Objective To investigate the effects of intrathecal morphine on cell-mediated immunity. Methods Forty male SD rats weighing 250-300 g were randomly divided into 5 groups ( n = 8 in each group) : sham-operated group (F); saline group (NS) and 3 morphine groups (M1, M2, M3). The animals were anesthetized with intraperitoneal chloral hydrate 300-350 mg?kg-1 . Microspinal catheter was inserted into the subarachnoid space at the lumbar region according to modified Yaksh technique. Correct implantation of the spinal catheter was confirmed by aspiration of CSF. In the morphine groups, after 5 days morphine was continuously infused through the spinal catheter at 2.5 (Ml), 5.0 (M2) and 10 ?g?h-1(M3) for 7 days. In NS group normal saline was continuously infused instead of morphine. On the 7th day 5% formalin 50 fd was injected into the plantar surface of left hindpaw. The number of flinches, lickings and total time of licking were recorded for 60 min. Pain intensity scoring (PIS) (0-3, 0 = no pain, 3 = severe pain) was used to assess the antinociceptive effect of intrathecal morphine. The animals were killed after evaluation of pain intensity. Body weight and spleen weight were measured. Spleen index (spleen weight/body weight) was calculated. T-lymphocyte function was evaluated based on Concanavalin-A (Con A) induced splenocyte proliferation. Modified lactic acid dehydrogenase (LDH) release assay was used to assess NK cell activity. Results The PIS scores were significantly lower in group M1 , M2 and M3 than in F and NS group. The spleen index, splenocyte proliferation induced by Con A and NK cell activity were significantly suppressed in the 3 morphine groups Conclusion Intrathecal morphine has significant antinociceptive effect and suppresses T-lymphocyte proliferation and NK cell activity in a dose-dependent manner

Objective To investigate the effects of continuous spinal anesthesia with different concentrations and doses of ropivacaine on the ultrastructure of the spinal cord and nerve roots. Methods Twenty-four male SD rats weighing 220-280 g were anesthetized with intraperitoneal 10% chloral hydrate 300-350 mg/kg. A polyurethane microcatheter was inserted into the lumbar subarachnoid space according to the technique described by Yaksh. An 8-cm catheter segment was left in the subarachnoid space. The animals were randomized to receive normal saline, 0.5%, 0.75% or 1.0% ropivacaine 40 ? 1 intrathecally 3 times at 1.5 h interval. Six hours after the first intrathecal administration the animals were decapitated and L1 ,2 segment of the spinal cord and nerve roots were immediately removed for electron microscopic examination. Results Electron microscopic examination revealed that in animals which received intrathecal (i.t.) normal saline, 0.5% or 0.75% ropivacaine the neurolemma of the nerve roots and the mitochondria and endoplasmic reticulum of the neurons in the spinal cord were intact, while in animals which received i.t. 1.0% ropivacaine the neurolemma was stratified and partly disrupted and there were swelling of endoplasmic reticulum and vacuole degeneration. Conclusion Six hours continuous spinal anesthesia with 1.0% ropivacaine may be injurious to the spinal cord and nerve roots.

Objective: To investigate the function of the ATP-sensitive K+(KATP) channel activation on the protective effect of hypercarbonic acidosis preconditioning on rabbit myocardial cells. Methods: Thirty-two rabbits were randomly divided into 4 groups (n = 8 for each group): pseudo-operation group (group P), ischemia and reperfusion group(group IR), hypercarbonic acidosis group(group H) and hypercarbonic acidosis+ glybenzcyclamide group (group H+G). Animals were ventilated normally in group IR and group P, tidal volume 15 mL/kg, breathing rate 35 bpm .The PETCO_2 was maintained at the level of 40-50 mm Hg for 30 minutes. Animals received low-frequency, low volume ventilation in group H group H+G, tidal volume 10 ml/kg, breathing rate 25 bpm to achieve hypercarbonic acidosis. The target value of PETCO_2 was 75-85 mm Hg. This value was maintained for 5 minutes. The animals then were ventilated normally to make the PETCO_2 return to 40-50 mm Hg. Animals were injected with 0.3 mg/kg glybenzcyclamide 10min before achieving hypercarbonic acidosis with hypoventilation in group H+G. Animals received ligation of left anterior branch artery for 30 minutes and reperfusion for 180 minutes in each group except P group. The myocardial ischemia area, the myocardial infarction area and their ratios were calculated by the ismaeil methods. Results: The ratio of the myocardial infarction area to the myocardial ischemia was significantly less in group H than those of group IR and group H+G (P < 0.01). The value of the ratio was similar between group H+G and group IR(P > 0.05). Conclusion: Hypercarbonic acidosis preconditioning can protect the cardiomyocytes by activating the KATP channel.

