Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

The alterations in nerve growth factor concentration in plasma and synovial fluid before and after total knee arthroplasty.

Total knee arthroplasty (TKA) is an effective procedure for pain relief; however, the emergence of postsurgical pain remains a concern. In this study, we investigated the production of nerve growth factor (NGF) and mediators that affect NGF production and their function in the synovial fluid and plasma after TKA. This study included 19 patients (20 knees) who had rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and knee osteoarthritis (OA) who underwent TKA, categorized into OA and non-OA groups. The levels of NGF, inflammatory cytokines, and lipid mediators were analyzed before and after surgery. The intraoperative synovial fluid NGF concentration was more than seven times higher in the non-OA group than in the OA group. The intra-articular NGF levels increased significantly by more than threefold postoperatively in the OA group but not in the non-OA group. Moreover, the levels of inflammatory cytokines and lipid mediators were increased in the synovial fluid of both groups. The intra-articular cytokines or NGF concentrations positively correlated with postoperative pain. Targeted NGF control has the potential to alleviate postsurgical pain in TKA, especially in patients with OA, emphasizing the importance of understanding NGF dynamics under different knee conditions.

CXCL13/CXCR5 promote chronic postsurgical pain and astrocyte activation in rats by targeting NLRP3.

Chronic postsurgical pain (CPSP) with high incidence negatively impacts the quality of life. X-C motif chemokine 13 (CXCL13) has been associated with postsurgery inflammation and exacerbates neuropathic pain in patients with CPSP. This study was aimed to illustrate the relationship between CXCL13 and nod-like receptor protein-3 (NLRP3), which is also involved in CPSP. A CPSP model was constructed by skin/muscle incision and retraction (SMIR) in right medial thigh, and the rats were divided into three groups: Sham, SMIR, and SMIR + anti-CXCL13 (intrathecally injected with anti-CXCL13 antibody). Then, the paw withdrawal threshold (PWT) score of rats was recorded. Primary rat astrocytes were isolated and treated with recombinant protein CXCL13 with or without NLRP3 inhibitor INF39. The expressions of CXCL13, CXCR5, IL-1ß, IL-18, GFAP, NLRP3, and Caspase-1 p20 were detected by real-time quantitative reverse transcription PCR, western blot, ELISA, immunocytochemistry, and immunofluorescence analyses. The anti-CXCL13 antibody alleviated SMIR-induced decreased PWT and increased expression of GFAP, CXCL13, CXCR5, NLRP3, and Caspase-1 p20 in spinal cord tissues. The production of IL-1ß, IL-18, and expression of CXCL13, CXCR5, GFAP, NLRP3, and Caspase-1 p20 were increased in recombinant protein CXCL13-treated primary rat astrocytes in a dose-dependent manner. Treatment with NLRP3 inhibitor INF39 inhibited the function of recombinant protein CXCL13 in primary rat astrocytes. The CXCL13/CXCR5 signaling could promote neuropathic pain, astrocytes activation, and NLRP3 inflammasome activation in CPSP model rats by targeting NLRP3. NLRP3 may be a potential target for the management of CPSP.

Microglial exosome miR-124-3p in hippocampus alleviates cognitive impairment induced by postoperative pain in elderly mice.

