Analgesic Effect of SKI306X on Chronic Postischemic Pain and Spinal Nerve Ligation-Induced Neuropathic Pain in Mice.

Neuropathic pain (NP) results from lesions or diseases affecting the peripheral or central somatosensory system. However, there are currently no drugs that are particularly effective in treating this condition. SKI306X is a blend of purified extracts of three oriental herbs (Clematis mandshurica, Trichosanthes kirilowii, and Prunella vulgaris) commonly used to treat osteoarthritis for their chondroprotective effects. Chronic postischemic pain (CPIP) and spinal nerve ligation (SNL) models were created by binding the upper left ankle of mice with an O-ring for 3 h and ligating the L5 spinal nerve, respectively. Mice with allodynia were injected intraperitoneally with 0.9% normal saline (NS group) or different doses (25, 50, or 100 mg/kg) of SKI306X (SKI groups). We assessed allodynia using von Frey filaments before injection and 30, 60, 90, 120, 180, and 240 min and 24 h after injection to confirm the antiallodynic effect of SKI306X. We also measured glial fibrillary acidic protein (GFAP) levels in the spinal cord and dorsal root ganglia to confirm the change of SKI306X administration. Both models exhibited significant mechanical allodynia. The intraperitoneal injection of SKI306X significantly increased the paw withdrawal threshold in a dose-dependent manner, as the paw withdrawal threshold was significantly increased after SKI306X administration compared with at baseline or after NS administration. GFAP levels in the SKI group decreased significantly (p < 0.05). Intraperitoneal administration of SKI306X dose-dependently attenuated mechanical allodynia and decreased GFAP levels, suggesting that GFAP is involved in the antiallodynic effect of SKI306X in mice with CPIP and SNL-induced NP.

Spinal nerve ligation: An experimental model to study neuropathic pain in rats and mice.

Neuropathic pain, defined as the most terrible of all tortures, which a nerve wound may inflict, is a common chronic painful condition caused by gradual damage or dysfunction of the somatosensory nervous system. As with many chronic diseases, neuropathic pain has a profound economic and emotional impact worldwide and represents a major public health issue from a treatment standpoint. This condition involves multiple sensory symptoms including impaired transmission and perception of noxious stimuli, burning, shooting, spontaneous pain, mechanical or thermal allodynia and hyperalgesia. Current pharmacological options for the treatment of neuropathic pain are limited, ineffective and have unacceptable side effects. In this framework, a deeper understanding of the pathophysiology and molecular mechanisms associated with neuropathic pain is key to the development of promising new therapeutical approaches. For this purpose, a plethora of experimental models that mimic common clinical features of human neuropathic pain have been characterized in rodents, with the spinal nerve ligation (SNL) model being one of the most widely used. In this chapter, we provide a detailed surgical procedure of the SNL model used to induce neuropathic pain in rats and mice. We further describe the behavioral approaches used for stimulus-evoked and spontaneous pain assessment in rodents. Finally, we demonstrate that our SNL model induces multiple pain behaviors in rats and mice.

Intrathecal gastrodin alleviates allodynia in a rat spinal nerve ligation model through NLRP3 inflammasome inhibition.

BACKGROUND: Gastrodin (GAS), a main bioactive component of the herbal plant, Gastrodia elata Blume, has shown to have beneficial effects on neuroinflammatory diseases such as Alzheimer's disease in animal studies and migraine in clinical studies. Inflammasome is a multimeric protein complex having a core of pattern recognition receptor and has been implicated in the development of neuroinflammatory diseases. Gastrodin has shown to modulate the activation of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. This study investigated the effects of GAS on the intensity of mechanical allodynia and associated changes in NLRP3 inflammasome expression at the spinal level using L5/6 spinal nerve ligation model (SNL) in rats. METHODS: Intrathecal (IT) catheter implantation and SNL were used for drug administration and pain model in male Sprague-Dawley rats. The effect of gastrodin or MCC950 (NLRP3 inflammasome inhibitor) on mechanical allodynia was measured by von Frey test. Changes in NLRP3 inflammasome components and interleukin-1ß (IL-1ß) and cellular expression were examined in the spinal cord and dorsal root ganglion. RESULTS: The expression of NLRP3 inflammasome components was found mostly in the neurons in the spinal cord and dorsal root ganglion. The protein and mRNA levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and IL-1ß were upregulated in SNL animals compared to Sham animals. IT administration of GAS significantly attenuated the expression of NLRP3 inflammasome and the intensity of SNL-induced mechanical allodynia. NLRP3 inflammasome inhibitor, MCC950, also attenuated the intensity of allodynia, but the effect is less strong and shorter than that of GAS. CONCLUSIONS: Expression of NLRP3 inflammasome and IL-1ß is greatly increased and mostly found in the neurons at the spinal level in SNL model, and IT gastrodin exerts a significant anti-allodynic effect in SNL model partly through suppressing the expression of NLRP3 inflammasome.

