Objective:To evaluate the relationship between P2X 7 receptors and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3)/interleukin-1beta (IL-1β) pathway in spinal neurons in the development of inflammatory pain (IP) in rats. Methods:SPF healthy adult male Wistar rats, weighing 180-220 g, were used in this study.Forty rats in which intrathecal catheters were successfully implanted were divided into 5 groups ( n=8 each) using a random number table method: control group (group CON), group IP, IP plus dimethyl sulfoxide (DMSO) group (group IP-DMSO), IP plus P2X 7 receptor antagonist A740003 group (group IP-A) and IP plus P2X 7 receptor agonist ATP group (group IP-ATP). Rats were anesthetized with pentobarbital sodium 40 mg/kg.IP was induced by injecting complete Freund′s adjuvant 50 μl into the right ankle joint cavity, while group CON was injected with the equal volume of normal saline instead.On 1 day before establishing the model, immediately after establishing the model, and on 1, 2 and 3 days after establishing the model, 1% DMSO 10 μl was intrathecally injected once a day in group IP-DMSO, A740003 0.1 nmol(dissolved in DMSO 10 μl) was intrathecally injected once a day in group IP-A, and ATP 150 nmol(dissolved in DMSO 10 μl) was injected intrathecally once a day in group IP-ATP.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on 3 days after establishing the model.Enzyme-linked immunosorbent assay was used to determine the prostaglandin E2 (PGE2) concentrations in right ankle tissues and IL-1β concentrations in cerebrospinal fluid (CSF). Then rats were sacrificed, and the lumber segments (L 4-6) of the spinal cord were removed for determination of the expression of NLRP3, casepase-1, IL-1β (by Western blot) and co-expression of P2X 7 receptors with neuron-specific nucleoprotein (NeuN) and NLRP3 and with NeuN (by immunofluorescence). Results:Compared with group CON, PGE2 contents in ankle tissues were significantly increased in group IP, and the MWT was significantly decreased, the TWL was shortened, the concentrations of IL-1β in CSF were increased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in the other four groups ( P<0.05). Compared with group IP, the MWT was significantly increased, the TWL was prolonged, the concentrations of IL-1β in CSF were decreased, and the expression of NLRP3, caspase-1 and IL-1β was down-regulated in group IP-A ( P<0.05), the MWT was significantly decreased, TWL was shortened, the concentrations of IL-1β in CSF were increased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in group IP-ATP ( P<0.05), and no significant change was found in the parameters mentioned above in group IP-DMSO ( P>0.05). P2X 7 was co-expressed with NeuN, and NLRP3 was co-expressed with NeuN. Conclusion:P2X 7 receptors in spinal neurons are involved in the development of inflammatory pain by activating NLRP3/IL-1β signaling pathway in rats.

Objective:To evaluate the changes in glucose metabolism in the prefrontal cortex during long-term cognitive dysfunction induced by neuropathic pain in developing rats.Methods:SPF healthy male Sprague-Dawley rats, aged 4 weeks, weighing 80-100 g, were used in this study.The model of neuropathic pain was established by using spared nerve injury in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before establishing the model (T 0) and 1, 3, 7, 14, 28, 42 and 56 days after establishing the model (T 1-7). According to the results of MWT compared between T 5 and T 0, the rats were divided into neuropathic pain group (group NP) and non-neuropathic pain group (group NNP). Open field test and novel object recognition test were performed at T 7 to assess anxiety-like behavior and cognitive function.Positron emission tomography/computed tomography imaging was performed to determine the standard uptake value of 18F-fluorodeoxyglucose in the prefrontal cortex.Then the rats were sacrificed, and prefrontal cortex was removed for determination of the expression of glucose transporter 3 using Western blot and immunofluorescence. Results:Compared with the baseline at T 0, the MWT at T 1-2 in group NNP and at T 1-7 in group NP were significantly decreased ( P<0.05). Compared with group NNP, the MWT at T 1-7 were significantly decreased, the time of staying at the central region at T 7 was shortened, the percentage of time for exploring the novel object was decreased, the percentage of novel object exploration was decreased, the standard uptake value of 18F-fluorodeoxyglucose in prefrontal cortex was decreased, and the expression of glucose transporter 3 in prefrontal cortex was down-regulated in group NP ( P<0.05). Conclusion:Long-term cognitive dysfunction induced by neuropathic pain may be related to decreased glucose metabolism in the prefrontal cortex of the developing rats.