Cognitive impairment induced by postoperative pain severely deteriorates the rehabilitation outcomes in elderly patients. The present study focused on the relationship between microglial exosome miR-124-3p in hippocampus and cognitive impairment induced by postoperative pain. Cognitive impairment model induced by postoperative pain was constructed by intramedullary nail fixation after tibial fracture. Morphine intraperitoneally was carried out for postoperative analgesia. Morris water maze tests were carried out to evaluate the cognitive impairment, while mRNA levels of neurotrophic factors (BDNF, NG) and neurodegenerative biomarker (VILIP-1) in hippocampus were tested by q-PCR. Transmission electron microscope was used to observe the axon degeneration in hippocampus. The levels of pro-inflammatory factors (TNF-α, IL-1ß, IL-6), the levels of anti-inflammatory factors (Ym, Arg-1, IL-10) and microglia proliferation marker cyclin D1 in hippocampus were measured to evaluate microglia polarization. Bioinformatics analysis was conducted to identify key exosomes while BV-2 microglia overexpressing exosome miR-124-3p was constructed to observe microglia polarization in vitro experiments. Exogenous miR-124-3p-loaded exosomes were injected into hippocampus in vivo. Postoperative pain induced by intramedullary fixation after tibial fracture was confirmed by decreased mechanical and thermal pain thresholds. Postoperative pain induced cognitive impairment, promoted axon demyelination, decreased BDNF, NG and increased VILIP-1 expressions in hippocampus. Postoperative pain also increased pro-inflammatory factors, cyclin D1 and decreased anti-inflammatory factors in hippocampus. However, these changes were all reversed by morphine analgesia. Bioinformatics analysis identified the critical role of exosome miR-124-3p in cognitive impairment, which was confirmed to be down-regulated in hippocampus of postoperative pain mice. BV-2 microglia overexpressing exosome miR-124-3p showed decreased pro-inflammatory factors, cyclin D1 and increased anti-inflammatory factors. In vivo, stereotactic injection of exogenous miR-124-3p into hippocampus decreased pro-inflammatory factors, cyclin D1 and increased anti-inflammatory factors. The cognitive impairment, axon demyelination, decreased BDNF, NG and increased VILIP-1 expressions in hippocampus were all alleviated by exogenous exosome miR-124-3p. Microglial exosome miR-124-3p in hippocampus alleviates cognitive impairment induced by postoperative pain through microglia polarization in elderly mice.

SIRT2 overexpression decreases remifentanil-stimulated post-surgical hyperalgesia via microglia.

Remifentanil (Remi)-induced hyperalgesia is a serious but common postoperative clinical problem. Sirtuin 2 (SIRT2) is essential in the pathogenetic mechanisms of several neurological disorders. However, whether SIRT2 contributes to the modulation of Remi-induced postsurgical hyperalgesia (POH) is unknown. Here, we investigated the regulatory potential of SIRT2 in Remi-stimulated POH. A rat Remi-stimulated POH model was built by infusing Remi in the surgical incision. Mechanical allodynia and thermal hyperalgesia were separately assessed by paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) measurements. SIRT2 and binding adaptor molecule 1 (Iba1) protein expressions and localization in spinal cord samples were detected by western blot and immunofluorescence. The results revealed SIRT2 downregulation in the spinal cord of rats with Remi-stimulated POH. Intrathecal administration of the overexpression plasmid harboring SIRT2 remarkably relieved mechanical allodynia, along with thermal hyperalgesia in the model animals. Iba1 amounts were increased upon intraoperative incision or Remi infusion, and this effect was more pronounced upon combining both treatments. Furthermore, SIRT2 overexpression suppressed microglia activation in the spinal cord of model animals, and starkly relieved incision- and/or Remi-associated pronociceptive processes as well as spinal microglia activation. SIRT2 elevation relieved Remi-associated POH, likely by suppressing spinal microglia activation. Thus, SIRT2 could be a potent target for treating neuropathic pain.

Dehydrocorydaline alleviates sleep deprivation-induced persistent postoperative pain in adolescent mice through inhibiting microglial P2Y12 receptor expression in the spinal cord.

