Objective To evaluate the relationship between cold hyperalgesia and trafficking of transient receptor potential melastatin 8 (TRPM8) to cell membrane in the dorsal root ganglion (DRG) of rats with neuropathic pain (NP).Methods Ninety-six healthy male Sprague-Dawley rats,aged 10-12 weeks,weighing 250-280 g,were divided into sham operation group (S group,n=48) and NP group (n =48) using a random number table.NP was produced by chronic constriction injury to the sciatic nerve.The number of paw lifts on the cold plate and mechanical paw withdrawal threshold (MWT) were measured on 1 day before operation and 1,4,7,10 and 14 days after operation.Rats were sacrificed after behavioral testing,and ipsilateral DRGs of the lumbar segment (L46) were dissected tor detection of the expression of TRPM8 in total and membrane proteins by Western blot,and the ratio of TRPM8 expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group S,the number of paw lifts on the cold plate was significantly increased,the MWT was decreased,the expression of TRPM8 in total and membrane proteins was up-regulated,and m/t ratio was increased on postoperative days 4,7,10 and 14 in group NP (P＜0.05 or 0.01).In group NP,the number of paw lifts on the cold plate was gradually increased with the prolongation of time after operation and reached the peak on postoperative day 10,maintaining at the peak until postoperative day 14;the MWT was gradually decreased and reached the lowest level on postoperative day 10,maintaining at the lowest level until postoperative day 14;the expression of TRPM8 in total and membrane proteins and m/t ratio were gradually increased with the prolongation of time after operation and reached the peak on postoperative day 10,maintaining at the peak level until postoperative day 14 (P＜0.01).Conclusion The mechanism underlying the development of cold hyperalgesia is related to enhanced trafficking of TRPM8 to cell membrane in DRGs of rats with NP.

Objective To evaluate the changes in the expression of spinal divalent metal transporter 1 (DMT1) during remifentanil-induced hyperalgesia in a rat model of incisional pain.Methods Thirtytwo male Sprague-Dawley rats,weighing 240-260 g,aged 2-3 months,were randomly divided into 4 groups (n =8 each) using a random number table:control group (group C),incisional pain group (group I),remifentanil group (group R),and incisional pain + remifentanil group (group I+R).In group C,normal saline was infused for 60 min at a rate of 0.1 ml · kg-1 · min-1.A 1-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the left hindpaw in sevoflurane-anesthetized rats,and normal saline was infused intravenously for 60 min at a rate of 1.0 μg · kg-1 · min-1 at the same time in group I.In group R,remifentanil was infused for 60 min at a rate of 1.0 μg · kg 1 · min-1 In group I+R,the model of incisional pain was established,and remifentanil was simultaneously infused for 60 min at a rate of 1.0 μg · kg-1 · min-1.At 24 h before normal saline or remifentanil infusion and 6,24 and 48 h after the end of infusion (T0-3),the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.All the rats wcrc sacrificed after the last measurement of pain thresholds,and the spinal cord was removed for determination of DMT1 with/without iron-responsive element [DMT1 (+)IRE and DMT1 (-)IRE] expression (by Western blot analysis and immunohistochemistry).Results Compared with group C,the MWT was significantly decreased,and the TWL was significantly shortened at T1-3,and the expression of spinal DMT1 (-) IRE was significantly up-regulated in I,R and I+R groups (P＜0.05).Compared with I and R groups,the MWT was significantly decreased,and TWL was significantly shortened at T1-3,and the expression of spinal DMT1 (-) IRE was significantly upregulated in group I+R (P＜0.05).There was no significant difference in the expression of spinal DMT1 (+) IRE between the four groups (P＞ 0.05).Conclusion Spinal DMT1 (-)IRE activation may be involved in the mechanism underlying remifentanil-induced hyperalgesia in a rat model of incisional pain.

