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ObjectiveTo investigate the changes in the expression of T-cell death-associated gene 8(TD- AG8) in spinal cord in rats with bone cancer pain.MethodsTwo hundred and twenty-four female rats weighting 150-180 g were randomly divided into 3 groups:normal control group(group Ⅰ,n ＝ 64),normal saline group (group Ⅱ,n ＝ 64),bone cancer pain group(group Ⅲ],n ＝ 96).Bone cancer pain was induced by inoculating Walker256 mammary gland carcinoma cells into the tibia medullary cavity.Mechanical withdrawl threshold(MWT)was measured at 1 d before(baseline)and 1,3,6,9,12,15 and 18 d after inoculation.Sixteen rats were sacrificed at 1 day before(baseline)and 6,9,12,15 and 18 d after inoculation in group Ⅲ and 18 d after inoculation in groups Ⅰ and Ⅱ.The L4-6 spinal cord were removed,and the number of TDAG8 positive cell was counted,and the expression of TDAG8 mRNA was measured by RT-PCR.ResultsCompared with baseline value and group Ⅰ,MWT was decreased,and the number of TDAG8 positive cells and the expression of TDAG8 mRNA in spinal cord were increased at 6-18 d after inoculation in group Ⅲ ( P < 0.01 ).ConclusionThe expression of TDAG8 in spinal cord is up-regulated in rats with bone cancer pain,which may be involved in the mechanism of the development of bone cancer pain.
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Objective To evaluate the role of gliocytes in the spinal cord in the development of inflammatory pain (IP) in rats. Methods Adult male SD rats weighing 180-220 g were used in this experiment. A catheter was implanted in the subarachnoid space according to the method described by Yang. Animals with abnormal motor function of the hindlimb after intrathecal (IT) catheter implantation were excluded. IP was induced by subcutaneous (sc) injection of complete Freund's adjuvant (CFA) 50 μl at the lateral side of the ankle joint of the right hindpaw. Sixty-five rats were randomly divided into 5 groups ( n = 13 each): group I IP control normal saline (NS) 50μl was injected sc instead of CFA; group II IP; group IE PC (IT) + IP control fluorinated citric acid (FC, a gliocyte metabolism inhibitor) 1 nmol/10μl was injected IT at 15 min before NS 50 μl sc injection; group IV NS (IT) + IP and group V FC (IT) + IP. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured 2 d before induction of IP (T_0, baseline) .before and at 2, 4, 6, 8, 10, 12, 24 and 26 h (T_(1-9)) after sc NS or CFA injection. Five enimals in each group were killed at T_5 (8 h after sc NS/CFA injection) and the lumbar segment (L_(4,5)) was removed for determination of glial fibrillary acidic protein ( CFAP) and OX-42 expression by immuno-histochemistry. Results In group Ⅱ and Ⅳ sc CFA significantly decreased MWT and TWL. Mechanical and thermal hyperalgegia induced by sc CFA was significantly suppressed by intrathecal FC in group V . IP significantly increased GFAP and OX-42 expression in the spinal cord. Intrathecal FC significantly attenuated IP-induced up-regulation of GFAP and OX-42 expression in the spinal cord. Conclusion The activation of gliocytes in the spinal cord is involved in the development of CFA-induced hyperalgesia in rats.
