Objective To evaluate the effects of hydrogen-rich saline on inflammatory responses in the sciatic nerve and spinal cord of rats with neuropathic pain (NP).Methods Ninety male adult Sprague-Dawley rats,weighing 180-220 g,were randomly divided into three groups (n=30 each) using a random number table:sham operation group (group S);NP group;hydrogen-rich saline group (group H).NP was induced by chronic constrictive injury.The animals were anesthetized with intraperitoneal 10％ chloral hydrate 300 mg/kg.The left sciatic nerve was exposed and ligated with 4-0 silk at 1 mm intervals.After ligation of the sciatic nerve,hydrogen-rich saline (0.6 mmol/L) 10 ml/kg was injected intraperitoneally twice a day for 7 consecutive days in group H,and the equal volume of normal saline was given in S and NP groups.At 7,10 and 14 days after ligation,the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were then sacrificed,and the left sciatic nerve and L4-6 segments of the spinal cords were removed for detection of the contents of tumor necrosis factor-alpha (TNF-α),interleukin-1 beta (IL-1β) and high mobility group box-1 (HMGB1) at 7,10 and 14 days after ligation.Results Compared with group S,the MWT and TWL were significantly decreased,and the contents of TNF-α,IL-1β and HMGB1 in the left sciatic nerve and spinal cords were increased at each time point in NP and H groups.Compared with group NP,the MWT and TWL were significantly increased,and the contents of TNF-α,IL-1β and HMGB1 in the left sciatic nerve and spinal cords were decreased at each time point in group H.Conclusion Hydrogen-rich saline alleviates NP through inhibiting inflammatory responses in the sciatic nerve and spinal cord of rats.

Objective To evaluate the accuracy of bispectral index (BIS) in predicting outcomes in patients with cerebral hemorrhage.Methods A total of 103 patients of either sex with acute cerebral hemorrhage,aged 18-77 yr,with body mass index of 17-29 kg/m2,of American Society of Anesthesiologists physical status Ⅱ-Ⅳ,undergoing craniotomy for evacuation of hematoma,were enrolled in this prospective study.BIS electrodes were adhered to the forehead of patients,and the BIS value was continuously recorded for 20 min.The maximum BIS value (BISmax) and minimum BIS value (BISmin) were recorded,and the mean BIS value (BISmean) was calculated.The patients were divided into 2 groups according to Glasgow Outcome Scale (GOS) scores assessed at 6 months after surgery:good outcome group (GOS score≥ 3) and poor outcome group (GOS score 1 or 2).The receiver operating characteristic curve of BIS in predicting outcomes was plotted,and the area under the curve (AUC) and 95％ confidential interval (CI) were calculated.The best cut point and sensitivity and specificity were calculated according to the BIS value when Youden index reached the maximal value.Results The AUC (95％ CI) for BISmax in predicting outcomes was 0.903 (0.830-0.976),the best cut point 79,the sensitivity 84％,the specificity 86％,and Youden index 0.695.The AUC (95％ CI) tor BISmin in predicting outcomes was 0.841 (0.760-0.921),the best cut point 43,the sensitivity 86％,the specificity 71％,and Youden index 0.577.The AUC (95％ CI) for BISmean in predicting outcomes w() 0.883 (0.800-0.958),the best cut point 60,the sensitivity 90％,the specificity 76％,and Youden index 0 562.Conclusion BIS can accurately predict outcomes in patients with acute cerebral hemoorrhage,and BISmax provides the highest accuracy.

Objeetive To detect ERG11 gene mutations in clinical isolares of Candida albicans resistant to fluconazole.and discuss their relationship with formation of drug resistance.Methods Clinical specimens were collected.CHROMagar mediuln and amplification of the fragment spanning the conserved sequence of 25S rDNA including some transposable introns.were used to identify subtype Candida albicans isolates.FIuconazole sensitivity was detected in vitro through microdilution and Rosco tablets.The other three fragment of ERG11 gene were amplified and followed by sequencing with resistant type strain ATCC 76615-19 and Candida albicans Darlington strain with two sensitive isolates as controh.Results Fifteen resistant isolates of Candida albicans were found,all of which were type A.Sixteen silent mutations and 11 missense mutations were detected.Mutations in ATCC 76615-19 and Darlington strain were same with what had been reported.In the 2 sensitive strains.G640A(E165K),A945C(E266D)and G1609A/G(V488I)occurred,as well as the other 9 silent mutations.Only G487T(A114S)and T916C(Y257H)existed in each of 14 resistant isolates.In the other one resistant isolate,T541C(Y132H),T495A(D116E),A530C (K128T)and T1493A(F449Y)occurred Mong with 8 silent mutations.Conclusions The occurrence of G487T(A114S)and 1916C(Y257H)in 14 isolates from different sources suggested they may involve in fluconazole resistance.The novel mutation T1493A(F449Y)can appear in resistant isolves of Candida albicans.

