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, 28 (52), 14087-96

Activation of the Neuronal Extracellular Signal-Regulated Kinase 2 in the Spinal Cord Dorsal Horn Is Required for Complete Freund's Adjuvant-Induced Pain Hypersensitivity

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Activation of the Neuronal Extracellular Signal-Regulated Kinase 2 in the Spinal Cord Dorsal Horn Is Required for Complete Freund's Adjuvant-Induced Pain Hypersensitivity

Qinghao Xu et al. J Neurosci.

Abstract

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling in the spinal cord dorsal horn (SCDH) has been implicated in injury-induced pain hypersensitivity. Available ERK pathway inhibitors cannot distinguish between ERK1 and ERK2, nor can they differentially target the expression of neuronal or glial ERK1/2. We selectively inhibited the expression of ERK2 in neurons of the adult mouse SCDH by use of an ERK2 small interfering RNA (siRNA) delivered by a neurotropic adenoassociated viral vector. In situ hybridization revealed a siRNA vector-induced decrease in ERK2 mRNA in the ipsilateral SCDH. Immunohistochemistry showed a decreased neuronal phospho-ERK1/2 (pERK1/2), and Western blot analysis revealed that both ERK2 expression and phosphorylation were reduced by the siRNA vector. In contrast, basal ERK1 expression was not affected, although pERK1 was slightly increased. The siRNA vector-induced knockdown of ERK2 expression in the SCDH did not alter the baseline mechanical or thermal paw withdrawal thresholds. Hindpaw intraplantar injection of complete Freund's adjuvant (CFA) produced peripheral inflammation, mechanical allodynia, and thermal hyperalgesia in vector control animals that persisted for at least 96 h. It also caused an increase in SCDH ERK1 and ERK2 levels at 96 h and pERK1 and pERK2 levels at 1 and 96 h. The ERK2 siRNA vector prevented changes in ERK1, ERK2, and pERK2. In addition, the siRNA vector protected the animals from developing mechanical allodynia and thermal hyperalgesia throughout the 96 h after CFA. These findings indicate that ERK2 in the SCDH neurons is critical for the development of inflammatory pain hypersensitivity.

