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Vincristine-induced peripheral neuropathy is a common side effect of vincristine treatment, which is accompanied by pain and can be dose-limiting. The molecular mechanisms that underlie vincristine-induced pain are not well understood. We have established an animal model to investigate pathophysiological mechanisms of vincristine-induced pain. Our previous studies have shown that the tetrodotoxin-sensitive voltage-gated sodium channel Nav1.6 in medium-diameter dorsal root ganglion (DRG) neurons contributes to the maintenance of vincristine-induced allodynia. In this study, we investigated the effects of vincristine administration on excitability in small-diameter DRG neurons and whether the tetrodotoxin-resistant (TTX-R) Nav1.8 channels contribute to mechanical allodynia. Current-clamp recordings demonstrated that small DRG neurons become hyper-excitable following vincristine treatment, with both reduced current threshold and increased firing frequency. Using voltage-clamp recordings in small DRG neurons, we now show an increase in TTX-R current density and a -7.3 mV hyperpolarizing shift in the half-maximal potential (V1/2) of activation of Nav1.8 channels in vincristine-treated animals, which likely contributes to the hyperexcitability that we observed in these neurons. Notably, vincristine treatment did not enhance excitability of small DRG neurons from Nav1.8 knockout mice, and the development of mechanical allodynia was delayed but not abrogated in these mice. Together, our data suggest that sodium channel Nav1.8 in small DRG neurons contributes to the development of vincristine-induced mechanical allodynia.
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Ganglios Espinales , Hiperalgesia , Canal de Sodio Activado por Voltaje NAV1.8 , Neuronas , Vincristina , Animales , Vincristina/toxicidad , Vincristina/farmacología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/genética , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Masculino , Ratones Noqueados , Tetrodotoxina/farmacología , Potenciales de Acción/efectos de los fármacos , Ratones Endogámicos C57BL , Antineoplásicos Fitogénicos/toxicidad , Técnicas de Placa-ClampRESUMEN
BACKGROUND: The timing of surgery for patients with gastric cancer (GC) who undergo neoadjuvant chemotherapy (neoCT) was mainly guided by serial radiologic imaging. However, an earlier assessment was indispensable to avoid delayed treatment for nonresponders and excessive toxicity for responders. Our previous study has identified circulating extracellular vesicles-derived lncRNA-GC1 as a biomarker for early detection and monitoring progression of GC. However, the potential role of neoCT remains poorly understood. METHODS: In this explorative biomarker analysis, we conducted a multi-cohort study to examine longitudinal levels of circulating extracellular vesicles-derived lncRNA-GC1 in 798 patients enrolled in the RESONANCE study (NCT01583361). Both circulating extracellular vesicles-derived lncRNA-GC1 and traditional gastrointestinal biomarkers were assessed at defined time nodes. Computed tomography (CT) scans were performed before treatment and 8-10 weeks and assessed based on the RECIST criteria. RESULTS: Circulating extracellular vesicles-derived lncRNA-GC1 could be detected in 96.3% of patients at baseline, and significant reductions were observed before cycle 2 (P<0.0001). Levels of circulating extracellular vesicles-derived lncRNA-GC1 showed a stronger correlation with tumor burden and exhibited earlier dynamic changes than the traditional gastrointestinal biomarkers during the first cycle of neoCT. Strong agreement was observed between circulating extracellular vesicles-derived lncRNA-GC1 response (reduction >50%) and radiographic response (Cohen's κ, 0.704). Importantly, circulating extracellular vesicles-derived lncRNA-GC1 maintained predictive value in two external cohorts. Patients with circulating extracellular vesicles-derived lncRNA-GC1 response showed superior disease-free survival [hazard ratio (HR), 0.6238; 95% CI, 0.4095-0.9501; P=0.0118] and overall survival (HR, 0.6131; 95% CI, 0.4016-0.9358; P=0.0090). CONCLUSION: Circulating extracellular vesicles-derived lncRNA-GC1 is an early marker of neoCT efficacy and predicts superior survival in GC patients treated with neoCT.
