Comparison of Nocifensive Behavior in NaV1.7-, NaV1.8-, and NaV1.9-Channelrhodopsin-2 Mice by Selective Optogenetic Activation of Targeted Sodium Channel Subtype-Expressing Afferents.

Voltage-gated sodium channels, including NaV1.7, NaV1.8, and NaV1.9, play important roles in pain transmission and chronic pain development. However, the specific mechanisms of their action remain unclear, highlighting the need for in vivo stimulation studies of these channels. Optogenetics, a novel technique for targeting the activation or inhibition of specific neural circuits using light, offers a promising solution. In our previous study, we used optogenetics to selectively excite NaV1.7-expressing neurons in the dorsal root ganglion of mice to induce nocifensive behavior. Here, we further characterize the impact of nocifensive behavior by activation of NaV1.7, NaV1.8, or NaV1.9-expressing neurons. Using CRISPR/Cas9-mediated homologous recombination, NaV1.7-iCre, NaV1.8-iCre, or NaV1.9-iCre mice expressing iCre recombinase under the control of the endogenous NaV1.7, NaV1.8, or NaV1.9 gene promoter were produced. These mice were then bred with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7-ChR2, NaV1.8-ChR2, or NaV1.9-ChR2 mice. Blue light exposure triggered paw withdrawal in all mice, with the strongest response in NaV1.8-ChR2 mice. These light sensitivity differences observed across NaV1.x-ChR2 mice may be dependent on ChR2 expression or reflect the inherent disparities in their pain transmission roles. In conclusion, we have generated noninvasive pain models, with optically activated peripheral nociceptors. We believe that studies using optogenetics will further elucidate the role of sodium channel subtypes in pain transmission.

Interplay of Nav1.8 and Nav1.7 channels drives neuronal hyperexcitability in neuropathic pain.

While voltage-gated sodium channels Nav1.7 and Nav1.8 both contribute to electrogenesis in dorsal root ganglion (DRG) neurons, details of their interactions have remained unexplored. Here, we studied the functional contribution of Nav1.8 in DRG neurons using a dynamic clamp to express Nav1.7L848H, a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, and demonstrate a profound functional interaction of Nav1.8 with Nav1.7 close to the threshold for AP generation. At the voltage threshold of -21.9 mV, we observed that Nav1.8 channel open-probability exceeded Nav1.7WT channel open-probability ninefold. Using a kinetic model of Nav1.8, we showed that a reduction of Nav1.8 current by even 25-50% increases rheobase and reduces firing probability in small DRG neurons expressing Nav1.7L848H. Nav1.8 subtraction also reduces the amplitudes of subthreshold membrane potential oscillations in these cells. Our results show that within DRG neurons that express peripheral sodium channel Nav1.7, the Nav1.8 channel amplifies excitability at a broad range of membrane voltages with a predominant effect close to the AP voltage threshold, while Nav1.7 plays a major role at voltages closer to resting membrane potential. Our data show that dynamic-clamp reduction of Nav1.8 conductance by 25-50% can reverse hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes pain in humans and suggests, more generally, that full inhibition of Nav1.8 may not be required for relief of pain due to DRG neuron hyperexcitability.

Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-ß (TGF-ß) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.

The single-cell transcriptomic atlas iPain identifies senescence of nociceptors as a therapeutical target for chronic pain treatment.

Chronic pain remains a significant medical challenge with complex underlying mechanisms, and an urgent need for new treatments. Our research built and utilized the iPain single-cell atlas to study chronic pain progression in dorsal root and trigeminal ganglia. We discovered that senescence of a small subset of pain-sensing neurons may be a driver of chronic pain. This mechanism was observed in animal models after nerve injury and in human patients diagnosed with chronic pain or diabetic painful neuropathy. Notably, treatment with senolytics, drugs that remove senescent cells, reversed pain symptoms in mice post-injury. These findings highlight the role of cellular senescence in chronic pain development, demonstrate the therapeutic potential of senolytic treatments, and underscore the value of the iPain atlas for future pain research.

Regulatory role of electroacupuncture on satellite glial cell activity in the colon and dorsal root ganglion of rats with irritable bowel syndrome.

