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A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10-8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.
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Dolor Crónico , Neuralgia , Anticuerpos de Cadena Única , Animales , Ratones , Simulación del Acoplamiento Molecular , Biblioteca de Péptidos , Receptor de Colecistoquinina B , Dolor Crónico/terapia , Ribosomas/metabolismoRESUMEN
High-impact chronic pain is suffered by 1 in 5 patients in the USA and globally. Effective, non-addictive, non-opioid therapeutics are urgently needed for the treatment of chronic pain. Slc7a5 (Lat1), also known as system L-neutral amino acid transporter, is involved in a number of physiological processes related to inflammation. Transcriptomics studies have shown that Slc7a5 and its binding partner Slc3a2 are expressed in neurons of the dorsal root ganglia (DRG) and spinal dorsal horn, which are critical to the initiation and maintenance of nociception and pathophysiology of chronic pain. In addition, Slc7a5 is a transporter for the first-line anti-allodynic gabapentinoid drugs and binds to ion channels implicated in nociception and chronic pain including the voltage-gated sodium channel Nav1.7 and the voltage-gated potassium channels Kv1.1 and Kv1.2. We found that blocking Slc7a5 with intrathecal administration of the drug JPH203 alleviated allodynia in the spared nerve injury (SNI) rodent model of neuropathic pain. Western blot and immunohistochemistry studies revealed an increase in Slc7a5 protein levels in the spinal cord and DRGs of SNI mice compared to control mice. Using whole-cell current-clamp electrophysiology, we observed that JPH203 treatment reduced excitability of small-diameter (< 30 µm) DRG neurons from SNI mice, in agreement with its behavioral effects. Voltage-clamp recordings from JPH203-treated naïve rat DRGs identified an effect on tetrodotoxin-resistant (TTX-R) sodium currents. Altogether, these results demonstrate that Slc7a5 is dysregulated in chronic neuropathic pain and can be targeted to provide relief of hypersensitivity.
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Hiperalgesia , Transportador de Aminoácidos Neutros Grandes 1 , Neuralgia , Animales , Ganglios Espinales/metabolismo , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/patología , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Ratones , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Neuralgia/patología , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Roedores , Asta Dorsal de la Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/patologíaRESUMEN
Injury to or disease of the nervous system can invoke chronic and sometimes intractable neuropathic pain. Many parallel, interdependent, and time-dependent processes, including neuroimmune interactions at the peripheral, supraspinal, and spinal levels, contribute to the etiology of this "disease of pain." Recent work emphasizes the roles of colony-stimulating factor 1, ATP, and brain-derived neurotrophic factor. Excitatory processes are enhanced, and inhibitory processes are attenuated in the spinal dorsal horn and throughout the somatosensory system. This leads to central sensitization and aberrant processing such that tactile and innocuous thermal information is perceived as pain (allodynia). Processes involved in the onset of neuropathic pain differ from those involved in its long-term maintenance. Opioids display limited effectiveness, and less than 35% of patients derive meaningful benefit from other therapeutic approaches. We thus review promising therapeutic targets that have emerged over the last 20 years, including Na+, K+, Ca2+, hyperpolarization-activated cyclic nucleotide-gated channels, transient receptor potential channel type V1 channels, and adenosine A3 receptors. Despite this progress, the gabapentinoids retain their status as first-line treatments, yet their mechanism of action is poorly understood. We outline recent progress in understanding the etiology of neuropathic pain and show how this has provided insights into the cellular actions of pregabalin and gabapentin. Interactions of gabapentinoids with the α2δ-1 subunit of voltage-gated Ca2+ channels produce multiple and neuron type-specific actions in spinal cord and higher centers. We suggest that drugs that affect multiple processes, rather than a single specific target, show the greatest promise for future therapeutic development.
