p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain

Abstract

The possible involvement of p38 mitogen-activated protein kinase activation in spinal cord and dorsal root ganglion (DRG) cells in the development of peripheral neuropathic pain has been explored. Ligation of the L5 spinal nerve (SNL) on one side in adult rats produces an early onset and long-lasting mechanical allodynia. This lesion results in activation of p38 in the L5 segment of the spinal cord, most prominently in the ipsilateral dorsal horn, starting soon after the lesion (<1 d) and persisting for >3 weeks. The activated p38 in the spinal cord is restricted entirely to microglia; phospho-p38 colocalizes only with the microglial marker OX-42 and not with either the neuronal marker neuronal-specific nuclear protein or the astrocyte marker GFAP. In contrast, SNL induces a delayed (>3 d) activation of p38 in the L5 DRG that occurs predominantly in neurons. Continuous injection of the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) via the intrathecal route, starting before the SNL surgery, reduces SNL-induced mechanical allodynia from day 1 to day 10, with maximal effects at early time points. Post-treatment with SB203580 starting on day 1 or on day 10 after surgery also reduces established mechanical allodynia. Because the reduction in neuropathic pain by p38 inhibition occurs before the appearance of p38 activation in DRG neurons, p38 activation in spinal cord microglia is likely to have a substantial role in the earliest phase of neuropathic pain. Coactivation of p38 in DRG neurons and spinal microglia may contribute to later phases of neuropathic pain.

Figure 1.

a–g, SNL induces p38 activation in the dorsal horn. a, b, Western blot analysis reveals rapid and persistent p38 activation in the L5 dorsal horn after L5 SNL. b, Quantification of a. p-p38 level is normalized against total p38 level. *p < 0.05 and **p < 0.01 versus control; ANOVA (n = 4 or 5). c, d, p-p38 immunostaining in the dorsal horn of naive control rats (c) and sham control rats (d) at day 3. e–g, Immunohistochemistry indicates a gradual increase in the number of p-p38 immunoreactive cells in the ipsilateral spinal cord after SNL. Scale bars: f, g, 200 μm. Cont, Control.

Figure 2.

a–i, SNL induces p38 activation in the spinal microglia. a–c, Double immunofluorescence in the dorsal horn for p-p38 (red) and NeuN, a neuronal marker (green; a); GFAP, an astrocyte marker (green; b); and OX-42, a microglia marker (green; c). Two images are merged in a–c. a and b were obtained from ipsilateral dorsal horn 3 d after SNL, and c was obtained from the contralateral dorsal horn as a control. d–f, Double immunofluorescence indicates a complete colocalization (f) between p-p38 (d) and OX-42 (e) in the dorsal horn 3 d after SNL. f, Overlay of d and e. g–i, OX-42 immunostaining in the spinal cord. h, i, High-magnification images showing the transformation of microglia from ramified shape to amoeboid shape 3 d after SNL. Scale bars: (in f) a–f,20μm; g, 200 μm; (in h) h, i, 20 μm. Cont, Control.

Figure 3.

a–e, SNL induces p38 activation in DRG neurons. a–c, Immunohistochemistry shows p-p38 staining in the L5 DRG of naive control rats (a) and SNL rats at 3 d (b). c, Quantification of p-p38 levels in the L5 DRG. **p < 0.01 and ***p < 0.001 (ANOVA) versus naive control; n = 3. d, e, Double immunofluorescence shows colocalization of p-p38 with NF-200 in DRG neurons 3 d after SNL. Arrows indicate small satellite cells. Scale bars: (in b) a, b, 100 μm; (in e) d, e, 50 μm. Cont, Control.

Figure 4.

a–c, p38 activation contributes to neuropathic pain. a, Before treatment, a continuous infusion of the p38 inhibitor SB203580 (SB) partially prevents the development of mechanical allodynia. SB203580 (2 μg) was administered intrathecally twice per day for 2 weeks, with the first injection 30 min before the SNL. The mechanical allodynia was tested in the morning before the daily SB203580 infusion. *p < 0.05; **p < 0.01; ***p < 0.001; Student's t test (compared with corresponding saline control). b, c, After treatment, a single injection of SB203580 (10μg) intrathecally decreases SNL-induced mechanical allodynia at both day 1 (b) and day 10 (c). *p < 0.05 and **p < 0.01 (ANOVA) compared with SNL baseline. n = 7 and 11 for mechanical test at day 1 and 10, respectively.