RESUMEN
Objective:To evaluate the effect of sleep fragmentation on postoperative cognitive dysfunction (POCD) and hippocampal glutaminergic metabolism in aged mice anesthetized with isoflurane.Methods:Forty healthy SPF-grade male C57BL/6J mice, aged 18 months, weighing 20-30 g, were divided into 4 groups ( n= 10 each) by the random number table method: normal control group (group C), sleep fragmentation group (group SF), isoflurane anesthesia/surgery group (group I/S), and sleep fragmentation plus isoflurane anesthesia/surgery group (group SF+ I/S). Group C did not received any treatment. Group SF received sleep fragmentation for 24 h. The right carotid artery exposure was performed under isoflurane anesthesia in group I/S. Group SF+ I/S received isoflurane anesthesia/right carotid artery exposure at 24 h after sleep fragmentation. The metabolic levels of glutamate (Glu), glutamine (Gln), Glu/Gln complex (Glx), and N-acetylaspartate (NAA) and their ratio to creatine (Cr) were measured by in vivo 9.4T hydrogen proton magnetic resonance spectroscopy at 2 h after anaesthesia. Y maze and Morris water maze tests were used to evaluate the cognitive function at 1-7 days after surgery. The mice were sacrificed after the behavioral testing, brain tissues were immediately obtained, and the number of Nissl bodies and density of dendritic spines in the hippocampal CA1 region were measured by Nissl staining and Golgi staining, respectively. Results:Compared with group C, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr, Gln/Cr and Glx/Cr in the hippocampal CA1 region were increased, and the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in SF, I/S and SF+ I/S groups ( P<0.05). Compared with group SF and group I/S, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr and Glx/Cr in hippocampal CA1 region was increased, the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in group SF+ I/S ( P<0.05). Conclusions:Sleep fragmentation exacerbates POCD in aged mice anesthetized with isoflurane, and the mechanism is related to nerve injury induced by abnormality in hippocampal glutaminergic metabolism excitability.
RESUMEN
Objective:To evaluate the effect of gender on anesthetic potency of ciprofol for gastroscopy when combined with fentanyl.Methods:American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ patients, aged 18-50 yr, with body mass index of 18-25 kg/m 2, undergoing elective gastroscopy with intravenous anesthesia, were divided into 2 groups according to gender: male group (M group) and female group (F group). After fentanyl 1.5 μg/kg was intravenously injected, ciprofol was given by the Dixon′s up-and-down method, with the initial dose of 0.4 mg/kg followed by dose increment/decrement of 0.04 mg/kg. The ED 50 and 95% confidence interval of ciprofol for gastroscopy anesthesia were calculated by the probit regression analysis. Results:The ED 50 (95% confidence interval) of ciprofol for gastroscopy was 0.33 (0.32-0.34) mg/kg in F group and 0.27 (0.26-0.28) mg/kg in M patients when combined with fentanyl 1.5 μg/kg. There was no significant difference between the two groups ( P>0.05). Conclusions:There is no significant gender difference in the anesthetic potency of ciprofol for gastroscopy (ED 50: female 0.33 mg/kg, male 0.27 mg/kg) when combined with fentanyl (1.5 μg/kg).
