Publications
221 results found
Anand U, Anand P, Sodergren MH, 2023, Terpenes in Cannabis sativa inhibit capsaicin responses in rat DRG neurons via Na+/K+ ATPase activation, International Journal of Molecular Sciences, Vol: 24, ISSN: 1422-0067
Terpenes in Cannabis sativa exert analgesic effects, but the mechanisms are uncertain. We exam-ined the effects of 10 terpenes on capsaicin responses in an established model of neuronal hyper-sensitivity. Adult rat DRG neurons cultured with neurotrophic factors NGF and GDNF, were loaded with Fura2AM for calcium imaging, and treated with individual terpenes or vehicle for 5 minutes, followed by 1 µMol capsaicin. In vehicle treated control experiments, capsaicin elicited immediate and sustained calcium influx. Most neurons treated with terpenes responded to capsaicin after 6-8 minutes. Few neurons showed immediate capsaicin responses that were transient or normal. The delayed responses were found to be due to calcium released from the endoplasmic reticulum, as they were maintained in calcium/magnesium free media, but not after thapsigargin pre-treatment. Terpene inhibition of calcium influx was reversed after washout of medium, in the absence of terpenes, and in the presence of the Na+/K+ ATPase inhibitor ouabain, but not CB1 or CB2 receptor antagonists. Thus terpenes inhibit capsaicin evoked calcium influx by Na+/K+ ATPase activation. Immunofluorescence showed TRPV1 co-expression with α1β1 Na+/K+ ATPase in most neurons while others were either TRPV1 or α1β1 Na+/K+ ATPase positive.
Anand U, Pacchetti B, Anand P, et al., 2022, The endocannabinoid analgesic entourage effect: investigations in cultured DRG neurons, Journal of Pain Research, Vol: 15, Pages: 3493-3507, ISSN: 1178-7090
Background: The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the “entourage effect”. We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors.Methods: Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 μM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 μMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3– 7 rats, controls N=16), and analysed.Results: In controls, 1 μMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10– 100 μMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001– 100 μMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P< 0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 μMol (P< 0.001), in 60% neurons, as did 2-LG at 0.1– 100 μMol (P< 0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 μMol 2-AG, 5 μMol 2-PG and 10 μMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P< 0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist.Conclusion: 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are r
Anand P, Privitera R, Donatien P, et al., 2022, Reversing painful and non-painful diabetic neuropathy with the capsaicin 8% patch: Clinical evidence for pain relief and restoration of function via nerve fiber regeneration, Frontiers in Neurology, Vol: 13, ISSN: 1664-2295
Introduction: Current oral treatments for pain in diabetic peripheral neuropathy (DPN) do not affect the progression of DPN i.e.“disease modification”. We assessed whether Capsaicin 8% patch treatment can provide pain relief and also restore nerve densityand function via nerve regeneration, in both painful (PDPN) and non-painful (NPDPN) diabetic peripheral neuropathy.Methods: 50 participants with PDPN were randomized to receive Capsaicin 8% patch Qutenza with Standard of Care (SOC) (PDPNQ+SOC group), or SOC alone (PDPN SOC group). Pain symptoms were assessed with a diary (Numerical Pain Rating Scale, NRPS) andquestionnaires. Investigations included quantitative sensory testing (QST) and distal calf skin biopsies, at baseline and 3 monthsafter baseline visit; subsequent options were 3-monthly visits over 1 year. 25 participants with NPDPN had tests at baseline, and3 months after all received Capsaicin 8% patch treatment.Results: At 3 months after baseline, PDPN Q+SOC group had reduction in NPRS score (p=0.0001), but not PDPN SOC group.Short-Form McGill Pain Questionnaire (SF-MPQ) showed significant reductions in scores for overall and other pain descriptors onlyin the PDPN Q+SOC group. Warm perception thresholds were significantly improved only in the PDPN Q+SOC group (p=0.02), andcorrelated with reduction in SF-MPQ overall pain score (p=0.04). NPDPN Q+SOC group did not report pain during the entire study.Density of intra-epidermal nerve fibres (IENF) with PGP9.5 was increased at 3 months in PDPN Q+SOC (p=0.0002) and NPDPN Q+SOC(p=0.002) groups, but not in the PDPN SOC group. Increased sub-epidermal nerve fibres (SENF) were observed with GAP43 (markerof regenerating nerve fibres) only in PDPN Q+SOC (p=0.003) and NPDPN Q+SOC (p=0.0005) groups. Pain relief in the PDPN Q+SOCgroup was correlated with the increased PGP9.5 IENF (p=0.0008) and GAP43 (p=0.004), whereas those with lack of pain reliefshowed no such increase; in some subjects pain relief and increas