During adolescence, a second period of central nervous system (CNS) plasticity that follows the fetal period, which involves sleep deprivation (SD), becomes apparent. SD during adolescence may result in abnormal development of neural circuits, causing imbalance in neuronal excitation and inhibition, which not only results in pain, but increases the chances of developing emotion disorders in adulthood, such as anxiety and depression. The quantity of surgeries during adolescence is also consistently on the rise, yet the impact and underlying mechanism of preoperative SD on postoperative pain remain unexplored. This study demonstrates that preoperative SD induces upregulation of the P2Y12 receptor, which is exclusively expressed on spinal microglia, and phosphorylation of its downstream signaling pathway p38Mitogen-activated protein/Nuclear transcription factor-κB (p38MAPK/NF-κB)in spinal microglia, thereby promoting microglia activation and microglial transformation into the proinflammatory M1 phenotype, resulting in increased expression of proinflammatory cytokines that exacerbate persisting postoperative incisional pain in adolescent mice. Both intrathecal minocycline (a microglia activation inhibitor) and MRS2395 (a P2Y12 receptor blocker) effectively suppressed microglial activation and proinflammatory cytokine expression. Interestingly, supplementation with dehydrocorydaline (DHC), an extract of Rhizoma Corydalis, inhibited the P2Y12/p38MAPK/NF-κB signaling pathway, microglia activation, and expression of pro-inflammatory cytokines in the model mice. Taken together, the results indicate that the P2Y12 receptor and microglial activation are important factors in persistent postoperative pain caused by preoperative SD in adolescent mice and that DHC has analgesic effects by acting on these targets.

NETO2-GluK2 interaction contributes to postoperative pain hypersensitivity through inducing PKCγ activation and synaptic incorporation of AMPA receptor GluR1 subunits in rat dorsal horn.

Important roles in the initiation and maintenance of postoperative pain are played by the functional control of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the rat dorsal horn (DH). However, the mechanisms underpinning the cross-talk between spinal KA and AMPA receptors in postoperative pain are poorly understood. We hypothesized that after the rat's plantar incision, the synaptic incorporation of AMPA receptor GluR1 subunits in the DH ipsilateral to the incision would increase due to the interaction between GluK2 and neuropilin tolloid-like 2 (NETO2). Our findings showed that incision stimuli caused severe pain responses, as measured by cumulative pain scores. GluK2-NETO2 but not GluK2-NETO1interaction was upregulated in ipsilateral dorsal horn neurons (DHNs) at 6 h post-incision. At 6 h post-incision, NETO2 small interfering ribonucleic acid (siRNA) intrathecal pretreatment increased mechanical withdrawal thresholds to von Freys and decreased ipsilateral paw cumulative pain scores. Further, PKCγactivation and synaptic abundance of GluK2 and GluR1 subunits in the ipsilateral DH were decreased by intrathecal pretreatment with NETO2 siRNA at 6 h post-incision. In conclusion, our findings imply that GluK2-NETO2 interaction could trigger PKCγactivation and the synaptic incorporation of AMPA receptor GluR1 subunits in rat DHs, which in turn led to the enhanced pain hypersensitivity after surgery. It sheds light on the interplay between KA and AMPA receptors in DHNs, which is thought to contribute to postoperative pain.

Role of magnesium-L-Threonate in alleviating skin/muscle incision and retraction induced mechanical allodynia and anxiodepressive-like behaviors in male rats.

Chronic postsurgical pain (CPSP) and its emotional comorbidities poses health burden to patients who have received the surgical treatment. However, its underlying mechanism remains unclear. Emerging studies indicate that magnesium deficiency is associated with neurological diseases, and magnesium supplement confers protection under these disease conditions. In this study, we examined the role and mechanism of magnesium deficiency in the pathology of surgery-induced allodynia and negative emotion using a rat model of skin/muscle incision and retraction (SMIR) and investigated the therapeutic effects of magnesium supplementation by oral magnesium-L-Threonate (L-TAMS) in SMIR-injured rats. In the SMIR model, rats developed mechanical allodynia and anxiodepressive-like behaviors. Further, SMIR caused microglia and astrocyte activation and enhanced expression of pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) in the anterior cingulate cortex (ACC). Importantly, magnesium ion (Mg2+) levels decreased in the serum and cerebrospinal fluid (CSF) of SMIR-injured rats, which exhibited high correlation with pain and emotion behavioral phenotypes in these rats. Repeated oral administration of L-TAMS increased serum and CSF levels of Mg2+ in SMIR-injured rats. Notably, L-TAMS administration reversed SMIR-induced mechanical allodynia and anxiodepressive-like behaviors but did not affect pain and emotional behaviors in sham rats. Moreover, L-TAMS administration suppressed SMIR-caused glial activation and proinflammatory cytokine expression in the ACC but had no such effect in sham rats. Together, our study demonstrates the contributing role of magnesium deficiency in the pathology of surgery-induced chronic pain and negative emotion. Moreover, we suggest that L-TAMS might be a novel approach to treat CPSP and its emotional comorbidities.