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ARN Largo no Codificante , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Terapia Neoadyuvante , ARN Largo no Codificante/genética , Estudios de Cohortes , Supervivencia sin EnfermedadRESUMEN
Gain-of-function mutations in Scn9a, which encodes the peripheral sensory neuron-enriched voltage-gated sodium channel Nav1.7, cause paroxysmal extreme pain disorder (PEPD), inherited erythromelalgia (IEM), and small fiber neuropathy (SFN). Conversely, loss-of-function mutations in the gene are linked to congenital insensitivity to pain (CIP). These mutations are evidence for a link between altered sodium conductance and neuronal excitability leading to somatosensory aberrations, pain, or its loss. Our previous work in young adult mice with the Nav1.7 gain-of-function mutation, I228M, showed the expected DRG neuron hyperexcitability, but unexpectedly the mice had normal mechanical and thermal behavioral sensitivity. We now show that with aging both male and female mice with this mutation unexpectedly develop a profound insensitivity to noxious heat and cold, as well skin lesions that span the body. Electrophysiology demonstrates that, in contrast to young mice, aged I228M mouse DRGs have a profound loss of sodium conductance and changes in activation and slow inactivation dynamics, representing a loss-of-function. Through RNA sequencing we explored how these age-related changes may produce the phenotypic changes and found a striking and specific decrease in C-low threshold mechanoreceptor- (cLTMR) associated gene expression, suggesting a potential contribution of this DRG neuron subtype to Nav1.7 dysfunction phenotypes. A GOF mutation in a voltage-gated channel can therefore produce over a prolonged time, highly complex and unexpected alterations in the nervous system beyond excitability changes.
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Mutación con Ganancia de Función , Canal de Sodio Activado por Voltaje NAV1.7 , Masculino , Femenino , Ratones , Animales , Mutación con Ganancia de Función/genética , Canal de Sodio Activado por Voltaje NAV1.7/genética , Nocicepción , Mutación/genética , Sodio , Ganglios Espinales/patologíaRESUMEN
BACKGROUND & AIMS: Diagnosing gastric cancer (GC) while the disease remains eligible for surgical resection is challenging. In view of this clinical challenge, novel and robust biomarkers for early detection thus improving prognosis of GC are necessary. The present study is to develop a blood-based long noncoding RNA (LR) signature for the early-detection of GC. METHODS: The present 3-step study incorporated data from 2141 patients, including 888 with GC, 158 with chronic atrophic gastritis, 193 with intestinal metaplasia, 501 healthy donors, and 401 with other gastrointestinal cancers. The LR profile of stage I GC tissue samples were analyzed using transcriptomic profiling in discovery phase. The extracellular vesicle (EV)-derived LR signature was identified with a training cohort (n = 554) and validated with 2 external cohorts (n = 429 and n = 504) and a supplemental cohort (n = 69). RESULTS: In discovery phase, one LR (GClnc1) was found to be up-regulated in both tissue and circulating EV samples with an area under the curve (AUC) of 0.9369 (95% confidence interval [CI], 0.9073-0.9664) for early-stage GC (stage I/II). The diagnostic performance of this biomarker was further confirmed in 2 external validation cohorts (Xi'an cohort, AUC: 0.8839; 95% CI: 0.8336-0.9342; Beijing cohort, AUC: 0.9018; 95% CI: 0.8597-0.9439). Moreover, EV-derived GClnc1 robustly distinguished early-stage GC from precancerous lesions (chronic atrophic gastritis and intestinal metaplasia) and GC with negative traditional gastrointestinal biomarkers (CEA, CA72-4, and CA19-9). The low levels of this biomarker in postsurgery and other gastrointestinal tumor plasma samples indicated its GC specificity. CONCLUSIONS: EV-derived GClnc1 serves as a circulating biomarker for the early detection of GC, thus providing opportunities for curative surgery and improved survival outcomes.