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Analgésicos/uso terapéutico , Neuralgia , Traumatismos de los Nervios Periféricos , Analgésicos Opioides/uso terapéutico , Animales , Gabapentina/uso terapéutico , Humanos , Canales Iónicos/metabolismo , Terapia Molecular Dirigida/métodos , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Neuralgia/metabolismo , Manejo del Dolor/métodos , Traumatismos de los Nervios Periféricos/complicaciones , Traumatismos de los Nervios Periféricos/tratamiento farmacológicoRESUMEN
Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.
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Dolor Crónico/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Anticuerpos de Cadena Única/uso terapéutico , Animales , Afinidad de Anticuerpos , Células Cultivadas , Dolor Crónico/inmunología , Femenino , Masculino , Ratones , Biblioteca de Péptidos , Antagonistas del Receptor Purinérgico P2X/química , Antagonistas del Receptor Purinérgico P2X/inmunología , Receptores Purinérgicos P2X4/química , Receptores Purinérgicos P2X4/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Migraine is characterized by severe headaches that can be preceded by an aura likely caused by cortical spreading depression (SD). The antiepileptic pregabalin (Lyrica) shows clinical promise for migraine therapy, although its efficacy and mechanism of action are unclear. As detected by diffusion-weighted MRI (DW-MRI) in wild-type (WT) mice, the acute systemic administration of pregabalin increased the threshold for SD initiation in vivo. In familial hemiplegic migraine type 1 mutant mice expressing human mutations (R192Q and S218L) in the CaV2.1 (P/Q-type) calcium channel subunit, pregabalin slowed the speed of SD propagation in vivo. Acute systemic administration of pregabalin in vivo also selectively prevented the migration of SD into subcortical striatal and hippocampal regions in the R192Q strain that exhibits a milder phenotype and gain of CaV2.1 channel function. At the cellular level, pregabalin inhibited glutamatergic synaptic transmission differentially in WT, R192Q, and S218L mice. The study describes a DW-MRI analysis method for tracking the progression of SD and provides support and a mechanism of action for pregabalin as a possible effective therapy in the treatment of migraine.
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Analgésicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/genética , Ataxia Cerebelosa/tratamiento farmacológico , Depresión de Propagación Cortical/efectos de los fármacos , Trastornos Migrañosos/tratamiento farmacológico , Migraña con Aura/tratamiento farmacológico , Pregabalina/farmacología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Canales de Calcio Tipo N/metabolismo , Ataxia Cerebelosa/diagnóstico por imagen , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/patología , Imagen de Difusión por Resonancia Magnética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Trastornos Migrañosos/diagnóstico por imagen , Trastornos Migrañosos/metabolismo , Trastornos Migrañosos/patología , Migraña con Aura/diagnóstico por imagen , Migraña con Aura/metabolismo , Migraña con Aura/patología , Mutación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Transmisión SinápticaRESUMEN
Trigeminal neuralgia is a heterogeneous disorder with likely multifactorial and complex etiology; however, trigeminal nerve demyelination and injury are observed in almost all patients with trigeminal neuralgia. The current management strategies for trigeminal neuralgia primarily involve anticonvulsants and surgical interventions, neither of which directly address demyelination, the pathological hallmark of trigeminal neuralgia, and treatments targeting demyelination are not available. Demyelination of the trigeminal nerve has been historically considered a secondary effect of vascular compression, and as a result, trigeminal neuralgia is not recognized nor treated as a primary demyelinating disorder. In this article, we review the evolution of our understanding of trigeminal neuralgia and provide evidence to propose its potential categorization, at least in some cases, as a primary demyelinating disease by discussing its course and similarities to multiple sclerosis, the most prevalent central nervous system demyelinating disorder. This proposed categorization may provide a basis in investigating novel treatment modalities beyond the current medical and surgical interventions, emphasizing the need for further research into demyelination of the trigeminal sensory pathway in trigeminal neuralgia. PERSPECTIVE: This article proposes trigeminal neuralgia as a demyelinating disease, supported by histological, clinical, and radiological evidence. Such categorization offers a plausible explanation for controversies surrounding trigeminal neuralgia. This perspective holds potential for future research and developing therapeutics targeting demyelination in the condition.
