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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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A rat model of bone pain from metastatic bone cancer. (A) Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain. The PWL progressively decreased and the ambulatory score progressively increased on days 6 (n = 12, ANOVA2w, P < 0.05 pos-hoc Bonferroni, df = 2, F = 1.07), 9, 12, 14, 16, 18, and 21 (ANOVA2w, P < 0.01, df = 2, F = 1.13) after inoculation (AI) in the tumor-bearing group compared with sham-injected controls and normal rats. Results are given as means ± S.D, *p < 0.05 **P < 0.01 vs. normal group; ▲p < 0.05 ▲ ▲p < 0.01 vs. sham group. (B) Radiographs of the left tibiae 18 days after inoculation with Walker 256 cells and the right normal tibiae. There was bilateral cortical bone damage and large bone defects at the 18 days' tumor-bearing proximal epiphysis of the tibiae.(C) HE staining of normal (upper panel) and 18 days of tumor-bearing (lower) proximal epiphysis of the tibiae. Note that tumor cells were densely packed in the marrow cavity and induced the destruction of trabeculae. Arrows indicate tumor cells. Bar = 1 mm in B and 40 μm in C.
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Figure 1: A rat model of bone pain from metastatic bone cancer. (A) Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain. The PWL progressively decreased and the ambulatory score progressively increased on days 6 (n = 12, ANOVA2w, P < 0.05 pos-hoc Bonferroni, df = 2, F = 1.07), 9, 12, 14, 16, 18, and 21 (ANOVA2w, P < 0.01, df = 2, F = 1.13) after inoculation (AI) in the tumor-bearing group compared with sham-injected controls and normal rats. Results are given as means ± S.D, *p < 0.05 **P < 0.01 vs. normal group; ▲p < 0.05 ▲ ▲p < 0.01 vs. sham group. (B) Radiographs of the left tibiae 18 days after inoculation with Walker 256 cells and the right normal tibiae. There was bilateral cortical bone damage and large bone defects at the 18 days' tumor-bearing proximal epiphysis of the tibiae.(C) HE staining of normal (upper panel) and 18 days of tumor-bearing (lower) proximal epiphysis of the tibiae. Note that tumor cells were densely packed in the marrow cavity and induced the destruction of trabeculae. Arrows indicate tumor cells. Bar = 1 mm in B and 40 μm in C.

Mentions: Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain (Fig. 1A). Before Walker 256 cells inoculation, there were no significant differences in overall mean baseline paw withdrawal latency (PWL) to tactile allodynia and ambulatory score(AS) to spontaneous pain among the normal group, sham group, and bone cancer pain group(n = 12, ANOVA2w, PWL, P = 0.13 and AS, P = 0.39). Initially, ipsilateral tactile allodynia was observed in the sham and cancer pain groups after inoculation, but by two to three days post-surgery, thresholds in both groups returned to the baseline. In contrast, six days after inoculation, marked decreases in the PWL to tactile allodynia were observed in more than 90% of the tumor-bearing rats compared with sham-injected controls (ANOVA2w, P < 0.05, pos-hoc Bonferroni); at the same time, the ambulatory score began to rise (ANOVA2w, P < 0.05, pos-hoc Bonferroni). Subsequently, the PWL progressively decreased and the ambulatory score progressively increased in the tumor-bearing group(ANOVArm, P < 0.01, pos-hoc Bonferroni).


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)

A rat model of bone pain from metastatic bone cancer. (A) Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain. The PWL progressively decreased and the ambulatory score progressively increased on days 6 (n = 12, ANOVA2w, P < 0.05 pos-hoc Bonferroni, df = 2, F = 1.07), 9, 12, 14, 16, 18, and 21 (ANOVA2w, P < 0.01, df = 2, F = 1.13) after inoculation (AI) in the tumor-bearing group compared with sham-injected controls and normal rats. Results are given as means ± S.D, *p < 0.05 **P < 0.01 vs. normal group; ▲p < 0.05 ▲ ▲p < 0.01 vs. sham group. (B) Radiographs of the left tibiae 18 days after inoculation with Walker 256 cells and the right normal tibiae. There was bilateral cortical bone damage and large bone defects at the 18 days' tumor-bearing proximal epiphysis of the tibiae.(C) HE staining of normal (upper panel) and 18 days of tumor-bearing (lower) proximal epiphysis of the tibiae. Note that tumor cells were densely packed in the marrow cavity and induced the destruction of trabeculae. Arrows indicate tumor cells. Bar = 1 mm in B and 40 μm in C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819998&req=5

Figure 1: A rat model of bone pain from metastatic bone cancer. (A) Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain. The PWL progressively decreased and the ambulatory score progressively increased on days 6 (n = 12, ANOVA2w, P < 0.05 pos-hoc Bonferroni, df = 2, F = 1.07), 9, 12, 14, 16, 18, and 21 (ANOVA2w, P < 0.01, df = 2, F = 1.13) after inoculation (AI) in the tumor-bearing group compared with sham-injected controls and normal rats. Results are given as means ± S.D, *p < 0.05 **P < 0.01 vs. normal group; ▲p < 0.05 ▲ ▲p < 0.01 vs. sham group. (B) Radiographs of the left tibiae 18 days after inoculation with Walker 256 cells and the right normal tibiae. There was bilateral cortical bone damage and large bone defects at the 18 days' tumor-bearing proximal epiphysis of the tibiae.(C) HE staining of normal (upper panel) and 18 days of tumor-bearing (lower) proximal epiphysis of the tibiae. Note that tumor cells were densely packed in the marrow cavity and induced the destruction of trabeculae. Arrows indicate tumor cells. Bar = 1 mm in B and 40 μm in C.
Mentions: Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain (Fig. 1A). Before Walker 256 cells inoculation, there were no significant differences in overall mean baseline paw withdrawal latency (PWL) to tactile allodynia and ambulatory score(AS) to spontaneous pain among the normal group, sham group, and bone cancer pain group(n = 12, ANOVA2w, PWL, P = 0.13 and AS, P = 0.39). Initially, ipsilateral tactile allodynia was observed in the sham and cancer pain groups after inoculation, but by two to three days post-surgery, thresholds in both groups returned to the baseline. In contrast, six days after inoculation, marked decreases in the PWL to tactile allodynia were observed in more than 90% of the tumor-bearing rats compared with sham-injected controls (ANOVA2w, P < 0.05, pos-hoc Bonferroni); at the same time, the ambulatory score began to rise (ANOVA2w, P < 0.05, pos-hoc Bonferroni). Subsequently, the PWL progressively decreased and the ambulatory score progressively increased in the tumor-bearing group(ANOVArm, P < 0.01, pos-hoc Bonferroni).

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