Objective To evaluate the role of extracelluar signal-regulated kinase (ERK)-cyclic AMP response element binding protein (CREB) signaling pathway in the spinal cord in naloxone-induced withdrawal response in morphine-dependent rats.Methods Fifty male adult SD rats,aged 2 months,weighing 200-250 g,in which intrathecal catheters were successfully implanted without complications,were randomly divided into 5 groups (n =10 each):group control (group C); group morphine dependence (group MD); group morphine withdrawal (group MW); group U0126 (ERK signaling pathway blocker); group dimethyl sulfoxide (DMSO,solvent for U0126).Morphine dependence was induced by increasing doses of subcutaneous morphine for 6 days.The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg twice every other day until 50 mg/kg on 6th day in groups MD,MW,U0126 and DMSO.Morphine withdrawal response was induced by intraperitoneal naloxone 4 mg/kg at 4 h after last morphine administration in groups MW,U0126 and DMSO.U0126 150μg (in DMSO 10 μl) and DMSO 10 μl were administered intrathecally at 30 min before naloxone administration in groups U0126 and DMSO respectively.Morphine withdrawal response (0=no withdrawal response,3 =severe response)and touch evoked agitation (0 =no agitation,2 =severe agitation) were observed and scored during 1 h after naloxone administration.The animals were then sacrificed and the spinal cord was removed for determination of the expression of phosphorylated ERK (p-ERK) and phosphorylated CREB (p-CREB) by immuno-histochemistry and Western blot.Results Morphine withdrawal significantly up-regulated the p-ERK and p-CREB expression in group MW compared with group C ( P ＜ 0.05).Withdrawal response score and touch evoked agitation score were significantly increased in groups MW,U0126 and DMSO as compared with group MD ( P ＜ 0.05).U0126 pretreatment significantly attenuated naloxone-induced increase in withdrawal response score and touch evoked agitation score and down-regulated p-ERK and p-CREB expression in group U0126 as compared with group MW ( P ＜ 0.05).Conclusion ERK-CREB signaling pathway in the spinal cord is involved in morphine withdrawal response in morphine-dependent rats.

Actovegin is a drug,which can improve cellular energy metabolism,especially glycometabolism.It has an insulin-like effect,such as increasing the uptake of glucose and activating glucose metabolic enzyme.In addition,it can inhibit the production of lactic acid.Actovegin was mainly applied to the treatment of Alzheimer disease initially,but now it is indicated more and more.

