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, 14 (6), 638-47

Protein Kinase B/Akt Is Required for Complete Freund's Adjuvant-Induced Upregulation of Nav1.7 and Nav1.8 in Primary Sensory Neurons

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Protein Kinase B/Akt Is Required for Complete Freund's Adjuvant-Induced Upregulation of Nav1.7 and Nav1.8 in Primary Sensory Neurons

Lingli Liang et al. J Pain.

Abstract

Voltage-gated sodium channels (Nav) are essential for the generation and conduction of action potentials. Peripheral inflammation increases the expression of Nav1.7 and Nav1.8 in dorsal root ganglion (DRG) neurons, suggesting that they participate in the induction and maintenance of chronic inflammatory pain. However, how Nav1.7 and Nav1.8 are regulated in the DRG under inflammatory pain conditions remains unclear. Using a complete Freund's adjuvant (CFA)-induced chronic inflammatory pain model and Western blot analysis, we found that phosphorylated Akt (p-Akt) was significantly increased in the ipsilateral L4/5 DRGs of rats on days 3 and 7 after intraplantar CFA injection. Immunohistochemistry showed that the percentage of p-Akt-positive neurons in the DRG was also significantly increased in the ipsilateral L4/5 DRGs at these time points. Moreover, CFA injection increased the colocalization of p-Akt with Nav1.7 and Nav1.8 in L4/5 DRG neurons. Pretreatment of rats with an intrathecal injection of Akt inhibitor IV blocked CFA-induced thermal hyperalgesia and CFA-induced increases in Nav1.7 and Nav1.8 in the L4/5 DRGs on day 7 after CFA injection. Our findings suggest that the Akt pathway participates in inflammation-induced upregulation of Nav1.7 and Nav1.8 expression in DRG neurons. This participation might contribute to the maintenance of chronic inflammatory pain.

Perspective: This article presents that inhibition of Akt blocks CFA-induced thermal hyperalgesia and CFA-induced increases in dorsal root ganglion Nav1.7 and Nav1.8. These findings have potential implications for use of Akt inhibitors to prevent and/or treat persistent inflammatory pain.

Conflict of interest statement

Disclosures

The authors do not have any conflicts of interest.