Anand U, Oldfield C, Pacchetti B, et al., 2021, Dose-related inhibition of capsaicin responses by cannabinoids CBG, CBD, THC and their combination in cultured sensory neurons, Journal of Pain Research, Vol: 14, Pages: 3603-3614, ISSN: 1178-7090
AbstractBackground: The analgesic effects of Cannabis sativa are mediated by ∆9 tetrahydrocannabinol (THC), but the contributions of other bioactive complex components, including cannabigerol (CBG) and cannabidiol (CBD), are unclear. We describe the individual and combined effects of CBG, CBD and THC, on blocking capsaicin responses in dorsal root ganglion (DRG), neurons, in an in vitro model of nociceptor hypersensitivity.Materials and Methods: Adult rat DRG were dissected and enzyme digested to obtain a neuronal suspension in BSF2 medium containing 2% fetal calf serum, and the neurotrophic factors NGF and GDNF. After 48 h, cultured neurons were loaded with Fura-2 AM, to determine the effects of cannabinoids on capsaicin responses using calcium imaging. In control experiments, neurons were treated with vehicle, followed by 1 µM capsaicin. In cannabinoid treated cultures, CBG, CBD or THC were applied individually, or combined (1:1:1 ratio), followed by 1 µM capsaicin. Data from n=6 experiments were analysed with Student’s t-test, and Pearson’s correlation coefficient. Results: CBG, CBD and THC, applied individually, elicited dose-related calcium influx in a subset of DRG neurons, and a corresponding dose-related reduction of subsequent responses to capsaicin. Maximum inhibition of capsaicin responses was observed at 30 µM CBG, 100 µM CBD, and 100 µM THC individually, and with combined CBD+CBG+THC (1:1:1) at 90 µM. THC+CBD+CBG combined in a 1:1:1 proportion has the potential to enhance the potency of these compounds applied individually. There was a high correlation between cannabinoid-mediated calcium influx and reduction of capsaicin responses: CBG = -0.88, THC = -0.97, CBD = -0.99 and combined CBG + THC + CBD = -1.00. Conclusions: CBG, CBD and THC demonstrated potent dose-related inhibition of capsaicin responses in DRG neurons when applied individually in vitro, and enhanced when applied in combination
Shillo P, Yiangou Y, Donatien P, et al., 2021, Nerve and Vascular Biomarkers in Skin Biopsies Differentiate Painful From Painless Peripheral Neuropathy in Type 2 Diabetes, FRONTIERS IN PAIN RESEARCH, Vol: 2
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Rice ASC, Dworkin RH, Finnerup NB, et al., 2021, Efficacy and safety of EMA401 in peripheral neuropathic pain: results of 2 randomised, double-blind, phase 2 studies in patients with postherpetic neuralgia and painful diabetic neuropathy, PAIN, Vol: 162, Pages: 2578-2589, ISSN: 0304-3959
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Privitera R, Anand P, Donatien P, et al., 2021, Capsaicin 8% patch treatment in non-freezing cold injury : evidence for pain relief and nerve regeneration., Frontiers in Neurology, Vol: 12, Pages: 1-10, ISSN: 1664-2295
Introduction: Neuropathic pain associated with Non-freezing Cold Injury (NFCI) is a major burden to military service personnel. A key feature of NFCI is reduction of the intra-epidermal nerve fibre density in skin biopsies, in keeping with painful neuropathy. Current oral treatments are generally ineffective and have undesirable side effects. Capsaicin 8% patch (Qutenza) has been shown to be well-tolerated and effective for reducing neuropathic pain, for up to 3 months after a single 30-minute application.Methods: In this single-centre open label study, 16 military participants with NFCI (mean duration 49 months) received 30-minute Capsaicin 8% patch treatment to the feet and distal calf. Pain symptoms were assessed using a pain diary (with the 11-point Numerical Pain Rating Scale, NPRS) and questionnaires, the investigations included skin biopsies, performed before and three months after treatment.Results: Participants showed significant decrease in spontaneous pain (mean NPRS: -1.1, 95% CI: 0.37 to 1.90; p=0.006), and cold-evoked pain (-1.2, 95% CI 0.40 to 2.04; p=0.006). The time-course of pain relief over 3 months was similar to other painful neuropathies. Patient Global Impression of Change showed improvement (p=0.0001).Skin punch biopsies performed 3 months after the patch application showed significant increase of nerve fibres with structural marker PGP9.5 (intra-epidermal nerve fibres [IENFs], p<0.0001; sub-epidermal nerve fibres [SENFs]; p=<0.0001), and of regenerating nerve fibres with their selective marker GAP43 (p=0.0001). The increase of IENFs correlated with reduction of spontaneous (p=0.027) and cold-evoked pain (p=0.019).Conclusions: Capsaicin 8% patch provides an exciting new prospect for treatment of NFCI, with regeneration and restoration of nerve fibres, for the first time, in addition to pain relief.
