Nav1.8在骨癌痛大鼠痛觉过敏维持机制中的作用研究
|
摘要
癌痛是长期困扰肿瘤病人的问题之一,不仅影响病人的情绪和生活质量,而且可能成为病人及家属决定停止积极治疗的一个重要因素。因而研究癌痛的机制,解决癌痛之苦成为临床上的迫切需要。临床最常见的三大肿瘤为肺癌、乳腺癌、前列腺癌,此类肿瘤晚期很容易发生骨转移,而骨癌痛则是慢性癌痛最为常见的一种,因此从研究骨癌痛的发生发展机制入手揭开癌症痛的机制有重要的临床意义。
由于缺乏合适的研究模型,癌痛的研究进展远远落后于炎症痛和神经病理性疼痛。近年来先后出现了一批成功的骨癌痛模型,模型制作多采用骨髓腔内直接接种肿瘤细胞的方法。骨癌痛模型将肿瘤局限在骨髓腔内,从而最大程度地减少了肿瘤广泛的系统性影响。在这些骨癌痛动物模型上可以观察到许多与临床症状类似的疼痛行为学表现,如自发性疼痛、痛觉过敏等。这些模型的出现大大加速了癌痛机制研究的进程,为寻找有效的癌痛治疗手段提供了良好的契机,骨保护素等在模型上已被证明能有效抑制癌痛。目前研究认为,骨癌痛是一种机制独特而复杂的慢性疼痛,尽管癌痛含有炎症痛以及神经病理性痛的成分,但是癌痛决不是炎症疼痛以及神经病理性疼痛的简单总和。因此,有必要深入研究癌痛机制并探索有效的癌痛治疗方法。
而电压门控性钠通道被认为在疼痛通路中起重要的作用,钠通道各亚型的运输,分布和密度,以及通道本身内在特性在相当程度上影响了外周感觉神经元的兴奋性,而伤害性感受神经元的电活动促成了疼痛的产生。河豚毒素不敏感(TTX-R)的Nav1.8钠通道专一表达在外周感觉神经元上,产生慢失活TTX-R钠电流,此通道是其所在细胞动作电位去极化期的钠电流的主要通道,被认为在伤害性感受器外周致敏中发挥了重要作用,因此Nav1.8在疼痛通路特别是在外周敏化的作用是疼痛机制研究的热点。Nav1.8在炎症痛模型DRG神经元上表达上调,TTX-R钠电流明显增加。此外,Nav1.8也参与了神经病理性疼痛的维持。鞘内注射反义寡核苷酸选择性降低Nav1.8表达,可有效的阻止由慢性神经或组织损伤引起的痛觉过敏和异常性疼痛。遗传学的方法技术则更有力的支持了Nav1.8在疼痛通路中不可或缺的作用,Nav1.8基因缺失小鼠对伤害性机械性刺激表现出明显的痛觉缺失,炎症性痛觉过敏延迟发展。
目前癌痛模型上电压门控性钠通道表达调节的相关研究还罕见报道。本研究着眼于电压门控性钠通道Nav1.8在骨癌痛外周敏化机制中的作用,通过Walker 256乳腺癌肉瘤细胞建立大鼠胫骨骨癌痛模型,从不同层面探索Nav1.8参与的骨癌痛维持机制。研究内容主要包括以下四个方面:⑴大鼠胫骨内注射walker256乳腺癌细胞建立骨癌痛模型及进行疼痛行为学评估;⑵通过REAL-TIME PCR、免疫印迹和免疫荧光染色观察DRG神经元中Nav1.8钠通道mRNA和蛋白表达情况;⑶利用全细胞膜片钳技术记录DRG神经元TTX-R钠电流,观察TTX-R钠通道的功能变化;⑷鞘内给予Nav1.8反义寡核苷酸对骨癌痛大鼠疼痛行为的影响。通过以上实验探讨钠通道Nav1.8是否和如何参与了骨癌痛维持过程中的痛觉过敏。结果如下:
1.骨癌痛模型建立
雌性Wistar大鼠胫骨内接种2×105个Walker 256细胞,术后第10天起观察到自发性运动痛评分进展性增高;诱发痛行为主要表现为机械性痛觉过敏而无明显的热痛觉过敏,与文献报道一致。在骨癌晚期(术后第17天起)可以观察到镜像痛行为。影像学结果提示,术后第21天骨质破坏明显,部分大鼠出现病理性骨折。病理检查可见晚期胫骨外观呈膨胀性增生,胫骨内有肿瘤生长,骨质破坏和增生同时存在。脊髓切片GFAP染色见患侧全层星形胶质细胞明显肥大增生。以上结果表明大鼠骨癌痛模型成功建立,模型主要行为学特征为自发性运动疼痛机械性痛觉过敏和机械性痛觉过敏。
2. Nav1.8钠通道在骨癌痛大鼠DRG神经元中的表达下调
骨癌痛大鼠在术后16-19天疼痛行为明确且稳定。在此期间,骨癌痛大鼠双侧腰段DRG神经元中Nav1.8 mRNA水平显著降低。Western blot结果显示,骨癌痛大鼠双侧DRG中Nav1.8蛋白水平明显降低,术侧蛋白表达比对侧更低。免疫荧光染色发现,癌痛大鼠双侧DRG神经元Nav1.8免疫荧光强度均有所降低,Nav1.8在小细胞上的表达显著减少。以上结果提示在骨癌痛大鼠疼痛维持期,Nav1.8在DRG神经元表达的显著降低可能与癌痛相关。骨癌痛大鼠手术对侧DRG神经元Nav1.8的变化提示骨癌痛发展维持过程中肿瘤源性的因素对钠通道表达的调节作用。
3.骨癌痛大鼠DRG神经元TTX-R钠电流密度下降
骨癌痛组DRG神经元的形态及被动膜电生理特性包括膜电容、静息电位等与对照组相比均无显著差异。癌痛组术侧DRG神经元总钠电流和TTX-R钠电流密度大量降低,提示Nav1.8的表达降低引起了TTX-R钠电流的减少。而骨癌痛对侧钠电流与假手术组相比无明显变化,提示肿瘤引起钠通道亚型的可塑性变化和功能代偿。
4.鞘内给予Nav1.8反义寡核苷酸可有效减轻骨癌痛大鼠疼痛行为
鞘内注射Nav1.8反义寡核苷酸可显著降低癌痛大鼠的自发性运动痛和机械性痛觉过敏。免疫荧光染色结果显示,鞘内注射Nav1.8反义寡核苷酸可以显著降低大鼠双侧DRG神经元上Nav1.8的表达。提示鞘内注射Nav1.8反义寡核苷酸效果可靠,通过干扰Nav1.8基因转录进而减少Nav1.8的表达减轻了骨癌痛大鼠的疼痛行为。
根据以上结果可以得出如下结论:
1.胫骨内接种Walker 256乳腺癌肉瘤细胞可以成功构建大鼠胫骨骨癌痛模型,其行为学特征主要表现为自发性疼痛和机械性痛觉过敏。
2.Nav1.8钠通道参与了骨癌痛大鼠疼痛维持机制。
(1)骨癌痛大鼠的疼痛维持与其脊髓背根神经节中Nav1.8钠通道在小细胞上的分布显著减低有关,而后者可能与Nav1.8 mRNA水平降低有密切关系。
(2)骨癌痛大鼠DRG神经元上总钠电流和TTX-R钠电流密度大量降低,Nav1.8钠通道的表达下调引起了TTX-R钠电流密度的下降。
(3)反义寡核苷酸法特异性敲低Nav1.8钠通道的表达,可以显著减轻骨癌痛大鼠的疼痛行为。
(4)骨癌痛大鼠对侧DRG神经元上Nav1.8钠通道表达及钠电流的特征提示肿瘤源性因素可能参与了钠通道亚型表达的调控。
Cancer pain is one of cancer’s most common symptoms and cancer patients have pain severe enough to require treatment at some point during the course of their disease. The quality of life of these patients is frequently diminished and pain can be a major contributor to this decrease in the quality of life. The greatest obstacles for developing new treatments for cancer pain are our limited knowledge of the basic neurobiological mechanisms that generate cancer pain. Cancer-induced bone pain remains a clinically challenging to treat rapidly and effectively. Metastasis of tumor cells to bone is particularly common in patients with lung, breast, and prostate cancer and patients with bone metastasis are more likely to experience severe pain. Understanding the neurobiological mechanisms underlying cancer pain is critical for improved pain management.
Until relatively recently it was difficult to study the pain associated with bone metastases, as the systemic models of cancer have much more widespread effects, making the underlying bone cancer pain difficult to evaluate. Cancer-induced pain models use direct inoculation of tumor cells into the medullary cavity of bone. The emergence of these focal bone metastases models, displaying behavioural signs compatible with the clinical syndrome including spontaneous pain and mechanical hyperalgesia, has meant that our understanding of the underlying mechanism of this chronic pain syndrome has advanced significantly in the last decade. Cancer pain was considered not simply to be a type of inflammatory or neuropathic pain but different types of cancer pain may be unique persistent pain states that change with the evolution of the disease. Thus, it is necessary to explore the underlying mechanisms of cancer-induced bone pain and direct the development of new therapies.