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Esclerosis Múltiple , Neuralgia del Trigémino , Humanos , Neuralgia del Trigémino/etiología , Neuralgia del Trigémino/terapia , Nervio Trigémino/patología , Nervio Trigémino/cirugía , Esclerosis Múltiple/complicacionesRESUMEN
Identifying and resolving molecular complexities underlying chronic neuropathic pain is a significant challenge. Among the numerous classes of histone deacetylases, Class I (HDAC 1-3) and Class III (sirtuins) have been best studied in experimental pain models where inhibitor pre-treatments but not post-treatments abrogate the development of pain-related behaviors. Post-treatment here in week 3 with less well-studied Class IIa HDAC4/5 selective inhibitor LMK235 diminishes the trigeminal ganglia increases of HDAC5 RNA and protein in two chronic orofacial neuropathic pain models to levels measured in naïve mice at week 10 post-model induction. HDAC4 RNA reported in lower limb inflammatory pain models is not evident in the trigeminal models. Many other gene alterations persisting at week 10 in the trigeminal ganglia (TG) are restored to naïve levels in mice treated with LMK235. Important pain-related upregulated genes Hoxc8,b9,d8; P2rx4, Cckbr, growth hormone (Gh), and schlafen (Slfn4) are greatly reduced in LMK235-treated mice. Fold increase in axon regeneration/repair genes Sostdc1, TTr, and Folr1 after injury are doubled by LMK235 treatment. LMK235 reduces the excitability of trigeminal ganglia neurons in culture isolated from nerve injured mice compared to vehicle-treated controls, with no effect on neurons from naïve mice. Electrophysiological characterization profile includes a shift where â¼20% of the small neurons recorded under LMK235-treated conditions are high threshold, whereas none of the neurons under control conditions have high thresholds. LMK235 reverses long-standing mechanical and cold hypersensitivity in chronic trigeminal neuropathic pain models in males and females (5,10 mg/kg), preventing development of anxiety- and depression-like behaviors. PERSPECTIVE: Data here support HDAC5 as key epigenetic factor in chronic trigeminal neuropathic pain persistence, validated with the study of RNA alterations, TG neuronal excitability, and pain-related behaviors. HDAC5 inhibitor given in week 3 restores RNA balance at 10 weeks, while upregulation remains for response to wound healing and chronic inflammation RNAs.
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Benzamidas , Inhibidores de Histona Desacetilasas , Neuralgia , Animales , Masculino , Ratones , Axones , Epigénesis Genética , Histona Desacetilasas/metabolismo , Regeneración Nerviosa , Neuralgia/tratamiento farmacológico , Ganglio del Trigémino/metabolismo , Inhibidores de Histona Desacetilasas/administración & dosificación , Benzamidas/administración & dosificaciónRESUMEN
Human and mouse dorsal root ganglia (hDRG and mDRG) neurons are important tools in understanding the molecular and electrophysiological mechanisms that underlie nociception and drive pain behaviors. One of the simplest differences in firing phenotypes is that neurons are single-firing (exhibit only one action potential) or multi-firing (exhibit 2 or more action potentials). To determine if single- and multi-firing hDRG exhibit differences in intrinsic properties, firing phenotypes, and AP waveform properties, and if these properties could be used to predict multi-firing, we measured 22 electrophysiological properties by whole-cell patch-clamp electrophysiology of 94 hDRG neurons from 6 male and 4 female donors. We then analyzed the data using several machine learning models to determine if these properties could be used to predict multi-firing. We used 1000 iterations of Monte Carlo Cross Validation to split the data into different train and test sets and tested the Logistic Regression, k-Nearest Neighbors, Random Forest, Supported Vector Classification, and XGBoost machine learning models. All models tested had a greater than 80% accuracy on average, with Supported Vector Classification and XGBoost performing the best. We found that several properties correlated with multi-firing hDRG neurons and together could be used to predict multi-firing neurons in hDRG including a long decay time, a low rheobase, and long first spike latency. We also found that the hDRG models were able to predict multi-firing with 90% accuracy in mDRG. Targeting the neuronal properties that lead to multi-firing could elucidate better targets for treatment of chronic pain.