Objective To investigate the effect of high concentration carbon dioxide preconditioning on lipid peroxidation during myocardial ischemia-reperfusion (I/R) in rabbits. Methods Twenty-four New Zealand white rabbits weighing 2.0-3.9 kg were randomly divided into 3 groups ( n = 8 each): sham operation group (group S) , I/R group, high concentration carbon dioxide preconditioning group (group H) . The amimals were tracheal intubated and mechanically ventilated. In groups S and I/R, fresh gas flow was set at 0.3 L/min (100% O2 ), respiratory rate 30-40 bpm and tidal volume IS ml/kg, and PETCO2 was maintained at 40-50 mm Hg for 30 min. In group H, fresh gas flow was set at 0.3 L/min (100% O2), respiratory rate 20-30 bpm and tidal volume 10 ml/kg, PETO2 was maintained at 75-85 mm Hg for 5 min, and then all the ventilatory parameters were adjusted to the same as those in groups S and I/R. Myocardial I/R was produced by occlusion of left anterior descending branch of coronary artery for 30 min followed by 3 h reperfusion after preconditioning in groups I/R and H. The animals were sacrificed at the end of reperfusion and myocardial tissues obtained for determination of the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content and examination of the ultrastnicture of myocardium with the transmission electron microscope. Results The SOD activity was significantly lower, while MDA content higher in group I/R than in group S ( P ＜ 0.01) . The SOD activity was significantly higher, while MDA content lower in group H than in group I/R ( P ＜ 0.01) . The myocardial injury was attenuated in group H compared with group I/R. ConclusionHigh concentration carbon dioxide preconditioning can reduce myocardial I/R injury in rabbits through inhibiting lipid peroxidation.

Objective To investigate the effect of lentivirus-mediated RNA interference(RNAi) on the expression of PKCγ mRNA and protein in rat neurons.Methods Primary cultured cortical neurons from SD rat (16 days of pregnency) embryos were randomly divided into 3 groups with 6 wells in each group:control group (group C),negative group(group NC)and RNAi group.Group C received no treatment.Each well in group NC was given negative lentivirus 3 × 105.Each well in group RNAi was given the recombinant lentiviral vector containing PKCγ shRNA(LV-PKC7 shRNA).The expression of PKCT mRNA and protein in rat neurons was detected by RT-PCR and Western blot 5 days later.The interference efficiency was ealculated.Results Compared with group NC,the expression of PKCγ mRNA and protein was down-regulatedin group RNAi(P<0.05),but no significant change Was found in group NC(P>0.05).The interference efficiency of gene and protein were 99.3%and 85.2%respectively.Conclusion Lentivirus-mediated RNAi can down restate the expression of PKC7 gene and protein in rat neurons.

Objective To investigate the effect of intrathecal injection of lentiviral vector-mediated up-regulation of glial cell line-derived neurotrophicfactor (GDNF) on neuropathic pain of chronic constriction injury (CCI) rats.Methods The CCI model was prepared by ligating the sciatic nerve of Sprague-Dawley (SD) rats.Seven days after CCI modeling,a single intrathecal injection of lentiviral vectors (LV)-GDNF was given.Before CCI and 3,5,7,14,and 21 days after CCI modeling,the mechanical pain threshold was tested in rats,and 21 days after surgery,Western blot was used to detect the expression of GDNF protein.Results On 21 days after CCI modeling,GDNF expression was reduced compared to sham group.After intrathecal injection of LV-GDNF,GDNF expression was up-regulated in the spinal cord,and CCI-induced mechanical hyperalgesia in rats was alleviated.Conclusions Intrathecal injection LV-GDNF can up-regulate the expression of GDNF and alleviate neuropathic pain in CCI rats.

Objective To investigate the role of cyclooxygenases (COXs) in the up-regulation of the expression of P2X3 receptors in the dorsal root ganglion (DRG) in rats with neuropsthic pain. Methods Twenty-four male SD rats, weighing 250-280 g, were randomly divided into 4 groups ( n = 6 each): sham operation group (group S), chronic constrictive injury (CCI) group, COX-1 inhibitor ibuprofen group (group Ⅰ), and COX-2 inhibitor celecoxib group (group C). Neuropathic pain was induced by CCI. The animals were anesthetized with intraperitoneal 10% chloral hydrate 300-500 mg/kg. CCI was produced by placing 4 ligatures on the left sciatic nerve at 1 mm intervals. In group S, the left sciatic nerve was only exposed but not ligated. In groups Ⅰ and C, ibuprofen 40 mg·kg-1 ·d-1 and celecoxib 30 mg·kg-1 ·d-1 were given through a gastric tube into the stomach at day 3-14 after operation respectively. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured before operation (baseline), and at 3, 5, 7, 10 and 14 days after operation. Then the rats were sacrificed and their L()-6 DRGs were removed to detect the expression of P2X3 mRNA and protein. Results Compared with group S, PWL was significantly shortened, PWT decreased, and P2X3 mRNA and protein expression up-regulated in group CCI ( P ＜ 0.05＝. Compared with group CCI, PWL was significantly prolonged, PWT increased, and P2X3 mRNA and protein expression down-regulated in groups Ⅰ and C (P ＜0.05＝. Compared with group Ⅰ, PWL was significantly prolonged, PWT increased, and P2X3 mRNA and protein expression up-regulated in group C ( P ＜0.05＝. Conclusion COXs are involved in the up-regulation of the expression of P2X3 receptors in the DRG in rats with neuropathic pain, and the effect of COX-1 is stronger than that of COX-2.

