Hyperexcitability Of Sensory Neurons Research Articles

Interleukin 33-mediated inhibition of A-type K+ channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice.

Background: Interleukin-33 (IL-33) has been implicated in nociceptive pain behaviors. However, the underlying molecular and cellular mechanisms remain unclear.Methods: Using electrophysiological recording, immunoblot analysis, immunofluorescence labeling, reverse transcription-PCR, siRNA-mediated knockdown approach and behavior tests, we determined the role of IL-33 in regulating sensory neuronal excitability and pain sensitivity mediated by A-type K+ channels.Results: IL-33 decreased A-type transient outward K+ currents (IA) in small-sized DRG neurons in a concentration-dependent manner, whereas the delayed rectifier currents (IDR) remained unaffected. This IL-33-induced IA decrease was dependent on suppression of the tumorigenicity 2 (ST2) receptor and was associated with a hyperpolarizing shift in the steady-state inactivation. Antagonism of Syk abrogated the IL-33-induced IA response, while inhibition of JAK2 and PKA elicited no such effect. Exposure of DRG cells to IL-33 increased the activity of Akt, but surprisingly, neither Akt nor PI3K influenced the IL-33-induced IA response. IL-33 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK). Chemical inhibition of p38 and genetic siRNA knockdown of p38 beta (p38β), but not p38α, abrogated the IA response induced by IL-33. Moreover, IL-33 increased neuronal excitability of DRG neurons and facilitated peripheral pain sensitivity in mice; both of these effects were occluded by IA blockade.Conclusions: Our present study reveals a novel mechanism by which IL-33/ST2 suppresses IA via a Syk-dependent p38β signaling pathway. This mechanism thereby increases DRG neuronal excitability and pain sensitivity in mice. Targeting IL-33/ST2-mediated p38β signaling may represent a therapeutic approach to ameliorate pain behaviors.

Hyperexcitability of Sensory Neurons in Fragile X Mouse Model.

Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model.

Identification of Novel Regulators of Zalcitabine-Induced Neuropathic Pain.

Neuropathic pain is one of the foremost adverse effects that worsens quality of life for patients undergoing an antiretroviral treatment. Currently, there are no effective analgesics for relieving it; thus, there is an urgent need to develop novel treatments for neuropathic pain. Previously, we described and validated F11 cells as a model of DRG (dorsal root ganglia) neurons. In the current work, we employed F11 cells to identify regulators of antiretroviral-induced neuropathic pain combining functional and transcriptomic analysis. The antiretroviral zalcitabine (ddC) increased the excitability of differentiated F11 cells associated with calcium signaling without morphological changes in the neuronal phenotype, mimicking the observed increase of painful signaling in patients suffering from antiretroviral-induced neuropathic pain. Employing RNA sequencing, we observed that zalcitabine treatment upregulated genes related with oxidative stress and calcium homeostasis. The functional impact of the transcriptomic changes was explored, finding that the exposure to zalcitabine significantly increased intracellular oxidative stress and reduced store-operated calcium entry (SOCE). Because the functional and transcriptomic evidence points toward fundamental changes in calcium signaling and oxidative stress upon zalcitabine exposure, we identified that NAD(P)H quinone dehydrogenase and the sarcoplasmic/endoplasmic reticulum calcium ATPase 3 were involved in zalcitabine-induced hyperexcitability of F11 cells. Overexpression of those genes increases the calcium-elicited hyperexcitability response and reduces SOCE, as well as increases intracellular ROS levels. These data do not only mimic the effects of zalcitabine but also highlight the relevance of oxidative stress and of calcium-mediated signaling in antiretroviral-induced hyperexcitability of sensory neurons, shedding light on new therapeutic targets for antiviral-induced neuropathic pain.

Two independent mouse lines carrying the Nav1.7 I228M gain-of-function variant display dorsal root ganglion neuron hyperexcitability but a minimal pain phenotype.

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.

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

SummaryThe immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in Pd1−/− mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.

