Robust spinal neuroinflammation mediates mechanical allodynia in Walker 256 induced bone cancer rats

Abstract

It has been reported that remarkable and sustained activation of astrocytes and/or microglia occurs in cancer induced pain (CIP), which is different from neuropathic and inflammatory pain. The present study was designed to investigate the role of spinal Toll-like receptor 4 (TLR4) induced glial neuroinflammation in cancer induced pain using a modified rat model of bone cancer. The rat model of CIP consisted of unilateral intra-tibial injection with Walker 256 mammary gland carcinoma. Nine days after Walker 256 inoculation, a robust activation of both astrocytes and microglia in bilateral spinal dorsal horn was observed together with significant bilateral mechanical allodynia. This neuroinflammation was characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker) and OX-42 (microglia marker), and an elevated level of IL-1β, IL-6 and TNF-α mRNA. I.t. administration of fluorocitrate (an inhibitor of glial metabolism, 1 nmol) or minocycline (an inhibitor of microglia, 100 μg) has significant anti-allodynic effects on day 12 after Walker 256 inoculation. Naloxone (a nonstereoselective TLR4 signaling blocker, 60 μg, i.t.) also significantly alleviated mechanical allodynia and simultaneously blocked the increased inflammatory cytokine mRNA. The results suggested that spinal TLR4 might play an important role in the sustained glial activation that critically contributed to the robust and sustained spinal neuroinflammation in CIP. This result could potentially help clinicians and researchers to better understand the mechanism of complicated cancer pain.

Figure 1

Tibia bone destruction by Walker 256 inoculation. Radiographs of the Contralateral (A) and Ipsilateral (B) tibia bone 20 days after Walker 256 inoculation. (A’-B’) showing the proximal end of the bones with a higher magnification. (C) showing the tibia bone from normal and tumor bearing rats 20 days after Walker 256 inoculation. (D) histology of tibial bone destruction stained by Hematoxylin-eosin. The hollow arrow showing normal marrow cavity in PBS rats instead tumor cells in Walker 256 inoculated rats. The black arrow indicates the trabecular bone.

Figure 2

Changes of mechanical response thresholds to von Frey hair (A) and paw withdrawal latency to radiant heat (B) of the ipsilateral and contralateral hind paw in normal rats and Walker 256 (4 × 10⁵cells), heat-killed cells and PBS -inoculated rats. Data are expressed as means ± SEM. *p < 0.05, **p <0.01 vs. normal rats.

Figure 3

Changes of expression of GFAP and OX-42 protein in the dorsal horn of the spinal cord detected by immunohistochemistry after the rats received carcinoma cells. (A) and (B) showing representative images for GFAP and OX-42 immunostaining respectively. (C) showing the selected area marked with red square in the spinal cord for quantification. (D, E) Quantification of astrocyte and microglia expression in the spinal dorsal horn after intra-tibial inoculation of Walker 256. Percentages of immunostaining are expressed as mean ± SEM (3 ~ 5 for each group). **p <0.01 vs. normal rats.

Figure 4

Changes of inflammatory cytokines IL-1β, IL-6, TNF-α mRNA level detected by Semiquantitative RT-PCR analysis. (A) and (B) showing the mRNA levels in spinal dorsal horn 8 days and 16 days after intra-tibial inoculation of Walker 256 respectively. The relative mRNA level was expressed as a ratio relative to the normal control. *p < 0.05, **p < 0.01 vs. normal rats. (C) showing the protein level of IL-1β, IL-6, TNF-α in the spinal dorsal horn at 12 days after Walker 256 inoculation. * p < 0.05, vs. PBS rats.

Figure 5

Anti-allodynic effects by intrathecal administration of fluorocitrate (A) or minocycline (B). On 12 days after Walker 256 inoculation, fluorocitrate (1 nmol), a glia metabolic inhibitor, or minocycline (100 μg), a kind of microglia activation inhibitor significantly increased the mechanical thresholds to von Frey hair stimulation 3 hrs after i.t. treatment. *p < 0.05 vs. normal saline treatment; #p < 0.05 vs. before treatment.

Figure 6

Spinal Toll-like receptor (TLR4) is involved in bone cancer pain. (A) showing the changes of TLR4 mRNA levels in bilateral dorsal horn of spinal cord 8 and 16 days after intra-tibial injection of PBS, Heat-killed or live Walker 256 cells. (B) Showing the blocking effect of single intrathecal administration of naloxone (60 μg) on elevated spinal IL-1β and TNF-α mRNA expression induced by intra-tibial inoculation of Walker 256 cells. (C) Anti-allodynic effects induced by single intrathecal administration of naloxone (60 μg). On the day 8 after Walker 256 inoculation, naloxone (60 μg), a nonsteroselective TLR4 antagonist, significantly increased the mechanical thresholds to von Frey hair stimulation 45, 135 mins after i.t. treatment. &&p < 0.01 vs. normal rats; *p < 0.05, **p < 0.01 vs. Walker 256 inoculated rats; #p < 0.05, ##p < 0.01 vs. normal saline treatment.