Spinal Nrf2 translocation may inhibit neuronal NF-κB activation and alleviate allodynia in a rat model of bone cancer pain.

Bone cancer pain (BCP) is a clinical pathology that urgently needs to be solved, but research on the mechanism of BCP has so far achieved limited success. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) has been shown to be involved in pain, but its involvement in BCP and the specific mechanism have yet to be examined. This study aimed to test the hypothesis that BCP induces the transfer of Nrf2 from the cytoplasm to the nucleus and further promotes nuclear transcription to activate heme oxygenase-1 (HO-1) and inhibit the activation of nuclear factor-kappa B (NF-κB) signalling, ultimately regulating the neuroinflammatory response. Von-Frey was used for behavioural analysis in rats with BCP, whereas western blotting, real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect molecular expression changes, and immunofluorescence was used to detect cellular localization. We demonstrated that BCP induced increased Nrf2 nuclear protein expression with decreased cytoplasmic protein expression in the spinal cord. Further increases in Nrf2 nuclear protein expression can alleviate hyperalgesia and activate HO-1 to inhibit the expression of NF-κB nuclear protein and inflammatory factors. Strikingly, intrathecal administration of the corresponding siRNA reversed the above effects. In addition, the results of double immune labelling revealed that Nrf2 and NF-κB were coexpressed in spinal cord neurons of rats with BCP. In summary, these findings suggest that the entry of Nrf2 into the nucleus promotes the expression of HO-1, inhibiting activation of the NF-κB signalling pathway, reducing neuroinflammation and ultimately exerting an anti-nociceptive effect.

Astrocyte activation in the periaqueductal gray promotes descending facilitation to cancer-induced bone pain through the JNK MAPK signaling pathway.

Descending nociceptive modulation from the supraspinal structures has an important role in cancer-induced bone pain (CIBP). Midbrain ventrolateral periaqueductal gray (vlPAG) is a critical component of descending nociceptive circuits; nevertheless, its precise cellular and molecular mechanisms involved in descending facilitation remain elusive. Our previous study has shown that the activation of p38 MAPK in vlPAG microglia is essential for the neuropathic pain sensitization. However, the existence of potential connection between astrocytes and c-Jun N-terminal kinase (JNK) pathway in CIBP has not yet been elucidated. The following study examines the involvement of astrocyte activation and upregulation of p-JNK in vlPAG, using a CIBP rat model. Briefly, CIBP was mimicked by an intramedullary injection of Walker 256 mammary gland carcinoma cells into the animal tibia. A significant increase in expression levels of astrocytes in the vlPAG of CIBP rats was observed. Furthermore, stereotaxic microinjection of the astrocytic cytotoxin L-α-aminoadipic acid decreased the mechanical allodynia as well as established and reversed the astrocyte activation in CIBP rats. A significant increase in expression levels of p-JNK in astrocytes in vlPAG of CIBP rats was also observed. Moreover, the intrathecal administration of JNK inhibitors SP600125 reduced the expression of glial fibrillary acidic protein, while microinjection of the SP600125 decreased the mechanical allodynia of CIBP rats. These results suggested that CIBP is associated with astrocyte activation in the vlPAG that probably participates in driving descending pain facilitation through the JNK MAPK signaling pathway. To sum up, these findings reveal a novel site of astrocytes modulation of CIBP.

Glial activation in the periaqueductal gray promotes descending facilitation of neuropathic pain through the p38 MAPK signaling pathway.

The midbrain ventrolateral periaqueductal gray (VL-PAG) is a key component that mediates pain modulation. Although spinal cord glial cells appear to play an important role in chronic pain development, the precise mechanisms involving descending facilitation pathways from the PAG following nerve injury are poorly understood. This study shows that cellular events that occur during glial activation in the VL-PAG may promote descending facilitation from the PAG during neuropathic pain. Chronic constriction nerve injury (CCI) was induced by ligature construction of the sciatic nerve in male Sprague-Dawley rats. Behavioral responses to noxious mechanical (paw withdrawal threshold; PWT) and thermal (paw withdrawal latency; PWL) stimuli were evaluated. After CCI, immunohistochemical and Western blot analysis of microglia and astrocytes in the VL-PAG showed morphological and quantitative changes indicative of activation in microglia and astrocytes. Intra-VL-PAG injection of microglial or astrocytic inhibitors attenuated PWT and PWL at days 7 and 14, respectively, following CCI. We also evaluated the effects of intra-VL-PAG administration of the phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) inhibitor SB 203580 at day 7 after CCI. This treatment abolished microglial activation and produced a significant time-dependent attenuation of PWT and PWL. Western blot analysis showed localized expression of p-p38 in the VL-PAG after CCI. P-p38 was expressed in labeled microglia of the VL-PAG but was not present in astrocytes and neurons on day 7 after CCI. These results demonstrate that CCI-induced neuropathic pain is associated with glial activation in the VL-PAG, which likely participates in descending pain facilitation through the p38 MAPK signaling pathway.