Metformin protects from oxaliplatin induced peripheral neuropathy in rats

Oxaliplatin is a commonly used drug to treat cancer, extending the rate of disease-free survival by 20% in colorectal cancer. However, oxaliplatin induces a disabling form of neuropathy resulting in more than 60% of patients having to reduce or discontinue oxaliplatin, negatively impacting their chance of survival. Oxaliplatin-induced neuropathies are accompanied by degeneration of sensory fibers in the epidermis and hyperexcitability of sensory neurons. These morphological and functional changes have been associated with sensory symptoms such as dysesthesia, paresthesia and mechanical and cold allodynia. Various strategies have been proposed to prevent or treat oxaliplatin-induced neuropathies without success. The anti-diabetic drug metformin has been recently shown to exert neuroprotection in other chemotherapy-induced neuropathies, so here we aimed to test if metformin can prevent the development of oxaliplatin-induced neuropathy in a rat model of this condition. Animals treated with oxaliplatin developed significant intraepidermal fiber degeneration, a mild gliosis in the spinal cord, and mechanical and cold hyperalgesia. The concomitant use of metformin prevented degeneration of intraepidermal fibers, gliosis, and the altered sensitivity. Our evidence further supports metformin as a new approach to prevent oxaliplatin-induced neuropathy with a potential important clinical impact.

Interleukin-10 resolves pain hypersensitivity induced by cisplatin by reversing sensory neuron hyperexcitability.

Understanding the mechanisms that drive transition from acute to chronic pain is essential to identify new therapeutic targets. The importance of endogenous resolution pathways acting as a "brake" to prevent development of chronic pain has been largely ignored. We examined the role of interleukin-10 (IL-10) in resolution of neuropathic pain induced by cisplatin. In search of an underlying mechanism, we studied the effect of cisplatin and IL-10 on spontaneous activity (SA) in dorsal root ganglia neurons. Cisplatin (2 mg/kg daily for 3 days) induced mechanical hypersensitivity that resolved within 3 weeks. In both sexes, resolution of mechanical hypersensitivity was delayed in Il10 mice, in WT mice treated intrathecally with neutralizing anti-IL-10 antibody, and in mice with cell-targeted deletion of IL-10R1 on advillin-positive sensory neurons. Electrophysiologically, small- to medium-sized dorsal root ganglia neurons from cisplatin-treated mice displayed an increase in the incidence of SA. Cisplatin treatment also depolarized the resting membrane potential, and decreased action potential voltage threshold and rheobase, while increasing ongoing activity at -45 mV and the amplitude of depolarizing spontaneous fluctuations. In vitro addition of IL-10 (10 ng/mL) reversed the effect of cisplatin on SA and on the depolarizing spontaneous fluctuation amplitudes, but unexpectedly had little effect on the other electrophysiological parameters affected by cisplatin. Collectively, our findings challenge the prevailing concept that IL-10 resolves pain solely by dampening neuroinflammation and demonstrate in a model of chemotherapy-induced neuropathic pain that endogenous IL-10 prevents transition to chronic pain by binding to IL-10 receptors on sensory neurons to regulate their activity.

Activation and functional modulation of satellite glial cells by oxaliplatin lead to hyperexcitability of sensory neurons in vitro

Platinum-based chemotherapeutics still play an important role in cancer therapy, however, severe side effects, such as painful neuropathy, occur frequently. The pathophysiologic mechanisms depend on the applied chemotherapeutic agent and are still controversial. In addition to neuronal damage, disturbance of glial cell activity may contribute to neurotoxicity. Here, we focused on the effect of oxaliplatin on satellite glial cell (SGC) function and on the activity of the dorsal root ganglion (DRG) neurons. SGCs were isolated as high-purity cultures and treated with 1 and 10 μM oxaliplatin for 2, 4 and 24 h. Subsequently, glial fibrillary acid protein (GFAP), reactive oxygen species (ROS), Connexin-43 (Cx-43), and inward rectifier potassium channel 4.1 (Kir4.1) expression was determined by immunocytochemical staining (ICC) and Western blot analyses. Immunochemical staining and Western blot analysis showed an increase in the immune reactivity (IR) and protein levels of ROS, GFAP, and Cx-43. Furthermore, reduction of the IR and protein levels and current density were demonstrated using patch-clamp measurements, of Kir4.1 channels after oxaliplatin exposure. Cytokine release in SGCs was measured using enzyme-linked immunosorbent assays (ELISA) after oxaliplatin exposure and indicated an increased release of IL-6 and TNFα, while IL-1β was decreased. The direct influence of SGC-secreted factors in the supernatant after oxaliplatin treatment led to the hyperexcitability of cultured DRG neurons. In summary, oxaliplatin has a direct impact on the modulation and function of different SGC proteins. Furthermore, SGC-released factors influence the excitability of sensory neurons, qualifying SGCs as potential targets for the prevention and treatment of oxaliplatin-induced polyneuropathy.