Voltage-gated sodium channels play a specialized role in pain pathways. They are critical determinants of the electrical excitability of sensory neurons and play a key role in pain sensation by controlling afferent impulse discharge. Nav1.8 is a TTX-R sensory neuron-specific channel mainly expressed in nociceptive neurons. This channel contributes a majority of the sodium current underlying the depolarizing phase of the action potential in cells in which it is present. Nav1.8 thus underlies the inactivating TTX-r sodium currents that have been found to play a critical role in many aspects of nociceptor function. Functional expression of TTX-R currents encoded by this channel is regulated by inflammatory mediators in rats. Both antisense and knock-out studies support a role for the channel in contributing to inflammatory pain, as PGE2-induced hyperalgesia is inhibited by antisense oligonucleotides. Antisense studies have also suggested a role for this protein in the development of neuropathic pain. A late-onset deficit in ectopic action propagation has been described in the Nav1.8 null mutant mouse together with deficits in mechanohypersensitivity.
However, there are few reports on the expression and roles of voltage-gated sodium channels. Here, we established a rat model of bone cancer pain and investigated the potential role of the sodium channel Nav1.8 in cancer-induce bone pain. This study started with this four steps:⑴Establishment of bone caner pain model in rats induced by inoculation of Walker 256 breast carcinosarcoma cells into the tibia;⑵Observation of Nav1.8 mRNA level and protein expression and distribution in DRGs from bone cancer pain rats;⑶Recording of TTX-R sodium currents in isolated DRG neurons by whole-cell patch clamp;⑷Effects of intrathecal injection of Nav1.8 antisense oligonucleotides on pain-related behaviors of bone cancer pain rats. The results were as follows:
1. Establishment of bone caner pain model in rats
Walker 256 mammary gland carcinoma cells (2×105) were injected into the tibia medullary cavity of female Wistar rats. The spontaneous ambulatory pain score (SPAS) increased significantly from day 10 after surgery. Mechanical hyperalgesia rather than thermal hyperalgesia was observed in tumor-bearing rats. Mirror-image pain was also observed in contralateral paws at the advanced stage of cancer. Radiological results showed signs of radiolucent lesion in the proximal epiphysis, medullary bone loss and cortical bone loss at day 21 after surgery. Sections obtained from the proximal end of tibia 21 days after the intra-tibia injection were stained with hematoxylin and eosin, tumor growth and various degrees of bone destruction and formation were observed in the animals received live Walker 256 carcinoma cells. In a few cases of severe bone destruction, the tumor destroyed bone matrix and periosteum and grew outside of the bone. Immunofluorescence of GFAP in spinal cord showed increased fluorescence intensity ipsilaterally, suggesting hypertrophy of astrocytes. In conclusion, the rat model of bone cancer pain has been succesfuly established.
2. Down-regulation of Nav1.8 in DRGs from rats with bone cancer pain
In the advanced stage of cancer pain (day 16-19 after surgery), the normalized Nav1.8 mRNA level, which was assessed by the Real-time RT-PCR assay, showed a significant decrease in bilateral L4/L5 dorsal root ganglia (DRG) of tumor-bearing rats compared with sham group. Western-blot showed that the total expression of Nav1.8 protein significantly decreased bilaterally in DRGs of tumor-bearing rats. Furthermore, as revealed by immunofluorescence, only the expression of Nav1.8 protein in small neurons downregulated significantly in bilateral DRGs of cancer pain rats. These results indicated that the down-regulation of Nav1.8 in DRGs may be related to the maintenance of cancer pain in the advanced stage.
3. TTX-R sodium currents decreased in the ipsilateral DRG neurons after injection of tumor cells
Results from whole-cell recording showed no changes of membrane capacity and resting membrane potential in bilateral DRG neurons from cancer rats compared with sham rats. There was a significant decrease in total sodium current density and TTX-R sodium current density of ipsilateral DRG neurons from cancer rats compared with sham group, resulting from the down-expression of Nav1.8 sodium channel. No changes of sodium current density in contralateral DRG neurons from cancer rats were observed, suggesting a potential role of tumor growth in expression patterns of voltage-gated sodium channels.
4. Intrathecal injection of Nav1.8 antisense ODN alleviated pain-related behaviors in rats with bone cancer pain
In animals that received antisense ODN, DRGs showed a significantly decrease in the number of Nav1.8 positive neurons compared with that in animals received mismatch ODN. Mechanical hyperalgesia were reversed by approximately 48 h after the antisense ODN administration and returned on the second day after the last Nav1.8 antisense ODN injection. These results suggested a Nav1.8 involved mechanism in the maintenance of cancer pain.
Conclusions:
1. The rat model of bone cancer pain has been succesfully established. The main behavior characters of this model are spontaneous ambulatory pain and mechanical hyperalgesia.
2. Nav1.8 is involved in the maintenance mechanisms of cancer pain.
(1) The hyperalgesia in rats with bone cancer pain may be related to the down-expression of Nav1.8 sodium channel in DRGs which due to the decrease of Nav1.8 mRNA.
(2) TTX-R sodium currents decreased in the ipsilateral DRG neurons after injection of tumor cells, resulting from the down-expression of Nav1.8 sodium channel.
(3) Antisense, but not mismatch ODN to Nav1.8 administered by intrathecal injections to cancer rats alleviated established mechanical hyperalgesia.
(4) Expression of Nav1.8 and TTX-R currents in DRG neurons from cancer rats suggested a potential role of tumor growth in expression patterns of voltage-gated sodium channels
由于缺乏合适的研究模型,癌痛的研究进展远远落后于炎症痛和神经病理性疼痛。近年来先后出现了一批成功的骨癌痛模型,模型制作多采用骨髓腔内直接接种肿瘤细胞的方法。骨癌痛模型将肿瘤局限在骨髓腔内,从而最大程度地减少了肿瘤广泛的系统性影响。在这些骨癌痛动物模型上可以观察到许多与临床症状类似的疼痛行为学表现,如自发性疼痛、痛觉过敏等。这些模型的出现大大加速了癌痛机制研究的进程,为寻找有效的癌痛治疗手段提供了良好的契机,骨保护素等在模型上已被证明能有效抑制癌痛。目前研究认为,骨癌痛是一种机制独特而复杂的慢性疼痛,尽管癌痛含有炎症痛以及神经病理性痛的成分,但是癌痛决不是炎症疼痛以及神经病理性疼痛的简单总和。因此,有必要深入研究癌痛机制并探索有效的癌痛治疗方法。
而电压门控性钠通道被认为在疼痛通路中起重要的作用,钠通道各亚型的运输,分布和密度,以及通道本身内在特性在相当程度上影响了外周感觉神经元的兴奋性,而伤害性感受神经元的电活动促成了疼痛的产生。河豚毒素不敏感(TTX-R)的Nav1.8钠通道专一表达在外周感觉神经元上,产生慢失活TTX-R钠电流,此通道是其所在细胞动作电位去极化期的钠电流的主要通道,被认为在伤害性感受器外周致敏中发挥了重要作用,因此Nav1.8在疼痛通路特别是在外周敏化的作用是疼痛机制研究的热点。Nav1.8在炎症痛模型DRG神经元上表达上调,TTX-R钠电流明显增加。此外,Nav1.8也参与了神经病理性疼痛的维持。鞘内注射反义寡核苷酸选择性降低Nav1.8表达,可有效的阻止由慢性神经或组织损伤引起的痛觉过敏和异常性疼痛。遗传学的方法技术则更有力的支持了Nav1.8在疼痛通路中不可或缺的作用,Nav1.8基因缺失小鼠对伤害性机械性刺激表现出明显的痛觉缺失,炎症性痛觉过敏延迟发展。
目前癌痛模型上电压门控性钠通道表达调节的相关研究还罕见报道。本研究着眼于电压门控性钠通道Nav1.8在骨癌痛外周敏化机制中的作用,通过Walker 256乳腺癌肉瘤细胞建立大鼠胫骨骨癌痛模型,从不同层面探索Nav1.8参与的骨癌痛维持机制。研究内容主要包括以下四个方面:⑴大鼠胫骨内注射walker256乳腺癌细胞建立骨癌痛模型及进行疼痛行为学评估;⑵通过REAL-TIME PCR、免疫印迹和免疫荧光染色观察DRG神经元中Nav1.8钠通道mRNA和蛋白表达情况;⑶利用全细胞膜片钳技术记录DRG神经元TTX-R钠电流,观察TTX-R钠通道的功能变化;⑷鞘内给予Nav1.8反义寡核苷酸对骨癌痛大鼠疼痛行为的影响。通过以上实验探讨钠通道Nav1.8是否和如何参与了骨癌痛维持过程中的痛觉过敏。结果如下:
1.骨癌痛模型建立
雌性Wistar大鼠胫骨内接种2×105个Walker 256细胞,术后第10天起观察到自发性运动痛评分进展性增高;诱发痛行为主要表现为机械性痛觉过敏而无明显的热痛觉过敏,与文献报道一致。在骨癌晚期(术后第17天起)可以观察到镜像痛行为。影像学结果提示,术后第21天骨质破坏明显,部分大鼠出现病理性骨折。病理检查可见晚期胫骨外观呈膨胀性增生,胫骨内有肿瘤生长,骨质破坏和增生同时存在。脊髓切片GFAP染色见患侧全层星形胶质细胞明显肥大增生。以上结果表明大鼠骨癌痛模型成功建立,模型主要行为学特征为自发性运动疼痛机械性痛觉过敏和机械性痛觉过敏。
2. Nav1.8钠通道在骨癌痛大鼠DRG神经元中的表达下调
骨癌痛大鼠在术后16-19天疼痛行为明确且稳定。在此期间,骨癌痛大鼠双侧腰段DRG神经元中Nav1.8 mRNA水平显著降低。Western blot结果显示,骨癌痛大鼠双侧DRG中Nav1.8蛋白水平明显降低,术侧蛋白表达比对侧更低。免疫荧光染色发现,癌痛大鼠双侧DRG神经元Nav1.8免疫荧光强度均有所降低,Nav1.8在小细胞上的表达显著减少。以上结果提示在骨癌痛大鼠疼痛维持期,Nav1.8在DRG神经元表达的显著降低可能与癌痛相关。骨癌痛大鼠手术对侧DRG神经元Nav1.8的变化提示骨癌痛发展维持过程中肿瘤源性的因素对钠通道表达的调节作用。
3.骨癌痛大鼠DRG神经元TTX-R钠电流密度下降
骨癌痛组DRG神经元的形态及被动膜电生理特性包括膜电容、静息电位等与对照组相比均无显著差异。癌痛组术侧DRG神经元总钠电流和TTX-R钠电流密度大量降低,提示Nav1.8的表达降低引起了TTX-R钠电流的减少。而骨癌痛对侧钠电流与假手术组相比无明显变化,提示肿瘤引起钠通道亚型的可塑性变化和功能代偿。
4.鞘内给予Nav1.8反义寡核苷酸可有效减轻骨癌痛大鼠疼痛行为
鞘内注射Nav1.8反义寡核苷酸可显著降低癌痛大鼠的自发性运动痛和机械性痛觉过敏。免疫荧光染色结果显示,鞘内注射Nav1.8反义寡核苷酸可以显著降低大鼠双侧DRG神经元上Nav1.8的表达。提示鞘内注射Nav1.8反义寡核苷酸效果可靠,通过干扰Nav1.8基因转录进而减少Nav1.8的表达减轻了骨癌痛大鼠的疼痛行为。
