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, 155 (3), 948-58

Inflammatory Pain-Induced Signaling Events Following a Conditional Deletion of the N-methyl-D-aspartate Receptor in Spinal Cord Dorsal Horn

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Inflammatory Pain-Induced Signaling Events Following a Conditional Deletion of the N-methyl-D-aspartate Receptor in Spinal Cord Dorsal Horn

H T Cheng et al. Neuroscience.

Abstract

The N-methyl-d-aspartate (NMDA) receptor in the spinal cord dorsal horn (SCDH) is one of the mechanisms involved in central sensitization during chronic pain. Previously, this laboratory created a spatio-temporal knockout (KO) of the N-methyl-d-aspartate receptor I (NR1) subunit in the mouse SCDH. The NR1 KO completely blocks NR1 gene and subsequent NMDA receptor expression and function in SCDH neurons. In the NR1 KO mice, the mechanical and cold allodynia induced at 24 h after complete Freund's adjuvant (CFA) was reduced. However, the protective effects of KO were transient and were not seen at 48 h after CFA. These observations suggest the presence of NMDA-independent pathways that contribute to CFA-induced pain. CFA induces the activation of several signaling cascades in the SCDH, including protein kinase C (PKC)gamma and extracellular signal-regulated kinases (ERK1/2). The phosphorylation of PKCgamma and ERK1/2 was inhibited in the SCDH of NR1 KO mice up to 48 h after CFA treatment, suggesting that these pathways are NMDA receptor-dependent. Interestingly, neuronal cyclooxygenase (COX) -2 expression and microglial p38 phosphorylation were induced in the SCDH of the NR1 KO at 48 h after CFA. Our findings provide evidence that inflammatory reactions are responsible for the recurrence of pain after NR1 KO in the SCDH.