STING-IFN-I pathway relieves incision induced acute postoperative pain via inhibiting the neuroinflammation in dorsal root ganglion of rats.

BACKGROUND: The purpose of this study was to study the effect of STING-IFN-I pathway on incision induced postoperative pain in rats and its possible mechanisms. METHODS: The pain thresholds were evaluated by measuring the mechanical withdrawal threshold and the thermal withdrawal latency. The satellite glial cell and macrophage of DRG were analyzed. The expression of STING, IFN-a, P-P65, iNOS, TNF-α, IL-1ß and IL-6 in DRG was evaluated. RESULTS: The activation of STING-IFN-I pathway can reduce the mechanical hyperalgesia, thermal hyperalgesia, down-regulate the expression of P-P65, iNOS, TNF-α, IL-1ß and IL-6, and inhibit the activation of satellite glial cell and macrophage in DRG. CONCLUSIONS: The activation of STING-IFN-I pathway can alleviate incision induced acute postoperative pain by inhibiting the activation of satellite glial cell and macrophage, which reducing the corresponding neuroinflammation in DRG.

Sestrin2 prevents neonatal incision pain and re-incision enhanced hyperalgesia in adult rats.

BACKGROUND: Improving the methods for recognizing pain is important for infants admitted to the neonatal intensive care unit. Sestrin2 is a novel stress-inducible protein with a neuroprotective role that functions as a molecular mediator of hormesis. Nevertheless, the role of sestrin2 in the pain process is still unclear. The following study examined the role of sestrin2 on mechanical hypersensitivity after pups incision, as well as enhanced pain hyperalgesia after adulthood re-incision in rats. METHODS: The experiment was divided into two parts: (1) studying the effect of sestrin2 in the neonatal incision; (2) studying the priming effect in adulthood re-incision. An animal model was established in seven-day-old rat pups with a right hind paw incision. Pups were intrathecally administrated rh-sestrin2 (exogenous sestrin2). Paw withdrawal threshold testing was performed to assay mechanical allodynia; tissue was analyzed in ex vivo using Western blot and immunofluorescence. SB203580 was further used to inhibit microglial function and evaluate the sex-dependent effect in adulthood. RESULTS: Sestrin2 expression increased transitorily in the spinal dorsal horn in pups after incision. Administration of rh-sestrin2 improved pups' mechanical hypersensitivity by regulating the AMPK/ERK pathway and alleviated re-incision-induced enhanced hyperalgesia in male and female adult rats. After administration of SB203580 in pups, the mechanical hyperalgesia following re-incision in adult rats was prevented in males but not females; however, the protective effect of SB203580 in males was counteracted by silencing sestrin2. CONCLUSIONS: These data suggest that sestrin2 prevents neonatal incision pain and re-incision enhanced hyperalgesia in adult rats. Moreover, microglia inhibition affects enhanced hyperalgesia only in adult males, which may be regulated through the sestrin2 mechanism. To sum up, these sestrin2 data may be a potential common molecular target for treating re-incision hyperalgesia in different sexes.

Hydrogen attenuates postoperative pain through Trx1/ASK1/MMP9 signaling pathway.