根据以上结果可以得出如下结论:
1.胫骨内接种Walker 256乳腺癌肉瘤细胞可以成功构建大鼠胫骨骨癌痛模型,其行为学特征主要表现为自发性疼痛和机械性痛觉过敏。
2.Nav1.8钠通道参与了骨癌痛大鼠疼痛维持机制。
(1)骨癌痛大鼠的疼痛维持与其脊髓背根神经节中Nav1.8钠通道在小细胞上的分布显著减低有关,而后者可能与Nav1.8 mRNA水平降低有密切关系。
(2)骨癌痛大鼠DRG神经元上总钠电流和TTX-R钠电流密度大量降低,Nav1.8钠通道的表达下调引起了TTX-R钠电流密度的下降。
(3)反义寡核苷酸法特异性敲低Nav1.8钠通道的表达,可以显著减轻骨癌痛大鼠的疼痛行为。
(4)骨癌痛大鼠对侧DRG神经元上Nav1.8钠通道表达及钠电流的特征提示肿瘤源性因素可能参与了钠通道亚型表达的调控。
Cancer pain is one of cancer’s most common symptoms and cancer patients have pain severe enough to require treatment at some point during the course of their disease. The quality of life of these patients is frequently diminished and pain can be a major contributor to this decrease in the quality of life. The greatest obstacles for developing new treatments for cancer pain are our limited knowledge of the basic neurobiological mechanisms that generate cancer pain. Cancer-induced bone pain remains a clinically challenging to treat rapidly and effectively. Metastasis of tumor cells to bone is particularly common in patients with lung, breast, and prostate cancer and patients with bone metastasis are more likely to experience severe pain. Understanding the neurobiological mechanisms underlying cancer pain is critical for improved pain management.
Until relatively recently it was difficult to study the pain associated with bone metastases, as the systemic models of cancer have much more widespread effects, making the underlying bone cancer pain difficult to evaluate. Cancer-induced pain models use direct inoculation of tumor cells into the medullary cavity of bone. The emergence of these focal bone metastases models, displaying behavioural signs compatible with the clinical syndrome including spontaneous pain and mechanical hyperalgesia, has meant that our understanding of the underlying mechanism of this chronic pain syndrome has advanced significantly in the last decade. Cancer pain was considered not simply to be a type of inflammatory or neuropathic pain but different types of cancer pain may be unique persistent pain states that change with the evolution of the disease. Thus, it is necessary to explore the underlying mechanisms of cancer-induced bone pain and direct the development of new therapies.
Voltage-gated sodium channels play a specialized role in pain pathways. They are critical determinants of the electrical excitability of sensory neurons and play a key role in pain sensation by controlling afferent impulse discharge. Nav1.8 is a TTX-R sensory neuron-specific channel mainly expressed in nociceptive neurons. This channel contributes a majority of the sodium current underlying the depolarizing phase of the action potential in cells in which it is present. Nav1.8 thus underlies the inactivating TTX-r sodium currents that have been found to play a critical role in many aspects of nociceptor function. Functional expression of TTX-R currents encoded by this channel is regulated by inflammatory mediators in rats. Both antisense and knock-out studies support a role for the channel in contributing to inflammatory pain, as PGE2-induced hyperalgesia is inhibited by antisense oligonucleotides. Antisense studies have also suggested a role for this protein in the development of neuropathic pain. A late-onset deficit in ectopic action propagation has been described in the Nav1.8 null mutant mouse together with deficits in mechanohypersensitivity.
However, there are few reports on the expression and roles of voltage-gated sodium channels. Here, we established a rat model of bone cancer pain and investigated the potential role of the sodium channel Nav1.8 in cancer-induce bone pain. This study started with this four steps:⑴Establishment of bone caner pain model in rats induced by inoculation of Walker 256 breast carcinosarcoma cells into the tibia;⑵Observation of Nav1.8 mRNA level and protein expression and distribution in DRGs from bone cancer pain rats;⑶Recording of TTX-R sodium currents in isolated DRG neurons by whole-cell patch clamp;⑷Effects of intrathecal injection of Nav1.8 antisense oligonucleotides on pain-related behaviors of bone cancer pain rats. The results were as follows:
1. Establishment of bone caner pain model in rats
Walker 256 mammary gland carcinoma cells (2×105) were injected into the tibia medullary cavity of female Wistar rats. The spontaneous ambulatory pain score (SPAS) increased significantly from day 10 after surgery. Mechanical hyperalgesia rather than thermal hyperalgesia was observed in tumor-bearing rats. Mirror-image pain was also observed in contralateral paws at the advanced stage of cancer. Radiological results showed signs of radiolucent lesion in the proximal epiphysis, medullary bone loss and cortical bone loss at day 21 after surgery. Sections obtained from the proximal end of tibia 21 days after the intra-tibia injection were stained with hematoxylin and eosin, tumor growth and various degrees of bone destruction and formation were observed in the animals received live Walker 256 carcinoma cells. In a few cases of severe bone destruction, the tumor destroyed bone matrix and periosteum and grew outside of the bone. Immunofluorescence of GFAP in spinal cord showed increased fluorescence intensity ipsilaterally, suggesting hypertrophy of astrocytes. In conclusion, the rat model of bone cancer pain has been succesfuly established.
2. Down-regulation of Nav1.8 in DRGs from rats with bone cancer pain
In the advanced stage of cancer pain (day 16-19 after surgery), the normalized Nav1.8 mRNA level, which was assessed by the Real-time RT-PCR assay, showed a significant decrease in bilateral L4/L5 dorsal root ganglia (DRG) of tumor-bearing rats compared with sham group. Western-blot showed that the total expression of Nav1.8 protein significantly decreased bilaterally in DRGs of tumor-bearing rats. Furthermore, as revealed by immunofluorescence, only the expression of Nav1.8 protein in small neurons downregulated significantly in bilateral DRGs of cancer pain rats. These results indicated that the down-regulation of Nav1.8 in DRGs may be related to the maintenance of cancer pain in the advanced stage.
3. TTX-R sodium currents decreased in the ipsilateral DRG neurons after injection of tumor cells
Results from whole-cell recording showed no changes of membrane capacity and resting membrane potential in bilateral DRG neurons from cancer rats compared with sham rats. There was a significant decrease in total sodium current density and TTX-R sodium current density of ipsilateral DRG neurons from cancer rats compared with sham group, resulting from the down-expression of Nav1.8 sodium channel. No changes of sodium current density in contralateral DRG neurons from cancer rats were observed, suggesting a potential role of tumor growth in expression patterns of voltage-gated sodium channels.
4. Intrathecal injection of Nav1.8 antisense ODN alleviated pain-related behaviors in rats with bone cancer pain
In animals that received antisense ODN, DRGs showed a significantly decrease in the number of Nav1.8 positive neurons compared with that in animals received mismatch ODN. Mechanical hyperalgesia were reversed by approximately 48 h after the antisense ODN administration and returned on the second day after the last Nav1.8 antisense ODN injection. These results suggested a Nav1.8 involved mechanism in the maintenance of cancer pain.
Conclusions:
1. The rat model of bone cancer pain has been succesfully established. The main behavior characters of this model are spontaneous ambulatory pain and mechanical hyperalgesia.
2. Nav1.8 is involved in the maintenance mechanisms of cancer pain.
(1) The hyperalgesia in rats with bone cancer pain may be related to the down-expression of Nav1.8 sodium channel in DRGs which due to the decrease of Nav1.8 mRNA.
(2) TTX-R sodium currents decreased in the ipsilateral DRG neurons after injection of tumor cells, resulting from the down-expression of Nav1.8 sodium channel.
(3) Antisense, but not mismatch ODN to Nav1.8 administered by intrathecal injections to cancer rats alleviated established mechanical hyperalgesia.
