that which is implementable, thereby expediating the benefit to the FMS patient population. These recommendations are intended for patients, international professionals and grant-giving bodies concerned with research into causes and management of patients with fibromyalgia syndrome.

Fibromyalgia syndrome (FMS) is characterized by widespread pain and tenderness, and patients typically experience fatigue and emotional distress. The etiology and pathophysiology of fibromyalgia are not fully explained and there are no effective drug treatments. Here we show that IgG from FMS patients produced sensory hypersensitivity by sensitizing nociceptive neurons. Mice treated with IgG from FMS patients displayed increased sensitivity to noxious mechanical and cold stimulation, and nociceptive fibers in skin-nerve preparations from mice treated with FMS IgG displayed an increased responsiveness to cold and mechanical stimulation. These mice also displayed reduced locomotor activity, reduced paw grip strength, and a loss of intraepidermal innervation. In contrast, transfer of IgG-depleted serum from FMS patients or IgG from healthy control subjects had no effect. Patient IgG did not activate naive sensory neurons directly. IgG from FMS patients labeled satellite glial cells and neurons in vivo and in vitro, as well as myelinated fiber tracts and a small number of macrophages and endothelial cells in mouse dorsal root ganglia (DRG), but no cells in the spinal cord. Furthermore, FMS IgG bound to human DRG. Our results demonstrate that IgG from FMS patients produces painful sensory hypersensitivities by sensitizing peripheral nociceptive afferents and suggest that therapies reducing patient IgG titers may be effective for fibromyalgia.

Introduction: Serum progesterone sulfate (P4S) concentrations increase in pregnancy and bind receptors that influence glucose homeostasis. We hypothesized that P4S modulate glucose homeostasis in pregnancy. Methods: Serum P4S were assayed using ultra-performance liquid chromatography- tandem mass spectrometry. Samples were studied in three separate patient groups: women with GDM (n=20) and matched healthy controls (n=38); participants (average 28 weeks’ gestation) from the upper (n=93) and lower (n=94) quartiles of fasting plasma glucose in the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) Study; 11-13 week pregnant women with BMI≤25 or BMI≥35 who subsequently had uncomplicated pregnancies or developed GDM (n=50/group). Glucose-stimulated insulin secretion (GSIS) was quantified in isolated wild-type, Fxr-/- and Tgr5-/- mouse and human islets in the presence of P4S and TRPM3 inhibitor isosakuranetin (ISO). Intracellular calcium concentrations were measured after treatment with P4S in TRPM3 transfected HEK293. Computer modelling using Molecular Operating Environment generated 3D structures of TRPM3 and P4S binding. Results: Epiallopregnanolone sulfate (PM5S) concentrations were reduced in serum samples from the HAPO study participants with higher fasting plasma glucose (p<0.01), in women with GDM (p<0.05) and in early pregnancy samples from women with BMI ≥35 who subsequently developed GDM (P<0.05). In wild-type and human islets, 50μM PM5S increased GSIS by at least 2-fold at 20mM glucose concentrations (P<0.001). This effect was not abolished from islets obtained from Fxr-/- or Tgr-/- mice, however it was abolished by ISO. PM5S elicited Ca2+ influx in TRPM3-expressing HEK293 cells. Computer modelling and docking showed identical positioning of PM5S to cholesterol hemisuccinate in TRPM3. Conclusion: PM5S increases GSIS and concentrations are reduced in the serum of women with GDM. The increased GSIS is mediated by TRPM3. Disclosure H. Fan: None. F. Fraternali: None. K. F. Hunt: None. J. Bowe: None. C. Williamson: Consultant; Self; GlaxoSmithKline plc., Mirum Pharmaceuticals. A. Mitchell: None. M. Giorgi: None. P. M. Jones: None. D. R. Mccance: None. D. A. Andersson: None. S. Bevan: None. H. Marschall: None. I. Eberini: None. Funding Tommy’s; Guy’s and St Thomas’ Biomedical Research Centre; National Health Service

Animal models are important tools in diabetes research as ethical and logistical constraints limit access to human tissue. Beta cell dysfunction is a common contributor to the pathogenesis of most types of diabetes. Spontaneous hyperglycaemia developed in a colony of C57BL/6J mice at King’s College London (KCL). Sequencing identified a mutation in the Ins2 gene, causing a glycine to serine substitution at position 32 on the B chain of the preproinsulin2 molecule. Mice with the Ins2+/G32S mutation were named KCL Ins2 G32S (KINGS) mice. The same mutation in humans (rs80356664) causes dominantly inherited neonatal diabetes. Mice were characterised and beta cell function was investigated. Male mice became overtly diabetic at around 5 weeks of age whereas female mice had only slightly elevated non-fasting glycaemia. Islets showed decreased insulin content and impaired glucose-induced insulin secretion, which was more severe in males. Transmission electron microscopy and studies of gene and protein expression showed beta cell endoplasmic reticulum (ER) stress in both sexes. Despite this, beta cell numbers were only slightly reduced in older animals. In conclusion, the KINGS mouse is a novel model of a human form of diabetes that may be useful to study beta cell responses to ER stress.

SUMMARYFibromyalgia syndrome (FMS) is a chronic pain condition characterized by widespread pain and tenderness1,2. The etiology and pathophysiology of fibromyalgia are unknown and there are no effective treatments. Here we show that sensory hypersensitivity in FMS is caused by autoantibodies that act by sensitizing nociceptive sensory neurons. Administration of IgG from FMS patients increased mouse pain sensitivities to stimulation with mechanical pressure and cold. In contrast, transfer of IgG depleted samples from FMS patients or IgG from healthy control subjects had no effect on pain sensitivity. Sensory nerve fibres in ex vivo skin-nerve preparations from mice treated with FMS IgG were hypersensitive to mechanical stimulation. Immunohistochemical analysis revealed that IgG from FMS patients specifically labeled satellite glial cells and myelinated fibre tracts, as well as a small number of macrophages and endothelial cells in mouse dorsal root ganglia but not skin, muscle, spinal cord and brain. Our results demonstrate that fibromyalgia pain is caused by IgG autoantibodies that sensitize peripheral nociceptive afferents neurons and suggest that therapies that reduce patient IgG titres may be effective treatments of fibromyalgia pain.