RESUMEN
Objective:To evaluate the relationship between CCL21 and triggering receptor expressed on myeloid cells 2 (TREM2)/DNAX-activating protein of 12 kDa (DAP12) signaling pathways in the spinal dorsal horn in remifentanil-induced hyperalgesia in mice with incisional pain.Methods:Thirty-two SPF healthy male C57BL/6J mice, weighing 18-22 g, aged 8-10 weeks, were divided into 4 groups ( n=8 each) using a random number table method: control group (group C), CCL21 neutralizing antibody group (group anti-CCL21), remifentanil + incisional pain group (group R+ I), and CCL21 neutralizing antibody + remifentanil + incisional pain group (group anti-CCL21+ R+ I).A CCL21 neutralizing antibody 0.3 μg (diluted to 10 μl in normal saline) was intrathecally injected in anti-CCL21 and anti-CCL21+ R+ I groups twice a day.Normal saline 10 μl was intrathecally injected at the same time point twice a day in C and R+ I groups.Fifteen min after intrathecal injection, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at an interval of 15 min in C and anti-CCL21 groups, and remifentanil 10 μg/kg (diluted to 0.1 ml in normal saline) was injected via the caudal vein for 4 consecutive times at an interval of 15 min in R+ I and anti-CCL21+ R+ I groups.The tail-flick latency (TFL) and mechanical paw withdrawal threshold (MWT) were measured at 24 h before remifentanil or normal saline injection (T 0) and 3, 6, 24 and 48 h after stopping injection of remifentanil or normal saline (T 1-4).The mice were sacrificed after the last measurement of pain threshold, and L 4-6 segments of the spinal cord were removed for determination of the expression of TREM2 and DAP12 protein and mRNA (by Western blot or quantitative real-time polymerase chain reaction). Results:Compared with group C, TFL was significantly shortened and MWT was decreased at T 1-4, and the expression of TREM2 and DAP12 protein and mRNA was up-regulated in group R+ I and R+ I+ anti-CCL21 ( P<0.05), and no significant change was found in the parameters mentioned above in group anti-CCL21 ( P>0.05).Compared with group R+ I, TFL was significantly prolonged and MWT was increased at T 1-4, and the expression of TREM2 and DAP12 protein and mRNA was down-regulated in group anti-CCL21+ R+ I ( P<0.05). Conclusions:CCL21 is involved in remifentanil-induced hyperalgesia by activating TREM2/DAP12 signaling pathways in the spinal dorsal horn of mice with incisional pain.
RESUMEN
Hepatocellular carcinoma (HCC) is a malignant tumor with a high incidence in China. Bone is a common extra hepatic metastasis site of HCC. Bone metastasis of HCC not only has a serious impact on the quality of life of patients, but also shortens their survival time. However, the pathogenesis of bone metastasis of HCC remains unclear. This review summarizes the clinical features, pathogenesis, prognostic factors, diagnosis and treatment progress and other aspects of bone metastasis of HCC, in order to provide new ideas for the clinical diagnosis and treatment.
RESUMEN
The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.
RESUMEN
SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for a cells ability to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.
RESUMEN
Remifentanil is widely used to control intraoperative pain. However, its analgesic effect is limited by the generation of postoperative hyperalgesia. In this study, we investigated whether the impairment of transmembrane protein 16C (TMEM16C)/Slack is required for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) activation in remifentanil-induced postoperative hyperalgesia. Remifentanil anesthesia reduced the paw withdrawal threshold from 2 h to 48 h postoperatively, with a decrease in the expression of TMEM16C and Slack in the dorsal root ganglia (DRG) and spinal cord. Knockdown of TMEM16C in the DRG reduced the expression of Slack and elevated the basal peripheral sensitivity and AMPAR expression and function. Overexpression of TMEM16C in the DRG impaired remifentanil-induced ERK1/2 phosphorylation and behavioral hyperalgesia. AMPAR-mediated current and neuronal excitability were downregulated by TMEM16C overexpression in the spinal cord. Taken together, these findings suggest that TMEM16C/Slack regulation of excitatory synaptic plasticity via GluA1-containing AMPARs is critical in the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats.
RESUMEN
Objective:To evaluate the relationship between the euchromatic histone-lysine N-methyltransferase (G9a) and sodium-dependent activation of potassium channel (Slack) in the dorsal root ganglia (DRG) of rats with neuropathic pain (NP).Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 1 month, weighing 100-120 g, were divided into 4 groups ( n=12 each) by a random number table method: sham operation group (S group), vector plus sham operation group (VS group), vector plus NP group (VN group), and G9a CRISPR/Cas9 knockout plus NP group (GN group). Sham operation was performed at the age of 2 months in group S. In group VS, AAV5 1 μl was microinjected into L 4 and L 5 DRG at the age of 1 month, and sham operation was performed at the age of 2 months.In VN group and GN group, AAV5 and G9a CRISPR/Cas9 knockout plasmid 1 μl were microinjected into L 4 and L 5 DRG at the age of 1 month, and NP model was established by spinal nerve ligation (SNL) at the age of 2 months.Six rats in each group were selected to measure the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) before microinjection (T 0), before SNL (T 1), and at 3, 5 and 7 days after SNL (T 2-4). The animals were sacrificed after the last behavioral testing, the DRGs of lumbar segment (L 4, 5) were removed for determination of the expression of G9a, dimethylation of histone H3 at lysine 9(H3K9me2) and Slack (by Western blot). At 7 days after establishing the model, 6 rats from each group were selected to culture the primary DRG neurons.The frequency and amplitude of Slack current in DRG neurons and miniature excitatory post-synaptic currents (mEPSCs) in the spinal dorsal horn were measured by whole-cell patch-clamp technique. Results:Compared with group S, the TWL was significantly shortened and the MWT was decreased at T 2-4, the expression of G9a and H3K9me2 in the spinal dorsal horn was up-regulated, the expression of Slack was down-regulated, the amplitude and frequency of Slack currents in DRG neurons were decreased, and the frequency of mEPSCs was increased in group VN ( P<0.05), and no significant change was found in the parameters mentioned above in group VS ( P>0.05). Compared with group VN, the TWL was significantly prolonged and the MWT was increased at T 2-4, the expression of G9a and H3K9me2 in the spinal dorsal horn was down-regulated, the expression of Slack was up-regulated, the amplitude and frequency of Slack currents in DRG neurons were increased, and the frequency of mEPSCs was decreased in group GN ( P<0.05). Conclusion:The mechanism of NP is related to up-regulating the expression of G9a in DRG, thus inhibiting the expression and opening of Slack channels in rats.
