Improved Pain Behaviors Research Articles

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Network pharmacology and pharmacological evaluation of Wenzheng Jiedu Powder Modified Formula for neuropathic pain relief.

This study aimed to elucidate the mechanism of Wenzheng Jiedu Powder Modified Formula (WJPMF) in treating neuropathic pain (NP). Network pharmacology and experimental verification were integrated to explore the therapeutic effects and key targets of WJPMF. Active components, corresponding target genes, and absorption, distribution, metabolism, and excretion (ADME) genes of WJPMF against NP were screened from public databases. Network analysis and molecular docking were conducted to identify key targets and verify binding abilities. In vivo experiments were performed on spared nerve injury (SNI) rats to assess the analgesic effects and regulatory mechanisms of WJPMF. WJPMF significantly improved pain behaviors in SNI rats by regulating ATP-binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor alpha (PPARA), peroxisome proliferator-activated receptor gamma (PPARG), and superoxide dismutase 2 (SOD2) expression, which were key targets involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. WJPMF shows promising therapeutic potential for NP through the modulation of specific targets, offering a novel therapeutic strategy for managing NP.

Mocetinostat activates Krüppel-like factor 4 and protects against tissue destruction and inflammation in osteoarthritis.

Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat - a class I selective histone deacetylase (HDAC) inhibitor - had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.

Growth differentiation factor-6 attenuates inflammatory and pain-related factors and degenerated disc-induced pain behaviors in rat model.

Previous studies have indicated that growth differentiation factor 6 (GDF6) is a potential candidate for intervertebral disc (IVD) degeneration (IDD) treatment. Here, we investigated the effect of GDF6 on IDD by examining changes in disc structure and the expression of inflammatory and pain-related factors. A rat posterior disc puncture model of single segments and three consecutive segments was constructed, and GDF6 or phosphate-buffered solution was administered via intradiscal injection 1 or 2 weeks after surgery. Magnetic resonance imaging showed a clear degeneration signal in the punctured disc, which was inhibited by GDF6. Histological staining revealed that GDF6 did not significantly improve the structure of IVDs in rats 8 weeks after puncture surgery, but it had an inhibitory effect on expression of the tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β in the IVD. Furthermore, GDF6 was found to protect the morphology and structure of the IVD 32 weeks after surgery. Mechanical and thermal hyperalgesia tests suggested that GDF6 injection can significantly improve mechanical and thermal-stimulated pain behavior in rats and inhibit the expression of inflammatory factors TNF-α and IL-1β and the pain factor calcitonin gene-related peptide in the dorsal root ganglion. A rat protein array test indicated that GDF6 could reduce the expression of cytokines IL-6, intercellular cell adhesion molecule-1, matrix metalloproteinase-13, IL-1β, and TNF-α and increase the expression of tissue inhibitor of metalloproteinases 1, Transforming growth factor-beta 2, IL-10, and resistin in a TNF-α-induced IDD cell model. Thus, our study demonstrates that GDF6 can improve the structure of the IVD, inhibit the expression of inflammatory and pain-related factors, and improve pain behavior in rats. Clinical Significance: To establish further preclinical research and clinical trials, comprehensive data are needed to validate the regenerative properties of GDF6. Ideally, a regenerative agent should also be able to relieve discogenic pain, achieving the best clinical outcomes.

Optogenetic stimulation of the motor cortex alleviates neuropathic pain in rats of infraorbital nerve injury with/without CGRP knock-down