Objective To investigate the effects of maternal behaviors in the rats with neuropathic pain (NP) on emotions of offspring rats and the relationship with DNA methylation in the amygdala.Methods Forty-eight healthy adult Sprague-Dawley rats (24 males and 24 females),weighing 200-250 g,were used in the study.Twelve female and 12 male rats were randomly selected,and NP was induced by chronic constriction injury (CCI).Each female rat was mated with one male rat at 10 days after CCI.Fortyeight F1 generation rats of maternal rats with NP were randomly divided into 2 groups (n =24 each) using a random number table:NP1 group and NP2 group.Forty-eight F1 generation rats of normal maternal rats were randomly divided into 2 groups (n=24 each) using a random number table:S1 group and S2 group.The F1 generation rats were cross-fed immediately after birth between group NP2 and group S2,and fed by their own mother rats in NP1 and S1 groups.All the offspring rats were fed to 21 days after birth by the maternal rats selected,and separately fed to 30 days after birth,and then subjected to behavioral testing.Retrieving and licking pups were recorded after delivery in maternal rats to evaluate the maternal behaviors.The mechanical and thermal paw withdrawal thresholds were measured in the offspring rats.Elevated plus maze and open field tests were conducted to detect anxiety and depression behaviors in the offspring rats.At 1 day after completion of behavioral testing,the expression of DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a and 3b in the amygdala was detected by Western blot analysis.Results Compared with S1 or S2 groups,the latency to lick pups,latency to retrieve pups,and total retrieval time were significantly prolonged,and the total time spent licking pups was significantly shortened in NP1 group or NP2 group (P＜0.05 or 0.01).There was no significant difference in the mechanical and thermal paw withdrawal thresholds in the offspring rats between the four groups (P＞0.05).Compared with group S1,the ratios of time spent in the open arm to the closed arm and of time spent in the central square to the peripheral square were significantly decreased,DNMT1 expression in the amygdala was significantly up-regulated,and the total DNA methylation was increased in the offspring rats in S2 and NP1 groups (P＜0.05).Compared with group NP2,the ratios of time spent in the open arm to the closed arm and of time spent in the central square to the peripheral square were significantly decreased,DNMT1 expression in the amygdala was significantly up-regulated,and the total DNA methylation was increased in the offspring rats in S2 and NP1 groups (P＜0.05).Conclusion Decreased maternal behaviors in the rats with NP results in negative emotions including anxiety and depression in the offspring rats,and the mechanism is related to increased DNA methylation in the amygdala of the offspring rats.

Objective To construct a lentiviral vector of RNA interference (RNAi) of PKCγ gene. Methods The effective sequence of siRNA targeting PKCγ gene was confirmed in our previous study. The complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, which contained U_6 promoter and green fluorescent protein (GFP) . The resulting lentiviral vector containing PKCγshRNA was named lentivinis RNAi vector of PKCγ, and it was confirmed by realtime PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGCSIL-CTP, pHelper 1.0 and pHelper 2.0. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of PKCγ producing PKCγshRNA was constructed successfully. The titer of concentrated virus was 1 ×10~9 TU/ml. Conclusion The lentivinis RNAi vector of PKCy was constructed successfully.

This study observed the protective effect of hypercapnic acidosis preconditioning on rabbit heart suffered from ischemia-reperfusion injury. Hypercapnic acidosis was established in animals with mechanical hypoventilation before ischemia-reperfusion. Thirty-two rabbits were randomly divided into 4 groups, with each having 8 animals in term of the degree of acidification: hypercapnic acidosis group A (group A), hypercapnic acidosis group B (group B), hypercapnic acidosis group C (group C), ischemia and reperfusion group (group IR). Animals in group IR were ventilated normally (tidal volume: 15 mL/kg, breathing rate 35 bpm). The PETCO(2) was maintained at the level of 40-50 mmHg for 30 min. Animals in groups A, B, C received low-frequency, low-volume ventilation to achieve hypercarbonic acidosis and the target levels of PETCO(2) were 75-85,65-75, 55-65 mmHg, respectively, with levels being maintained for 5 min. The animals then were ventilated normally to lower PETCO(2) to 40-50 mmHg. The left anterior branch artery of all the animals was ligated for 30 min and reperfused for 180 min. Then the infarct size was calculated. The cardiomyocytes were morphologically observed and ECG and hemodynamics were monitored on continuous basis. Acid-base balance was measured during procedure. Our results showed that the infarct size was (48.5+/-11.5)% of the risk area in the control group and (42.4+/-7.9)% in group C (P>0.05). Mean infarct size was significantly smaller in group B (34.5%+/-9.4%) (P<0.05 vs control group) and group A (31.0%+/-9.1%) (P<0.01 vs control group). It is concluded that HA-preconditioning can effectively protect the myocardium.
Acidosis, Respiratory/*physiopathology ; Hypercapnia/*physiopathology ; Ischemic Preconditioning, Myocardial/*methods ; Myocardial Reperfusion Injury/*prevention & control ; Random Allocation