RESUMEN
Objective:To evaluate the effects of propofol on α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor expression in the hippocampus of neonatal rats.Methods:Eighty-four clean-grade healthy Sprague-Dawley rats of either sex, aged 7 days, weighing 14-18 g, were divided into 2 groups ( n=42 each) using a random number table method: control group (group C) and propofol group (group P). Propofol 30 mg/kg was intraperitoneally injected in group P, fat emulsion 3 mg/kg was intraperitoneally injected in group C, 1/2 of the initial dose was given at a 20 min interval, 3 times in total, for 3 consecutive days.The arterial blood samples were taken for blood gas analysis after administration on 1st day.The rats were sacrificed at 3, 7 and 28 days after the last administration of propofol, and the bilateral hippocampus was obtained for detection of the expression of AMPA receptors containing GluR1, GluR2 and GluR3 subunits in total and membrane protein (by Western blot), and the ratio of membrane protein to total protein (M/T) was calculated.The concentrations of free calcium ion were measured.The learning and memory ability was evaluated by Morris water maze test on 28 days after the last administration. Results:Compared with group C, the expression of AMPA receptor containing GluR1 subunit in total and membrane protein was significantly up-regulated, M/T was increased, the expression of AMPA receptor containing GluR2 subunit in total and membrane protein was down-regulated, and M/T was decreased at each time point ( P<0.05), no significant change was found in the expression of AMPA receptor containing GluR3 subunits ( P>0.05), the concentrations of free calcium ion in hippocampal cells were increased, and the escape latency was prolonged, the number of crossing the original platform was decreased, and the time of staying at the target quadrant was shortened at 2-4 days of training in group P ( P<0.05). Conclusion:The mechanism by which propofol reduces cognitive function is related to up-regulation of the expression of AMPA receptors containing GluR1 subunit in the hippocampus and down-regulation of the expression of AMPA receptors containing GluR2 subunits, which increases the concentration of free calcium ions in nerve cells of neonatal rats.
RESUMEN
Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SignificanceSARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.
RESUMEN
BackgroundRapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse Transcription and Loop-Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. ResultsHere we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. ConclusionsLAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
RESUMEN
Objective:To evaluate the role of secreted protein acidic and rich in cysteine like protein 1 (SPARCL1) in spinal dorsal horns in the development of remifentanil-induced hyperalgesia in mice with incisional pain.Methods:Forty-eight healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 6 groups ( n=8 each) using a random number table method: control group (group C), incisional pain group (group I), remifentanil group (group R), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus negative control group (group I+ R+ N), and incisional pain plus remifentanil plus SPARCL1-siRNA group (group I+ R+ S). In I+ R+ N and I+ R+ S groups, 1×10 8 IFU/ml negative control siRNA and SPARCL1-siRNA 10 μl were intrathecally injected, respectively, once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected once a day for 3 consecutive days in C, I, R and I+ R groups.After transfection was stable, normal saline 0.1 ml was intravenously injected through the tail vein for 4 consecutive times at 15 min interval in C and I groups, and remifentanil 10 μg/kg (diluted to 0.1 ml in normal saline) was intravenously injected via the tail vein for 4 consecutive times at 15 min interval in R, I+ R, I+ R+ N and I+ R+ S groups.The incisional pain model was established after the first administration via the tail vein in R, I+ R, I+ R+ N and I+ R+ S groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusing normal saline or remifentanil (T 0) and 3, 6, 24 and 48 h after stopping infusion (T 1-4). Animals were sacrificed after measuring pain threshold at T 4, and L 4-6 segments of the spinal cord were removed for determination of the expression of SPARCL1 protein and mRNA by Western blot and quantitative real-time polymerase chain reaction, respectively. Results:Compared with group C, MWT was significantly decreased and TWL was shortened at T 1-4 in I+ R and I+ R+ N groups and at T 2-4 in I, R and I+ R+ S groups, and the expression of SPARCL1 protein and mRNA was significantly up-regulated in R, I+ R and I+ R+ C groups ( P<0.05 or 0.01). Compared with group I and group R, MWT was significantly decreased, TWL was shortened, and the expression of SPARCL1 protein and mRNA was up-regulated in group I+ R ( P<0.01). Compared with group I+ R, MWT was significantly increased and TWL was prolonged at T 1-4, and the expression of SPARCL1 protein and mRNA was down-regulated in group I+ R+ S ( P<0.05 or 0.01). Conclusion:Enhanced activity of SPARCL1 in the spinal dorsal horns is involved in the development of remifentanil-induced hyperalgesia in mice with incisional pain.
