Nociceptive Dorsal Root Ganglion Research Articles

Human birth tissue products as a non-opioid medicine to inhibit post-surgical pain.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain. Moreover, we unravel the underlying mechanisms of pain inhibition induced by FLO and HC-HA/PTX3.

Interleukin-6 induces nascent protein synthesis in human dorsal root ganglion nociceptors primarily via MNK-eIF4E signaling

Plasticity of dorsal root ganglion (DRG) nociceptors in the peripheral nervous system requires new protein synthesis. This plasticity is believed to be responsible for the physiological changes seen in DRG nociceptors in animal models of chronic pain. Experiments in human DRG (hDRG) neurons also support this hypothesis, but a direct observation of nascent protein synthesis in response to a pain promoting substance, like interleukin-6 (IL-6), has not been measured in these neurons. To fill this gap in knowledge, we used acutely prepared human DRG explants from organ donors. These explants provide a physiologically relevant microenvironment, closer to in vivo conditions, allowing for the examination of functional alterations in DRG neurons reflective of human neuropathophysiology. Using this newly developed assay, we demonstrate upregulation of the target of the MNK1/2 kinases, phosphorylated eIF4E (p-eIF4E), and nascently synthesized proteins in a substantial subset of hDRG neurons following exposure to IL-6. To pinpoint the specific molecular mechanisms driving this IL-6-driven increase in nascent proteins, we used the specific MNK1/2 inhibitor eFT508. Treatment with eFT508 resulted in the inhibition of IL-6-induced increases in p-eIF4E and nascent proteins. Additionally, using TRPV1 as a marker for nociceptors, we found that these effects occurred in a large number of human nociceptors. Our findings provide clear evidence that IL-6 drives nascent protein synthesis in human TRPV1+ nociceptors primarily via MNK1/2-eIF4E signaling. The work links animal findings to human nociception, creates a framework for additional hDRG signaling experiments, and substantiates the continued development of MNK inhibitors for pain

STINGing away the pain: the role of interferon-stimulated genes.

Pain and inflammation are biologically intertwined responses that warn the body of potential danger. In this issue of the JCI, Defaye, Bradaia, and colleagues identified a functional link between inflammation and pain, demonstrating that inflammation-induced activation of stimulator of IFN genes (STING) in dorsal root ganglia nociceptors reduced pain-like behaviors in a rodent model of inflammatory pain. Utilizing mice with a gain-of-function STING mutation, Defaye, Bradaia, and colleagues identified type I IFN regulation of voltage-gated potassium channels as the mechanism of this pain relief. Further investigation into mechanisms by which proinflammatory pathways can reduce pain may reveal druggable targets and insights into new approaches for treating persistent pain.

An Overview of the Effects of Tenapanor on Visceral Hypersensitivity in the Treatment of Irritable Bowel Syndrome with Constipation.

Patients with irritable bowel syndrome with constipation (IBS-C) experience persistent abdominal pain, a common symptom leading to greater healthcare utilization and reports of treatment non-response. Clinically significant improvements in abdominal pain were observed in clinical trials of tenapanor, a first-in-class inhibitor of sodium/hydrogen exchanger isoform 3 (NHE3), for the treatment of IBS-C in adults. This narrative review reports the current knowledge about visceral hypersensitivity as a mechanism for abdominal pain in patients with IBS-C and explores the published evidence for hypothesized mechanisms by which tenapanor may reduce visceral hypersensitivity leading to the observed clinical response of decreased abdominal pain. Abdominal pain is experienced through activation and signaling of nociceptive dorsal root ganglia that innervate the gut. These sensory afferent neurons may become hypersensitized through signaling of transient receptor potential cation channel subfamily V member 1 (TRPV1), resulting in reduced action potential thresholds. TRPV1 signaling is also a key component of the proinflammatory cascade involving mast cell responses to macromolecule exposure following permeation through the intestinal epithelium. Indirect evidence of this pathway is supported by observations of higher pain in association with increased intestinal permeability in patients with IBS. Tenapanor reduces intestinal sodium absorption, leading to increased water retention in the intestinal lumen, thereby improving gastrointestinal motility. In animal models of visceral hypersensitivity, tenapanor normalized visceromotor responses and normalized TRPV1-mediated nociceptive signaling. By improving gastrointestinal motility, decreasing intestinal permeability and inflammation, and normalizing nociception through decreased TRPV1 signaling, tenapanor may reduce visceral hypersensitivity, leading to less abdominal pain in patients with IBS-C. Therapies that have demonstrated effects on visceral hypersensitivity may be the future direction for meaningful abdominal pain relief for patients with IBS-C.

