Possible role of spinal astrocytes in maintaining chronic pain sensitization: review of current evidence with focus on bFGF/JNK pathway

Abstract

Although pain is regarded traditionally as neuronally mediated, recent progress shows an important role of spinal glial cells in persistent pain sensitization. Mounting evidence has implicated spinal microglia in the development of chronic pain (e.g. neuropathic pain after peripheral nerve injury). Less is known about the role of astrocytes in pain regulation. However, astrocytes have very close contact with synapses and maintain homeostasis in the extracellular environment. In this review, we provide evidence to support a role of spinal astrocytes in maintaining chronic pain. In particular, c-Jun N-terminal kinase (JNK) is activated persistently in spinal astrocytes in a neuropathic pain condition produced by spinal nerve ligation. This activation is required for the maintenance of neuropathic pain because spinal infusion of JNK inhibitors can reverse mechanical allodynia, a major symptom of neuropathic pain. Further study reveals that JNK is activated strongly in astrocytes by basic fibroblast growth factor (bFGF), an astroglial activator. Intrathecal infusion of bFGF also produces persistent mechanical allodynia. After peripheral nerve injury, bFGF might be produced by primary sensory neurons and spinal astrocytes because nerve injury produces robust bFGF upregulation in both cell types. Therefore, the bFGF/JNK pathway is an important signalling pathway in spinal astrocytes for chronic pain sensitization. Investigation of signaling mechanisms in spinal astrocytes will identify new molecular targets for the management of chronic pain.

Fig. 1. Spinal infusion of astroglial toxin alpha-aminoadipate (L-α-AA) blocks neuropathic pain

L-α-AA (10, 50, 150 nmol) was injected intrathecally 10 days after SNL. Mechanical allodynia, a major feature of chronic pain, is dose-dependently suppressed by the toxin. *, P<0.05; **, P<0.01, compared to vehicle (saline) control; ANOVA; n=6. Mechanical allodynia was tested using von Frey hairs. Reproduced, with permission, from (Zhuang et al., 2006a).

Fig. 2. SNL induces persistent JNK activation in spinal astroglia

(a,b) Immunohistochemistry reveals an increase in pJNK in the ipsilateral spinal dorsal horn (L5) 10 days after SNL. White lines indicate the border of the dorsal horn. Scale, 50 μm. (c,d) High-magnification images of (a) and (b), respectively, showing pJNK staining in the medial superficial dorsal horn. Scale, 50 μm. (e) Double immunofluorescence shows that pJNK (red) colocalizes with the astroglial marker GFAP (green) in the medial superficial dorsal horn. Two single-stained images are merged. c, d, e have the same magnification. (f) High-magnification image of (e) demonstrates colocalization of pJNK and GFAP. Note that some fine processes of astrocytes are labeled by pJNK but not by GFAP antibody. Scale bar, 25 μm. Reproduced, with permission, from (Zhuang et al., 2006a).

Fig. 3. Spinal infusion of the JNK inhibitors reverses neuropathic pain after SNL

(a) Reversal of SNL-induced mechanical allodynia by intrathecal infusion of the peptide inhibitor of JNK D-JNKI-1 (50 μM) via an osmotic pump (0.5 μl hr⁻¹ for 3 days) starting 10 days after SNL. *, P<0.05, compared to corresponding saline controls; t-test; n=5. (b) Reversal of SNL-induced mechanical allodynia by a bolus intrathecal injection of D-JNKI-1 (4 nmol) and SP600125 (50 nmol) 10 days after SNL. *, P<0.05; **, P<0.01, compared to corresponding pre-injection baseline; ANOVA; n=6. Note that the peptide inhibitor D-JNKI-1 is more potent than the small molecule inhibitor SP600125 in reversing allodynia. Reproduced, with permission, from (Zhuang et al., 2006a).

Fig. 4. Nerve injury induces expression of bFGF mRNA in the dorsal root ganglion (DRG)

(a,b) Dark-field images following in situ hybridization show expression of bFGF mRNA in the control, noninjured DRG (a) and 3-day axotomized DRG (b). There are much fewer bFGF-positive cells in the control DRG than in the DRG after nerve injury. Scale bar, 100 μm. (c) Bright-field image folowing in situ hybridization shows expression of bFGF mRNA in the injured DRG neurons. The section is counterstained with toluidine blue. Small, medium and large arrowheads indicate small, medium and large neurons, respectively. bFGF-positive cells are labeled with silver grains. The oligodeoxynucleotide probe for bFGF mRNA is labeled with ³⁵S-dATP. Scale bar, 25 μm. Reproduced, with permission, from (Ji et al., 1995).

Fig. 5. Spinal infusion of bFGF induces delayed but persistent mechanical allodynia

Either bFGF or saline was infused intrathecally via an osmotic pump (10 ng μl⁻¹ h⁻¹) for one week. **, P<0.01 compared to saline control; unpaired t-test; n=6. Mechanical allodynia does not develop until day 4 and is maintained on day 8.

Fig. 6. Activation of JNK by bFGF in astrocytes

(a) Upper panel: Western blotting in the upper panel reveals an increase in pJNK1 levels in the spinal dorsal horn after bFGF infusion intrathecally via an osmotic pump (10 ng μl⁻¹h⁻¹) for one week. The spinal tissues were collected on day 8 after final behavioral testing (see Fig. 5). Note that only pJNK1 is expressed in the spinal cord. Lower panel: total concentration of JNK does not change after bFGF infusion. (b) Density of pJNK1 bands in (a), normalized to JNK1 loading control. **, P<0.01 compared to saline control. (c) Western blotting shows that bFGF induces pJNK1, pERK1 and pERK2 in astrocytic cultures. By contrast, plasminogen (PMG) and ciliary neurotrophic factor (CNTF) do not activate JNK. p-p38 levels are barely detected in astroglial cultures following all the reagents. Astroglial cultures were prepared from brains of neonatal rats and maintained for 2–3 weeks. Cultures were stimulated with bFGF, PMG and CNTF (each at 100 ng ml⁻¹) for 2 hours.