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The nervous system is shielded by special barriers. Nerve injury results in blood-nerve barrier breakdown with downregulation of certain tight junction proteins accompanying the painful neuropathic phenotype. The dorsal root ganglion (DRG) consists of a neuron-rich region (NRR, somata of somatosensory and nociceptive neurons) and a fibre-rich region (FRR), and their putative epi-/perineurium (EPN). Here, we analysed blood-DRG barrier (BDB) properties in these physiologically distinct regions in Wistar rats after chronic constriction injury (CCI). , , and (rats) mRNA were downregulated 1 week after traumatic nerve injury. Claudin-1 immunoreactivity (IR) found in the EPN, claudin-19-IR in the FRR, and ZO-1-IR in FRR-EPN were unaltered after CCI. However, laser-assisted, vessel specific qPCR, and IR studies confirmed a significant loss of claudin-5 in the NRR. The NRR was three-times more permeable compared to the FRR for high and low molecular weight markers. NRR permeability was not further increased 1-week after CCI, but significantly more CD68 macrophages had migrated into the NRR. In summary, NRR and FRR are different in naïve rats. Short-term traumatic nerve injury leaves the already highly permeable BDB in the NRR unaltered for small and large molecules. Claudin-5 is downregulated in the NRR. This could facilitate macrophage invasion, and thereby neuronal sensitisation and hyperalgesia. Targeting the stabilisation of claudin-5 in microvessels and the BDB barrier could be a future approach for neuropathic pain therapy.
Learn More >Effective and safe analgesics represent an unmet medical need for the treatment of acute and chronic pain. A series of N-cyclopropylmethyl-7α-phenyl-6,14-endoethano-tetrahydronorthebaines were designed, synthesized and assayed, leading to the discovery of a benzylamine derivative (compound 4, SLL-039) as a highly selective and potent κ opioid agonist (κ, Ki=0.47 nM, κ/μ=682, κ/δ=283), which was confirmed by functional assays in vitro and antinociceptive assays in vivo. The in vivo effect could be blocked by pretreatment with the selective κ antagonist nor-BNI. Moreover, this compound did not induce sedation, a common dose limiting effect of κ opioid receptor agonists, at its analgesic dose compared to U50,488H. The dissociation of sedation/antinociception found in SLL-039 was assumed to be correlated with the occupation of its benzamide motif in a unique subsite involving V1182.63, W124EL1 and E209EL2.
Learn More >Spinal gastrin-releasing peptide receptor-expressing (GRPR) neurons play an essential role in itch signal processing. However, the circuit mechanisms underlying the modulation of spinal GRPR neurons by direct local and long-range inhibitory inputs remain elusive. Using viral tracing and electrophysiological approaches, we dissected the neural circuits underlying the inhibitory control of spinal GRPR neurons. We found that spinal galanin GABAergic neurons form inhibitory synapses with GRPR neurons in the spinal cord and play an important role in gating the GRPR neuron-dependent itch signaling pathway. Spinal GRPR neurons also receive inhibitory inputs from local neurons expressing neuronal nitric oxide synthase (nNOS). Moreover, spinal GRPR neurons are gated by strong inhibitory inputs from the rostral ventromedial medulla. Thus, both local and long-range inhibitory inputs could play important roles in gating itch processing in the spinal cord by directly modulating the activity of spinal GRPR neurons.
Learn More >High rates of acute and chronic pain are associated with traumatic brain injury (TBI), but mechanisms responsible for the association remain elusive. Recent data suggest dysregulated descending pain modulation circuitry could be involved. Based on these and other observations, we hypothesized that serotonin (5-HT)-dependent activation of spinal CXC Motif Chemokine Receptor 2 (CXCR2) may support TBI-related nociceptive sensitization in a mouse model of mild TBI (mTBI). We observed that systemic 5-HT depletion with p-chlorophenylalanine attenuated mechanical hypersensitivity seen after mTBI. Likewise, selective spinal 5-HT fiber depletion with 5,7-dihydroxytryptamine (5,7-DHT) reduced hypersensitivity after mTBI. Consistent with a role for spinal 5-HT serotonin receptors, intrathecal ondansetron administration after TBI dose-dependently attenuated nociceptive sensitization. Also, selective CXCR2 antagonist SCH527123 treatment attenuated mechanical hypersensitivity after mTBI. Furthermore, spinal CXCL1 and CXCL2 mRNA and protein levels were increased after mTBI as were GFAP and IBA-1 markers. Spinal 5,7-DHT application reduced both chemokine expression and glial activation. Our results suggest dual pathways for nociceptive sensitization after mTBI, direct 5-HT effect through 5-HT receptors and indirectly through upregulation of chemokine signaling. Designing novel clinical interventions against either the 5-HT mediated component or chemokine pathway may be beneficial in treating pain frequently seen in patients after mTBI.