Induction of antiviral interferon-stimulated genes by neuronal STING promotes the resolution of pain in mice.

Inflammation and pain are intertwined responses to injury, infection, or chronic diseases. While acute inflammation is essential in determining pain resolution and opioid analgesia, maladaptive processes occurring during resolution can lead to the transition to chronic pain. Here we found that inflammation activates the cytosolic DNA-sensing protein stimulator of IFN genes (STING) in dorsal root ganglion nociceptors. Neuronal activation of STING promotes signaling through TANK-binding kinase 1 (TBK1) and triggers an IFN-β response that mediates pain resolution. Notably, we found that mice expressing a nociceptor-specific gain-of-function mutation in STING exhibited an IFN gene signature that reduced nociceptor excitability and inflammatory hyperalgesia through a KChIP1-Kv4.3 regulation. Our findings reveal a role of IFN-regulated genes and KChIP1 downstream of STING in the resolution of inflammatory pain.

Syntaxin1A overexpression and pain insensitivity in individuals with 7q11.23 duplication syndrome.

Genetic modifications leading to pain insensitivity phenotypes, while rare, provide invaluable insights into the molecular biology of pain and reveal targets for analgesic drugs. Pain insensitivity typically results from Mendelian loss-of-function mutations in genes expressed in nociceptive (pain-sensing) dorsal root ganglion (DRG) neurons that connect the body to the spinal cord. We document a pain insensitivity mechanism arising from gene overexpression in individuals with the rare 7q11.23 duplication syndrome (Dup7), who have 3 copies of the approximately 1.5-megabase Williams syndrome (WS) critical region. Based on parental accounts and pain ratings, people with Dup7, mainly children in this study, are pain insensitive following serious injury to skin, bones, teeth, or viscera. In contrast, diploid siblings (2 copies of the WS critical region) and individuals with WS (1 copy) show standard reactions to painful events. A converging series of human assessments and cross-species cell biological and transcriptomic studies identified 1 likely candidate in the WS critical region, STX1A, as underlying the pain insensitivity phenotype. STX1A codes for the synaptic vesicle fusion protein syntaxin1A. Excess syntaxin1A was demonstrated to compromise neuropeptide exocytosis from nociceptive DRG neurons. Taken together, these data indicate a mechanism for producing "genetic analgesia" in Dup7 and offer previously untargeted routes to pain control.

Chemotherapy-induced peripheral neuropathy models constructed from human induced pluripotent stem cells and directly converted cells: a systematic review.

Developments in human cellular reprogramming now allow for the generation of human neurons for in vitro disease modelling. This technique has since been used for chemotherapy-induced peripheral neuropathy (CIPN) research, resulting in the description of numerous CIPN models constructed from human neurons. This systematic review provides a critical analysis of available models and their methodological considerations (ie, used cell type and source, CIPN induction strategy, and validation method) for prospective researchers aiming to incorporate human in vitro models of CIPN in their research. The search strategy was developed with assistance from a clinical librarian and conducted in MEDLINE (PubMed) and Embase (Ovid) on September 26, 2023. Twenty-six peer-reviewed experimental studies presenting original data about human reprogrammed nonmotor neuron cell culture systems and relevant market available chemotherapeutics drugs were included. Virtually, all recent reports modeled CIPN using nociceptive dorsal root ganglion neurons. Drugs known to cause the highest incidence of CIPN were most used. Furthermore, treatment effects were almost exclusively validated by the acute effects of chemotherapeutics on neurite dynamics and cytotoxicity parameters, enabling the extrapolation of the half-maximal inhibitory concentration for the 4 most used chemotherapeutics. Overall, substantial heterogeneity was observed in the way studies applied chemotherapy and reported their findings. We therefore propose 6 suggestions to improve the clinical relevance and appropriateness of human cellular reprogramming-derived CIPN models.