Ursolic acid molecules dock MAPK1 to modulate gut microbiota diversity to reduce neuropathic pain.

To investigate the efficacy of Ursolic acid in alleviating neuropathic pain in rats with spinal nerve ligation (SNL), the SNL rat model was surgically induced. Different concentrations of Ursolic acid and manipulated target mitogen-activated protein kinase 1 (MAPK1) were administered to the SNL rats. Fecal samples were collected from each group of rats for 16S rDNA analysis to examine the impact of gut microbiota. Molecular docking experiments were conducted to assess the binding energy between Ursolic acid and MAPK1. In vivo studies were carried out to evaluate the expression of inflammatory factors and signaling pathways in spinal cord and colon tissues. Ursolic acid was found to have a beneficial effect on pain reduction in rats by increasing plantar withdrawal latency (PWL) and paw withdrawal threshold (PWT). Comparing the Ursolic acid group with the control group revealed notable differences in the distribution of Staphylococcus, Allobaculum, Clostridium, Blautia, Bifidobacterium, and Prevotella species. Network pharmacology analysis identified MAPK1 and intercellular adhesion molecule-1 (ICAM1) as common targets for Ursolic acid, SNL, and neuropathic pain. Binding sites between Ursolic acid and these targets were identified. Additionally, immunofluorescent staining showed a decrease in GFAP and IBA1 intensity in the spinal cord along with an increase in NeuN following Ursolic acid treatment. Overexpression of MAPK1 in SNL rats led to an increase in inflammatory factors and a decrease in PWL and PWT. Furthermore, MAPK1 counteracted the pain-relieving effects of Ursolic acid in SNL rats. Ursolic acid was found to alleviate neuropathic pain in SNL rats by targeting MAPK1 and influencing gut microbiota homeostasis.

Neuropathy, its Profile and Experimental Nerve Injury Neuropathic Pain Models: A Review.

Neuropathy is a terrible disorder that has a wide range of etiologies. Drug-induced neuropathy, which happens whenever a chemical agent damages the peripheral nerve system, has been linked here to the iatrogenic creation of some drugs. It is potentially permanent and causes sensory impairments and paresthesia that typically affects the hands, feet, and stockings; motor participation is uncommon. It might appear suddenly or over time, and the long-term outlook varies. The wide range of chronic pain conditions experienced by people has been one of the main obstacles to developing new, more effective medications for the treatment of neuropathic pain. Animal models can be used to examine various neuropathic pain etiologies and symptoms. Several models investigate the peripheral processes of neuropathic pain, whereas some even investigate the central mechanisms, such as drug induce models like vincristine, cisplatin, bortezomib, or thalidomide, etc., and surgical models like sciatic nerve chronic constriction injury (CCI), sciatic nerve ligation through spinal nerve ligation (SNL), sciatic nerve damage caused by a laser, SNI (spared nerve injury), etc. The more popular animal models relying on peripheral nerve ligatures are explained. In contrast to chronic sciatic nerve contraction, which results in behavioral symptoms of less reliable stressful neuropathies, (SNI) spared nerve injury generates behavioral irregularities that are more feasible over a longer period. This review summarizes the latest methods models as well as clinical ideas concerning this mechanism. Every strongest current information on neuropathy is discussed, along with several popular laboratory models for causing neuropathy.