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Human induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) and human dorsal root ganglia neurons (hDRG-N) are popular tools in the field of pain research; however, few groups make use of both approaches. For screening and analgesic validation purposes, important characterizations can be determined of the similarities and differences between hDRG-N and hiPSC-SNs. This study focuses specifically on the electrophysiology properties of hDRG-N in comparison to hiPSC-SNs. We also compared hDRG-N and hiPSC-SNs from both male and female donors to evaluate potential sex differences. We recorded neuronal size, rheobase, resting membrane potential, input resistance, and action potential waveform properties from 83 hiPSCs-SNs (2 donors) and 108 hDRG-N neurons (8 donors). We observed several statistically significant electrophysiological differences between hDRG-N and hiPSC-SNs, such as size, rheobase, input resistance, and several action potential waveform properties. Correlation analysis also revealed many properties that were positively or negatively correlated, some of which were differentially correlated between hDRG-N and hiPSC-SNs. This study shows several differences between hDRG-N and hiPSC-SNs and allows a better understanding of the advantages and disadvantages of both for use in pain research. We hope this study will be a valuable resource for pain researchers considering the use of these human in vitro systems for mechanistic studies and/or drug development projects. PERSPECTIVE: hiPSC-SNs and hDRG-N are popular tools in the field of pain research. This study allows for a better functional understanding of the pros and cons of both tools.
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Ganglios Espinales , Células Madre Pluripotentes Inducidas , Células Receptoras Sensoriales , Humanos , Femenino , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Ganglios Espinales/fisiología , Ganglios Espinales/citología , Células Receptoras Sensoriales/fisiología , Adulto , Potenciales de Acción/fisiología , Caracteres Sexuales , Persona de Mediana Edad , Células Cultivadas , Fenómenos Electrofisiológicos/fisiologíaRESUMEN
Chronic pain affects approximately one-fifth of people worldwide and reduces quality of life and in some cases, working ability. Ion channels expressed along nociceptive pathways affect neuronal excitability and as a result modulate pain experience. Several ion channels have been identified and investigated as potential targets for new medicines for the treatment of a variety of human diseases, including chronic pain. Voltage-gated channels Na+ and Ca2+ channels, K+ channels, transient receptor potential channels (TRP), purinergic (P2X) channels and acid-sensing ion channels (ASICs) are some examples of ion channels exhibiting altered function or expression in different chronic pain states. Pharmacological approaches are being developed to mitigate dysregulation of these channels as potential treatment options. Since natural compounds of plant origin exert promising biological and pharmacological properties and are believed to possess less adverse effects compared to synthetic drugs, they have been widely studied as treatments for chronic pain for their ability to alter the functional activity of ion channels. A literature review was conducted using Medline, Google Scholar and PubMed, resulted in listing 79 natural compounds/extracts that are reported to interact with ion channels as part of their analgesic mechanism of action. Most in vitro studies utilized electrophysiological techniques to study the effect of natural compounds on ion channels using primary cultures of dorsal root ganglia (DRG) neurons. In vivo studies concentrated on different pain models and were conducted mainly in mice and rats. Proceeding into clinical trials will require further study to develop new, potent and specific ion channel modulators of plant origin.
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A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks. Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model. Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the "Global Year about Back Pain" and 2022 the "Global Year for Translating Pain Knowledge to Practice." One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The features of this model are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP's naming 2022 as the Global Year for Translating Pain Knowledge to Practice.