BackgroundPrevious studies have reported that electrical stimulation of the motor cortex is effective in reducing trigeminal neuropathic pain; however, the effects of optical motor cortex stimulation remain unclear.ObjectiveThe present study aimed to investigate whether optical stimulation of the primary motor cortex can modulate chronic neuropathic pain in rats with infraorbital nerve constriction injury.MethodsAnimals were randomly divided into a trigeminal neuralgia group, a sham group, and a control group. Trigeminal neuropathic pain was generated via constriction of the infraorbital nerve and animals were treated via selective inhibition of calcitonin gene-related peptide in the trigeminal ganglion. We assessed alterations in behavioral responses in the pre-stimulation, stimulation, and post-stimulation conditions. In vivo extracellular recordings were obtained from the ventral posteromedial nucleus of the thalamus, and viral and α-CGRP expression were investigated in the primary motor cortex and trigeminal ganglion, respectively.ResultsWe found that optogenetic stimulation significantly improved pain behaviors in the trigeminal neuralgia animals and it provided more significant improvement with inhibited α-CGRP state than active α-CGRP state. Electrophysiological recordings revealed decreases in abnormal thalamic firing during the stimulation-on condition.ConclusionOur findings suggest that optical motor cortex stimulation can alleviate pain behaviors in a rat model of trigeminal neuropathic pain. Transmission of trigeminal pain signals can be modulated via knock-down of α-CGRP and optical motor cortex stimulation.

Chrysin Attenuates the NLRP3 Inflammasome Cascade to Reduce Synovitis and Pain in KOA Rats.

PurposeOur recent reports have revealed that inhibiting NLRP3 activation reduces synovial inflammation and fibrosis in knee osteoarthritis (KOA). Synovial inflammation is involved the entire process of KOA and promotes the progression of KOA. Natural flavonoid Chrysin from Scutellariae Radix, a traditional Chinese medicine, exhibits multifarious biological activities and potentially has protective activity against osteoarthritis. However, the mechanism of Chrysin in the treatment of synovial inflammation remains elusive. The purpose of our research was to explore the anti-inflammatory effects of Chrysin on KOA, which was induced by monoiodoacetic acid (MIA) in rats by targeting the NLRP3 inflammasome in the hopes of identifying an effective drug to treat KOA.MethodsThe MIA-induced KOA model was used to evaluate the cold pain threshold and paw withdrawal threshold (PWT) of joints after MIA (40 mg/mL) injection into the knee joints. Microscopically, we used LPS (5 ug/mL) and ATP (4 mmol/L) to stimulate fibroblast-like synovial cells (FLSs) to explore the underlying mechanisms and effects of Chrysin. Two staining methods, H&E and Sirius Red, were applied to assess histopathological changes in synovial membranes. Cellular signal transduction was determined by qRT-PCR and WB. Cytokine expression (inflammatory cytokines and pain-related cytokines) was detected by ELISA. The degree of chronic inflammatory pain was evaluated by c-Fos immunofluorescence.ResultsThe results showed that Chrysin not only attenuated synovial inflammation but also reduced the secretion of pain-related factors and increased the PWT and cold pain threshold in rats. Chrysin also inhibited NLRP3 inflammasome activation and increased IL-1β levels to alleviate the synovitis.ConclusionChrysin can relieve knee synovial inflammation and improve pain behavior in KOA rats, which may be related to the ability of Chrysin to inhibit NLRP3 inflammasome activation. Therefore, Chrysin may be developed as a new drug for the treatment of KOA.

Optical Modulation on the Nucleus Accumbens Core in the Alleviation of Neuropathic Pain in Chronic Dorsal Root Ganglion Compression Rat Model

The role of the nucleus accumbens (NAc) in chronic neuropathic pain has been suggested, but the role of the NAc in dorsal root ganglion (DRG) neuropathic pain remains unclear. The objective of this study was to determine whether optogenetic stimulation of the NAc influences DRG compression-induced neuropathic pain. We established sham or DRG lesions in female Sprague-Dawley rats by L4-5 DRG root compression, and the animals received unilateral injections of optogenetic virus in the NAc core. We employed reflexive pain tests to assess the alterations between the groups at the light on/off states. To determine thalamic firing, we performed single-unit in vivo extracellular recording. For statistical analysis, we used one- or two-way repeated-measures analysis of variance. Compared to sham-operated rats, chronic compressed DRG rats showed elevated behavioral sensitivity and sustained neuronal hyperexcitability in the thalamus. NAc optic stimulation improved pain behaviors and lowered thalamic discharge from ventral posterolateral thalamic nuclei. The NAc core impacts the reward and motivational aspects of chronic neuropathic pain influenced by limbic behaviors to thalamic discharge. Increased thalamic firing activity may result in chronic compressed DRG-induced neuropathic pain, and optogenetic neuromodulation of the NAc can ease chronic pain and thalamic discharge.

