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Down-regulation of Toll-like receptor 4 gene expression by short interfering RNA attenuates bone cancer pain in a rat model.

Lan LS, Ping YJ, Na WL, Miao J, Cheng QQ, Ni MZ, Lei L, Fang LC, Guang RC, Jin Z, Wei L - Mol Pain (2010)

Bottom Line: We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls.Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.

ABSTRACT

Background: This study demonstrates a critical role in CNS innate immunity of the microglial Toll-like receptor 4 (TLR4) in the induction and maintenance of behavioral hypersensitivity in a rat model of bone cancer pain with the technique of RNA interference (RNAi). We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.

Results: We assessed tactile allodynia and spontaneous pain in female Sprague-Dawley (SD) rats after intramedullary injection of Walker 256 cells into the tibia. In a complementary study, TLR4 small interfering RNA(siRNA) was administered intrathecally to bone cancer pain rats to reduce the expression of spinal TLR4. The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls. Only intrathecal injection of TRL4 siRNA at post-inoculation day 4 could prevent initial development of bone cancer pain; intrathecal injection of TRL4 siRNA at post-inoculation day 9 could attenuate, but not completely block, well-established bone cancer pain.

Conclusions: TLR4 might be the main mediator in the induction of bone cancer pain. Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

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Intrathecal TLR4 siRNA439 attenuates bone cancer pain in the rat model. (A, B) In the siRNA-treated IBCP group, the PWTs were significantly higher and the ambulatory scores were significantly lower than those observed in mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.02), and there were no significant differences compared to normal rats. This indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could prevent the initial development of bone cancer pain. (C, D) In the siRNA-treated WBCP group, the PWTs were significantly elevated compared with the mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 0.96), but still lower than in normal rats (P < 0.05) Meanwhile, spontaneous pain was attenuated compared with mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.05). However, the ambulatory score was still higher than in normal rats (P < 0.05), which indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could alleviate, but not reverse, well-established bone cancer pain. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. normal group; ▲ P < 0.05, ▲ ▲ P < 0.01 vs. vehicle-treated group; #P < 0.05, ##P < 0.01 vs. bone cancer pain group; ◆P < 0.05, ◆◆P < 0.01 vs. mismatch siRNA-treated group. Arrows indicate the siRNA injection times.
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Figure 5: Intrathecal TLR4 siRNA439 attenuates bone cancer pain in the rat model. (A, B) In the siRNA-treated IBCP group, the PWTs were significantly higher and the ambulatory scores were significantly lower than those observed in mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.02), and there were no significant differences compared to normal rats. This indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could prevent the initial development of bone cancer pain. (C, D) In the siRNA-treated WBCP group, the PWTs were significantly elevated compared with the mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 0.96), but still lower than in normal rats (P < 0.05) Meanwhile, spontaneous pain was attenuated compared with mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.05). However, the ambulatory score was still higher than in normal rats (P < 0.05), which indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could alleviate, but not reverse, well-established bone cancer pain. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. normal group; ▲ P < 0.05, ▲ ▲ P < 0.01 vs. vehicle-treated group; #P < 0.05, ##P < 0.01 vs. bone cancer pain group; ◆P < 0.05, ◆◆P < 0.01 vs. mismatch siRNA-treated group. Arrows indicate the siRNA injection times.

Mentions: In the siRNA-treated IBCP group, the PWTs were significantly higher and the ambulatory scores were significantly lower than those observed in mismatch siRNA- and vehicle-treated rats (n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni), and there were no significant differences compared to normal rats (ANOVA2w, PWT, P = 0.93 and AS, P = 0.3) (Fig. 5A, B). No significant difference was observed between mismatch siRNA- and vehicle-treated rats, which indicated that intrathecal injection of TLR4 siRNA439 could prevent the initial development of bone cancer pain. In the siRNA-treated WBCP group, the PWTs were significantly elevated compared with the mismatch siRNA- and vehicle-treated rats (n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni), but still lower than in normal rats (P < 0.05) (Fig. 5C, D). Meanwhile, spontaneous pain was attenuated compared with mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni). However, the ambulatory score was still higher than normal rats (P < 0.05), which indicated that intrathecal injection of TLR4 siRNA439 could alleviate, but not reverse, well-established bone cancer pain.


Down-regulation of Toll-like receptor 4 gene expression by short interfering RNA attenuates bone cancer pain in a rat model.

