Spinal astrocytic activation contributes to mechanical allodynia in a rat chemotherapy-induced neuropathic pain model

Abstract

Chemotherapy-induced neuropathic pain (CNP) is the major dose-limiting factor in cancer chemotherapy. However, the neural mechanisms underlying CNP remain enigmatic. Accumulating evidence implicates the involvement of spinal glia in some neuropathic pain models. In this study, using a vincristine-evoked CNP rat model with obvious mechanical allodynia, we found that spinal astrocyte rather than microglia was dramatically activated. The mechanical allodynia was dose-dependently attenuated by intrathecal administratration of L-α-aminoadipate (astrocytic specific inhibitor); whereas minocycline (microglial specific inhibitor) had no such effect, indicating that spinal astrocytic activation contributes to allodynia in CNP rat. Furthermore, oxidative stress mediated the development of spinal astrocytic activation, and activated astrocytes dramatically increased interleukin-1β expression which induced N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal neurons to strengthen pain transmission. Taken together, our findings suggest that spinal activated astrocytes may be a crucial component of the pathophysiology of CNP and "Astrocyte-Cytokine-NMDAR-neuron" pathway may be one detailed neural mechanisms underlying CNP. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for treating CNP.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Mechanical allodynia occurred in vincristine treated rats.

With regard to (A) body weight, (B) motor function and plasma levels of (C) aspartate aminotransferase and (D) alanine aminotransferase, there was no difference between Sham group and Vincristine group. (E) Compared with Sham group, the paw withdrawal threshold of Vincristine group was significantly decreased. (F) No significant changes in thermal withdrawal thresholds were observed between Sham group and Vincristine group. All data were calculated as mean ± SEM (n = 10/group/week). *P

Figure 2. Spinal astrocyte was activated in vincristine treated rats.

(A–D) Compared to Sham group, GFAP-like immunoreactivity in spinal dorsal horn of Vincristine group was significantly increased. Bar = 200 µm. (E) Compared to Naive group and Sham group, Western blot analysis showed that spinal GFAP expression was significantly increased in Vincristine group. A systemic treatment with PBN (scavenger for reactive oxygen species) significantly reduced GFAP overexpression in Vincristine group at 1 week post osmotic pump implantation (1 w). All data were calculated as mean ± SEM (n = 10/group/week). *P

Figure 3. Spinal microglia was not activated in vincristine treated rats, astrocytic specific inhibitor LAA but not microglial specific inhibitor minocycline attenuated mechanical allodynia.

(A and B) With regard to OX42-like immunoreactivity in spinal dorsal horn, there was no difference between Sham group and Vincristine group. Bar = 200 µm. (C) No significant difference in OX42 expression in spinal cord was observed among Naive group, Sham group and Vincristine group. In Vincristine group, OX42 expression was unchanged through the period tested. (D) Intrathecal injection of LAA significantly attenuated the allodynia. However, minocycline did not influence the allodynia. (E) Allodynia was attenuated by LAA in a dose-dependent manner. All data were calculated as mean ± SEM (n = 10/group). *P<0.05, **P<0.01 vs. vincristine+Saline group or vincristine+minocycline group in D. *P<0.05 vs. LAA 50 nmol group, △P<0.05 vs. LAA 100 nmol group in E. Vin: vincristine; d: day; w: week; min: minute; OX42: microglial marker; LAA: L-α-aminoadipate.

Figure 4. IL-1β overexpression in spinal cord was related to mechanical allodynia in vincristine treated rats, and activated astrocytes were the only source of IL-1β.

(A) IL-1β expression was significantly upregulated in spinal cord of Vincristine group compared to Naive group and Sham group. Intrathecal treatment with LAA (astrocytic specific toxin) significantly reduced IL-1β overexpression in Vincristine group. (B) Intrathecal injection of Pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) significantly attenuated the allodynia. (C–K) Double immunofluorescent staining showed that IL-1β-immunoreactivity was localized in GFAP-immunopositive cells but not in OX42-immunopositive cells or NeuN-immunopositive cells in spinal cord of Vincristine group. Bar = 20 µm. All data were calculated as mean ± SEM (n = 10/group). *P

Figure 5. IL-1β released from astrocyte induced NMDA receptor phosphorylation in spinal dorsal horn neurons in vincristine treated rats, which was related to mechanical allodynia.

(A) The phosphorylated NR1 subunit of NMDA receptor (P-NR1) was significantly increased in Vincristine group compared to Naive group and Sham group. Intrathecal treatment with LAA (astrocytic specific toxin), PF (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) significantly reduced P-NR1 in Vincristine group. (B) Intrathecal injection of AP5 (NMDA receptor antagonist) or MK-801 (non-competitive NMDA receptor antagonist) significantly attenuated the allodynia. (C–E) Double immunofluorescent staining showed that P-NR1-immunoreactivity and IL-1RI-immunoreactivity were totally co-localized in spinal dorsal horn of Vincristine group. Bar = 50 µm. All data were calculated as mean ± SEM (n = 10/group). *P

Figure 6. Schematic drawing showed the development of mechanical allodynia in vincristine treated rats.

In the situation of chemotherapy, mitochondrial dysfunction may induce oxidative stress which dramatically activates astrocytes in spinal cord. Cytokines like IL-1β are then released from astrocyte and act on spinal dorsal horn neurons to produce chemotherapy-induced neuropathic pain. ROS: reactive oxygen species.