Objective To evaluate the role of spinal peroxynitrite in remifentanil-induced hyperalgesia in rats.Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 2-3 months, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C), remifentanil group (group R), hydrogen-rich saline group (group C + H), and remifentanil + hydrogen-rich saline group (group R+H).In group C, normal saline was infused for 60 min at a rate of 0.1 ml · kg-1 · min-1.In group R, remifentanil was infused for 60 min at a rate of 1.0 μg · kg-1 · min-1.In group R+H, remifentanil was infused for 60 min at a rate of 1.0 μg · kg-1 · min-1 ,and hydrogen-rich saline 10 ml/kg was intraperitoneally injected at 10 min before iv infusion.At 24 h before iv infusion and 6, 24 and 48 h after iv infusion (T0-3) , the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.All the rats were sacrificed after the last measurement of pain thresholds, the lumbar segment (L4-6) of the spinal cord was removed for determination of the nitrated manganese superoxide dismutase (MnSOD) expression (by immunoprecipitation and Western blot analysis), 3-nitrotyrosine (3-NT) expression (by Western blot) and spinal MnSOD activity (by spectrophotometer).Results Compared with group C, the MWT was significantly decreased, and TWL was shortened at T1-T3, the expression of spinal 3-NT and nitrated MnSOD was up-regulated, and MnSOD activity was decreased in R and R+H groups, and no significant change was found in the parameters mentioned above in group C+H.Compared with group R, the MWT was significantly increased, and TWL was prolonged at T1-T3, the expression of spinal 3-NT and nitrated MnSOD was down-regulated, and MnSOD activity was increased in group R + H.Conclusion Spinal peroxynitrite is involved in the mechanism underlying remifentanil-induced hyperalgesia through inhibiting the activity of MnSOD in rats.

Objective To evaluate the effects of hydrogen-rich saline on incisional pain-remifentanil-induced hyperalgesia in rats.Methods Thirty-two male Sprague-Dawley rats,aged 2-3 months,weighing 240-260 g,in which the catheter was successfully inserted into the caudal vein,were randomly divided into 4 groups (n =8 each) using a random number table:control gourp (group C),remifentanil + incisional pain group (group R + I) and different doses of hydrogen-rich saline groups (H1 and H2 groups).A l-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the left hindpaw in chloral hydrate-anesthetized rats.Remifentanil 1 μg· kg-1 · min-1 was infused intravenously for 60 min sarting from beginning of establishment of incisional pain model in R + I,H1 and H2 groups.The equal volume of normal saline was infused intravenóusly for 60 rin instead of remifentanil group C.Hydrogen-rich saline 5 and 10 ml/kg were injected intraperitoneally at 10 min before establishment of incisional pain model in H1 and H2 groups,respectively.Paw withdrawal threshold (PWT) to yon Frey hair stimulation and paw withdrawal latency (PWL) to thermal stimulation were measured at 24 h before remifentanil infusion and 2,6,24 and 48 h after remifentanil infusion (T0-T4).The rats were sacrificed after measuremnt of pain threshold,and L4-6 segments of the spinal cord were removed for determination of the expression of R1 and 2B subunits-containing N-methyl-D-aspartate receptors (NR1 and NR2B) in total and membrane proteins by Western blot.The ratio between the expression of NR1 in membrane protein and in total protein (mNR1/tNR1) and NR2B in membrane protein and in total protein (mNR2B/tNR2B) was calculated.Results Compared with group C,PWT was significantly decreased,PWL was shortened,the expression of mNR1,mNR2B,tNR1 and tNR2B was up-regulated,and the ratios of mNR1/tNR1 and mNR2B/tNR2B were increased in R + I,H1 and H2 groups.Compared with group R + I,PWT was significantly increased,PWL was prolonged,the expression of mNR1 and mNR2B was down-regulated,and the ratios of mNR1/tNR1 and mNR2B/tNR2B were decreased in Ht and H2 groups.Compared with group H1,PWT was significantly increased,PWL was prolonged,the expression of mNR1 and mNR2B was down-regulated,and the ratios of mNR1/tNR1 and mNR2B/tNR2B were decreased in group H2.Conclusion Hydrogen-rich saline can attenuate incisional pain-remifentanil-induced hyperalgesia in rats and inhibition of trafficking of spinal neuronal NR1 and NR2B from cytoplasm to cell membrane may be involved in the mechanism.