BACKGROUND: Postoperative pain is a serious clinical problem with a poorly understood mechanism, and lacks effective treatment. Hydrogen (H2) can reduce neuroinflammation; therefore, we hypothesize that H2 may alleviate postoperative pain, and aimed to investigate the underlying mechanism. METHODS: Mice were used to establish a postoperative pain model using plantar incision surgery. Mechanical allodynia was measured using the von Frey test. Cell signaling was assayed using gelatin zymography, western blotting, immunohistochemistry, and immunofluorescence staining. Animals or BV-2 cells were received with/without ASK1 and Trx1 inhibitors to investigate the effects of H2 on microglia. RESULTS: Plantar incision surgery increased MMP-9 activity and ASK1 phosphorylation in the spinal cord of mice. MMP-9 knockout and the ASK1 inhibitor, NQDI-1, attenuated postoperative pain. H2 increased the expression of Trx1 in the spinal cord and in BV-2 cells. H2 treatment mimicked NQDI1 in decreasing the phosphorylation of ASK1, p38 and JNK. It also reduced MMP-9 activity, downregulated pro-IL-1ß maturation and IBA-1 expression in the spinal cord of mice, and ameliorated postoperative pain. The protective effects of H2 were abolished by the Trx1 inhibitor, PX12. In vitro, in BV-2 cells, H2 also mimicked NQDI1 in inhibiting the phosphorylation of ASK1, p38, and JNK, and also reduced MMP-9 activity and decreased IBA-1 expression induced by LPS. The Trx1 inhibitor, PX12, abolished the protective effects of H2 in BV-2 cells. CONCLUSIONS: For the first time, the results of our study confirm that H2 can be used as a therapeutic agent to alleviate postoperative pain through the Trx1/ASK1/MMP9 signaling pathway. MMP-9 and ASK1 may be the target molecules for relieving postoperative pain.

Pretreatment of the ROS Inhibitor Phenyl-N-tert-butylnitrone Alleviates Sleep Deprivation-Induced Hyperalgesia by Suppressing Microglia Activation and NLRP3 Inflammasome Activity in the Spinal Dorsal Cord.

Sleep deprivation, a common perioperative period health problem, causes ocular discomfort and affects postsurgical pain. However, the mechanism of sleep deprivation-induced increased pain sensitivity is elusive. This study aims to explore the role of ROS in sleep deprivation (SD)-induced hyperalgesia and the underlying mechanism. A 48-h continuous SD was performed prior to the hind paw incision pain modeling in mice. We measured ROS levels, microglial activation, DNA damage and protein levels of iNOS, NLRP3, p-P65 and P65 in mouse spinal dorsal cord. The involvement of ROS in SD-induced prolongation of postsurgical pain was further confirmed by intrathecal injection of ROS inhibitor, phenyl-N-tert-butylnitrone (PBN). Pretreatment of 48-h SD in mice significantly prolonged postsurgical pain recovery, manifesting as lowered paw withdrawal mechanical threshold and paw withdrawal thermal latency. It caused ROS increase and upregulation of iNOS on both Day 1 and 7 in mouse spinal dorsal cord. In addition, upregulation of NLRP3 and p-P65, microglial activation and DNA damage were observed in mice pretreated with 48-h SD prior to the incision. Notably, intrathecal injection of PBN significantly reversed the harmful effects of SD on postsurgical pain recovery, hyperalgesia, microglial activation and DNA damage via the NF-κB signaling pathway. Collectively, ROS increase is responsible for SD-induced hyperalgesia through activating microglial, triggering DNA damage and enhancing NLRP3 inflammasome activity in the spinal dorsal cord.

Intraoperative abobotulinumtoxinA alleviates pain after surgery and improves general wellness in a translational animal model.