Privitera R, Anand P, 2021, Capsaicin 8% patch Qutenza and other current treatments for neuropathic pain in chemotherapy-induced peripheral neuropathy (CIPN), CURRENT OPINION IN SUPPORTIVE AND PALLIATIVE CARE, Vol: 15, Pages: 125-131, ISSN: 1751-4258
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Anand U, Pacchetti B, Anand P, et al., 2021, Cannabis-based medicines and pain: a review of potential synergistic and entourage effects, Pain Management, Vol: 11, Pages: 395-403, ISSN: 1758-1869
The recent legalization of medicinal cannabis in several jurisdictions has spurred the development of therapeutic formulations for chronic pain. Unlike pure delta-9-tetrahydrocannabinol (THC), full-spectrum products contain naturally occurring cannabinoids and have been reported to show improved efficacy or tolerability, attributed to synergy between cannabinoids and other components in the cannabis plant. Although ‘synergy’ indicates that two or more active compounds may produce an additive or combined effect greater than their individual analgesic effect, potentiation of the biological effect of a compound by related but inactive compounds, in combination, was termed the ‘entourage effect’. Here, we review current evidence for potential synergistic and entourage effects of cannabinoids in pain relief. However, definitive clinical trials and in vitro functional studies are still required.
Anand U, Jones B, Korchev Y, et al., 2020, CBD effects on TRPV1 signaling pathways in cultured DRG neurons, Journal of Pain Research, Vol: 2020, Pages: 2269-2278, ISSN: 1178-7090
Introduction: Cannabidiol (CBD) is reported to produce pain relief, but the clinically relevant cellular and molecular mechanisms remain uncertain. The TRPV1 receptor integrates noxious stimuli and plays a key role in pain signaling. Hence, we conducted in vitro studies, to elucidate the efficacy and mechanisms of CBD for inhibiting neuronal hypersensitivity in cultured rat sensory neurons, following activation of TRPV1. Methods: Adult rat dorsal root ganglion (DRG) neurons were cultured, and supplemented with the neurotrophic factors NGF and GDNF, in an established model of neuronal hypersensitivity. 48 h after plating, neurons were stimulated with CBD (Adven 150, EMMAC Life Sciences) at 1, 10, 100 nMol/L and 1, 10 and 50 µMol/L. In separate experiments, DRG neurons were also stimulated with capsaicin with or without CBD (1 nMol/L to10 µMol/L), in a functional calcium imaging assay. The effects of the adenylyl cyclase activator forskolin and the calcineurin inhibitor cyclosporin were determined. We also measured forskolin-stimulated cAMP levels, without and after treatment with CBD, using a homogenous time resolved fluorescence (HTRF) assay. The results were analysed using Student’s t-test. Results: DRG neurons treated with 10 and 50 µMol/L CBD showed calcium influx, but not at lower doses. Neurons treated with capsaicin demonstrated robust calcium influx, which was dose-dependently reduced in the presence of low dose CBD (IC50 = 100 nMol/L). The inhibition or desensitization by CBD was reversed in the presence of forskolin and cyclosporin. Forskolin stimulated cAMP levels were significantly reduced in CBD treated neurons.Conclusions: CBD at low doses corresponding to plasma concentrations observed physiologically, inhibits or desensitizes neuronal TRPV1 signalling by inhibiting the adenylyl cyclase – cAMP pathway, which is essential for maintaining TRPV1 phosphorylation and sensitization. CBD also facilitated calcineurin-med
Anand P, Dickenson A, Finco G, et al., 2019, Novel insights on the management of pain: highlights from the 'Science of Relief' meeting., Pain Management, Vol: 9, Pages: 521-533, ISSN: 1758-1869
The 'Science of Relief' event, held in Milan on 10-11 May 2019, was aimed at promoting dialog between different stakeholders among scientific associations, pharma industry, healthcare services and related institutions. The goal was to renew interest and attention on the management of pain, sharing new solutions in order to bring the patients and their quality of life to the center of attention. An international group of scientists and clinicians presented and discussed new and known evidence in the field of chronic pain, from physiopathology and diagnosis to the choice of appropriate and timely pharmacological treatments. This paper reports the highlights of those presentations.
