Thermal Hyperalgesia In Rats Research Articles

(+)-Catechin attenuates CCI-induced neuropathic pain in male rats by promoting the Nrf2 antioxidant pathway to inhibit ROS/TLR4/NF-κB-mediated activation of the NLRP3 inflammasome.

The objective of this study was to investigate the potential mechanisms by which (+)-catechin alleviates neuropathic pain. Thirty-two male Sprague-Dawley rats were divided into four groups: the sham group, the chronic constriction injury (CCI)group, the CCI+ ibuprofen group, and the CCI+ (+)-catechin group. CCI surgery induces thermal hyperalgesia in rats and (+)-catechin ameliorated CCI-induced thermal hyperalgesia and repaired damaged sciatic nerve in rats. CCI decreased SOD levels in male rat spinal cord dorsal horn and promoted MDA production, induced oxidative stress by increasing NOX4 levels and decreasing antioxidant enzyme HO-1 levels, and also increased protein levels of TLR4, p-NF-κB, NLRP3 inflammasome components, and IL-1β. In contrast, (+)-catechin reversed the above results. In i vitro experiments, (+)-catechin reduced the generation of reactive oxygen species (ROS) in GMI-R1 cells after LPS stimulation and attenuated the co-expression of IBA-1 and NLRP3. It also showed significant inhibition of the NF-κB and NLRP3 inflammatory pathways and activation of the Nrf2-mediated antioxidant system. Overall, these findings suggest that (+)-catechin inhibits the activation of the NLRP3 inflammasome through the triggering of the Nrf2-induced antioxidant system, the inhibition of the TLR4/NF-κB pathway, and the production of ROS to alleviate CCI-induced neuropathic pain in male rats.

Potential analgesic effect of a novel cannabidiol nanocrystals powder for the treatment of neuropathic pain.

The current analgesics often prevent patients from getting effective treatment due to their adverse effects. Cannabidiol (CBD) is well tolerated, has few side effects and has been extensively investigated in analgesia. However, its oral bioavailability is extremely low. In order to solve this problem, we developed the cannabidiol nanocrystals (CBD-NC) in the earlier stage. In this study, we evaluated the nociceptive behaviours associated with neuropathic pain (NP) induced by the spared nerve injury (SNI) model. Assessment of pain threshold was evaluated by paw withdraw threshold (PWT) and paw withdrawal latency (PWL). The improving effect on the motor dysfunction was determined by rota-rod testing. To assess the neuroprotective effect, nerve demyelination and expression of peripheral myelin protein PMP22 were measured with myelin sheath staining and western blotting. Protein expressions in microglia of spinal cord were tested by western blot to explore the underlying mechanism. Compared with the CBD oil solution, CBD-NC significantly reduced mechanical allodynia and thermal hyperalgesia in rats. CBD-NC could improve motor dysfunction induced by SNI in rats, significantly reverse the demyelination and increase the expression of the marker protein of peripheral myelin. Underlying spinal analgesic mechanism of microglia and related factors were preliminarily confirmed. CBD-NC administration is an effective treatment for NP associated with SNI, and the analgesic effect of CBD-NC was significantly better than that of CBD oil sol. By contrast, CBD-NC has a fast-acting and long-term effect in the treatment of NP. Our study further supports the potential therapeutic effect of CBD-NC on NP. The absolute bioavailability of the CBD-NC intramuscular injection formulation can reach 203.31%, which can solve the problem of low oral bioavailability. This research evaluated the therapeutic effect of CBD-NC on NP associated with the SNI model for the first time. All available date showed that whatever the analgesic or neuroprotective effect of CBD-NC, it was significantly better than that of CBD oil sol., which was consistent with the results of the pharmacokinetic. This research supports the initiation of more trials testing the efficacy of CBD-NC for treating NP.

Differential Regulation of Intracisternally Injected Angiotensin II-Induced Mechanical Allodynia and Thermal Hyperalgesia in Rats.