Pain after surgery remains a significant healthcare challenge. Here, abobotulinumtoxinA (aboBoNT-A, DYSPORT) was assessed in a post-surgical pain model in pigs. Full-skin-muscle incision and retraction surgery on the lower back was followed by intradermal injections of either aboBoNT-A (100, 200, or 400 U/pig), vehicle (saline), or wound infiltration of extended-release bupivacaine. We assessed mechanical sensitivity, distress behaviors, latency to approach the investigator, and wound inflammation/healing for 5-6 days post-surgery. We followed with immunohistochemical analyses of total and cleaved synaptosomal-associated protein 25 kD (SNAP25), glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor protein-1(Iba1), calcitonin gene-related peptide (CGRP) and substance P (SP) in the skin, dorsal root ganglia (DRG) and the spinal cord of 400 U aboBoNT-A- and saline-treated animals. At Day 1, partial reversal of mechanical allodynia in aboBoNT-A groups was followed by a full reversal from Day 3. Reduced distress and normalized approaching responses were observed with aboBoNT-A from 6 h post-surgery. Bupivacaine reversed mechanical allodynia for 24 h after surgery but did not affect distress or approaching responses. In aboBoNT-A-treated animals cleaved SNAP25 was absent in the skin and DRG, but present in the ipsilateral dorsal horn of the spinal cord. In aboBoNT-A- versus saline-treated animals there were significant reductions in GFAP and Iba1 in the spinal cord, but no changes in CGRP and SP. Analgesic efficacy of aboBoNT-A appears to be mediated by its activity on spinal neurons, microglia and astrocytes. Clinical investigation to support the use of aboBoNT-A as an analgesic drug for post-surgical pain, is warranted.

Thalamocortical circuits drive remifentanil-induced postoperative hyperalgesia.

Remifentanil-induced hyperalgesia (RIH) is a severe but common postoperative clinical problem with elusive underlying neural mechanisms. Here, we discovered that glutamatergic neurons in the thalamic ventral posterolateral nucleus (VPLGlu) exhibited significantly elevated burst firing accompanied by upregulation of Cav3.1 T-type calcium channel expression and function in RIH model mice. In addition, we identified a glutamatergic neuronal thalamocortical circuit in the VPL projecting to hindlimb primary somatosensory cortex glutamatergic neurons (S1HLGlu) that mediated RIH. In vivo calcium imaging and multi-tetrode recordings revealed heightened S1HLGlu neuronal activity during RIH. Moreover, preoperative suppression of Cav3.1-dependent burst firing in VPLGlu neurons or chemogenetic inhibition of VPLGlu neuronal terminals in the S1HL abolished the increased S1HLGlu neuronal excitability while alleviating RIH. Our findings suggest that remifentanil induces postoperative hyperalgesia by upregulating T-type calcium channel-dependent burst firing in VPLGlu neurons to activate S1HLGlu neurons, thus revealing an ion channel-mediated neural circuit basis for RIH that can guide analgesic development.

Preoperative Acute Sleep Deprivation Causes Postoperative Pain Hypersensitivity and Abnormal Cerebral Function.

Preoperative sleep loss can amplify post-operative mechanical hyperalgesia. However, the underlying mechanisms are still largely unknown. In the current study, rats were randomly allocated to a control group and an acute sleep deprivation (ASD) group which experienced 6 h ASD before surgery. Then the variations in cerebral function and activity were investigated with multi-modal techniques, such as nuclear magnetic resonance, functional magnetic resonance imaging, c-Fos immunofluorescence, and electrophysiology. The results indicated that ASD induced hyperalgesia, and the metabolic kinetics were remarkably decreased in the striatum and midbrain. The functional connectivity (FC) between the nucleus accumbens (NAc, a subregion of the ventral striatum) and the ventrolateral periaqueductal gray (vLPAG) was significantly reduced, and the c-Fos expression in the NAc and the vLPAG was suppressed. Furthermore, the electrophysiological recordings demonstrated that both the neuronal activity in the NAc and the vLPAG, and the coherence of the NAc-vLPAG were suppressed in both resting and task states. This study showed that neuronal activity in the NAc and the vLPAG were weakened and the FC between the NAc and the vLPAG was also suppressed in rats with ASD-induced hyperalgesia. This study highlights the importance of preoperative sleep management for surgical patients.

CAV-1 Overexpression Exacerbates the Manifestation in EPAC-1-Induced Chronic Postsurgical Pain in Rats.