Objective To investigate the role of glucocorticoid receptor (GR) in the induction of neuronal apoptosis in spinal dorsal horn in chronic morphine tolerant rats. Methods Twenty healthy male SD rats weighing 300-350 g in which intrathecal (IT) catheter was successfully implanted without complication were randomized into 4 groups ( n = 5 each): group Ⅰ control ( group C);group Ⅱ morphine ( group M );group Ⅲ morphine +RU38486 (GR antagonist) (group MR) and group Ⅳ morphine + dexamethasone (GR agonist) (group MD).Normal saline 10 μl, morphine 10 μg, morphine 10 μg + RU38486 2 μg and morphine 10μg + dexamethasone 4 μg were injected IT twice a day at 8:00 and 20:00 for6 consecutive days in group C, M, MR and MD respectively. Tail flick latency (TFL) to a thermal nociceptive stimulus was measured every day at 30 min after IT administration in the morning (8:00). Hyperalgesia was considered to be a sign of morphine tolerance. The animals were killed at 7 days after IT drug administration. The L3-5 segment of the spinal cord was isolated for determination of neuronal apoptosis in spinal dorsal horn by TUNEL staining. Apoptotic index was calculated ( the number of apoptotic neurons/the total number of neurons × 100% ). Results TFL was significantly prolonged at day 1 and 3 of IT morphine 10 μg twice a day and returned to baseline at day 5 and 7 indicating morphine tolerance. RU38486 inhibited while dexamethasone enhanced morphine tolerance. IT morphine significantly increased the number of apoptotic neurons in spinal dorsal horn. Morphine-induced neuronal apoptosis was decreased by IT RU38486 and increased by IT dexamethasone. Conclusion Glucocorticoid receptors may be involved in morphine tolerance by inducing neuronal apoptosis in spinal dorsal horn.

Objective To explore the role of excitatory amino acid carrier 1 (EAAC1)in dorsal root ganglion (DRG) in the mechanism of developing morphine tolerance. Methods Thirty male SD rats were implanted intrathecal catheters and randomized into 6 groups with 5 rats each. The rats of 4 groups were made into the model of adjuvant-induced arthritis in the left hind limb and were administered intrathecally, morphine 10 μg(group M_(10)), morphine 20μg(group M_(20)), morphine 20 μg plus naloxone 10 μg(group MN) ,or saline(group C) respectively. The other 2 groups without were administered intrathecally saline (group C_0) or morphine 20 μg (group M0). The drugs were administered twice daily for 7 days. Mechanical withdrawl threshold(MWT) of the left hind limb was examined to evaluate the behavior. Immunohistochemistry was used to detect the expression of EAAC1 in the left L_(3-4) and L_(4-5) DRG. Results Morphine tolerance was formmed stably in the arthritis rats of group M_(10) and group M_(20) after administering morphine for 7 days. The expression of EAAC1 in DRG was downregulated. Conclusion DRG EAAC1 may be involved in the mechanism of developing morphine tolerance in rats with inflammatory pain.

Objective To evaluate the effect of compound K ( CK) on spinal Toll?like receptor 4 ( TLR4) expression during morphine?induced hyperalgesia in rats. Methods Thirty?six healthy male Spra?gue?Dawley rats in which intrathecal catheters were successfully implanted were randomly divided into 3 groups ( n=12 each) using a random number table: control group ( group C) , morphine?induced hyperal?gesia group ( group M) , and morphine + CK group ( group M+CK) . Starting from 5 days after successful implantation, normal saline 10 μl, morphine 10 μg, and morphine 10 μg + CK 10 μg were injected in?trathecally twice a day for 7 consecutive days. Tail?flick latency ( TFL) to a thermal nociceptive stimulus was measured at 1 day before administration ( T0 , baseline) and at 30 min after the initial administration on 1st, 3rd, 5th and 7th days (T1?4), and the percentage of maximum possible effect (MPE) was calculated. The rats were sacrificed after the last measurement of tail?flick latency, and the lumbar segment ( L3?5 ) of the spinal cord was removed for determination of the expression of TLR4 by Western blot. Results MPE was significantly lower at T3,4 than at T1 in M and M+CK groups ( P<0?05) . Compared with group C, MPE was significantly lower at T2?4 , and the expression of TLR4 was up?regulated in M and M+CK groups ( P<0?05). Compared with group M, MPE was significantly increased at T1?4, and the expression of TLR4 was down?regulated in group M+CK ( P<0?05 ) . Conclusion The mechanism by which CK alleviates mor?phine?induced hyperalgesia is associated with down?regulation of TLR4 expression in rats.

