Modulation of thalamic nociceptive processing after spinal cord injury through remote activation of thalamic microglia by cysteine cysteine chemokine ligand 21

Abstract

Spinal cord injury (SCI) results in the generation and amplification of pain caused in part by injury-induced changes in neuronal excitability at multiple levels along the sensory neuraxis. We have previously shown that activated microglia, through an ERK (extracellular signal-regulated kinase)-regulated PGE(2) (prostaglandin E(2)) signaling mechanism, maintain neuronal hyperexcitability in the lumbar dorsal horn. Here, we examined whether microglial cells in the thalamus contribute to the modulation of chronic pain after SCI, and whether microglial activation is governed by spinally mediated increases in the microglial activator cysteine-cysteine chemokine ligand 21 (CCL21). We report that CCL21 is upregulated in dorsal horn neurons, that tissue levels are increased in the dorsal horn and ventral posterolateral (VPL) nucleus of the thalamus 4 weeks after SCI, and that the increase can be differentially reduced by spinal blockade at T1 or L1. In intact animals, electrical stimulation of the spinothalamic tract induces increases in thalamic CCL21 levels. Recombinant CCL21 injected into the VPL of intact animals transiently activates microglia and induces pain-related behaviors, effects that could be blocked with minocycline. After SCI, intra-VPL antibody-mediated neutralization of CCL21 decreases microglial activation and evoked hyperexcitability of VPL neurons, and restores nociceptive thresholds to near-normal levels. These data identify a novel pathway by which SCI triggers upregulation of the neuroimmune modulator CCL21 in the thalamus, which induces microglial activation in association with pain phenomena.

Figure 1.

Progressive microglial activation in the VPL after SCI and pain behaviors. b, C11b/c (OX-42) immunostaining revealed the presence of microglia in the VPL that exhibit the resting morphotype. Approximate positions of relevant anatomical structures are shown in an atlas overlay (Paxinos and Watson, 1998): M1, motor cortex; Hc, hippocampus; Po, posterior thalamic nucleus; ic, internal capsule. c–f, One to 4 weeks after T9 SCI, microglia progressively demonstrated the activated morphotype. g, Quantification of percentage field area for Cd11b/c revealed significant (*p < 0.05) microglial activation starting at 2 weeks after injury. Error bars indicate SD. h, Correlation analysis at 4 weeks after SCI showed a strong relationship between Cd11b/c field area and mechanical nociceptive thresholds for individual animals. Plot regression line and key values are shown.

Figure 2.

CCL21 upregulation in lumbar dorsal horn neurons after SCI. a, In sham-operated animals, CCL21 signal was low within lumbar dorsal horn neurons. b, In tissue collected 4 weeks after SCI, CCL21 signal was increased in dorsal horn neurons within laminae I–V (Paxinos and Watson, 1998). c, CCL21 signal was eliminated with preabsorption antibody treatment after SCI. d, e, After SCI, CCL21 signal colocalized with NeuN, a marker of neurons (d), but only in a subset of cells (arrows) (e). f, g, CCL21 also colocalized with glutamate. h, Enzyme immunoassay revealed significantly elevated dorsal horn tissue levels of CCL21 in the T9 (*p < 0.05) and L4 (⁺p < 0.05) segments 4 weeks after SCI, compared with T9 and L4 segments of sham-operated animals. Error bars indicate SD.

Figure 3.

CCL21 content in the VPL after SCI is reduced by spinal blockade. a, b, In intact animals a, levels of CCL21 signal were low, whereas after SCI (b) levels were higher in parenchyma and neuronal cell bodies. c, e, Spinal cord interruption with 2% lidocaine followed by surgical transection at either T1 or L1 spinal segments (c) resulted in decreases in CCL21 signal after interruption of spinal afferent barrage (d, e). f, Quantification of CCL21 levels in the ventrobasal complex of the thalamus revealed a significant increase after SCI when compared with intact animals (*p < 0.05), which was significantly (⁺p < 0.05) attenuated after rostral (T1) or caudal (L1) spinal blockade. Rostral blockade was significantly more effective than caudal block in reducing CCL21 levels (^@p < 0.05). Error bars indicate SD.

Figure 4.

Unilateral stimulation of the STT elicits increases in thalamic CCL21 in intact animals. a, b, Electrical stimulation of the ventrolateral funiculus (a) resulted in an increase in CCL21 levels within the ventrobasal thalamus on the ipsilateral side, but not the contralateral side [*p < 0.05 ipsilateral (I) vs contralateral (C) sides], measured by enzyme immunoassay (b). The horizontal line indicates CCL21 levels after SCI. The site of stimulation is shown (asterisk). Error bars indicate SD.

Figure 5.

Intra-VPL injection of rmCCL21 elicits microglial activation and pain-related behaviors. a, In intact animals, rmCCL21 was stereotaxically injected into the VPL bilaterally. M1, Motor cortex; Hc, hippocampus. b, In intact animals, Cd11b/c staining identified microglia exhibiting the resting morphology. c–i, In rmCCL21-receiving animals (c–i), microglia demonstrated a transient activation, which peaked at 8 h after injection (f). j, This activation was significant compared with animals receiving vehicle at 4–24 h. k, At the peak of microglial activation (8 h), mechanical and thermal nociceptive thresholds on the hindpaw were evaluated. When compared with vehicle, rmCCL21 produced a strong lowering of mechanical nociceptive thresholds (*p < 0.05). Simultaneous delivery of the microglial inhibitor minocycline partially blocked this reduction in mechanical threshold. Similarly, withdrawal latencies to radiant thermal stimulation were reduced with rmCCL21 (*p < 0.05), whereas coadministration of minocycline attenuated this effect. Error bars indicate SD.

Figure 6.

CCL21 neutralization abrogates microglial activation, neuronal hyperexcitability, and pain-related behaviors. a–c, Microglial activation in the VPL 4 weeks after SCI was unaffected by IgG injection (a), whereas neutralizing antibodies directed against CCL21 (αCCL21; b) robustly deactivated microglia at 8 h (b) and 24 h (c). d, Quantification of Cd11b/c field area revealed significant decreases in microglial activation in animals receiving minocycline (⁺p < 0.05) as well as αCCL21 (*p < 0.05) at both 8 and 24 h after injection. e, Extracellular unit recordings of VPL multireceptive neurons with peripheral receptive fields in the hindpaw from intact animals exhibited low evoked activity in response to PB, PR, and PI stimulation. After SCI, evoked responses were elevated and unaffected by IgG administration at the 8 h time point. f, In contrast, at the 8 h time point, αCCL21 resulted in reduced hyperexcitability of multireceptive neurons in the VPL, which was significantly decreased compared with IgG to all peripheral stimuli. Analysis of mechanical nociceptive thresholds (g) demonstrated the development of mechanical allodynia 4 weeks after SCI in all animals before drug delivery. In animals receiving minocycline (⁺p < 0.05) and αCC21 (*p < 0.05), paw withdrawal thresholds were significantly elevated compared with vehicle and IgG. h, Thermal nociceptive thresholds were also significantly reduced after SCI. Administration of minocycline (⁺p < 0.05) and αCCL21 (*p < 0.05) significantly elevated paw withdrawal latencies compared with vehicle and IgG. Mechanical and thermal thresholds were restored to predrug levels by the 24 h time point, indicating only a transient relief from mechanical allodynia and thermal hyperalgesia. Error bars indicate SD.