Pre-oxygenation with high-flow oxygen through the nasopharyngeal airway compared to facemask on carbon dioxide clearance in emergency adults: a prospective randomized non-blinded clinical trial.

INTRODUCTION: Before tracheal intubation, it is essential to provide sufficient oxygen reserve for emergency patients with full stomachs. Recent studies have demonstrated that high-flow nasal oxygen (HFNO) effectively pre-oxygenates and prolongs apneic oxygenation during tracheal intubation. Despite its effectiveness, the use of HFNO remains controversial due to concerns regarding carbon dioxide clearance. The air leakage and unknown upper airway obstruction during HFNO therapy cause reduced oxygen flow above the vocal cords, possibly weaken the carbon dioxide clearance. METHODS: Patients requiring emergency surgery who had fasted < 8 h and not drunk < 2 h were randomly assigned to the high-flow group, who received 100% oxygen at 30-60 L/min through nasopharyngeal airway (NPA), or the mask group, who received 100% oxygen at 8 L/min. PaO2 and PaCO2 were measured immediately before pre-oxygenation (T0), anesthesia induction (T1), tracheal intubation (T2), and mechanical ventilation (T3). The gastric antrum's cross-sectional area (CSA) was measured using ultrasound technology at T0, T1, and T3. Details of complications, including hypoxemia, reflux, nasopharyngeal bleeding, postoperative pulmonary infection, postoperative nausea and vomiting (PONV), and postoperative nasopharyngeal pain, were recorded. The primary outcomes were PaCO2 measured at T1, T2, and T3. The secondary outcomes included PaO2 at T1, T2, and T3, CSA at T1 and T3, and complications happened during this trial. RESULTS: Pre-oxygenation was administered by high-flow oxygen through NPA (n = 58) or facemask (n = 57) to 115 patients. The mean (SD) PaCO2 was 32.3 (6.7) mmHg in the high-flow group and 34.6 (5.2) mmHg in the mask group (P = 0.045) at T1, 45.0 (5.5) mmHg and 49.4 (4.6) mmHg (P < 0.001) at T2, and 47.9 (5.1) mmHg and 52.9 (4.6) mmHg (P < 0.001) at T3, respectively. The median ([IQR] [range]) PaO2 in the high-flow and mask groups was 404.5 (329.1-458.1 [159.8-552.9]) mmHg and 358.9 (274.0-413.3 [129.0-539.1]) mmHg (P = 0.007) at T1, 343.0 (251.6-428.7 [73.9-522.1]) mmHg and 258.3 (162.5-347.5 [56.0-481.0]) mmHg (P < 0.001) at T2, and 333.5 (229.9-411.4 [60.5-492.4]) mmHg and 149.8 (87.0-246.6 [51.2-447.5]) mmHg (P < 0.001) at T3, respectively. The CSA in the high-flow and mask groups was 371.9 (287.4-557.9 [129.0-991.2]) mm2 and 386.8 (292.0-537.3 [88.3-1651.7]) mm2 at T1 (P = 0.920) and 452.6 (343.7-618.4 [161.6-988.1]) mm2 and 385.6 (306.3-562.0 [105.5-922.9]) mm2 at T3 (P = 0.173), respectively. The number (proportion) of complications in the high-flow and mask groups is shown below: hypoxemia: 1 (1.7%) vs. 9 (15.8%, P = 0.019); reflux: 0 (0%) vs. 0 (0%); nasopharyngeal bleeding: 1 (1.7%) vs. 0 (0%, P = 1.000); pulmonary infection: 4 (6.9%) vs. 3 (5.3%, P = 1.000); PONV: 4 (6.9%) vs. 4 (7.0%, P = 1.000), and nasopharyngeal pain: 0 (0%) vs. 0 (0%). CONCLUSIONS: Compared to facemasks, pre-oxygenation with high-flow oxygen through NPA offers improved carbon dioxide clearance and enhanced oxygenation prior to tracheal intubation in patients undergoing emergency surgery, while the risk of gastric inflation had not been ruled out. TRIAL REGISTRATION: This trial was registered prospectively at the Chinese Clinical Research Registry on 26/4/2022 (Registration number: ChiCTR2200059192).