Cognitive impairment in a classical rat model of chronic migraine may be due to alterations in hippocampal synaptic plasticity and N-methyl-D-aspartate receptor subunits

Although migraine is a major global public health problem, its impact on cognitive abilities remains controversial. Thus, the present study investigated the effects of repeated administration of inflammatory soup to the dura of rats, over three weeks, on spatial cognition, hippocampal synaptic plasticity, and the expression of N-methyl-D-aspartate receptor subunits. Additionally, low doses of amitriptyline (5 mg/kg) were applied to assess its therapeutic effects. The inflammatory soup group exhibited significant reductions in the cutaneous stimulation threshold, presence of mild cognitive impairment, and decreased long-term potentiation in right hippocampus. However, amitriptyline improved pain behaviors, enhanced cognitive function, and increased synaptic plasticity in the inflammatory soup rats. On the other hand, the administration of amitriptyline to normal rats negatively influenced synaptic plasticity and reduced the expression of N-methyl-D-aspartate receptor subunits. The present results indicate that inflammatory soup-induced dural nociception led to impairments in spatial cognition that could be attributed to reductions in hippocampal long-term potentiation and the decreased expression of N-methyl-D-aspartate receptor subunits.

Suppression of WNK1-SPAK/OSR1 Attenuates Bone Cancer Pain by Regulating NKCC1 and KCC2

Our preliminary experiment indicated the activation of with-nolysine kinases 1 (WNK1) in bone cancer pain (BCP) rats. This study aimed to investigate the underlying mechanisms via which WNK1 contributed to BCP. A rat model of BCP was induced by Walker-256 tumor cell implantation. WNK1 expression and distribution in the lumbar spinal cord dorsal horn and dorsal root ganglion were examined. SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress-responsive kinase 1 (OSR1), sodium-potassium-chloride cotransporter 1 (NKCC1), and potassium-chloride cotransporter 2 (KCC2) expression were assessed. Pain behaviors including mechanical allodynia and movement-evoked pain were measured. BCP rats exhibited significant mechanical allodynia, with increased WNK1 expression in the dorsal horn and dorsal root ganglion neurons, elevated SPAK/OSR1 and NKCC1 expression in the dorsal root ganglion, and decreased KCC2 expression in the dorsal horn. WNK1 knock-down by small interfering alleviated mechanical allodynia and movement-evoked pain, inhibited WNK1-SPAK/OSR1-NKCC1 activities, and restored KCC2 expression. In addition, closantel (a WNK1-SPAK/OSR1 inhibitor) improved pain behaviors, downregulated SPAK/OSR1 and NKCC1 expression, and upregulated KCC2 expression in BCP rats. Activation of WNK1-SPAK/OSR1 signaling contributed to BCP in rats by modulating NKCC1 and KCC2 expression. Therefore, suppression of WNK1-SPAK/OSR1 may serve as a potential target for BCP therapy. PerspectiveOur findings demonstrated that the WNK1-SPAK/OSR1 signaling contributed to BCP in rats via regulating NKCC1 and KCC2. Suppressing this pathway reduced pain behaviors. Based on these findings, the WNK1-SPAK/OSR1 signaling may be a potential target for BCP therapy.

Substance P expression in the DRG of three murine models of arthritis pain: correlation with pain measures and effect of local neurotoxin analgesia