Anand U, Korchev Y, Anand P, 2019, The role of urea in neuronal degeneration and sensitization: an in vitro model of uremic neuropathy, Molecular Pain, Vol: 15, ISSN: 1744-8069
Background: Uremic neuropathy commonly affects patients with chronic kidney disease (CKD), with painful sensations in the feet, followed by numbness and weakness in the legs and hands. The symptoms usually resolve following kidney transplantation, but the mechanisms of uremic neuropathy and associated pain symptoms remain unknown. As blood urea levels are elevated inpatients with CKD, we examined the morphological and functional effects of clinically observed levels of urea on sensory neurons. Methods: Rat DRG neurons were treated with 10or50 mMol/L urea for 48 hours, fixed and immunostained for PGP9.5 and βIII tubulin immunofluorescence, ,. Neurons were also immunostained for TRPV1, TRPM8 and Gap43 expression, and the capsaic insensitivity of urea or vehicle treated neurons was determined.Results: Urea treated neurons had degenerating neurites with diminished PGP9.5 immunofluorescence,and swollen, retracted growth cones. βIII tubulin appeared clumped after urea treatment. Neurite lengths were significantly reduced to 60 ± 2.6%(10 mMol/L, **P<0.01), and to 56.2± 3.3 %, (50 mMol/L, **P<0.01),urea treatmentfor 48 hours, compared with control neurons. Fewer neurons survived urea treatment,with 70.08 ± 13.3% remaining after 10 mMol/L (*P<0.05), and 61.49 ± 7.4 % after 50 mMol/L ureatreatment (**P<0.01), compared with controls. The proportion of neurons expressing TRPV1 wasreduced after urea treatment, but not TRPM8 expressing neurons. In functional studies, treatment with urea resulted in dose-dependent neuronal sensitization.Capsaicinresponses were significantly increased to 115.29 ± 3.4%(10 mMol/L, **P<0.01) and 125.3 ± 4.2%(50 mMol/L,**P<0.01), compared with controls. Sensitization due to urea was eliminated in the presence of the TRPV1 inhibitor SB705498, the MEKinhibitor PD98059,the PI3 kinase inhibitor LY294002, and the TRPM8 inhibitor AMTB. ConclusionNeurite degenerationandsensitization a
Anand P, Elsafa E, Privitera R, et al., 2019, Rational treatment of chemotherapy-induced peripheral neuropathy with capsaicin 8% patch: from pain relief towards disease modification, Journal of Pain Research, Vol: 12, Pages: 2039-2052, ISSN: 1178-7090
Purpose: Chemotherapy-induced peripheral neuropathy (CIPN) with associated chronic pain is a common and disabling condition. Current treatments for neuropathic pain in CIPN are largely ineffective, with unfavorable side-effects. The capsaicin 8% patch (capsaicin 179 mg patch) is approved for the treatment of neuropathic pain: a single topical cutaneous application can produce effective pain relief for up to 12 weeks. We assessed the therapeutic potential of capsaicin 8% patch in patients with painful CIPN, and its mechanism of action.Patients and methods: 16 patients with chronic painful CIPN (mean duration 2.5 years), in remission for cancer and not receiving chemotherapy, were treated with 30 min application of capsaicin 8% patch to the feet. Symptoms were monitored using the 11-point numerical pain rating scale (NPRS), and questionnaires. Investigations were performed at baseline and three months after patch application, including skin biopsies with a range of markers, and quantitative sensory testing (QST).Results: Patients reported significant reduction in spontaneous pain (mean NPRS: −1.27; 95% CI 0.2409 to 2.301; p=0.02), touch-evoked pain (−1.823; p=0.03) and cold-evoked pain (−1.456; p=0.03). Short-Form McGill questionnaire showed a reduction in neuropathic (p=0.0007), continuous (p=0.01) and overall pain (p=0.004); Patient Global Impression of Change showed improvement (p=0.001). Baseline skin biopsies showed loss of intra-epidermal nerve fibers (IENF), and also of sub-epidermal nerve fibers quantified by image analysis. Post-patch application skin biopsies showed a significant increase towards normalization of intra-epidermal and sub-epidermal nerve fibers (for IENF: structural marker PGP9.5, p=0.009; heat receptor TRPV1, p=0.027; regenerating nerve marker GAP43, p=0.04). Epidermal levels of Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), and Langerhans cells were also normalized. QST remained unchanged and there were no systemic side-
Shillo P, Selvarajah D, Greig M, et al., 2019, Reduced vitamin D levels in painful diabetic peripheral neuropathy, DIABETIC MEDICINE, Vol: 36, Pages: 44-51, ISSN: 0742-3071
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Sloan G, Shillo P, Selvarajah D, et al., 2018, A new look at painful diabetic neuropathy, DIABETES RESEARCH AND CLINICAL PRACTICE, Vol: 144, Pages: 177-191, ISSN: 0168-8227
