Increased expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 within dorsal root ganglia in a rat model of bone cancer pain

doi:10.1016/j.neulet.2012.01.069

Neuroscience Letters

Volume 512, Issue 2, 23 March 2012, Pages 61-66

https://doi.org/10.1016/j.neulet.2012.01.069 Get rights and content

Abstract

In attempt to understand the underlying mechanisms of cancer-induced bone pain, we investigated the presence of two tetrodotoxin-resistant sodium channels, Nav1.8 (SNS/PN3) and Nav1.9 (SNS2/NaN), in dorsal root ganglia (DRG) neurons in an animal model of bone cancer pain. Thirty-six female Sprague-Dawley rats were randomized into three groups: Sham operation group (Sham), cancer-bearing animals killed after 7 days (C7) and cancer-bearing animals killed after 14 days group (C14). After establishment of bone cancer pain model, behavioral tests were carried out to determine the paw withdrawal threshold (PWT) of mechanical and thermal hyperalgesia, respectively. Real-time RT-PCR, Western bolt and Immunofluorescence were used to determine the mRNA and protein expression of Nav1.8 and Nav1.9 in ipsilateral lumbar 4-5 DRG. Compared to Sham group, PWT of mechanical and thermal hyperalgesia in C14 group displayed a significant decrease (P < 0.01) from post-operation day (POD) 5 and POD7 to the end point of the observation, respectively. Compared to Sham group, the relative mRNA expression of Nav1.8 and Nav1.9 exhibited a significant up-regulation in C14 group (8.9 times and 9 times, respectively, P < 0.01) but not C7 group (1.5 times and 2.4 times, respectively). Western blot and Immunofluorescence revealed an apparent increase of Nav1.8 (P < 0.05) and Nav1.9 (P < 0.05) protein in C14 group compared with Sham group. The up-regulation of mRNA and protein levels of Nav1.8 and Nav1.9 suggested their potential involvement in the development and maintenance of bone cancer pain.

Highlights

► A rat model of bone cancer pain was established as previously described with minor revision. ► We examined pain-related behavioral changes and expression of Nav1.8 and Nav1.9. ► Expression of Nav1.8 and Nav1.9 was up-regulated in lumbar 4-5 DRG. ► The potential involvement of Nav1.8 and Nav1.9 in bone cancer pain was indicated.

Introduction

Bone cancer pain, which impairs patients’ physical and psychological function, is a malignant disease-related pain state dominated by a battery of symptoms including tonic background pain at rest, spontaneous pain at rest and movement-evoked pain [32]. While the background pain at rest could be temporarily handled by opioid analgesia now, the other two components are still difficult to deal with. Most of our current pharmacological treatments are non-selective relief of pain syndromes. Even for the gold standard therapy–palliative radiotherapy, there is only 24% of patients obtain complete pain relief following one month of treatment [23] and many late-stage individuals are too frail to benefit from current therapeutic regimens.

Our knowledge about pathophysiological mechanisms of pain – obtained from studies on ion channels, receptors and signal transduction – has expanded considerably in recent years [28]. Rearrangement of voltage-gated sodium channels (VGSC) in the neuronal membrane has been proposed as a critical molecular event that participate in the processing of pain signal after peripheral nerve or tissue injury.

Two tetrodotoxin-resistant (TTX-R) VGSC, Nav1.8 (SNS/PN3) and Nav1.9 (NaN/SNS2), are preferentially localized in small and medium size nociceptive dorsal root ganglia (DRG) neurons [19]. Both electrophysiological and pharmacological results have demonstrated an indispensable role for Nav1.8 in many types of chronic pain states [15], [16]. Moreover, this hypothesis is also supported by genetic engineering approaches: selective knock-down of Nav1.8 protein prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury; in contrast, knock-down of Nav1.9 protein, seems to have no effect on nerve injury-induced behavioral responses [27]. However, according to recent report, increases of the persistent TTX-R current (dominated by Nav1.9 channel) has also been indicated in the genesis of inflammatory pain [18] and application of a soup of inflammatory mediators rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability [20].

However, it is still unknown if these subunits that are confined to the DRG neurons will exert a specialized role in the molecular mechanism of bone cancer pain. Given that inflammation and nerve injury are two elements that involved in the pathophysiologic mechanisms of bone cancer pain [14] and the two sodium channels also exert their functions in inflammatory and/or neuropathic pain, we deduced that there may be some potential implication of Nav1.8 and/or Nav1.9 in the mechanism of bone cancer pain.

Section snippets

Animals and surgery

All procedures were approved by the Institutional Animal Care and Use Committee of Chinese PLA General Hospital (Number: 2010-X3-23) and were in accordance with NIH Guide to the Care and Use of Laboratory Animals.