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Objective To evaluate the role of brain-derived neurotrophic factor (BDNF) in inflammatory pain in rats. Methods Sixty female SD rats weighing 150-180 g in which intrathecal (IT) catheters were successfully placed without complication were randomly divided into 5 groups (n= 12 each): group Ⅰ sham operation; group Ⅱ sham operation + IT anti-BDNF antibody; group Ⅲ inflammatory pain; group Ⅳinflammatory pain + IT control IgG and group Ⅴ inflammatory pain + IT anti-BDNF antibody. Inflammatory pain was induced by injecting complete Freund's adjuvant (CFA) into ankle joint cavity of left hindpaw, while in sham operation group equal volume of normal saline was injected instead of CFA. Anti-BDNF antibody or control IgG 15 μg/10 μl was injected IT once a day for 3 days after inflammatory pain was induced. Paw withdrawal latency to thermal stimuli (PWTL) was measured one day before and at 3, 5, 7, 10 and 14 d after inflammatory pain was induced. The rat was sacrificed on 3 rd day of IT anti-BDNF antibody or control IgG injection. The lumbar segment L4-6 of the spinal cord was removed for detection of the expression of BDNF and p-ERK1/2 by immunohistochemistory and Western blot. Results Intra-articular CFA injection significantly increased the expression of BDNF and p-ERK1/2 in the spinal cord in group Ⅲ as compared with sham-operated animals in group Ⅰ . IT antiBDNF antibody injection significantly suppressed the expression of BDNF and p-ERK1/2. PWTL was significantly shortened after intra-articular CFA injection in group Ⅲ as compared with group Ⅰ . IT anti-BDNF antibody reversed the inflammation-induced thermal hyperalgesia in group Ⅴ but IT control IgG did not. Conclusion BDNF in the spinal cord may be involved in inflammatory pain through p-ERK1/2 signal transduction pathway.
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Objective To investigate the role of P2Y1 purinergic receptors in the spinal cord in a rat model of bone cancer pain. Methods Ninety female SD rats weighing 150-180 g were randomly divided into 5 groups (n = 18 each): Ⅰ group sham operation; Ⅱ group bone cancer pain; Ⅲ group sham operation + MRS2179 (a specific P2Y1 purinergic receptor antagonist); Ⅳ group BCP + vehicle (group NS); Ⅴ group BCP+ MRS2179.Bone cancer pain was induced by inoculating Walker 256 mammary gland carcinoma cells into medullary cavity of tibia. In group Ⅲ, Ⅳ, Ⅴ MRS2179 100 pmol/10 μl or NS 10 μl was injected intrathecally once a day for 3 days starting from the 7th day after operation. Mechanical pain threshold to von Frey stimuli was measured before and every other day after operation. The anirnals were sacrificed on the 9th day after operation. The L4-6 segment of the spinal cord was removed for detection of expression of P2Y1 receptor and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in the spinal dorsal horn. Results P2Y1 receptors and p-ERK1/2 coexisted in spinal dorsal horn. Inoculation of cancer cells into tibia significantly decreased mechanical pain threshold at postoperative day 6-18 and increased the expression of P2Y1 receptor and p-ERK1/2 on the 9th day after operation in group Ⅱ and Ⅳ as compared with group Ⅰ . Intrathecal MRS 2179 significantly increased pain threshold and decreased expression of P2Y1 receptor and p-ERK1/2 in group Ⅴ compared with group Ⅱ and Ⅳ. Conclusion P2Y1 receptors in the spinal cord are involved in the development of bone cancer pain, which may be related to the activation of ERK1/2.
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Aim To pick out the siRNA which could most effectively inhibit the expression of brain-derived neurotrophic factor( BDNF) in microglial cells,to detect the cytotoxicity of the transfection complex,and to ob-serve the change of OX-42 expression,the microglial marker,after BDNF siRNA treatment. Methods Four siRNAs were chemically synthesized: three of them were used to inhibit BDNF expression in microglial cells,the rest was fluorescence-labeled mismatch siRNA as a negative control. They were all transfected into microglial cells,respectively. BDNF mRNA was detected 24 h after transfection by Real-Time PCR and itsprotein expression was observed done by Western blot 48 h later. The Sulforhodamine B( SRB) assay was used to investigate the drug-induced cytotoxicity. Co-expres-sion pattern of BDNF and OX-42 was determined by double-labeling immunofluorescence. Results ① The BDNF siRNA1588 was the most effective siRNA,compared with the vehicle or mismatch siRNA-treated group( P