Figures

Figure 1.
Figure 1.
Expression of GFP and the knockdown of ERK2 mRNA in the mouse SCDH at 3 weeks after the IPI of a rAAV vector expressing an ERK2 siRNA. The expression of GFP in the ipsilateral SCDH after administration of a rAAV vector expressing a control siRNA (MM) (A) or an active ERK2 siRNA, 2-5 (B), 2-7 (C), or 2-8 (D). E–H, In situ hybridization revealed no change in ERK2 mRNA level in the ipsilateral SCDH compared with the contralateral side in animals treated with vector MM (E). In contrast, each of the three active siRNA vectors induces a significant decrease in ERK2 mRNA in the ipsilateral SCDH (F–H). Scale bar, 500 μm. I, ERK2 mRNA expression was estimated from in situ images. The ratio of the integrated optical density of in situ labeling in the ipsilateral SCDH to that in the contralateral SCDH was calculated. Data are mean ± SEM (n = 3). Compared with the control vector MM, vector 2-5, 2-7, and 2-8 induced a significant decrease (*p < 0.05) in the ERK2 mRNA expression in the ipsilateral SCDH. There was no difference in the ratio among the three active siRNA vectors.
Figure 2.
Figure 2.
Inhibition of ERK2 expression and phosphorylation in the ipsilateral SCDH of mice treated with vector 2-7, an active ERK2 siRNA. An example of the blot is shown in A, the relative protein levels of ERK2 are shown in B, and ERK1 is shown in C. A, B, Western blot analysis shows a reduction (*p < 0.05 vs the control vector MM group) of ERK2 expression only on the ipsilateral side of the SCDH after vector 2-7 treatment. A, C, ERK1 expression was unaffected by vector 2-7. Western blot analysis (D, E) showed a decrease (*p < 0.05 vs the control vector MM group) in pERK2 and an increase (*p < 0.05 vs the control vector MM group) in pERK1 (D, F) in the ipsilateral SCDH after vector 2-7 treatment. A blot is shown in D, and the relative levels of pERK2 and pERK1 are shown in E and F, respectively. The data are the averages from four separate gel determinations. Error bars indicate SEM. C, Contralateral; I, ipsilateral.
Figure 3.
Figure 3.
The cellular localization of pERK1/2 immunolabeling in the ipsilateral SCDH at 3 weeks after rAAV vector administration. A, pERK1/2 immunolabeling was detected in the ipsilateral SCDH after the control vector, MM (red). B, NeuN labeling revealed typically distributed neuronal morphologies (green). C, Merged image shows that pERK1/2 was strongly colocalized with NeuN (yellow, arrows). D, pERK1/2 immunolabeling was significantly reduced by vector 2-7 compared with vector MM (A). E, The NeuN labeling pattern is similar to that seen with vector MM (B). F, Merged image shows almost no colocalization of pERK1/2 and NeuN labeling after vector 2-7 compared with vector MM (C). Scale bar, 100 μm.
Figure 4.
Figure 4.
Vector 2-7 but not vector MM reduces ERK2 expression in the ipsilateral SCDH and prevents the phosphorylation of ERK2 induced by intraplantar CFA. A, B, Western blot analysis shows a significant reduction in ERK2 in the ipsilateral SCDH after vector 2-7 compared with the control, vector MM in the absence of intraplantar treatment (NT) (*p < 0.05 vs vector MM/NT). This reduction in ERK2 persists at 1 and 96 h after CFA (*p < 0.05 vs vector MM). At 96 h after CFA, ERK2 is increased in the ipsilateral SCDH of the vector MM group NT or 1 h after CFA (#p < 0.05). A, C, ERK1 expression is also increased at 96 h after CFA (#p < 0.05), and this increase is prevented by vector 2-7 treatment. D, E, pERK2 is decreased after vector 2-7 and the reduction persists at 1 and 96 h after CFA (*p < 0.05 vs vector MM). For the vector MM, pERK2 was increased at 1 and 96 h after CFA compared with the NT control (#p < 0.05). D, F, pERK1 was increased after vector 2-7 (*p < 0.05 vs vector MM/NT). This increase in pERK1 was not altered after CFA in the vector 2-7 group. In contrast, pERK1 was increased at 1 and 96 h in the vector MM group (#p < 0.05 vs vector MM/NT). The data are the averages from four separate gel determinations. Error bars indicate SEM.
Figure 5.
Figure 5.
Vector 2-7 prevents the increased phosphorylation of ERK1/2 that is induced in the ipsilateral SCDH by intraplantar CFA. A, The basal level expression of pERK1/2 can be seen mainly in lamina I–II neurons in the control mice after vector MM and in the absence of intraplantar CFA treatment (NT). B, C, pERK1/2 immunolabeling was increased at 1 h (B) and 96 h (C) after CFA. D–F, pERK1/2 immunolabeling in the SCDH of mice treated with vector 2-7 is reduced compared with MM control, before (NT) and after CFA. G, H, Quantification of pERK1/2 as labeled neuron density (G) or percentage of field (H) in laminas I–II revealed a significant increase at 1 and 96 h after CFA in the MM control mice (#p < 0.05 vs vector MM/NT). vector 2-7 reduced pERK1/2 immunolabeling at each corresponding time point compared with vector MM (G, H) (*p < 0.05 vs vector MM). CFA induced an increase in pERK1/2 labeling as measured by neuron density or percentage of field at 1 and 96 h in the vector MM group (G, H) (#p < 0.05). For the vector 2-7 group, CFA produced increases in labeling although less than the corresponding increases in the vector MM group. Scale bar, 500 μm. Error bars indicate SEM.
Figure 6.
Figure 6.
A spatial knockdown of ERK2 in the SCDH by vector 2-7 does not affect thermal (A) or mechanical (B) paw withdrawal thresholds. A brief thermal stimulus was applied to the paw (A), or mechanical (tactile) stimuli were applied using von Frey hairs (B). These thresholds were measured before and 3 weeks after the ipsilateral IPI vector administration into the right SCDH of either the control vector (MM) or the siRNA vector 2-7 (n = 10 per treatment group). Error bars indicate SEM.
Figure 7.
Figure 7.
A spatial knockdown of ERK2 in the SCDH by vector 2-7 significantly reduces thermal hyperalgesia (B) and mechanical allodynia (C) resulting from the intraplantar injection of the inflammatory agent, CFA. A, The CFA resulted in equivalent inflammation as measured by paw size at 24, 48, and 96 h (*p < 0.05 vs baseline; n = 10 per treatment group). The control vector MM group showed thermal hyperalgesia as measured as a reduction in the paw withdrawal threshold using a thermal stimulus (B) and mechanical allodynia as measured as a reduction in the mechanical threshold (50% G threshold) using von Frey hairs (C) applied to the paw at 24, 48, and 96 h after intraplantar CFA compared with the baseline (*p < 0.05 vs baseline). The mice that received vector 2-7 were protected from CFA-induced thermal hyperalgesia (B) and mechanical allodynia (C) (n = 10 per treatment group). Error bars indicate SEM.

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