Antinociceptive Effect of the Combination of a Novel α4ß2* Agonist with Donepezil in a Chronic Pain Model.

Chronic pain presents a major challenge in contemporary medicine, given the limited effectiveness and numerous adverse effects linked to available treatments. Recognizing the potential of the cholinergic pathway as a therapeutic target, the present work evaluates the antinociceptive activity of a combination of Cris-104, a novel α4ß2* receptor agonist, and donepezil, a central anticholinesterase agent. Isobolographic analysis revealed that equimolar combination was approximately 10 times more potent than theoretically calculated equipotent additive dose. Administration of Cris-104 and donepezil combination (3 µmol/kg) successfully reversed hyperalgesia and mechanical allodynia observed in rats subjected to spinal nerve ligation (SNL). The combination also modulated neuroinflammation by reducing astrocyte activation, evident in the decreased expression of glial fibrillary acidic protein (GFAP) in the spinal cord. The observed synergism in combining a nicotinic receptor agonist with an anticholinesterase agent underscores its potential for treating chronic pain. This alternative therapeutic distinct advantage, including dose reduction and high selectivity for the receptor, contribute to a more favorable profile with minimized adverse effects.

CoREST1 in primary sensory neurons regulates neuropathic pain in male mice.

AIMS: Epigenetic regulation is implicated in the neurogenesis of neuropathic pain. The repressor element 1 (RE1) silencing transcription factor (REST) corepressor (CoREST) proteins function as corepressors in the REST complex and/or LSD1 epigenetic complex. In the current study, we aimed to find the expression profile of CoREST1 in the dorsal root ganglion (DRG) and investigate whether it plays a role in neuropathic pain. MAIN METHODS: The evoked pain behaviors in mice were examined by the von Frey test and thermal test in a spinal nerve ligation (SNL)-induced neuropathic pain mice model. CoREST1 siRNA or virus was administered by DRG microinjection or intrathecal injection. The CoREST1 expression in DRGs was examined by immunofluorescence, quantitative PCR, Western blotting, and co-immunoprecipitation. KEY FINDINGS: CoREST1 was non-selectively expressed in large, medium, and small DRG neurons, and it exclusively colocalized with LSD1. In neuropathic pain models, peripheral nerve injury induced the upregulation of CoREST1 and increased binding of CoREST1 with LSD1 in injured DRGs in male mice. Furthermore, CoREST1 siRNA prevented the development of SNL-induced pain hypersensitivity as well as led to the reduction of established pain hypersensitivity during the maintenance period in SNL mice. Conversely, the overexpression of CoREST1 in DRGs by in vivo transfection of virus-induced pain hypersensitivity in naive mice. SIGNIFICANCE: Our study demonstrated that CoREST1, along with LSD1, was expressed in primary sensory neurons specifically in response to nerve injury, and promoted nociceptive pain hypersensitivity in mice. Thus, CoREST1 might serve as a potential target for treating neuropathic pain.

Neuralized1-Mediated CPEB3 Ubiquitination in the Spinal Dorsal Horn Contributes to the Pathogenesis of Neuropathic Pain in Rats.