Objective To evaluate the changes in the expression of CC-chemokine ligand 3 (CCL3) and CC-chemokine receptor 5 (CCR5) in the spinal cord during hyperalgesia induced by remifentanil in rats with incisional pain.Methods Thirty-two male Sprague-Dawley rats,aged 2-3 months,weighing 240-260 g,were randomly divided into 4 groups (n=8 each) using a random number table:control group (group C),incisional pain group (group Ⅰ),remifentanil group (group R) and remifentanil+incisional pain group (group R+I).A 1-cm longitudinal incision was made in the plantar surface of the left hindpaw in anesthetized rats.While the model of incisional pain was established,remifentanil was infused for 60 min at 1 μg · kg-1 · min-1.At 24 h before infusion of remifentanil (baseline) and 2,6,24 and 48 h after the end of infusion,the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were sacrificed after the last measurement of pain threshold,the lumbar segment (L4-6) of the spinal cord was removed for determination of CL3 and CCR5 mRNA expression (by real-time PCR) and CL3 and CCR5 expression (by Western blot).Results Compared with group C,the MWT was significantly decreased,the TWL was shortened,and the expression of CCL3 and CCR5 mRNA and protein was up-regulated in I,R and R+ I groups.Compared with I and R groups,the MWT was significantly dccreascd,the TWL was shortened,and the expression of CCL3 and CCR5 mRNA and protein was up-regulated in group R+I.Conclusion The mechanism by which remifentanil induces hyperalgesia is related to up-regulated expression of CCL3 and CCR5 in the spinal cord of rats with incisional pain.

Objective To evaluate the effects of different ratios of medicine dosage for sevoflurane and propofol on postoperative cognitive function in aged rats.Methods Sixty healthy male Wistar rats,aged 18-20 months,weighing 600-650 g,were divided into 6 groups (n =10 each) using a random number table:control group (group C),sevoflurane anesthesia group (group S),propofol anesthesia group (group P) and different ratios of mmedicine dosage for sevoflurane and propofol groups (group SP1,group SP2 and group SP3).Normal saline 10 ml was infused intravenously in group C.Group S inhaled 3.1％ sevoflurane.Propofol was infused intravenously at a rate of 40 mg · kg-1 · h-1 in group P.In group SP1,2.4％ sevoflurane was inhaled,and propofol was intravenously infused at a rate of 10 mg · kg-1 · h-1.In group SP2,1.8％ sevoflurane was inhaled,and propofol was intravenously infused at a rate of 20 mng · kg-1 · h-1.In group SP3,1.2％ sevoflurane was inhaled,and propofol was intravenously infused at a rate of 30 mg · kg-1 · h-1.Duration of anesthesia was 2 h in all the groups.After loss of righting reflex,open reduction and internal fixation was performed after tibial fracture was induced in the other five groups except group C.At 1 day after anesthesia,Y-maze test was performed,and the time spent on the novel arm and total number of entries into each arm were recorded,and fear conditioning test was carried out for determination of the rate of freezing time to reflect the cognitive function.The rats were sacrificed after behavioral testing,and the hippocampus was removed for determination of occludin,matrix metalloproteinase-2 (MMP-2) and MMP-9 expression by Western blot.Results Compared with group C,the time spent on the novel arm was significantly shortened,the total number of entries into each arm and rate of freezing time were decreased,the expression of occludin was down-regulated,the expression of MMP-2 and MMP-9 was up-regulated in the other five groups,and the most unmarked change in the indices mentioned above was found in group SP2 (P＜ 0.05 or 0.01).Conclusion Different ratios of medicine dosage for sevoflurane and propofol can induce postoperative cognitive decline,and inhaling 1.8％ sevoflurane and infusing propofol 20 mg · kg-1 · h-1 produce little influence on postoperative cognitive function in aged rats.

