Mitochondrial Dysfunction of Peripheral Platelets as a Predictive Biomarker for Postoperative Delirium in Elderly Patients.

OBJECTIVE: To determine the association between the preoperative Bioenergetic Health Index (BHI) of platelets and the occurrence of postoperative delirium (POD) in elderly patients. METHODS: Elderly patients scheduled for major abdominal surgery under general anesthesia were included. The presence of POD was assessed within the 3 days after surgery. Seahorse XF analysis and transmission electron microscopy were utilized to evaluate the mitochondrial metabolism and morphology of platelets. RESULTS: A total of 20 out of 162 participants developed POD. Participants with POD showed lower preoperative Mini-Mental State Examination scores and total protein levels, fewer educational years, longer surgery duration, higher mean platelet volume, and lower platelet BHI compared with those without POD. Damaged mitochondria with swollen appearance and distorted cristae was detected in platelets from participants with POD. Preoperative platelet BHI was independently associated with the occurrence of POD after adjusting for age, education, preoperative Mini-Mental State Examination score, preoperative mean platelet volume and total protein levels, surgical type and duration, and lymphocyte counts on the first postoperative day (OR 0.11, 95% CI 0.03-0.37, p < 0.001). The areas under the receiver operating curves for predicting POD were 0.83 (95% CI 0.76-0.88) for platelet BHI. It showed a sensitivity of 85.00% and specificity of 73.24%, with an optimal cutoff value of 1.61. Using a serial combination (mean platelet volume followed by BHI) yielded a sensitivity of 80.00% and specificity of 82.39%. INTERPRETATION: Preoperative platelet BHI was independently associated with the occurrence of POD in elderly patients and has the potential as a screening biomarker for POD risk. ANN NEUROL 2024.

Microglia Promote Inhibitory Synapse Phagocytosis in the Spinal Cord Dorsal Horn and Modulate Pain-Like Behaviors in a Murine Cancer-Induced Bone Pain Model.

BACKGROUND: The microglial activation has been implicated in cancer-induced bone pain. Recent studies have revealed that microglia mediate synaptic pruning in the central nervous system, where the cluster of differentiation 47-signal regulatory protein α (CD47-SIRPα) axis creates a "don't eat me" signal and elicits an antiphagocytic effect to protect synapses against elimination. To date, the synaptic phagocytosis in microglia has never been investigated in the murine cancer-induced bone pain model. The present experiments sought to explore whether microglia phagocytize synapses in mice with bone cancer pain as well as the possible mechanisms. METHODS: Male C3H/HeN mice were used to induce bone cancer pain. Minocycline and S-ketamine were injected into D14. The number of spontaneous flinches (NSF) and paw withdrawal mechanical thresholds (PWMT) were measured on D0, D4, D7, D10, D14, D21, and D28. Hematoxylin and eosin staining presented bone lesions. Western blotting examined the Gephyrin, CD47, and SIRPα expression. Flow cytometry evaluated the proportion of SIRPα+ cells in the spine. Immunofluorescence and 3-dimensional reconstruction showed the Gephyrin puncta inside microglial lysosomes. RESULTS: Mice embedded with tumor cells induced persistent spontaneous pain and mechanical hyperalgesia. Hematoxylin and eosin staining revealed bone destruction and tumor infiltration in marrow cavities. Microglia underwent a responsive and proliferative burst (t = -16.831, P < .001). Western blotting manifested lowered Gephyrin expression in the tumor group (D4, D7, D10, D14, D21, and D28: P < .001). Immunofluorescence and 3-dimensional reconstruction showed larger volumes of Gephyrin puncta inside microglial lysosomes (t = -23.273, P < .001; t = -27.997, P < .001). Treatment with minocycline or S-ketamine exhibited pain relief and antiphagocytic effects (t = -6.191, P < .001, t = -7.083, P < .001; t = -20.767, P < .001, t = -17.080, P < .001; t = 11.789, P < .001, t = 16.777, P < .001; t = 8.868, P < .001, t = 21.319, P < .001). Last but not least, the levels of CD47 and SIRPα proteins were downregulated (D10: P = .004, D14, D21, and D28: P < .001; D10, D14, D21, and D28: P < .001). Flow cytometry and immunofluorescence substantiated reduced microglial SIRPα (t = 11.311, P < .001; t = 12.189, P < .001). CONCLUSIONS: Microglia-mediated GABAergic synapse pruning in the spinal cord dorsal horn in bone cancer pain mice, which might be associated with the declined CD47-SIRPα signal. Our research uncovered an innovative mechanism that highlighted microglia-mediated synaptic phagocytosis in a murine cancer-induced bone pain model.