3'-O-Methylorobol Inhibits the Voltage-Gated Sodium Channel Nav1.7 with Anti-Itch Efficacy in A Histamine-Dependent Itch Mouse Model.

An itch is a clinical complication that affects millions of patients. However, few treatment options are available. The voltage-gated sodium channel Nav1.7 is predominantly expressed in peripheral sensory neurons and is responsible for the rising phase of action potentials, thereby mediating nociceptive conduction. A gain-of-function mutation of Nav1.7 results in the hyperexcitability of sensory neurons and causes the inherited paroxysmal itch. Conversely, a monoclonal antibody that selectively inhibits Nav1.7 is able to effectively suppress the histamine-dependent itch in mice. Therefore, Nav1.7 inhibitors may possess the potential to relieve the itch. In the present study, using whole-cell voltage-clamp recordings, we demonstrated that 3’-O-methylorobol inhibited Na+ currents in Nav1.7-CHO cells and tetrodotoxin-sensitive Na+ currents in mouse dorsal root ganglion (DRG) neurons with IC50 (half-maximal inhibitory concentration) values of 3.46 and 6.60 μM, respectively. 3’-O-methylorobol also suppressed the tetrodotoxin-resistant Na+ currents in DRG neurons, though with reduced potency (~43% inhibition at 30 µM). 3’-O-methylorobol (10 µM) affected the Nav1.7 by shifting the half-maximal voltage (V1/2) of activation to a depolarizing direction by ~6.76 mV, and it shifted the V1/2 of inactivation to a hyperpolarizing direction by ~16.79 mV. An analysis of 3’-O-methylorobol activity toward an array of itch targets revealed that 3’-O-methylorobol was without effect on histamine H1 receptor, TRPV1, TRPV3, TRPV4, TRPC4 and TRPM8. The intrathecal administration of 3’-O-methylorobol significantly attenuated compound 48/80-induced histamine-dependent spontaneous scratching bouts and the expression level of c-fos in the nuclei of spinal dorsal horn neurons with a comparable efficacy to that of cyproheptadine. Our data illustrated the therapeutic potential for 3’-O-methylorobol for histamine-dependent itching, and the small molecule inhibition of Nav1.7 may represent a useful strategy to develop novel therapeutics for itching.

MNK-eIF4E signalling is a highly conserved mechanism for sensory neuron axonal plasticity: evidence from Aplysia californica.

Injury to sensory neurons causes an increase in the excitability of these cells leading to enhanced action potential generation and a lowering of spike threshold. This type of sensory neuron plasticity occurs across vertebrate and invertebrate species and has been linked to the development of both acute and persistent pain. Injury-induced plasticity in sensory neurons relies on localized changes in gene expression that occur at the level of mRNA translation. Many different translation regulation signalling events have been defined and these signalling events are thought to selectively target subsets of mRNAs. Recent evidence from mice suggests that the key signalling event for nociceptor plasticity is mitogen-activated protein kinase-interacting kinase (MNK) -mediated phosphorylation of eukaryotic translation initiation factor (eIF) 4E. To test the degree to which this is conserved in other species, we used a previously described sensory neuron plasticity model in Aplysia californica. We find, using a variety of pharmacological tools, that MNK signalling is crucial for axonal hyperexcitability in sensory neurons from Aplysia. We propose that MNK-eIF4E signalling is a core, evolutionarily conserved, signalling module that controls nociceptor plasticity. This finding has important implications for the therapeutic potential of this target, and it provides interesting clues about the evolutionary origins of mechanisms important for pain-related plasticity. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Role of NaV1.6 and NaVβ4 Sodium Channel Subunits in a Rat Model of Low Back Pain Induced by Compression of the Dorsal Root Ganglia

