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, 35 (20), 7950-63

Delayed Activation of Spinal Microglia Contributes to the Maintenance of Bone Cancer Pain in Female Wistar Rats via P2X7 Receptor and IL-18

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Delayed Activation of Spinal Microglia Contributes to the Maintenance of Bone Cancer Pain in Female Wistar Rats via P2X7 Receptor and IL-18

Yan Yang et al. J Neurosci.

Abstract

Accumulating evidence suggests that activation of spinal microglia contributes to the development of inflammatory and neuropathic pain. However, the role of spinal microglia in the maintenance of chronic pain remains controversial. Bone cancer pain shares features of inflammatory and neuropathic pain, but the temporal activation of microglia and astrocytes in this model is not well defined. Here, we report an unconventional role of spinal microglia in the maintenance of advanced-phase bone cancer pain in a female rat model. Bone cancer elicited delayed and persistent microglial activation in the spinal dorsal horn on days 14 and 21, but not on day 7. In contrast, bone cancer induced rapid and persistent astrocytic activation on days 7-21. Spinal inhibition of microglia by minocycline at 14 d effectively reduced bone cancer-induced allodynia and hyperalgesia. However, pretreatment of minocycline in the first week did not affect the development of cancer pain. Bone cancer increased ATP levels in CSF, and upregulated P2X7 receptor, phosphorylated p38, and IL-18 in spinal microglia. Spinal inhibition of P2X7/p-38/IL-18 pathway reduced advanced-phase bone cancer pain and suppressed hyperactivity of spinal wide dynamic range (WDR) neurons. IL-18 induced allodynia and hyperalgesia after intrathecal injection, elicited mechanical hyperactivity of WDR neurons in vivo, and increased the frequency of mEPSCs in spinal lamina IIo nociceptive synapses in spinal cord slices. Together, our findings demonstrate a novel role of microglia in maintaining advanced phase cancer pain in females via producing the proinflammatory cytokine IL-18 to enhance synaptic transmission of spinal cord nociceptive neurons.

Keywords: IL-18; P2X7 receptor; bone cancer pain; female rats; microglia; neuron-glial interaction.