Learn More >The development of opioid-induced analgesic tolerance is a clinical challenge in long-term use for managing chronic pain. The mechanisms of morphine tolerance are poorly understood. Mitochondria-derived reactive oxygen species (ROS) is a crucial signal inducing analgesic tolerance and pain. Chronic administration of morphine leads to robust ROS production and accumulation of damaged mitochondria, which are immediately removed by mitophagy. Here, we show that morphine inhibits mitochondria damage-induced accumulation of PTEN-induced putative kinase 1 (PINK1) in neurons. It interrupts the recruitment of Parkin to the impaired mitochondria and inhibits the ubiquitination of mitochondrial proteins catalyzed by Parkin. Consequently, morphine suppresses the recognition of autophagosomes to the damaged mitochondria mediated by LC3 and SQSTM1/p62 (sequestosome-1). Thus, morphine inhibits autophagy flux and leads to the accumulation of SQSTM1/p62. Finally, the impaired mitochondria cannot be delivered to lysosomes for degradation and ultimately induces robust ROS production and morphine tolerance. Our findings suggest that the dysfunction of mitophagy is involved in morphine tolerance. The deficiency of PINK1/Parkin-mediated clearance of damaged mitochondria is crucial for the generation of excessive ROS and important to the development of analgesic tolerance. These findings suggest that the compounds capable of stabilizing PINK1 or restoring mitophagy may be utilized to prevent or reduce opioid tolerance during chronic pain management.
Learn More >The non-selective activation of central and peripheral opioid receptors is a major shortcoming of currently available opioids. Targeting peripheral opioid receptors is a promising strategy to preclude side effects. Recently, we showed that fentanyl-derived μ-opioid receptor (MOR) agonists with reduced acid dissociation constants (pK) due to introducing single fluorine atoms produced injury-restricted antinociception in rat models of inflammatory, postoperative and neuropathic pain. Here, we report that a new double-fluorinated compound (FF6) and fentanyl show similar pK, MOR affinity and [S]-GTPγS binding at low and physiological pH values. In vivo, FF6 produced antinociception in injured and non-injured tissue, and induced sedation and constipation. The comparison of several fentanyl derivatives revealed a correlation between pK values and pH-dependent MOR activation, antinociception and side effects. An opioid ligand's pK value may be used as discriminating factor to design safer analgesics.
Learn More >Naringenin (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one is a natural flavonoid found in fruits from the citrus family. Since (2S)-Naringenin is known to racemize, its bioactivity might be related to one or both enantiomers. Computational studies predicted that (2R)-Naringenin may act on voltage-gated ion channels, particularly the N-type calcium channel (CaV2.2) and the NaV1.7 sodium channel – both key for pain signaling. Here we set out to identify the possible mechanism of action of Naringenin. Naringenin inhibited depolarization-evoked Ca2+ influx in acetylcholine-, ATP- and capsaicin-responding rat dorsal root ganglion (DRG) neurons. This was corroborated in electrophysiological recordings from DRG neurons. Pharmacological dissection of each of the voltage-gated Ca2+ channels subtypes could not pinpoint any selectivity of Naringenin. Instead, Naringenin inhibited NaV1.8-dependent, tetrodotoxin (TTX)-resistant while sparing tetrodotoxin sensitive (TTX-S) voltage-gated Na+ channels as evidenced by the lack of further inhibition by the NaV1.8 blocker A-803467. The effects of the natural flavonoid were validated ex vivo in spinal cord slices where Naringenin decreased both the frequency and amplitude of sEPSC recorded in neurons within the substantia gelatinosa. The antinociceptive potential of Naringenin was evaluated in male and female mice. Naringenin had no effect on the nociceptive thresholds evoked by heat. Naringenin's reversed allodynia was in mouse models of post-surgical and neuropathic pain. Here, driven by a call by the NCCIH's strategic plan to advance fundamental research into basic biological mechanisms of action of natural products, we advance the antinociceptive potential of the flavonoid Naringenin.
Learn More >Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches.
Learn More >Neuropathic pain is one of the most debilitating of all chronic pain syndromes. Intrathecal (i.t.) bone marrow stromal cell (BMSC) injections have a favorable safety profile; however, results have been inconsistent, and complete understanding of how BMSCs affect neuropathic pain remains elusive.
Learn More >While comorbid pain in depression (CP) occurs at a high rate worldwide, the neural connections underlying the core symptoms of CP have yet to be elucidated. Here, we define a pathway whereby GABAergic neurons from the central nucleus of the amygdala (GABA) project to glutamatergic neurons in the parafascicular nucleus (Glu). These Glu neurons relay directly to neurons in the second somatosensory cortex (S2), a well-known area involved in pain signal processing. Enhanced inhibition of the GABA→Glu→S2 pathway is found in mice exhibiting CP symptoms. Reversing this pathway using chemogenetic or optogenetic approaches alleviates CP symptoms. Together, the current study demonstrates the putative importance of the GABA→Glu→S2 pathway in controlling at least some aspects of CP.
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