RESUMEN
Objective@#To evaluate the role of spinal COX-1 and COX-2 in remifentanil-induced hyperalgesia in mice with incisional pain.@*Methods@#Thirty-two male C57BL/6J mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), incisional pain plus remifentanil group (group IR), incisional pain plus remifentanil plus selective COX-1 inhibitor group (group IR+ SC560), and incisional pain plus remifentanil plus selective COX-2 inhibitor group (group IR+ SC236). In IR, IR+ SC560 and IR+ SC236 groups, normal saline 10 μl, SC560 25 μg and SC236 25 μg were intrathecally injected, respectively, 15 min later remifentanil 10 μg/kg was injected via the tail vein for 4 times at 15 min intervals.An incisional pain model was established after the first injection of remifentanil.The mechanical paw withdrawal threshold (MWT) was measured at 24 h before normal saline or remifentanil injection and 3, 6, 24 and 48 h after the last injection (T0-T4). The mice were sacrificed after the last measurement of pain threshold, and the L4-6 segments of the spinal cord were removed for determination of the expression of COX-1 and COX-2 (by Western blot) and expression of COX-1 and COX-2 mRNA (by quantitative real-time polymerase chain reaction).@*Results@#Compared with group C, the MWT was significantly decreased, and the expression of COX-2 protein and mRNA was up-regulated in IR, IR+ SC560 and IR+ SC236 groups (P<0.05). Compared with group IR, the MWT was significantly increased in IR+ SC560 and IR+ SC236 groups (P<0.05). There was no significant difference in the MWT at each time point between IR+ SC560 and IR+ SC236 (P>0.05) .There was no significant difference in the expression of COX-2 protein and mRNA among group IR, group IR+ SC560 and group IR+ SC236 (P>0.05). There was no significant difference in the expression of COX-1 protein and mRNA among the four groups (P>0.05).@*Conclusion@#Compared with COX-1, spinal COX-2 plays a major role in the pathophysiological mechanism of remifentanil-induced hyperalgesia in mice with incisional pain.
RESUMEN
Objective@#To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.@*Methods@#Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), NL1-shRNA plasmid group (group NL), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus blank vector group (group I+ R+ B), and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+ R+ NL). Negative lentivirus was intrathecally injected in group I+ R+ B.In NL and I+ R+ NL groups, 10 μl NL-1-shRNA lentivirus at 1×108 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+ R groups.After transfection was stable, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+ R, I+ R+ B and I+ R+ NL groups, 0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals, and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3, 6, 24 and 48 h after the end of administration (T1-4). The animals were sacrificed after measurement of pain threshold at T4, and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors, and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.@*Results@#Compared with group C, the MWT was significantly decreased, and TFL was shortened at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated, and m/t ratio was increased in I+ R and I+ R+ B groups (P<0.05). Compared with I+ R and I+ R+ B groups, the MWT was significantly increased and TFL was prolonged at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated, and m/t ratio was decreased in group I+ R+ NL (P<0.05).@*Conclusion@#NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane, which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.