Effect of Dexmedetomidine on Postoperative Plasma Neurofilament Light Chain in Elderly Patients Undergoing Thoracoscopic Surgery: A Prospective, Randomized Controlled Trial.

Purpose: Dexmedetomidine exerts a neuroprotective effect, however, the mechanism underlying this effect remains unclear. This study aimed to explore whether dexmedetomidine can reduce the increase in neurofilament light chain (NfL) protein concentration to play a neuroprotective role during thoracoscopic surgery. Patients and Methods: Patients aged ≥60 years undergoing general anesthesia for thoracoscopic surgery were randomly assigned to receive dexmedetomidine (group D) or not receive dexmedetomidine (group C). Patients in group D received a loading dose of dexmedetomidine 0.5 µg/kg before anesthesia induction and a continuous infusion at 0.5 µg·kg-1·h-1 until the end of the surgery. Dexmedetomidine was not administered in group C. The primary outcome was the NfL concentration on postoperative day 1. The concentrations of procalcitonin (PCT), serum amyloid A (SAA), and high-sensitivity C-reactive protein (hs-CRP) were detected preoperatively and on postoperative day 1. In addition, the numerical rating scale (NRS) and quality of recovery-40 (QoR-40) scores were evaluated. Results: A total of 38 patients in group D and 37 in group C were included in the analysis. No differences were observed between the groups in terms of the plasma concentration of NfL preoperatively and on postoperative day 1 (11.17 [8.86, 13.93] vs 13.15 [10.76, 15.56] pg/mL, P > 0.05; 16.70 [12.23, 21.15] vs 19.48 [15.25, 22.85] pg/mL, P > 0.05, respectively). However, the postoperative plasma NfL concentration was significantly higher than the preoperative value in both groups (both P < 0.001). The groups exhibited no differences in PCT, SAA, hs-CRP, NRS, and QoR-40 (all P > 0.05). Conclusion: Intraoperative administration of dexmedetomidine at a conventional dose does not appear to significantly reduce the increase in postoperative plasma NfL concentration in elderly patients undergoing thoracoscopic surgery. This finding suggests that the neuroprotective effect of dexmedetomidine at a conventional dose was not obvious during general anesthesia.

[Influence of P2Y12 receptor inhibitor on pain threshold and spinal p38MAPK in rat bone cancer pain model].

OBJECTIVE: To investigate the role of P2Y12 receptor in rat bone cancer pain model and its influence on p38MAPK (Mitogen-activated protein kinase). METHOD: A total of forty female SD rats, weighting 200 - 250 g, were randomly divided into 5 groups (n = 8): normal group (group N), sham group (group S), vehicle group (group DA), cancer group (group A), and analgesia group (group MA). Rats in group N were untreated, rats in group S were injected with Hank's solution 10 µl into the left tibial metaphysis; rats in group DA, group A and group MA were injected with Walker 256 cancer cells (10 µl, 2×107 cells/ml) into the left tibial metaphysic to establish the model of bone cancer pain. Catheterization was simultaneously made in four groups between L3 and L4 vertebra except group N. Saline (0.9%, 15 µl) was injected in group S and group A, DMSO (5%, 15 µl) was injected in group DA, and MRS2395 (400 pmol/µl, 15 µl) was injected in group MA on day 9 to 12 post-inoculation. Mechanical withdrawal thresholds were measured on left hind paw before and every 10 min after intrathecal injection. Rats were euthanized after measuring mechanical withdrawal threshold at day 12 post-inoculation. L4-6 sections of spinal cord were collected to determine the expression of p-p38MAPK by immunohistochemistry and immunofluorescent. RESULT: Compared to that in group N (36.1 g ± 4.0 g) and group S (38.9 g ± 5.2 g), mechanical withdrawal thresholds in group MA (19.8 g ± 5.0 g) were decreased on day 9 post-inoculation (P < 0.01), and the expression of p-p38MAPK in spinal cord was increased on day 12 (P < 0.01). Compared to that in group DA (17.7 g ± 3.0 g) and group A (19.1 g ± 2.5 g), mechanical withdrawal threshold in group MA was obviously increased after intrathecal injection, peaked at (26.5 g ± 4.7 g) (P < 0.05); compared with group DA (number 43.4 ± 3.8, IOD 569 ± 27) and group A(number 45.0 ± 2.6, IOD 594 ± 22), the expression level of p-p38MAPK in spinal cord in group MA at day 12 was significantly decreased (number 20.9 ± 2.2, IOD 246 ± 25) (P < 0.01); Mechanical withdrawal threshold in group MA was still lower than group N and group S, while the expression of p-p38MAPK was higher than group N (number 9.9 ± 2.4, IOD 82 ± 28) and group S (number 10.9 ± 2.2, IOD 109 ± 25) (P < 0.01). Immunofluorescent showed that p-p38MAPK was colocalized with microglia in spinal dorsal horn, but not with neurons and astrocytes. CONCLUSIONS: These results demonstrate rat bone cancer pain could partially relieved after intrathecal injection of P2Y12 receptor inhibitor MRS2395 through inhibiting the phosphorylation of p38MAPK in spinal dorsal horn.