Purpose: Caveolae (CAV) are an invaginated microcapsule with the shape of Ω on the surface of the cell membrane. Caveolin-1 (CAV-1) is involved in neuropathic pain, and adenosine monophosphate (AMP)-exchange protein directly activated by cAMP1 (EPAC-1) is a potential therapeutic target for chronic pain. However, whether EPAC-1 promotes chronic postsurgical pain (CPSP) through CAV-1 has not been reported. Here, we aim to investigate the underlying mechanism of CAV in CPSP. Methods: All the rats were divided into 9 groups, including the Naive group, Sham group, skin/muscle incision and retraction (SMIR) group, SMIR + CAV-1 siRNA group, SMIR + control siRNA group, SMIR (7 days)+Saline group, SMIR (7 days)+CE3F4 group, 8-PCPT group, and Saline group. The CPSP rat model was established after SMIR. A mechanical withdrawal threshold (MWT) was recorded to evaluate the animal's behavior. Western blotting and immunofluorescent were performed to detect the protein expression levels of EPAC-1 and P-CAV-1. Results: EPAC-1 and CAV-1 were both overexpressed after operation, particularly in astrocytes, microglia, and neurons of spinal marrow (all P < 0.05). Interestingly, CAV-1 siRNA can partly reverse the SMIR-induced hypersensitivity, but there was no effect on EPAC-1. Besides, EPAC-1 blockage partly reversed the SMIR-induced hypersensitivity and CAV-1 overexpression, and EPAC-1 activation promoted CAV-1 overexpression and hypersensitivity in normal rats (all P < 0.05). Conclusion: CAV-1 mediates the functional coupling of microglia, astrocytes, and neurons, and thus EPAC-1/CAV-1 plays an important role in CPSP exacerbation.

Upregulation of Hevin contributes to postoperative pain hypersensitivity by inducing neurexin1ß/neuroligin1-mediated synaptic targeting of GluA1-containing AMPA receptors in rat dorsal horn.

The astrocytes-secreted active molecule, Hevin considerably contributes in the transsynaptic bridge of neurexin1ß/neuligin1 in excitatory synapse. Previous studies have demonstrated that activity-dependent synaptic recruitment of spinal neuroligin1 and GluA1-containing AMPA receptors (AMPARs) is involved in incisional, inflammatory and neuropathic pain. Here, we hypothesized that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1ß/neuroligin1-mediated synaptic targeting of GluA1-containing AMPARs in spinal dorsal horns (DH). Our results showed that plantar incision induced significant postoperative pain behavior, which was described by the cumulative pain scores. At 1 d and 3 d post-incision, Hevin expression was considerably elevated in ipsilateral DHs, although it recovered to baseline value at 5 d following the incision. At 1 d post plantar incision, the neurexin1ß/neuroligin1 interactions significantly increased in ipsilateral DHs in rats subjected to incision when compared with those in control rats. Intrathecal pretreatments of small interference RNA targeting Hevin substantially suppressed postoperative pain hypersensitivity and reduced the neurexin1ß/neurolgin1 interaction as well as the synaptic targeting of GluA1 in ipsilateral spinal DHs. These data suggest that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1ß/neuroligin1 interaction and subsequent synaptic targeting of GluA1-containing AMPARs in ipsilateral spinal DHs. It provides new insights into the role of Hevin-mediated trans-synaptic regulation in postoperative pain hypersensitivity, which would help develop a novel therapeutic strategy.