Thirty-six female Sprague-Dawley rats (180–200 g) were randomized into three groups: Sham-operation group (Sham), cancer-bearing animals killed after 7 days (C7) and cancer-bearing animals killed after 14 days group (C14). The surgery was performed as previously described [22]. 5 μL of

Decreased PWT in mechanical and thermal hyperalgesia test

As to mechanical stimulation, rats in C14 group displayed a considerably decreased (P < 0.01) PWT from POD7 (29.8 ± 4.2 g) to the termination of the experiment (9.0 ± 0.3 g) compared with Sham group (67.2 ± 6.4 g at POD7; 63.3 ± 3.3 g at POD14) (Fig. 1A). Rats treated with radiant heat in C14 group presented a decreased (P < 0.01) PWT from POD5 (42.9 ± 1.1 ms) to POD14 (36.3 ± 1.0 ms) compared with their counterparts in Sham group (48.3 ± 1.4 ms at POD7; 50 ± 1.0 ms at POD14) (Fig. 1B).

Up-regulation of Nav1.8 mRNA and protein expression in ipsilateral DRG

There was an up-regulation of Nav1.8

Discussion

Using behavioral measurement and molecular biological assays, we have observed a decreased PWT of mechanical and thermal hyperalgesia and an up-regulation of Nav1.8 and Nav1.9 mRNA and protein expression within lumbar 4-5 DRG in an animal model of bone cancer pain. The pain-related behavioral changes emerged almost concurrently with the up-regulation of Nav1.8 and Nav1.9, which suggested that altered expression of the two subunits may contribute to bone cancer pain.

Acknowledgments

Our research is funded by National Natural Science Foundation of China (Grant 30901398). The authors thank Yuejuan Li and Fang Li for their revision.

References (34)

S.R. Chaplan et al.
Quantitative assessment of tactile allodynia in the rat paw
J. Neurosci. Methods
(1994)
L. Colvin et al.
Challenges in cancer pain management—bone pain
Eur. J. Cancer
(2008)
S. Dib-Hajj et al.
NaN/Nav1.9: a sodium channel with unique properties
Trends Neurosci.
(2002)
S.D. Dib-Hajj et al.
Plasticity of sodium channel expression in DRG neurons in the chronic constriction injury model of neuropathic pain
Pain
(1999)
J. Fjell et al.
Differential role of GDNF and NGF in the maintenance of two TTX-resistant sodium channels in adult DRG neurons
Brain Res. Mol. Brain Res.
(1999)
E.M. Garry et al.
Varicella zoster virus induces neuropathic changes in rat dorsal root ganglia and behavioral reflex sensitisation that is attenuated by gabapentin or sodium channel blocking drugs
Pain
(2005)
K. Hargreaves et al.
A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia
Pain
(1988)
S.K. Joshi et al.
Additive antinociceptive effects of the selective Nav1.8 blocker A-803467 and selective TRPV1 antagonists in rat inflammatory and neuropathic pain models
J. Pain
(2009)
S.K. Joshi et al.
Involvement of the TTX-resistant sodium channel Nav 1.8 in inflammatory and neuropathic, but not post-operative, pain states
Pain
(2006)
S.G. Khasar et al.
A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat
Neurosci. Lett.
(1998)

S. Leo et al.

Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice

Behav. Brain Res.

(2010)

Q.L. Mao-Ying et al.

A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells

Biochem. Biophys. Res. Commun.

(2006)

K. Obata et al.

Contribution of injured and uninjured dorsal root ganglion neurons to pain behavior and the changes in gene expression following chronic constriction injury of the sciatic nerve in rats

Pain

(2003)

M. Rogers et al.

The role of sodium channels in neuropathic pain

Semin. Cell. Dev. Biol.

(2006)

C.F. Villarreal et al.

The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception

Neurosci. Lett.

(2005)

J.S. Choi et al.

Physiological interactions between Nav1.7 and Nav1.8 sodium channels: a computer simulation study

J. Neurophysiol.

(2011)

T.R. Cummins et al.

Glial-derived neurotrophic factor upregulates expression of functional SNS and NaN sodium channels and their currents in axotomized dorsal root ganglion neurons

J. Neurosci.

(2000)

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Plenary ArticleIncreased expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 within dorsal root ganglia in a rat model of bone cancer pain

Abstract

Highlights

Introduction

Section snippets

Animals and surgery

Decreased PWT in mechanical and thermal hyperalgesia test

Up-regulation of Nav1.8 mRNA and protein expression in ipsilateral DRG

Discussion

Acknowledgments

J. Neurosci. Methods

Eur. J. Cancer

Trends Neurosci.

Pain

Brain Res. Mol. Brain Res.

Pain

Pain

J. Pain

Pain

Neurosci. Lett.

Behav. Brain Res.

Biochem. Biophys. Res. Commun.

Pain

Semin. Cell. Dev. Biol.

Neurosci. Lett.

Physiological interactions between Nav1.7 and Nav1.8 sodium channels: a computer simulation study

J. Neurophysiol.

Glial-derived neurotrophic factor upregulates expression of functional SNS and NaN sodium channels and their currents in axotomized dorsal root ganglion neurons

J. Neurosci.

Plenary Article
Increased expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 within dorsal root ganglia in a rat model of bone cancer pain