Lan LS, Ping YJ, Na WL, Miao J, Cheng QQ, Ni MZ, Lei L, Fang LC, Guang RC, Jin Z, Wei L - Mol Pain (2010)

Intrathecal TLR4 siRNA439 attenuates bone cancer pain in the rat model. (A, B) In the siRNA-treated IBCP group, the PWTs were significantly higher and the ambulatory scores were significantly lower than those observed in mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.02), and there were no significant differences compared to normal rats. This indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could prevent the initial development of bone cancer pain. (C, D) In the siRNA-treated WBCP group, the PWTs were significantly elevated compared with the mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 0.96), but still lower than in normal rats (P < 0.05) Meanwhile, spontaneous pain was attenuated compared with mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.05). However, the ambulatory score was still higher than in normal rats (P < 0.05), which indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could alleviate, but not reverse, well-established bone cancer pain. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. normal group; ▲ P < 0.05, ▲ ▲ P < 0.01 vs. vehicle-treated group; #P < 0.05, ##P < 0.01 vs. bone cancer pain group; ◆P < 0.05, ◆◆P < 0.01 vs. mismatch siRNA-treated group. Arrows indicate the siRNA injection times.
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: Intrathecal TLR4 siRNA439 attenuates bone cancer pain in the rat model. (A, B) In the siRNA-treated IBCP group, the PWTs were significantly higher and the ambulatory scores were significantly lower than those observed in mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.02), and there were no significant differences compared to normal rats. This indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could prevent the initial development of bone cancer pain. (C, D) In the siRNA-treated WBCP group, the PWTs were significantly elevated compared with the mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 0.96), but still lower than in normal rats (P < 0.05) Meanwhile, spontaneous pain was attenuated compared with mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni, df = 4, F = 1.05). However, the ambulatory score was still higher than in normal rats (P < 0.05), which indicated that intrathecal injection of TLR4 siRNA439 (SITLR4) could alleviate, but not reverse, well-established bone cancer pain. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. normal group; ▲ P < 0.05, ▲ ▲ P < 0.01 vs. vehicle-treated group; #P < 0.05, ##P < 0.01 vs. bone cancer pain group; ◆P < 0.05, ◆◆P < 0.01 vs. mismatch siRNA-treated group. Arrows indicate the siRNA injection times.
Mentions: In the siRNA-treated IBCP group, the PWTs were significantly higher and the ambulatory scores were significantly lower than those observed in mismatch siRNA- and vehicle-treated rats (n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni), and there were no significant differences compared to normal rats (ANOVA2w, PWT, P = 0.93 and AS, P = 0.3) (Fig. 5A, B). No significant difference was observed between mismatch siRNA- and vehicle-treated rats, which indicated that intrathecal injection of TLR4 siRNA439 could prevent the initial development of bone cancer pain. In the siRNA-treated WBCP group, the PWTs were significantly elevated compared with the mismatch siRNA- and vehicle-treated rats (n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni), but still lower than in normal rats (P < 0.05) (Fig. 5C, D). Meanwhile, spontaneous pain was attenuated compared with mismatch siRNA- and vehicle-treated rats(n = 10, ANOVA2w, P < 0.01, pos-hoc Bonferroni). However, the ambulatory score was still higher than normal rats (P < 0.05), which indicated that intrathecal injection of TLR4 siRNA439 could alleviate, but not reverse, well-established bone cancer pain.

Bottom Line: We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls.Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.

ABSTRACT

Background: This study demonstrates a critical role in CNS innate immunity of the microglial Toll-like receptor 4 (TLR4) in the induction and maintenance of behavioral hypersensitivity in a rat model of bone cancer pain with the technique of RNA interference (RNAi). We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.

Results: We assessed tactile allodynia and spontaneous pain in female Sprague-Dawley (SD) rats after intramedullary injection of Walker 256 cells into the tibia. In a complementary study, TLR4 small interfering RNA(siRNA) was administered intrathecally to bone cancer pain rats to reduce the expression of spinal TLR4. The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls. Only intrathecal injection of TRL4 siRNA at post-inoculation day 4 could prevent initial development of bone cancer pain; intrathecal injection of TRL4 siRNA at post-inoculation day 9 could attenuate, but not completely block, well-established bone cancer pain.

Conclusions: TLR4 might be the main mediator in the induction of bone cancer pain. Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

Show MeSH
Related in: MedlinePlus