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Donatien P, Anand U, Yiangou Y, et al., 2018, Granulocyte-macrophage colony-stimulating factor receptor expression in clinical pain disorder tissues and role in neuronal sensitization, PAIN Reports, Vol: 3, Pages: e676-e676, ISSN: 2471-2531
Introduction: Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is highly expressed in peripheral macrophages and microglia, and is involved in arthritis and cancer pain in animal models. However, there is limited information on GM-CSFR expression in human central nervous system (CNS), peripheral nerves, or dorsal root ganglia (DRG), particularly in chronic pain conditions. Objectives: Immunohistochemistry was used to quantify GM-CSFR expression levels in human tissues, and functional sensory effects of GM-CSF were studied in cultured DRG neurons. Results: Granulocyte-macrophage colony-stimulating factor receptor was markedly increased in microglia at lesional sites of multiple sclerosis spinal cords (P = 0.01), which co-localised with macrophage marker CD68 (P = 0.009). In human DRG, GM-CSFR was expressed in a subset of small/medium diameter cells (30%) and few large cells (10%), with no significant change in avulsion-injured DRG. In peripheral nerves, there was a marked decrease in axonal GM-CSFR after chronic painful nerve injury (P = 0.004) and in painful neuromas (P = 0.0043); CD-68-positive macrophages were increased (P = 0.017) but did not appear to express GM-CSFR. Although control synovium showed absent GM-CSFR immunostaining, this was markedly increased in macrophages of painful osteoarthritis knee synovium. Granulocyte-macrophage colony-stimulating factor receptor was expressed in 17 ± 1.7% of small-/medium-sized cultured adult rat DRG neurons, and in 27 ± 3.3% of TRPV1-positive neurons. Granulocyte-macrophage colony-stimulating factor treatment sensitized capsaicin responses in vitro, which were diminished by p38 MAPK or TrkA inhibitors. Conclusion: Our findings support GM-CSFR as a therapeutic target for pain and hypersensitivity in clinical CNS and peripheral inflammatory conditions. Although GM-CSFR was decreased in chronic painful injured peripheral nerves, it could mediate CNS neuroinflammatory effects, which deserves
Anand U, Yiangou Y, Akbar A, et al., 2018, Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons, PLoS ONE, Vol: 13, ISSN: 1932-6203
IntroductionGlucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD).ObjectivesThe aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons.MethodsGLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging.ResultsSignificantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons.ConclusionOur results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of
Anand P, Privitera R, Yiangou Y, et al., 2017, Trench Foot or Non-Freezing Cold Injury As a Painful Vaso-Neuropathy: Clinical and Skin Biopsy Assessments., Frontiers in Neurology, Vol: 8, ISSN: 1664-2295
BACKGROUND: Trench foot, or non-freezing cold injury (NFCI), results from cold exposure of sufficient severity and duration above freezing point, with consequent sensory and vascular abnormalities which may persist for years. Based on observations of Trench foot in World War II, the condition was described as a vaso-neuropathy. While some reports have documented nerve damage after extreme cold exposure, sensory nerve fibres and vasculature have not been assessed with recent techniques in NFCI. OBJECTIVE: To assess patients with chronic sensory symptoms following cold exposure, in order to diagnose any underlying small fibre neuropathy, and provide insight into mechanisms of the persistent pain and cold hypersensitivity. METHODS: Thirty soldiers with cold exposure and persistent sensory symptoms (>4 months) were assessed with quantitative sensory testing, nerve conduction studies, and skin biopsies. Immunohistochemistry was used to assess intraepidermal (IENF) and subepidermal (SENF) nerve fibres with a range of markers, including the pan-neuronal marker protein gene product 9.5 (PGP 9.5), regenerating fibres with growth-associated protein 43 (GAP43), and nociceptor fibres with transient receptor potential cation channel subfamily V member 1 (TRPV1), sensory neuron-specific receptor (SNSR), and calcitonin gene-related peptide (CGRP). von Willebrand factor (vWF), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF) were used for assessing blood vessels, and transient receptor potential cation channel, subfamily A member 1 (TRPA1) and P2X purinoceptor 7 (P2X7) for keratinocytes, which regulate nociceptors via release of nerve growth factor. RESULTS: Clinical examination showed pinprick sensation was abnormal in the feet of 20 patients (67%), and between 67 and 83% had abnormalities of thermal thresholds to the different modalities. 7 patients (23%) showed reduced sensory action potential amplitude of plantar nerves. 27 patie