BACKGROUND: Recently, lithium was reported shown neuroprotective effect against apoptosis induced by a variety of insults in vitro and in vitro,but the precise mechanisms underlying its neuroprotective effect remain unknown.OBJECTIVE: To observe the effect of lithium chloride on neuronal apoptosis and the expression of P53 or nuclear factor kappa B (NF-κB) protein in the CA1 region of the hippocampus after global ischemia in gerbils.DESIGN: A randomized controlled experimental research.SETTING: Department of Anatomy of Nanjing Medical University.MATERIALS: Fifty-four healthy male gerbils weighing 50-70 g, clearing grade, were purchased from Experimental Animal Center of Zhejiang Province.METHODS: Totally 54 gerbils were randomly divided into three groups namely: sham-operation group (SH group), ischemia-reperfusion group (IR group) and lithium chloride group (LI group), with 18 in each group. SH group, IR group and LI group were further divided into 3 subgroups respectively (SH1d, SH3d, SH7d; IR1d, IR3d, IR7d; LI1d, LI3d, LI7d), according to the time of reperfusion, with 6 gerbils in each. Gerbils in LI group were injected intraperitoneally with lithium chloride 3 mEq /kg, once a day for 7consecutive days before operation. Normal saline was used instead of lithium in SH group and IR group as vehicle control. Forebrain ischemia was induced at 24 hours after the last injection of lithium chloride. After gerbils being anesthetized, the bilateral common carotid arteries were blocked with micro aneurysm clips for 5 minutes, and the micro aneurysm clips were removed and the cerebral blood flow restored. Sham-operation animals were underwent the same operation except occlusion of bilateral common carotid arteries. Gerbils in each group were killed at every time points.4 μm coronal sections at 1.7-4.0 mm visual cross were cut at the level of the dorsal hippocampus. The apoptosis cells were assayed with in situ Cell Death Detection Kit, and assay of positive cell in cell apoptosis, P53 and positive NF-κB was performed with immunohistochemistry staining. The total number of TUNEL positive cells, P53 or NF-κB positive cells per image (area of 1 mm2) was counted.MAIN OUTCOME MEASURES: Neuronal apoptosis and expression of P53 or NF-κB protein in the CA1 region after cerebral ischemic reperfusion.apoptosis cell in cerebral hippocampus CA1 region: No TUNEL positive cells were detected in SH group, a large majority of TUNEL positive cells were detected in the CA1 region in IR group on the 3rd day after reperfusion [(552.0±145.5, 142.4±103.5) pcs/mm2, t= 5.623, P ＜ 0.01], and TUNEL positive cells declined on the 7th day after reperfusion. The numbers of TUNEL positive cells in the CA1 region of LI3d, LI7d group were significantly lower than those of IR3d, IR7d group [(408.0±119.8, 156.0±108.2) pcs/mm2,CA1 region: In IR group, the expression of P53 protein was increased on the 1st, 3rd and 7th day after reperfusion compared with that in SH group and cerebral hippocampus CA1 region: No NF-κB protein was expressed in SH group. In IR group, the expression of NF-κB protein was increased on the 1st day after reperfusion (78.5±25.2)/mm2, significantly increased on the 3rd day after reperfusion (176.5±35.5)/mm2 and on the 7th day after reperfusion, the expression of NF-κB protein disappeared. There were no significant statistical difference between LI group and IR group on the 1st day after reperfusion. The expression of NF-κB protein in LI group was significantly lower than that in IR group on the 3rd day after reperfusion [(64.5±30.8)/mm2,t=5.824, P ＜ 0.01].CONCLUSION: Lithium chloride can significantly suppress neuronal apoptosis after global ischemia in gerbils. The down-regulation of expression of P53 or NF-κB protein is one of the mechanisms of the neuroprotective effect by lithium chloride.

Objective To investigate the effects of thoracic epidural anesthesia and analgesia on cellular immune function and erythrocytes glycometabolism in the patients undergoing thoracic surgery.Methods Forty esophageal carcinoma patients,classified as ASA Ⅰ or Ⅱ,scheduled for elective thoracic surgery were randomly divided into two groups with 20 cases each：group A underwent general anesthesia plus thoracic epidural anesthesia （TEA） during thoracic surgery and received patient-controlled epidural analgesia （PCEA） with fentanyl and ropivacaine postoperatively;group B received general anesthesia during thoracic surgery and patient-controlled intravenous analgesia （PCIA） postoperatively. Venous blood samples were collected for the measurement of Th1,Th2 and the activities of PFK,G-6PD and AR before the induction（T0）,2 h after the initiation of the incision（T1）,and 4 h（T2）,24 h（T3）and 48 h（T4）after surgery. Results The Th1/Th2 ratio in both groups were decreased significantly after completion of surgery compared with baseline levels （P0.05）. At T2,T3 and T4 the Th1/Th2 ratio in group A were higher than group B. Compared with these before operation,the activity of PFK was decreased significantly and the activities of G-6PD and AR in erythrocytes were increased markedly at T3 in group B（P0.05）.But erythrocytes PFK,G-6PD and AR activity slightly changed in group A.Conclusion These findings show that thoracic epidural anesthesia and PCEA may inhibit Th0 cells to differentiate into Th2 cells,protect cellular immune function and moderate erythrocyte glucose metabolism changes.