Low back pain is a common cause of chronic pain and disability. It is modeled in rodents by chronically compressing the lumbar dorsal root ganglia (DRG) with small metal rods, resulting in ipsilateral mechanical and cold hypersensitivity, and hyperexcitability of sensory neurons. Sodium channels are implicated in this hyperexcitability, but the responsible isoforms are unknown. In this study, we used siRNA-mediated knockdown of the pore-forming NaV1.6 and regulatory NaVβ4 sodium channel isoforms that have been previously implicated in a different model of low back pain caused by locally inflaming the L5 DRG. Knockdown of either subunit markedly reduced spontaneous pain and mechanical and cold hypersensitivity induced by DRG compression, and reduced spontaneous activity and hyperexcitability of sensory neurons with action potentials <1.5 msec (predominately cells with myelinated axons, based on conduction velocities measured in a subset of cells) 4 days after DRG compression. These results were similar to those previously obtained in the DRG inflammation model and some neuropathic pain models, in which sensory neurons other than nociceptors seem to play key roles. The cytokine profiles induced by DRG compression and DRG inflammation were also very similar, with upregulation of several type 1 pro-inflammatory cytokines and downregulation of type 2 anti-inflammatory cytokines. Surprisingly, the cytokine profile was largely unaffected by NaVβ4 knockdown in either model. The NaV1.6 channel, and the NaVβ4 subunit that can regulate NaV1.6 to enhance repetitive firing, play key roles in both models of low back pain; targeting the abnormal spontaneous activity they generate may have therapeutic value.

Increased Resurgent Sodium Currents in Nav1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies.

Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the Possum transgenic mouse strain. We show that, in male DRG neurons, these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents, and the T790A mutation increased them fourfold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel β4 subunits fails to significantly alter T790A current density but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer early afterdepolarizations and narrower action potentials after β4 knockdown. Together, our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN.SIGNIFICANCE STATEMENT This work demonstrates that two disease mutations in the voltage-gated sodium channel Nav1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization, they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open-channel block is a major factor in Nav1.8-associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.

Two-pore-domain potassium channels and molecular mechanisms underlying migraine

Migraine is a common, disabling neurological disorder with genetic, environmental and hormonal components and a prevalence estimated at ∼15%. Migraine episodes are notably related, among several factors, to electric hyperexcitability in sensory neurons. Their electrical activity is controlled by ion channels that generate current, specifically by the two-pore-domain potassium, K2P, channels, which inhibit electrical activity. Mutation in the gene encoding TRESK, a K2P channel, causes the formation of TRESK-MT1, the expected non-functional C-terminal truncated TRESK channel, and an additional unexpected protein, TRESK-MT2, which corresponds to a non-functional N-terminal truncated TRESK channel, through a mechanism called frameshift mutation-induced Alternative Translation Initiation (fsATI). TRESK-MT1 is inactive but TRESK-M2 targets two other ion channels, TREK1 and TREK2, inducing a great stimulation of the neuronal electrical activity that may cause migraines. These findings identify TREK1 and TREK2 as potential molecular targets for migraine treatment and suggest that fsATI should be considered as a distinct class of mutations.

Inhibition of A-Type K+ Channels by Urotensin-II Induces Sensory Neuronal Hyperexcitability Through the PKCα-ERK Pathway.