OBJECTIVE: To investigate the role of satellite glial cells in irritable bowel syndrome (IBS) and the effect of electroacupuncture (EA) at the Tianshu (ST25) and Shangjuxu (ST37) combination. METHODS: A model for visceral hypersensitivity in IBS was induced through colorectal distension (CRD) stimulation. Clean-grade male Sprague-Dawley (SD) rats were randomly divided into four groups: a normal group (NG), a model group (MG), an electroacupuncture group (EA), and a glial cell inhibitor group (FCA). Bilateral EA (2/100 Hz, 1 mA, 30 min) was administered at the Tianshu (ST25) and Shangjuxu (ST37) in week 6. Abdominal withdrawal reflex (AWR) scores were used to assess the behavioral response associated with visceral hyperalgesia, while hematoxylin-eosin staining was employed to evaluate pathological changes in the colon. The protein and mRNA levels of glial fibrillary acidic protein (GFAP) in the colon and colon-related dorsal root ganglion (DRG) were analyzed using immun-ofluorescence, immun-ohistochemistry, Western blotting, real-time polymerase chain reaction. The impact of EA on electrophysiological properties of colon-related DRG neurons was observed through whole-cell patch clamp analysis. RESULTS: EA significantly reduced the visceral pain behavior scores in rats with IBS in response to graded (20, 40, 60, 80 mm Hg) CRD stimulation. Additionally, EA downregulated the protein and mRNA expression levels of GFAP in the colon and colon-related DRG of rats with IBS. EA also regulated the resting membrane potential, rheobase and action potential of colon-related DRG neurons in rats with IBS. CONCLUSIONS: EA can regulate the excitatory properties of colon-related DRG neurons by downregulating the protein and mRNA expression of GFAP in the colon and colon-related DRG, indicating a potential neurobiological mechanism by which EA relieves visceral hypersensitivity in rats with IBS.

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OBJECTIVES: To explore the relationship between sensitization points of the body surface and the expression of pituitary adenylate cyclase activating polypeptides (PACAP) in myocardial ischemia (MI) mice, so as to reveal the underlying mechanisms of acupoint sensitization from the perspective of molecular biology. METHODS: Male C57BL/6J mice were randomly divided into control and model groups (28 mice/group). The MI-induced visceral pain model was established by intraperitoneal injection of isoprenaline (ISO, 160 mg/kg). The mice of the control group received intraperitoneal injection of the same dose of normal saline. Six days after modeling, the Evans blue (EB) dye was injected into the tail vein of mice to observe the distribution and quantity of the plasma extravasated EB points at the body surface. Meanwhile, the mechanical pain threshold (MPT) was measured to evaluate the level of pain sensitivity in the activated area on their body surface and left forelimb and hindlimb, respectively. Hematoxylin-eosin (H.E.) staining was used to evaluate the morphologic and pathological changes of the heart tissue in the two groups. Then, the expressions of PACAP in the thoracic (T)1-T5 dorsal root ganglia (DRGs), spinal cord and skin in the dominant area of body surface were detected by Western blot and immunofluorescence staining, respectively. RESULTS: Compared with the control group, the heart tissue of the model group was hypertrophic and the myocardial tissue showed obvious inflammatory cell infiltration and fibrosis. In addition to these pathologic changes, the number of EB exudation points on the body surface was significantly increased (P<0.01), and was mainly distributed in the innervated region of T1-T5 segments of the spinal cord, and the MPT of these EB exudation points was lower than that of non-exudation points (P<0.01). At the same time, the MPTs of left forelimb and hindlimb were significantly decreased in the model group (P<0.001). More importantly, the level of protein and positive expression of PACAP were significantly higher in the model group than those in the control group, which was observed in the innervated body surface, spinal cord and its DRG neurons of T1-T5 segments (P<0.01, P<0.001, P<0.05). CONCLUSIONS: ISO injection resulted in histological lesions and cardiogenic referred pain on the body surface after the formation of MI in mice. The expression of PACAP in the body surface of the sensitization points, the corresponding T1-T5 segments of spinal cord and DRG neurons were significantly increased, which may partly explain the reason for acupoint sensitization in the animal model of visceral pain.

Thermosensing ability of TRPC5: current knowledge and unsettled questions.