The Mas-related G protein-coupled receptor d (Mrgprd) mediates pain hypersensitivity in painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, axonal degeneration, and changes in cutaneous innervation. However, the complete molecular profile underlying the hyperexcitable cellular phenotype of DRG nociceptors in PDN has not been elucidated. This gap in our knowledge is a critical barrier to developing effective, mechanism-based, and disease-modifying therapeutic approaches that are urgently needed to relieve the symptoms of PDN. Using single-cell RNA sequencing of DRGs, we demonstrated an increased expression of the Mas-related G protein-coupled receptor d (Mrgprd) in a subpopulation of DRG neurons in the well-established high-fat diet (HFD) mouse model of PDN. Importantly, limiting Mrgprd signaling reversed mechanical allodynia in the HFD mouse model of PDN. Furthermore, in vivo calcium imaging allowed us to demonstrate that activation of Mrgprd-positive cutaneous afferents that persist in diabetic mice skin resulted in an increased intracellular calcium influx into DRG nociceptors that we assess in vivo as a readout of nociceptors hyperexcitability. Taken together, our data highlight a key role of Mrgprd-mediated DRG neuron excitability in the generation and maintenance of neuropathic pain in a mouse model of PDN. Hence, we propose Mrgprd as a promising and accessible target for developing effective therapeutics currently unavailable for treating neuropathic pain in PDN.

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells

PurposeThis study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization.MethodshAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model.ResultsIL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation.ConclusionCxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

134 Retrograde Adeno-Associated Viruses-mediated Gene Transfer to Primary Nociceptors via Paw Injection Modulates Pain-Related Phenotypes

INTRODUCTION: Primary nociceptors are the first neurons involved in processing normal and pathological pain. Novel biological approaches such as gene therapies targeting these neurons have great potential, but delivery can be challenging when open or percutaneous infusion into the dorsal root ganglion (DRG) is needed. METHODS: Primary nociceptors were first targeted unilaterally via right hind paw injection of a retrograde adeno-associated virus serotype 2 (AAVRetro) or a mosaic AAVRetro/rh10 vector encoding a constitutive hM3Dq chemogenetic transgene. A second cohort of mice received the same vectors via unilateral intraneural injection in the right sciatic nerve. Changes in pain-related behaviors were assessed 45-60 min following intraperitoneal administration of the ligand clozapine-n-oxide (CNO) using the Von Frey and Hargreaves tests every other week for 6 weeks post-AAVRetro injection. RESULTS: Immunofluorescence and RNA in-situ hybridization of paw injected mice revealed transduction of primary nociceptors in the DRG from L4 to L6 segments, including TrkA, Trpv1, CGRP, and NaV1.8 positive neurons. Chemogenetic activation elicited robust mechanical allodynia responses at 4-week post-RetroAAV delivery in paw injected mice, whilst in sciatic injected mice we observed an earlier pain phenotype starting at 2-week post-RetroAAV administration. No thermal hyperalgesia responses were observed. CONCLUSIONS: We demonstrate for the first time that AAVRetro effectively transduces DRG nociceptors via paw injection. Furthermore, chemogenetic activation caused a strong pain phenotype similar to mice injected in the sciatic nerve. These results support further exploration of non-invasive delivery of retrograde viral vectors to modulate pain neurons for potential therapeutic applications.

Kv1 activator mechanism of action in action potential inhibition.

Delayed-rectifier, voltage-gated potassium channels (Kv), such as the Kv1 family, are found in nociceptive dorsal root ganglion (DRG) neurons and represent a potential target for novel analgesics. Activating Kv1 channels in nociceptive neurons may work to decrease neuronal excitability and diminish the sensation of pain. Here, we explored the mechanism by which a Kv1 activator decreases action potential firing in DRG neurons. The small molecules compound A (cmpd-A) and B (cmpd-B) induced a hyperpolarizing shift in the conductance-voltage relationship of a heterologously-expressed Kv1 channel while cmpd-A additionally slowed the deactivation rate in a voltage-independent manner. Cmpd-A, but not cmpd-B, caused a frequency-dependent inhibition of AP firing in rat DRG neurons. We examined the effects of both compounds on Kv1 biophysics and neuronal membrane properties to explain the mechanism of action underlying cmpd-A AP inhibition.

The contribution of endocytosis to sensitization of nociceptors and synaptic transmission in nociceptive circuits.

Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.

Shared nociceptive dorsal root ganglion neurons participating in acupoint sensitization.

