Objective To determine the change in PSD95 mRNA expression in spinal dorsal horn in a rat model of neuropathic pain. Methods Twelve female SD rats weighing 150-200 g were randomized into 2 groups (n = 6 each) : control group in which left sciatic nerve and its branches were exposed but not cut; SNI group in which the branches of the left sciatic nerve-tibial and common fibular nerves were ligated and cut. Pain threshold was measured by foot-lift response to mechanical stimulation of ipsilateral hindpaw with 12 g and 2 g produced by plantar touch stimulator (Ugo Basile Co. Italy) representing hyperalgesia and allodynia respectively, 3 days before, immediately after and on the 1st, 3rd, 5th, 7th, 9th and 11th day after operation. On the 11th day the animals were killed after measurement of pain threshold and L4-6 segment of the spinal cord was removed for determination of expression of NR2B and nNOS by immuno-histochemistry and expression of PSD95 mRNA by RT-PCR.Results Starting from the 5th day after operation all rats in SNI group developed a relative mechanical allodynia. The expression of NR2B and nNOS was mainly distributed in Ⅰ or Ⅱ laminae of dorsal horn. The expression of NR2B and nNOS was significantly higher in SNI group than in control group. The expression of PSD95 mRNA in SNI group was significantly decreased when compared to control group (P

Objective To investigate the changes in the expression of protein kinase C?and C?(PKC?, PKC?) in the dorsal horn of spinal cord in a rat with neuropathic pain. Methods Twenty-four healthy SD rats weighing 150-250 g were randomly divided into 3 groups (n = 8 each): groupⅠsham operation; groupⅡchronic constructive injury (CCI) and groupⅢspinal nerve ligation (SNL) . CCI was produced by placing 4 loose ligatures on the sciatic nerve at 1 mm intervals with 4-0 catgut and SNL by ligation and transaction of L5 spinal nerve. The threshold to von Frey hair stimulation and radiant heat were measured before operation (baseline) and on the 2nd, 4th and 7th day after operation. The animals were killed after measurement of pain threshold. The lumbar segment (L4-6) of the spinal cord was removed for determination of the expression of PKC?, PKC?and Fos protein by immuno-histochemistry. Results The mechanical and thermal withdrawal latencies were significantly decreased on the 4th and 7th day after operation as compared to the baselines values before operation in CCI and SNL groups. But there was no significant difference in the withdrawal latencies to mechanical and thermal stimuli between CCI and SNL groups. The expression of PKC?, PKC?and Fos protein was significantly higher in CCI and SNL groups than in sham operation group. The expression of PKC?and Fos protein was not significantly different between CCI and SNL groups, while PKC?expression was significantly higher in group SNL than in group CCI.Conclusion The increase in the expression of PKC?and PKC?in the dorsal horn of spinal cord is involved in the mechanism of central sensitization in the neuropathic pain.