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Gastritis Atrófica , Neoplasias Gástricas , Humanos , Biomarcadores de Tumor/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/cirugía , Gastritis Atrófica/diagnóstico , Gastritis Atrófica/genética , Antígeno CA-19-9 , Detección Precoz del Cáncer , MetaplasiaRESUMEN
BACKGROUND: A previous validated study has identified the diagnostic value of circulating exosomal lncRNA-GC1 for detecting and monitoring gastric cancer. We aimed to further determine the predictive role of circulating exosomal lncRNA-GC1 for prognosis and chemotherapy response. METHODS: We retrospectively conducted a multi-phase analysis with four independent cohorts of 981 patients. A training cohort was used to generate the predictive model. One internal and two external cohorts were recruited as validation cohorts. Patients with stage II or III gastric cancer in the combined cohort were used to evaluate the predictive value of circulating exosomal lncRNA-GC1 for chemotherapy response. FINDINGS: In the training cohort, circulating exosomal lncRNA-GC1 was identified as an independent prognostic predictor for disease-free and overall survival. A prognostic risk stratification model based on circulating exosomal lncRNA-GC1 and AJCC stage revealed better predictive accuracy for disease-free and overall survival than the traditional AJCC stage system alone (C-index: DFS 0.701 vs 0.614; OS 0.720 vs 0.611, both P<0.05). And it has been further verified in the validation cohorts. In interaction analysis, for stage II and III GC, patients with low-level of circulating exosomal lncRNA-GC1 derived more survival benefit from adjuvant chemotherapy (P < 0.05); while those with high-level did not. INTERPRETATION: Measurement of circulating exosomal lncRNA-GC1 provides clinically important prognostic information and could complement the AJCC stage to optimize decision-making for selecting patients who could benefit more from fluorouracil-based chemotherapy after surgery. FUNDING: The funders are listed in the Acknowledgement.
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ARN Largo no Codificante , Neoplasias Gástricas , Biomarcadores de Tumor , Humanos , Estadificación de Neoplasias , Pronóstico , ARN Largo no Codificante/genética , Estudios Retrospectivos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genéticaRESUMEN
Background: The current model for predicting prognosis and chemotherapy response of patients with gastric adenocarcinoma is the TNM staging system, which may lack adequate accuracy and evaluations of molecular features at the individual level. We aimed to develop a prediction model to assess the individualized prognosis and responsiveness to fluorouracil-based adjuvant chemotherapy. Method: This retrospective study concluded 2 independent cohorts of patients with GAC. The expression of dysbindin was quantified and evaluated the association with the overall survival for GAC patients. A prediction model for postoperative overall survival was generated and internally and externally validated. The interaction between dysbindin expression and PACT was detected in advanced GAC patients. Results: Of the 637 patients enrolled in the study, 425 were men (66.7%) with a mean (SD) age of 59.79 (9.81) years. High levels of dysbindin expression predicted a poor prognosis in patients with GAC. Multivariate analysis demonstrated dysbindin expression was an independent prognostic predictor of overall survival in the test, validation and combined cohorts. A prognostic predictive model incorporating age, dysbindin expression, pathological differentiation, Lauren's classification and the TNM staging system was established. This model had better predictive accuracy for overall survival than the traditional TNM staging system and was internally and externally validated. More importantly, advanced GAC patients with low dysbindin expression were likely to benefit from fluorouracil-based PACT. Conclusion: The risk stratification model incorporating dysbindin expression and TNM staging system showed better predictive accuracy. Advanced GAC patients with low dysbindin expression revealed better response of fluorouracil-based adjuvant chemotherapy.