Shillo P, Selvarajah D, Greig M, et al., 2017, Nerve and vascular biomarkers in skin biopsies differentiate painful from painless advanced diabetic peripheral neuropathy, 53rd Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), Publisher: SPRINGER, Pages: S71-S72, ISSN: 0012-186X
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Privitera R, Birch R, Sinisi M, et al., 2017, Capsaicin 8% patch treatment for amputation stump and phantom limb pain: a clinical and functional MRI study, Journal of Pain Research, Vol: 10, Pages: 1623-1634, ISSN: 1178-7090
Purpose: The aim of this study was to measure the efficacy of a single 60 min application of capsaicin 8% patch in reducing chronic amputation stump and phantom limb pain, associated hypersensitivity with quantitative sensory testing, and changes in brain cortical maps using functional MRI (fMRI) scans.Methods: A capsaicin 8% patch (Qutenza) treatment study was conducted on 14 patients with single limb amputation, who reported pain intensity on the Numerical Pain Rating Scale ≥4/10 for chronic stump or phantom limb pain. Pain assessments, quantitative sensory testing, and fMRI (for the lip pursing task) were performed at baseline and 4 weeks after application of capsaicin 8% patch to the amputation stump. The shift into the hand representation area of the cerebral cortex with the lip pursing task has been correlated with phantom limb pain intensity in previous studies, and was the fMRI clinical model for cortical plasticity used in this study.Results: The mean reduction in spontaneous amputation stump pain, phantom limb pain, and evoked stump pain were −1.007 (p=0.028), −1.414 (p=0.018), and −2.029 (p=0.007), respectively. The areas of brush allodynia and pinprick hypersensitivity in the amputation stump showed marked decreases: −165 cm2, −80% (p=0.001) and −132 cm2, −72% (p=0.001), respectively. fMRI analyses provided objective evidence of the restoration of the brain map, that is, reversal of the shift into the hand representation of the cerebral cortex with the lip pursing task (p<0.05).Conclusion: The results show that capsaicin 8% patch treatment leads to significant reduction in chronic pain and, particularly, in the area of stump hypersensitivity, which may enable patients to wear prostheses, thereby improving mobility and rehabilitation. Phantom limb pain (“central” pain) and associated brain plasticity may be modulated by peripheral inputs, as they can be ameliorated by the peripherally restricted
Shillo P, Selvarajah D, Greig M, et al., 2017, Nerve and vascular biomarkers in skin biopsies differentiate painful from painless advanced diabetic peripheral neuropathy, Diabetic Medicine, Vol: 34, Pages: 32-32, ISSN: 0742-3071
Anand P, Yiangou Y, Anand U, et al., 2016, Nociceptin/Orphanin FQ receptor expression in clinical pain disorders and functional effects in cultured neurons, Pain, Vol: 157, Pages: 1960-1969, ISSN: 1872-6623
The Nociceptin/Orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand Nociceptin/Orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry, and assessed functional effects of NOP and [micro] opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder sub-urothelium revealed a remarkable several-fold increase in Detrusor Overactivity (p<0.0001) and Painful Bladder Syndrome patient specimens (p=0.0014), compared to controls. In post-mortem control human DRGs, 75-80% of small/medium neurons (<=50 [micro]m diameter) in the lumbar (somatic) and sacral (visceral) DRG were positive for NOP, and fewer large neurons; avulsion-injured cervical human DRG neurons showed similar numbers. NOP-immunoreactivity was significantly decreased in injured peripheral nerves (p=0.0004), and also in painful neuromas (p=0.025). Calcium imaging studies in cultured rat DRG neurons demonstrated dose-dependent inhibition of capsaicin responses in the presence of N/OFQ, with an IC50 of 8.6 pM. In cultured human DRG neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (p<0.001). The maximum inhibition of capsaicin responses was greater with N/OFQ than [mu]-opioid receptor agonist DAMGO. Our findings highlight the potential of NOP agonists, particularly in urinary bladder overactivity and pain syndromes. The regulation of NOP expression in visceral and somatic sensory neurons by target-derived neurotrophic factors deserves further study, and the efficacy of NOP selective agonists in clinical trials.