SAP102 contributes to hyperalgesia formation in the cancer induced bone pain rat model by anchoring NMDA receptors.

The pathogenesis of cancer induced bone pain (CIBP) is extremely complex, and glutamate receptor dysfunction plays an important role in the formation of CIBP. Synapse-associated protein 102 (SAP102) anchors glutamate receptors in the postsynaptic membrane. However, its effect on hyperalgesia formation in CIBP has not been clarified. This study investigated the role of SAP102 in the formation of hyperalgesia in rats with CIBP SAP102 is present in spinal dorsal horn neurons, but not in astrocytes or microglia. NMDAR-NR2B is localized with neurons. In addition, SAP102 and NMDAR-NR2B expression levels in spinal dorsal horn tissues were detected by Western blot and co-immunoprecipitation. Intrathecal injection of lentiviral vector of RNAi to knockdown SAP102 expression in the spinal dorsal horn significantly attenuated abnormal mechanic pain when compared to non-coding lentiviral vector. These findings indicate that SAP102 can anchor NMDA receptors to affect hyperalgesia formation in bone cancer pain.

Proteomic profiling reveals Arl6ip-1 as a candidate target in cancer-induced bone pain rat model after oxycodone treatment.

Treatment of cancer-induced bone pain (CIBP) is challenging in clinics. Oxycodone is used to treat CIBP. However, the lack of understanding of the mechanism of CIBP limits the application of oxycodone. In this study, proteomic profiling of oxycodone-treated spinal dorsal cord of rats with CIBP was performed. Briefly, a total of 3519 proteins were identified in the Sham group; 3505 proteins in the CIBP group; and 3530 proteins in the CIBP-OXY treatment group. The 2-fold cut-off value was used as the differential protein standard for abundance reduction or increase (p < 0.05). Significant differences were found in the abundance of 16 proteins between Sham and CIBP group; 11 proteins in the CIBP group had increased abundance while 5 proteins had reduced abundance. Furthermore, fifteen proteins with differential abundance were identified between the CIBP group and the OXY group. Compared with the CIBP group, there were six increased abundances and nine reduced abundances in the OXY group. In addition, a reduced expression of ADP-ribosylation factor-like 6 binding factor 1 (Arl6ip-1), an endoplasmic reticulum protein that has an important role in cell conduction and material transport, was found in the CIBP group compared with the Sham group. Its expression increased after the administration of OXY. Proteomics results were further verified by Western-blot. Fluorescent staining revealed that Arl6ip-1 co-localized with spinal dorsal horn neurons, but not with astrocytes or microglia. Based on the observed results, we believe that Arl6ip-1 may be a potential drug target for OXY treatment of CIBP rats.

Phosphoproteomic profiling of oxycodone­treated spinal cord of rats with cancer­induced bone pain.

Treatment of cancer­induced bone pain (CIBP) is challenging in clinical settings. Oxycodone (OXY) is used to treat CIBP; however, a lack of understanding of the mechanisms underlying CIBP limits the application of OXY. In the present study, all rats were randomly divided into three groups: The sham group, the CIBP group, and the OXY group. Then, a rat model of CIBP was established by inoculation of Walker 256 tumor cells from rat tibia. Phosphoproteomic profiling of the OXY­treated spinal dorsal cords of rats with CIBP was performed, and 1,679 phosphorylated proteins were identified, of which 160 proteins were significantly different between the CIBP and sham groups, and 113 proteins were significantly different between the CIBP and OXY groups. Gene Ontology analysis revealed that these proteins mainly clustered as synaptic­associated cellular components; among these, disks large homolog 3 expression was markedly increased in rats with CIBP and was reversed by OXY treatment. Subsequent domain analysis of the differential proteins revealed several significant synaptic­associated domains. In conclusion, synaptic­associated cellular components may be critical in OXY­induced analgesia in rats with CIBP.