The c-Jun N-terminal kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition

Abstract

Peripheral inflammation induces persistent central sensitization characterized by mechanical allodynia and heat hyperalgesia that are mediated by distinct mechanisms. Compared to well-demonstrated mechanisms of heat hyperalgesia, mechanisms underlying the development of mechanical allodynia and contralateral pain are incompletely known. In this study, we investigated the distinct role of spinal JNK in heat hyperalgesia, mechanical allodynia, and contralateral pain in an inflammatory pain model. Intraplantar injection of complete Freund's adjuvant (CFA) induced bilateral mechanical allodynia but unilateral heat hyperalgesia. CFA also induced a bilateral activation (phosphorylation) of JNK in the spinal cord, and the phospho JNK1 (pJNK1) levels were much higher than that of pJNK2. Notably, both pJNK and JNK1 were expressed in GFAP-positive astrocytes. Intrathecal infusion of a selective peptide inhibitor of JNK, D-JNKI-1, starting before inflammation via an osmotic pump, reduced CFA-induced mechanical allodynia in the maintenance phase but had no effect on CFA-induced heat hyperalgesia. A bolus intrathecal injection of D-JNKI-1 or SP600126, a small molecule inhibitor of JNK also reversed mechanical allodynia bilaterally. In contrast, peripheral (intraplantar) administration of D-JNKI-1 reduced the induction of CFA-induced heat hyperalgesia but did not change mechanical allodynia. Finally, CFA-induced bilateral mechanical allodynia was attenuated in mice lacking JNK1 but not JNK2. Taken together, our data suggest that spinal JNK, in particular JNK1 plays an important role in the maintenance of persistent inflammatory pain. Our findings also reveal a unique role of JNK1 and astrocyte network in regulating tactile allodynia and contralateral pain.

Figure 1

Time course of CFA-induced mechanical allodynia (A) and heat hypealgesia (B) in rats. CFA produces rapid (<1 hour) and persistent (>2 weeks) mechanical allodynia in both ipsilateral and contralateral paws (A). CFA only induces ipsilateral heat hyperalgesia, which largely recovers after 2 weeks (B). *, P<0.05; **, P<0.01, compared to pre-CFA baseline, n = 6.

Figure 2

Western blot analysis showing time course of CFA-induced expression of pJNK1, pJNK2, JNK1, and JNK2 in the spinal cord of rats. JNK1 and JNK2 are rapidly activated and maintained for >14 days in both ipsilateral (A) and contralateral (B) lumbar spinal cord. Low panels in A and B show densities of pJNK1 and pJNK2 bands after being normalized to tubulin. Total levels of JNK1 and JNK2 do not change after inflammation (C). Low panels in C shows densities of JNK1 and JNK2 against GAPDH. *, P<0.05; compared to naive animals, n = 4–6.

Figure 3

Immunohistochemistry showing pJNK and JNK1 expression in spinal cord astrocytes of rats after CFA injection. (A, B) CFA increases expression of pJNK in the spinal cord 3 days after inflammation. (C) Colocalization of pJNK and GFAP (marker for astroglia) 3 days after CFA injection. (D–F) Double staining shows a colocalization of JNK1 (D) and GFAP (E) in the superficial spinal cord (laminae I–III) 3 days after CFA injection. Scale bars, 50 μm.

Figure 4

Intrathecal infusion of D-JNKI-1 via an osmotic pump reduces CFA-induced mechanical allodynia in the maintenance phase in rats. (A) Structural organization of the D-JNKI-1 peptide. The D-JNKI-1 (30 amino acids, aa) is a chimeric peptide starting with a 10-aa TAT transporter sequence, followed by a 20-aa minimal JBD (JNK binding domain) sequence from the JIP1 molecule. (B) Pretreatment of D-JNKI-1, starting 2 days before inflammation, inhibits CFA-induced mechanical allodynia in the maintenance phase (day 1 to day 4) but not in the induction phase (6 h). (C) Pretreatment of D-JNKI-1 fails to alter CFA-induced heat hyperalgesia. D-JNKI-1 (100 μM) or vehicle (PBS) infusion (1 μl/h, 1 week duration) started 2 days before CFA injection and ended 5 days after CFA injection. *, P<0.05, compared to vehicle control, n = 5.

Figure 5

Reversal of CFA-induced mechanical allodynia in rats by intrathecal D-JNKI-1 and SP600125. (A–C) A bolus injection of D-JNKI-1, given 3 days after CFA injection, dose-dependently reduces mechanical allodynia on the ipsilateral (A) and contralateral (B) paws. Notice that the anti-allodynic effects of D-JNKI-1 is more robust in the contralateral paws. D-JNKI-1 only reduces CFA-induced unilateral heat hyperalgesia at the highest dose (C). *, P<0.05; **, P<0.01; compared to corresponding saline control, n = 6–9. (D–F) A bolus injection of SP600125 (20 nmol), given 3 days after CFA injection, reduces ipsilateral (D) and contralateral (E) mechanical allodynia but not unilateral heat hyperalgesia (F). *, P<0.05, compared to corresponding vehicle (10% DMSO) control, n = 5–6. (G) Western blotting showing a reduction of spinal p-c-Jun levels, 3 h after SP600125 treatment. Right panel shows the density of p-c-Jun band after being normalized to GAPDH. *, P<0.05, n = 6.

Figure 6

Peripheral (intraplantar) inhibition of JNK in rats reduces CFA-induced heat hyperalgesia in the induction phase. (A, B) Pretreatment of D-JNKI-1 (10 nmol), 30 minutes before CFA injection, attenuates CFA-induced heat hyperalgesia (A) but not mechanical allodynia (B). (C, D) Posttreatment of intraplantar D-JNKI-1, 3 days after CFA, fails to reverse established heat hyperalgesia (C) or mechanical allodynia (D). *, P<0.05, compared to saline control, n = 5.

Figure 7

Western blotting showing time course of CFA-induced pJNK1 and pJNK2 expression in the spinal cord of mice. (A) CFA induced persistent increase (> 4 weeks) of JNK1 and JNK2 in both ipsilateral and contralateral spinal cord. (B, C) Densities of pJNK1 (B) and pJNK2 (C) in the spinal cord after being normalized to GAPDH. *, P<0.05, compared to naïve animals, n = 4–6.

Figure 8

CFA-induced mechanical allodynia is reduced in mice lacking JNK1. CFA produced rapid and persistent bilateral mechanical allodynia (A, B) and ipsilateral heat hyperalgesia (C, D) in wild-type mice. But in JNK1^−/− mice, mechanical allodynia is attenuated bilaterally, especially in the maintenance phase (A, B). CFA-induced heat hyperalgeisa is comparable in wild-type and JNK1^−/− mice (C, D). *, P<0.05; **, P<0.01; compared to wild type mice at the corresponding time points. Notice that the baseline pain sensitivity is normal in the knockout mice. n = 7–8.

Figure 9

CFA-induced inflammatory pain is normal in mice lacking JNK2. CFA-induced mechanical allodynia (A, B) and heat hyperalgesia (C, D) are comparable between JNK2^−/− and wild-type mice in all the time points examined. n = 5–6.