Compelling evidence has shown that Neuralized1 (Neurl1) facilitates hippocampal-dependent memory storage by modulating cytoplasmic polyadenylation element-binding protein 3 (CPEB3)-dependent protein synthesis. In the current study, we investigated the role of Neurl1 in the pathogenesis of neuropathic pain and the underlying mechanisms. The neuropathic pain was evaluated by lumbar 5 spinal nerve ligation (SNL) in rats. Immunofluorescence staining, Western blotting, qRT-PCR, and coimmunoprecipitation (Co-IP) were performed to investigate the underlying mechanisms. Our results showed that SNL led to an increase of Neurl1 in the spinal dorsal horn. Spinal microinjection of AAV-EGFP-Neurl1 shRNA alleviated mechanical allodynia; decreased the level of CPEB3 ubiquitination; inhibited the production of GluA1, GluA2, and PSD95; and reduced GluA1-containing AMPA receptors in the membrane of the dorsal horn following SNL. Knockdown of spinal CPEB3 decreased the production of GluA1, GluA2, and PSD95 in the dorsal horn and attenuated abnormal pain after SNL. Overexpression of Neurl1 in the dorsal horn resulted in pain-related hypersensitivity in naïve rats; raised the level of CPEB3 ubiquitination; increased the production of GluA1, GluA2, and PSD95; and augmented GluA1-containing AMPA receptors in the membrane in the dorsal horn. Moreover, spinal Neurl1 overexpression-induced mechanical allodynia in naïve rats was partially reversed by repeated intrathecal injections of CPEB3 siRNA. Collectively, our results suggest that SNL-induced upregulation of Neurl1 through CPEB3 ubiquitination-dependent production of GluA1, GluA2, and PSD95 in the dorsal horn contributes to the pathogenesis of neuropathic pain in rats. Targeting spinal Neurl1 might be a promising therapeutic strategy for the treatment of neuropathic pain.

CYP1B1-derived epoxides modulate the TRPA1 channel in chronic pain.

Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Effect and underlying mechanisms of spirocyclopiperazinium salt compound DXL-A-24 in rats following spinal nerve ligation.

PURPOSE: Neuropathic pain represents a significant public health problem and its effective management remains a challenge. The present study is designed to evaluate the analgesic effect of the spirocyclopiperazinium salt compound DXL-A-24 in spinal nerve ligation (SNL) model, and further to explore the possible molecular mechanisms. METHODS: SNL model was established on rats, and mechanical allodynia and thermal hyperalgesia were estimated with the von Frey and hot plate tests; the expression of CaMKIIα, CREB, JAK2, STAT3 and c-fos was determined by western blotting; the protein level of TNF-α was analysed by ELISA; the mRNA expression of TNF-α and c-fos was detected using qRT-PCR analysis and the receptor blocking test was used for target searching. RESULTS: Administration of DXL-A-24 (1, 0.5, 0.25 mg/kg, i.g.) obviously relieved SNL-induced mechanical allodynia and thermal hyperalgesia in rats (P < 0.01), with the percentage of pain threshold elevation (PTE%) was 103 %, 68 % and 47 %, respectively, in mechanical allodynia; the percentage of maximal possible effect (MPE%) was 56 %, 34 % and 21 %, respectively, in thermal hyperalgesia on day 7 after SNL. Pretreatment with peripheral α7 nicotinic or M4 muscarinic receptor antagonist, the effect of DXL-A-24 was completely blocked (P > 0.05). DXL-A-24 significantly reduced the upregulated pCaMKIIα, pCREB, pJAK2, pSTAT3 and TNF-α protein (P < 0.01), which could be blocked by α7 nicotinic receptor or M4 muscarinic receptor antagonist. In addition, administration of DXL-A-24 attenuated the mRNA and protein expression of c-fos and TNF-α mRNA in DRG of SNL rat. We did not observe significant acute toxicity and chronic hepatorenal impairment at effective dose and high dose. CONCLUSIONS: We report firstly that administration of DXL-A-24 displays obvious antineuropathic pain effects in SNL rats. The underlying mechanism may involve the reduction of the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and the suppression of TNF-α and c-fos expression, which may be mediated by activating peripheral α7 nicotinic and M4 muscarinic receptors. This study may provide a new perspective for developing new antineuralgic drug.

Bestrophin-1 Participates in Neuropathic Pain Induced by Spinal Nerve Transection but not Spinal Nerve Ligation.

Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCCinh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCCinh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCCinh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCCinh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL. PERSPECTIVE: SNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.

CYP1B1-derived epoxides modulate the TRPA1 channel in chronic pain

Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Radiocaine: An Imaging Marker of Neuropathic Injury.

