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Widespread or ectopic sensitization is a hallmark symptom of chronic pain, characterized by aberrantly enhanced pain sensitivity in multiple body regions remote from the site of original injury or inflammation. The central mechanism underlying widespread sensitization remains unidentified. The central nucleus of the amygdala (also called the central amygdala, CeA) is well situated for this role because it receives nociceptive information from diverse body sites and modulates pain sensitivity in various body regions. In this study, we examined the role of the CeA in a novel model of ectopic sensitization of rats. Injection of formalin into the left upper lip resulted in latent bilateral sensitization in the hind paw lasting >13 days in male Wistar rats. Chemogenetic inhibition of gamma-aminobutyric acid-ergic neurons or blockade of calcitonin gene-related peptide receptors in the right CeA, but not in the left, significantly attenuated this sensitization. Furthermore, chemogenetic excitation of gamma-aminobutyric acid-ergic neurons in the right CeA induced de novo bilateral hind paw sensitization in the rats without inflammation. These results indicate that the CeA neuronal activity determines hind paw tactile sensitivity in rats with remote inflammatory pain. They also suggest that the hind paw sensitization used in a large number of preclinical studies might not be simply a sign of the pain at the site of injury but rather a representation of the augmented CeA activity resulting from inflammation/pain in any part of the body or from activities of other brain regions, which has an active role of promoting defensive/protective behaviors to avoid further bodily damage.
Learn More >Sex differences in pain severity, response, and pathological susceptibility are widely reported, but the neural mechanisms that contribute to these outcomes remain poorly understood. Here we show that dopamine (DA) neurons in the ventrolateral periaqueductal gray/dorsal raphe (vlPAG/DR) differentially regulate pain-related behaviors in male and female mice through projections to the bed nucleus of the stria terminalis (BNST). We find that activation of vlPAG/DR neurons or vlPAG/DR terminals in the BNST reduces nociceptive sensitivity during naive and inflammatory pain states in male mice, whereas activation of this pathway in female mice leads to increased locomotion in the presence of salient stimuli. We additionally use slice physiology and genetic editing approaches to demonstrate that vlPAG/DR projections to the BNST drive sex-specific responses to pain through DA signaling, providing evidence of a novel ascending circuit for pain relief in males and contextual locomotor response in females.
Learn More >Migraineurs experience increased oxidative stress which drives the initiation and maintenance of migraine-related pain in animal models and, by extension, migraine in humans. Oxidative stress augments calcitonin gene-related peptide (CGRP) levels, a mediator of migraine pain. Insulin-like growth factor-1 (IGF-1), a neuroprotective growth factor, reduces susceptibility to spreading depression, a preclinical model of migraine, in cultured brain slices by blocking oxidative stress and neuroinflammation from microglia. Similarly, nasal delivery of IGF-1 inhibits spreading depression in vivo. After recurrent cortical spreading depression, nasal administration of IGF-1 also significantly reduces trigeminal ganglion oxidative stress and CGRP levels as well as trigeminocervical c-Fos activation. Here, we probed for the impact of nasal IGF-1 pretreatment on trigeminal system activation using a second well-established preclinical model of migraine, systemic nitroglycerin injection. Adult male rats were treated with one of three doses of IGF-1 (37.5, 75 or 150 μg) and the optimal dose found in males was subsequently used for treatment of female rats. One day later, animals received an intraperitoneal injection of nitroglycerin. Measurements taken two hours later after nitroglycerin alone showed increased surrogate markers of trigeminal activation – oxidative stress and CGRP in the trigeminal ganglion and c-Fos in the trigeminocervical complex compared to vehicle control. These effects were significantly reduced at all doses of IGF-1 for trigeminal ganglion metrics of oxidative stress and CGRP and only at the lowest dose in both males and females for c-Fos. The latter inverted U-shaped or hormetic response is seen in enzyme-targeting drugs. While the specific mechanisms remain to be explored, our data here supports the ability of IGF-1 to preserve mitochondrial and antioxidant pathway homeostasis as means to prevent nociceptive activation in the trigeminal system produced by an experimental migraine model.
Learn More >TRAF6 Contributes to CFA-Induced Spinal Microglial Activation and Chronic Inflammatory Pain in Mice.
Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be expressed in spinal astrocytes and is involved in neuropathic pain. In this study, we investigated the role and mechanism of TRAF6 in complete Freund's adjuvant (CFA)-evoked chronic inflammatory hypersensitivity and the effect of docosahexaenoic acid (DHA) on TRAF6 expression and inflammatory pain. We found that TRAF6 was dominantly increased in microglia at the spinal level after intraplantar injection of CFA. Intrathecal TRAF6 siRNA alleviated CFA-triggered allodynia and reversed the upregulation of IBA-1 (microglia marker). In addition, intrathecal administration of DHA inhibited CFA-induced upregulation of TRAF6 and IBA-1 in the spinal cord and attenuated CFA-evoked mechanical allodynia. Furthermore, DHA prevented lipopolysaccharide (LPS)-caused increase of TRAF6 and IBA-1 in both BV2 cell line and primary cultured microglia. Finally, intrathecal DHA reduced LPS-induced upregulation of spinal TRAF6 and IBA-1, and alleviated LPS-induced mechanical allodynia. Our findings indicate that TRAF6 contributes to pain hypersensitivity via regulating microglial activation in the spinal dorsal horn. Direct inhibition of TRAF6 by siRNA or indirect inhibition by DHA may have therapeutic effects on chronic inflammatory pain.
Learn More >Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in Na1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between Na subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of Na1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of Na1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of Na1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of Na1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.
Learn More >Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.
Learn More >Neuropathic pain patients often experience innocuous cooling as excruciating pain. The cell and molecular basis of this cold allodynia is little understood. We used in vivo calcium imaging of sensory ganglia to investigate how the activity of peripheral cold-sensing neurons was altered in three mouse models of neuropathic pain: Oxaliplatin-induced neuropathy, partial sciatic nerve ligation and ciguatera poisoning. In control mice, cold-sensing neurons were few in number and small in size. In neuropathic animals with cold allodynia, a set of normally silent large-diameter neurons became sensitive to cooling. Many of these silent cold-sensing neurons responded to noxious mechanical stimuli and expressed the nociceptor markers NaV1.8 and CGRPα. Ablating neurons expressing NaV1.8 resulted in diminished cold allodynia. The silent cold-sensing neurons could also be activated by cooling in control mice through blockade of KV1 voltage-gated potassium channels. Thus silent cold-sensing neurons are unmasked in diverse neuropathic pain states and cold allodynia results from peripheral sensitization caused by altered nociceptor excitability.
Learn More >The resolution of inflammation is a biosynthetically active process controlled by the interplay between oxygenated polyunsaturated mediators and G-protein coupled receptor-signaling pathways. These enzymatically oxygenated polyunsaturated fatty acids belong to distinct families of specialized pro-resolving autacoids. The protectin family of mediators has attracted an interest because of their potent pro-resolving and anti-inflammatory actions verified in several in vivo disease models. Herein, we present the stereoselective synthesis and biological evaluations of 3-oxa-PD1n-3 DPA, a protectin D1 analog. Results from mouse models indicate that the mediators protectin D1, PD1n-3 DPA and the new analog 3-oxa-PD1n-3 DPA all relieved streptozotocin-induced diabetic neuropathic pain at doses of 90 and 300 pmol, equivalent to 30 and 100 ng, respectively, following intrathecal (I.T.) injection. Of interest, at a low dose of only 30 pmol (10 ng; I.T.) only 3-oxa PD1n-3 DPA was able to alleviate neuropathic pain, directly compared to vehicle controls. Moreover, using a chronic itch model of cutaneous T-cell lymphoma (CTCL), all three compounds at 300 pmol (100 ng) showed a significant reduction in itching for several hours. The biomolecular information on the structure-functions of the protectins and the new synthetic analog 3-oxa-PD1n-3 DPA is of interest towards developing new immunoresolvents.
Learn More >In humans, tissue injury and depression can both cause pain hypersensitivity, but whether this involves distinct circuits remains unknown. Here, we identify two discrete glutamatergic neuronal circuits in male mice: a projection from the posterior thalamic nucleus (PO) to primary somatosensory cortex glutamatergic neurons (S1) mediates allodynia from tissue injury, whereas a pathway from the parafascicular thalamic nucleus (PF) to anterior cingulate cortex GABA-containing neurons to glutamatergic neurons (ACC) mediates allodynia associated with a depression-like state. In vivo calcium imaging and multi-tetrode electrophysiological recordings reveal that PO and PF populations undergo different adaptations in the two conditions. Artificial manipulation of each circuit affects allodynia resulting from either tissue injury or depression-like states, but not both. Our study demonstrates that the distinct thalamocortical circuits PO→S1 and PF→ACC subserve allodynia associated with tissue injury and depression-like states, respectively, thus providing insights into the circuit basis of pathological pain resulting from different etiologies.
Learn More >Acute pancreatitis (AP) and recurring AP are serious health care problems causing excruciating pain and potentially lethal outcomes due to sepsis. The validated caerulein- (CAE) induced mouse model of acute/recurring AP produces secondary persistent hypersensitivity and anxiety-like behavioral changes for study.
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