BACKGROUND: It is demonstrated that ropivacaine has less toxicity than bupivacaine, but bupivacaine has higher liposolubility and efficacy, so a less dose of bupivacaine is needed in clinical comparing with ropivacaine. Serious convulsion is usually followed by cardiotoxicity induced by local anesthetics. The ratio of medial lethal dose (CD50) and median convulsant doses (LD50) is usually used to assess the comparative safety of local anesthetics.OBJECTIVE: To establish CD50 and LD50 of 2% lidocaine, 0.75% bupivacaine and 0.75% ropivacaine for Kunming mice and select proper indicator for neurotoxicity, then to compare neurotoxicity of the three local anesthetics on central nervous system.DESIGN: Randomized and controlled study.SETTING: Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: This study was carried out from July to December in 2002 in the Laboratory of Anesthesiology, Tongji Hospital, Tonji Medical College, Huazhong University of Science and Technology. Totally 310Kunming mice aged of 1-month with clean grade were enrolled in this study.METHODS: ① To determine the relation of dose of local anesthetics with conclusion rate and death rate in mice. Todetermine the dose-effect relationship for ropivacaine, 50 mice were selected and divided into 5 groups with 10rates in each group who received dose of 76.80, 68.69, 61.44,49.15, 31.46 mg/kg respectively. For bupivacaine, 90 mice were divided into 9 groups, with 10 rates in each group who received intraperitoneal dose of 50.00, 47.29, 44.72, 42.29, 40.00, 35.78, 32.00, 28.62, 25.60 mg/kg respectively. For lidocaine, 100 mice were divided into 10 groups,with10rates in each group who received dose of 183.11, 163.77, 146.48,131.02, 117.19, 93.75, 75.00, 60.00, 48.00, 38.40 mg/kg respectively. For each local anes thetic, the rates of convulsion or death were tried to distribute on both sides of 50% symmetrically. On the dose-response curve, 4or 5 well-spaced points were obtained for probit analysis to determine CD50 and LD50 of each agent. ② The effect of different dose of lidocaine on conclusion duration and c-fos expression in brain with different doses.Forty mice were divided into 4 groups with 10 rates in each group who received 0, 30%, 60% and 90% convulsant doses of lidocaine intraperitoneally. The duration of convulsion were recorded carefully for the convulsant mice that should be marked correctly for next procedure. Two hours later, the convulsant mice were anesthetized deeply and fixed by transcardiac perfusion for Immunohistochemistry to detect c-Fos expression. ③ Comparison of neurotoxicity induced by CD50 of three agents.Thirty mice were randomly divided into 3 groups with 10 rates in each group who received intraperitoneally CD50 of lidocaine, bupivacaine and ropivacaine respectively. The duration of convulsion and the number of neurons expressed with c-Fos in mice brain were compared among these three groups.MAIN OUTCOME MEASURES: The response of mice to intraperitoneal local anesthestics, duration of convusion and c-Fos expression using immunohistochemistry methods.RESULTS: Date of totally 310 mice was entered into final results analysis. ① The relation of dose of local anesthetics and conclusion rate or death rate in mice. The therapeutic index (LD50/CD50) of 2% lidocaine,0.75% bupivacaine and 0.75% ropivacaine were 2.89, 1.48 and 1.34, respectively. ② c-Fos expression induced by lidocaine in mice brain: The cFos expression in mice brain was mainly distributed in three zones-thalamencephal, hypothalamus, amydyla and pyriform cortex. ③ Compare of the duration of convulsion and number of neurons with c-Fos expression induced by different dose oflidocaine. Compared with control group, the duration of convulsion and number of neurons with c-Fos expression in amydyla and pyriform cortex all increased significantly in CD30, CD60and CD90 group (P ＜ 0.05). ④ Neurotoxicity induced by CD50 of lidoacaine, bupivacaine and ropivacasine The duration of convulsion and expression of c-fos in amydyla and pyriform cortex were significantly increased in ropivacaine group compared to bupivacaine or lidocaine group intraperitoneally (P ＜ 0.05). There were no significant differences in the duration of convulsion and expression of c-Fos between lidocaine and bupivacaine group (P ＞ 0.05).CONCLUSION: Compared with bupivacaine, ropivacaine produced less toxicity when identical dose was used in clinic. It is indicated that if an accidental convulsion induced by ropivacaine, it may be more severe than that induced by correspondent either lidocaine or bupivacaine. It may be the reason that ropivacaine have less lipid solubility, absorbed easily from this tissue compartment, and to get a high concentration in blood.

Objective To investigate the signaling pathways involved in the activation of neuron and glia in spinal cord in rats with neuropathic pain. Methods Twelve female SD rats (weighted 150 to 200 g) were randomized into two groups of spared nerve injury(group SNI) and control(group C). Surgery was performed to build model of SNI neuropathic pain in group SNI. Foot-lift response frequency to mechanical stimulation for ipsilateral hindpaw was assessed by 12 g and 2 g touch stimulator at different times. On the 11~(th) day after operation, 3 rats from each group were fixed by perfusion and the expressions of mitogen-activated/extracellular signal-regulated kinase (MEK), p-mitogen-activated/extracellular signal-regulated kinase (p-MEK), p-extracellular regulated protein kinase(p-ERK) and nuclear factor kappa B (NF-κB) were detected by immunohistochemistry method. And proteins from ipsilateral LA-6 spinal cord in other 3 rats from each group were extracted for Western Blot analysis. Western Blot and immunohistochemistry were performed with antibodies specific for MEK, p-MEK, pERK and NF-κB. Results All rats in group SNI developed a relative unchangeable mechanical allodynia since the 5~(th) day after operation. The results of immunohistochemistry method showed that the expression of MEK was mainly in cytoplasm, p-MEK in cell nuclear, p-ERK in astrocyte and NF-κB in neuron according to morphologic observation. Western Blot analysis indicated that the expressions of p-MEK, p-ERK and NF-κB in group SNI were increased significantly compared with those in group C(P<0. 05). Conclusion In the spinal cord of rats with neuropathic pain, MEK-ERK signaling pathway is activated in astrocytes and NF-κB in neurons, which may contribute to the development of neuropathic pain.