ObjectiveTo investigate the effect of propofol on IL-1β and TNF-α release from BV-2 microglia cells induced by lipopolysaccharide (LPS) and the role of Toll-like receptor 4 (TLR4).MethodsBV-2 microglia cells were seeded in 96-well plates and randomly divided into 4 groups ( n =12 each):control group,LPS group,propofol group (group P) and LPS + propofol group.In group LPS,the cells were incubated with LPS 1 μg/ml for 24 h.In group P,the cells were incubated with propofol 30 μmol/L for 24 h.In group LPS + propofol,the cells were incubated with LPS 1 μg/ml and propofol 30 μmol/L for 24 h.The concentrations of TNF-α ( at 6 h of incubation) and IL-1β (at 24 h of incubation) in the supernatant were detected by ELISA.TLR4 mRNA expression was detectedat at 6 h of incubation by RT-PCR.TLR4 protein expression was detected at 24 h of incubation by Western blot.ResultsCompared with control group,IL-1β and TNF-α release was significantly increased,and the expression of TLR4 mRNA and protein up-regulated in groups LPS and LPS + propofol ( P ＜ 0.05).Compared with group LPS,IL-1β and TNF-α release was significantly decreased,and the expression of TLR4 mRNA and protein down-regulated in group LPS + propofol (P ＜ 0.05 ).Conclusion Propofol can inhibit IL-1β and TNF-α release from BV-2 microglia cells induced by LPS and inhibition of TLR4 expression may be involved in the mechanism.

Objective To evaluate the effect of minocycline on the long-term cognitive function after partial hepatectomy in mice.Methods Sixty-four male C57BL/6 mice,aged 3 months,weighing about 20 g,were randomly divided into 4 groups (n =16 each) using a random number table:control group (group C),partial hepatectomy group (group O),PBS group,and minocycline group (Mino group).At 3 days after partial hepatectomy,the rats underwent Morris water maze test.The animals were sacrificed and their hippocampi were immediately removed for determination of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) mRNA (using real-time fluorescent quantitative PCR) and nuclear factor kappa B (NF-κB) expression in the nuclcar protein of the cells (using Western blot).After the mice regained consciousness after operation,minocycline 50 mg· kg-1 · d-1 was injected intraperitoneally for 30 consecutive days in Mino group,while the equal volume of PBS was given instead of minocycline in group PBS.The parameters mentioned above were then recorded.Results Compared with group C,the escape latency and swimming distance were significantly prolonged,and the expression of TNF-α mRNA,IL-6 mRNA and NF-κB was up-regulated in group O (P ＜ 0.05).Compared with group PBS,the escape latency and swimming distance were significantly shortened,and the expression of TNF-α mRNA,IL-6 mRNA and NF-κB was down-regulated in group Mino (P ＜ 0.05).Conclusion Minocycline can improve the long-term cognitive function after partial hepatectomy in mice and inhibition of inflammatory responses in hippocampi is involved in the mechanism.

Objective To evaluate the relationship between protein kinase B (AKT) and protein kinase (PKCθ) and morphine-induced inhibition of differentiation of T helper (Th) cells.Methods Peripheral venous blood samples were taken from 20 healthy volunteers..Peripheral blood mononuclear cells (PBMCs) were extracted by density gradient centrifugation method.CD4+ T lymphocytes extracted were purified by magnetic bead separation.CD4+ T lymphocytes were randomly assigned into 5 groups (n =3 each) using a random number table.CD4 + T lymphocytes were incubated routinely in group C.CD4 + T lymphocytes were incubated in the presence of PMA 25 ng/ml + ionomycin 1 μg/ml (group PI),PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μμg/ml (group M),or PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μg/ml + naloxone 50 μμg/ml (group N).The cells were incubated for 4 h in the incubator containing 5％ CO2 at 37 ℃.The expression of interferon (IFN)-γ and interleukin-4 (IL-4) was detected by flow cytometry.The expression of IFN-γ and IL-4 was used to reflect the percentage of Th1 and Th2 cells,respectively.The ratio of Th1/Th2 was calculated.The expression of AKT,phosphorylated AKT (p-AKT),PKCθ and phosphorylated PKCθ (p-PKCθ) was detected by Western blot,and the ratio of p-AKT/AKT and p-PKCθ/PKCθ was calculated to reflect the activities of AKT and PKCθ,respectively.Results Compared with group C,the percentage of Th1 and Th2 cells and ratio of Th1/Th2 were significantly increased in PI,M and N groups,the activities of AKT and PKCθ were increased in PI and N groups,and the activity of PKCθ was increased in group M (P ＜ 0.05).Compared with group PI,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly decreased in group M,the ratio of Th1/Th2 was decreased in group N (P ＜0.05),and no significant change in the activity of PKCθ was found in M and N groups (P ＞ 0.05).Compared with group M,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly increased (P ＜ 0.05),while no significant change in the activity of PKCθ was observed in group N (P ＞ 0.05).Conclusion The mechanism by which morphine inhibits the differentiation of Th cells through activating opioid receptors is related to inhibition of AKT activation,but not related to PKCθ.