(4) Expression of Nav1.8 and TTX-R currents in DRG neurons from cancer rats suggested a potential role of tumor growth in expression patterns of voltage-gated sodium channels
引文
1. Jimenez-Andrade JM, Martin CD, Koewler NJ, Freeman KT, Sullivan LJ, Halvorson KG, Barthold CM, Peters CM, Buus RJ, Ghilardi JR, Lewis JL, Kuskowski MA, Mantyh PW: Nerve growth factor sequestering therapy attenuates non-malignant skeletal pain following fracture. Pain 2007; 133: 183-96
2. Zhang RX, Liu B, Li A, Wang L, Ren K, Qiao JT, Berman BM, Lao L: Interleukin 1beta facilitates bone cancer pain in rats by enhancing NMDA receptor NR-1 subunit phosphorylation. Neuroscience 2008; 154: 1533-8
3. Nagamine K, Ozaki N, Shinoda M, Asai H, Nishiguchi H, Mitsudo K, Tohnai I, Ueda M, Sugiura Y: Mechanical allodynia and thermal hyperalgesia induced by experimental squamous cell carcinoma of the lower gingiva in rats. J Pain 2006; 7: 659-70
4. Zhang RX, Liu B, Wang L, Ren K, Qiao JT, Berman BM, Lao L: Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain 2005; 118: 125-36
5. Shimoyama M, Tatsuoka H, Ohtori S, Tanaka K, Shimoyama N: Change of dorsal horn neurochemistry in a mouse model of neuropathic cancer pain. Pain 2005; 114: 221-30
6. Sabino MA, Mantyh PW: Pathophysiology of bone cancer pain. J Support Oncol 2005; 3: 15-24
7. Halvorson KG, Kubota K, Sevcik MA, Lindsay TH, Sotillo JE, Ghilardi JR, Rosol TJ, Boustany L, Shelton DL, Mantyh PW: A blocking antibody to nerve growth factor attenuates skeletal pain induced by prostate tumor cells growing in bone. Cancer Res 2005; 65: 9426-35
8. Sevcik MA, Luger NM, Mach DB, Sabino MA, Peters CM, Ghilardi JR, Schwei MJ, Rohrich H, De Felipe C, Kuskowski MA, Mantyh PW: Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain 2004; 111: 169-80
9. Mantyh PW: A mechanism-based understanding of bone cancer pain. Novartis Found Symp 2004; 261: 194-214; discussion 214-9, 256-61
10. Clohisy DR, Mantyh PW: Bone cancer pain and the role of RANKL/OPG. J Musculoskelet Neuronal Interact 2004; 4: 293-300
11. Sabino MA, Luger NM, Mach DB, Rogers SD, Schwei MJ, Mantyh PW: Different tumors in bone each give rise to a distinct pattern of skeletal destruction,bone cancer-related pain behaviors and neurochemical changes in the central nervous system. Int J Cancer 2003; 104: 550-8
12. Wacnik PW, Eikmeier LJ, Ruggles TR, Ramnaraine ML, Walcheck BK, Beitz AJ, Wilcox GL: Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 2001; 21: 9355-66
13. Honore P, Rogers SD, Schwei MJ, Salak-Johnson JL, Luger NM, Sabino MC, Clohisy DR, Mantyh PW: Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience 2000; 98: 585-98
14. Wacnik PW, Kehl LJ, Trempe TM, Ramnaraine ML, Beitz AJ, Wilcox GL: Tumor implantation in mouse humerus evokes movement-related hyperalgesia exceeding that evoked by intramuscular carrageenan. Pain 2003; 101: 175-86
15. Clohisy DR, Mantyh PW: Bone cancer pain. Cancer 2003; 97: 866-73
16. Luger NM, Sabino MA, Schwei MJ, Mach DB, Pomonis JD, Keyser CP, Rathbun M, Clohisy DR, Honore P, Yaksh TL, Mantyh PW: Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain. Pain 2002; 99: 397-406
17. Tanaka M, Cummins TR, Ishikawa K, Dib-Hajj SD, Black JA, Waxman SG: SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. Neuroreport 1998; 9: 967-72
18. Coggeshall RE, Tate S, Carlton SM: Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats. Neurosci Lett 2004; 355: 45-8
19. Sleeper AA, Cummins TR, Dib-Hajj SD, Hormuzdiar W, Tyrrell L, Waxman SG, Black JA: Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy. J Neurosci 2000; 20: 7279-89
20. Decosterd I, Ji RR, Abdi S, Tate S, Woolf CJ: The pattern of expression of the voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models. Pain 2002; 96: 269-77
21. Zhang XF, Zhu CZ, Thimmapaya R, Choi WS, Honore P, Scott VE, Kroeger PE, Sullivan JP, Faltynek CR, Gopalakrishnan M, Shieh CC: Differential actionpotentials and firing patterns in injured and uninjured small dorsal root ganglion neurons after nerve injury. Brain Res 2004; 1009: 147-58
22. Porreca F, Lai J, Bian D, Wegert S, Ossipov MH, Eglen RM, Kassotakis L, Novakovic S, Rabert DK, Sangameswaran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci U S A 1999; 96: 7640-4
23. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN: The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 1999; 2: 541-8
24. Zhang RX, Li A, Liu B, Wang L, Xin J, Ren K, Qiao JT, Berman BM, Lao L: Electroacupuncture attenuates bone cancer-induced hyperalgesia and inhibits spinal preprodynorphin expression in a rat model. Eur J Pain 2008; 12: 870-8
25. Freeman KT, Koewler NJ, Jimenez-Andrade JM, Buus RJ, Herrera MB, Martin CD, Ghilardi JR, Kuskowski MA, Mantyh PW: A fracture pain model in the rat: adaptation of a closed femur fracture model to study skeletal pain. Anesthesiology 2008; 108: 473-83
26. Schwei MJ, Honore P, Rogers SD, Salak-Johnson JL, Finke MP, Ramnaraine ML, Clohisy DR, Mantyh PW: Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci 1999; 19: 10886-97
27. Honore P, Luger NM, Sabino MA, Schwei MJ, Rogers SD, Mach DB, O'Keefe P F, Ramnaraine ML, Clohisy DR, Mantyh PW: Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nat Med 2000; 6: 521-8
28. Kehl LJ, Hamamoto DT, Wacnik PW, Croft DL, Norsted BD, Wilcox GL, Simone DA: A cannabinoid agonist differentially attenuates deep tissue hyperalgesia in animal models of cancer and inflammatory muscle pain. Pain 2003; 103: 175-86
29. Medhurst SJ, Walker K, Bowes M, Kidd BL, Glatt M, Muller M, Hattenberger M, Vaxelaire J, O'Reilly T, Wotherspoon G, Winter J, Green J, Urban L: A rat model of bone cancer pain. Pain 2002; 96: 129-40
30. Buffon A, Ribeiro VB, Wink MR, Casali EA, Sarkis JJ: Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties. Life Sci 2007; 80: 950-8
31. Buffon A, Wink MR, Ribeiro BV, Casali EA, Libermann TA, Zerbini LF,Robson SC, Sarkis JJ: NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor. Biochim Biophys Acta 2007; 1770: 1259-65
32. Mao-Ying QL, Zhao J, Dong ZQ, Wang J, Yu J, Yan MF, Zhang YQ, Wu GC, Wang YQ: A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. Biochem Biophys Res Commun 2006; 345: 1292-8
33. Beyreuther BK, Callizot N, Brot MD, Feldman R, Bain SC, Stohr T: Antinociceptive efficacy of lacosamide in rat models for tumor- and chemotherapy-induced cancer pain. Eur J Pharmacol 2007; 565: 98-104
34. Nagae M, Hiraga T, Yoneda T: Acidic microenvironment created by osteoclasts causes bone pain associated with tumor colonization. J Bone Miner Metab 2007; 25: 99-104
35. Colvin L, Fallon M: Challenges in cancer pain management--bone pain. Eur J Cancer 2008; 44: 1083-90
36. Mercadante S: Malignant bone pain: pathophysiology and treatment. Pain 1997; 69: 1-18
37. Rodan GA, Martin TJ: Therapeutic approaches to bone diseases. Science 2000; 289: 1508-14
38. Peters CM, Ghilardi JR, Keyser CP, Kubota K, Lindsay TH, Luger NM, Mach DB, Schwei MJ, Sevcik MA, Mantyh PW: Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain. Exp Neurol 2005; 193: 85-100
39. Ono K, Harano N, Nagahata S, Seta Y, Tsujisawa T, Inenaga K, Nakanishi O: Behavioral characteristics and c-Fos expression in the medullary dorsal horn in a rat model for orofacial cancer pain. Eur J Pain 2008
40. Mantyh PW, Clohisy DR, Koltzenburg M, Hunt SP: Molecular mechanisms of cancer pain. Nat Rev Cancer 2002; 2: 201-9
41. Brigatte P, Sampaio SC, Gutierrez VP, Guerra JL, Sinhorini IL, Curi R, Cury Y: Walker 256 tumor-bearing rats as a model to study cancer pain. J Pain 2007; 8: 412-21
42. Djouhri L, Fang X, Okuse K, Wood JN, Berry CM, Lawson SN: The TTX-resistant sodium channel Nav1.8 (SNS/PN3): expression and correlation with membrane properties in rat nociceptive primary afferent neurons. J Physiol 2003; 550: 739-52
43. Fang X, Djouhri L, McMullan S, Berry C, Okuse K, Waxman SG, Lawson SN: trkA is expressed in nociceptive neurons and influences electrophysiological properties via Nav1.8 expression in rapidly conducting nociceptors. J Neurosci 2005; 25: 4868-78
44. Clohisy DR, Perkins SL, Ramnaraine ML: Review of cellular mechanisms of tumor osteolysis. Clin Orthop Relat Res 2000: 104-14
45. Luger NM, Honore P, Sabino MA, Schwei MJ, Rogers SD, Mach DB, Clohisy DR, Mantyh PW: Osteoprotegerin diminishes advanced bone cancer pain. Cancer Res 2001; 61: 4038-47
