35 results found
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
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.
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 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-
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, 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
Zhang Y, Clausmeyer J, Babakinejad B, et al., 2016, Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis., ACS Nano, Vol: 10, Pages: 3214-3221, ISSN: 1936-086X
Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.
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
Payne CE, Brown AR, Theile JW, et al., 2015, A novel selective and orally bioavailable Na(v)1.8 channel blocker, PF-01247324, attenuates nociception and sensory neuron excitability, BRITISH JOURNAL OF PHARMACOLOGY, Vol: 172, Pages: 2654-2670, ISSN: 0007-1188
Lopez-Cordoba A, Joensson P, Babakinejad B, et al., 2015, SICM-Based Nanodelivery System for Local TRPV1 Stimulation, 59th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 332A-332A, ISSN: 0006-3495
Roblin D, Yosipovitch G, Boyce B, et al., 2015, Topical TrkA Kinase Inhibitor CT327 is an Effective, Novel Therapy for the Treatment of Pruritus due to Psoriasis: Results from Experimental Studies, and Efficacy and Safety of CT327 in a Phase 2b Clinical Trial in Patients with Psoriasis, ACTA DERMATO-VENEREOLOGICA, Vol: 95, Pages: 542-548, ISSN: 0001-5555
Babakinejad B, Joensson P, Lopez Cordoba A, et al., 2013, Local Delivery of Molecules from a Nanopipette for Quantitative Receptor Mapping on Live Cells, ANALYTICAL CHEMISTRY, Vol: 85, Pages: 9333-9342, ISSN: 0003-2700
Anand U, Facer P, Yiangou Y, et al., 2013, Angiotensin II type 2 receptor (AT(2)R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons, European Journal of Pain, Vol: 17, Pages: 1012-1026, ISSN: 1532-2149
BackgroundThe angiotensin II (AngII) receptor subtype 2 (AT2R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration.MethodsWe used immunostaining with characterized antibodies to study the localization of AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence.ResultsAT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1R antagonist losartan had no effect on capsaicin responses. AT2R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas.ConclusionsAT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.
Babakinejad B, Yasufumi T, Joensson P, et al., 2012, Quantitative Characterization of Local Chemical Delivery through Nanopipette, BIOPHYSICAL JOURNAL, Vol: 102, Pages: 313A-313A, ISSN: 0006-3495
Yiangou Y, Anand U, Otto WR, et al., 2011, Increased levels of SV2A botulinum neurotoxin receptor in clinical sensory disorders and functional effects of botulinum toxins A and E in cultured human sensory neurons, Journal of Pain Research, Vol: 2011, Pages: 347-355, ISSN: 1178-7090
Background: There is increasing evidence that botulinum neurotoxin A may affect sensory nociceptor fibers, but the expression of its receptors in clinical pain states, and its effects in human sensory neurons, are largely unknown.Methods: We studied synaptic vesicle protein subtype SV2A, a receptor for botulinum neurotoxin A, by immunostaining in a range of clinical tissues, including human dorsal root ganglion sensory neurons, peripheral nerves, the urinary bladder, and the colon. We also determined the effects of botulinum neurotoxins A and E on localization of the capsaicin receptor, TRPV1, and functional sensitivity to capsaicin stimuli in cultured human dorsal root ganglion neurons.Results: Image analysis showed that SV2A immunoreactive nerve fibers were increased in injured nerves proximal to the injury (P = 0.002), and in painful neuromas (P = 0.0027); the ratio of percentage area SV2A to neurofilaments (a structural marker) was increased proximal to injury (P = 0.0022) and in neuromas (P = 0.0001), indicating increased SV2A levels in injured nerve fibers. In the urinary bladder, SV2A nerve fibers were found in detrusor muscle and associated with blood vessels, with a significant increase in idiopathic detrusor overactivity (P = 0.002) and painful bladder syndrome (P = 0.0087). Colon biopsies showed numerous SV2A-positive nerve fibers, which were increased in quiescent inflammatory bowel disease with abdominal pain (P = 0.023), but not in inflammatory bowel disease without abdominal pain (P = 0.77) or in irritable bowel syndrome (P = 0.13). In vitro studies of botulinum neurotoxin A-treated and botulinum neurotoxin E-treated cultured human sensory neurons showed accumulation of cytoplasmic vesicles, neurite loss, and reduced immunofluorescence for the heat and capsaicin receptor, TRPV1. Functional effects included dose-related inhibition of capsaicin responses on calcium imaging after acute treatment with botulinum neurotoxins A and E.Conclusion: Differential
Anand U, Otto WR, Anand P, 2010, Sensitization of capsaicin and icilin responses in oxaliplatin treated adult rat DRG neurons, Molecular Pain, Vol: 6, ISSN: 1744-8069
