Experimental Studies: Role Of Spinal Glia And Proinflammatory Cytokines In A Rat Metastatic Bone Cancer Pain Model

Objective To investigate the role of spinal glia and proinflammatory cytokines in a rat model of bone cancer pain induced by breast cancer cells.Methods (1) Intra-tibial injection of MADB-106 breast cancer cells to establish a rat model of bone cancer pain, the thermal hyperalgesia (PWL) and mechanical hyperalgesia (PWT) of the cancer and vehicle rats were measured, radiology and histochemical staining were used to confirm cancer development in the rat tibia, immunoreactivity for glial fibrillary acidic protein (GFAP) and OX-42 (labels complement type 3 receptors,CD11b), astrocyte and microglial activation markers, respectively, was assessed. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbant assay (ELISA) were used to compare the spinal levels of IL-1βand TNF-αin the rats of bone cancer pain and that of controls. (2) The rats of bone cancer pain were treated by intrathecal SB203580 injection, the PWL, histology and the spinal levels of IL-1βand TNF-αwere detected. (3) Chronic morphine tolerance models were produced in the rats of bone cancer pain, the PWL and PWT were used to evaluate pain behavior changes with the time-course in all rats. RT-PCR and ELISA were used to compare the spinal levels of IL-1βand TNF-αin the rats between morphine tolerance and that of controls. Immunohistochemistry were used to compare the spinal levels of GFAP and OX-42 in the rats between morphine tolerance and that of controls. (4) The rats of bone cancer pain were treated with radiotherapy. The PWL and PWT were used to evaluate pain behavior changes with the time-course in all rats. X-ray were used to evaluate the bone destruction . Tunel were used to observe the cancer cell apoptosis between two groups. RT-PCR were used to compare the spinal levels of GFAP, OX-42, IL-1βand TNF-α, ELISA were used to compare the spinal levels of IL-1βand TNF-αin the rats between radiotherapy and that of controls. Results (1) The injection of the cancer cells led to progressive thermal and mechanical hyperalgesia associated with the ongoing bone destruction; the GFAP and OX-42 optical density ratios of ipsilateral side to the contralateral side in the spinal dorsal horn were increased with time course after cancer cell injection; the levels of IL-1βand TNF-αwere up-regulated in the ipsilateral spinal cord with time course. (2) Following intrathecal injection of SB203580, no effect of SB203580 on the bone destruction was observed, the left PWLs were significant higher than that of controls, Meanwhile, intrathecal injection of SB203580 evidently reduced the levels of spinal IL-1βand TNF-α, P<0.05. (3) In the rats of morphine tolerance, the levels of PWL and PWT were significant increased at 14,16 and 18 days than controls, P<0.05.The spinal mRNA or protein levels of IL-1βand TNF-αin ipsilateral side in morphine tolerance rats were higher than that of controls , P < 0.05. (4) The rats in radiotherapy group were detected in higer PWL and PWT at 13, 16, 19 days after cancer cell injection than that of controls, P<0.05. The cancer cell apoptosis and bone destruction were significantly reduced between radiotherapy group and that of controls. The spinal mRNA levels of GFAP , OX-42, IL-1βand TNF-αin ipsilateral side in radiotherapy rats were lower than that of controls , P < 0.05. The spinal protein levels of IL-1βand TNF-αin ipsilateral side of the radiotherapy rats were lower than that of controls, P < 0.05.Conclusion These results demonstrated that a novel bone cancer pain model could be successfully established through inoculation of MADB-106 breast cancer cells of the tibia. In this model, bone cancer induced spinal microglia and astrocyte activations, which promoted the release of IL-1βand TNF-α, thus contributing to the maintenance of bone cancer pain. Lessen bone destruction, inhibiting spine glia activation and the expression of proinflammatory cytokines could reduce the bone cancer pain. Meanwhile, the activation of spinal astrocyte and microglia and release of the proinflammatory cytokines were related to morphine tolerance.