骨癌痛大鼠吗啡耐受后脊髓胶质细胞及炎性细胞因子的变化

目的 评价骨癌痛大鼠吗啡耐受后脊髓胶质细胞及炎性细胞因子的变化.方法 雌性SD大鼠,体重180～220 g,采用MADB-106鼠源性乳腺癌细胞注入大鼠左侧胫骨建立骨癌痛模型.14d后,选择一般状况良好、体重无减轻、进食好、活动正常、热痛阈<18.5 s、机械痛阈<27.8 g的大鼠16只,随机分为2组:对照组(n=7)注射等容量生理盐水;吗啡耐受组(n=9)皮下注射吗啡3次/d,连续5 d,每天单次注射剂量按10、20、40、50、60 mg/kg递增.于造模前和造模后10 d开始隔日1次测定热痛阈和机械痛阈;造模后19 d皮下注射吗啡3 mg/ks,30 min后测定热痛阈和机械痛阈.随后处死动物,取腰段脊髓,采用RT-PCR法测定脊髓白细胞介素-1β(IL-1β)mRNA和肿瘤坏死因子α(TNF-α)mRNA的表达;采用ELISA法测定脊髓IL-1β和TNF-α的含量;采用免疫组化法测定脊髓星形胶质细胞和小胶质细胞的活力.结果 与造模前比较.对照组造模后14、16、18 d时热痛阈和机械痛阈降低(P<0.05);与对照组比较,吗啡耐受组造模后14、16、18 d时热痛阈和机械痛阈升高,造模后19 d时热痛阈和机械痛阈降低,左侧脊髓IL-1β mRNA和TNF-αmRNA表达升高,脊髓IL-1β和TNF-α的含量增加,脊髓星形胶质细胞和小胶质细胞的活力增强(P<0.05).结论 骨癌痛大鼠连续递增剂量腹腔注射吗啡可引起脊髓星形胶质细胞和小胶质细胞进一步活化,IL-1β和TNF-α释放增加,从而发生吗啡耐受.

Changes in spinal gliocytes and inflammatory cytokines in rats with metastatic bone cancer pain after development of morphine tolerance

Objective To investigate the changes in the spinal gliocytes and inflammatory cytokines in rats with metastatic bone cancer pain after development of morphine tolerance. Methods Female SD rats weighing 180-220 g were used in this study. Bone cancer pain was produced by inoculation of MADB-I06 breast cancer ceils into the left tibia. Pain threshold to noxious thermal and mechanical stimulation were measured before (baseline) and after inoculation of cancer cells. Fourteen days after inoculation of cancer cells 16 rats with cancer pain the latency from the onset of radiant heat to withdrawal of hindpaw (PWL) <18.5 s; the paw withdrawal threshold to yon Frey filament stimulation (PWT) <27.8 g] were randomized into 2 groups: group I control (C,n=7) and group Ⅱ chronic morphine tolerance (MT, n=9). Morpine tolerance was induced by intraperitoneal injection of increasing doses of morphine. Morphine was injected 3 times/day×5 days. The doses for each injection were increased everyday from 10 mg/kg on the first day to 20, 40, 50 and 60 mg/kg. Equal volumes of normal saline was injected instead of morphine in control group. The animals were killed on the 19th day after cancer cell inoculation. The lumbar segments were removed for determination of the expression of IL-1β and TNF-αmRNA (by RT-PCR) ,the content of IL-1β and TNF-α (by ELISA) and the levels of GFAP (an astrocyte marker) and OX-42 (a microglial marker) (by immuno-histochemistry ) in the spinal cord. Resulls The PWL and PWT were significantly decreased on the 14, 16 and 18 d after cancer cell inoculation as compared with the baseline before inoculation. PWL and PWT were significantly higher on 14, 16 and 18 d but lower on 19 d after inoculation in morphine tolerance group than in control group. The expression of IL-1β and TNF-αmRNA and protein and the levels of GFAP and OX-42 in the left side of spinal cord were significantly higher in morphine tolerance group than in control group. Conclusion lntraperitoneal injection of increasing doses of morphine/ can activate spinal astrocyte and microglia and increase the release of inflammatory cytokines, leading to morphine tolerance in rats with metastatic bone cancer pain.