The present study examined the underlying mechanisms of mechanical allodynia and thermal hyperalgesia induced by the intracisternal injection of angiotensin (Ang) II. Intracisternal Ang II injection decreased the air puff threshold and head withdrawal latency. To determine the operative receptors for each distinct type of pain behavior, we intracisternally injected Ang II receptor antagonists 2 h after Ang II injection. Losartan, an Ang II type 1 receptor (AT1R) antagonist, alleviated mechanical allodynia. Conversely, PD123319, an Ang II type 1 receptor (AT2R) antagonist, blocked only thermal hyperalgesia. Immunofluorescence analyses revealed the co-localization of AT1R with the astrocyte marker GFAP in the trigeminal subnucleus caudalis and co-localization of AT2R with CGRP-positive neurons in the trigeminal ganglion. Intracisternal pretreatment with minocycline, a microglial inhibitor, did not affect Ang II-induced mechanical allodynia, whereas L-α-aminoadipate, an astrocyte inhibitor, significantly inhibited Ang II-induced mechanical allodynia. Furthermore, subcutaneous pretreatment with botulinum toxin type A significantly alleviated Ang II-induced thermal hyperalgesia, but not Ang II-induced mechanical allodynia. These results indicate that central Ang II-induced nociception is differentially regulated by AT1R and AT2R. Thus, distinct therapeutic targets must be regulated to overcome pain symptoms caused by multiple underlying mechanisms.

Effect of spirocyclopiperazinium salt compound LXM-15 on spinal nerve injury in rats.

Currently available therapies for neuropathic pain show limited efficacy. This study aimed to investigate the anti-nociceptive effect of the spirocyclopiperazinium salt compound LXM-15 in spinal nerve ligation (SNL) rats and to explore the potential mechanisms. Mechanical allodynia and thermal hyperalgesia tests were used to evaluate the effects of LXM-15 in SNL rats. The expression of CaMKIIα, CREB, JAK2, STAT3, c-fos and TNF-α was detected by western blotting, ELISA or qRT-PCR analysis. Receptor blocking test was performed to explore possible target. Administration of LXM-15 (1, 0.5, 0.25 mg/kg, i.g.) dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in rats subjected to SNL (p < 0.01, p < 0.05), and the effects were completely blocked by peripheral α7 nicotinic or M4 muscarinic receptor antagonist (p > 0.05). LXM-15 significantly decreased the overexpression of phosphorylated CaMKIIα, CREB, JAK2 and STAT3 proteins and the mRNA levels of TNF-α and c-fos (p < 0.01, p < 05). All of the effects could be blocked by α7 or M4 receptor antagonist. Furthermore, LXM-15 reduced the protein expression of TNF-α and c-fos (p < 0.01, p < 0.05). No significant acute toxicity or abnormal hepatorenal function was observed. This is the first study to report that LXM-15 exerts significant anti-nociceptive effect on SNL rats. This effect may occur by activating peripheral α7 nicotinic and M4 muscarinic receptors, further inhibiting the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and finally inhibiting the expression of TNF-α and c-fos. Existing treatments for neuropathic pain show limited efficacy with severe adverse reactions. This paper is the first to report that LXM-15, a new spirocyclopiperazinium salt compound, exerts a significant anti-nociception in SNL rats without obvious toxicity. The underlying mechanisms include activating peripheral α7 nicotinic and M4 muscarinic receptors, then inhibiting the signalling pathways of CaMKIIα/CREB and JAK2/STAT3 and the expressions of TNF-α and c-fos. This study sheds new light on the development of novel analgesic drugs with fewer side effects.

Synergistic effect of ellagic acid and gabapentin in a rat model of neuropathic pain.

Neuropathic pain is a subtype of chronic pain characterized by a primary lesion or dysfunction of the peripheral or central nervous system. The current pain management of neuropathic pain is inadequate and needs new medications. We studied the effects of 14 days of intraperitoneal ellagic acid (EA) and gabapentin administration in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the right sciatic nerve. Rats were divided into six groups: (1) control, (2) CCI, (3) CCI + EA (50mg/kg), 4) CCI + EA (100mg/kg), 5) CCI + gabapentin (100mg/kg), and 6) CCI + EA (100mg/kg) + gabapentin (100mg/kg). Behavioral tests, including mechanical allodynia, cold allodynia, and thermal hyperalgesia, were conducted on days - 1(pre-operation), 7, and 14 post-CCI. In addition, at day 14 post-CCI, spinal cord segments were collected to measure the expression of inflammatory markers, including tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), and oxidative stress markers, including malondialdehyde (MDA) and thiol. CCI increased mechanical allodynia, cold allodynia, and thermal hyperalgesia in rats which were reduced by treatment with EA (50 or 100mg/kg), gabapentin, or their combination. CCI increased TNF-α, NO, and MDA levels and decreased thiol content in the spinal cord, which all were reverted by administration of EA (50 or 100mg/kg), gabapentin, or their combination. This is the first report on ellagic acid's ameliorative effect in rats' CCI-induced neuropathic pain. This effect can be attributed to its anti-oxidative and anti-inflammatory, thus making it potentially useful as an adjuvant to conventional treatment.