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There is a pressing need for understanding of factors that confer resilience to pain. Gain-of-function mutations in sodium channel Nav1.7 produce hyperexcitability of dorsal root ganglion neurons underlying inherited erythromelalgia, a human genetic model of neuropathic pain. While most individuals with erythromelalgia experience excruciating pain, occasional outliers report more moderate pain. These differences in pain profiles in blood-related erythromelalgia subjects carrying the same pain-causative Nav1.7 mutation and markedly different pain experience provide a unique opportunity to investigate potential genetic factors that contribute to inter-individual variability in pain. We studied a patient with inherited erythromelalgia and a Nav1.7 mutation (c.4345T>G, p. F1449V) with severe pain as is characteristic of most inherited erythromelalgia patients, and her mother who carries the same Nav1.7 mutation with a milder pain phenotype. Detailed six-week daily pain diaries of pain episodes confirmed their distinct pain profiles. Electrophysiological studies on subject-specific induced pluripotent stem cell-derived sensory neurons from each of these patients showed that the excitability of these cells paralleled their pain phenotype. Whole-exome sequencing identified a missense variant (c.2263C>T, p. D755N) in KCNQ3 (Kv7.3) in the pain resilient mother. Voltage-clamp recordings showed that co-expression of Kv7.2-wild type (WT)/Kv7.3-D755N channels produced larger M-currents than that of Kv7.2-WT/Kv7.3-WT. The difference in excitability of the patient-specific induced pluripotent stem cell-derived sensory neurons was mimicked by modulating M-current levels using the dynamic clamp and a model of the mutant Kv7.2-WT/Kv7.3-D755N channels. These results show that a 'pain-in-a-dish' model can be used to explicate genetic contributors to pain, and confirm that KCNQ variants can confer pain resilience via an effect on peripheral sensory neurons.
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ABSTRACT: Small-fiber neuropathy (SFN), characterized by distal unmyelinated or thinly myelinated fiber loss, produces a combination of sensory dysfunction and neuropathic pain. Gain-of-function variants in the sodium channel Nav1.7 that produce dorsal root ganglion (DRG) neuron hyperexcitability are present in 5% to 10% of patients with idiopathic painful SFN. We created 2 independent knock-in mouse lines carrying the Nav1.7 I228M gain-of-function variant, found in idiopathic SFN. Whole-cell patch-clamp and multielectrode array recordings show that Nav1.7 I228M knock-in DRG neurons are hyperexcitable compared with wild-type littermate-control neurons, but despite this, Nav1.7 I228M mice do not display mechanical or thermal hyperalgesia or intraepidermal nerve fiber loss in vivo. Therefore, although these 2 Nav1.7 I228M knock-in mouse lines recapitulate the DRG neuron hyperexcitability associated with gain-of-function mutations in Nav1.7, they do not recapitulate the pain or neuropathy phenotypes seen in patients. We suggest that the relationship between hyperexcitability in sensory neurons and the pain experienced by these patients may be more complex than previously appreciated and highlights the challenges in modelling channelopathy pain disorders in mice.
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Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.7 , Animales , Mutación con Ganancia de Función , Humanos , Ratones , Canal de Sodio Activado por Voltaje NAV1.7/genética , Fenotipo , Células Receptoras SensorialesRESUMEN
Vincristine, a widely used chemotherapeutic agent, produces painful peripheral neuropathy. The underlying mechanisms are not well understood. In this study, we investigated whether voltage-gated sodium channels are involved in the development of vincristine-induced neuropathy. We established a mouse model in which repeated systemic vincristine treatment results in the development of significant mechanical allodynia. Histological examinations did not reveal major structural changes at proximal sciatic nerve branches or distal toe nerve fascicles at the vincristine dose used in this study. Immunohistochemical studies and in vivo two-photon imaging confirmed that there is no significant change in density or morphology of intra-epidermal nerve terminals throughout the course of vincristine treatment. These observations suggest that nerve degeneration is not a prerequisite of vincristine-induced mechanical allodynia in this model. We also provided the first detailed characterization of tetrodotoxin-sensitive (TTX-S) and resistant (TTX-R) sodium currents in dorsal root ganglion neurons following vincristine treatment. Accompanying the behavioural hyperalgesia phenotype, voltage-clamp recordings of small and medium dorsal root ganglion neurons from vincristine-treated animals revealed a significant upregulation of TTX-S Na+ current in medium but not small neurons. The increase in TTX-S Na+ current density is likely mediated by Nav1.6, because in the absence of Nav1.6 channels, vincristine failed to alter TTX-S Na+ current density in medium dorsal root ganglion neurons and, importantly, mechanical allodynia was significantly attenuated in conditional Nav1.6 knockout mice. Our data show that TTX-S sodium channel Nav1.6 is involved in the functional changes of dorsal root ganglion neurons following vincristine treatment and it contributes to the maintenance of vincristine-induced mechanical allodynia.