Objective:To compare the effects of desflurane and sevoflurane anesthesia on the sleep quality of sleep-deprived mice.Methods:Thirty-two clean-grade healthy male C57BL/6 mice, aged 10 weeks, weighing 20-25 g, were divided into 4 groups ( n=8 each) by the random number table method: control group (C group), sleep deprivation group (SD group), sleep deprivation+ sevoflurane group (SD+ SEV group), and sleep deprivation+ desflurane group (SD+ DES group). In the four groups, EEG-EMG electrodes were implanted for recording EEG and EMG, and sleep deprivation model was developed by the gentle stimulation method with a brush for 12 h (6: 00-18: 00) after 7 days of adaptation. The 6 h after sleep deprivation was divided into 2 time periods: T 1 period (18: 00-20: 00) and T 2 period (20: 00-24: 00). T 1 period In SD group, mice were allowed ad libitum recovery sleep after sleep deprivation. C group and SD group were exposed to 60% oxygen 1.5 L/min. In SD+ DES group and SD+ SEV group, mice were exposed to 6% desflurane and 2.5% sevoflurane, respectively, for 2 h in 60% oxygen 1.5 L/min following sleep deprivation. T 2 period Four groups were allowed ad libitum recovery sleep with the EEG-EMG signal recording. The percentages and number of wakefulness time, rapid eye movement time and non-rapid eye movement time during each time period were calculated using Lunion Data software. Results:Compared with C group, the percentage of non-rapid eye movement time and the percentage of rapid eye movement time were significantly decreased, and the percentage of wakefulness time was increased during 12 h sleep deprivation in SD group, SD+ SEV group and SD+ DES group ( P<0.05). Compared with T 1 period, the percentage of non-rapid eye movement time was significantly increased, and the percentage of wakefulness time and percentage of rapid eye movement time were decreased in T 2 period in SD group ( P<0.05). Compared with SD group, the percentage of non-rapid eye movement time and percentage of rapid eye movement time were significantly decreased, and the percentage of wakefulness time was increased in T 2 period in SD+ SEV group and SD+ DES group ( P<0.05). There was no significant difference in the percentage of non-rapid eye movement, rapid eye movement and wakefulness time in T 2 period between SD+ SEV group and SD+ DES group ( P>0.05). Compared with SD+ SEV group, the number of non-rapid eye movement in T 2 period was significantly reduced in SD+ DES group ( P<0.05). Conclusions:The effect of desflurane anesthesia in improving sleep quality is better than sevoflurane anesthesia in sleep-deprived mice.

Objective:To evaluate the effect of propofol on neuronal activity in medial prefrontal cortex (mPFC) during social behavior in sleep-deprived rats.Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 8 weeks, weighing 200-250 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group Con), chronic sleep deprivation plus natural sleep group (group CSD+ NS), and chronic sleep deprivation plus propofol group (CSD+ Pro). Sleep deprivation model was developed by the modified multiple platform method, and the rats were placed in the sleep-deprivation tank for 20 h a day (14: 00-10: 00) and allowed to sleep naturally for 4 h (10: 00-14: 00) a day for 28 consecutive days.Propofol 40 mg/kg was intraperitoneally injected after sleep deprivation for 28 consecutive days in group CSD+ Pro, while the equal volume of 10% fat emulsion was given instead in group C and group CSD+ NS.Electroencephalographic recordings in cerebral cortical regions were performed on the days 1st, 14th and 28th after sleep deprivation.The apoptotic neurons in mPFC were detected using TUNEL method after the end of sleep deprivation, and the apoptosis index was calculated.A three chamber sociability test was used to detect the social behavior of rats, and local field potential signals in mPFC were collected. Results:Compared with group Con, the percentage of rapid eye movement sleep was significantly increased, the sniffing time preference coefficients in the 2 stages were reduced, the percentage of the β waves and θ waves-band power in mPFC during the social sniffing process was decreased, and the apoptosis index of neurons in mPFC was increased in group CSD+ NS ( P<0.05). Compared with group CSD+ NS, the percentage of rapid eye movement sleep was significantly increased, the sniffing time preference coefficient in the 2 stages was increased, and the percentage of β waves and θ waves-band power in mPFC during the social sniffing process was increased, and the apoptosis index of neurons in mPFC was decreased in group CSD+ Pro ( P<0.05). Conclusions:Propofol inhibits the apoptosis in neurons in mPFC and increases β and θ waves in the mPFC during social interaction after sleep deprivation in sleep-deprived rats, which is helpful in improving sleep deprivation-induced social disorder.