Cerium Oxide Nanoparticles Ameliorate Oxidative Stress, Inflammation, and Pain Behavior in Neuropathic Rats.

Neuropathic pain originating from a dysfunction in the nervous system is often intractable and chronic. Recently, several studies using nanoparticles suggested a new way to control neuropathic pain. This study intended to explore the potential neuroprotective effect of Cerium Oxide Nanoparticles (CNPs) synthesized by pullulan in neuropathic pain in rats. On the right common sciatic nerve of male Wistar rats, the chronic constriction injury (CCI) procedure was used to establish a neuropathic pain model. CNPs were injected into the caudal vein of the rat. Behavioral methods were used to detect mechanical allodynia, cold allodynia, and thermal hyperalgesia in rats. Besides, inflammation factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitric oxide (NO), and markers of oxidative stress, including Malondialdehyde (MDA) and total thiol, were measured in the spinal cord segment of rats. In rats with CCI, mechanical allodynia, cold allodynia, and thermal hyperalgesia developed, which improved when the rats were administered CNPs. Spinal cord specimens of CCI rats had elevated inflammation and oxidative stress status (↑IL-1β, ↑TNF-α, ↑NO, ↑MDA) and decreased antioxidative levels (↓total thiol). As a result of CNPs treatment, these changes were reversed in the spinal cord specimens. CNPs alleviate neuropathic pain by exhibiting antioxidative and anti-inflammatory activities.

Targeting the vascular endothelial growth factor A/neuropilin 1 axis for relief of neuropathic pain.

Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between the membrane receptor neuropilin 1 (NRP1) and VEGF-A-blocked VEGF-A-mediated sensory neuron hyperexcitability and reduced mechanical hypersensitivity in a rodent chronic neuropathic pain model. These findings identified the NRP1-VEGF-A signaling axis for therapeutic targeting of chronic pain. In an in-silico screening of approximately 480 K small molecules binding to the extracellular b1b2 pocket of NRP1, we identified 9 chemical series, with 6 compounds disrupting VEGF-A binding to NRP1. The small molecule with greatest efficacy, 4'-methyl-2'-morpholino-2-(phenylamino)-[4,5'-bipyrimidin]-6(1H)-one, designated NRP1-4, was selected for further evaluation. In cultured primary sensory neurons, VEGF-A enhanced excitability and decreased firing threshold, which was blocked by NRP1-4. In addition, NaV1.7 and CaV2.2 currents and membrane expression were potentiated by treatment with VEGF-A, and this potentiation was blocked by NRP1-4 cotreatment. Neuropilin 1-4 reduced VEGF-A-mediated increases in the frequency and amplitude of spontaneous excitatory postsynaptic currents in dorsal horn of the spinal cord. Neuropilin 1-4 did not bind to more than 300 G-protein-coupled receptors and receptors including human opioids receptors, indicating a favorable safety profile. In rats with spared nerve injury-induced neuropathic pain, intrathecal administration of NRP1-4 significantly attenuated mechanical allodynia. Intravenous treatment with NRP1-4 reversed both mechanical allodynia and thermal hyperalgesia in rats with L5/L6 spinal nerve ligation-induced neuropathic pain. Collectively, our findings show that NRP1-4 is a first-in-class compound targeting the NRP1-VEGF-A signaling axis to control voltage-gated ion channel function, neuronal excitability, and synaptic activity that curb chronic pain.

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

PurposeNeuropathic 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. MethodsSNL 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. ResultsAdministration 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. ConclusionsWe 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.

