Objective To evaluate the role of spinal RhoA/ROCK signaling pathway in the development of lipopolysaccharide (IP)-induced inflammatory pain(IP) in rats.Methods Fifty-two male Sprague-Dawley rats, weighing 180-220 g, were equally randomized into 4 groups using a random number table: normal saline group (group NS) , LPS group, RhoA inhibitor C3 exoenzyme group (group LC) , and ROCK inhibitor Y27632 group (group LY).Inflammatory pain was induced by injecting LPS 25 μl (300 ng) into the plantar surface of hindpaws in IP, LC and LY groups, while the equal volume of normal saline was injected instead in NS group.C3 exoenzyme 10 pg and Y27632 10 nmol were injected intrathecally at 30 min prior to LPS administration in LC and LY groups, respectively.Before LPS injection (T0) , and at 1, 3, 5, 12 and 24 h after LPS injection (T1-5) , the mechanical and thermal pain thresholds were measured.Five rats in each group were sacrificed after pain thresholds were measured at T3, and L4.5 segments of the spinal cord were removed for determination of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) mRNA expression in spinal dorsal horns by real-time reverse transcriptase-polymerase chain reaction.Results Compared with group NS, the mechanical and thermal pain thresholds were significantly decreased at T2-5in IP, LC and LY groups, and TNF-α and IL-1β mRNA expression was up-regulated at T3 in IP group.Compared with group IP, the mechanical and thermal pain thresholds were significantly increased at T2-5, and TNF-α and IL-1β mRNA expression was down-regulated at T3 in LC and LY groups.Conclusion Spinal RhoA/ROCK signaling pathway is involved in the development of LPS-induced IP in rats.

Objective To evaluate the role of spinal IKK2/NF-κB pathway in the maintenance of bone cancer pain (BCP) in rats.Methods Twenty-eight unmated adult female Sprague-Dawley rats,weighing 160-200 g,were randomly divided into 4 groups (n =7 each) using a random number table:sham operation group (group S),BCP group (group BP),BCP + normal saline group (group BN),and BCP + BMS345541 group (group BB).BCP was induced by injecting Walker 256 mammary gland cancer cell suspension 5 μl (4 × 105 cells/ml) into the bone marrow of the right tibia of rats anesthetized with chloral hydrate in BP,BN and BB groups,while the equal volume of normal saline was injected in group S.On 10-12 days after operation,selective IKK2 inhibitor BMS345541 (50 μg/10 μl) was intrathecally injected once a day in group BB,and the equal volume of normal saline (10μl) was given once a day in group BN.The mechanical paw withdrawal threshold (MWT) was measured before intra-tibia injection (T0),on 7 days after intra-tibia injection (T1),at 1 h before drug administration and 1,2,4,12 and 24 h after drug administration on day 10 after operation,and at 4 h after drug administration on day 12 after operation (T2-8).The rats were sacrificed after MWT was measured at Ts and L4-6 segments of the spinal cord were removed for determination of phosphorylated NF-κB (p-NF-κB) expression (using Western blot analysis).Results Compared with group S,MWT was significantly decreased at T1-2,and the expression of p-NF-κB was up-regulated in BP,BN,and BB groups.Compared with group BP,MWT was significantly increased at T4-6,and the expression of p-NF-κB in the spinal cord was down-regulated in group BB.Conclusion Spinal IKK2/NF-κB signaling pathway is involved in the maintenance of bone cancer pain in rats.