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Antineoplásicos Fitogénicos/toxicidad , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Células Receptoras Sensoriales/metabolismo , Vincristina/toxicidad , Animales , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
Importance: The gastric cancer (GC)-associated long noncoding RNA1 (lncRNA-GC1) plays an important role in gastric carcinogenesis. However, exosomal lncRNA-GC1 and its potential role in GC are poorly understood. Objective: To evaluate the diagnostic value of circulating exosomal lncRNA-GC1 for early detection and monitoring progression of GC. Design, Setting, and Participants: We performed a multiphase investigation of circulating exosomal lncRNA-GC1 for early detection of GC involving consecutive patients with GC (n = 522), patients with gastric precancerous lesions (n = 85), and healthy donor individuals (HDs; n = 219) from December 2016 to February 2019 at Chinese People's Liberation Army General Hospital, China. LncRNA-GC1 was measured by reverse transcription-polymerase chain reaction by independent researchers who had no access to patients' information. Receiver operating characteristic curves were used to calculate diagnostic efficiency in comparison between lncRNA-GC1 and 3 traditional biomarkers (carcinoembryonic antigen [CEA], cancer antigen 72-4 [CA72-4], and CA19-9). Main Outcomes and Measures: Assessment of diagnostic efficiency on the basis of area under curve (AUC), specificity, and sensitivity. Results: Of the 826 patients included in the study, 508 were men (61.5%), and the median age of all patients was 60 years (range, 28-82 years). In the test phase, lncRNA-GC1 achieved better diagnostic performance than the standard biomarkers CEA, CA72-4, and CA19-9 (AUC = 0.9033) for distinguishing between the patients with GC and HDs. Additionally, exosomal lncRNA-GC1 levels were significantly higher in culture media from GC cells compared with those of normal gastric epithelial cells (t = 5.310; P = .002). In the verification phase, lncRNA-GC1 retained its diagnostic efficiency in discriminating patients with GC from those with gastric precancerous lesions as well from HDs. Moreover, lncRNA-GC1 exhibited a higher AUC compared with those of CEA, CA72-4, and CA19-9 for early detection of GC with sufficient specificity and sensitivity, especially for patients with GC with negative standard biomarkers. Moreover, the levels of circulating exosomal lncRNA-GC1 were significantly associated with GC from early to advanced stages (HD vs stage I, t = 20.98; P < .001; stage I vs stage II, t = 2.787; P = .006; stage II vs stage III, t = 4.471; P < .001; stage III vs stage IV, t = 1.023; P = .30), independent of pathological grading and Lauren classification (pathological grading: HD vs G1, t = 21.09; P < .001; G1 vs G2, t = 0.3718; P = .71; G2 vs G3, t = 0.3598; P = .72; Lauren classification: t = 24.81; P <.001). In the supplemental phase, the levels of circulating exosomal lncRNA-GC1 were consistent with those in GC tissues and cells and were higher compared with those in normal tissues and cells. Furthermore, the levels of circulating lncRNA-GC1 were unchanged after exosomes were treated with RNase and remained constant after prolonged exposure to room temperature or after repeated freezing and thawing (t = 1.443; P = .39). Total circulating lncRNA-GC1 was nearly all packaged within exosomes rather than a free form in plasma. Conclusions and Relevence: Circulating exosomal lncRNA-GC1 may serve as a noninvasive biomarker for detecting early-stage GC and for monitoring disease progression. Combining circulating exosomal lncRNA-GC1 detection with endoscopy could improve the early diagnostic rate of GC.