46. Strickland IT, Martindale JC, Woodhams PL, Reeve AJ, Chessell IP, McQueen DS: Changes in the expression of NaV1.7, NaV1.8 and NaV1.9 in a distinct population of dorsal root ganglia innervating the rat knee joint in a model of chronic inflammatory joint pain. Eur J Pain 2008; 12: 564-72
47. Coste B, Crest M, Delmas P: Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons. J Gen Physiol 2007; 129: 57-77
48. Goldberg YP, MacFarlane J, MacDonald ML, Thompson J, Dube MP, Mattice M, Fraser R, Young C, Hossain S, Pape T, Payne B, Radomski C, Donaldson G, Ives E, Cox J, Younghusband HB, Green R, Duff A, Boltshauser E, Grinspan GA, Dimon JH, Sibley BG, Andria G, Toscano E, Kerdraon J, Bowsher D, Pimstone SN, Samuels ME, Sherrington R, Hayden MR: Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations. Clin Genet 2007; 71: 311-9
49. Hoyt SB, London C, Ok H, Gonzalez E, Duffy JL, Abbadie C, Dean B, Felix JP, Garcia ML, Jochnowitz N, Karanam BV, Li X, Lyons KA, McGowan E, Macintyre DE, Martin WJ, Priest BT, Smith MM, Tschirret-Guth R, Warren VA, Williams BS, Kaczorowski GJ, Parsons WH: Benzazepinone Nav1.7 blockers: potential treatments for neuropathic pain. Bioorg Med Chem Lett 2007; 17: 6172-7
50. Catterall WA: From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000; 26: 13-25
51. Onkal R, Mattis JH, Fraser SP, Diss JK, Shao D, Okuse K, Djamgoz MB: Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue. J Cell Physiol 2008; 216: 716-26
52. Fraser SP, Diss JK, Chioni AM, Mycielska ME, Pan H, Yamaci RF, Pani F, Siwy Z, Krasowska M, Grzywna Z, Brackenbury WJ, Theodorou D, Koyuturk M, Kaya H, Battaloglu E, De Bella MT, Slade MJ, Tolhurst R, Palmieri C, Jiang J, Latchman DS, Coombes RC, Djamgoz MB: Voltage-gated sodium channel expression and potentiation of human breast cancer metastasis. Clin Cancer Res 2005; 11: 5381-9
53. Gold MS, Weinreich D, Kim CS, Wang R, Treanor J, Porreca F, Lai J: Redistribution of Na(V)1.8 in uninjured axons enables neuropathic pain. J Neurosci 2003; 23: 158-66
54. Elliott JR: Slow Na+ channel inactivation and bursting discharge in a simple model axon: implications for neuropathic pain. Brain Res 1997; 754: 221-6
55. Schild JH, Kunze DL: Experimental and modeling study of Na+ current heterogeneity in rat nodose neurons and its impact on neuronal discharge. J Neurophysiol 1997; 78: 3198-209
56. Scroggs RS: Evidence of a physiological role for use-dependent inactivation of NaV1.8 sodium channels. J Physiol 2008; 586: 923
57. Maingret F, Coste B, Padilla F, Clerc N, Crest M, Korogod SM, Delmas P: Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism. J Gen Physiol 2008; 131: 211-25
58. Luo S, Perry GM, Levinson SR, Henry MA: Nav1.7 expression is increased in painful human dental pulp. Mol Pain 2008; 4: 16
59. Jarecki BW, Sheets PL, Jackson JO, 2nd, Cummins TR: Paroxysmal extreme pain disorder mutations within the D3/S4-S5 linker of Nav1.7 cause moderate destabilization of fast inactivation. J Physiol 2008; 586: 4137-53
60. Schmalhofer W, Calhoun J, Burrows R, Bailey T, Kohler MG, Weinglass AB, Kaczorowski GJ, Garcia ML, Koltzenburg M, Priest BT: ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors. Mol Pharmacol 2008
61. Zhang ZN, Li Q, Liu C, Wang HB, Wang Q, Bao L: The voltage-gated Na+ channel Nav1.8 contains an ER-retention/retrieval signal antagonized by the {beta}3 subunit. J Cell Sci 2008; 121: 3243-52
62. Chattopadhyay M, Mata M, Fink DJ: Continuous delta-opioid receptor activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy. J Neurosci 2008; 28: 6652-8
63. Diss JK, Calissano M, Gascoyne D, Djamgoz MB, Latchman DS: Identification and characterization of the promoter region of the Nav1.7 voltage-gated sodium channel gene (SCN9A). Mol Cell Neurosci 2008; 37: 537-47
64. Hains BC, Klein JP, Saab CY, Craner MJ, Black JA, Waxman SG: Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury. J Neurosci 2003; 23: 8881-92
65. Lindia JA, Kohler MG, Martin WJ, Abbadie C: Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats. Pain 2005; 117: 145-53
66. Villarreal CF, Sachs D, Cunha FQ, Parada CA, Ferreira SH: The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception. Neurosci Lett 2005; 386: 72-7
67. Storkson RV, Kjorsvik A, Tjolsen A, Hole K: Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 1996; 65: 167-72
68. Wu WP, Xu XJ, Hao JX: Chronic lumbar catheterization of the spinal subarachnoid space in mice. J Neurosci Methods 2004; 133: 65-9
69. Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, de Groat WC: The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 2001; 21: 8690-6
70. Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F: Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 2002; 95: 143-52
71. Krafte DS, Chapman M, Marron B, Atkinson R, Liu Y, Ye F, Curran M, Kort M, Jarvis MF: Block of Nav1.8 by small molecules. Channels (Austin) 2007; 1: 152-3
72. Gaida W, Klinder K, Arndt K, Weiser T: Ambroxol, a Nav1.8-preferring Na(+) channel blocker, effectively suppresses pain symptoms in animal models of chronic, neuropathic and inflammatory pain. Neuropharmacology 2005; 49: 1220-7
73. Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A,Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR, Krafte DS: A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A 2007; 104: 8520-5
74. Kort ME, Drizin I, Gregg RJ, Scanio MJ, Shi L, Gross MF, Atkinson RN, Johnson MS, Pacofsky GJ, Thomas JB, Carroll WA, Krambis MJ, Liu D, Shieh CC, Zhang X, Hernandez G, Mikusa JP, Zhong C, Joshi S, Honore P, Roeloffs R, Marsh KC, Murray BP, Liu J, Werness S, Faltynek CR, Krafte DS, Jarvis MF, Chapman ML, Marron BE: Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the Nav1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain. J Med Chem 2008; 51: 407-16
75. Kerr BJ, Souslova V, McMahon SB, Wood JN: A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain. Neuroreport 2001; 12: 3077-80
76. Abrahamsen B, Zhao J, Asante CO, Cendan CM, Marsh S, Martinez-Barbera JP, Nassar MA, Dickenson AH, Wood JN: The cell and molecular basis of mechanical, cold, and inflammatory pain. Science 2008; 321: 702-5
77. Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, Kobayashi J, Nau C, Wood JN, Reeh PW: Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 2007; 447: 855-8
1. Catterall WA: From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000; 26: 13-25
2. Lindia JA, Kohler MG, Martin WJ, Abbadie C: Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats. Pain 2005; 117: 145-53
3. Black JA, Liu S, Tanaka M, Cummins TR, Waxman SG: Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain. Pain 2004; 108: 237-47
4. Okuse K, Chaplan SR, McMahon SB, Luo ZD, Calcutt NA, Scott BP, Akopian AN, Wood JN: Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain. Mol Cell Neurosci 1997; 10: 196-207
5. Dib-Hajj S, Black JA, Cummins TR, Waxman SG: NaN/Nav1.9: a sodium channel with unique properties. Trends Neurosci 2002; 25: 253-9
6. Akopian AN, Sivilotti L, Wood JN: A tetrodotoxin-resistant voltage-gatedsodium channel expressed by sensory neurons. Nature 1996; 379: 257-62
7. Casula MA, Facer P, Powell AJ, Kinghorn IJ, Plumpton C, Tate SN, Bountra C, Birch R, Anand P: Expression of the sodium channel beta3 subunit in injured human sensory neurons. Neuroreport 2004; 15: 1629-32
8. Hains BC, Saab CY, Klein JP, Craner MJ, Waxman SG: Altered sodium channel expression in second-order spinal sensory neurons contributes to pain after peripheral nerve injury. J Neurosci 2004; 24: 4832-9
9. Hains BC, Klein JP, Saab CY, Craner MJ, Black JA, Waxman SG: Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury. J Neurosci 2003; 23: 8881-92
10. Hains BC, Saab CY, Waxman SG: Changes in electrophysiological properties and sodium channel Nav1.3 expression in thalamic neurons after spinal cord injury. Brain 2005; 128: 2359-71
11. Shah BS, Stevens EB, Pinnock RD, Dixon AK, Lee K: Developmental expression of the novel voltage-gated sodium channel auxiliary subunit beta3, in rat CNS. J Physiol 2001; 534: 763-76
12. Cummins TR, Aglieco F, Renganathan M, Herzog RI, Dib-Hajj SD, Waxman SG: Nav1.3 sodium channels: rapid repriming and slow closed-state inactivation display quantitative differences after expression in a mammalian cell line and in spinal sensory neurons. J Neurosci 2001; 21: 5952-61