Anand U, Sinisi M, Fox M, et al., 2016, Mycolactone mediated neurite degeneration and functional effects in cultured human and rat DRG neurons: mechanisms underlying hypoalgesia in Buruli Ulcer, Molecular Pain, Vol: 12, ISSN: 1744-8069
Background: Mycolactone (ML) is a polyketide toxin secreted by the mycobacterium M.ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients withBuruli Ulcer. A recent pre-clinical study proposed that ML may produce analgesia via activationof the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shownanalgesic efficacy in animal models and clinical trials for neuropathic pain. We thereforeinvestigated the morphological and functional effects of ML in cultured human and rat dorsal rootganglia (DRG) neurons, and the role of AT2R using EMA401. Primary sensory neurons wereprepared from avulsed cervical human DRG, and rat DRG. 24 hours after plating, neurons wereincubated for 24 to 96 hours with synthetic ML A/B, followed by immunostaining with antibodiesto PGP9.5, Gap43, tubulin, or Mitotracker dye staining. Acute functional effects wereexamined by measuring capsaicin responses with calcium imaging in DRG neuronal culturestreated with ML.Results: Morphological effects: ML treated cultures showed dramatically reduced numbers ofsurviving neurons and non-neuronal cells, reduced Gap43 and tubulin expression, degeneratingneurites and reduced cell body diameter, compared with controls. Dose related reduction ofneurite length was observed in ML treated cultures. Mitochondria were distributed throughout thelength of neurites and soma of control neurons, but clustered in the neurites and soma of MLtreated neurons. Functional effects: ML treated human and rat DRG neurons showed doserelated inhibition of capsaicin responses, which were reversed by calcineurin inhibitorcyclosporine and phosphodiesterase inhibitor IBMX, indicating involvement of cAMP/ATPreduction. The morphological and functional effects of ML were not altered by Angiotensin II orAT2R antagonist EMA401.3Conclusion: ML induces toxic effects in DRG neurons, leading to impaired nociceptor function,neurite degeneration and cell death, resembling the cuta
Anand P, Privitera R, Yiangou Y, et al., 2016, Angiotensin II Type 2 receptor antagonist EMA401 for the treatment of pain in patients with chemotherapy-induced peripheral neuropathy: efficacy and nerve biomarkers in skin biopsies., Publisher: WILEY, Pages: 634-634, ISSN: 1351-5101
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Van Acker N, Ragé M, Sluydts E, et al., 2016, Automated PGP9.5 immunofluorescence staining: a valuable tool in the assessment of small fiber neuropathy?, BMC Research Notes, Vol: 9, ISSN: 1756-0500
BackgroundIn this study we explored the possibility of automating the PGP9.5 immunofluorescence staining assay for the diagnosis of small fiber neuropathy using skin punch biopsies. The laboratory developed test (LDT) was subjected to a validation strategy as required by good laboratory practice guidelines and compared to the well-established gold standard method approved by the European Federation of Neurological Societies (EFNS). To facilitate automation, the use of thinner sections. (16 µm) was evaluated. Biopsies from previously published studies were used. The aim was to evaluate the diagnostic performance of the LDT compared to the gold standard. We focused on technical aspects to reach high-quality standardization of the PGP9.5 assay and finally evaluate its potential for use in large scale batch testing.ResultsWe first studied linear nerve fiber densities in skin of healthy volunteers to establish reference ranges, and compared our LDT using the modifications to the EFNS counting rule to the gold standard in visualizing and quantifying the epidermal nerve fiber network. As the LDT requires the use of 16 µm tissue sections, a higher incidence of intra-epidermal nerve fiber fragments and a lower incidence of secondary branches were detected. Nevertheless, the LDT showed excellent concordance with the gold standard method. Next, the diagnostic performance and yield of the LDT were explored and challenged to the gold standard using skin punch biopsies of capsaicin treated subjects, and patients with diabetic polyneuropathy. The LDT reached good agreement with the gold standard in identifying small fiber neuropathy. The reduction of section thickness from 50 to 16 µm resulted in a significantly lower visualization of the three-dimensional epidermal nerve fiber network, as expected. However, the diagnostic performance of the LDT was adequate as characterized by a sensitivity and specificity of 80 and 64 %, respectively.ConclusionsThis study, desi
Granovsky Y, Anand P, Nakae A, et al., 2016, Normative data for A delta contact heat evoked potentials in adult population: a multicenter study, Pain, Vol: 157, Pages: 1156-1163, ISSN: 0304-3959
There has been a significant increase over recent years in the use of contact heat evoked potentials (CHEPs) for the evaluation of small nerve fiber function. Measuring CHEP amplitude and latency has clinical utility for the diagnosis and assessment of conditions with neuropathic pain. This international multicenter study aimed to provide reference values for CHEPs to stimuli at 5 commonly examined body sites. Contact heat evoked potentials were recorded from 226 subjects (114 females), distributed per age decade between 20 and 79 years. Temperature stimuli were delivered by a thermode (32°C-51°C at a rate of 70°C/s). In phase I of the study, we investigated side-to-side differences and reported the maximum normal side-to-side difference in A[delta] CHEP peak latency and amplitude for leg, forearm, and face. In phase II, we obtained normative data for 3 CHEP parameters (N2P2 amplitude, N2 latency, and P2 latency), stratified for gender and age decades from face, upper and lower limbs, and overlying cervical and lumbar spine. In general, larger CHEP amplitudes were associated with higher evoked pain scores. Females had CHEPs of larger amplitude and shorter latency than males. This substantive data set of normative values will facilitate the clinical use of CHEPs as a rapid, noninvasive, and objective technique for the assessment of patients presenting with neuropathic pain.