RESUMEN
Objective To evaluate the relationship between remifentanil-induced hyperalgesia and Antamins family member membrane protein 16C ( TMEM16C) and Slack channel in the spinal dorsal horn of rats with incisional pain. Methods Forty-eight male Sprague-Dawley rats, aged 1 month, were studied. The study was performed in two parts. Experiment Ⅰ Twenty-four rats were divided into 4 groups ( n=6 each) using a random number table method: normal saline group ( S group ) , virus vector group ( V group) , virus vector plus remifentanil plus incisional pain group ( VRI group) , and AAV5-TMEM16C o-ver-expression plus remifentanil plus incisional pain group (ORI group). Normal saline (S group), virus vector (V group and VRI group) or AAV5-TMEM16C (ORI group) 1 μl was injected via the L4,5 spinal dorsal horn. Remifentanil 1 μg ·kg-1 ·min-1 was infused for 60 min via the caudal vein at 30 days, and the incisional pain model was simultaneously established in VRI and ORI groups. The thermal paw withdrawal latency ( TWL) and mechanical paw withdrawal threshold ( MWT) were measured at 24 h before remifen-tanil infusion ( T0 ) and 2, 6, 24, and 48 h after stopping infusion ( T1-4 ) . The L4,5 segments of spinal dorsal horns were obtained at the end of the behavioral testing, and the expression of TMEM16C and Slack in total and membrane proteins was determined by Western blot. ExperimentⅡ Twenty-four rats were di-vided into 4 groups ( n=6 each) using a random number table method: normal saline plus artificial cerebro-spinal fluid (ACSF) group (SA group), virus vector plus ACSF group (VA group), virus vector plus remifentanil group ( VR group ) , and AAV5-TMEM16C over-expression plus remifentanil group ( OR group). Normal saline (SA group), virus vector (VA group and VR group) or AAV5-TMEM16C (OR group) 1μl were injected via the spinal dorsal horn at the level of L4,5 . L4,5 spinal cord slices were obtained at 30 days and incubated for 60 min in ACSF ( SA and VA groups ) or in ACSF containing 4 nmol∕L remifentanil ( VR group and OR group) . The whole-cell patch-clamp was used to measure the frequency and amplitude of the Slack channel current after the end of incubation in each group. Results ExperimentⅠCompared with S group, the TWL was significantly shortened and the MWT was decreased at T1-4 , and the expression of TMEM16C and Slack in total and membrane proteins was down-regulated in VRI group ( P<0. 05) . Compared with VRI group, the TWL was significantly prolonged and the MWT was increased at T1-4 , and the expression of TMEM16C and Slack in total and membrane proteins was up-regulated in ORI group ( P<0. 05) . Experiment Ⅱ Compared with SA group, the amplitude and frequency of Slack chan-nel current in spinal dorsal horns were significantly decreased in VR group ( P<0. 05) . Compared with VR group, the amplitude and frequency of the Slack current were significantly increased in OR group ( P<0. 05) . Conclusion The mechanism of remifentanil-induced hyperalgesia is related to down-regulating the expression of TMEM16C and further down-regulating the expression of the Slack channel in the spinal dorsal horn of rats with incisional pain.
RESUMEN
Objective To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.Methods Forty SPF healthy male C57BL/6J mice,aged 8-10 weeks,weighing 18-22 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),NL1-shRNA plasmid group (group NL),incisional pain plus remifentanil group (group I+R),incisional pain plus remifentanil plus blank vector group (group I+R+B),and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+R+NL).Negative lentivirus was intrathecally injected in group I+R+B.In NL and I+R+NL groups,10 μl NL-1-shRNA lentivirus at 1×10s IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+R groups.After transfection was stable,normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+R,I+R+B and I+R+NL groups,0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals,and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3,6,24 and 48 h after the end of administration (T1-4).The animals were sacrificed after measurement of pain threshold at T4,and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors,and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group C,the MWT was significantly decreased,and TFL was shortened at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated,and m/t ratio was increased in I+R and I+R+B groups (P＜0.05).Compared with I+R and I+R+B groups,the MWT was significantly increased and TFL was prolonged at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated,and m/t ratio was decreased in group I+R+NL (P＜0.05).Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane,which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.