Objective To evaluate the effects of intrathecal 2-PMPA on chronic inflammatory pain in rats . Methods Twenty four male Sprague-Dawley rats ,aged 4-6 months ,weighing 200-250 g ,were randomly divided into 3 groups (n=8 each) using a random number table :normal saline (NS) group ,complete Freund′s adjuvant (CFA ) group and N-acetylaspartylglutamate peptidase inhibitor 2-PMPA group (group 2-PMPA ) . Chronic inflammatory pain was induced by injecting 100μl of CFA into the plantar surface of the left hindpaw .Immediately after injection of CFA ,2-PMPA 100 μg was injected intrathecally once a day for 3 consecutive days in group 2-PMPA ,while the equal volume of NS was given instead of 2-PMPA in NS and CFA groups .The paw withdrawal latency to thermal nociceptive stimulus (TWL ) and paw withdrawal threshold (PWT ) to von Frey filament stimulation were measured before injection of CFA (baseline ,T1 ) and after the last injection of CFA (T2 ) .Then the rats were sacrificed and the L4 ,5 segments of the spinal cord were removed for determination of NR2B expression by Western blot .Results Compared with group NS ,TWL and PWT were significantly decreased at T2 and the expression of NR2B was up-regulated in CFA and 2-PMPA groups ( P<0.05 ) .Compared with group CFA ,TWL and PWT were significantly increased at T2 and the expression of NR2B was down-regulated in group 2-PMPA ( P<0.05) .Conclusion Intrathecal 2-PMPA can alleviate CFA-induced chronic inflammatory pain in rats ,and inhibition of NR2B expression in the spinal cord is involved in the mechanism .

Objective To evaluate the role of chemokine receptor CXCR4 in spinal dorsal horn in the development of morphine tolerance in rats with bone cancer pain (BCP).Methods Forty female Sprague-Dawley rats,weighing 180-220 g,were equally randomized into 5 groups using a random number table:sham operation group (group S),BCP group (group B),BCP + AMD3100 (specific CXCR4 antagonist) group (group BA),BCP + morphine group (group BM),BCP + morphine + AMD3100 group (group BMA).BCP was induced by injecting Walker 256 mammary gland cancer cell suspension (4 × 105 cells/ml) 5 μl into the bone marrow of the right tibia of rats anesthetized with chloral hydrate.On 6 days after injection of mammary gland cancer cells,AMD3100 2 mg/kg was intraperitoneally injected twice a day for 9 days in BA group,and morphine 10 mg/kg was subcutaneously injected twice a day for 9 days in BM group.AMD3100 was intraperitoneally injected and morphine was subcutaneously injected as previously described at the corresponding time point in BMA group.Before injection of mammary gland cancer cells (T0) and on 4,6,8,10,12 and 14 days after injection of mammary gland cancer cells (T1-6),paw withdrawal threshold to von Frey hair mechanical stimulation (PWMT) was measured.The rats were then sacrificed and L3-5 segments of the spinal cord were removed for determination of c-fos expression in spinal dorsal horn using immunofluorescence.Results Compared with S group,PMWT was significantly decreased at T2-6 in B and BA groups and at T4-6 in BM group,and c-fos expression was up-regulated at T6 in BM group (P ＜0.01).PMWT was significantly higher at T3-5 in BM group and at T3-6 in BMA group than in group B (P ＜ 0.01).Compared with BM group,PMWT was significantly increased at T5,6 and c-fos expression was down-regulated at T6 in BMA group (P ＜ 0.01).Conclusion Chemokine receptor CXCR4 in spinal dorsal horn is involved in the development of morphine tolerance in rats with BCP and the mechanism may be related to activation of c-fos.

Objective To evaluate the changes in the expression of Piezo2 in spinal cord neurons in a rat model of bone cancer pain.Methods Sixty-four pathogen-free healthy adult female unmated Sprague-Dawley rats,weighing 160-200 g,were divided into 2 groups (n=32 each) using a random number table:sham operation group (group S) and bone cancer pain group (group BP).Bone cancer pain was induced by injecting breast cancer cells into the abdominal cavity.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before inoculating breast cancer cells and 7,14 and 21 days after inoculation (T0-3).Eight rats were sacrificed after measurement of MWT,and their lumbar enlargement segments of the spinal cord were removed for determination of Piezo2 expression by Western blot and immunofluorescence.The coexpression of Piezo2 with the neuronal marker NeuN,microglial marker Iba-1 and astrocyte marker GFAP was detected at T2 using double immunofluorescent staining.Results Compared with group S,the MWT was significandy decreased at T1-3,and the Piezo2 expression in the lumbar enlargement segment of the spinal cord was up-regulated in group BP (P ＜ 0.05).Piezo2 was mainly expressed in the spinal lamina Ⅰ and Ⅱ and co-expressed with NeuN and rarely co-expressed with GFAP or Iba-1.Conclusion The development and maintenance of bone cancer pain are related to up-regulated expression of Piezo2 in the lumbar enlargement segment of the spinal cord in rats.