When the body is under pathological stress (injury or disease), the status of associated acupoints changes, including decreased pain threshold. Such changes in acupoint from a “silent” to an “active” state are considered “acupoint sensitization,” which has become an important indicator of acupoint selection. However, the mechanism of acupoint sensitization remains unclear. In this study, by retrograde tracing, morphological, chemogenetic, and behavioral methods, we found there are some dorsal root ganglion (DRG) neurons innervating the ST36 acupoint and ipsilateral hind paw (IHP) plantar simultaneously. Inhibition of these shared neurons induced analgesia in the complete Freund’s adjuvant (CFA) pain model and obstruction of nociceptive sensation in normal mice, and elevated the mechanical pain threshold (MPT) of ST36 acupoint in the CFA model. Excitation of shared neurons induced pain and declined the MPT of ST36 acupoint. Furthermore, most of the shared DRG neurons express TRPV1, a marker of nociceptive neurons. These results indicate that the shared nociceptive DRG neurons participate in ST36 acupoint sensitization in CFA-induced chronic pain. This raised a neural mechanism of acupoint sensitization at the level of primary sensory transmission.

Investigating Peripheral Regional Anesthesia Using Induced Pluripotent Stem Cell Technology: Exploring Novel Terrain.

Investigating Peripheral Regional Anesthesia Using Induced Pluripotent Stem Cell Technology: Exploring Novel Terrain.

Etv4 regulates nociception by controlling peptidergic sensory neuron development and peripheral tissue innervation.

The perception of noxious environmental stimuli by nociceptive sensory neurons is an essential mechanism for the prevention of tissue damage. Etv4 is a transcriptional factor expressed in most nociceptors in dorsal root ganglia (DRG) during the embryonic development. However, its physiological role remains unclear. Here, we show that Etv4 ablation results in defects in the development of the peripheral peptidergic projections in vivo, and in deficits in axonal elongation and growth cone morphology in cultured sensory neurons in response to NGF. From a mechanistic point of view, our findings reveal that NGF regulates Etv4-dependent gene expression of molecules involved in extracellular matrix (ECM) remodeling. Etv4-null mice were less sensitive to noxious heat stimuli and chemical pain, and this behavioral phenotype correlates with a significant reduction in the expression of the pain-transducing ion channel TRPV1 in mutant mice. Together, our data demonstrate that Etv4 is required for the correct innervation and function of peptidergic sensory neurons, regulating a transcriptional program that involves molecules associated with axonal growth and pain transduction.

Exploring pharmacological inhibition of Gq/11 as an analgesic strategy.

Misuse of opioids has greatly affected our society. One potential solution is to develop analgesics that act at targets other than opioid receptors. These can be used either as stand-alone therapeutics or to improve the safety profile of opioid drugs. Previous research showed that activation of Gq/11 proteins by G-protein coupled receptors has pro-nociceptive properties, suggesting that blockade of Gq/11 signalling could be beneficial for pain control. The aim of this study was to test this hypothesis pharmacologically by using potent and selective Gq/11 inhibitor YM-254890. We used a series of behavioural assays to evaluate the acute responses of mice to painful thermal stimulation while administering YM-254890 alone and in combination with morphine. We then used electrophysiological recordings to evaluate the effects of YM-254890 on the excitability of dorsal root ganglion (DRG) nociceptor neurons. We found that systemic administration of YM-254890 produced anti-nociceptive effects and also augmented morphine analgesia in both hotplate and tail flick paradigms. However, it also caused substantial inhibition of locomotion, which may limit its therapeutic utility. To circumvent these issues, we explored the local administration of YM-254890. Intrathecal injections of YM-254890 produced lasting analgesia in a tail flick test and greatly augmented the anti-nociceptive effects of morphine without any significant effects on locomotor behaviour. Electrophysiological studies showed that YM-254890 reduced the excitability of DRG nociceptors and augmented their opioid-induced inhibition. These findings indicate that pharmacological inhibition of Gq/11 could be explored as an analgesic strategy.

The Antiseizure Drug Perampanel Is a Subunit-Selective Negative Allosteric Modulator of Kainate Receptors.