Purpose: Substance P (SP) release and binding to NK-1 produces pain transmission. Pain from arthritis is expressed as both tenderness and as loss of function. Different arthritis pain models express pain differently. This study evaluated the relationship of dorsal root ganglion (DRG) SP expression to pain using both measures of tenderness and function in 3 different models of arthritis pain. Neurotoxins (NT) that prevent release of SP, such as onabotulinum toxin (BoNT/A), and those that deplete SP, such as vanilloids (VAN), can produce analgesia in these models. We correlated effects on SP expression in the DRG with analgesic responses. Methods: C57/Bl6 male mice received intra-articular (IA) carrageenan, Complete Freund’s Adjuvant (CFA) or Collagenase (COL) to produce acute inflammatory, chronic inflammatory or chronic noninflammatory arthritis respectively. IA therapies were given at appropriate intervals before examination. Twelve-week-old mice were examined after arthritis induction. Evoked (tenderness) and spontaneous (functional) pain was quantitated. DRGs were harvested for immunohistochemistry (IHC) after pain assessment. SP expression was measured as % DRG neurons expressing SP. Results: Both Evoked and Spontaneous pain in arthritic and naïve mice correlated with SP expression (R2=0.799, β=1.242 and R2=0.751, β=0.649 respectively). IA vanilloid agonists and antagonist reduced SP expression in chronic inflammatory arthritis (CFA) but did not improve pain behavior. IA BoNT/A reduced SP expression in CFA arthritis but significantly increased SP expression in COL arthritis at 4 weeks after induction but not at 6 weeks. Vanilloid (VAN) treatment normalized evoked pain and weightbearing in mice with acute arthritis pain but did not change SP expression in the DRG. None of the neurotoxins altered pain expression or SP expression in non-arthritic mice. Conclusions: SP expression correlates with both evoked and spontaneous pain behaviors in murine models of arthritis pain. Depending on the NT mechanism of action, SP expression may either increase (BoNT/A) or decrease (VAN). The effect of NT on SP expression may also depend on pathophysiology of pain production and chronicity. SP expression does not necessarily correlate with NT-induced analgesia.

Effects of intrathecally postsynaptic density protein-95 antisense oligodeoxynucleotide on pain behaviors in mice model of neuropathic pain

Objective To investigate the effects of intrathecally postsynaptic density protein-95 (PSD-95) antisense oligodeoxynucleotide on neuropathic pain behaviors of sciatic nerve ligation mouse. Methods Forty-eight male C57BL/6 mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups (n=12): Sham group, normal saline group (group NS), PSD-95 antisense oligodeoxynucleotide group (group A) and PSD-95 missense oligodeoxynucleotide group (group M). In groups NS, A and M, normal saline 5 μl, antisense oligodeoxynucleotide 5 μg/5 μl and missense oligodeoxynucleotide 5 μg/5 μl were injected intrathecally once a day for 14 d, starting from the 1st day after chronic constriction injury(CCI), respectively. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWL) were measured on day 1 before CCI and on day 1, 3, 5, 7, 14, 17, 21 after CCI. Results During day 1-day 14, mice in group A maintained the pain thresholds consistent with Sham group [14 d, PWMT(5.69±1.34) g, P>0.05, PWL (9.65±1.44) s, P>0.05]. The withdrawal thresholds in the ipsilateral hind paws of the group A were significantly lower than sham group on day 17 after CCI[PWMT(4.24±1.83) g, P<0.05, PWL(7.18±0.41) s, P<0.05]. However, compared with group NS [PWMT (1.77±0.38) g, P<0.05, PWL (4.33±1.21) s, P<0.05]and group M[PWMT (1.6±0.37) g, P<0.05, PWL(4.38± 0.95) s, P<0.05], the withdrawal thresholds of group A was significantly elevated on day 17 after CCI. Conclusions Intrathecally treated with PSD-95 antisense oligodeoxynucleotide in the development of neuropathic pain induced by CCI completely improved pain behaviors during the course of injection, and the effects of pain relief lasted at least 7 d after no injection. Key words: Neuropathic pain; postsynaptic density protein-95; Antisense oligonucleotide; N-methyl-D-aspartate

Regulation of the NR2B-CREB-CRTC1 Signaling Pathway Contributes to Circadian Pain in Murine Model of Chronic Constriction Injury.