Our understanding of how the mammalian somatosensory system detects noxious cold is still limited. While the role of TRPM8 in signaling mild non-noxious coolness is reasonably understood, the molecular identity of channels transducing painful cold stimuli remains unresolved. TRPC5 was originally described to contribute to moderate cold responses of dorsal root ganglia neurons in vitro, but mice lacking TRPC5 exhibited no change in behavioral responses to cold temperature. The question of why a channel endowed with the ability to be activated by cooling contributes to the cold response only under certain conditions is currently being intensively studied. It seems increasingly likely that the physiological detection of cold temperatures involves multiple different channels and mechanisms that modulate the threshold and intensity of perception. In this review, we aim to outline how TRPC5 may contribute to these mechanisms and what molecular features are important for its role as a cold sensor.

Exogenous Mitochondrial Transplantation Facilitates the Recovery of Autologous Nerve Grafting in Repairing Nerve Defects.

Autologous nerve transplantation (ANT) remains the gold standard for treating nerve defects. However, its efficacy in nerve repair still requires improvement. Mitochondrial dysfunction resulting from nerve injury may be a significant factor limiting nerve function restoration. This study investigated the impact of supplementing exogenous mitochondria (EM) in ANT and explored its effect on the efficacy of ANT in nerve repair. SD rats were used to prepare a model of a 10 mm sciatic nerve defect repaired by ANT (Auto group) and a model of ANT supplemented with EM (Mito group). At 12 weeks post-operation, functional, neurophysiological, and histological evaluations of the target organ revealed that the Mito group exhibited significantly better outcomes compared with the Auto group, with statistically significant differences (P < 0.05). In vitro experiments demonstrated that EM could be endocytosed by Schwann cells (SCs) and dorsal root ganglion neurons (DRGs) when co-cultured. After endocytosis by SCs, immunofluorescence staining of autophagy marker LC3II and mitochondrial marker Tomm20, as well as adenoviral fluorescence labeling of lysosomes and mitochondria, revealed that EM could promote autophagy in SCs. CCK8 and EDU assays also indicated that EM significantly promoted SCs proliferation and viability. After endocytosis by DRGs, EM could accelerate axonal growth rate. A sciatic nerve defect repair model prepared using Thy1-YFP-16 mice also revealed that EM could accelerate axonal growth in vivo, with statistically significant results (P < 0.05). This study suggests that EM enhances autophagy in SCs, promotes SCs proliferation and viability, and increases the axonal growth rate, thereby improving the efficacy of ANT. This research provides a novel therapeutic strategy for enhancing the efficacy of ANT in nerve repair.

Transcriptome analysis of rheumatoid arthritis uncovers genes linked to inflammation-induced pain.

Autoimmune diseases such as rheumatoid arthritis (RA) can promote states of chronic inflammation with accompanying tissue destruction and pain. RA can cause inflammatory synovitis in peripheral joints, particularly within the hands and feet, but can also sometimes trigger temporomandibular joint (TMJ) arthralgia. To better understand the effects of ongoing inflammation-induced pain signaling, dorsal root ganglia (DRGs) were acquired from individuals with RA for transcriptomic study. We conducted RNA sequencing from the L5 DRGs because it contains the soma of the sensory neurons that innervate the affected joints in the foot. DRGs from 5 RA patients were compared with 9 non-arthritic controls. RNA-seq of L5 DRGs identified 128 differentially expressed genes (DEGs) that were dysregulated in the RA subjects as compared to the non-arthritic controls. The DRG resides outside the blood brain barrier and, as such, our initial transcriptome analysis detected signs of an autoimmune disorder including the upregulated expression of immunoglobulins and other immunologically related genes within the DRGs of the RA donors. Additionally, we saw the upregulation in genes implicated in neurogenesis that could promote pain hypersensitivity. Overall, our DRG analysis suggests that there are upregulated inflammatory and pain signaling pathways that can contribute to chronic pain in RA.

Comprehensive mapping of sensory and sympathetic innervation of the developing kidney.