Perampanel (PMP) is a third-generation antiseizure drug reported to be a potent and selective noncompetitive negative allosteric modulator of one subfamily of ionotropic glutamate receptor (iGluR), the α-amino-3-hydroxy-S-methylisoxazole-4-propionic acid receptors (AMPARs). However, the recent structural resolution of AMPARs in complex with PMP revealed that its binding pocket is formed from residues that are largely conserved in two members of another family of iGluRs, the GluK4 and GluK5 kainate receptor (KAR) subunits. We show here that PMP inhibits both recombinant and neuronal KARs, contrary to the previous reports, and that the negative allosteric modulator (NAM) activity requires GluK5 subunits to be channel constituents. PMP inhibited heteromeric GluK1/GluK5 and GluK2/GluK5 KARs at IC50 values comparable to that for AMPA receptors but was much less potent on homomeric GluK1 or GluK2 KARs. The auxiliary subunits Neto1 or Neto2 also made GluK2-containing KARs more sensitive to inhibition. Finally, PMP inhibited mouse neuronal KARs containing GluK5 subunits and Neto proteins in nociceptive dorsal root ganglia neurons and hippocampal mossy fiber-CA3 pyramidal neuron synapses. These data suggest that clinical actions of PMP could arise from differential inhibition of AMPAR or KAR signaling and that more selective drugs might maintain antiseizure efficacy while reducing adverse effects.SIGNIFICANCE STATEMENT PMP is a regulatory approved antiseizure drug used for refractory partial-onset and generalized tonic-clonic seizures that acts as a selective negative allosteric modulator of AMPARs. Here, we demonstrate that PMP inhibits KARs, a second family of ionotropic glutamate receptors, in addition to AMPARs. NAM activity on KARs required GluK5 subunits or Neto auxiliary subunits as channel constituents. KAR inhibition, therefore, could contribute to PMP antiseizure action or the adverse effects that are significant with this drug. Drug discovery aimed at more selective allosteric modulators that discriminate between AMPARs and KARs could yield next-generation drugs with improved therapeutic profiles for treatment of epilepsy.

Regulation of Eukaryotic Elongation Factor 2 Kinase in Mouse Sensory Neurons and Anti-nociceptive Effects of a Selective Inhibitor

Eukaryotic elongation factor 2 (eEF2) is an essential component of the translation machinery that catalyzes translocation of the ribosome along the mRNA transcript. Phosphorylation of eEF2 causes a transient suspension of ongoing translation. eEF2 kinase, the only enzyme known to phosphorylate eEF2, is activated by calcium/calmodulin and AMP-activated kinase (AMP kinase), a key sensor of ATP availability, among other mechanisms. Using an antibody selective for phosphorylated eEF2 (peEF2), we examined eEF2 regulation in sensory neurons of the mouse dorsal root ganglion (DRG). Immunohistochemistry for peEF2 revealed tonically high levels of peEF2 in a subset of primarily small-diameter DRG neurons, particularly putative nociceptors positive for either TRPV1 or IB4. In dissociated DRG neurons, application of 50 mM K+ for 1 or 10 minutes induced intense peEF2 staining in virtually all neurons; this staining remained elevated above baseline 60 minutes after stimulation and was prevented by a selective eEF2 kinase inhibitor, A484954 (30 micromolar). Both peEF2 and eEF2K levels in DRG increased in response to hindpaw inflammatory insult (complete Freud's adjuvant injection), as measured by western blot. Therefore, A484954 bioavailability and pharmacokinetics were evaluated by mass spectrometry in plasma and brain 30, 60 and 90 minutes after oral administration of 1 or 7.5 mg/kg by gavage. Peak plasma and brain concentrations were observed 60 minutes after administration. Daily oral administration of A484954 in the carrageenan model of inflammatory pain significantly reduced hyperalgesia to radiant heat throughout the behavioral response to carrageenan, suggesting that suppression of eEF2 phosphorylation is anti-nociceptive. L3-5 DRG, lumbar spinal dorsal horn and whole brain were analyzed to confirm inhibition of eEF2 phosphorylation by oral A484954. These results indicate that peEF2 is rapidly induced in response to sensory neuron activation and sustained during inflammatory hyperalgesia. Suppression of eEF2 phosphorylation is anti-nociceptive, possibly through a direct action on DRG nociceptors. Grant support from NIH NIGMS R01102346 and P20GM103643.

Sexually Dimorphic Effects of Nociceptor LKB1 on Mitochondrial Metabolism and Mechanical Allodynia After Fasting