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Dolor Crónico , Hipersensibilidad , Dolor de la Región Lumbar , Humanos , Animales , Ratones , Activador de Plasminógeno de Tipo Uroquinasa , Dolor de Espalda , Serina Proteasas , Modelos Animales de EnfermedadRESUMEN
Human induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) and human dorsal root ganglia (hDRG) neurons are popular tools in the field of pain research; however, few groups make use of both approaches. For screening and analgesic validation purposes, important characterizations can be determined of the similarities and differences between hDRG and hiPSC-SNs. This study focuses specifically on electrophysiology properties of hDRG in comparison to hiPSC-SNs. We also compared hDRG and hiPSC-SNs from both male and female donors to evaluate potential sex differences. We recorded neuronal size, rheobase, resting membrane potential, input resistance, and action potential waveform properties from 83 hiPSCs-SNs (2 donors) and 108 hDRG neurons (9 donors). We observed several statistically significant electrophysiological differences between hDRG and hiPSC-SNs, such as size, rheobase, input resistance, and several actional potential (AP) waveform properties. Correlation analysis also revealed many properties that were positively or negatively correlated, some of which were differentially correlated between hDRG and hiPSC-SNs. This study shows several differences between hDRG and hiPSC-SNs and allows better understanding of the advantages and disadvantages of both for use in pain research. We hope this study will be a valuable resource for pain researchers considering the use of these human in vitro systems for mechanistic studies and/or drug development projects.
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Familial hemiplegic migraine type-1 (FHM-1) is a form of migraine with aura caused by mutations in the P/Q-type (Cav2.1) voltage-gated calcium channel. Pregabalin, used clinically in the treatment of chronic pain and epilepsy, inhibits P/Q-type calcium channel activity and recent studies suggest that it may have potential for the treatment of migraine. Spreading Depolarization (SD) is a neurophysiological phenomenon that can occur during migraine with aura by propagating a wave of silenced neuronal function through cortex and sometimes subcortical brain structures. Here, utilizing an optogenetic stimulation technique optimized to allow for non-invasive initiation of cortical SD, we demonstrate that chronic pregabalin administration [12 mg/kg/day (s.c.)] in vivo increased the threshold for cortical spreading depolarization in transgenic mice harboring the clinically-relevant Cav2.1S218L mutation (S218L). In addition, chronic pregabalin treatment limited subcortical propagation of recurrent spreading depolarization events to the striatum and hippocampus in both wild-type and S218L mice. To examine contributing underlying mechanisms of action of chronic pregabalin, we performed whole-cell patch-clamp electrophysiology in CA1 neurons in ex vivo brain slices from mice treated with chronic pregabalin vs vehicle. In WT mice, chronic pregabalin produced a decrease in spontaneous excitatory postsynaptic current (sEPSC) amplitude with no effect on frequency. In contrast, in S218L mice chronic pregabalin produced an increase in sEPSC amplitude and decreased frequency. These electrophysiological findings suggest that in FHM-1 mice chronic pregabalin acts through both pre- and post-synaptic mechanisms in CA1 hippocampal neurons to elicit FHM-1 genotype-specific inhibitory action. The results highlight the potential of chronic pregabalin to limit recurrent SD to subcortical brain structures during pathophysiological events in both the genetically-normal and FHM-1 brain. The work further provides insights into FHM-1 pathophysiology and the potential for chronic pregabalin treatment to prevent SD in migraineurs.
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Trastornos Migrañosos , Migraña con Aura , Ratones , Animales , Migraña con Aura/tratamiento farmacológico , Migraña con Aura/genética , Pregabalina/farmacología , Pregabalina/uso terapéutico , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/genética , Ratones Transgénicos , HipocampoRESUMEN
The cholecystokinin receptor system, specifically cholecystokinin 2 receptor (CCK2R) is a historic target for pain management that has shown limited success. However, new approaches to target CCK2R have incited fresh enthusiasm for this target. In this mini-review, we discuss what is known about CCK2R in peripheral and central circuits under naïve physiological conditions and under conditions of chronic pain, the interactions of CCK2Rs with opioids and briefly, recent efforts to develop new treatments targeting CCK2R for chronic pain.