Figures

Figure 1
Figure 1
CFA-induced thermal hypersensitivity and Akt activation in L4 and L5 DRGs. (A) Thermal hypersensitivity was demonstrated by significant decreases in paw withdrawal latency (PWL) on the side ipsilateral (Ipsi) to intraplantar CFA injection. **P < 0.01 vs the corresponding baseline (0 h). n = 5–6/group; Contra, contralateral. (B) The level of p-Akt, but not total Akt, was significantly increased in the ipsilateral L4/5 DRGs on days 3 and 7 after CFA injection. The upper panels depict representative Western blots. The lower panel shows the statistical summary of the densitometric analysis expressed relative to basal level (0 h) after normalization to the corresponding β-actin. *P < 0.05 vs the corresponding basal level. n = 3/group. (C) The expression of p-Akt did not change significantly in the contralateral DRGs after intraplantar CFA injection at any time point examined. The upper panels depict representative Western blots. The lower panel shows the statistical summary of the densitometric analysis expressed relative to basal level after normalization to the corresponding β-actin. n = 3/group.
Figure 2
Figure 2
Effect of intraplantar CFA injection on number of p-Akt–labeled neurons in L4 and L5 DRGs. (A) Co-localization of p-Akt with NeuN (a neuronal marker) in L5 DRG. Scale bar: 50 μm. (B) Representative images depict p-Akt–labeled neurons in the ipsilateral L4 and L5 DRGs on days 3 and 7 after saline or CFA injection. Scale bar: 50 μm. (C) Statistical analysis shows that the percentage of p-Akt–labeled neurons was significantly greater in the ipsilateral L4 and L5 DRGs of CFA-injected rats than in those of saline-injected rats on days 3 and 7 after injection. ** P < 0.01 vs the corresponding saline-treated group. n = 3/group.
Figure 3
Figure 3
Effects of intrathecal Akt inhibitor on CFA-induced thermal hypersensitivity. Intrathecal treatment with Akt inhibitor IV (Akt IV) significantly attenuated the CFA-induced decrease in paw withdrawal latency (PWL) on day 7 after CFA injection. Intrathecal injection of Akt IV in the absence of CFA injection had no effect on basal PWL. **P < 0.01 vs the group injected with saline and treated with vehicle (DMSO). #P < 0.05 vs the group injected with CFA and treated with vehicle. n = 5–6/group.
Figure 4
Figure 4
Effects of intrathecal Akt inhibitor on the CFA-induced increase in Nav1.7 expression in the ipsilateral L4/5 DRGs on day 7 after CFA injection. (A) The amount of Nav1.7 was time-dependently increased in the ipsilateral L4/5 DRGs after CFA injection. The upper panels depict representative Western blots. The lower panel shows the statistical summary of the densitometric analysis expressed relative to basal level (0 h) after normalization to the corresponding β-actin. *P < 0.05, **P < 0.01 vs the corresponding basal level. n = 3/group. (B) Colocalization of Nav1.7 with NeuN in L5 DRG. Scale bar: 50 μm. (C) Representative images depicting Nav1.7-labeled neurons in the ipsilateral L5 DRG on day 7 after intraplantar injection with saline (S) or CFA. Rats were treated with intrathecal injections of vehicle (V) or Akt inhibitor IV (AI). Scale bar: 50 μm. (D) Statistical summary showing the percentages of Nav1.7-labeled neurons in the ipsilateral L4 and L5 DRGs of each group. **P < 0.01 vs the group injected with saline and treated with vehicle. #P < 0.05 vs the group injected with CFA and treated with vehicle. n = 3/group. (E) Intrathecal Akt inhibitor IV significantly blocked the CFA-induced increase in Nav1.7 expression in the ipsilateral L4/5 DRGs on day 7 after CFA injection. The upper panels depict representative Western blots. The lower panel shows the statistical summary of the densitometric analysis expressed relative to the group injected with saline and treated with vehicle after normalization to the corresponding β-actin. *P < 0.05 vs the group injected with saline and treated with vehicle. #P < 0.05 vs the group injected with CFA and treated with vehicle. n = 3/group.
Figure 5
Figure 5
Effects of intrathecal Akt inhibitor on the CFA-induced increase in Nav1.8 expression in the ipsilateral L4 and L5 DRGs on day 7 after CFA injection. (A) The level of Nav1.8 was time-dependently increased in the ipsilateral L4/5 DRGs after CFA injection. The upper panels depict representative Western blots. The lower panel shows the statistical summary of the densitometric analysis expressed relative to basal level (0 h) after normalization to the corresponding β-actin. **P < 0.01 vs the corresponding basal level. n = 3/group. (B) Colocalization of Nav1.8 with NeuN in L5 DRG. Scale bar: 50 μm. (C) Representative images depicting Nav1.8-labeled neurons in the ipsilateral L5 DRG on day 7 after intraplantar injection with saline (S) or CFA. Rats were treated with intrathecal injections of vehicle (V) or Akt inhibitor IV (AI). Scale bar: 50 μm. (D) Statistical summary showing the percentages of Nav1.8-labeled neurons in the ipsilateral L4 and L5 DRGs of each group. *P < 0.01 vs the group injected with saline and treated with vehicle. #P < 0.05 vs the group injected with CFA and treated with vehicle. n = 3/group. (E) Intrathecal Akt inhibitor IV significantly blocked the CFA-induced increase in Nav1.8 expression in the ipsilateral L4/5 DRGs on day 7 after CFA injection. The upper panels depict representative Western blots. The lower panel shows the statistical summary of the densitometric analysis expressed relative to the group injected with saline and treated with vehicle after normalization to the corresponding β-actin. *P < 0.05 vs the group injected with saline and treated with vehicle. #P < 0.05 vs the group injected with CFA and treated with vehicle. n = 3/group.
Figure 6
Figure 6
Colocalization of p-Akt with Nav1.7 and Nav1.8 in L4 and L5 DRGs. (A) The left panels depict representative labeling for p-Akt and Nav1.7 and their merged images in the ipsilateral L5 DRG on day 7 after saline or CFA injection. The right panel shows the statistical summary of the percentage of neurons that double labeled for p-Akt and Nav1.7 on day 7 after saline or CFA injection. n = 3/group. Scale bar: 50 μm. (B) The left panels depict representative labeling for p-Akt and Nav1.8 and their merged images in the ipsilateral L5 DRG on day 7 after saline or CFA injection. The right panel shows the statistical summary of the percentage of neurons that double labeled for p-Akt and Nav1.8 on day 7 after saline or CFA injection. *P < 0.05 vs the corresponding saline-treated group. n = 3/group. Scale bar: 50 μm.

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