Previous studies have implicated urotensin-II in the nociception of sensory neurons. However, to date the relevant mechanisms remain unknown. In the current study we determined the role of urotensin-II in the regulation of transient outward A-type potassium currents (IA) and neuronal excitability in trigeminal ganglion (TG) neurons. We found that application of urotensin-II to small-diameter TG neurons decreased IA in a dose-dependent manner, whereas the delayed rectifier potassium current was unaffected. The IA decrease induced by urotensin-II depended on the urotensin-II receptor (UT-R) and was associated with a hyperpolarizing shift in the steady-state inactivation curve. Exposure of TG cells to urotensin-II markedly increased protein kinase C (PKC) activity, and PKC inhibition eliminated the UT-R-mediated IA decrease. Antagonism of PKCα, either pharmacologically or genetically, but not of PKCβ prevented the decrease in IA induced by urotensin-II. Analysis of phospho-extracellular signal-regulated kinase (p-ERK) revealed that urotensin-II significantly increased the expression level of p-ERK, whereas p-p38 and p-c-Jun N-terminal kinase remained unchanged. Inhibition of mitogen-activated protein kinase/ERK signaling by the kinase antagonist U0126 and PD98059 completely abolished the UT-R-mediated IA decrease. Moreover, urotensin-II significantly increased the action potential firing rate of small TG neurons; pretreatment with 4-aminopyridine prevented this effect. In summary, our findings suggest that urotensin-II selectively attenuated IA through stimulation of the PKCα-dependent ERK1/2 signaling pathway. This UT-R-dependent mechanism might contribute to neuronal hyperexcitability in TG neurons.

Neuronal Transforming Growth Factor beta Signaling via SMAD3 Contributes to Pain in Animal Models of Chronic Pancreatitis

Chronic pancreatitis (CP) is characterized by pancreatic inflammation and fibrosis, associated with increased pancreatic expression of transforming growth factor beta (TGFB). It is not clear how these might contribute to pain. We investigated whether TGFB signaling via SMAD induces sensitization of pancreatic sensory neurons to increase nociception. CP was induced in Sprague-Dawley rats by infusion of trinitrobenzene sulfonic acid; some rats were given intrathecal infusions of TGFB1. CP was induced in control mice by administration of cerulein; we also studied β1glo/Ptf1acre-ER mice, which on induction overexpress TGFB1 in pancreatic acinar cells, and TGFBr1f/f-CGRPcreER mice, which have inducible disruption of TGFBr1 in calcitonin gene-related peptide-positive neurons. Dominant negative forms of human TGFBR2 and SMAD3 were overexpressed from viral vectors in rat pancreas. Some rats were given the SMAD3 inhibitors SIS3 or halofuginone. After induction of CP, mice were analyzed for pain in behavior tests or electrophysiologic studies of sensory neurons. Pancreatic nociceptor excitability was examined by patch-clamp techniques and nociception was measured by Von Frey Filament tests for referred somatic hyperalgesia and behavioral responses to pancreatic electrical stimulation. Pancreata were collected from mice and rats and analyzed histologically and by enzyme-linked immunosorbent assay and immunohistochemistry. Overexpression of TGFB in pancreatic acinar cells of mice and infusion of TGFB1 into rats resulted in sensory neuron hyperexcitability, SMAD3 activation, and increased nociception. This was accompanied by a reduction in the transient A-type current in pancreas-specific sensory neurons in rats, a characteristic of nociceptive sensitization in animal models of CP. Conversely, pancreata from TGFBr1f/f-CGRPcreER mice, rats with pancreatic expression of dominant negative forms of human TGFBR2 or SMAD3, and rats given small molecule inhibitors of SMAD3 had attenuated neuronal sensitization and pain behavior following induction of CP. In contrast to findings from peripheral administration of TGFB1, intrathecal infusion of TGFB1 reduced hyperalgesia in rats with CP. In pancreata of mice and rats, TGFB promotes peripheral nociceptive sensitization via a direct effect on primary sensory neurons mediated by intra-neuronal SMAD3. This is distinct from the central nervous system, where TGFB reduces nociception. These results provide an explanation for the link between fibrosis and pain in patients with CP. This signaling pathway might be targeted therapeutically to reduce pain in patients with CP.

Anti-nociceptive effects of bupivacaine-encapsulated PLGA nanoparticles applied to the compressed dorsal root ganglion in mice