BackgroundPain induces metabolic stress in pain sensing neurons (nociceptors); alleviating this stress represents an avenue for treating pain. Liver kinase B1 (LKB1) is involved in maintaining energy homeostasis and is a key mediator of the cellular response to metabolic stress. If cellular energy modulation and pain relief is LKB1‐dependent, then its removal could lead to metabolic imbalance and innocuous stimuli becoming painful (allodynia).HypothesisIn a novel mouse transgenic model where LKB1 is removed from Nav1.8+ nociceptors (Nav1.8LKB1‐), we predict nociceptors will have reduced abilities to cope with metabolic stress, leading to energy imbalance and subsequent allodynia.MethodsAfter baselining for mechanical allodynia, animals were fasted for 24hr to induce metabolic stress. A subset of animals had their primary dorsal root ganglia nociceptors cultured for 6 hours for the Seahorse MitoStress assay. The remainder of animals were tested for mechanical allodynia once a day for 5 days after food was returned.ResultsAs expected, a 24hr fast induced mechanical allodynia in a genotype dependent manner, with Nav1.8LKB1‐ animals responding to the fast in a sexually dimorphic manner. While fasting initially caused allodynia in males of both genotypes, Nav1.8LKB1‐ animals took longer to recover. However, in females, only Nav1.8LKB1‐animals exhibited allodynia after fasting. A sexually dimorphic and genotype dependent effect was also seen in neuronal mitochondrial function. In control females, fasting increased neuronal oxidative metabolism, suggesting the LKB1 pathway is necessary to mitigate fasting‐induced energy depletion. Neither fasting nor genotype changed neuronal mitochondrial function in males, suggesting an alternate mechanism was responsible for their initial allodynia.ConclusionsAlthough Nav1.8LKB1‐ animals of both sexes developed genotype‐dependent allodynia in response to a metabolic stressor (24hr fast), only females exhibited a difference in mitochondrial function in response to this fast. These data support existing literature that suggests neuronal processes are responsible for the development of allodynia in females but not males and suggests that these differences are metabolic in nature. However, additional studies are needed to determine how other sex‐ and cell‐specific metabolic processes can contribute to pain development. This includes how the metabolism of immune cells, such as microglia and macrophages, which have been shown contribute to pain in males but not females, can be affected in an LKB1‐dependent manner to reduce a painful phenotype. Acknowledgements: We would like to thank Luz Barron and Han Jeong for their contributions.

F11 dorsal root ganglion nociceptor neurons release calcitonin gene related peptide in response to pathogen associated molecular patterns

The immune and nervous systems co‐evolved to direct and mount coordinated behaviors, reflexes and immune responses to protect the host. In the neuro‐immune paradigm sensory nociceptor neurons, located in multiple peripheral ganglia, have been shown to be sensitive to pattern associated molecular patterns (PAMPs). Upon stimulation, these sensory nociceptors release the neurotransmitter calcitonin gene related peptide (CGRP) which acts as a cytokine to assist with the coordination of the innate immune response. However, it remains unclear which PAMPs are sensed by these sensory c‐fiber nerves and what specific substances are released in response to the specific PAMPs. Additionally, how long does this stimulation by PAMPs last? These are critical knowledge gaps in our understanding of how sensory neurons shape the innate immune response. To answer these questions, we cultured immortalized F11 dorsal root ganglion (DRG) neurons and exposed them to lipopolysaccharide (LPS 500 ng/mL, Gram‐negative PAMP), lipoteichoic acid (LTA 50 μg/mL, Gram‐positive PAMP), tumor necrosis factor alpha (TNFα 500 ng/mL, inflammatory cytokine) and capsaicin (1 μM, known C‐fiber agonist, serving as a positive control). The exposures to the stimuli lasted for 0.5, 1, 2, 4 or 8 hours. The experiments were run in triplicate on three separate days where a control condition was run on each day for each time point. We measured the release of CGRP with ELISA, known to be released by DRG neurons in response to LPS. We hypothesized that CGRP would be released early on as neural excitation occurs quickly. All conditions and time points were normalized to the average of respective controls for specific time points on respective days. In contrast to our hypothesis, no condition increased CGRP concentration at 0.5 hours. However, at 1‐hour LPS, LTA, TNFα and capsaicin increased CGRP release significantly (p<0.05). At 2 hours, LPS and LTA retained their neural stimulation as CGRP was still increased in comparison to controls (P<0.01). Stimulation subsided at 4 and 8 hours as CGRP concentration returned close to control conditions at respective time points. Our data demonstrate that Gram‐positive and Gram‐negative PAMPs stimulate the release of a known neurotransmitter from sensory nociceptor cells. Additional experiments with fungal PAMPs will be run as well as identifying the complete secrotome of the neuronal culture in response to a variety of PAMPs. These data coincide with the notion that neurons are stimulated early during infection by PAMPs and inflammation directly, as indicated by TNFα stimulation, in order to quickly assist with the innate immune response.