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Chemotherapy-induced neuropathic pain (CINP) is a debilitating and difficult-to-treat side effect of chemotherapeutic drugs. CINP is marked with oxidative stress and neuronal hypersensitivities. The peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates genes involved in oxidative stress and inflammation. We hypothesize that PPARγ agonists are protective against CIPN by reducing oxidative stress and inhibiting neuronal hypersensitivities. To test our hypothesis, acute or chronic CIPN was introduced by short or long-term treatment of oxaliplatin in BALB/c mice. CIPN mice were treated with either a novel blood-brain barrier (BBB) penetrable PPARγ agonist ELB00824, or a BBB non-penetrable PPARγ agonist pioglitazone, or vehicle. Cold allodynia, mechanical allodynia, motor coordination, sedation and addiction were measured with dry ice, von Frey filaments, beam-walking tests, and conditioned place preference, respectively. Oxidative stress was accessed by measuring byproducts of protein oxidation (carbonyl and 3-Nitrotyrosine) and lipid peroxidation [Thiobarbituric acid reactive substances (TBARS)], as wells as gene expression of Cat, Sod2, Ppargc1a. The effects of ELB00824 on nociceptor excitability were measured using whole-cell electrophysiology of isolated dorsal root ganglion neurons. Preemptive ELB00824, but not pioglitazone, reduced oxaliplatin-induced cold and mechanical allodynia and oxidative stress. ELB0824 suppressed oxaliplatin-induced firing in IB4- neurons. ELB00824 did not cause motor discoordination or sedation/addiction or reduce the antineoplastic activity of oxaliplatin (measured with an MTS-based cell proliferation assay) in a human colon cancer cell line (HCT116) and a human oral cancer cell line (HSC-3). Our results demonstrated that ELB00824 prevents oxaliplatin-induced pain, likely via inhibiting neuronal hypersensitivities and oxidative stress.
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Antineoplásicos , Hipersensibilidad , Neuralgia , Animales , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/prevención & control , Hipersensibilidad/tratamiento farmacológico , Ratones , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/prevención & control , Neuronas/metabolismo , Oxaliplatino , Estrés Oxidativo , PPAR gamma/metabolismoRESUMEN
The persistence of increased excitability and spontaneous activity in injured peripheral neurons is imperative for the development and persistence of many forms of neuropathic pain. This aberrant activity involves increased activity and/or expression of voltage-gated Na+ and Ca2+ channels and hyperpolarization activated cyclic nucleotide gated (HCN) channels as well as decreased function of K+ channels. Because they display limited central side effects, peripherally restricted Na+ and Ca2+ channel blockers and K+ channel activators offer potential therapeutic approaches to pain management. This review outlines the current status and future therapeutic promise of peripherally acting channel modulators. Selective blockers of Nav1.3, Nav1.7, Nav1.8, Cav3.2, and HCN2 and activators of Kv7.2 abrogate signs of neuropathic pain in animal models. Unfortunately, their performance in the clinic has been disappointing; some substances fail to meet therapeutic end points whereas others produce dose-limiting side effects. Despite this, peripheral voltage-gated cation channels retain their promise as therapeutic targets. The way forward may include (i) further structural refinement of K+ channel activators such as retigabine and ASP0819 to improve selectivity and limit toxicity; use or modification of Na+ channel blockers such as vixotrigine, PF-05089771, A803467, PF-01247324, VX-150 or arachnid toxins such as Tap1a; the use of Ca2+ channel blockers such as TTA-P2, TTA-A2, Z 944, ACT709478, and CNCB-2; (ii) improving methods for assessing "pain" as opposed to nociception in rodent models; (iii) recognizing sex differences in pain etiology; (iv) tailoring of therapeutic approaches to meet the symptoms and etiology of pain in individual patients via quantitative sensory testing and other personalized medicine approaches; (v) targeting genetic and biochemical mechanisms controlling channel expression using anti-NGF antibodies such as tanezumab or re-purposed drugs such as vorinostat, a histone methyltransferase inhibitor used in the management of T-cell lymphoma, or cercosporamide a MNK 1/2 inhibitor used in treatment of rheumatoid arthritis; (vi) combination therapy using drugs that are selective for different channel types or regulatory processes; (vii) directing preclinical validation work toward the use of human or human-derived tissue samples; and (viii) application of molecular biological approaches such as clustered regularly interspaced short palindromic repeats (CRISPR) technology.