Bupivacaine is a commonly used local anesthetic in postoperative pain management. We evaluated the effects of a prolonged, local delivery of bupivacaine on pain behavior accompanying a chronic compression of the dorsal root ganglion (CCD) – an animal model of radicular pain. Poly(lactide-coglycolide) (PLGA) nanoparticles encapsulating bupivacaine were injected unilaterally into the L3 and L4 DRGs of mice just before producing CCD by implanting a stainless-steel rod in the intervertebral foramen of each ganglion. Behavioral sensitivity to punctate mechanical stimuli (Von Frey filaments) of different forces of indentation, delivered to each hind paw, was measured before and on subsequent days of testing after the CCD. Nanoparticles were spherical in morphology and 150 ± 10 nm in diameter. Bupivacaine was steadily released as measured in vitro over 35 days. A dye that was encapsulated in the nanoparticles was found in the intact DRG after 2 weeks. CCD alone or with injection of blank (control) nanoparticles produced a behavioral hypersensitivity to the punctate stimuli on the ipsilateral paw without affecting sensitivity on the contralateral, over a period of 7–14 days. The hypersensitivity was manifested as an increased incidence of paw-withdrawal to indentation forces normally below threshold (allodynia) and an increased shaking to a filament force that always elicited withdrawal prior to CCD (hyperalgesia). In contrast, nanoparticles with bupivacaine prevented any manifestation of allodynia or hyperalgesia on the ipsilateral hind paw while leaving normal nociceptive responses largely intact on both hind paws. CCD induced behavioral hypersensitivity to nociceptive stimuli is known to be associated with a hyperexcitability of sensory neurons originating in the compressed ganglion. We hypothesize that bupivacaine-loaded PLGA nanoparticles may prevent the occurrence of this neuronal hyperexcitability without reducing the nociceptive information normally conducted from the periphery to the central nervous system. The slow, sustained delivery of bupivacaine by nanoparticles may provide a means of preventing the occurrence of postoperative neuronal hyperexcitability that could develop into chronic neuropathic pain.

The G2A receptor (GPR132) contributes to oxaliplatin-induced mechanical pain hypersensitivity

Chemotherapy-induced peripheral neuropathic pain (CIPN) is a common and severe debilitating side effect of many widely used cytostatics. However, there is no approved pharmacological treatment for CIPN available. Among other substances, oxaliplatin causes CIPN in up to 80% of treated patients. Here, we report the involvement of the G-protein coupled receptor G2A (GPR132) in oxaliplatin-induced neuropathic pain in mice. We found that mice deficient in the G2A-receptor show decreased mechanical hypersensitivity after oxaliplatin treatment. Lipid ligands of G2A were found in increased concentrations in the sciatic nerve and dorsal root ganglia of oxaliplatin treated mice. Calcium imaging and patch-clamp experiments show that G2A activation sensitizes the ligand-gated ion channel TRPV1 in sensory neurons via activation of PKC. Based on these findings, we conclude that targeting G2A may be a promising approach to reduce oxaliplatin-induced TRPV1-sensitization and the hyperexcitability of sensory neurons and thereby to reduce pain in patients treated with this chemotherapeutic agent.

Activation of KCNQ Channels Suppresses Spontaneous Activity in Dorsal Root Ganglion Neurons and Reduces Chronic Pain after Spinal Cord Injury.

A majority of people who have sustained spinal cord injury (SCI) experience chronic pain after injury, and this pain is highly resistant to available treatments. Contusive SCI in rats at T10 results in hyperexcitability of primary sensory neurons, which contributes to chronic pain. KCNQ channels are widely expressed in nociceptive dorsal root ganglion (DRG) neurons, are important for controlling their excitability, and their activation has proven effective in reducing pain in peripheral nerve injury and inflammation models. The possibility that activators of KCNQ channels could be useful for treating SCI-induced chronic pain is strongly supported by the following findings. First, SCI, unlike peripheral nerve injury, failed to decrease the functional or biochemical expression of KCNQ channels in DRG as revealed by electrophysiology, real-time quantitative polymerase chain reaction, and Western blot; therefore, these channels remain available for pharmacological targeting of SCI pain. Second, treatment with retigabine, a specific KCNQ channel opener, profoundly decreased spontaneous activity in primary sensory neurons of SCI animals both in vitro and in vivo without changing the peripheral mechanical threshold. Third, retigabine reversed SCI-induced reflex hypersensitivity, adding to our previous demonstration that retigabine supports the conditioning of place preference after SCI (an operant measure of spontaneous pain). In contrast to SCI animals, naïve animals showed no effects of retigabine on reflex sensitivity or conditioned place preference by pairing with retigabine, indicating that a dose that blocks chronic pain-related behavior has no effect on normal pain sensitivity or motivational state. These results encourage the further exploration of U.S. Food and Drug Administration-approved KCNQ activators for treating SCI pain, as well as efforts to develop a new generation of KCNQ activators that lack central side effects.