Voltage-gated sodium channels (Navs) play a crucial electrical signaling role in neurons. Nav-isoforms present in peripheral sensory neurons and dorsal root ganglia of the spinal cord are critically involved in pain perception and transmission. While these isoforms, particularly Nav1.7, are implicated in neuropathic pain disorders, changes in the functional state and expression levels of these channels have not been extensively studied in vivo. Radiocaine, a fluorine-18 radiotracer based on the local anesthetic lidocaine, a non-selective Nav blocker, has previously been used for cardiac Nav1.5 imaging using positron-emission tomography (PET). In the present study, we used Radiocaine to visualize changes in neuronal Nav expression after neuropathic injury. In rats that underwent unilateral spinal nerve ligation, PET/MR imaging demonstrated significantly higher uptake of Radiocaine into the injured sciatic nerve, as compared to the uninjured sciatic nerve, for up to 32 days post-surgery. Radiocaine, due to its high translational potential, may serve as a novel diagnostic tool for neuropathic pain conditions using PET imaging.

NS5806 inhibits ERK activation to attenuate pain induced by peripheral nerve injury.

Neuropathic pain is a serious health problem, but optimal drug treatments remain lacking. It has been known that the compound NS5806 is a Kv4.3 activator, which increases Kv4.3-mediated K+ current to reduce neuronal excitability. In this study, we investigated the molecular and cellular mechanisms underlying the analgesic effect of NS5806 in neuropathic pain induced by peripheral nerve injury. Using lumbar (L)5/L6 spinal nerve ligation (SNL) in rats, we found that, without changing the basal nociception, the analgesic effect of NS5806 (220 µg/kg) peaked at 4 h and lasted for 8 h after intraperitoneal injection. Multiple doses of NS5806 reduced not only SNL-upregulated proinflammatory mediators in the DRG and spinal cord on day 1 and day 4 after L5/L6 SNL, but also SNL-evoked expansion of DRG macrophages and spinal microglia on day 4. Furthermore, at 10 min after L5 SNL, NS5806 pretreatment for 4 h suppressed SNL-induced phosphorylated extracellular signal-regulated kinase (pERK) in both Kv4.3+ and Kv4.3- neurons in the dorsal root ganglion (DRG) and superficial spinal dorsal horn, indicating that the action of NS5806 is not restricted to Kv4.3+ neurons. In vitro kinase activity assays revealed that NS5806 weakly inhibited ERK2, MEK1, MEK2, and c-Raf in the ERK pathway. Since NS5806 and the ERK pathway inhibitors have similar antinociceptive characteristics, this study suggests that NS5806 also acts as an ERK pathway inhibitor to attenuate neuropathic pain.

Development of a PET radioligand for α2δ-1 subunit of calcium channels for imaging neuropathic pain.

Neuropathic pain affects 7-10% of the adult population. Being able to accurately monitor biological changes underlying neuropathic pain will improve our understanding of neuropathic pain mechanisms and facilitate the development of novel therapeutics. Positron emission tomography (PET) is a noninvasive molecular imaging technique that can provide quantitative information of biochemical changes at the whole-body level by using radiolabeled ligands. One important biological change underlying the development of neuropathic pain is the overexpression of α2δ-1 subunit of voltage-dependent calcium channels (the target of gabapentin). Thus, we hypothesized that a radiolabeled form of gabapentin may allow imaging changes in α2δ-1 for monitoring the underlying pathophysiology of neuropathic pain. Here, we report the development of two 18F-labeled derivatives of gabapentin (trans-4-[18F]fluorogabapentin and cis-4-[18F]fluorogabapentin) and their evaluation in healthy rats and a rat model of neuropathic pain (spinal nerve ligation model). Both isomers were found to selectively bind to the α2δ-1 receptor with trans-4-[18F]fluorogabapentin having higher affinity. Both tracers displayed around 1.5- to 2-fold increased uptake in injured nerves over the contralateral uninjured nerves when measured by gamma counting ex vivo. Although the small size of the nerves and the signal from surrounding muscle prevented visualizing these changes using PET, this work demonstrates that fluorinated derivatives of gabapentin retain binding to α2δ-1 and that their radiolabeled forms can be used to detect pathological changes in vitro and ex vivo. Furthermore, this work confirms that α2δ-1 is a promising target for imaging specific features of neuropathic pain.

MAO-B Inhibitor, KDS2010, Alleviates Spinal Nerve Ligation-induced Neuropathic Pain in Rats Through Competitively Blocking the BDNF/TrkB/NR2B Signaling.