Postsurgical Latent Pain Sensitization Is Driven by Descending Serotonergic Facilitation and Masked by µ-Opioid Receptor Constitutive Activity in the Rostral Ventromedial Medulla.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MORCA) in the dorsal horn of the spinal cord. To demonstrate LS, we conducted plantar incision in mice and then waited 3-4 weeks for hypersensitivity to resolve. At this time (remission), systemic administration of the opioid receptor antagonist/inverse agonist naltrexone reinstated mechanical and heat hypersensitivity. We first tested the hypothesis that LS extends to serotonergic neurons in the rostral ventral medulla (RVM) that convey pronociceptive input to the spinal cord. We report that in male and female mice, hypersensitivity was accompanied by increased Fos expression in serotonergic neurons of the RVM, abolished on chemogenetic inhibition of RVM 5-HT neurons, and blocked by intrathecal injection of the 5-HT3R antagonist ondansetron; the 5-HT2AR antagonist MDL-11 939 had no effect. Second, to test for MORCA, we microinjected the MOR inverse agonist d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and/or neutral opioid receptor antagonist 6ß-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws; 6ß-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MORCA, rather than an opioid ligand-dependent mechanism, maintains LS in remission. We conclude that incision establishes LS in descending RVM 5-HT neurons that drives pronociceptive 5-HT3R signaling in the dorsal horn, and this LS is tonically opposed by MORCA in the RVM. The 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.SIGNIFICANCE STATEMENT Surgery leads to latent pain sensitization and a compensatory state of endogenous pain control that is maintained long after tissue healing. Here, we show that either chemogenetic inhibition of serotonergic neuron activity in the RVM or pharmacological inhibition of 5-HT3 receptor signaling at the spinal cord blocks behavioral signs of postsurgical latent sensitization. We conclude that MORCA in the RVM opposes descending serotonergic facilitation of LS and that the 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.

The transcription factor CCAAT/enhancer-binding protein ß in spinal microglia contributes to pre-operative stress-induced prolongation of postsurgical pain.

Prolongation of postsurgical pain caused by pre-operative stress is a clinically significant problem, although the mechanisms are not fully understood. Stress can promote the pro-inflammatory activation of microglia, and the transcription factor CCAAT/enhancer-binding protein (C/EBP) ß regulates pro-inflammatory gene expression in microglia. Therefore, we speculated that C/EBPß in spinal microglia may have critical roles in the development of chronic postsurgical pain. Accordingly, in this study, we used a single prolonged stress (SPS) procedure and plantar incisions to evaluate the roles of C/EBPß in postsurgical pain. Our experiments showed that SPS exposure prolonged mechanical allodynia, increased the expression of C/EBPß and pro-inflammatory cytokines, and potentiated the activation of spinal microglia. Subsequently, microinjection of C/EBPß siRNA attenuated the duration of SPS-prolonged postoperative mechanical allodynia and inhibited microglial activation in the spinal cord. Conversely, mimicking this increase in C/EBPß promoted microglial activation via pretreatment with a pre-injection of AAV5-C/EBPß, leading to prolongation of postsurgical pain. Overall, these results suggested that spinal microglia may play key roles in prolongation of postsurgical pain induced by pre-operative stress and that C/EBPß may be a potential target for disease treatment.

Dezocine relieves the postoperative hyperalgesia in rats through suppressing the hyper-action of Akt1/GSK-3ß pathway.

The relieving role of dezocine in pain after surgery was previously reported, while the potential mechanism was not completely clear. Therefore, the current research probed into the regulatory mechanism of dezocine in pain after surgery. A postoperative pain model was established by performing plantar incision surgery on the juvenile Sprague-Dawley rats. After the rats were treated with dezocine or SC79 (Akt1 activator), the paw withdrawal threshold and paw withdrawal latency of rats were detected to evaluate the mechanical allodynia and thermal hyperalgesia. After the plantar tissue, dorsal root ganglions, and spinal cord of rats were collected, the expressions of Akt1, p-Akt1, GSK-3ß, and p-GSK-3ß in the tissues were determined by western blot to evaluate the activation state of the Akt1/GSK-3ß pathway. After surgery, the paw withdrawal threshold and paw withdrawal latency of rats were lessened, whereas the ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß were augmented in rat plantar tissue, dorsal root ganglions, and spinal cord. After treatment with dezocine alone, the paw withdrawal threshold and paw withdrawal latency of postoperative rats were elevated, but ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß were reduced. After co-treatment with dezocine and SC79, SC79 reversed the effects of dezocine on elevating the paw withdrawal threshold and paw withdrawal latency, and reducing the ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß in postoperative rats. Dezocine ameliorated the postoperative hyperalgesia in rats via repressing the hyper-action of Akt1/GSK-3ß pathway.