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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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Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast-like morphology were present in both nerve and DRG cultures, whereas neurons were only present in the DRG culture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the DRG sample with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures.
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Figure 5: Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast-like morphology were present in both nerve and DRG cultures, whereas neurons were only present in the DRG culture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the DRG sample with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures.

Mentions: We hypothesized that if sciatic nerve NaV1.8 mRNA was of non-neuronal origin, it would be detectable in cultures of dissociated sciatic nerves where there are no neuronal somata and neuronal axons have degenerated. No real-time PCR signal was detected for NaV1.8 in sciatic nerve cultures (Fig 5A, C), whereas robust NaV1.8 expression was observed in DRG cultures that served as a positive control (Fig. 5B, C). However, similar signals for 18s rRNA were detected in the sciatic nerve and DRG cultures (Fig. 5D), suggesting similar concentrations of template cDNA. Additionally, we were able to detect strong expression of s100B, a Schwann cell marker, in both sciatic nerve and DRG cultures, with Ct values ranging from 26–30. Interestingly, although s100B expression was strong in nerve cultures, it was still stronger in DRG cultures (Fig. 5E).


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)

Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast-like morphology were present in both nerve and DRG cultures, whereas neurons were only present in the DRG culture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the DRG sample with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast-like morphology were present in both nerve and DRG cultures, whereas neurons were only present in the DRG culture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the DRG sample with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures.
Mentions: We hypothesized that if sciatic nerve NaV1.8 mRNA was of non-neuronal origin, it would be detectable in cultures of dissociated sciatic nerves where there are no neuronal somata and neuronal axons have degenerated. No real-time PCR signal was detected for NaV1.8 in sciatic nerve cultures (Fig 5A, C), whereas robust NaV1.8 expression was observed in DRG cultures that served as a positive control (Fig. 5B, C). However, similar signals for 18s rRNA were detected in the sciatic nerve and DRG cultures (Fig. 5D), suggesting similar concentrations of template cDNA. Additionally, we were able to detect strong expression of s100B, a Schwann cell marker, in both sciatic nerve and DRG cultures, with Ct values ranging from 26–30. Interestingly, although s100B expression was strong in nerve cultures, it was still stronger in DRG cultures (Fig. 5E).

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