Complex Topology of Ubiquitin Chains Mediates Lysosomal Degradation of MrgC Proteins.

BACKGROUND: Activation of Mas-related G protein-coupled receptor C (MrgC) receptors relieves pain, but also leads to ubiquitination of MrgC receptors. Ubiquitination mediates MrgC receptor endocytosis and degradation. However, MrgC degradation pathways and ubiquitin-linked chain types are not known. METHODS: N2a cells were treated with cycloheximide (CHX, protein synthesis inhibitor), Mg132 (proteasome inhibitor), 3-Methyladenine (3MA, autophagy lysosome inhibitor) and Chloroquine (CQ, autophagy lysosome inhibitor) to observe the half-life and degradation pathway of MrgC. The location of internalized MrgC receptors and lysosomes (Lyso-Tracker) was observed by immunofluorescence staining. N2a cells were transfected with Myc-MrgC and a series of HA-tagged ubiquitin mutants to study the ubiquitin-linked chain type of MrgC. RESULTS: The amount of MrgC protein decreased with time after CHX treatment of N2a cells. Autophagy lysosome inhibitors can inhibit the degradation of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. 3-MA and CQ inhibited the degradation of MrgC protein, whereas Mg-132 did not inhibit it. Partially internalized MrgC receptors were co-labeled with lysosomes. MrgC proteins have multiple topologies of ubiquitin-modified chains. CONCLUSION: As a member of the G protein-coupled receptor family, MrgC receptors can be degraded over time. The complex topology of the ubiquitin-linked chain mediates the lysosomal degradation of MrgC proteins.

Association between single nucleotide variants and severe chronic pain in older adult patients after lower extremity arthroplasty.

BACKGROUND: Hip or knee osteoarthritis (OA) is one of the main causes of disability worldwide and occurs mostly in the older adults. Total hip or knee arthroplasty is the most effective method to treat OA. However, severe postsurgical pain leading to a poor prognosis. So, investigating the population genetics and genes related to severe chronic pain in older adult patients after lower extremity arthroplasty is helpful to improve the quality of treatment. METHODS: We collected blood samples from elderly patients who underwent lower extremity arthroplasty from September 2020 to February 2021 at the Drum Tower Hospital Affiliated to Nanjing University Medical School. The enrolled patients provided measures of pain intensity using the numerical rating scale on the 90th day after surgery. Patients were divided into the case group (Group A) and the control group (Group B) including 10 patients respectively by the numerical rating scale. DNA was isolated from the blood samples of the two groups for whole-exome sequencing. RESULTS: In total, 661 variants were identified in the 507 gene regions that were significantly different between both groups (P < 0.05), including CASP5, RASGEF1A, CYP4B1, etc. These genes are mainly involved in biological processes, including cell-cell adhesion, ECM-receptor interaction, metabolism, secretion of bioactive substances, ion binding and transport, regulation of DNA methylation, and chromatin assembly. CONCLUSIONS: The current study shows some variants within genes are significantly associated with severe postsurgical chronic pain in older adult patients after lower extremity arthroplasty, indicating a genetic predisposition for chronic postsurgical pain. The study was registered according to ICMJE guidelines. The trial registration number is ChiCTR2000031655 and registration date is April 6th, 2020.

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.