Objective To investigate the role of extracellular signal-regulated kinase(ERK)signal pathway in the spinal cord in the development of chronic morphine tolerance.Methods Thirty healthy male SD rats weighing 300-350 g in which intrathecal(IT)catheters were successfully implanted without complication were randomly divided into 3 groups(n = 10 each): group Ⅰ control(group C);group Ⅱ morphine tolerance(group M)and group Ⅲ morphine tolerance + PD98059(ERK upstream kinase MEK inhibitor)(group P).Morphine tolerance was induced with IT morphine 10 μg twice a day for 7 consecutive days.In group P PD98059 10 μg was injected IT at 30 min before each morphine administration.Tail flick latency(TFL)(the time between the onset of heat stimulus and voluntary tail withdrawal)was measured once a day at 30 min after first IT morphine administration and at 1 day after last IT morphine.Maximum analgesic effect(MPE)was calculated.MPE =(TFL after IT morphine - baseline TFL)/(12 - baseline TFL)× 100%.The animals were sacrificed after last TFL measurement.The L3-5 segment of the spinal cord was isolated for determination of the expression of μ-receptor and phosphorylated ERK 1/2(p-ERK1/2)by Western blot analysis and fluoroimmunoassay.Results Morphine tolerance was induced in group M and M + P.MPE was higher in group P than in group M.The expression of μ-receptor in spinal dorsal horn was significantly lower while the p-ERK1/2 expression was higher in group M than in group C.IT PD98059 significantly up-regulated μ-receptor expression and down-regulated p-ERK expression in group P as compared with group M.Conclusion ERK signal pathway is involved in the development of chronic morphine tolerance in rats.

Objective To investigate the changes in the expression of glutamate-aspartste transporter in spinal dorsal horn in rats with inflammatory pain and chronic morphine tolerance. Methods Thirty healthy male SD rats in which intrathecal (IT) catheters were successfully placed without complications were randomized into 3 groups ( n = 10 each): normal saline group ( group NS), arthritis group ( group A), and arthritis + morphine group (group AM). NS 50 μl was injected into the ankle joint of the left hindlimb in group NS, while complete Freund's adjuvant was injected in the other two groups instead. After 3 days, group NS and A received IT NS 10 μl twice a day for 7 consecutive days, group AM IT morphine 10 μg (10 μl) twice a day for 7 consecutive days. Mechanical pain threshold (MPT) was measured before IT administration and at day 2, 4, 6 and 8 after IT administration (T0-4). The animals were sacrificed after the last MPT measurement. The spinal cords were isolated for determination of GLAST expression in spinal dorsal horn. Results Compared with group NS, MPT was significantly decreased in the other groups and GLAST expression was down-regulated in group AM (P ＜ 0.05). Compared with group A, no significant change was found in MPT at T3,4 (P ＞ 0.05), while GLAST expression was down-regulated in group AM ( P ＜ 0.05). Conclusion The development of chronic morphine tolerance is related to the decrease in the function of GLAST in spinal dorsal horn in rats with inflammatory pain.