Background: Oxaliplatin chemotherapy induced neuropathy is a dose related cumulative toxicity that manifests astingling, numbness, and chronic pain, compromising the quality of life and leading to discontinued chemotherapy.Patients report marked hypersensitivity to cold stimuli at early stages of treatment, when sensory testing revealscold and heat hyperalgesia. This study examined the morphological and functional effects of oxaliplatin treatmentin cultured adult rat DRG neurons.Results: 48 hour exposure to oxaliplatin resulted in dose related reduction in neurite length, density, and numberof neurons compared to vehicle treated controls, using Gap43 immunostaining. Neurons treated acutely with 20μg/ml oxaliplatin showed significantly higher signal intensity for cyclic AMP immunofluorescence (160.5 ± 13 a.u.,n = 3, P < 0.05), compared to controls (120.3 ± 4 a.u.). Calcium imaging showed significantly enhanced capsaicin(TRPV1 agonist), responses after acute 20 μg/ml oxaliplatin treatment where the second of paired capsaicinresponses increased from 80.7 ± 0.6% without oxaliplatin, to 171.26 ± 29% with oxaliplatin, (n = 6 paired t test,P < 0.05); this was reduced to 81.42 ± 8.1% (P < 0.05), by pretretreatment with the cannabinoid CB2 receptoragonist GW 833972. Chronic oxaliplatin treatment also resulted in dose related increases in capsaicin responses.Similarly, second responses to icilin (TRPA1/TRPM8 agonist), were enhanced after acute (143.85 ± 7%, P = 0.004,unpaired t test, n = 3), and chronic (119.7 ± 11.8%, P < 0.05, n = 3) oxaliplatin treatment, compared to control(85.3 ± 1.7%). Responses to the selective TRPM8 agonist WS-12 were not affected.Conclusions: Oxaliplatin treatment induces TRP sensitization mediated by increased intracellular cAMP, which maycause neuronal damage. These effects may be mitigated by co-treatment with adenylyl cyclase inhibitors, like CB2agonists, to alleviate the n
Anand U, Otto WR, Sanchez-Herrera D, et al., 2008, Cannabinoid receptor CB2 localisation and agonist-mediated inhibition of capsaicin responses in human sensory neurons, PAIN, Vol: 138, Pages: 667-680, ISSN: 0304-3959
Sánchez D, Johnson N, Li C, et al., 2008, Noncontact measurement of the local mechanical properties of living cells using pressure applied via a pipette., Biophysical journal, Vol: 95, Pages: 3017-27, ISSN: 1542-0086
Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.
Anand U, Otto WR, Bountra C, et al., 2008, Cytosine arabinoside affects the heat and capsaicin receptor TRPV1 localisation and sensitivity in human sensory neurons, JOURNAL OF NEURO-ONCOLOGY, Vol: 89, Pages: 1-7, ISSN: 0167-594X
Anand U, Otto WR, Facer P, et al., 2008, TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons, NEUROSCIENCE LETTERS, Vol: 438, Pages: 221-227, ISSN: 0304-3940
De Beule PAA, Dunsby C, Galletly NP, et al., 2007, A hyperspectral fluorescence lifetime probe for skin cancer diagnosis, REVIEW OF SCIENTIFIC INSTRUMENTS, Vol: 78, ISSN: 0034-6748
Kumar S, Dunsby C, De Beule PAA, et al., 2007, Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging, Optics Express, Vol: 15, Pages: 12548-12561
We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movementinduced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.
Sanchez D, Arand U, Gorelik J, et al., 2007, Localized and non-contact mechanical stimulation of dorsal root ganglion sensory neurons using scanning ion conductance microscopy, JOURNAL OF NEUROSCIENCE METHODS, Vol: 159, Pages: 26-34, ISSN: 0165-0270
De Beule PAA, Dunsby C, Owen DM, et al., 2007, A novel hyperspectral lifetime probe for autofluorescence - art. no. 643303, Conference on Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VII, Pages: 43303-43303
The application of autofluorescence in non-invasive medical diagnostics could have great potential. Two major drawbacks inherent to this approach are low signal levels compared to those from exogenous fluorescent probes and complexity caused by the multiplicity of fluorescent biomolecules in tissue. Here we present a new optical system that is based on single channel detection via all optical fiber and call measure the fluorescence emission spectrum and fluorescence lifetime simultaneously for excitation wavelengths of 355 and 435nm. Single channel measurements integrate the signal normally available in all imaging setup and therefore have a better signal-to-noise ratio. Resolving both the fluorescence emission spectrum and fluorescence lifetime provides the opportunity to discriminate multiple fluorophores. This instrument is intended for NAD(P)H and flavin measurements for the dynamic monitoring of cellular metabolism and optical measurements of cancerous tissue. Initial results from a study of live cells and a clinical study of human skin lesions are presented.
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