RESUMEN
Objective To evaluate the role of spinal COX-1 and COX-2 in remifentanil-induced hyperalgesia in mice with incisional pain.Methods Thirty-two male C57BL/6J mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups (n =8 each) using a random number table method:control group (group C),incisional pain plus remifentanil group (group IR),incisional pain plus remifentanil plus selective COX-1 inhibitor group (group IR+SC560),and incisional pain plus remifentanil plus selective COX-2 inhibitor group (group IR+SC236).In IR,IR+SC560 and IR+SC236 groups,normal saline 10 μl,SC560 25 μg and SC236 25 μg were intrathecally injected,respectively,15 min later remifentanil 10 μg/kg was injected via the tail vein for 4 times at 15 min intervals.An incisional pain model was established after the first injection of remifentanil.The mechanical paw withdrawal threshold (MWT) was measured at 24 h before normal saline or remifentanil injection and 3,6,24 and 48 h after the last injection (T0-T4).The mice were sacrificed after the last measurement of pain threshold,and the L4-6 segments of the spinal cord were removed for determination of the expression of COX-1 and COX-2 (by Western blot)and expression of COX-1 and COX-2 mRNA (by quantitative real-time polymerase chain reaction).Results Compared with group C,the MWT was significantly decreased,and the expression of COX-2 protein and mRNA was up-regulated in IR,IR+SC560 and IR+SC236 groups (P＜0.05).Compared with group IR,the MWT was significantly increased in IR+SC560 and IR+SC236 groups (P＜0.05).There was no significant difference in the MWT at each time point between IR+SC560 and IR+SC236 (P＞0.05).There was no significant difference in the expression of COX-2 protein and mRNA among group IR,group IR+SC560 and group IR+SC236 (P＞0.05).There was no significant difference in the expression of COX-1 protein and mRNA among the four groups (P＞0.05).Conclusion Compared with COX-1,spinal COX-2 plays a major role in the pathophysiological mechanism of remifentanil-induced hyperalgesia in mice with incisional pain.
RESUMEN
Objective To investigate the relationship remifentanil-induced hyperalgesia and func-tion of nitrated glutamate transportor-1 ( GLT-1) and glutamine synthetase ( GS) in the spinal cord of rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 260-280 g, aged 2-3 months, in which caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) u-sing a random number table method: control group ( group C) , incisional pain group ( group I) , remifen-tanil group ( group R) and remifentanil plus incisional pain group ( group RI) . Normal saline was intrave-nously infused for 60 min at 0. 1 ml · kg-1 · min-1 in group C. The model of incisional pain was estab-lished, and normal saline was simultaneously infused for 60 min via the tail vein at 0. 1 ml·kg-1 ·min-1 in group I. Remifentanil was infused for 60 min via the tail vein at 1. 0 μg· kg-1 ·min-1 in group R. The model of incisional pain was established, and remifentanil was infused for 60 min via the tail vein at 1. 0μg· kg-1 ·min-1 in group RI. The mechanical paw withdrawal threshold ( MWT) and thermal paw with-drawal latency ( TWL) were measured at 24 h before infusion of remifentanil or normal saline ( T0 ) and at 2, 6, 24 and 48 h after infusion ( T1-4 ) . The rats were sacrificed after the last measuremnet of pain thresh-old, and the L4-6 segment of the spinal cord was removed for determination of the expression of GLT-1 and GS (by Western blot) and expression of nitrated GLT-1 (nGLT-1) and nitrated GS (nGS) (by Western blot) . Ratios of nGLT-1∕GLT-1 and nGS∕GS were calculated. Results Compared with group C, the MWT was significantly decreased and TWL was shortened at T1-4 , the expression of GLT-1 and GS was down-regu-lated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1∕GLT-1 and nGS∕GS were increased in I, R and RI groups ( P<0. 05) . Compared with group I and group R, the MWT was signifi-cantly decreased and TWL was shortened at T1-4 , the expression of GLT-1 and GS was down-regulated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1∕GLT-1 and nGS∕GS were increased in group RI ( P<0. 05) . Conclusion The mechanism of remifentanil-induced hyperalgesia may be related to impaired function of GLT-1 and GS in the spinal cord of rats with incisional pain.