Figures

Figure 1.
Figure 1.
Bone cancer induces behavioral hypersensitivity and bone destruction. A, B, Intratibia inoculation with Walker 256 mammary gland carcinoma cells (4 × 105) induces significant mechanical allodynia (A) and thermal hyperalgesia (B) in the ipsilateral hindpaw. C, Intratibia inoculation with Walker 256 mammary gland carcinoma cells induces spontaneous flinches in the hindpaw ipsilateral to the affected limb. The number of times the hindpaw was counted per 5 min during a 10 min observation period, and the average number of flinches per 5 min were calculated. *p < 0.05; **p < 0.01 versus sham rats. D, Radiographs represent robust radiolucent lesions of the tibia ipsilateral to the tumor-bearing limb on PTDs 14 and 21. Contr., Contralateral; Ipsi., ipsilateral.
Figure 2.
Figure 2.
Time course of astrocytic and microglial activation on the lumbar spinal cord following bone cancer. A, Western blot analysis reveals an early upregulation of GFAP from PTD 7 and a delayed upregulation of IBA-1 from PTD 14 in the ipsilateral L3–L5 spinal dorsal horn. B–D, Immunohistochemisty data show astrogliosis, as indicated by intense GFAP immunoreactivity and hypertrophied astrocytes with thick processes, and microgliosis, as indicated by intense IBA-1 immunoreactivity and large cell bodies and short or thick processes of microglia in the ipsilateral spinal dorsal horn. Astrogliosis and microgliosis occurred from early phase (PTD 7) and advanced phase (PTD 14), respectively. *p < 0.05; **p < 0.01 versus sham rats.
Figure 3.
Figure 3.
Delayed activation of microglia is involved in the maintenance, but not the induction, of bone cancer pain. A, B, Development of mechanical allodynia (A) and thermal hyperalgesia (B) is blocked by repeated intrathecal injections of astrocyte metabolic inhibitor fluorocitrate (FC; 0.8 μg = 1 nmol) but not by microglia inhibitor minocycline (Mino; 100 μg) during the early phase of bone cancer pain. FC or Mino is given once daily for 6 d with the first injection occurring 30 min before intratibia inoculation. **p < 0.01 versus baseline. ##p < 0.01 versus vehicle (Veh)- and Mino-treated groups. C, Repeated intrathecal injections of FC (0.8 μg = 1 nmol, once daily for 6 d) do not influence motor coordination in normal rats in a rotarod test. D, Few fluorescein-labeled apoptotic nuclei were detected in the lumbar spinal dorsal horns from normal or bone cancer rats with repeated intrathecal injections of FC (0.8 μg = 1 nmol, once daily for 6 d). HL-60 cells treated by 0.5 μg/ml actinomycin D for 19 h to induce apoptosis were used as a positive control. E, F, Intrathecal injection of either FC or Mino on PTD 14 significantly reduces bone cancer-induced mechanical allodynia (E) and thermal hyperalgesia (F). *p < 0.05; **p < 0.01 versus vehicle controls.
Figure 4.
Figure 4.
Bone cancer induces the upregulation of P2X7R in the spinal dorsal horn and increases ATP release in spinal CSF. A, Double immunofluorescence reveals that P2X7R-IR is predominantly colocalized with CD11b-IR (microglial marker), but not with NeuN-IR (neuronal marker), on the ipsilateral spinal dorsal horn. Inset, The antibody to P2X7 detected a band corresponding to P2X7R in wild-type mice but not in P2X7R knock-out mice. B, Western blot analysis reveals significant upregulation of P2X7R levels on PTDs 14 and 21. C, ATP concentration in spinal CSF was significantly increased on PTD 14. *p < 0.05, **p < 0.01 versus sham rats.
Figure 5.
Figure 5.
P2X7R is involved in microglial activation, and bone cancer-induced allodynia and hyperalgesia in advanced phase. A, Blockade of P2X7R by BBG (a selective P2X7R antagonist; 0.3 μg, i.t.) significantly suppressed bone cancer-induced IBA-1 upregulation on PTD 14. *p < 0.01 versus vehicle control (Veh). B, C, Knockdown of P2X7R by siRNA (5 μg) targeted against P2X7R blocks bone cancer-induced IBA-1 (B) and P2X7R (C) upregulation on PTD 14. *p < 0.05, **p < 0.01 versus control siRNA (Cntr.). D, Increased GFAP level on PTD 14 is not suppressed by P2X7R siRNA. E, F, Intrathecal injection of BBG on PTD 14 significantly reduces bone cancer-induced mechanical allodynia (E) and thermal hyperalgesia (F). *p < 0.05; **p < 0.01 versus vehicle controls. G, H, Knockdown P2X7R by siRNA blocks bone cancer-induced allodynia (G) and hyperalgesia (H). **p < 0.01 versus control siRNA.
Figure 6.
Figure 6.
