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Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy.

Thakor DK, Lin A, Matsuka Y, Meyer EM, Ruangsri S, Nishimura I, Spigelman I - Mol Pain (2009)

Bottom Line: Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves.Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves.The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oral Biology and Medicine, School of Dentistry, University of California, Los Angeles, CA 90095-1668, USA. dthakor@ucla.edu

ABSTRACT

Background: Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.

Results: After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.

Conclusion: Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

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Detection of NaV1.8 mRNA in the sciatic nerve. A. NaV1.8 mRNA levels were significantly increased in the sciatic nerve ipsilateral to the injury site as compared to contralateral and uninjured DRG. (n = 3; p < 0.05, one-way ANOVA with Tukey post-hoc). B. NaV1.8 mRNA levels in SNE-injured DRG were not significantly different from those of uninjured DRG (n = 4 for uninjured DRG and n = 3 for all other samples). C: Southern blot of the Nav1.8 3' RACE product from contralateral and ipsilateral sciatic nerve. Note the presence of a distinct band representing the expected 3'RACE product of 830 bp. Sequencing of the RACE product confirmed the presence of the 3' end of the NaV1.8 coding region as well as the NaV1.8 3' un-translated region and polyA tail. Tissue was harvested 2 weeks after SNE.
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Figure 4: Detection of NaV1.8 mRNA in the sciatic nerve. A. NaV1.8 mRNA levels were significantly increased in the sciatic nerve ipsilateral to the injury site as compared to contralateral and uninjured DRG. (n = 3; p < 0.05, one-way ANOVA with Tukey post-hoc). B. NaV1.8 mRNA levels in SNE-injured DRG were not significantly different from those of uninjured DRG (n = 4 for uninjured DRG and n = 3 for all other samples). C: Southern blot of the Nav1.8 3' RACE product from contralateral and ipsilateral sciatic nerve. Note the presence of a distinct band representing the expected 3'RACE product of 830 bp. Sequencing of the RACE product confirmed the presence of the 3' end of the NaV1.8 coding region as well as the NaV1.8 3' un-translated region and polyA tail. Tissue was harvested 2 weeks after SNE.

Mentions: Real-time PCR detected the presence of NaV1.8 mRNA in the sciatic nerve and revealed a significant up-regulation in the ipsilateral nerve as compared to the contralateral and uninjured nerve (p < 0.05, one-way ANOVA with Tukey post-hoc; Fig. 4A). In contrast, ipsilateral, contralateral, and uninjured DRG showed no significant differences in NaV1.8 mRNA levels (Fig. 4B). To confirm that the NaV1.8 PCR signals represent mRNA and not genomic DNA contamination, 3'RACE PCR was performed. Southern blot hybridization analysis showed a positive band at the predicted size of 830 bp in both contralateral and ipsilateral sciatic nerve samples (Fig. 4C). After agarose gel separation and cloning of amplicons, DNA sequencing positively identified the 3' end of the coding region and the 3' untranslated region of rat Nav1.8 as well as an additional poly A tail (data not shown).


Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy.

Thakor DK, Lin A, Matsuka Y, Meyer EM, Ruangsri S, Nishimura I, Spigelman I - Mol Pain (2009)

Detection of NaV1.8 mRNA in the sciatic nerve. A. NaV1.8 mRNA levels were significantly increased in the sciatic nerve ipsilateral to the injury site as compared to contralateral and uninjured DRG. (n = 3; p < 0.05, one-way ANOVA with Tukey post-hoc). B. NaV1.8 mRNA levels in SNE-injured DRG were not significantly different from those of uninjured DRG (n = 4 for uninjured DRG and n = 3 for all other samples). C: Southern blot of the Nav1.8 3' RACE product from contralateral and ipsilateral sciatic nerve. Note the presence of a distinct band representing the expected 3'RACE product of 830 bp. Sequencing of the RACE product confirmed the presence of the 3' end of the NaV1.8 coding region as well as the NaV1.8 3' un-translated region and polyA tail. Tissue was harvested 2 weeks after SNE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2667430&req=5

Figure 4: Detection of NaV1.8 mRNA in the sciatic nerve. A. NaV1.8 mRNA levels were significantly increased in the sciatic nerve ipsilateral to the injury site as compared to contralateral and uninjured DRG. (n = 3; p < 0.05, one-way ANOVA with Tukey post-hoc). B. NaV1.8 mRNA levels in SNE-injured DRG were not significantly different from those of uninjured DRG (n = 4 for uninjured DRG and n = 3 for all other samples). C: Southern blot of the Nav1.8 3' RACE product from contralateral and ipsilateral sciatic nerve. Note the presence of a distinct band representing the expected 3'RACE product of 830 bp. Sequencing of the RACE product confirmed the presence of the 3' end of the NaV1.8 coding region as well as the NaV1.8 3' un-translated region and polyA tail. Tissue was harvested 2 weeks after SNE.
Mentions: Real-time PCR detected the presence of NaV1.8 mRNA in the sciatic nerve and revealed a significant up-regulation in the ipsilateral nerve as compared to the contralateral and uninjured nerve (p < 0.05, one-way ANOVA with Tukey post-hoc; Fig. 4A). In contrast, ipsilateral, contralateral, and uninjured DRG showed no significant differences in NaV1.8 mRNA levels (Fig. 4B). To confirm that the NaV1.8 PCR signals represent mRNA and not genomic DNA contamination, 3'RACE PCR was performed. Southern blot hybridization analysis showed a positive band at the predicted size of 830 bp in both contralateral and ipsilateral sciatic nerve samples (Fig. 4C). After agarose gel separation and cloning of amplicons, DNA sequencing positively identified the 3' end of the coding region and the 3' untranslated region of rat Nav1.8 as well as an additional poly A tail (data not shown).

Bottom Line: Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves.Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves.The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oral Biology and Medicine, School of Dentistry, University of California, Los Angeles, CA 90095-1668, USA. dthakor@ucla.edu

ABSTRACT

Background: Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.

Results: After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.

Conclusion: Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

Show MeSH
Related in: MedlinePlus