Microglia inflammatory response contributes to chronic constriction injury-induced neuropathic pain via miR-339/PFKFB3 axis

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the effect of miR-339 on neuropathic pain.&#x0D; Methods: A rat neuropathic pain model was established through chronic constriction injury (CCI). Expression of miR-339 in spinal cord was determined 14 days later. Microglial inflammatory response was evaluated using immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), while IL-6 and TNF-α were assessed by enzyme-linked immunosorbent assay (ELISA). Pain- associated behavioral effects and microglia-related inflammation were investigated after intrathecal administration of miR-339 agomir into rats post-CCI. The target gene of miR-339 involved in neuropathic pain was evaluated by a luciferase reporter assay. Microglia cells were isolated from rats, then treated with lipopolysaccharide (LPS). The LPS-induced inflammatory response in microglia cells was determined using quantitative reverse transcription PCR analysis of IL-6 and TNF-α.&#x0D; Results: CCI decreased mechanical allodynia and thermal hyperalgesia thresholds, but increased Iba1, IL-6, and TNF-α in rats. MiR-339 was reduced in rat spinal cord after CCI induction while intrathecal injection of miR-339 agomir alleviated CCI-induced changes in mechanical and thermal hyperalgesia in rats, and reversed expression of Iba1, IL-6, and TNF-α. Furthermore, 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) was identified as a miR-339 target gene, and over- expression of miR-339 decreased the expression of PFKFB3, IL-6, and TNF-α in LPS-induced microglia cells.&#x0D; Conclusion: The miR-339/PFKFB3 axis ameliorates CCI-induced neuropathic pain by suppression of microglia inflammatory response, suggesting a novel strategy for neuropathic pain management.&#x0D; &#x0D; &#x0D; &#x0D;

MiR-30b-5p attenuates neuropathic pain by the CYP24A1-Wnt/β-catenin signaling in CCI rats.

MicroRNAs (miRNAs) has been reported to act as key regulators of neuronal function. Increasing evidence has showed that miRNAs exert significant effects in neuropathic pain. We explored the role of miR-30b-5p in neuropathic pain by establishing a rat model of chronic constrictive injury (CCI). The sciatic nerve of CCI rats was used to induce chronic neuropathic pain. The expression and cellular distribution of miR-30b-5p were determined by RT-qPCR and FISH. The mRNA level, protein level, and cellular distribution of CYP24A1 were detected by RT-qPCR, western blot, and immunofluorescence staining assays, respectively. The interaction between miR-30b-5p and CYP24A1 was examined by a luciferase reporter assay. The behavioral effects of miR-30b-5p were assessed after intrathecal administration. Mechanical stimuli and radiant heat were applied to assess mechanical allodynia and thermal hyperalgesia of rats. ELISA was performed to measure the concentration of inflammatory cytokines. MiR-30b-5p expression was significantly downregulated in the spinal cord tissues and of CCI rats. Overexpression of miR-30b-5p attenuated symptoms of neuropathic pain, including mechanical allodynia and thermal hyperalgesia. Additionally, miR-30b-5p overexpression suppressed neuroinflammation by reducing the levels of IL-6, TNF-α and COX2 and elevating the levels of IL-10 in CCI rats. Mechanistically, CYP24A1 was a target of miR-30b-5p, and its expression was negatively regulated by miR-30b-5p. Moreover, CYP24A1 expression was upregulated in CCI rats and knockdown of CYP24A1 attenuated neuropathic pain and neuroinflammation. Furthermore, miR-30b-5p reduced the levels of the Wnt pathway-related genes in CCI rats by downregulating CYP24A1. Rescue assays showed that overexpression of CYP24A1 or activation of Wnt pathway reduced the alleviative effects of miR-30b-5p overexpression on neuropathic pain in CCI rats. Overall,miR-30b-5p inhibits neuropathic pain progression in CCI rats by inhibiting the CYP24A1-Wnt/β-catenin pathway.

Effect of apple peel extract on diabetes-induced peripheral neuropathy and wound injury.