P2X7R mediates bone cancer-induced IL-18 upregulation via p38 MAPK in the spinal dorsal horn. A, Western blot analysis reveals significant upregulation of p-p38 levels on PTDs 14 and 21. **p < 0.01 versus sham rats. B, Knockdown P2X7R significantly reduces bone cancer-induced activation of p38 on PTD 14. **p < 0.01 versus control siRNA (Cntr.). C, Western blot analysis reveals significant upregulation of IL-18 levels on PTDs 14 and 21. **p < 0.05 versus sham rats. D, Knockdown P2X7R significantly suppressed bone cancer-induced upregulation of IL-18 on PTD 14. **p < 0.01 versus control siRNA. E, Selective p38 inhibitor SB239063 (SB; 10 μg, i.t.) markedly suppressed the bone cancer-induced increase in IL-18 levels on PTD 14. F, G, Intrathecal injection of SB239063 or IL-18BP (an endogenous inhibitor of IL-18 activity; 1 μg, i.t.) reversed bone cancer-induced mechanical allodynia (F) and hyperalgesia (G) on PTD 14. *p < 0.05, **p < 0.01 versus vehicle controls (PBS or DMSO).
Figure 7.
Figure 7.
Colocalization of P2X7R/p-p38 and IL-18 in spinal microglia. A, Double immunofluorescence of p-p38 with IL-18-IR, CD11b-IR, NeuN-, IR, and GFAP-IR on the ipsilateral spinal dorsal horn on PTD 14. Note the heavy colocalization of p-p38-IR with IL-18-IR and CD11b-IR. B, Double immunofluorescence shows colocalization of IL-18 with P2X7R and IBA-1 on the ipsilateral spinal dorsal horn on PTD 14.
Figure 8.
Figure 8.
P2X7R/IL-18 signaling regulates neuronal activity in the spinal dorsal horn. A, Histograms show typical responses of a WDR neuron to brush and pinch delivered to the receptive fields in a PTD 14 rat. Oscilloscope recording shows a single sweep (inset). B, C, Histograms show the typical responses of WDR neurons to von Frey filament stimuli delivered to the receptive fields in a naive rat (B) and a PTD 14 rat (C). D, E, The average number of discharges evoked by brush and pinch (D) and von Frey hairs (E) in PTD 14 rats is significantly higher than in sham rats. **p < 0.01 versus sham controls. F, G, Blockade of P2X7R by BBG or the inhibition of IL-18 by IL-18BP significantly suppresses the evoked responses of WDR neurons by von Frey hairs (F), and brush and pinch (G) stimuli in PTD 14 rats. *p < 0.05, **p < 0.01 versus vehicle (Veh) control. H, Histograms show the facilitatory effect of IL-18 on responses of WDR neurons to von Frey stimuli delivered to the receptive fields in a naive rats. I, J, Intrathecal injection of exogenous IL-18 (3 μg) enhances the evoked response of WDR neurons by von Frey hairs (I), and brush and pinch (J) stimuli in naive rats. K, Intrathecal injection of IL-18 of different doses (0.03, 0.3, and 3 μg) induces allodynia in naive rats. L, Pretreatment of fluorocitrate (FC; 0.8 μg = 1 nmol, i.t., 30 min before IL-18 injection) did not block IL-18-induced allodynia. *p < 0.05, **p < 0.01 versus vehicle control.
Figure 9.
Figure 9.
IL-18 modulates mEPSCs in the superficial dorsal horn neurons. A, Patch-clamp recording of mEPSC shows an increase in the frequency of mEPSCs after perfusion of IL-18 (10 ng/ml). B, C, Corresponding cumulative distributions. D, Quantification of mEPSC frequency. E, Quantification of mEPSC amplitudes. **p < 0.01 versus control. F, mEPSC traces show an increase in the frequency of mEPSCs after the perfusion of IL-18 in the presence of fluorocitrate (FC; 10 μm). G, H, Corresponding cumulative distributions. I, Ratio of the frequency and amplitude of mEPSCs following treatment with IL-18, FC, and FC plus IL-18. Dashed line indicates baseline. Rats were pretreated with FC for 30 min before the application of IL-18. *p < 0.05, **p < 0.01 versus control; #p < 0.05, ##p < 0.01 versus FC alone.
Figure 10.
Figure 10.
Schematic illustration of neuron–glia and glia–glia interactions in the spinal cord dorsal horn in bone cancer pain. Bone cancer-induced hyperexcitability of primary sensory neurons (peripheral sensitization) causes excessive release of neurotransmitters or neuromodulators from central afferent terminals to activate adjacent glia and postsynaptic neurons in the spinal dorsal horn. P2X7R is upregulated in microglia and activated by ATP, which is produced by activated astrocytes, as well as by primary afferent terminals and spinal cord neurons. Upon activation, microglia synthesize and release IL-18 via phosphorylation of p38 MAPK, leading to enhanced excitatory synaptic transmission and neuronal hyperactivity in the dorsal horn (central sensitization). As a result of this IL-18-mediated neuromodulation in the spinal cord pain circuit, pain sensitivity is enhanced. Solid lines indicate the pathways and mechanisms demonstrated in this study. Dashed lines indicate other possible pathways and mechanisms.

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