The kidneys act as finely tuned sensors to maintain physiological homeostasis. Both sympathetic and sensory nerves modulate kidney function through precise neural control. However, how the kidneys are innervated during development to support function remains elusive. Using light-sheet and confocal microscopy, we generated anatomical maps of kidney innervation across development. Kidney innervation commences on embryonic day 13.5 (E13.5) as network growth aligns with arterial differentiation. Fibers are synapsin I+, highlighting ongoing axonogenesis and potential signaling crosstalk. By E17.5, axons associate with nephrons, and the network continues to expand postnatally. CGRP+, substance P+, TRPV1+, and PIEZO2+ sensory fibers and TH+ sympathetic fibers innervate the developing kidney. TH+ and PIEZO2+ axons similarly innervate the human kidney, following the arterial tree to reach targets. Retrograde tracing revealed the primary dorsal root ganglia, T10-L2, from which sensory neurons project to the kidneys. Together, our findings elucidate the temporality and neuronal diversity of kidney innervation.

Nuclear Magnetic Resonance Treatment Induces ßNGF Release from Schwann Cells and Enhances the Neurite Growth of Dorsal Root Ganglion Neurons In Vitro.

Peripheral nerve regeneration depends on close interaction between neurons and Schwann cells (SCs). After nerve injury, SCs produce growth factors and cytokines that are crucial for axon re-growth. Previous studies revealed the supernatant of SCs exposed to nuclear magnetic resonance therapy (NMRT) treatment to increase survival and neurite formation of rat dorsal root ganglion (DRG) neurons in vitro. The aim of this study was to identify factors involved in transferring the observed NMRT-induced effects to SCs and consequently to DRG neurons. Conditioned media of NMRT-treated (CM NMRT) and untreated SCs (CM CTRL) were tested by beta-nerve growth factor (ßNGF) ELISA and multiplex cytokine panels to profile secreted factors. The expression of nociceptive transient receptor potential vanilloid 1 (TRPV1) channels was assessed and the intracellular calcium response in DRG neurons to high-potassium solution, capsaicin or adenosine triphosphate was measured mimicking noxious stimuli. NMRT induced the secretion of ßNGF and pro-regenerative-signaling factors. Blocking antibody experiments confirmed ßNGF as the main factor responsible for neurotrophic/neuritogenic effects of CM NMRT. The TRPV1 expression or sensitivity to specific stimuli was not altered, whereas the viability of cultured DRG neurons was increased. Positive effects of CM NMRT supernatant on DRG neurons are primarily mediated by increased ßNGF levels.

Piezo1 mediates mechanical signals in TRPV1-positive nociceptors in mice.

AIM: This investigation addresses Piezo1's expression and mechanistic role in dorsal root ganglion (DRG) neurons and delineates its participation in mechanical and inflammatory pain modulation. METHODS: We analyzed Piezo1's expression patterns in DRG neurons and utilized Piezo1-specific shRNA to modulate its activity. Electrophysiological assessments of mechanically activated (MA) currents in DRG neurons and behavioral analyses in mouse models of inflammatory pain were conducted to elucidate Piezo1's functional implications. Additionally, we investigated the excitability of TRPV1-expressing DRG neurons, particularly under inflammatory conditions. RESULTS: Piezo1 was preferentially expressed in DRG neurons co-expressing the TRPV1 nociceptor marker. Knockdown of Piezo1 attenuated intermediately adapting MA currents and lessened tactile pain hypersensitivity in models of inflammatory pain. Additionally, silencing Piezo1 modified the excitability of TRPV1-expressing neurons under inflammatory stress. CONCLUSION: Piezo1 emerges as a key mediator in the transmission of mechanical and inflammatory pain, indicating its potential as a novel target for pain management therapies. Our finding not only advances the understanding of nociceptive signaling but also emphasizes the therapeutic potential of modulating Piezo1 in the treatment of pain.

Mitochondrial dysfunction and disulfidptosis co-regulate neuronal cell in neuropathic pain based on bioinformatics analysis.