13. Shah BS, Rush AM, Liu S, Tyrrell L, Black JA, Dib-Hajj SD, Waxman SG: Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface. J Neurosci 2004; 24: 7387-99
14. Nassar MA, Stirling LC, Forlani G, Baker MD, Matthews EA, Dickenson AH, Wood JN: Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc Natl Acad Sci U S A 2004; 101: 12706-11
15. Yeomans DC, Levinson SR, Peters MC, Koszowski AG, Tzabazis AZ, Gilly WF, Wilson SP: Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents. Hum Gene Ther 2005; 16: 271-7
16. Nassar MA, Levato A, Stirling LC, Wood JN: Neuropathic pain develops normally in mice lacking both Nav1.7 and Nav1.8. Mol Pain 2005; 1: 24
17. Waxman SG, Dib-Hajj S: Erythermalgia: molecular basis for an inheritedpain syndrome. Trends Mol Med 2005; 11: 555-62
18. Cummins TR, Dib-Hajj SD, Waxman SG: Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J Neurosci 2004; 24: 8232-6
19. Vijayaragavan K, Boutjdir M, Chahine M: Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C. J Neurophysiol 2004; 91: 1556-69
20. Benn SC, Costigan M, Tate S, Fitzgerald M, Woolf CJ: Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons. J Neurosci 2001; 21: 6077-85
21. Tanaka M, Cummins TR, Ishikawa K, Dib-Hajj SD, Black JA, Waxman SG: SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. Neuroreport 1998; 9: 967-72
22. Coggeshall RE, Tate S, Carlton SM: Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats. Neurosci Lett 2004; 355: 45-8
23. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN: The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 1999; 2: 541-8
24. Kerr BJ, Souslova V, McMahon SB, Wood JN: A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain. Neuroreport 2001; 12: 3077-80
25. Villarreal CF, Sachs D, Cunha FQ, Parada CA, Ferreira SH: The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception. Neurosci Lett 2005; 386: 72-7
26. Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, de Groat WC: The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 2001; 21: 8690-6
27. Laird JM, Souslova V, Wood JN, Cervero F: Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice. J Neurosci 2002; 22: 8352-6
28. Beyak MJ, Ramji N, Krol KM, Kawaja MD, Vanner SJ: Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role incolitis-induced hyperexcitability. Am J Physiol Gastrointest Liver Physiol 2004; 287: G845-55
29. Sleeper AA, Cummins TR, Dib-Hajj SD, Hormuzdiar W, Tyrrell L, Waxman SG, Black JA: Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy. J Neurosci 2000; 20: 7279-89
30. Decosterd I, Ji RR, Abdi S, Tate S, Woolf CJ: The pattern of expression of the voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models. Pain 2002; 96: 269-77
31. Zhang XF, Zhu CZ, Thimmapaya R, Choi WS, Honore P, Scott VE, Kroeger PE, Sullivan JP, Faltynek CR, Gopalakrishnan M, Shieh CC: Differential action potentials and firing patterns in injured and uninjured small dorsal root ganglion neurons after nerve injury. Brain Res 2004; 1009: 147-58
32. Porreca F, Lai J, Bian D, Wegert S, Ossipov MH, Eglen RM, Kassotakis L, Novakovic S, Rabert DK, Sangameswaran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci U S A 1999; 96: 7640-4
33. Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F: Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 2002; 95: 143-52
34. Roza C, Laird JM, Souslova V, Wood JN, Cervero F: The tetrodotoxin-resistant Na+ channel Nav1.8 is essential for the expression of spontaneous activity in damaged sensory axons of mice. J Physiol 2003; 550: 921-6
35. Black JA, Langworthy K, Hinson AW, Dib-Hajj SD, Waxman SG: NGF has opposing effects on Na+ channel III and SNS gene expression in spinal sensory neurons. Neuroreport 1997; 8: 2331-5
36. Dib-Hajj SD, Black JA, Cummins TR, Kenney AM, Kocsis JD, Waxman SG: Rescue of alpha-SNS sodium channel expression in small dorsal root ganglion neurons after axotomy by nerve growth factor in vivo. J Neurophysiol 1998; 79: 2668-76
37. Fjell J, Cummins TR, Dib-Hajj SD, Fried K, Black JA, Waxman SG: 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; 67: 267-82
38. Fjell J, Cummins TR, Fried K, Black JA, Waxman SG: In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons. J Neurophysiol 1999; 81: 803-10
39. Cummins TR, Black JA, Dib-Hajj SD, Waxman SG: 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; 20: 8754-61
40. Fang X, Djouhri L, McMullan S, Berry C, Okuse K, Waxman SG, Lawson SN: trkA is expressed in nociceptive neurons and influences electrophysiological properties via Nav1.8 expression in rapidly conducting nociceptors. J Neurosci 2005; 25: 4868-78
41. Vijayaragavan K, Powell AJ, Kinghorn IJ, Chahine M: Role of auxiliary beta1-, beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium channel. Biochem Biophys Res Commun 2004; 319: 531-40
42. Okuse K, Malik-Hall M, Baker MD, Poon WY, Kong H, Chao MV, Wood JN: Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature 2002; 417: 653-6
43. Malik-Hall M, Poon WY, Baker MD, Wood JN, Okuse K: Sensory neuron proteins interact with the intracellular domains of sodium channel NaV1.8. Brain Res Mol Brain Res 2003; 110: 298-304
44. Fang X, Djouhri L, Black JA, Dib-Hajj SD, Waxman SG, Lawson SN: The presence and role of the tetrodotoxin-resistant sodium channel Na(v)1.9 (NaN) in nociceptive primary afferent neurons. J Neurosci 2002; 22: 7425-33
45. Maruyama H, Yamamoto M, Matsutomi T, Zheng T, Nakata Y, Wood JN, Ogata N: Electrophysiological characterization of the tetrodotoxin-resistant Na+ channel, Na(v)1.9, in mouse dorsal root ganglion neurons. Pflugers Arch 2004; 449: 76-87
46. Blum R, Kafitz KW, Konnerth A: Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9. Nature 2002; 419: 687-93
47. Priest BT, Murphy BA, Lindia JA, Diaz C, Abbadie C, Ritter AM, Liberator P, Iyer LM, Kash SF, Kohler MG, Kaczorowski GJ, MacIntyre DE, Martin WJ: Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior. Proc Natl Acad Sci U S A 2005; 102: 9382-7
48. Baker MD, Chandra SY, Ding Y, Waxman SG, Wood JN: GTP-induced tetrodotoxin-resistant Na+ current regulates excitability in mouse and rat small diameter sensory neurones. J Physiol 2003; 548: 373-82
49. Rush AM, Waxman SG: PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G-proteins. Brain Res 2004; 1023: 264-71
50. Klein JP, Tendi EA, Dib-Hajj SD, Fields RD, Waxman SG: Patterned electrical activity modulates sodium channel expression in sensory neurons. J Neurosci Res 2003; 74: 192-8
51. Keizer DW, West PJ, Lee EF, Yoshikami D, Olivera BM, Bulaj G, Norton RS: Structural basis for tetrodotoxin-resistant sodium channel binding by mu-conotoxin SmIIIA. J Biol Chem 2003; 278: 46805-13
2. Zhang RX, Liu B, Li A, Wang L, Ren K, Qiao JT, Berman BM, Lao L: Interleukin 1beta facilitates bone cancer pain in rats by enhancing NMDA receptor NR-1 subunit phosphorylation. Neuroscience 2008; 154: 1533-8
3. Nagamine K, Ozaki N, Shinoda M, Asai H, Nishiguchi H, Mitsudo K, Tohnai I, Ueda M, Sugiura Y: Mechanical allodynia and thermal hyperalgesia induced by experimental squamous cell carcinoma of the lower gingiva in rats. J Pain 2006; 7: 659-70
4. Zhang RX, Liu B, Wang L, Ren K, Qiao JT, Berman BM, Lao L: Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain 2005; 118: 125-36
5. Shimoyama M, Tatsuoka H, Ohtori S, Tanaka K, Shimoyama N: Change of dorsal horn neurochemistry in a mouse model of neuropathic cancer pain. Pain 2005; 114: 221-30
6. Sabino MA, Mantyh PW: Pathophysiology of bone cancer pain. J Support Oncol 2005; 3: 15-24
7. Halvorson KG, Kubota K, Sevcik MA, Lindsay TH, Sotillo JE, Ghilardi JR, Rosol TJ, Boustany L, Shelton DL, Mantyh PW: A blocking antibody to nerve growth factor attenuates skeletal pain induced by prostate tumor cells growing in bone. Cancer Res 2005; 65: 9426-35
8. Sevcik MA, Luger NM, Mach DB, Sabino MA, Peters CM, Ghilardi JR, Schwei MJ, Rohrich H, De Felipe C, Kuskowski MA, Mantyh PW: Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain 2004; 111: 169-80
9. Mantyh PW: A mechanism-based understanding of bone cancer pain. Novartis Found Symp 2004; 261: 194-214; discussion 214-9, 256-61
10. Clohisy DR, Mantyh PW: Bone cancer pain and the role of RANKL/OPG. J Musculoskelet Neuronal Interact 2004; 4: 293-300