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Biomarcadores de Tumor/sangre , Detección Precoz del Cáncer/métodos , ARN Largo no Codificante/sangre , Neoplasias Gástricas/sangre , Neoplasias Gástricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Progresión de la Enfermedad , Exosomas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Sodium channel Nav1.7, encoded by the SCN9A gene, is a well-validated target that plays a key role in controlling pain sensation. Loss-of-function mutations of Nav1.7 can cause a syndrome of profound congenital insensitivity to pain in humans. Better understanding of how the loss of Nav1.7 leads to loss of pain sensibility would help to decipher the fundamental mechanisms of nociception and inform strategies for development of novel analgesics. Using a recently described rat Nav1.7 loss-of-function model with deficient nociception but intact olfactory function, we investigated the involvement of endogenous opioid and cannabinoid systems in this rodent model of Nav1.7-related congenital insensitivity to pain. We found that both the opioid receptor antagonist naloxone and cannabinoid receptor blockers SR141716A (rimonabant) and SR144528 fail to restore acute pain sensitivity in Nav1.7 loss-of-function rats. We observed, however, that after rimonabant administration, Nav1.7 loss-of-function but not WT rats displayed abnormal behaviours, such as enhanced scratching, caudal self-biting, and altered facial expressions; the underlying mechanism is still unclear. Dorsal root ganglion neurons from Nav1.7 loss-of-function rats, although hypoexcitable compared with WT neurons, were still able to generate action potentials in response to noxious heat and capsaicin. Our data indicate that complete loss of dorsal root ganglion neuron excitability is not required for insensitivity to pain and suggest that endogenous opioid and cannabinoid systems are not required for insensitivity to pain in the absence of Nav1.7 channels in this rat Nav1.7 loss-of-function model.
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Canal de Sodio Activado por Voltaje NAV1.7 , Insensibilidad Congénita al Dolor , Potenciales de Acción , Animales , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/tratamiento farmacológico , Dolor/genética , Insensibilidad Congénita al Dolor/tratamiento farmacológico , Insensibilidad Congénita al Dolor/genética , RatasRESUMEN
Despite pain prevalence altering with age, the effects of aging on the properties of nociceptors are not well understood. Nociceptors, whose somas are located in dorsal root ganglia, are frequently divided into two groups based on their ability to bind isolectin B4 (IB4). Here, using cultured neurons from 1-, 3-, 5-, 8-, 12-, and 18-month-old mice, we investigate age-dependent changes in IB4-positive and IB4-negative neurons. Current-clamp experiments at physiological temperature revealed nonlinear changes in firing frequency of IB4-positive, but not IB4-negative neurons, with a peak at 8 months. This was likely due to the presence of proexcitatory conductances activated at depolarized membrane potentials and significantly higher input resistances found in IB4-positive neurons from 8-month-old mice. Repetitive firing in nociceptors is driven primarily by the TTX-resistant sodium current, and indeed, IB4-positive neurons from 8-month-old mice were found to receive larger contributions from the TTX-resistant window current around the resting membrane potential. To further address the mechanisms behind these differences, we performed RNA-seq experiments on IB4-positive and IB4-negative neurons from 1-, 8-, and 18-month-old mice. We found a larger number of genes significantly affected by age within the IB4-positive than IB4-negative neurons from 8-month-old mice, including known determinants of nociceptor excitability. The above pronounced age-dependent changes at the cellular and molecular levels in IB4-positive neurons point to potential mechanisms behind the reported increase in pain sensitivity in middle-aged rodents and humans, and highlight the possibility of targeting a particular group of neurons in the development of age-tailored pain treatments.
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Senescencia Celular/genética , Glicoproteínas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Nociceptores/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica/genética , Glicoproteínas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Debilidad Muscular/genética , Nociceptores/citologíaRESUMEN
Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specific contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, inflammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, significantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma. Together, NaV1.6 in NaV1.8-positive neurons does not influence pain thresholds under normal or pathological conditions, but NaV1.6 in large NaV1.8-negative DRG neurons plays an important role in neuropathic pain.