Basolateral Amygdala Reactive Microglia May Contribute to Synaptic Impairment and Depressive-Like Behavior in Mice with Bone Cancer Pain.

Anxiety and depression induced by cancer-related pain disturb quality of life and willingness to survive. As a component of the limbic system, the basolateral amygdala (BLA) is critical for processing negative emotions. The reactive microglial engulfment of synapses may promote depression during adolescence. However, whether microglia phagocytose synapses to mediate cancer pain-induced depression remains unclear. The present study established a bone cancer-pain model to investigate the association between dendritic spine synapses and depressive-like behavior and explore the phagocytic function of microglia in the BLA. We found that tumor-bearing mice experienced postoperative pain-related depression, and their BLAs exhibited reactive microglia, as well as phagocytic synapses. The microglial inhibitor minocycline effectively mitigated depressive behavior, synaptic damage, and the phagocytic function of microglia. Our study implicates microglia-mediated synaptic loss in the BLA may act as the pathological basis of depressive-like behavior in bone cancer pain model.

Systemic immune-inflammation index within the first postoperative hour as a predictor of severe postoperative complications in upper abdominal surgery: a retrospective single-center study.

BACKGROUND: Systemic pro-inflammatory factors play a critical role in mediating severe postoperative complications (SPCs) in upper abdominal surgery (UAS). The systemic immune-inflammation index (SII) has been identified as a new inflammatory marker in many occasions. The present study aims to determine the association between SII and the occurrence of SPCs after UAS. METHODS: Included in this study were 310 patients with upper abdominal tumors who received UAS and subsequently were transferred to the anesthesia intensive care unit between November 2020 and November 2021 in Nanjing Drum Hospital. SPCs, including postoperative pulmonary complications (PPCs), major adverse cardiac and cardiovascular events, postoperative infections and delirium, were recorded during the hospital stay. The clinical features of the patients with and without SPCs were compared by Student's t-test or Fisher's exact test as appropriate. Risk factors associated with SPC occurrence were evaluated by univariable and multivariable logistic regression analyses. Receiver operating characteristic (ROC) curve analysis was used to establish a cut-off level of SII value to predict SPCs. RESULTS: Of the 310 patients receiving UAS, 103 patients (33.2%) presented at least one SPC, including PPCs (n = 62), adverse cardiovascular events (n = 22), postoperative infections (n = 51), and delirium (n = 5). Both preoperative SII and 1-h postoperative SII in patients with SPCs were significantly higher than those in patients without SPCs. Multivariate analysis showed that 1-h postoperative SII was an independent predictor for SPC occurrence (OR = 1.000, 95% CI 1.000-1.000, P = 0.007), together with postoperative C-reactive protein, postoperative arterial lactate, postoperative oxygenation-index and older age. The ROC curve showed that the optimal cutoff value of 1-h postoperative SII to predict SPCs was 754.6078 × 109/L, with an 88.3% sensitivity and a 29% specificity. Multivariate analysis also confirmed that 1-h postoperative SII > 754.6078 × 109/L was associated with increased SPC occurrence (OR = 2.656, 95% CI 1.311-5.381, P = 0.007). CONCLUSION: Our findings demonstrated an association between the higher level of 1-h postoperative SII and SPCs, suggesting that 1-h postoperative SII, especially categorized 1-h postoperative SII using cutoff value, may be a useful tool for identifying patients at risk of developing SPCs.

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.

Risk factors of postoperative nausea and vomiting following ambulatory surgery: A retrospective case-control study.