Shillo PR, Selvarajah D, Greig M, et al., 2016, Clinical neuropathy phenotyping and assessment of biomarkers in painful diabetic peripheral neuropathy, DIABETIC MEDICINE, Vol: 33, Pages: 162-162, ISSN: 0742-3071
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Smith MT, Anand P, Rice AS, 2016, Selective small molecule angiotensin II type 2 receptor antagonists for neuropathic pain: preclinical and clinical studies., Pain, Vol: 157, Pages: S33-S41, ISSN: 0304-3959
Neuropathic pain affects up to 10% of the general population, but drug treatments recommended for the treatment of neuropathic pain are associated with modest efficacy and/or produce dose-limiting side effects. Hence, neuropathic pain is an unmet medical need. In the past 2 decades, research on the pathobiology of neuropathic pain has revealed many novel pain targets for use in analgesic drug discovery programs. However, these efforts have been largely unsuccessful as molecules that showed promising pain relief in rodent models of neuropathic pain generally failed to produce analgesia in early phase clinical trials in patients with neuropathic pain. One notable exception is the angiotensin II type 2 (AT2) receptor that has clinical validity on the basis of a successful double-blind, randomized, placebo-controlled, clinical trial of EMA401, a highly selective, orally active, peripherally restricted AT2 receptor antagonist in patients with postherpetic neuralgia. In this study, we review research to date on target validation, efficacy, and mode of action of small molecule AT2 receptor antagonists in rodent models of peripheral neuropathic pain and in cultured human sensory neurons, the preclinical pharmacokinetics of these compounds, and the outcome of the above clinical trial.
Jochmann E, Boettger MK, Anand P, et al., 2015, Antigen-induced arthritis in rats is associated with increased growth-associated protein 43-positive intraepidermal nerve fibres remote from the joint., Arthritis Research and Therapy, Vol: 17, ISSN: 1478-6362
INTRODUCTION: Pain in arthritis may be experienced in regions outside the affected joint, and hyperalgesia may even be widespread. The spread of pain is usually attributed to mechanisms in the central nervous system. We investigated whether rats with antigen-induced arthritis (AIA) exhibit peripheral changes in skin innervation remote from the inflamed joint. METHODS: After immunization, unilateral AIA in the knee joint was induced in rats. Intraepidermal nerve fibre density was determined by immunohistochemical staining for protein gene product 9.5 (PGP 9.5) and for nerve fibres expressing calcitonin gene-related peptide (CGRP), substance P (SP), transient receptor potential vanilloid 1 (TRPV1; the heat and capsaicin receptor), β-tubulin, and growth-associated protein 43 (GAP-43; a marker of regenerating nerve fibres) in paw pad skin and back skin. Cluster of differentiation 11b (CD11b)-positive macrophages and CD3-positive T cells were quantified in skin, and macrophages were quantified in the lumbar dorsal root ganglia. In addition, pain-related behaviour was assessed. RESULTS: Intraepidermal nerve fibre density (PGP 9.5) and the numbers of fibres expressing CGRP, SP, TRPV1, or β-tubulin did not show a significant change in the acute (3 days) or chronic phase (21 days) of AIA compared with control rats that were only immunized. However, paw skin and back skin revealed a significantly higher number of nerve fibres expressing GAP-43 at both the acute and chronic stages of AIA. The skin of arthritic rats in these regions did not contain a greater density of CD11b and CD3 immune cells than the skin of control rats. Enhanced expression of GAP-43 in nerve fibres of the skin was not related to hyperalgesia in the joint, but it accompanied persistent secondary cutaneous hyperalgesia in the skin remote from the inflamed joint. CONCLUSIONS: Although the innervation of the skin remote from the joint did not show significant abnormalities of the other ner
Anand U, Yiangou Y, Sinisi M, et al., 2015, Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT(2)R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies, Molecular Pain, Vol: 11, ISSN: 1744-8069
Background: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherallyrestrictedanalgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2Ris expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRGneurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, andtheir signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons.AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nervegrowth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 andp42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calciumimaging.Results: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control(n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised withAT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference betweencontrol (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves(4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g,n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths weresignificantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGFsignificantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitizationof capsaicin responses was not obs
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