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Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Neuralgia/genética , Animales , Femenino , Ganglios Espinales/metabolismo , Hiperalgesia/genética , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/fisiología , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Tejido Nervioso/metabolismo , Neuralgia/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Pharmacotherapy for pain currently involves trial and error. A previous study on inherited erythromelalgia (a genetic model of neuropathic pain due to mutations in the sodium channel, Nav 1.7) used genomics, structural modelling and biophysical and pharmacological analyses to guide pharmacotherapy and showed that carbamazepine normalizes voltage dependence of activation of the Nav 1.7-S241T mutant channel, reducing pain in patients carrying this mutation. However, whether this approach is applicable to other Nav channel mutants is still unknown. EXPERIMENTAL APPROACH: We used structural modelling, patch clamp and multi-electrode array (MEA) recording to assess the effects of carbamazepine on Nav 1.7-I234T mutant channels and on the firing of dorsal root ganglion (DRG) sensory neurons expressing these mutant channels. KEY RESULTS: In a reverse engineering approach, structural modelling showed that the I234T mutation is located in atomic proximity to the carbamazepine-responsive S241T mutation and that activation of Nav 1.7-I234T mutant channels, from patients who are known to respond to carbamazepine, is partly normalized with a clinically relevant concentration (30 µM) of carbamazepine. There was significantly higher firing in intact sensory neurons expressing Nav 1.7-I234T channels, compared with neurons expressing the normal channels (Nav 1.7-WT). Pre-incubation with 30 µM carbamazepine also significantly reduced the firing of intact DRG sensory neurons expressing Nav 1.7-I234T channels. Although the expected use-dependent inhibition of Nav 1.7-WT channels by carbamazepine was confirmed, carbamazepine did not enhance use-dependent inhibition of Nav 1.7-I234T mutant channels. CONCLUSION AND IMPLICATIONS: These results support the utility of a pharmacogenomic approach to treatment of pain in patients carrying sodium channel variants. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
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Carbamazepina/farmacología , Mutación , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Farmacogenética , Células Receptoras Sensoriales/efectos de los fármacos , Temperatura , Células HEK293 , Humanos , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.7/química , Células Receptoras Sensoriales/metabolismoRESUMEN
Neuropathic pain is a significant complication following spinal cord injury (SCI) with few effective treatments. Drug development for neuropathic pain often fails because preclinical studies do not always translate well to clinical conditions. Identification of biological characteristics predictive of disease state or drug responsiveness could facilitate more effective clinical translation. Emerging evidence indicates a strong correlation between dendritic spine dysgenesis and neuropathic pain. Because dendritic spines are located on dorsal horn neurons within the spinal cord nociceptive system, dendritic spine remodeling provides a unique opportunity to understand sensory dysfunction after SCI. In this study, we provide support for the postulate that dendritic spine profiles can serve as biomarkers for neuropathic pain. We show that dendritic spine profiles after SCI change to a dysgenic state that is characteristic of neuropathic pain in a Rac1-dependent manner. Suppression of the dysgenic state through inhibition of Rac1 activity is accompanied by attenuation of neuropathic pain. Both dendritic spine dysgenesis and neuropathic pain return when inhibition of Rac1 activity is lifted. These findings suggest the utility of dendritic spines as structural biomarkers for neuropathic pain.
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Espinas Dendríticas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Animales , Catéteres de Permanencia , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/patología , Masculino , Microelectrodos , Neuralgia/metabolismo , Neuralgia/patología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Células del Asta Posterior/patología , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Factores de Tiempo , Tacto/fisiología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The rate of action potential firing in nociceptors is a major determinant of the intensity of pain. Possible modulators of action potential firing include the HCN ion channels, which generate an inward current, I(h), after hyperpolarization of the membrane. We found that genetic deletion of HCN2 removed the cyclic adenosine monophosphate (cAMP)-sensitive component of I(h) and abolished action potential firing caused by an elevation of cAMP in nociceptors. Mice in which HCN2 was specifically deleted in nociceptors expressing Na(V)1.8 had normal pain thresholds, but inflammation did not cause hyperalgesia to heat stimuli. After a nerve lesion, these mice showed no neuropathic pain in response to thermal or mechanical stimuli. Neuropathic pain is therefore initiated by HCN2-driven action potential firing in Na(V)1.8-expressing nociceptors.