Neuropathic pain (NP) affects approximately 6.9-10% of the world's population and necessitates the development of novel treatments. Mitochondria are essential in the regulation of cell death. Neuroimmune mechanisms are implicated in various forms of cell death associated with NP. However, the specific involvement of mitochondrial dysfunction and disulfidptosis in NP remains uncertain. Further research is required to gain a better understanding of their combined contribution. Our comprehensive study employs a variety of bioinformatic analysis methods, including differential gene analysis, weighted gene co-expression network analysis, machine learning, functional enrichment analysis, immune infiltration, sub-cluster analysis, single-cell dimensionality reduction and cell-cell communication to gain insight into the molecular mechanisms behind these processes. Our study rationally defines a list of key gene sets for mitochondrial dysfunction and disulfidptosis. 6 hub mitochondrial genes and 3 disulfidptosis-related genes (DRGs) were found to be associated with NP. The key genes were predominantly expressed in neurons and were lowly expressed in the NP group compared to SHAM. In addition, our macrophages used the APP (Amyloid precursor protein)-CD74 (MHC class II invariant chain) pathway to interact with neurons. These results suggest that NP is interconnected with the mechanistic processes of mitochondrial dysfunction and disulfidptosis, which may contribute to clinically targeted therapies.

Inhibition of KIF5b-mediated Nav1.8 transport by ropivacaine contributes to axonal regeneration following sciatic nerve injury in rats.

Peripheral nerve injury (PNI), typically caused by traumatic accidents or medical events, is currently one of the most common diseases that leads to limb disability. After PNI, tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 is upregulated at the lesion site. Our earlier study suggested that ropivacaine promotes axon regrowth by regulating Nav1.8-mediated macrophage signaling. Nevertheless, the mechanism of ropivacaine in regulation of Nav1.8 expression remains incompletely understood. Kinesin family 5b (KIF5b) was reported to mediate the Nav1.8 axonal transport from dorsal root ganglia (DRGs) to lesion site. Herein, we investigated whether ropivacaine promotes axon regeneration through inhibition of KIF5b-mediated Nav1.8 transport. Reduced levels of KIF5b and Nav1.8 in DRGs coincide with their increase at the lesion site. Nav1.8 mRNA was significantly increased at the lesion site but not in DRGs. Surprisingly, ropivacaine reversed the alterations of Nav1.8 and KIF5b protein expression without affecting Nav1.8 mRNA level. Due to KIF5b overexpression in DRGs, Nav1.8 protein level was significantly decreased in DRGs and increased at the lesion site. We also found KIF5b overexpression significantly impaired behavioral functions, reduced the recovery index of compound muscle action potential (CMAP) amplitude, inhibited axonal regrowth, slowed M1 macrophage infiltration and shift to M2 phenotype, and delayed myelin debris clearance. Notably, all aforementioned results caused by KIF5b overexpression were alleviated by ropivacaine. Furthermore, we demonstrated that inhibition of Nav1.8 transport by A-803467 produced mitigating effects on the impairment of regenerative capacity induced by KIF5b overexpression similar to ropivacaine. These results suggest that ropivacaine promotes axonal regeneration at least partially by inhibiting KIF5b-mediated Nav1.8 forward transport.

Mu-Opioid Receptor (MOR) Dependence of Pain in Chemotherapy-Induced Peripheral Neuropathy.

We recently demonstrated that transient attenuation of Toll-like receptor 4 (TLR4) in dorsal root ganglion (DRG) neurons, can both prevent and reverse pain associated with chemotherapy-induced peripheral neuropathy (CIPN), a severe side effect of cancer chemotherapy, for which treatment options are limited. Given the reduced efficacy of opioid analgesics to treat neuropathic, compared with inflammatory pain, the cross talk between nociceptor TLR4 and mu-opioid receptors (MORs), and that MOR and TLR4 agonists induce hyperalgesic priming (priming), which also occurs in CIPN, we determined, using male rats, whether (1) antisense knockdown of nociceptor MOR attenuates CIPN, (2) and attenuates the priming associated with CIPN, and (3) CIPN also produces opioid-induced hyperalgesia (OIH). We found that intrathecal MOR antisense prevents and reverses hyperalgesia induced by oxaliplatin and paclitaxel, two common clinical chemotherapy agents. Oxaliplatin-induced priming was also markedly attenuated by MOR antisense. Additionally, intradermal morphine, at a dose that does not affect nociceptive threshold in controls, exacerbates mechanical hyperalgesia (OIH) in rats with CIPN, suggesting the presence of OIH. This OIH associated with CIPN is inhibited by interventions that reverse Type II priming [the combination of an inhibitor of Src and mitogen-activated protein kinase (MAPK)], an MOR antagonist, as well as a TLR4 antagonist. Our findings support a role of nociceptor MOR in oxaliplatin-induced pain and priming. We propose that priming and OIH are central to the symptom burden in CIPN, contributing to its chronicity and the limited efficacy of opioid analgesics to treat neuropathic pain.

