Light touch induces ERK activation in superficial dorsal horn neurons after inflammation: involvement of spinal astrocytes and JNK signaling in touch-evoked central sensitization and mechanical allodynia

Abstract

Activation of extracellular signal-regulated kinase (ERK) in spinal cord neurons could serve as a marker for sensitization of dorsal horn neurons in persistent pain. ERK is normally activated by high-threshold noxious stimuli. We investigated how low-threshold mechanical stimuli could activate ERK after complete Freund's adjuvant (CFA)-induced inflammation. Unilateral injection of CFA induced ipsilateral heat hyperalgesia and bilateral mechanical allodynia. CFA-induced ERK activation in ipsilateral dorsal horn neurons declined after 2 days. Interestingly, low-threshold mechanical stimulation given by light touch either on the inflamed paw or the contralateral non-inflamed paw dramatically increased ERK phosphorylation in the dorsal horn ipsilateral to touch stimulation. Notably, light touch induced ERK phosphorylation mainly in superficial neurons in laminae I-IIo. Intrathecal administration of the astroglial toxin L-α-aminoadipate on post-CFA day 2 reversed CFA-induced bilateral mechanical allodynia but not heat hyperalgesia. Furthermore, L-α-aminoadipate, the glial inhibitor fluorocitrate, and a peptide inhibitor of c-Jun N-terminal Kinase all reduced light touch-evoked ERK activation ipsilateral to touch. Collectively, these data suggest that (i) ERK can be activated in superficial dorsal horn neurons by low-threshold mechanical stimulation under pathological condition and (ii) ERK activation by light touch is associated with mechanical allodynia and requires an astrocyte network.

Fig. 1

Light touch on the inflamed paw induces ERK activation 2 days after CFA injection. (A–C) CFA induces heat hyperalgesia (A), mechanical allodynia (B), and edema on the ipsilateral paw (C). ***P<0.001, compared to baseline. (D) pERK was not induced by light touch on non-inflamed naïve paw. (E) Low level of pERK in the ipsilateral dorsal horn on post-CFA day 2. (F) Light touch on the inflamed paw induces dramatic increase of pERK expression in the dorsal horn. (G) Histogram shows the number of pERK-positive cells in the superficial dorsal horn (laminae I–II). (H) Double staining of pERK and PKCγ shows the lamina distribution of pERK in the spinal cord. (I) Histogram shows the number of pERK positive cells in different lamina. ** P<0.01, compared to control; ++ P<0.01, compared to CFA only. Scale bar, 100 μm

Fig. 2

Cellular distribution of light touch-induced pERK. pERK positive cells are colocalized with NeuN (A–C), a marker for neurons, but not with GFAP (E–G), a marker for astrocytes, or Iba1(I–K), a marker for microglia (I). Scale bar, 100 μm. D, H, and L are higher magnification images of C, G, and K, respectively. Scale bar, 50 μm.

Fig. 3

Light touch on the contralateral non-inflamed paw induces ERK expression in the superficial dorsal horn 2 days after CFA injection. (A) CFA does not induce heat hyperalgesia in the contralateral paw. (B) CFA induces contralateral mechanical allodynia. (C) CFA does not elicit edema in the contralateral paw. *P<0.05, compared to baseline. (D) Very few cells express pERK in the contralateral spinal cord on post-CFA day 2. (E) Light touch on the non-inflamed paw induces pERK expression in the superficial dorsal horn. (F) Histogram shows the number of pERK expression in the dorsal horn with or without light touch (LT). Scale bar, 100 μm

Fig. 4

Intrathecal injection of the astroglial toxin L-alpha aminoadipate (L-α-AA) reverses CFA-induced mechanical allodynia but not heat hyperalgesia. (A, B) Intrathecal injection of L-α-AA (50 nmol) on post-CFA day 2 reduces mechanical allodynia on the ipsilateral paw (A) and contralateral paw (B). (C–D) L-α-AA fails to change the paw withdrawal latency on the ipsilateral paw (C) and contralateral paw (D). * P<0.05; ** P<0.01 compared to vehicle control.

Fig. 5

Intrathecal injection of astrocyte toxins and JNK inhibitor decreases ERK activation following light touch on the inflamed paw. Light touch of the inflamed paw induces pERK expression in the superficial dorsal horn (A), which is inhibited by pretreatment with L-α-AA (B), fluorocitrate (FC, C) and D-JNKI-1 (D). (E) Histogram shows that light touch-induced pERK expression is decreased by pretreatment with L-α-AA, fluorocitrate (FC) or D-JNKI-1 given 3 hours before light touch (LT). ** P<0.01; *** P<0.001 compared to light touch group. Scale bar, 100 μm

Fig. 6

Intrathecal injection of astrocyte toxins and JNK inhibitor decreases ERK activation following light touch (LT) on the non-inflamed paw. Light touch on the non-inflamed paw induces pERK expression (A), which is decreased by pretreatment with Lα-AA (B), fluorocitrate (FC, C) and D-JNKI-1 (D). E. Histogram shows the number of pERK-positive cells in the superficial dorsal horn. *** P<0.001 compared to light touch group. Scale bar, 100 μm