Objective: To explore potential risk factors of postoperative nausea and vomiting (PONV) following ambulatory surgery. Method: Clinical data of 1670 cases receiving ambulatory surgery in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from September 2017 to December 2019 were retrospectively analyzed. They were categorized to PONV group and non-PONV group, and perioperative data in both groups were analyzed for assessing risk factors of PONV following ambulatory laparoscopy. Results: There were 156/1,670 (9.3%) PONV cases, and the female and male incidence in recruited cases was 12.0% and 6.0%, respectively. Analyses on perioperative data of them identified that female gender [adjusted odds ratio (aOR) = 2.060, P < 0.001], operation time >1 h (aOR = 1.554, P = 0.011), postoperative pain at rest (aOR = 1.909, P = 0.013) and postoperative pain during activities (aOR = 3.512, P < 0.001) were independent risk factors of PONV following ambulatory surgery. Furthermore, postoperative pain at rest and during activities were linearly, positively correlated to the incidence of PONV. Conclusion: Female gender, operation time >1 h and postoperative pain are closely related with the incidence of PONV following ambulatory surgery. Alleviating postoperative pain properly is one of the methods to reduce risk factors of PONV following ambulatory surgery.

Neuroinflammation-mediated mitochondrial dysregulation involved in postoperative cognitive dysfunction.

Neuroinflammation following peripheral surgery is a pivotal pathogenic mechanism of postoperative cognitive dysfunction (POCD). However, the key site of inflammation-mediated neural damage remains unclear. Impaired mitochondrial function is a vital feature of degenerated neurons. Dynamin-related protein 1 (DRP1), a crucial regulator of mitochondrial dynamics, has been shown to play an essential role in synapse formation. Here, we designed experiments to assess whether Drp1-regulated mitochondrial dynamics and function are involved in the pathological processes of POCD and elucidate its relationship with neuroinflammation. Aged mice were subjected to experimental laparotomy under isoflurane anesthesia. Primary neurons and SH-SY5Y cells were exposed to tumor necrosis factor (TNF). We found an increase in Drp1 activation as well as mitochondrial fragmentation both in the hippocampus of mice after surgery and primary neurons after TNF exposure. Pretreatment with Mdivi-1, a Drp1 specific inhibitor, reduced this mitochondrial fragmentation. Drp1 knockdown with small interfering RNA blocked TNF-induced mitochondrial fragmentation in SH-SY5Y cells. However, the application of Mdivi-1 exhibited a negative impact on mitochondrial function and neurite growth in primary neurons. Calcineurin activity was increased in primary neurons after TNF exposure and contributed to the Drp1 activation. The calcineurin inhibitor FK506 exhibited a Drp1-independent function that mitigated mitochondrial dysfunction. Finally, we found that FK506 pretreatment ameliorated the neurite growth in neurons treated with TNF and the learning ability of mice after surgery. Overall, our research indicated a crucial role of mitochondrial function in the pathological processes of POCD, and neuronal metabolic modulation may represent a novel and important target for POCD.

The analgesic effects of pioglitazone in the bone cancer pain rats via regulating the PPARγ/PTEN/mTOR signaling pathway in the spinal dorsal horn.

BACKGROUND AND OBJECTIVES: Bone cancer pain (BCP) remains a difficult clinical problem. This study examined whether pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, is effective for attenuating BCP, and investigated the interaction between activation of PPARγ and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) / mammalian target of rapamycin (mTOR) signal in the spinal dorsal horn (SDH) of BCP rats. METHODS: We tested the effects of intrathecal (i.t.) injection of adenovirus-mediated PTEN (Ad-PTEN), PTEN antisense oligonucleotide (Ad-antisense PTEN), mTOR inhibitor rapamycin, pioglitazone and PPARγ antagonist GW9662 on bone cancer-induced mechanical allodynia by measuring the paw withdrawal threshold (PWT). Western blot or immunofluorescence examined the expression of spinal PPARγ, PTEN, mTOR, p-mTOR and p-S6K1. RESULTS: Bone cancer did not alter total mTOR expression but caused significant downregulation of PTEN and upregulation of p-mTOR and p-S6K1 in spinal neurons. Rapamycin markedly reduced BCP. Upregulation of spinal PTEN by i.t. Ad-PTEN significantly relieved BCP and downregulated p-mTOR and p-S6K1; while i.t. Ad-antisense PTEN led to the opposite effects of Ad-PTEN. Spinal PPARγ expression increased in BCP rats, co-localizing mainly with neurons and a few astrocytes, but not in microglia. Pioglitazone (500 µg/day i.t. for one week, from 7 days after surgery) attenuated BCP, further increased expression of PPARγ, and inhibited downregulation of PTEN and upregulation of p-mTOR and p-S6K1 in the SDH. Pioglitazone's analgesic effect was enhanced by Ad-PTEN and attenuated by Ad-antisense PTEN. Blockade of PPARγ with GW9662 (300 µg i.t. 15 min prior to pioglitazone) reversed the effects of pioglitazone on BCP and regulations of PPARγ/PTEN/mTOR signal. CONCLUSIONS: Intrathecal pioglitazone administration alleviates BCP by regulating the PPARγ/PTEN/mTOR signal in the SDH. Our data provided new insight in the therapeutic strategy in BCP management.