Diabetic peripheral neuropathy (DPN) affects up to 50 % diabetic patients. Moreover, uncontrolled diabetes associated with impaired wound healing. The present study was aimed at exploring the effect of apple peel extract (APE) on type 2 diabetes (T2D)-induced DPN and delayed wound healing. In adult male Sprague-Dawley rats on high-fat diet, a single low dose streptozotocin (STZ, 35mg/kg) was administeredvia intraperitoneal route to induce T2D. Plantar test using Hargreaves apparatus was used to evaluate the DPN. Six different groups of rats were treated orally with saline (naïve control and DPN control), APE (100, 200 and 400mg/kg) and gabapentin (30mg/kg) daily for 7 consecutive days and thermal paw withdrawal latency (PWL) was measured. To elucidate the underlying antioxidant effect of APE, the catalase (CAT), glutathione (GSH) and malonaldehyde (MDA) levels were measured. To evaluate the wound healing potential of APE, excision ischemic open wound model was used. Six different groups of rats were applied with 2 % gum acacia (naïve control and diabetic control), 1 % silver sulfadiazine (SSD) cream and APE cream (5, 10 and 20 %)twice daily for 28 days. Dry connective tissue parameters like hydroxyproline and hexosamine were also measured to further confirm the wound healing activity. Diabetes produced thermal hyperalgesia in rats with a significant decrease in PWL as compared to naive controls indicating induction of DPN. APE and gabapentin significantly improved PWL in diabetic animals. Biochemical analysis revealed a significant improvement in oxidative stress parameters such as catalase, GSH and MDA. Wound closure was significantly more after day 15 of topical application of APE and SSD as compared to control group. APE significantly increased hydroxyproline and hexosamine levels as compared to standard cream. Moreover, histopathology revealed that, topical application of APE cream showed an enhanced healing process. On the basis of the findings, we conclude that APE has a potential to be used as a therapeutic intervention for the management of DPN and delayed wound healing in the diabetic condition.

Role of central endothelin-1 in hyperalgesia, anhedonia, and hypolocomotion induced by endotoxin in male rats.

Sickness syndrome is an adaptive response that can be distinguished by specific signs and symptoms, such as fever and generalized hyperalgesia. Endothelin-1 (ET-1) is produced by inflammatory stimuli, including lipopolysaccharide, and involved in the pathogenesis of inflammation and pain by acting through ETA and ETB receptors. ET-1 also induces fever by acting on the central nervous system. The present study investigated the role of ET-1 in sickness syndrome responses, including hyperalgesia, anhedonia, and hypolocomotion. Intracerebroventricular ET-1 administration induced mechanical and thermal hyperalgesia in rats, which was ameliorated by the ETA receptor antagonist BQ123 and exacerbated by the ETB receptor antagonist BQ788. A cyclooxygenase blocker did not alter hyperalgesia that was induced by ET-1. Lipopolysaccharide administration induced hyperalgesia, and both BQ123 and BQ788 abolished this mechanical hyperalgesia, but the thermal response was only partially blocked. The blockade of ETA receptors in the hypothalamus also abolished lipopolysaccharide-induced mechanical hyperalgesia, and the ETB receptor antagonist did not influence this response. Lipopolysaccharide also induced anhedonia, reflected by lower sucrose preference, and reduced locomotor activity. Both antagonists restored locomotor activity, but only BQ788 reversed the reduction of sucrose preference. These results indicate that ET-1 and both ETA and ETB receptors are involved in various responses that are related to sickness syndrome, including hyperalgesia, anhedonia, and hypolocomotion, that is induced by LPS. Hypothalamic ETA but not ETB receptors are involved in mechanical hyperalgesia that is observed during lipopolysaccharide-induced sickness syndrome.

Role of the CXCR4/ALK5/Smad3 Signaling Pathway in Cancer-Induced Bone Pain.

PurposeThe chemokine receptor, CXCR4, and the transforming growth factor-beta receptor, ALK5, both contribute to various processes associated with the sensation of pain. However, the relationship between CXCR4 and ALK5 and the possible mechanisms promoted by ALK5 in the development of pain have not been evaluated.Materials and MethodsTumor cell implantation (TCI) technology was used to generate a model of cancer-induced bone pain (CIBP) in rats; intrathecal (i.t.) injections of small interfering (si) RNAs targeting CXCR4 and the ALK5-specific inhibitor, RepSox, were performed. Behavioral outcomes, Western blotting, and immunofluorescence techniques were used to evaluate the expression of the aforementioned specific target proteins in the CIBP model.ResultsThe results revealed that i.t. administration of siRNAs targeting CXCR4 resulted in significant reductions in both mechanical and thermal hyperalgesia in rats with CIBP and likewise significantly reduced the expression of ALK5 in the spinal cord. Similarly, i.t. administration of RepSox also resulted in significant reductions in mechanical and thermal hyperalgesia in rats with CIBP together with diminished levels of spinal p-Smad3.ConclusionTaken together, our results suggest that CXCR4 expression in the spinal cord may be a critical mediator of CIBP via its capacity to activate ALK5 and downstream signaling pathways.