11. Sabino MA, Luger NM, Mach DB, Rogers SD, Schwei MJ, Mantyh PW: Different tumors in bone each give rise to a distinct pattern of skeletal destruction,bone cancer-related pain behaviors and neurochemical changes in the central nervous system. Int J Cancer 2003; 104: 550-8
12. Wacnik PW, Eikmeier LJ, Ruggles TR, Ramnaraine ML, Walcheck BK, Beitz AJ, Wilcox GL: Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 2001; 21: 9355-66
13. Honore P, Rogers SD, Schwei MJ, Salak-Johnson JL, Luger NM, Sabino MC, Clohisy DR, Mantyh PW: Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience 2000; 98: 585-98
14. Wacnik PW, Kehl LJ, Trempe TM, Ramnaraine ML, Beitz AJ, Wilcox GL: Tumor implantation in mouse humerus evokes movement-related hyperalgesia exceeding that evoked by intramuscular carrageenan. Pain 2003; 101: 175-86
15. Clohisy DR, Mantyh PW: Bone cancer pain. Cancer 2003; 97: 866-73
16. Luger NM, Sabino MA, Schwei MJ, Mach DB, Pomonis JD, Keyser CP, Rathbun M, Clohisy DR, Honore P, Yaksh TL, Mantyh PW: Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain. Pain 2002; 99: 397-406
17. Tanaka M, Cummins TR, Ishikawa K, Dib-Hajj SD, Black JA, Waxman SG: SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. Neuroreport 1998; 9: 967-72
18. Coggeshall RE, Tate S, Carlton SM: Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats. Neurosci Lett 2004; 355: 45-8
19. Sleeper AA, Cummins TR, Dib-Hajj SD, Hormuzdiar W, Tyrrell L, Waxman SG, Black JA: Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy. J Neurosci 2000; 20: 7279-89
20. Decosterd I, Ji RR, Abdi S, Tate S, Woolf CJ: The pattern of expression of the voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models. Pain 2002; 96: 269-77
21. Zhang XF, Zhu CZ, Thimmapaya R, Choi WS, Honore P, Scott VE, Kroeger PE, Sullivan JP, Faltynek CR, Gopalakrishnan M, Shieh CC: Differential actionpotentials and firing patterns in injured and uninjured small dorsal root ganglion neurons after nerve injury. Brain Res 2004; 1009: 147-58
22. Porreca F, Lai J, Bian D, Wegert S, Ossipov MH, Eglen RM, Kassotakis L, Novakovic S, Rabert DK, Sangameswaran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci U S A 1999; 96: 7640-4
23. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN: The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 1999; 2: 541-8
24. Zhang RX, Li A, Liu B, Wang L, Xin J, Ren K, Qiao JT, Berman BM, Lao L: Electroacupuncture attenuates bone cancer-induced hyperalgesia and inhibits spinal preprodynorphin expression in a rat model. Eur J Pain 2008; 12: 870-8
25. Freeman KT, Koewler NJ, Jimenez-Andrade JM, Buus RJ, Herrera MB, Martin CD, Ghilardi JR, Kuskowski MA, Mantyh PW: A fracture pain model in the rat: adaptation of a closed femur fracture model to study skeletal pain. Anesthesiology 2008; 108: 473-83
26. Schwei MJ, Honore P, Rogers SD, Salak-Johnson JL, Finke MP, Ramnaraine ML, Clohisy DR, Mantyh PW: Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci 1999; 19: 10886-97
27. Honore P, Luger NM, Sabino MA, Schwei MJ, Rogers SD, Mach DB, O'Keefe P F, Ramnaraine ML, Clohisy DR, Mantyh PW: Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nat Med 2000; 6: 521-8
28. Kehl LJ, Hamamoto DT, Wacnik PW, Croft DL, Norsted BD, Wilcox GL, Simone DA: A cannabinoid agonist differentially attenuates deep tissue hyperalgesia in animal models of cancer and inflammatory muscle pain. Pain 2003; 103: 175-86
29. Medhurst SJ, Walker K, Bowes M, Kidd BL, Glatt M, Muller M, Hattenberger M, Vaxelaire J, O'Reilly T, Wotherspoon G, Winter J, Green J, Urban L: A rat model of bone cancer pain. Pain 2002; 96: 129-40
30. Buffon A, Ribeiro VB, Wink MR, Casali EA, Sarkis JJ: Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties. Life Sci 2007; 80: 950-8
31. Buffon A, Wink MR, Ribeiro BV, Casali EA, Libermann TA, Zerbini LF,Robson SC, Sarkis JJ: NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor. Biochim Biophys Acta 2007; 1770: 1259-65
32. Mao-Ying QL, Zhao J, Dong ZQ, Wang J, Yu J, Yan MF, Zhang YQ, Wu GC, Wang YQ: A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. Biochem Biophys Res Commun 2006; 345: 1292-8
33. Beyreuther BK, Callizot N, Brot MD, Feldman R, Bain SC, Stohr T: Antinociceptive efficacy of lacosamide in rat models for tumor- and chemotherapy-induced cancer pain. Eur J Pharmacol 2007; 565: 98-104
34. Nagae M, Hiraga T, Yoneda T: Acidic microenvironment created by osteoclasts causes bone pain associated with tumor colonization. J Bone Miner Metab 2007; 25: 99-104
35. Colvin L, Fallon M: Challenges in cancer pain management--bone pain. Eur J Cancer 2008; 44: 1083-90
36. Mercadante S: Malignant bone pain: pathophysiology and treatment. Pain 1997; 69: 1-18
37. Rodan GA, Martin TJ: Therapeutic approaches to bone diseases. Science 2000; 289: 1508-14
38. Peters CM, Ghilardi JR, Keyser CP, Kubota K, Lindsay TH, Luger NM, Mach DB, Schwei MJ, Sevcik MA, Mantyh PW: Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain. Exp Neurol 2005; 193: 85-100
39. Ono K, Harano N, Nagahata S, Seta Y, Tsujisawa T, Inenaga K, Nakanishi O: Behavioral characteristics and c-Fos expression in the medullary dorsal horn in a rat model for orofacial cancer pain. Eur J Pain 2008
40. Mantyh PW, Clohisy DR, Koltzenburg M, Hunt SP: Molecular mechanisms of cancer pain. Nat Rev Cancer 2002; 2: 201-9
41. Brigatte P, Sampaio SC, Gutierrez VP, Guerra JL, Sinhorini IL, Curi R, Cury Y: Walker 256 tumor-bearing rats as a model to study cancer pain. J Pain 2007; 8: 412-21
42. Djouhri L, Fang X, Okuse K, Wood JN, Berry CM, Lawson SN: The TTX-resistant sodium channel Nav1.8 (SNS/PN3): expression and correlation with membrane properties in rat nociceptive primary afferent neurons. J Physiol 2003; 550: 739-52
43. Fang X, Djouhri L, McMullan S, Berry C, Okuse K, Waxman SG, Lawson SN: trkA is expressed in nociceptive neurons and influences electrophysiological properties via Nav1.8 expression in rapidly conducting nociceptors. J Neurosci 2005; 25: 4868-78
44. Clohisy DR, Perkins SL, Ramnaraine ML: Review of cellular mechanisms of tumor osteolysis. Clin Orthop Relat Res 2000: 104-14
45. Luger NM, Honore P, Sabino MA, Schwei MJ, Rogers SD, Mach DB, Clohisy DR, Mantyh PW: Osteoprotegerin diminishes advanced bone cancer pain. Cancer Res 2001; 61: 4038-47
46. Strickland IT, Martindale JC, Woodhams PL, Reeve AJ, Chessell IP, McQueen DS: Changes in the expression of NaV1.7, NaV1.8 and NaV1.9 in a distinct population of dorsal root ganglia innervating the rat knee joint in a model of chronic inflammatory joint pain. Eur J Pain 2008; 12: 564-72
47. Coste B, Crest M, Delmas P: Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons. J Gen Physiol 2007; 129: 57-77
48. Goldberg YP, MacFarlane J, MacDonald ML, Thompson J, Dube MP, Mattice M, Fraser R, Young C, Hossain S, Pape T, Payne B, Radomski C, Donaldson G, Ives E, Cox J, Younghusband HB, Green R, Duff A, Boltshauser E, Grinspan GA, Dimon JH, Sibley BG, Andria G, Toscano E, Kerdraon J, Bowsher D, Pimstone SN, Samuels ME, Sherrington R, Hayden MR: Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations. Clin Genet 2007; 71: 311-9
49. Hoyt SB, London C, Ok H, Gonzalez E, Duffy JL, Abbadie C, Dean B, Felix JP, Garcia ML, Jochnowitz N, Karanam BV, Li X, Lyons KA, McGowan E, Macintyre DE, Martin WJ, Priest BT, Smith MM, Tschirret-Guth R, Warren VA, Williams BS, Kaczorowski GJ, Parsons WH: Benzazepinone Nav1.7 blockers: potential treatments for neuropathic pain. Bioorg Med Chem Lett 2007; 17: 6172-7
50. Catterall WA: From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000; 26: 13-25
51. Onkal R, Mattis JH, Fraser SP, Diss JK, Shao D, Okuse K, Djamgoz MB: Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue. J Cell Physiol 2008; 216: 716-26
52. Fraser SP, Diss JK, Chioni AM, Mycielska ME, Pan H, Yamaci RF, Pani F, Siwy Z, Krasowska M, Grzywna Z, Brackenbury WJ, Theodorou D, Koyuturk M, Kaya H, Battaloglu E, De Bella MT, Slade MJ, Tolhurst R, Palmieri C, Jiang J, Latchman DS, Coombes RC, Djamgoz MB: Voltage-gated sodium channel expression and potentiation of human breast cancer metastasis. Clin Cancer Res 2005; 11: 5381-9
53. Gold MS, Weinreich D, Kim CS, Wang R, Treanor J, Porreca F, Lai J: Redistribution of Na(V)1.8 in uninjured axons enables neuropathic pain. J Neurosci 2003; 23: 158-66