Figures

Figure 1
Figure 1
IPI of rAAV-GFP-Cre into the SCDH of a floxed NR1 mouse results in viral transduction, Cre-mediated recombination, and a spatiotemporal knock-out of the NR1 gene. (A) On the side ipsilateral to the injection of rAAV-GFP-Cre, viral transduction results in the expression of GFP immunoreactivity in the SCDH (circled area). (B) Decreased NR1 gene expression as measured by in situ hybridization in the circled area of SCDH in an adjacent section, demonstrating successful NR1 KO. Bar = 250 μm.
Figure 2
Figure 2
Mechanical allodynia (A) and cold allodynia (B) resulting from the intraplantar injection of CFA are significantly attenuated at 24 h but not 48 h after CFA treatment in mice with a spatial KO of NR1 in the SCDH (Cre). (A) Mechanical allodynia was measured as a reduction in the baseline mechanical threshold (50% gm threshold) using von Frey hairs applied to the CFA treated paw while (B) Cold allodynia was measured as an increase in the number of responses after applying a drop of acetone to the CFA treated paw. Measurements were made before (baseline) and 24 and 48 hours after intraplantar CFA. Baseline values were not altered in GFP (n = 8) or Cre (n = 13) mice when measured before and after ipsilateral viral vector injection into the SCDH (data not shown). Data are the mean ± SEM (*p< 0.05).
Figure 3
Figure 3
CFA-induced activation of PKCγ and its phosphorylation in the SCDH are attenuated in NR1 KO (Cre) mice at 1h and 48 h after CFA treatments. Confocal images of lumbar SCDH sections show the expression of PKCγ (red) and NeuN (green) 10 min after intraplantar saline (A) or CFA (B) Bar = 50 μm. PKCγ expression was detected in the inner layer of lamina II (arrowheads) while neuronal PKCγ expression is show as yellow labeling (arrows). (C) Immunoblots of lumbar SCDH samples at 10 min after the injection of intraplantar saline or CFA into the right (R) paw of NR1 floxed mice show increased expression of pPKCγ and PKCγ ipsilateral to the CFA injected paw. (D) Immunoblots of samples of the right lumbar SCDH collected at 10 min after the injection of intraplantar saline (S) or CFA into the right paw of GFP or Cre mice show decreased expression of pPKCγ and PKCγ in the SCDH of Cre mice. Actin immunoblots serve as loading controls. (E) Densitometric analysis of pPKCγ immunoblots. The data were normalized to saline treated GFP mice. The CFA induced expression of pPKCγ in lumbar SCDH is significantly reduced at 10 min and 48 h after intraplantar CFA in Cre compared to GFP mice. Data are the mean ± SEM (n = 3) (* p< 0.05).
Figure 4
Figure 4
CFA-induced activation of phospho (p) ERK1/2 in the SCDH is attenuated in NR1 KO (Cre) mice. (A) The time course of ERK1/2 and pERK1/2 after intraplantar CFA. Immunoblots of pERK1/2 in samples of the right lumbar SCDH of NR1 floxed mice before and at 10 min, 1 h, and 24 h after no treatment (NT) or intraplantar CFA. (B) Densitometric analysis of the immunoblots for pERK1/2 after treatments as described in (A). Data are normalized to the band intensity of total ERK1/2 and presented as the fold change over the untreated control (NT). (C) Immunoblots of pERK1/2 and ERK1/2 in samples of the right lumbar SCDH at 1 h after the injection of intraplantar saline (S) or CFA into the right paw of GFP or Cre mice. (D) Densitometric analysis of the immunoblots for pERK1/2 at 1 h after treatment as described in (C) shows the attenuation of CFA-induced pERK1/2 activation in Cre compared to GFP mice. Data are normalized to the band intensity of total ERK1/2 and presented as the fold change over the saline treated control. Data are the mean ± SEM (n = 3) (* p< 0.05).
Figure 5
Figure 5
CFA-induced activation of phospho (p) ERK1/2 is attenuated in pERK1/2 labeled neurons in the SCDH of NR1 KO (Cre) mice 10 min and 48 h after CFA treatments. Immunolabeling of pERK1/2 in the right lumbar SCDH of GFP (A) and Cre (B) mice at 10 min after intraplantar CFA. pERK1/2 labeled neurons (arrows) were detected in the superficial layers of the SCDH in Control (A) but not Cre (B) mice, Bar = 50 μm. Representative images from n = 4 animals per group. (C) The number of CFA–induced pERK1/2 labeled neurons is significantly reduced in Cre compared to GFP mice after 10 min and 48 h after CFA treatments. Data are the mean ± SEM (* p< 0.05).
Figure 6
Figure 6
COX-2 expression is upregulated in the SCDH of NR1 KO (Cre) mice. Sections of the right lumbar SCDH were obtained from Cre mice at 48 h after the intraplantar injection of saline (A, Cre/saline) or CFA (B, Cre/CFA) and labeled for COX-2. Saline treated Cre mice show COX-2 expression in macrophages (arrows) (A), while CFA treated Cre mice show COX-2 expression in both macrophages (arrows) and neurons (arrowheads) (B), Bar = 50 μm. The neuronal expression of COX-2 in CFA-treated Cre mice (C, arrowheads) was confirmed by double labeling with anti-NSE (D, arrowheads). E: Immunoblots demonstrate an enhanced expression of COX-2 in Cre compared to GFP mice 48 h after CFA treatment as described above. F: Densitometric analysis of COX-2 immunoblots demonstrates increased COX-2 expression in Cre mice 48 h after CFA treatment. The highest level of COX-2 expression was seen in Cre mice after CFA injection. Data are the mean ± SEM (n = 3) (* p< 0.05).
Figure 7
Figure 7
Phospho(p)p38 is activated by CFA in the SCDH of NR1 KO (Cre) mice. (A) Immunoblots of pp38 and p38 in samples of the right lumbar SCDH at 48 h after the injection of intraplantar saline (S) or CFA into the right paws of GFP or Cre mice. (B) Densitometric analysis of the immunoblots shows that increased expression of pp38 was only seen in CFA-treated Cre mice. (C) The expression of GFP in the SCDH of a Cre mouse. (D) pp38 labeling of an adjacent section shows pp38 expression in the nuclei of microglia (arrows). (E) OX42 labels the cell membrane and processes of microglia on an adjacent section (arrows). (E) Confocal microscopy shows labeling of pp38 (red) and OX42 (green) and demonstrates that pp38 was activated in microglial nuclei (arrowheads). In contrast, OX42 labeled the cell processes (arrows). C, D, E, bar = 50 μm F, bar = 15 μm. Representative images from n = 4 animals per group.

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