Numerous clinical investigations have revealed the circadian rhythm changes in the perception of chronic pain, and most clinical chronic pain types peak in the night. However, it is still undiscovered whether circadian rhythm of pain exists in rodents and the specific mechanism that may underlie it. Our study was conducted to investigate the rhythmic changes of hyperalgesia behavior in a chronic constrictive injury (CCI) model of rodents and to explore the role of the N-methyl-d-aspartate receptor 2B (NR2B)-cAMP response element binding protein (CREB)-CREB-regulated transcription coactivator 1 (CRTC1) signaling pathway in this pain rhythm. A CCI operation was performed to mimic clinical chronic pain. Paw mechanical withdrawal threshold and paw withdrawal thermal latency were used to test pain behavior in rats; a von Frey cilia test was used to test mechanical hyperalgesia in mice at Zeitgeber time (ZT) 4, ZT10, ZT16, and ZT22 for 14 contiguous days. The relative mRNA and protein expression of NR2B, CREB and CRTC1 in the suprachiasmatic nuclei and the dorsal horn were measured by real-time polymerase chain reaction and Western blot. CRTC1 and CREB interference adenovirus vectors were injected intrathecally at 2 time points, respectively (ZT12 and ZT0), to further explore the proper time point for pain treatment. During the period of chronic pain state, the pain behavior of CCI rodents showed a circadian rhythm with the peak at ZT4 or ZT10 daily. The pain thresholds were significantly different between the activity period and the rest period. The expressions of NR2B, CRTC1, and CREB at the spinal level were consistent with the pain rhythm. The intrathecal treatment with CRTC1 or CREB interference adenovirus from day 7 to day 9 after CCI surgery markedly improved pain behaviors. Nevertheless, when given at ZT0, they were both more effective at relieving peak pain than drugs given at ZT12. Pain behavior in the chronic pain of CCI displayed circadian rhythm and was associated with circadian secretion of pain-related receptors. The NR2B-CREB-CRTC1 signaling pathway may play a crucial role in this rhythm. Moreover, our results suggest that measures to relieve pain should be taken before pain reaches its peak.

Improvements in impaired GABA and GAD65/67 production in the spinal dorsal horn contribute to exercise-induced hypoalgesia in a mouse model of neuropathic pain.

BackgroundPhysical exercise effectively attenuates neuropathic pain, and multiple events including the inhibition of activated glial cells in the spinal dorsal horn, activation of the descending pain inhibitory system, and reductions in pro-inflammatory cytokines in injured peripheral nerves may contribute to exercise-induced hypoalgesia. Since fewer GABAergic hypoalgesic interneurons exist in the dorsal horn in neuropathic pain model animals, the recovery of impaired GABAergic inhibition in the dorsal horn may improve pain behavior. We herein determined whether the production of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) in the dorsal horn is restored by treadmill running and contributes to exercise-induced hypoalgesia in neuropathic pain model mice. C57BL/6 J mice underwent partial sciatic nerve ligation (PSL). PSL-Runner mice ran on a treadmill at 7 m/min for 60 min/day, 5 days/week, from two days after PSL.ResultsMechanical allodynia and heat hyperalgesia developed in PSL-Sedentary mice but were significantly attenuated in PSL-Runner mice. PSL markedly decreased GABA and GAD65/67 levels in neuropils in the ipsilateral dorsal horn, while treadmill running inhibited these reductions. GABA+ neuronal nuclei+ interneuron numbers in the ipsilateral dorsal horn were significantly decreased in PSL-Sedentary mice but not in PSL-Runner mice. Pain behavior thresholds positively correlated with GABA and GAD65/67 levels and GABAergic interneuron numbers in the ipsilateral dorsal horns of PSL-Sedentary and -Runner mice.ConclusionsTreadmill running prevented PSL-induced reductions in GAD65/67 production, and, thus, GABA levels may be retained in interneurons and neuropils in the superficial dorsal horn. Therefore, improvements in impaired GABAergic inhibition may be involved in exercise-induced hypoalgesia.

Resveratrol Reduces Myometrial Infiltration, Uterine Hyperactivity, and Stress Levels and Alleviates Generalized Hyperalgesia in Mice With Induced Adenomyosis.