This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current (I(NMDA)) and the expression of cytochrome C in cultured developing rat hippocampal neurons. The hippocampi were dissected from newborn Sprague-Dawley rats. Hippocampal neurons were primarily cultured for 5 days and then treated with different concentrations of isoflurane [(0.25, 0.5, 0.75, 1 minimum alveolar concentration (MAC))]. The peak of I(NMDA) was recorded by means of the whole cell patch clamp technique. The cytochrome C level was detected by Western blotting and quantitative real-time PCR. Our results showed that isoflurane (0.25, 0.5, 0.75 and 1 MAC) potentiated the amplitude of I(NMDA) by (116±8.8)%, (122±11.7)%, (135±14.3)% and (132±14.6)%, respectively, and isoflurane increased the mRNA expression of cytochrome C in a concentration-dependent manner. The cytochrome C mRNA expression reached a maximum after 0.5 MAC isoflurane stimulation for 6 h (P<0.05). It was concluded that isoflurane enhances the expression of cytochrome C in cultured rat hippocampal neurons, which may be mediated by facilitation of NMDA receptor.

Objective To investigate the effects of isoflurane anenthesia on myocyte enhancer factor 2(MEF2) signaling pathway in neonatal rat hippocampus. Methods Twenty-four 5-day-old SD rats of both sexes,weighing 10-13 g, were randomly divided into 2 groups ( n = 12 each): control group (group C) and isoflurane group (group I). In group I, 1.5% isoflurane in 100% O2 was inhaled for 6 h. Group C received no treatment.Three rata in each group were sacrificed at 2, 4, 6 h of isoflurane anenthesia and 24 h after isoflurane anenthesia (T1-4), and the hippocampi removed for determination of MEF2 mRNA, synGAP Ⅰ mRNA, Arc mRNA and synapsinⅠ mRNA expression (by PT-PCR) and synapsin Ⅰ protein expression (by Western blot).Results Compared with group C, the expression of MEF2 mRNA, synGAP Ⅰ mRNA, Arc mRNA and synapsin Ⅰ mRNA at T1-3 and synapsin Ⅰ protein at T2-4 was up-regulated in group I ( P ＜ 0.05). Conclusion Inhalation of anaesthetic concentration of isoflurane may affect synapse formation during the development of central nervous system by actirating hippocampal MEF2 signaling pathways in neonatal rats.

This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current (INMDA) and the expression of cytochrome C in cultured developing rat hippocampal neurons.The hippocampi were dissected from newborn Sprague-Dawley rats.Hippocampal neurons were primarily cultured for 5 days and then treated with different concentrations of isoflurane [(0.25,0.5,0.75,1 minimum alveolar concentration (MAC))].The peak of INMDA was recorded by means of the whole cell patch clamp technique.The cytochrome C level was detected by Western blotting and quantitative real-time PCR.Our results showed that isoflurane (0.25,0.5,0.75 and 1 MAC) potentiated the amplitude of INMDA by (116±8.8)％,(122±11.7)％,(135±14.3)％ and (132±14.6)％,respectively,and isoflurane increased the mRNA expression of cytochrome C in a concentration-dependent manner.The cytochrome C mRNA expression reached a maximum after 0.5 MAC isoflurane stimulation for 6 h (P＜0.05).It was concluded that isoflurane enhances the expression of cytochrome C in cultured rat hippocampal neurons,which may be mediated by facilitation of NMDA receptor.