Spinal cannabinoid receptor 2 activation reduces hypersensitivity associated with bone cancer pain and improves the integrity of the blood-spinal cord barrier.

BACKGROUND: Disruption of the blood-spinal cord barrier (BSCB) can facilitate inflammation that results in pain hypersensitivity. Proinflammatory cytokines produced by activated microglia and astrocytes damage the BSCB. This study aims to explore whether the BSCB is damaged in the bone cancer pain (BCP) model and to investigate a potential role and mechanism of JWH015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone), a selective cannabinoid receptor 2 (CB2R) agonist, in preserving the BSCB integrity in the BCP model. METHODS: We used a male mouse model of BCP. Pain hypersensitivity was measured over time. Evans blue dye extravasation, transmission electron microscopy and Western blotting were performed to investigate the permeability and structural integrity of the BSCB. Immunofluorescence staining and western blotting were used to investigate the effect of JWH015 on the activation of glial cells and the levels of proinflammatory cytokines. RESULTS: A single intrathecal injection of JWH015 ameliorated pain hypersensitivity, the BSCB disruption and microglia and astrocyte activation. Decreases in the expression of ZO-1 and claudin-5 were partially restored by JWH015. The levels of the proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α and the enzyme MMP9 were reduced by JWH015. However, all effects were prevented by pretreatment with a CB2R-selective antagonist, AM630 ((6-iodo-2-methyl-1-(2-morpholinoethyl)-1H-indol-3-yl)(4-methoxyphenyl)methanone). CONCLUSIONS: JWH015 alleviates neuroinflammation and maintains the BSCB integrity and permeability in a mouse model of BCP, which is probably mediated by inhibiting glial cells activation. This study reveals the new analgesic mechanism of JWH015 on BCP and provides a perspective to explore novel drugs that target the BSCB to control BCP.

GPR30 receptor promotes preoperative anxiety-induced postoperative hyperalgesia by up-regulating GABAA-α4ß1δ subunits in periaqueductal gray in female rats.

BACKGROUND: G-protein coupled estrogen receptor 30 (GPR30) was proved the specific estrogen receptor relating to mechanical hyperalgesia. Studies have shown that the GABAA receptor subunits α4, ß1, and δ in the periaqueductal gray (PAG) neurons promote the descending facilitation system. This study inquired into whether and how GPR30 and GABAA-α4ß1δ in the PAG promote preoperative anxiety-induced postoperative hyperalgesia in female rats. METHODS: All the female rats were subjected to the single prolonged stress (SPS) to stimulate preoperative anxiety. Subsequently, mechanical allodynia was evaluated before and after the incision, based on the paw withdrawal mechanical threshold (PWMT). The selective GPR30 agonist G1 and antagonist G15 were locally microinjected into the PAG. The expression of GPR30, protein kinase A (PKA), and GABAA receptor subunits α4, ß1, and δ in the PAG neurons were detected using western blotting and immunofluorescence. RESULTS: Behavioral testing revealed that Group S and Group I decreased the nociceptive threshold levels of PWMT in female rats. PWMT in Group S + I decreased more than that of Group S and Group I. Further, results of western blotting showed the expression of GPR30, PKA, and GABAA α4, ß1, and δ subunits significantly up-regulated in Group S + I, and immunofluorescence indicated that the neurons of PAG in Group S + I appeared simultaneously immunopositive for GPR30 and GABAA α4, ß1, and δ receptors. After microinjection of G1 into the PAG, female rats with plantar incision continued to exhibit significant hyperalgesia until postoperative 48 h. On the other hand, microinjection of G15 with SPS and plantar incision procedure relieved postoperative hyperalgesia in female rats. Western blotting demonstrated that intra-PAG injection of G15 markedly decreased the GPR30, PKA, and GABAA α4, ß1, and δ levels in Group G15 + I. CONCLUSIONS: Our results indicate that the GPR30-PKA-GABAAα4ß1δ pathway in the PAG promotes preoperative anxiety-induced postoperative hyperalgesia in female rats. This mechanism might be a potential novel therapeutic target for hyperalgesia in females.