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The gabapentinoid drugs, gabapentin and pregabalin, are first-line treatments for neuropathic pain. The epidemics of chronic pain and opioid misuse have given rise to the widespread use of non-opioid drugs such as the gabapentinoids for treatment. Unfortunately, the widespread use of gabapentinoid drugs has resulted in reports of misuse and abuse. Here we summarize the clinical reports of gabapentinoid abuse in different patient populations to help inform clinical practice of chronic pain management.
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Dolor Crónico , Gabapentina , Pregabalina , Analgésicos/uso terapéutico , Dolor Crónico/tratamiento farmacológico , Gabapentina/uso terapéutico , Humanos , Pregabalina/uso terapéutico , Trastornos Relacionados con SustanciasRESUMEN
In this brief report, we demonstrate that the Cav3.3 T-type voltage-gated calcium channel subtype is involved in our FRICT-ION model of chronic trigeminal neuropathic pain. We first showed that the Cacna1i gene encoding Cav3.3 is significantly upregulated in whole trigeminal ganglia of FRICT-ION mice compared to controls at week 10 post-injury. We confirmed protein upregulation of Cav3.3 compared to controls using Western blot analysis of whole trigeminal ganglia tissues. Finally, we demonstrated that intraperitoneal injection of a selective TAT-based Cav3.3 blocking peptide in FRICT-ION mice significantly reduces Cav3.3 protein expression at the peak anti-allodynic effect (4 hrs post-injection) of the attenuated neuropathic pain behavior. We also suggest that blockade of Cav3.3 may be more effective in attenuating trigeminal neuropathic pain in female than male FRICT-ION mice. Therefore, blocking or attenuating Cav3.3 function may be an effective strategy for the treatment of trigeminal neuropathic pain.
Asunto(s)
Canales de Calcio Tipo T , Animales , Ganglios Espinales , Hiperalgesia , Ratones , Regulación hacia ArribaRESUMEN
Brain-derived neurotrophic factor (BDNF) is critically involved in the pathophysiology of chronic pain. However, the mechanisms of BDNF action on specific neuronal populations in the spinal superficial dorsal horn (SDH) requires further study. We used chronic BDNF treatment (200 ng/ml, 5-6 days) of defined-medium, serum-free spinal organotypic cultures to study intracellular calcium ([Ca2+]i) fluctuations. A detailed quantitative analysis of these fluctuations using the Frequency-independent biological signal identification (FIBSI) program revealed that BDNF simultaneously depressed activity in some SDH neurons while it unmasked a particular subpopulation of 'silent' neurons causing them to become spontaneously active. Blockade of gap junctions disinhibited a subpopulation of SDH neurons and reduced BDNF-induced synchrony in BDNF-treated cultures. BDNF reduced neuronal excitability assessed by measuring spontaneous excitatory postsynaptic currents. This was similar to the depressive effect of BDNF on the [Ca2+]i fluctuations. This study reveals novel regulatory mechanisms of SDH neuronal excitability in response to BDNF.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Células del Asta Posterior/fisiología , 1-Octanol/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Análisis por Conglomerados , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Modelos Neurológicos , Células del Asta Posterior/efectos de los fármacos , RatasRESUMEN
Deletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain, suggesting that the locus of analgesia is the nociceptor. Here we demonstrate, using in vivo calcium imaging and extracellular recording, that NaV1.7 knockout mice have essentially normal nociceptor activity. However, synaptic transmission from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also reversed substantially by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid independent. Male and female humans with NaV1.7-null mutations show naloxone-reversible analgesia. Thus, inhibition of neurotransmitter release is the principal mechanism of anosmia and analgesia in mouse and human Nav1.7-null mutants.