Objective:To evaluate the hemostatic effects of our self-designed pelvic band with inflatable balloon in a swine model of hemodynamically unstable pelvic fracture.Methods:"Open-book like" fractures were created with the external iliac blood vessels exposed in 24 12-month-old female Bama miniature pigs which were randomly divided into 4 groups ( n=6). Group C (the control group) was subjected to no treatment other than exposure of the external iliac blood vessels, group D to no treatment following destruction of the external iliac blood vessels, group T1 to fixation with simple pelvic band after destruction of the external iliac blood vessels, and group T2 to fixation with our self-designed pelvic band with inflatable balloon after destruction of the external iliac blood vessels. The 4 groups were compared in terms of 40-min survival rate, bladder pressure, peak lactate value, total blood loss, bleeding rate, infusion rate, and angiographic images. Results:There was no significant difference in the baseline indexes among the 4 groups before experiment, showing comparability ( P>0.05). The 40-min survival rate in group T2 was 83.3% (5/6), significantly higher than that in groups D and T1 [0% (0/6) and 0% (0/6)] ( P<0.05). There were no significant differences among groups C, D, T1 and T2 in bladder pressure [(6.67±1.03) mmHg, (5.83±1.94) mmHg, (6.00±1.55) mmHg, and (6.00±1.10) mmHg] or in total blood loss among groups D, T1 and T2[(1,198.0±182.9) mL, (1,252.0±148.4) mL, and (1,150.0±125.7) mL] (all P>0.05). The peak lactate value in group T2 [(2.26±0.24) mmol/L] was significantly lower than that in group D [(5.00±0.60) mmol/L] and group T1 [(3.86±0.57) mmol/L], and the bleeding rate and infusion rate in group T2 [(25.83±5.49) mL/min and (26.00±4.69) mL/min] were also significantly lower than those in group D [(83.50±19.85) mL/min and (71.50±29.11) mL/min] and group T1 [(54.17±15.59) mL/min and (54.17±8.98) mL/min] (all P<0.05). Angiography showed contrast agent extravasation in group T2, especially from the artery, but the extravasation speed in group T2 was significantly slower than that in group D. Conclusion:In a swine model of hemodynamically unstable pelvic fracture, our self-designed pelvic band with inflatable balloon has a definite hemostatic effect on vascular injury which is better than that of a simple pelvic band.

Objective:To determine the effects of preperitoneal balloon (PPB) tamponade with different volumes of fluid on hemodynamically unstable pelvic fracture-associated arterial and venous hemorrhage in a swine model.Methods:A model of open-book pelvic fracture with injuries to external iliac vessels was established in 18 female 12-month old Bama miniature pigs. After the successful establishment of hemodynamically unstable pelvic fracture with vascular injury was confirmed by contrast agent imaging, the animals were randomized into 3 even groups ( n=6): a control group (group C) subjected to PPB tamponade with 0 mL fluid injected, group T1 subjected to PPB tamponade with 500-mL fluid injected, and group T2 subjected to PPB tamponade with 1,000-mL fluid injected. The 3 groups were compared in terms of 60-min survival rate, balloon pressure, peritoneal pressure, bladder pressure, 70-min survival rate, blood loss, and infusion volume. Results:There was no statistically significant difference in the basic hemodynamic or other experimental indicators among the 3 groups before experiment, indicating comparability ( P>0.05). The 60-min survival rate in group T2 was 100.0% (6/6), significantly higher than those in group C and group T1 [0.0% (0/6), 0.0% (0/6)] ( P<0.05). After fluid injection, the balloon pressure and preperitoneal pressure in group T2 were respectively (127.2±4.7) mmHg and (34.5±3.6) mmHg, significantly higher than those in group T1 [(78.7±3.8) mmHg and (13.7±2.8) mmHg] and in group C [0 mmHg and (9.0±1.4) mmHg], and the 2 indicators in group T1 were significantly higher than those in group C (all P<0.05). After fluid injection, there was no statistically significant difference among groups C, T1, and T2 in bladder pressure [(6.7±1.0) mmHg, (5.8±1.9) mmHg, and (6.0±1.1) mmHg] or in bleeding volume [(1,163.0±191.3) mL, (1,212.0±148.4) mL, and (975.0±133.2) mL] (all P≥ 0.05). The infusion volume in group T1 [(1,250.0±225.8) mL] was significantly larger than that in group C [(951.7±177.8) mL] ( P<0.05). No colorectal or bladder injuries were found by the anatomy of the experimental animals in 3 groups. Conclusions:PPB tamponade with 1,000-mL fluid injected in a swine model can efficiently control pelvic fracture-associated arterial and venous hemorrhage, and increase the 60-min survival rate with no colorectal or bladder injuries.