Pioglitazone Attenuates Experimental Colitis-Associated Hyperalgesia through Improving the Intestinal Barrier Dysfunction.

Impaired intestinal mucosal integrity during colitis involves the peroxisome proliferator-activated receptor-γ (PPARγ), an important anti-inflammatory factor in intestinal mucosa homoeostasis, which is a potential target in colitis. Recurrent chronic pain is a vital pathogenetic feature of colitis. Nevertheless, potential functions of PPARγ in the colitis-associated hyperalgesia remain unclear. This study aimed to investigate biological roles of pioglitazone in relieving colitis-associated pain hypersensitivity by a PPARγ tight junction protein-dependent mechanism during the course of dextran sodium sulfate (DSS)-induced intestinal inflammation. The DSS-induced colitis model was generated in C57BL/6 mice. Changes in colitis induced the injury of intestinal mucosal barrier and hyperalgesia after a 6-day treatment of pioglitazone (25 mg/kg, IP injection) were assessed through immunofluorescent, hematoxylin and eosin (H&E) staining, western blot analysis, and determination of paw withdrawal mechanical threshold. A significant reduction of paw withdrawal mechanical threshold occurred after DSS treatment. Follow-up data showed that systematic administration of PPARγ agonist pioglitazone ameliorated the DSS-induced colitis and the development of colitis-associated hyperalgesia by repairing the intestinal mucosal barrier. The tight junction proteins ZO-1 and Claudin-5 were upregulated by PPARγ signaling, which in turn promoted the improvement of intestinal barrier function. Moreover, pioglitazone inhibited phosphorylation of ERK and NF-κB in the colon and decreased the levels of inflammatory cytokines in both colon spine tissues. Furthermore, systemically pioglitazone treatment inhibited the activation of microglia and astrocytes, as well as DSS-induced phosphorylation of NR2B subunit in spinal cord, which was correspondingly consistent with the pain behavior. Pioglitazone ameliorates DSS-induced colitis and attenuates colitis-associated mechanical hyperalgesia, with improving integrity of the intestinal mucosal barrier by directly upregulating tight junction proteins. The PPARγ-tight junction protein signaling might be a potential therapeutic target for the treatment of colitis-associated chronic pain.

The Ubiquitination of Spinal MrgC Alleviates Bone Cancer Pain and Reduces Intracellular Calcium Concentration in Spinal Neurons in Mice.

Mas-related G-protein-coupled receptor subtype C (MrgC) has been shown to play an important role in the development of bone cancer pain. Ubiquitination is reported to participate in pain. However, whether MrgC ubiquitination plays a role in bone cancer pain remains unclear. To answer this question, we designed and performed this study. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer pain. MrgC agonist bovine adrenal medulla 8-22 (BAM 8-22) or MrgC antagonist anti-MrgC antibody were injected intrathecally on day 14 after bone cancer pain was successfully induced. The pain behaviors, the MrgC ubiquitination levels and intracellular calcium concentration in spinal neurons were measured before and after injection, respectively. With comparison to normal and sham group, mice in tumor group exhibited serious bone cancer pain on day 14, and the level of MrgC ubiquitination and intracellular calcium concentration in spinal neurons was significantly higher. Intrathecal injection of BAM 8-22 significantly alleviated bone cancer pain, increased the MrgC ubiquitination level and decreased intracellular calcium concentration in spinal neurons; however, these effects were reversed by administration of anti-MrgC antibody. Our study reveals that MrgC ubiquitination participates in the production and maintenance of bone cancer pain in mice, possibly through the regulation of intracellular calcium concentration in mice spinal neurons.

Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor.

BACKGROUND: Preoperative anxiety is associated with postoperative hyperalgesia; however, few studies have investigated the mechanism underlying this association in female surgical patients. Research has suggested that ON cells in the rostral ventromedial medulla (RVM) receive nerve impulses via cholecystokinin 2 (CCK2) receptors, facilitating hyperalgesia. Additionally, the downstream serotonergic projection system from the RVM to the spinal cord has a dual regulating effect on pain responses, and the 5-hydoxytryptophan 2B (5-HT2B) receptor in spinal dorsal horn neurons is critically involved in mechanical allodynia. METHODS: Ovariectomized rats were treated with estrogen replacement, single prolonged stress (SPS), and plantar incision. Various receptor agonists and antagonists were then administered into the RVM and spinal cord to study the mechanism underlying postoperative hyperalgesia caused by preoperative anxiety in female rats. RESULTS: Behavioral testing revealed that preoperative SPS induced postoperative hyperalgesia, as well as the expression of the CCK2 receptor in the RVM and the expression of the 5-HT2B receptor, protein kinase Cγ (PKCγ), and phosphorylation of the N-methyl-d-aspartate receptor1 (p-NR1) in the spinal cord increased confirmed by Western blot. RVM microinjection of the CCK2 receptor agonist CCK-8 and intrathecal injection of the 5-HT2B receptor agonist BW723C86 both produced hyperalgesia in female rats after plantar incision, whereas the CCK2 receptor antagonist YM022, the 5-HT2B receptor antagonist RS127445, and the PKCγ inhibitor C37H65N9O13 decreased the rats' sensitivity to the same stimulus. Additionally, electrophysiological analysis suggested that activation of the 5-HT2B receptor increased the whole-cell current (IBa) in superficial dorsal horn neurons through the PKCγ pathway. CONCLUSION: Our study demonstrated that preoperative anxiety-induced postoperative hyperalgesia in female rats is associated with descending pain pathways. The CCK2 receptor in the RVM and spinal 5-HT2B receptor may play a role in this hyperalgesic effect.

Time-Efficient Allocation Mechanisms for Crowdsensing Tasks with Precedence Constraints.

Crowdsensing has emerged as an efficient and inexpensive way to perform specialized tasks by leveraging external crowds. In some crowdsensing systems, different tasks may have different requirements, and there may be precedence constraints among them, such as the Unmanned Aerial Vehicle (UAV) crowdsensing systems. Moreover, minimizing the total execution time is a regular target for finishing the crowdsensing tasks with precedence constraints. As far as we know, only a few existing studies consider the precedence constraints among crowdsensing tasks, and none of them can minimize the total execution time simultaneously. To tackle this challenge, an efficient allocation mechanism for tasks with precedence constraints is first proposed, which can minimize the total execution time. Then, a case study is given to show how to fit our mechanism in the UAV crowdsensing system. Finally, the simulation results show that the proposed mechanisms have good approximate optimal ratios under different parameter settings and are efficient for the UAV crowdsensing system as well.

Perioperative activation of spinal α7 nAChR promotes recovery from preoperative stress-induced prolongation of postsurgical pain.