Objective To investigate the effects of lithium chloride pretreatnent on cognitive function after laparotomy in aged rats. Methods Forty-eight male SD rats aged 18 months, weighing 550-700 g were randomly divided into 3 groups (n = 16 each): group Ⅰ control (group C); group Ⅱ operation (group O) and group Ⅲ lithium chloride preconditioning (group L). In group L lithium chloride 2 mmol/kg was injected intraperitoneally (IP) once a day for 7 consecutive days before exploratory laparotomy. In group C and group O equal volume of normal saline was injected instead of lithium chloride. The animals were anesthetized with IP 2 % pentobarbital 0.25 ml/100 g. Morris water-maze (MWM) test was performed at day 4-6 after operation in 8 animals in each group. Another 8 animals were killed at 24 h after operation and their brains were immediately removed for determination of IL-1β content and expression of total glycogen synthase kinase-3β (GSK-3β) and p-GSK-3β in hippocampus by ELISA and Western blot respectively. Results Compared with group C the escape latency and swimming distance were both significantly prolonged at day 4-6 after operation in group O, while in group L only swimming distance was prolonged at day 4 after operation. The IL-1β content in hippocampus was significantly higher and the expression of p-GSK-3β was significantly lower in group O than in group C and L. There was no significant difference in total GSK-3β among the 3 groups. Conclusion Lithium chloride pretreatment can improve the cognitive function after laparotomy in aged rats by inhibiting GSK-3β activity and attenuating inflammatory response in hippocampus.

Objective To investigate the effects of different doses of ketamine on spatial cognitive function and the expression of mRNA and protein of N-methyl-D-uspartate (NMDA) receptor subunits NR1 and NR2B in the hippocampus of aged rats.Methods Forty SD rats of both sexes aged 15 months weighing 470-570 g were randomly divided into 4 groups (n=10 each) : one control group (C) and three ketomine groups (K1 ,K2 ,K3).The animals in group K1 ,K2 and K3 received intraperiteneal(IP) ketaminc 10,20 and 100 mg/kg respectively once a day for 3 days,whereas the animals in control group received IP normal saline instead of ketamine.One day after the last drug administration,the animals underwent Morris water maze test 4 times a day for 3 consecutive days.The animals were killed within 1 h after the last test for determination of the expression of NR1 mRNA and NR2B mRNA (using RT-PCR) and protein (using immuno-histochemistry) in the hippocampus.Results The latency period and swimming distance of water maze test were significantly longer on the 2nd and 3rd days in group K3 than in control group.The NR2B mRNA and protein expression was significantly lower in group K3 than in control group.Conclusion Anesthetic dose of ketamine decreases spatial cognitive function and the expression of NR2B mRNA and protein in aged rats whereas subanesthetic doses of ketamine do not.

Objective To investigate the effects of different concentrations of ketamine on the differentiation of human T helper (Th) cells.Methods Twenty ASA Ⅰ patients (aged 20-60 years) undergoing elective operation under general anesthesia were enrolled in this study.Peripheral venous blood samples were taken before anesthesia.Peripheral blood mononuclear cells were isolated and divided randomly into three groups (n =20 each):being incubated in the presence of 0.9％ NaCl (group C),2.5 μg/ml ketamine (group K1) and 25.0 μg/ml ketamine (group K2),respectively,for 24 hours,and were then stimulated with phytohaemagglutinin for 48 hours,respectively.The percentages of Th1 and Th2 cells were detected by four-color fluorescence flow cytometry.The Th1/Th2 ratio was calculated.The levels of interleukin-2 (IL-2),IL-4,IL-6,IL-10,immunoreactive fibronectin-γ (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in the supernatant were determined by cytometric bead array.Results There was no significant difference in the levels of IL-2,IL-4,IL-6,IL-10,IFN-γ and TNF-α in the supernatant,the percentages of Th1 and Th2 cells and the ratio of Th1/Th2 among groups C,K1 and K2(P＞ 0.05).Conclusion The sedative and anesthetic concentrations of ketamine exert no effect on the differentiation of human Th cells in vitro.