Toll-like receptor 4 (TLR4) signaling in the trigeminal ganglion mediates facial mechanical and thermal hyperalgesia in rats

There is increasing evidence that the toll-like receptor 4 (TLR4) signaling pathway contribute to development of hyperalgesia in the trigeminal system. The aim of the present study was to investigate the role of TLR4 in the trigeminal ganglion (TG) in facial hyperalgesia induced by injection of Lipopolysaccharide (LPS) or intraoral mucosal incision, which is an orofacial postoperative pain model, in male Wistar rats. The TLR4 antagonist (LPS-RS, 20 µg/10 µL) was administrated 30 min before LPS injection into the TG (10 µg/10 µL) or oral mucosa (10 µg/50 µL). In the postoperative pain model, rats were treated with LPS-RS (20 µg/10 µL) into the TG for three consecutive days after the incision. Facial heat and mechanical hyperalgesia were assessed hourly after LPS injection or intraoral incision. In addition, expression of NFκB was assessed in the TG on day 3 after intraoral incision. Our results showed that blockade of TLR4 in the TG attenuated facial heat and mechanical hyperalgesia induced by LPS or by mucosal incision, and that both conditions are associated to increase of phosphorylated NFκB in the TG. In conclusion, the present study suggests that activation of TLR4-NFκB signaling pathway in the TG contributes to the development of facial heat and mechanical hyperalgesia and may contribute to pain in inflammatory oral conditions.

Antioxidant modulation of sirtuin 3 during acute inflammatory pain: The ROS control

Oxidative stress induced post-translational protein modifications are associated with the development of inflammatory hypersensitivities. At least 90% of cellular reactive oxygen species (ROS) are produced in the mitochondria, where the mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), is located. MnSOD’s ability to reduce ROS is enhanced by the mitochondrial NAD+-dependent deacetylase sirtuin (SIRT3). SIRT3 can reduce ROS levels by deacetylating MnSOD and enhancing its ability to neutralize ROS or by enhancing the transcription of MnSOD and other oxidative stress-responsive genes. SIRT3 can be post-translationally modified through carbonylation which results in loss of activity. The contribution of post-translational SIRT3 modifications in central sensitization is largely unexplored. Our results reveal that SIRT3 carbonylation contributes to spinal MnSOD inactivation during carrageenan-induced thermal hyperalgesia in rats. Moreover, inhibiting ROS with natural and synthetic antioxidants, prevented SIRT3 carbonylation, restored the enzymatic activity of MnSOD, and blocked the development of thermal hyperalgesia. These results suggest that therapeutic strategies aimed at inhibiting post-translational modifications of SIRT3 may provide beneficial outcomes in pain states where ROS have been documented to play an important role in the development of central sensitization.

Hydrogen sulfide attenuates diabetic neuropathic pain through NO/cGMP/PKG pathway and μ-opioid receptor.

There are currently approximately 425 million diabetic patients worldwide, of which approximately 90% of patients with diabetes suffer from neuropathy. Diabetic neuropathic pain (DNP) is a common complication of diabetic neuropathy. Nearly half of the patients hospitalized with diabetes have pain symptoms or symptoms related to neurological injury, and the incidence increases with age and diabetic duration. Anti-DNP analgesics have either limited therapeutic effects or serious side effects or lack of clinical trials, which has limited their application. Physiopathological mechanisms and treatment of DNP remain a significant challenge. The present confirmed that inhalation of H2S may attenuate the diabetic neuropathic pain through NO/cGMP/PKG pathway and μ-opioid receptor. It provides us the animal study foundation for the application of H2S on the treatment of DNP and clarifies some target molecules in the pain modulation of DNP.