Functional Role of Piezo1 in the Human Eosinophil Cell Line AML14.3D10: Implications for the Immune and Sensory Nervous Systems.

Mechanosensitive ion channels, particularly Piezo channels, are widely expressed in various tissues. However, their role in immune cells remains underexplored. Therefore, this study aimed to investigate the functional role of Piezo1 in the human eosinophil cell line AML14.3D10. We detected Piezo1 mRNA expression, but not Piezo2 expression, in these cells, confirming the presence of the Piezo1 protein. Activation of Piezo1 with Yoda1, its specific agonist, resulted in a significant calcium influx, which was inhibited by the Piezo1-specific inhibitor Dooku1, as well as other nonspecific inhibitors (Ruthenium Red, Gd3+, and GsMTx-4). Further analysis revealed that Piezo1 activation modulated the expression and secretion of both pro-inflammatory and anti-inflammatory cytokines in AML14.3D10 cells. Notably, supernatants from Piezo1-activated AML14.3D10 cells enhanced capsaicin and ATP-induced calcium responses in the dorsal root ganglion neurons of mice. These findings elucidate the physiological role of Piezo1 in AML14.3D10 cells and suggest that factors secreted by these cells can modulate the activity of transient receptor potential 1 (TRPV1) and purinergic receptors, which are associated with pain and itch signaling. The results of this study significantly advance our understanding of the function of Piezo1 channels in the immune and sensory nervous systems.

RNA-binding protein SYNCRIP contributes to neuropathic pain through stabilising CCR2 expression in primary sensory neurones.

BACKGROUND: Nerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to the genesis of neuropathic pain. SYNCRIP, an RNA-binding protein, is critical for the stabilisation of gene expression. Whether SYNCRIP participates in nerve injury-induced alterations in DRG gene expression and nociceptive hypersensitivity is unknown. METHODS: The expression and distribution of SYNCRIP in mouse DRG after chronic constriction injury (CCI) of the unilateral sciatic nerve were assessed. Effect of microinjection of Syncrip small interfering RNA into the ipsilateral L3 and L4 DRGs on the CCI-induced upregulation of chemokine (C-C motif) receptor 2 (CCR2) and nociceptive hypersensitivity were examined. Additionally, effects of microinjection of adeno-associated virus 5 expressing full length Syncrip mRNA (AAV5-Syncrip) on basal DRG CCR2 expression and nociceptive thresholds were observed. RESULTS: SYNCRIP is expressed predominantly in DRG neurones, where it co-exists with CCR2. Levels of Syncrip mRNA and SYNCRIP protein in injured DRG increased time-dependently on days 3-14 after CCI. Blocking this increase through microinjection of Syncrip small interfering RNA into injured DRG attenuated CCI-induced upregulation of DRG CCR2 and development and maintenance of nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of AAV5-Syncrip elevated CCR2 expression in microinjected DRGs, enhanced the responses to mechanical, heat, and cold stimuli, and induced ongoing pain in naive mice. Mechanistically, SYNCRIP bound to 3-UTR of Ccr2 mRNA and stabilised its expression in DRG neurones. CONCLUSIONS: SYNCRIP contributes to the induction and maintenance of neuropathic pain likely through stabilising expression of CCR2 in injured DRG. SYNCRIP may be a potential target for treating this disorder.

Antiallodynic and antihyperalgesic effects of decursin associated with correcting mitochondrial dysfunction and oxidative stress in type 1 diabetic mice.