Objective To evaluate the role of glial cell line-derived neurotrophic factor family re-ceptor alpha3(GFRα3)in the expression and membrane trafficking of transient receptor potential melasta-tin 8(TRPM8)in the dorsal root ganglion(DRG)during cold hyperalgesia in rats with neuropathic pain (NP). Methods Thirty-two healthy adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups(n=8 each) using a random number table: sham operation plus GFRα3 dsRNA group(Sham+dsRNA group), sham operation plus GFRα3 siRNA group(group Sham+siRNA), NP plus GFRα3 dsRNA group(group NP+dsRNA)and NP plus GFRα3 siRNA group(group NP+siRNA). NP was produced by chronic constriction injury to the sciatic nerve. At 10-30 days after operation, GFRα3 dsRNA 10 μg∕20 μl was intrathecally injected once a day for 4 consecutive days in Sham+dsRNA and NP+dsRNA groups, and 10 μg∕20 μl GFRα3 siRNA, of which the sense strand was modified with 2′-O-methyl and 5′-cholesterol, was intrathe-cally injected once a day for 4 consecutive days in Sham+siRNA and NP+siRNA groups. The number of paw lifts on the cold plate, mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency (TWL)were measured on 1 day before operation and 10, 11, 12, 13(before intrathecal injection)and 14 days after operation. The rats were sacrificed after the last behavioral testing, and ipsilateral DRGs of the lumbar segment(L4-6)were dissected for detection of the expression of GFRα3 and TRPM8 in total and membrane proteins by Western blot, and the ratio of TRPM8 expression in the membrane protein to that in the total protein(m∕t ratio)was calculated. Results Compared with group Sham+dsRNA, the number of paw lifts on the cold plate was significantly increased, the MWT was decreased, and TWL was shortened after operation in NP+dsRNA and NP+siRNA groups, the expression of GFRα3 and TRPM8 in total and membrane proteins was significantly up-regulated, and m∕t ratio was increased in group NP+dsRNA, and the expression of GFRα3 in DRGs was significantly down-regulated(P<001), and no significant change was found in the expression of TRPM8 in total and membrane proteins or m∕t ratio in group NP+siRNA(P>005). Compared with group NP+dsRNA, the number of paw lifts on the cold plate was significantly de-creased, the expression of GFRα3 and TRPM8 in total and membrane proteins was down-regulated, m∕t ra-tio was decreased(P<001), and no significant change was found in MWT or TWL in group NP+siRNA (P>005). Conclusion GFRα3 in DRGs can up-regulate the expression of TRPM8 and enhance the membrane trafficking of TRPM8, which may be involved in the maintenance mechanism of cold hyperalgesia in rats with NP.

Objective To investigate the relationship remifentanil-induced hyperalgesia and func-tion of nitrated glutamate transportor-1 ( GLT-1) and glutamine synthetase ( GS) in the spinal cord of rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 260-280 g, aged 2-3 months, in which caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) u-sing a random number table method: control group ( group C) , incisional pain group ( group I) , remifen-tanil group ( group R) and remifentanil plus incisional pain group ( group RI) . Normal saline was intrave-nously infused for 60 min at 0. 1 ml · kg-1 · min-1 in group C. The model of incisional pain was estab-lished, and normal saline was simultaneously infused for 60 min via the tail vein at 0. 1 ml·kg-1 ·min-1 in group I. Remifentanil was infused for 60 min via the tail vein at 1. 0 μg· kg-1 ·min-1 in group R. The model of incisional pain was established, and remifentanil was infused for 60 min via the tail vein at 1. 0μg· kg-1 ·min-1 in group RI. The mechanical paw withdrawal threshold ( MWT) and thermal paw with-drawal latency ( TWL) were measured at 24 h before infusion of remifentanil or normal saline ( T0 ) and at 2, 6, 24 and 48 h after infusion ( T1-4 ) . The rats were sacrificed after the last measuremnet of pain thresh-old, and the L4-6 segment of the spinal cord was removed for determination of the expression of GLT-1 and GS (by Western blot) and expression of nitrated GLT-1 (nGLT-1) and nitrated GS (nGS) (by Western blot) . Ratios of nGLT-1∕GLT-1 and nGS∕GS were calculated. Results Compared with group C, the MWT was significantly decreased and TWL was shortened at T1-4 , the expression of GLT-1 and GS was down-regu-lated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1∕GLT-1 and nGS∕GS were increased in I, R and RI groups ( P<0. 05) . Compared with group I and group R, the MWT was signifi-cantly decreased and TWL was shortened at T1-4 , the expression of GLT-1 and GS was down-regulated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1∕GLT-1 and nGS∕GS were increased in group RI ( P<0. 05) . Conclusion The mechanism of remifentanil-induced hyperalgesia may be related to impaired function of GLT-1 and GS in the spinal cord of rats with incisional pain.