MAO-B inhibitors have been implicated to reverse neuropathic pain behaviors. Our previous study has demonstrated that KDS2010 (KDS), a newly developed reversible MAO-B inhibitor, could attenuate Paclitaxel (PTX)-induced tactile hypersensitivity in mice through suppressing reactive oxidant species (ROS)-decreased inhibitory GABA synaptic transmission in the spinal cord. In this study, we evaluated the analgesic effect of KDS under a new approach, in which KDS acts on dorsal horn sensory neurons to reduce excitatory transmission. Oral administration of KDS effectively enhanced mechanical thresholds in the spinal nerve ligation (SNL) induced neuropathic pain in rats. Moreover, we discovered that although treatment with KDS increased brain-derived neurotrophic factor (BDNF) levels, KDS inhibited Tropomyosin receptor kinase B (TrkB) receptor activation, suppressing increased p-NR2B-induced hyperexcitability in spinal dorsal horn sensory neurons after nerve injury. In addition, KDS showed its anti-inflammatory effects by reducing microgliosis and astrogliosis and the activation of MAPK and NF-ᴋB inflammatory pathways in these glial cells. The levels of ROS production in the spinal cords after the SNL procedure were also decreased with KDS treatment. Taken together, our results suggest that KDS may represent a promising therapeutic option for treating neuropathic pain. PERSPECTIVE: Our study provides evidence suggesting the mechanisms by which KDS, a novel MAO-B inhibitor, can be effective in pain relief. KDS, by targeting multiple mechanisms involved in BDNF/TrkB/NR2B-related excitatory transmission and neuroinflammation, may represent the next future of pain medicine.

Plasticity of neuronal excitability and synaptic balance in the anterior nucleus of paraventricular thalamus after nerve injury.

The anterior nucleus of the paraventricular thalamus (aPVT) integrates various synaptic inputs and conveys information to the downstream brain regions for arousal and pain regulation. Recent studies have indicated that the PVT plays a crucial role in the regulation of chronic pain, but the plasticity mechanism of neuronal excitability and synaptic inputs for aPVT neurons in neuropathic pain remains unclear. Here, we report that spinal nerve ligation (SNL) significantly increased the neuronal excitability and reset the excitatory/inhibitory (E/I) synaptic inputs ratio of aPVT neurons in mice. SNL significantly increased the membrane input resistance, firing frequency, and the half-width of action potential. Additionally, SNL enlarged the area of afterdepolarization and prolonged the rebound low-threshold spike following a hyperpolarized current injection. Further results indicate that an inwardly rectifying current density was decreased in SNL animals. SNL also decreased the amplitude, but not the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), nor the amplitude or frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) of aPVT neurons. Moreover, SNL disrupted the E/I synaptic ratio, caused a decrease in weighted tau and half-width of averaged sIPSCs, but did not change these physiological properties of averaged sEPSCs. Finally, pharmacological activation of the GABAA receptor at aPVT could effective relieve SNL-induced mechanical allodynia in mice. These results reveal the plasticity of intrinsic neuronal excitability and E/I synaptic balance in the aPVT neurons after nerve injury and it may play an important role in the development of pain sensitization.

Activation of Double-Stranded RNA-Activated Protein Kinase in the Dorsal Root Ganglia and Spinal Dorsal Horn Regulates Neuropathic Pain Following Peripheral Nerve Injury in Rats.

