Animal Studies

Neuropathic pain affects up to 10% of the total population and no specific target is ideal for therapeutic need. The sodium leak channel (NALCN), a non-selective cation channel, mediates the background Na leak conductance and controls neuronal excitability and rhythmic behaviors. Here, we show that increases of NALCN expression and function in dorsal root ganglion (DRG) and dorsal spinal cord contribute to chronic constriction injury (CCI)-induced neuropathic pain in rodents. NALCN current and neuronal excitability in acutely isolated DRG neurons and spinal cord slices of rats were increased after CCI which were decreased to normal levels by NALCN-siRNA. Accordingly, pain-related symptoms were significantly alleviated by NALCN-siRNA-mediated NALCN knockdown and completely prevented by NALCN-shRNA-mediated NALCN knockdown in rats or by conditional NALCN knockout in mice. Our results indicate that increases in NALCN expression and function contribute to CCI-induced neuronal sensitization; therefore, NALCN may be a novel molecular target for control of neuropathic pain.

Diabetic neuropathic pain (DNP) is a common complication of diabetes characterized by persistent pain. Emerging evidence links astrocytes to mechanical nociceptive processing, and the motor cortex (MCx) is a cerebral cortex region that is known to play a key role in pain regulation. However, the association between MCx astrocytes and DNP pathogenesis remains largely unexplored. Here, we studied this association using designer receptors exclusively activated by designer drugs (DREADDs) to specifically manipulate MCx astrocytes. We proved that the selective inhibition of MCx astrocytes reduced DNP in streptozocin (STZ)-induced DNP models and discovered a potential mechanism by which astrocytes release cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β), to increase neuronal activation in the MCx, thereby regulating pain. Together, these results demonstrate a pivotal role for MCx astrocytes in DNP pathogenesis and provide new insight into DNP treatment strategies.Astrocytes are critical for maintaining CNS homeostasis. In recent years, astrocytes have been demonstrated to play roles in pain signaling modulation and neuropathic pain maintenance, with studies showing that they inhibit pain transmission at the spinal level. This work suggests that astrocytes also modulate pain at the supraspinal level. Indeed, we show that chemogenetically manipulated MCx astrocytes affect the mechanical withdrawal thresholds of rats and elucidate a potential mechanism by which astrocytes release inflammatory mediators to increase neuronal activation in the MCx, thereby regulating pain. Together, our data support that inhibition of astrocytes in the MCx region might have broad prospects for diabetic neuropathic pain treatment.

Obesity is one of the largest modifiable risk factors for the development of musculoskeletal diseases, including intervertebral disc (IVD) degeneration and back pain. Despite the clinical association, no studies have directly assessed whether diet-induced obesity accelerates IVD degeneration, back pain, or investigated the biological mediators underlying this association. In this study, we examine the effects of chronic consumption of a high-fat or high-fat/high-sugar (western) diet on the IVD, knee joint, and pain-associated outcomes.

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been any direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1β production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

Spinal cord injury (SCI) is one of the main causes leading to neuropathic pain. Here, we aim to explore the molecular mechanism and function of lncRNA PVT1 in neuropathic pain induced by SCI. The expression of lncRNA PVT1, microRNA (miR) - 186-5p was measured via quantitative reverse transcription PCR (qRT-PCR), and the activation of astrocytes (labeled by GFAP) was detected by immunohistochemistry. Western blot was conducted to detect the expression of chemokine ligand 13 (CXCL13), chemokine receptor 5 (CXCR5), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) in spinal cord injury lesions. The levels of inflammatory cytokines (including IL-1β and IL-6) and MDA in tissues were examined via Enzyme-linked immunosorbent assay (ELISA). In vitro experiments were also conducted in primary cultured astrocyte to explore the response of astrocyte to lipopolysaccharide (LPS). What's more, the PVT1-miR-186-5p interaction was verified via the dual luciferase activity assay and RNA immunoprecipitation (RIP) assay. The results demonstrated that the levels of PVT1, CXCL13 and CXCR5 were upregulated, while miR-186-5p were decreased in SCI rats' spinal cord and LPS-mediated astrocytes. In the SCI model, PVT1 depletion significantly alleviated neuropathic pain, astrocytic activation and reduced the expression of neuroinflammatory factors and proteins. The relevant mechanism studies confirmed that PVT1 is a competitive endogenous RNA (ceRNA) of miR-186-5p, targets and inhibits its expression and promotes the expression of CXCL13/CXCR5, while miR-186-5p targets CXCL13. In conclusion, inhibition of lncRNA PVT1 alleviates neuropathic pain in SCI rats by upregulating miR-186-5p and down-regulating CXCL13/CXCR5. The PVT1/miR-186-5p/CXCL13/CXCR5 axis can be used as a new therapeutic target for neuropathic pain.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

The nuclear factor erythroid 2-related factor (Nrf2) signaling pathway has recently emerged as a novel therapeutic target in treating various diseases. Therefore, the present study aimed to assess the protective role of the Nrf2 activator, dimethyl fumarate (DMF) in the complete Freund's adjuvant (CFA)- induced arthritis model. DMF (25, 50, and 100 mg/kg) and dexamethasone (2 mg/kg) were orally administered for 14 days. Pain-related tests, paw volume, and arthritic scores were measured weekly. Serum TNF-α, IL-1β, cyclic citrullinated peptide (CCP), C-reactive protein (CRP), and rheumatoid factor (RF) levels were estimated. Nitrite, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), catalase (CAT), and myeloperoxidase (MPO) levels were also evaluated. NF-κB, Nrf2, HO-1, and COX-2 levels were estimated in the joint tissue. DMF treatment exerted anti-arthritic activity by enhancing the nociceptive threshold, improving arthritis scores, and reducing paw edema. Also, DMF suppressed changes in oxidative stress markers and inflammatory mediators and enhanced Nrf2 and HO-1 levels in CFA-injected rats. These findings indicate that the anti-arthritic activity of DMF may be mediated by the activation of the Nrf2/HO-1 pathway, which reduced oxidative damage and inflammation.

Naturally occurring spine osteoarthritis is clinically associated with the manifestation of chronic inflammatory muscle (myofascial) disease. The purpose of this study was to investigate the causal association between experimentally induced spine osteoarthritis and neurogenic inflammatory responses within neurosegmentally linked myotomes. Wistar Kyoto rats were randomly assigned to spine facet compression surgery (L4-L6) or sham surgery. Animals exposed to facet compression surgery demonstrated radiographic signs of facet-osteoarthritis (L4-L6 spinal levels) and sensory changes (allodynia, thermal hyperalgesia) at 7, 14 and 21 days post-intervention, consistent with the induction of central sensitization; no radiologic or sensory changes were observed after sham surgery. Increased levels of proinflammatory biomarkers including substance P (SP), calcitonin gene related peptide (CGRP), protease-activated receptor-2 (PAR2) and calcium/calmodulin dependent protein kinase II (CaMKII) were observed post-surgery within neurosegmentally-linked rectus femoris (L2-L5) muscle when compared to the non-segmentally linked biceps brachii (C4-C7) muscle; no differences were observed between muscles in the sham surgery group. These findings offer novel insight into the potential role of spine osteoarthritis and neurogenic inflammatory mechanisms in the pathophysiology of chronic inflammatory muscle (myofascial) disease.