RESUMEN
Objective To evaluate the relationship between NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptors (NR2B receptors) and Ca2+/calmodulin-dependent protein kinase Ⅱ α (CaMK Ⅱ α) in the spinal cord during remifentanil-induced hyperalgesia in a rat model of incisional pain (IP).Methods Forty male Sprague-Dawley rats in which intrathecal and caudal catheters were successfully placed,weighing 260-280 g,aged 2-3 months,were divided into 4 groups (n=10 each) using a random number table method:control group (group C),remifentanil plus IP group (group RI),NR2B antagonist Ro 25-6981 group (group Ro) and remifentanil plus IP plus Ro 25-6981 group (group RI+Ro).In group C,normal saline 0.1 ml was intrathecally injected,and 10 min later normal saline was infused for 60 min via the tail vein at a rate of0.1 ml · kg-1 · min-1.In group RI,normal saline 0.1 ml was intrathecally injected,and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1.0 μg · kg-1 · min-1,and IP was established immediately after onset of remifentanil infusion.In group Ro,Ro 25-6981 (0.1 ml) 10 μg was intrathecally injected,and 10 min later normal saline was infused for 60 min via the tail vein at a rate of 0.1 ml · kg-1 · min-1.In group RI+Ro,Ro 25-6981 (0.1 ml) 10 μg was intrathecally injected,and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1.0 μg · kg-1 · min-1,and IP was established immediately after onset of remifentanil infusion.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before intravenously infusing normal saline or remifentanil and at 2,6,24 and 48 h after the end of infusion (T0-4).The rats were sacrificed after the last behavioral test,and the L4-6 segment of the spinal cord was removed for determination of the expression of NR2B in total and membrane protein (tNR2B and mNR2B) and expression of CaMK Ⅱ α in total protein (tCaMK Ⅱ α) and phosphorylated CaMK Ⅱ α (pCaMKⅡα).The ratios of mNR2B/tNR2B and pCaMKⅡα/tCaMK Ⅱα were calculated.Results Compared with group C,the MWT was significantly decreased,TWL was shortened,the expression of tNR2B,mNR2B,tCaMKⅡα and pCaMKⅡα was up-regulated,and the ratios of mNR2B/tNR2B and pCaMK Ⅱ α/tCaMK Ⅱ α were increased in group RI (P＜0.05 or 0.01).Compared with group RI,the MWT was significantly increased,TWL was prolonged,the expression of tNR2B,mNR2B,tCaMKⅡα and pCaMKⅡα was down-regulated,and the ratios of mNR2B/tNR2B and pCaMK Ⅱ α/tCaMK Ⅱ α were decreased in group RI+ Ro (P＜0.05 or 0.01).Conclusion Enhanced function of NR2B can activate CaMKⅡα during remifentanil-induced hyperalgesia,which may be involved in the mechanism of remifentanil-induced hyperalgesia in a rat model of IP.
RESUMEN
Objective To investigate the changes in the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ α (CaMK Ⅱ α) in the spinal cord and dorsal root ganglia (DRG) during remifentanil-induced hyperalgesia in a rat model of incisional pain (IP).Methods Thirty-two male Sprague-Dawley rats in which caudal vein catheter was successfully placed,aged 260-280 g,were divided into 4 groups (n=8 each) using a random number table method:control group (group C),IP group,remifentanil group (group R) and remifentanil plus IP group (group RIP).Normal saline was infused via the caudal vein for 60 min at a rate of 0.1 ml · kg-1 · min-1 in group C.Normal saline was infused via the caudal vein for 60 min at a rate of 0.1 ml · kg-1 · min-1,and the model of IP was simultaneously established in group IP.Remifentanil was infused via the caudal vein for 60 min at a rate of 1.0 μg · kg-1 · min-1 in group R.Remifentanil was infused via the caudal vein for 60 min at a rate of 1.0 μg · kg-1 · min-1,and the model of IP was simultaneously established in group RIP.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion and 2,6,24 and 48 h after infusion (T0-4).The rats were sacrificed after the last behavioral test,and L4-6 segment of the spinal cord and DRGs were removed for determination of the expression of total and phosphorylated CaMK Ⅱ α (tCaMK Ⅱ α,pCaMK Ⅱ α) by Western blot.The ratio of pCaMK Ⅱ /tCaMK Ⅱ α was calculated.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the expression of tCaMK Ⅱ α and pCaMK Ⅱ α in the spinal cord and DRGs was up-regulated,and the ratio of pCaMK Ⅱ α/tCaMK Ⅱ α was increased in I,R and RIP groups (P＜0.05 or 0.01).Compared with group IP and group R,MWT was significantly decreased,TWL was shortened,the expression of tCaMK Ⅱ α and pCaMK Ⅱ α in the spinal cord and DRGs was up-regulated,and the ratio of pCaMK Ⅱ α/tCaMK Ⅱ α was increased in group RIP (P＜0.05 or 0.01).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to upregulated expression of CaMK Ⅱ α in the spinal cord and DRGs in a rat model of IP.