Cyclic GMP-dependent protein kinase-I localized in nociceptors modulates nociceptive cortical neuronal activity and pain hypersensitivity.

Chronic pain represents a frequent and poorly understood public health issue. Numerous studies have documented the key significance of plastic changes along the somatosensory pain pathways in chronic pain states. Our recent study demonstrated that the cGMP-dependent protein kinase I (PKG-I) specifically localized in nociceptors constitutes a key mediator of hyperexcitability of primary sensory neurons and spinal synaptic plasticity after inflammation. However, whether PKG-I in nociceptors further affects the cortical plasticity in the ascending pain pathways under pathological states has remained elusive. The immediate-early gene c-fos and phosphorylated ERK1/2 (pERK1/2) are considered reliable indicators for the neuronal activation status and it permits a comprehensive and large-scale observation of nociceptive neuronal activity along the ascending pain pathways subjected to tissue injury. In the present study, we systemically demonstrated that peripheral injury in PKG-Ifl/fl mice produced a significant upregulation of c-Fos or pERK1/2 over from the periphery to the cortex along the pain pathways, including dorsal root ganglion, spinal dorsal horn, ventral posterolateral thalamus, primary somatosensory hindlimb cortex, anterior cingulate cortex, basolateral amygdala, periaqueductal gray, and parabrachial nucleus. In contrast, very few cells in the above regions showed c-Fos or pERK1/2 induction in nociceptor-specific knockout mice lacking PKG-I (SNS-PKG-I−/− mice). Our results indicate that PKG-I expressed in nociceptors is not only a key determinant of dorsal root ganglion hyperexcitability and spinal synaptic plasticity but also an important modulator of cortical neuronal activity in pathological pain states and represent what we believe to be novel targets in the periphery for pain therapeutics.

Localized Sympathectomy Reduces Mechanical Hypersensitivity by Restoring Normal Immune Homeostasis in Rat Models of Inflammatory Pain.

Some forms of chronic pain are maintained or enhanced by activity in the sympathetic nervous system (SNS), but attempts to model this have yielded conflicting findings. The SNS has both pro- and anti-inflammatory effects on immunity, confounding the interpretation of experiments using global sympathectomy methods. We performed a "microsympathectomy" by cutting the ipsilateral gray rami where they entered the spinal nerves near the L4 and L5 DRG. This led to profound sustained reductions in pain behaviors induced by local DRG inflammation (a rat model of low back pain) and by a peripheral paw inflammation model. Effects of microsympathectomy were evident within one day, making it unlikely that blocking sympathetic sprouting in the local DRGs or hindpaw was the sole mechanism. Prior microsympathectomy greatly reduced hyperexcitability of sensory neurons induced by local DRG inflammation observed 4 d later. Microsympathectomy reduced local inflammation and macrophage density in the affected tissues (as indicated by paw swelling and histochemical staining). Cytokine profiling in locally inflamed DRG showed increases in pro-inflammatory Type 1 cytokines and decreases in the Type 2 cytokines present at baseline, changes that were mitigated by microsympathectomy. Microsympathectomy was also effective in reducing established pain behaviors in the local DRG inflammation model. We conclude that the effect of sympathetic fibers in the L4/L5 gray rami in these models is pro-inflammatory. This raises the possibility that therapeutic interventions targeting gray rami might be useful in some chronic inflammatory pain conditions. Sympathetic blockade is used for many pain conditions, but preclinical studies show both pro- and anti-nociceptive effects. The sympathetic nervous system also has both pro- and anti-inflammatory effects on immune tissues and cells. We examined effects of a very localized sympathectomy. By cutting the gray rami to the spinal nerves near the lumbar sensory ganglia, we avoided widespread sympathetic denervation. This procedure profoundly reduced mechanical pain behaviors induced by a back pain model and a model of peripheral inflammatory pain. One possible mechanism was reduction of inflammation in the sympathetically denervated regions. This raises the possibility that therapeutic interventions targeting gray rami might be useful in some inflammatory conditions.