In this study, we sought to determine whether resveratrol (RSV), a nonhormonal compound, would suppress the myometrial infiltration, improve pain behavior, lower stress level, improve the expression of some proteins known to be involved in adenomyosis, and reduce uterine contractility in a mice model of adenomyosis. Adenomyosis was induced in 28 female ICR mice neonatally dosed with tamoxifen, while another 12 (group C) were dosed with solvent only, serving as a blank control. Starting from 4 weeks after birth, hotplate test was administrated to all mice every 4 weeks. At the 16th week, all mice with induced adenomyosis were randomly divided into 3 groups: low-dose RSV (2 mg/kg), high-dose RSV (3 mg/kg), and untreated. Group C received no treatment. After 3 weeks of treatment, they were hotplate tested again, their uterine horns and brains were harvested, and a blood sample was taken to measure the plasma corticosterone (CORT) level by enzyme-linked immunosorbent assay. The left uterine horn was used for immunohistochemistry analysis. The brain stem nucleus raphe magnus (NRM) sections were subjected to immunofluorescence staining for glutamic acid decarboxylase isoform 65 (GAD65). The depth of myometrial infiltration and uterine contractility was evaluated. We found that RSV is well tolerated and that it dose dependently suppressed myometrial infiltration, improved generalized hyperalgesia, reduced uterine contractility and lowered plasma CORT levels, and improved the expression of some proteins known to be involved in adenomyosis. It also elevated the number of GAD65-expressing neurons in the brain stem NRM, possibly boosting the GABAergic inhibition of pain due to adenomyosis. Therefore, RSV appears to be a promising compound for treating adenomyosis.

The effect of NR2B subunit palmitoylation at the spinal level after chronic dorsal root ganglia compression in rats.

The NR2B subunit (N-methyl-D-aspartate receptor 2B subunit) regulates the source of pain, and it participates in the formation of central sensitization. Palmitoylation was shown to be involved in the regulation of N-methyl-D-aspartate receptor internalization. In the present study, we investigated the effects of NR2B subunit palmitoylation in a chronic dorsal root ganglia compression (CCD) rat model. Paw mechanical withdrawal threshold and paw withdrawal thermal latency were used to assess mechanical allodynia and thermal hyperalgesia after a CCD operation and an intrathecal injection of the inhibitor of palmitoylation (2-bromopalmitate [2-BP]). The acyl-biotinyl exchange method, Western blotting, and coimmunoprecipitation were used to investigate the effects of pain processing and the expression of levels of NR2B palmitoylation and phosphorylation at the spinal level. CCD rats had long-lasting thermal hyperalgesia and mechanical allodynia, leading to upregulation of the level of NR2B palmitoylation and phosphorylation at the spinal level. An intrathecal treatment with 2-BP on day 14 after CCD surgery markedly improved pain behaviors and downregulated the expression of NR2B palmitoylation and phosphorylation. These data suggest that upregulated NR2B palmitoylation in CCD-induced neuropathic pain and intrathecal injection of 2-BP could reduce pain behaviors and NR2B phosphorylation. Our findings indicate that spinal NR2B palmitoylation is an important component of CCD-induced neuropathic pain, and it might be a potential target for chronic pain therapy.

Palliation of Bone Cancer Pain by Antagonists of Platelet-Activating Factor Receptors

Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF) receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC) model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2) protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients.

Epigallocatechin-3-Gallate Reduces Myometrial Infiltration, Uterine Hyperactivity, and Stress Levels and Alleviates Generalized Hyperalgesia in Mice Induced With Adenomyosis