A substantial proportion of diabetic patients suffer a debilitating and persistent pain state, known as peripheral painful neuropathy that necessitates improved therapy or antidote. Decursin, a major active ingredient from Angelica gigas Nakai, has been reported to possess antidepressant activity in preclinical studies. As antidepressants have been typically used as standard agents against persistent neuropathic pain, this study aimed to probe the effect of decursin on neuropathic pain associated with streptozotocin-induced type 1 diabetes in male C57BL6J mice. The Hargreaves test and the von Frey test were used to assess pain-like behaviors, shown as heat hyperalgesia and mechanical allodynia respectively. Chronic treatment of diabetic mice with decursin not only ameliorated the established symptoms of heat hyperalgesia and mechanical allodynia, but also arrested the development of these pain states given preemptively at low doses. Although decursin treatment hardly impacted on metabolic disturbance in diabetic mice, it ameliorated exacerbated oxidative stress in pain-associated tissues, improved mitochondrial bioenergetics in dorsal root ganglion neurons, and restored nerve conduction velocity and blood flow in sciatic nerves. Notably, the analgesic actions of decursin were modified by pharmacologically manipulating redox status and mitochondrial bioenergetics. These findings unveil the analgesic activity of decursin, an effect that is causally associated with its bioenergetics-enhancing and antioxidant effects, in mice with type 1 diabetes.

Stretch triggers microtubule stabilization and MARCKS-dependent membrane incorporation in the shaft of embryonic axons.

Neurons have a unique polarized nature that must adapt to environmental changes throughout their lifespan. During embryonic development, axon elongation is led by the growth cone,1 culminating in the formation of a presynaptic terminal. After synapses are formed, axons elongate in a growth cone-independent manner to accompany body growth while maintaining their ultrastructure and function.2,3,4,5,6 To further understand mechanical strains on the axon shaft, we developed a computer-controlled stretchable microfluidic platform compatible with multi-omics and live imaging. Our data show that sensory embryonic dorsal root ganglia (DRGs) neurons have high plasticity, with axon shaft microtubules decreasing polymerization rates, aligning with the direction of tension, and undergoing stabilization. Moreover, in embryonic DRGs, stretch triggers yes-associated protein (YAP) nuclear translocation, supporting its participation in the regulatory network that enables tension-driven axon growth. Other than cytoskeleton remodeling, stretch prompted MARCKS-dependent formation of plasmalemmal precursor vesicles (PPVs), resulting in new membrane incorporation throughout the axon shaft. In contrast, adolescent DRGs showed a less robust adaptation, with axonal microtubules being less responsive to stretch. Also, while adolescent DRGs were still amenable to strain-induced PPV formation at higher stretch rates, new membrane incorporation in the axon shaft failed to occur. In summary, we developed a new resource to study the biology of axon stretch growth. By unraveling cytoskeleton adaptation and membrane remodeling in the axon shaft of stretched neurons, we are moving forward in understanding axon growth.

Electroacupuncture inhibits TLR4/NF-κB signaling in the dorsal root ganglion of rats with spared nerve injury.

OBJECTIVE: Neuropathic pain can be provoked by high mobility group box 1 (HMGB1) activation of toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling in the dorsal root ganglion (DRG). Electroacupuncture (EA) has been reported to effectively alleviate neuropathic pain with few side effects, but its precise mechanism of action remains unknown. The aim of this study was to explore whether 2 Hz EA stimulation suppresses TLR4/NF-κB signaling in the DRG following spared nerve injury (SNI) in a rat model. METHODS: In this experiment, SNI rats were given 2 Hz EA once every other day for a total of 21 days. Paw withdrawal threshold (PWT) was measured to assess SNI-induced mechanical hypersensitivity, and western blotting and immunofluorescence staining were used to determine the levels of pain-related signaling molecules and pro-inflammatory mediators in the DRG. RESULTS: SNI up-regulated HMGB1, TLR4, myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB p65 protein expression in the DRG. In addition, immunofluorescence staining demonstrated that SNI induced higher levels of TLR4 and MyD88 in the DRG. We also demonstrated co-localization of TLR4 and MyD88 with both calcitonin gene-related peptide (CGRP) and isolectin GS-IB4 in the DRG of SNI rats, respectively. Meanwhile, 2 Hz EA stimulation effectively reversed the elevations of HMGB1, TLR4, MyD88 and NF-κB p65 induced by SNI in the DRG, which was coupled with amelioration of SNI-induced mechanical hypersensitivity. CONCLUSIONS: The results of this study suggested that inhibition of the TLR4/NF-κB signaling pathway in the DRG by 2 Hz EA might be exploited as a therapeutic option for neuropathic pain.