BACKGROUND: Magnetic labeling of stem cells is a recently developed stem cell in vitro labeling technique. Through in conjunction with magnetic resonance imaging (MRI), it can monitor transplanted stem cells in vivo. OBJECTIVE: To identify the method of superparamagnetic iron oxide (SPIO) labeling pig bone marrow mesenchymal stem cells (BMSCs), to investigate the characteristics of stem cells labeled by various SPIO following MRI, and to determine the minimum amount of labeled cells fer MRI. DESIGN, TIME AND SETTING: A control observation was performed at the laboratories of Department of Cardiovascular Surgery, Medical College of Soochow University, and Medical Imaging Centre, First Affiliated Hospital, Soochow University between September 2006 and March 2007. MATERIALS: Fresh porcine iliac bone marrow was collected from Taihu Meishan pigs. SPIO nanometer particles were purchased from Schedng, Germany. Ultramicro SPIO (USPIO) nanoparticles were provided by School of Chemistry and Chemical Engineering, Soochow University. For such particles, crystal nucleus surface was coated with dextran, and following coating, they were named 1#, 2#, 3# for short according to particle size. METHODS: Following isolation, purification, and culture, BMSCs were in vitro labeled by various kinds of SPIO nanoparticles. The labeled cells were subjected to Prussian blue staining and fluorescence microscope observation. The cell growth was observed using MTT method and the growth curve was plotted. For Feridex-labeled cells, 1×106), 5×105, and 1×105 three cell amount groups were set, for unlabeled cells, a 5×105 cell amount group was included, and for 1#, 2#, and 3# SPIO-labeled cells, only 5×105 cell amount group was used. MRI was conduced for measurement of signal intensity of cells labeled by different scanning sequences, followed by statistical analysis. MAIN OUTCOME MEASURES: Detection of SPIO nandparticles-labeled cells by Prussian blue staining; Growth curves of SPIO nanoparticles-labeled cells; Detection of cellular apoptosis by double staining; Determination of signal intensity of cell masses from different Ependoff tubes using MRI with T1WI, T2WI, and fast field echo (FFE) sequences. RESULTS: BMSCs could be labeled with SPIO and the labeling efficiency was 100%. Different amounts of blue-stained Fe particles could be observed in the cytoplasm by Prussian blue staining. SPIO labeling caused a significantly lower signal attenuation effect in T2WI and FFE (T2*WI) images than in T1WI images. In a labeling concentration of 25 mg/L Fe solution, the minimum cell amount for MRI was 1 x 105. The signal intensity exhibited significant difference in 2#, 3#USPIO- and Feridex-labeled cells in no matter T2WI or T2*Wl images(P < 0.01). But no significant difference was found between 1#USPIO- and Feridex-labeled cellss in no mater T2WI or T2*WI images(P > 0.05). There was significant difference in signal intensity of Feridex-labeled BMSCs between T2WI, T2*WI and T1Wl images (P < 0.01). CONCLUSION: BMSCs can be easily and efficiently labeled by SPIO nanoparticles without interference, at proper concentrations on cell viability and proliferation. MRI visualization of SPIO-labeled BMSCs is feasible in both T2WI and T2*WI images.

Objective To evaluate the role of extracellular signal-regulated kinase-cyclic AMP response element binding protein(ERK-CREB) signal pathway in glucocorticoid receptors-mediated chronic morphine tolerance in rats.Methods Male SD rats aged 2 months weighing 280-320 g were used in this study.A catheter was placed in subarachnoid space via foramen magnum according to Yaksh.Thirty-six rats in which intrathecal (IT) catheters were successfully implanted were randomly divided into 6 groups ( n =6 each):control group ( group C),chronic morphine tolerance group (group M),morphine + dexamethasone group (group MD),morphine + RU38486 group (group MR),dexamethasone group (group D),RU38486 group (group R).Normal saline 10 μl,morphine 10 μg,morphine 10 μg + dexamethasone 4 μg,morphine 10 μg + RU38486 2 μg,dexamethasone 4μg,RU38486 2 μg was administered IT twice a day(8:00 and 20:00)for 6 consecutive days in groups C,M,MD,MR,D,R respectively.Tail flick latency (TFL) was measured at 1 d before IT drug administration(baseline)and at 30 min after first IT drug administration during 1,3,5 d and at 1 d after last IT drug administration (T1～4).Maximum analgesic effect (MPE) was calculated.The animals were sacrificed after last TEL measurement.The L3～5 segment of the spinal cord was isolated for determination of the expression of phosphorylated ERK(pERK)and phosphorylated CREB(pCREB) by immunofluorescence staining.Results MPE was significantly T1 lower at T3,4 than at T1 in groups M and MD.Compared with group C,MPE was significantly increased,the expression of pERK and pCREB up-regulated in group M,but no significant change was found in the parameters mentioned above in groups R and D.Compared with group M,MPE was significantly increased,the expression of pERK and pCREB up-regulated in group MR,and MPE was significantly increased,the expression of pERK and pCREB down-regulated in group MD.Conclusion The mechanism by which glucocorticoid receptors-mediated chronic morphine tolerance may be associated with the inhibition of ERK-CREB pathway.