Preoperative stress could delay the recovery of postoperative pain and has been reported to be a risk factor for chronic postsurgical pain. As stress could facilitate the proinflammatory activation of microglia, we hypothesized that these cells may play a vital role in the development of preoperative stress-induced pain chronification after surgery. Our experiments were conducted in a rat model that consists of a single prolonged stress (SPS) procedure and plantar incision. A previous SPS exposure induced anxiety-like behaviors, prolonged incision-induced mechanical allodynia, and potentiated the activation of spinal microglia. Based on the results from ex vivo experiments, spinal microglia isolated from SPS-exposed rats secreted more proinflammatory cytokines upon challenge with LPS. Our results also demonstrated that microglia played a more important role than astrocytes in the initiation of SPS-induced prolongation of postsurgical pain. We further explored the therapeutic potential of agonism of α7 nAChR, an emerging anti-inflammatory target, for SPS-induced prolongation of postsurgical pain. Multiple intrathecal (i.t.) injections of PHA-543613 (an α7 nAChR agonist) or PNU-120596 (a type II positive allosteric modulator) during the perioperative period shortened the duration of postsurgical pain after SPS and suppressed SPS-potentiated microglia activation, but their effects were abolished by pretreatment with methyllycaconitine (an α7 nAChR antagonist; i.t.). Based on the results from ex vivo experiments, the anti-inflammatory effects of PHA-543613 and PNU-120596 may have been achieved by the direct modulation of microglia. In conclusion, stress-induced priming of spinal microglia played a key role in the initiation of preoperative stress-induced prolongation of postsurgical pain, and PHA-543613 and PNU-120596 may be potential candidates for preventing pain chronification after surgery.

Imbalanced spinal infiltration of Th17/Treg cells contributes to bone cancer pain via promoting microglial activation.

Increasing evidence suggests that T cells participate in the pathology of neuropathic pain, as well as the activation of microglia. However, whether T cells infiltrate into the spinal cord and contribute to the development of bone cancer pain (BCP) remains unknown. Here, we used a mouse model of BCP to show that numbers of T cells infiltrated into the spinal cord after sarcoma cell implantation with increased BCP, and most infiltrating T cells in the spinal cord were CD3+CD4+ T cells. Both Th17 and Treg subpopulations were analyzed by immunofluorescence. Treg cells in the spinal cord were transiently up-regulated, followed by an imbalance towards Th17 afterwards, and elevated IL-17/IL-17A levels were observed in both blood and spinal cord. Meanwhile, TGF-ß, IL-6, and IL-23, the factors which regulate Th17/Treg differentiation, increased their expressions during the development of BCP. Additionally, IL-17A receptor (IL-17AR) was found to be expressed on microglia, and the level of IL-17AR increased with activated microglia during BCP development. Furthermore, BCP was ameliorated when IL-17/IL-17A neutralizing antibodies were intrathecally injected, accompanied with inhibited Th17/Treg infiltration and suppressed microglial activation. In conclusion, T cells infiltrated into the spinal cord with the imbalance of Th17/Treg towards Th17 during the development of BCP, which could promote the microglial activation and further increased BCP, while neutralizing IL-17/IL-17A in the spinal cord could ameliorate BCP. Our results suggest that targeting the imbalanced Th17/Treg infiltration in the spinal cord could be a novel strategy for BCP therapy.

Characterization of antioxidant activity of sulfur compounds in black garlic.

Different types of sulfur compounds, namely S-allyl-L-cysteine (SAC), S-allyl-L-cysteine sulfoxide (ACSO) and a synthetic γ-L-glutamyl-S-allyl-L-cysteine (GSAC) were extracted from black garlic and their inhibition to the advanced glycation end-products (AGEs) were investigated. Upon addition of inhibitor, the amount of produced fructosamine was determined by UV visible spectroscopy. The change of pentosidine and fluorescent AGEs during reaction was detected by fluorescence method and the change of carboxymethyl lysine (CML) was detected by high performance liquid chromatography. It was found that the inhibitory effects of SAC and ACSO are stronger to the early and mid non-fluorescent products in glycosylation reaction, and GSAC has an obvious inhibitory effect on the later reaction products. All these three inhibitors can effectively inhibit advanced glycation reaction. Although their effects on glycation products are different due to different chemical structures, they have similar inhibitory effects on fluorescent products.