Tumor necrosis factor-alpha induces mechanical allodynia after spinal nerve ligation by activation of p38 MAPK in primary sensory neurons

Abstract

Tumor necrosis factor-alpha (TNF) is implicated in the initiation of neuropathic pain. In vitro, TNF activates p38 mitogen-activated kinase. Accordingly, we investigated whether TNF activates the p38 cascade in vivo to trigger pain behavior after spinal nerve ligation (SNL). Treatment starting 2 d before SNL with the TNF antagonist etanercept (1 mg, i.p., every third day) attenuated mechanical allodynia. Treatment starting 1 or 7 d after SNL was ineffective. Similarly, intrathecal infusion of a p38 inhibitor (SB203580, 4 mg/d) was effective only if it was started before but not 7 d after SNL. For both treatments, the cessation of therapy resulted in increased allodynia. In separate experiments using Western blots and immunohistochemistry, ipsilateral lumbar spinal cord and L5 and L6 DRG were analyzed for total and phosphorylated p38 after SNL alone or SNL combined with etanercept pretreatment. In DRG, activated p38 was transiently elevated 5 hr after SNL and returned to baseline by 1 d after SNL. Phosphorylated p38 was localized in small TNF-positive DRG neurons. In spinal cord, p38 was activated between 5 hr and 3 d after SNL and returned to baseline within 5 d. In DRG, but not spinal cord, etanercept pretreatment blocked p38 activation. These data indicate that after SNL treatment, phosphorylated p38 levels in spinal cord and DRG are transiently elevated. In DRG, p38 activation is blocked by systemic TNF inhibition. Parallel inhibition of p38 activation and allodynia may represent a clinically relevant therapeutic window. These data suggest a sequential role for TNF and p38 in the induction of neuropathic pain.

Fig. 1.

Mechanical allodynia induced by SNL is attenuated by the systemic inhibition of TNF and the intrathecal inhibition of p38. A, Treatment with the TNF antagonist etanercept (▴) starting 2 d before SNL (d-2) attenuates mechanical allodynia induced by SNL.B, Treatment with etanercept started 1 d after SNL (▴) has no effect on mechanical thresholds, even when the etanercept dose is doubled (dark shaded bar).C, Treatment with etanercept started 7 d after SNL has no effect. D, Treatment with the p38 inhibitor SB203580 (d-2) but not treatment starting 7 d after SNL attenuates mechanical allodynia induced by SNL. *p < 0.05, **p < 0.005, ***p < 0.001 versus vehicle-treated rats;n = 6–12 per treatment group.

Fig. 2.

SNL induces transient p38 activation in DRG neurons and spinal cord. A, Time course of p38 phosphorylation in DRG neurons after SNL. Phospho-p38 (p-p38) is transiently increased 5 hr after SNL.B, Time course of p38 phosphorylation in lumbar spinal cord after SNL; phospho-p38 is increased between 5 hr and 3 d after SNL. *p < 0.05, **p < 0.005 versus control; n = 6 per time point.

Fig. 3.

Phospho-p38 (p-p38) is activated in small, presumably nociceptive DRG neurons and not in activated satellite cells. A–C, p38 phosphorylation is increased in DRG neurons (arrows) 5 hr after SNL.D, Serial staining of phospho-p38 (red) with PGP 9.5 (green), a marker for neuronal cells, in DRG neurons indicates that p38 is predominantly activated in neuronal cells (arrows). E, Serial staining of phospho-p38 (red) with GFAP (green, open arrow), a marker for activated satellite cells, indicates that p38 (arrows) is not phosphorylated in activated satellite cells 5 hr after SNL.F, Double staining demonstrates colocalization (arrows) of phospho-p38 (red) with IB4 (green) in DRG neurons after SNL, suggesting p38 activation in small, presumably nociceptive DRG neurons. Scale bars, 30 μm. n = 4 per time point.

Fig. 4.

TNF induces p38 activation in DRG after SNL.A, Phospho-p38 (p-p38,red) is colocalized with TNF (green) in small DRG neurons after SNL (arrows). Scale bar, 30 μm. B, Systemic anti-TNF treatment with etanercept (Eta, 1 mg/rat, i.p.) suppresses the SNL-induced increase in phospho-p38 in DRG 5 hr after SNL. C, Systemic etanercept does not suppress the increase of phospho-p38 in lumbar spinal cord after SNL. *p < 0.05, **p < 0.005 versus control; n = 6 per group.