Lanthionine Synthetase C‐like Protein 2: A potential therapeutic target for treating neuropathic pain

Glial activation and over‐production of pro‐inflammatory cytokines and chemokines in the spinal cord are critical mechanisms underlying the genesis of neuropathic pain. While pro‐inflammatory signaling molecules have been extensively investigated, much less is known about anti‐inflammatory signaling pathways and systems. Studies in recent years have demonstrated that lanthionine synthetase C‐like 2 protein (LANCL2) is expressed in cells related to the immune function, like T cells, macrophages, endothelial and epithelial cells, and dendritic cells, suggesting its potential as a target for immunoregulation. In this study, we determined the role of LANCL2 in the genesis of neuropathic pain. Specifically, we show that LANCL2 protein is expressed in spinal dorsal horn and down‐regulated in rats with neuropathic pain. Knockdown of spinal LANCL2 induces mechanical allodynia and thermal hyperalgesia, which is concomitantly accompanied with increases in extracellular signal‐regulated kinase (ERK) activity, tumor necrosis factor alpha (TNF‐α) production, and the activation of astrocytes and microglia in the spinal dorsal horn. Activation of spinal LANCL2 by intrathecal administration of abscisic acid alleviates mechanical and thermal hypersensitivity induced by nerve injury. Pre‐emptive pharmacological activation of LANCL2 prevents the down‐regulation of LANCL2 protein expression, and significantly attenuates the development of mechanical allodynia and thermal hyperalgesia in rats induced by nerve injury. These behavioral changes were accompanied with the suppression of ERK activity and TNF‐α production, and astrocyte and microglia activation in the spinal dorsal horn of rats with nerve injury. Our study provides evidence that LANCL2 may be a potential molecular target for the development of analgesics for neuropathic pain and the management of a wide range of conditions involving neuroinflammation.Support or Funding InformationNS064289; NS107569

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

CDK5 inhibits the clathrin-dependent internalization of TRPV1 by phosphorylating the clathrin adaptor protein AP2μ2.

Transient receptor potential vanilloid 1 (TRPV1), a nonselective, ligand-gated cation channel, responds to multiple noxious stimuli and is targeted by many kinases that influence its trafficking and activity. Studies on the internalization of TRPV1 have mainly focused on that induced by capsaicin or other agonists. Here, we report that constitutive internalization of TRPV1 occurred in a manner dependent on clathrin, dynamin, and adaptor protein complex 2 (AP2). The μ2 subunit of AP2 (AP2μ2) interacted directly with TRPV1 and was required for its constitutive internalization. Cyclin-dependent kinase 5 (CDK5) phosphorylated AP2μ2 at Ser45, which reduced the interaction between TRPV1 and AP2μ2, leading to decreased TRPV1 internalization. Intrathecal delivery of a cell-penetrating fusion peptide corresponding to the Cdk5 phosphorylation site in AP2μ2, which competed with AP2μ2 for phosphorylation by Cdk5, increased the abundance of TRPV1 on the surface of dorsal root ganglion neurons and reduced complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia in rats. In addition to describing a mechanism of TRPV1 constitutive internalization and its inhibition by CDK5, these findings demonstrate that CDK5 promotes inflammatory thermal hyperalgesia by reducing TRPV1 internalization, providing previously unidentified insights into the search for drug targets to treat pain.

Down-regulation of miR-140-3p can alleviate neonatal repetitive pain in rats via inhibiting TGF-β3

MicroRNAs (miRNAs) have been shown to be involved in the pathophysiological processes of pain. At present, the roles and mechanisms of miRNAs in neonatal repetitive pain are largely unknown. In our research, the expression of miR-140-3p was increased in premature infants who received repetitive painful stimuli since admission, and in rat pups after repetitive needlestick stimulation. As a result of behavioral testing, the inhibition of miR-140-3p significantly suppressed abnormal mechanical and thermal hyperalgesia in rats after needlestick. Furthermore, the inhibition decreased the expression of the inflammatory cytokines IL-1β, TNF-α, and IL-6, as well as glucocorticoid receptor expression in rats after needlestick. Using bioinformatic analyses, the 3ʹ-untranslated region of TGF-β3 was predicted to be a target of miR-140-3p. Down-regulation of miR-140-3p significantly promoted the expression of TGF-β3 in vitro and in vivo. Mechanistic investigations revealed that TGF-β3 is a direct target of miR-140-3p, and is involved in the miR-140-3p-mediated effects on neonatal repetitive pain and neuroinflammation. In summary, our current research suggests that down-regulation of miR-140-3p can inhibit painful tactile stimulation of rat pups by inhibiting TGF-β3. Our results suggest that miR-140-3p may provide a new regulatory target for preventing the effects of neonatal repetitive pain.