In an effort to search for novel therapeutics for adenomyosis, we sought to determine whether treatment with epigallocatechin-3-gallate (EGCG) would suppress the myometrial infiltration, improve pain behavior, lower stress level, and reduce uterine contractility in a mice model of adenomyosis. Adenomyosis was induced in 28 female ICR mice neonatally dosed with tamoxifen, while another 12 (group C) were dosed with solvent only, which served as a blank control. Starting from 4 weeks after birth, hot plate test was administrated to all mice every 4 weeks. At the 16th week, all mice induced with adenomyosis were randomly divided into 3 groups: low-dose EGCG (5 mg/kg), high-dose EGCG (50 mg/kg), and untreated. Group C received no treatment. After 3 weeks of treatment, the hot plate test was administered again, a blood sample was taken to measure the plasma corticosterone level by enzyme-linked immunosorbent assay, and then all mice were sacrificed. The depth of myometrial infiltration and uterine contractility were also evaluated. We found that the induction of adenomyosis resulted in progressive generalized hyperalgesia, along with elevated amplitude and frequency of uterine contractions as well as elevated plasma corticosterone levels. The EGCG treatment dose dependently suppressed myometrial infiltration, improved generalized hyperalgesia, reduced uterine contractility, and lowered plasma corticosterone levels. These results suggest that induced adenomyosis causes pain and elevates stress levels in mice. Uterine hyperactivity may contribute to dysmenorrhea in women with adenomyosis who might also have elevated stress level due to pain. The EGCG appears to be a promising compound for treating adenomyosis.

The Retardation of Myometrial Infiltration, Reduction of Uterine Contractility, and Alleviation of Generalized Hyperalgesia in Mice With Induced Adenomyosis by Levo-Tetrahydropalmatine (l-THP) and Andrographolide

Adenomyosis is a tough disease to manage nonsurgically. Levo-tetrahydropalmatine (l-THP), a known analgesic, and andrographolide, a nuclear factor kappa B (NF-κB) inhibitor, are both active ingredients extracted from Chinese medicinal herbs. We sought to determine whether treatment of l-THP, andrographolide, and valproic acid (VPA) would suppress the myometrial infiltration, improve pain behavior, and reduce uterine contractility in a mice model of adenomyosis. Adenomyosis was induced in 55 female ICR mice neonatally dosed with tamoxifen, while another 8 (group C) were dosed with solvent only. Starting from 4 weeks after birth, hotplate test was administrated to all mice every 4 weeks. At the 16th week, all mice with induced adenomyosis were randomly divided into 6 groups, each receiving different treatment for 3 weeks: low- or high-dose l-THP, andrographolide, low-dose l-THP and andrographolide jointly, VPA, and untreated. Group C received no treatment. After treatment, the hotplate test was administered and all mice were killed. The depth of myometrial infiltration of ectopic endometrium and uterine contractility were measured and compared across groups. We found that induction of adenomyosis resulted in progressive generalized hyperalgesia, along with elevated amplitude and irregularity of uterine contractions. Treatment with either l-THP, andrographolide, VPA, or l-THP and andrographolide jointly suppressed myometrial infiltration, improved generalized hyperalgesia, and reduced the amplitude and irregularity of uterine contractions. These results suggest that increased uterine contractility, in the form of increased contractile amplitude and irregularity, may contribute to dysmenorrhea in women with adenomyosis. More importantly, l-THP, andrographolide, and VPA all seem to be promising compounds for treating adenomyosis.

Analgesia effects of intrathecally coadministered dexamethasone and Alt inhibitors on chronic dorsal root ganglion compression-induced pain in mouse