Objective:To evaluate the effect of propofol on parvalbumin (PV) neurons in the medical prefrontal cortex(mPFC)of rats with social behavior disorders induced by chronic sleep deprivation.Methods:Forty-two SPF male Sprague-Dawley rats, aged 8 weeks, weighing 200-250 g, were divided into 3 groups ( n=14 each) using a random number table method: control group (group Con), chronic sleep deprivation plus natural sleep group (group CSD+ NS), and chronic sleep deprivation plus propofol group (group CSD+ Pro). Sleep deprivation model was established by the modified multiple platform method, the rats were placed in the sleep-deprivation tank for 20 h a day (14: 00-10: 00), and allowed to sleep naturally for 4 h (10: 00-14: 00) a day for 28 consecutive days. Propofol 40 mg/kg was intraperitoneally injected for 28 consecutive days after sleep deprivation in CSD+ Pro group. While the equal volume of 10% fat emulsion was given in Con and CSD+ NS groups. After the end of sleep deprivation, a three-box social experiment was used to detect the social behavior of rats, and the number of the PV positive cells and density of the perineuronal network (PNN) in the mPFC area were measured by immunofluorescence. Results:Compared with group Con, the pertentage of rapid eye movement sleep and sniffing time preference coefficients for the strange rat 1 in the first stage and for the strange rat 2 in the second stage were significantly decreased, and the number of the PV positive cells and density of PNN in the mPFC area were decreased in group CSD+ NS ( P<0.05). Compared with group CSD+ NS, the sniffing time preference coefficients for the strange rat 1 in the first stage and for the strange rat 2 in the second stage were significantly increased, the number of the PV positive cells and density of PNN in the mPFC area were increased( P<0.05), and no significant change was found in the percentage of the rapid eye movement sleep in group CSD+ Pro. Conclusions:Propofol probably increases the number and function of PV neurons in the mPFC and ameliorates sleep deprivation-induced social behavior disorders in sleep-deprived rats.

Objective:To evaluate the role of activation of vesicular glutamate transporter 2 (VGLUT2) neurons in vagal nodose ganglion in dexmedetomidine-caused bradycardia in mice.Methods:Ninety-six SPF healthy male VGLUT2-cre mice, aged 10 weeks, weighing 20-25 g, were divided into 6 groups ( n=16 each) by the random number table method: normal saline control group (NS group), dexmedetomidine group (Dex group), viral control + chemogenetic control + dexmedetomidine group (eGFP-NS+ Dex group), viral transfection + chemogenetic control + dexmedetomidine group (hM4Di-NS+ Dex group), viral control + chemogenetic inhibition + dexmedetomidine group (eGFP-CNO+ Dex group) and viral transfection + chemogenetic inhibition + dexmedetomidine group (hM4Di-CNO+ Dex group). Dexmedetomidine 100 μg/kg was intraperitoneally injected in Dex group. The equal volume of normal saline was intraperitoneally injected in NS group. AAV2/9-hSyn-DIO-hM4Di-eGFP was injected in the right nodose ganglion in hM4Di-NS+ Dex group and hM4Di-CNO+ Dex group, and AAV2/9-hSyn-DIO-eGFP was injected in the right nodose ganglion in eGFP-NS+ Dex group and eGFP-CNO+ Dex group, allowing the virus expression for 21 days. On the 22nd day after virus injection, clozapine-n-oxide (CNO) 5 mg/kg was intraperitoneally injected in hM4Di-CNO+ Dex group and eGFP-CNO+ Dex group, the equal volume of normal saline was intraperitoneally injected in hM4Di-NS+ Dex group and eGFP-NS+ Dex group, 1 h later the efficacy of CNO reached the peak, and then dexmedetomidine 100 μg/kg was intraperitoneally injected. The respiratory rate, heart rate, SpO 2 and discharge frequency of the right vagal nodose ganglion were synchronously measured by multi-channel electrophysiology in vivo. The expression of phosphorylated extracellular signal-regulated kinase (pERK) and VGLUT2 and co-expression of pERK and VGLUT2 in the right vagal nodose ganglion were detected by immunofluorescence assay. Results:Compared with NS group, the percentage of heart rate variation and neuron firing frequency after administration were significantly increased, and pERK expression was up-regulated in the other five groups ( P<0.05). Compared with Dex group, the percentage of heart rate variation and neuron firing frequency after administration were significantly decreased, and pERK expression was down-regulated in hM4Di-CNO+ Dex group, and no significant change was found in the parameters mentioned above in hM4Di-NS+ Dex group, eGFP-NS+ Dex group and eGFP-CNO+ Dex group ( P>0.05). Compared with hM4Di-CNO+ Dex group, the percentage of heart rate variation and neuron firing frequency after administration were significantly increased, and pERK expression was up-regulated in eGFP-CNO+ Dex group ( P<0.05). There was no significant difference in the percentage of respiratory variation and SpO 2 among the six groups ( P>0.05). The expression of VGLUT2-positive neurons was abundant in nodose ganglia, and the co-expression rate of pERK and VGLUT2 was nearly 90%. The co-expression rate of pERK and VGLUT2 decreased to about 30% after inhibition of VGLUT2 neurons in ganglion. Conclusions:The mechanism by which dexmedetomidine causes bradycardia is associated with activation of VGLUT2 neurons in vagal nodose ganglia in mice.