Double-stranded RNA (dsRNA)-activated kinase (PKR) is an important component in inflammation and immune dysfunction. However, the role of PKR in neuropathic pain remains unclear. Here, we showed that lumbar 5 spinal nerve ligation (SNL) led to a significant increase in the level of phosphorylated PKR (p-PKR) in both the dorsal root ganglia (DRG) and spinal dorsal horn. Images of double immunofluorescence staining revealed that p-PKR was expressed in myelinated A-fibers, unmyelinated C-fibers, and satellite glial cells in the DRG. In the dorsal horn, p-PKR was located in neuronal cells, astrocytes, and microglia. Data from behavioral tests showed that intrathecal (i.t.) injection of 2-aminopurine (2-AP), a specific inhibitor of PKR activation, and PKR siRNA prevented the reductions in PWT and PWL following SNL. Established neuropathic pain was also attenuated by i.t. injection of 2-AP and PKR siRNA, which started on day 7 after SNL. Prior repeated i.t. injections of PKR siRNA prevented the SNL-induced degradation of IκBα and IκBß in the cytosol and the nuclear translocation of nuclear factor κB (NF-κB) p65 in both the DRG and dorsal horn. Moreover, the SNL-induced increase in interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) production was diminished by this treatment. Collectively, these results suggest that peripheral nerve injury-induced PKR activation via NF-κB signaling-regulated expression of proinflammatory cytokines in the DRG and dorsal horn contributes to the pathogenesis of neuropathic pain. Our findings suggest that pharmacologically targeting PKR might be an effective therapeutic strategy for the treatment of neuropathic pain.

Downregulation of Sp1 Inhibits the Expression of HDAC1/SOX10 to Alleviate Neuropathic Pain-like Behaviors after Spinal Nerve Ligation in Mice.

Specific protein 1 (Sp1) is a member of the Sp/Kruppel-like factor family, which regulates cellular processes of neurons in the nervous system. This study was performed to examine the regulatory role and the underlying mechanism of transcription factor Sp1 in neuropathic pain (NP)-like behaviors after spinal nerve ligation (SNL). Sp1 and histone deacetylase 1(HDAC1) expressions were determined in the C57BL6 mouse model with NP-like behaviors after SNL, which demonstrated that Sp1 and HDAC1 elevation occurred in neurons in the spinal dorsal horn of SNL mice. The chromatin immunoprecipitation assay verified that Sp1 was bound to the HDAC1 promoter region and HDAC1 to the SRY-box-containing gene 10 (SOX10) promoter region in the spinal dorsal horn. Immunofluorescence was performed to determine Sp1, HDAC1, and SOX10 in the spinal dorsal horn neurons as well as the neuronal marker (NeuN), microglial marker (Iba-1), and astrocyte marker (GFAP). The nociceptive test was performed to characterize the hindlimb paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of mice 0-10 days after model establishment. Loss- and gain-of-function assays revealed that Sp1 promoted HDAC1 expression, and HDAC1 in turn promoted SOX10 expression. HDAC1 elevation reversed the effects of Sp1 silencing, and the increased PWT and PWL of SNL mice were negated after SOX10 overexpression. Meanwhile, SOX10 also restored the results by Sp1 knockdown. Collectively, downregulating Sp1 alleviates NP-like behaviors after SNL via the HDAC1/SOX10 axis.

The Effects of Nuclear Factor Erythroid 2 (NFE2)-Related Factor 2 (Nrf2) Activation in Preclinical Models of Peripheral Neuropathic Pain.

Oxidative stress, resulting from an imbalance between the formation of damaging free radicals and availability of protective antioxidants, can contribute to peripheral neuropathic pain conditions. Reactive oxygen and nitrogen species, as well as products of the mitochondrial metabolism such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, are common free radicals. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor encoded by the NFE2L2 gene and is a member of the cap 'n' collar subfamily of basic region leucine zipper transcription factors. Under normal physiological conditions, Nrf2 remains bound to Kelch-like ECH-associated protein 1 in the cytoplasm that ultimately leads to proteasomal degradation. During peripheral neuropathy, Nrf2 can translocate to the nucleus, where it heterodimerizes with muscle aponeurosis fibromatosis proteins and binds to antioxidant response elements (AREs). It is becoming increasingly clear that the Nrf2 interaction with ARE leads to the transcription of several antioxidative enzymes that can ameliorate neuropathy and neuropathic pain in rodent models. Current evidence indicates that the antinociceptive effects of Nrf2 occur via reducing oxidative stress, neuroinflammation, and mitochondrial dysfunction. Here, we will summarize the preclinical evidence supporting the role of Nrf2 signaling pathways and Nrf2 inducers in alleviating peripheral neuropathic pain.