Objective To investigate the analgesic effects of intrathecal dexamethasone injection on pain induced by chronic compression of dorsal root ganglion in mouse.Methods Using rat model of radicular pain induced by chronic compression of dorsal root ganglion ( CCD), 40 male SD rats successfully received intrathecal catheter implantation and without motor dysfunction were randomly divided into 5 groups:Sham-operation group ( Sham group, n = 8 ), Control group ( CCD group, n = 8), Dexamethasone group ( D group, n = 8), Akt inhibitor V group (A group, n = 8 ) and Dexamethasone plus Akt inhibitor Ⅳ group (DA group, n = 8 ).Rats in D group, A group or DA group were intrathecally treated with dexamethasone (100μg/kg) ,Akt inhibitor Ⅳ (0.6μg/10μl) or dexamethasone ( 100 μg/kg) plus Akt inhibitor Ⅳ (0.6 μg/10 μl) on Day 3,13 after CCD respectively, while rats in C and Sham group received Vehicle (10% DMSO).Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were tested on 3 d before and 3 d,4 d,7 d,10 d,13 d,14 d and 15 d after operation.Results Compared with Sham group,both PWMT (P＜0.01) and PWTL (P＜0.01) were significantly decreased after CCD surgery on the ipsilateral side.After dexamethasone and Akt inhibitor were respectively intrathecally injected at 3 postoperative day,PWMT (7.33 ± 1.03 ) g, (5.67 ± 1.03 ) g, (2.67 ± 1.03 ) g (P ＜0.01 ) ,PWTL( 16.47 ±0.46)s, ( 14.48 ±0.84) s, ( 10.82 ±2.21 ) s(P＜0.01 ) ,then decreased gradually,and intrathecally injected again at 13 postoperative day, PWMT ( 7.33 ± 1.03 ) g, ( 5.67 ± 1.03 ) g, (2.33 ± 0.81 ) g (P ＜0.01 ), PWTL( 16.44 ±0.90) s, ( 14.01 ±0.82)s, ( 10.22 ± 1.28)s (P＜0.01).Coadministration dexamethasone and Akt inhibitor exhibit significant synergies, postoperative 4 d PWMT( 10.83 ± 2.04)g, (2.67 ± 1.03 )g (P ＜0.01),PWTL(19.11 ±2.01)s,(10.82 ±2.21)s (P＜0.01);14 d PWMT (7 ±0.82)g,(2.33 ±0.81)g (P ＜ 0.01 ), PWTL( 17.16 ± 1.14)s, ( 10.22 ± 1.28 ) s (P ＜ 0.01 ).Conclusion Intrathecal high-dose dexamethas one or PKB / Akt inhibitors can effectively improve pain behavior response induced by chronic compression of dorsal root ganglia,combination of these two drugs could generate significant synergies, and the effection is more obvious, more durable. Key words: Glucocorticoid ; PKB/Akt ; Neuropathic pain ;

Effects of intrathecally coadministered dexamethasone and spironolactone on pain behaviors in a rat model of radicular pain

Objective To investigate the effects of intrathecally coadministered dexamethasone and spironolactone in trathecally on radicular pain behaviors.Methods Using rat model of radicular pain induced by chronic compression of dorsal root ganglion (CCD) ,48 male SD rats successfully received intrathecal catheter implantation and without motor dysfunction were randomly divided into Sham-operation group (Sham group, n= 12),Control group ( C group, n = 12 ), Dexamethasone group ( D group, n = 8 ), Spironolactone group ( S group, n = 8 )and Dexamethasone plus spironolactone group (DS group, n=8).Rats in D group,S group or DS group were intrathecally treated with dexamethasone 4 μg, spironolactone 3 μg or dexamethasone 4 μg plus spironolactone 3 μg twice daily on day 2 ～4 after CCD respectively,while rats in C and Sham group received 10μl 10% alcohol.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency (PWTL) were tested on day 1 before CCD and day 1,4,7,10,14,17 and 21 after CCD.Results Compared with Sham group, both PWMT and PWTL were significantly decreased after CCD surgery on the ipsilateral side(P＜0.01 ＝.Intrathecally administrated with dexamethasone significantly improved pain behaviors (P＜0.01 ＝ and these therapeutic effects lasted up to 10 days after CCD surgery.As with dexamethasone,intrathecal spironolactone also significantly attenuated PWMT (P＜0.01 ＝ and PWTL (P＜0.01 ＝ and the change lasted up to 7 days after CCD surgery.Coadministration spironolactone and dexamethasone exhibited significant synergies( PWMT: ( 13.52 ± 0.72) g vs ( 11.58 ± 1.38 ) g, P ＜0.01; PWTL: ( 19.63 ± 1.68) s vs ( 14.14 ± 1.52) s, P ＜ 0.01 ＝.These effects lasted up to at least 10 days.Conclusion Both dexamethasone and spironolactone intrathecally have therapeutic effects on radicular pain behaviors, combination injection of these two drugs could generate significant synergies. Key words: Neuropathic pain; Dexamethasone; Spironolactone; Pro-inflammatory cytokine