Objective To evaluate the changes in the expression of artemin in skin around the inci-sion during remifentanil-induced hyperalgesia in the rats with incisional pain. Methods Thirty-two healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 4 groups (n = 8 each) using a random number table: control group (group C), incisional pain group (group I), remifen-tanil group (group R) and incisional pain plus remifentanil group (group I+R). Remifentanil was intrave-nously infused for 60 min at a rate of 1 μg·kg-1 ·min-1 in group R. In group I, the model of incisional pain was established, and the equal volume of normal saline was infused for 60 min via the tail vein at the same time. In group I+R, the model of incisional pain was established, and remifentanil was infused for 60 min via the tail vein at a rate of 1 μg·kg-1 ·min-1 at the same time. The equal volume of normal saline was infused for 60 min via the tail vein in group C. Mechanical paw withdrawal threshold (MWT) and ther-mal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline and 2, 6, 24 and 48 h after the end of infusion (T0-4 ). Rats were sacrificed following the last measurement of pain threshold, and ipsilateral plantar skin was removed for detection of the expression of artemin protein and mRNA (by fluorescent quantitative real-time polymerase chain reaction or Western blot). Results Compared with group C, MWT was significantly decreased and TWL was shorten at T1-4 , and the expression of artemin protein and mRNA in plantar skin was up-regulated in R, I and I+R groups (P<0. 01). Compared with R and I groups, MWT was significantly decreased and TWL was shorten at T1-4 , and the ex-pression of artemin protein and mRNA in plantar skin was up-regulated in group I+R (P<0. 01). Conclu-sion The peripheral mechanism by which remifentanil induces hyperalgesia may be related to up-regulated expression of artemin in skin around the incision in the rats with incisional pain.

Objective To evaluate the effect of remifentanil on iron metabolism in spinal dorsal horn neurons of rats.Methods The primary spinal dorsal horn neurons of rats were seeded in the culture plate at a density of 2× 105 cells/well and divided into 4 groups using a random number table method:control group (C group,n=40),remifentanil group (R group,n=40),divalent metal transporter 1 without iron-responsive element [DMT1 (-) IRE] siRNA group (siRNA group,n=32) and DMT1 (-) IRE siRNA plus remifentanil group (siRNA+R group,n=32).siRNA and siRNA+R groups were subjected to DMT1 (-) IRE siRNA transfection on day 3 of culture.R and siRNA+R groups were incubated for 60 min in the solution with remifentanil at a final concentration of 40 nmol/L.The contents of reactive oxygen species (ROS) and Fe2+ were determined by fluorescent probe method,the malondialdehyde (MDA) content was detected by TBA method,and the content of labile iron pool (LIP) was detected by calcein AM and iron chelator at the end of incubation with remifentanil in R and siRNA+R groups and at the corresponding time points in the other groups.The expression of DMT1 (-) IRE and DMT1 (+) IRE was determined by Westem blot in C and R groups.Results Compared with C group,the expression of DMT1 (-) IRE in the spinal dorsal horn neurons was significantly up-regulated,the contents of Fe2+,LIP,ROS and MDA were increased (P＜0.05),and no significant change was found in the expression of DMT1 (+) IRE in R group (P＞0.05).Compared with R group,the contents of Fe2+,LIP,ROS and MDA in the spinal dorsal horn neurons were significantly decreased in siRNA+R group (P＜0.05).Conclusion Remifentanil increases the iron content of spinal dorsal horn neurons by activating DMT1 (-) IRE,which may be associated with the mechanism of remifentanil-induced postoperative hyperalgesia in rats.