54. Elliott JR: Slow Na+ channel inactivation and bursting discharge in a simple model axon: implications for neuropathic pain. Brain Res 1997; 754: 221-6
55. Schild JH, Kunze DL: Experimental and modeling study of Na+ current heterogeneity in rat nodose neurons and its impact on neuronal discharge. J Neurophysiol 1997; 78: 3198-209
56. Scroggs RS: Evidence of a physiological role for use-dependent inactivation of NaV1.8 sodium channels. J Physiol 2008; 586: 923
57. Maingret F, Coste B, Padilla F, Clerc N, Crest M, Korogod SM, Delmas P: Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism. J Gen Physiol 2008; 131: 211-25
58. Luo S, Perry GM, Levinson SR, Henry MA: Nav1.7 expression is increased in painful human dental pulp. Mol Pain 2008; 4: 16
59. Jarecki BW, Sheets PL, Jackson JO, 2nd, Cummins TR: Paroxysmal extreme pain disorder mutations within the D3/S4-S5 linker of Nav1.7 cause moderate destabilization of fast inactivation. J Physiol 2008; 586: 4137-53
60. Schmalhofer W, Calhoun J, Burrows R, Bailey T, Kohler MG, Weinglass AB, Kaczorowski GJ, Garcia ML, Koltzenburg M, Priest BT: ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors. Mol Pharmacol 2008
61. Zhang ZN, Li Q, Liu C, Wang HB, Wang Q, Bao L: The voltage-gated Na+ channel Nav1.8 contains an ER-retention/retrieval signal antagonized by the {beta}3 subunit. J Cell Sci 2008; 121: 3243-52
62. Chattopadhyay M, Mata M, Fink DJ: Continuous delta-opioid receptor activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy. J Neurosci 2008; 28: 6652-8
63. Diss JK, Calissano M, Gascoyne D, Djamgoz MB, Latchman DS: Identification and characterization of the promoter region of the Nav1.7 voltage-gated sodium channel gene (SCN9A). Mol Cell Neurosci 2008; 37: 537-47
64. Hains BC, Klein JP, Saab CY, Craner MJ, Black JA, Waxman SG: Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury. J Neurosci 2003; 23: 8881-92
65. Lindia JA, Kohler MG, Martin WJ, Abbadie C: Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats. Pain 2005; 117: 145-53
66. Villarreal CF, Sachs D, Cunha FQ, Parada CA, Ferreira SH: The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception. Neurosci Lett 2005; 386: 72-7
67. Storkson RV, Kjorsvik A, Tjolsen A, Hole K: Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 1996; 65: 167-72
68. Wu WP, Xu XJ, Hao JX: Chronic lumbar catheterization of the spinal subarachnoid space in mice. J Neurosci Methods 2004; 133: 65-9
69. Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, de Groat WC: The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 2001; 21: 8690-6
70. Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F: Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 2002; 95: 143-52
71. Krafte DS, Chapman M, Marron B, Atkinson R, Liu Y, Ye F, Curran M, Kort M, Jarvis MF: Block of Nav1.8 by small molecules. Channels (Austin) 2007; 1: 152-3
72. Gaida W, Klinder K, Arndt K, Weiser T: Ambroxol, a Nav1.8-preferring Na(+) channel blocker, effectively suppresses pain symptoms in animal models of chronic, neuropathic and inflammatory pain. Neuropharmacology 2005; 49: 1220-7
73. Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A,Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR, Krafte DS: A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A 2007; 104: 8520-5
74. Kort ME, Drizin I, Gregg RJ, Scanio MJ, Shi L, Gross MF, Atkinson RN, Johnson MS, Pacofsky GJ, Thomas JB, Carroll WA, Krambis MJ, Liu D, Shieh CC, Zhang X, Hernandez G, Mikusa JP, Zhong C, Joshi S, Honore P, Roeloffs R, Marsh KC, Murray BP, Liu J, Werness S, Faltynek CR, Krafte DS, Jarvis MF, Chapman ML, Marron BE: Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the Nav1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain. J Med Chem 2008; 51: 407-16
75. Kerr BJ, Souslova V, McMahon SB, Wood JN: A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain. Neuroreport 2001; 12: 3077-80
76. Abrahamsen B, Zhao J, Asante CO, Cendan CM, Marsh S, Martinez-Barbera JP, Nassar MA, Dickenson AH, Wood JN: The cell and molecular basis of mechanical, cold, and inflammatory pain. Science 2008; 321: 702-5
77. Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, Kobayashi J, Nau C, Wood JN, Reeh PW: Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 2007; 447: 855-8
1. Catterall WA: From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000; 26: 13-25
2. Lindia JA, Kohler MG, Martin WJ, Abbadie C: Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats. Pain 2005; 117: 145-53
3. Black JA, Liu S, Tanaka M, Cummins TR, Waxman SG: Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain. Pain 2004; 108: 237-47
4. Okuse K, Chaplan SR, McMahon SB, Luo ZD, Calcutt NA, Scott BP, Akopian AN, Wood JN: Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain. Mol Cell Neurosci 1997; 10: 196-207
5. Dib-Hajj S, Black JA, Cummins TR, Waxman SG: NaN/Nav1.9: a sodium channel with unique properties. Trends Neurosci 2002; 25: 253-9
6. Akopian AN, Sivilotti L, Wood JN: A tetrodotoxin-resistant voltage-gatedsodium channel expressed by sensory neurons. Nature 1996; 379: 257-62
7. Casula MA, Facer P, Powell AJ, Kinghorn IJ, Plumpton C, Tate SN, Bountra C, Birch R, Anand P: Expression of the sodium channel beta3 subunit in injured human sensory neurons. Neuroreport 2004; 15: 1629-32
8. Hains BC, Saab CY, Klein JP, Craner MJ, Waxman SG: Altered sodium channel expression in second-order spinal sensory neurons contributes to pain after peripheral nerve injury. J Neurosci 2004; 24: 4832-9
9. Hains BC, Klein JP, Saab CY, Craner MJ, Black JA, Waxman SG: Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury. J Neurosci 2003; 23: 8881-92
10. Hains BC, Saab CY, Waxman SG: Changes in electrophysiological properties and sodium channel Nav1.3 expression in thalamic neurons after spinal cord injury. Brain 2005; 128: 2359-71
11. Shah BS, Stevens EB, Pinnock RD, Dixon AK, Lee K: Developmental expression of the novel voltage-gated sodium channel auxiliary subunit beta3, in rat CNS. J Physiol 2001; 534: 763-76
12. Cummins TR, Aglieco F, Renganathan M, Herzog RI, Dib-Hajj SD, Waxman SG: Nav1.3 sodium channels: rapid repriming and slow closed-state inactivation display quantitative differences after expression in a mammalian cell line and in spinal sensory neurons. J Neurosci 2001; 21: 5952-61
13. Shah BS, Rush AM, Liu S, Tyrrell L, Black JA, Dib-Hajj SD, Waxman SG: Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface. J Neurosci 2004; 24: 7387-99
14. Nassar MA, Stirling LC, Forlani G, Baker MD, Matthews EA, Dickenson AH, Wood JN: Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc Natl Acad Sci U S A 2004; 101: 12706-11
15. Yeomans DC, Levinson SR, Peters MC, Koszowski AG, Tzabazis AZ, Gilly WF, Wilson SP: Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents. Hum Gene Ther 2005; 16: 271-7
16. Nassar MA, Levato A, Stirling LC, Wood JN: Neuropathic pain develops normally in mice lacking both Nav1.7 and Nav1.8. Mol Pain 2005; 1: 24
17. Waxman SG, Dib-Hajj S: Erythermalgia: molecular basis for an inheritedpain syndrome. Trends Mol Med 2005; 11: 555-62
18. Cummins TR, Dib-Hajj SD, Waxman SG: Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J Neurosci 2004; 24: 8232-6
19. Vijayaragavan K, Boutjdir M, Chahine M: Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C. J Neurophysiol 2004; 91: 1556-69
20. Benn SC, Costigan M, Tate S, Fitzgerald M, Woolf CJ: Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons. J Neurosci 2001; 21: 6077-85
21. Tanaka M, Cummins TR, Ishikawa K, Dib-Hajj SD, Black JA, Waxman SG: SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. Neuroreport 1998; 9: 967-72
22. Coggeshall RE, Tate S, Carlton SM: Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats. Neurosci Lett 2004; 355: 45-8
23. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J, Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC, Dickenson AH, Wood JN: The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 1999; 2: 541-8
24. Kerr BJ, Souslova V, McMahon SB, Wood JN: A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain. Neuroreport 2001; 12: 3077-80
25. Villarreal CF, Sachs D, Cunha FQ, Parada CA, Ferreira SH: The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception. Neurosci Lett 2005; 386: 72-7
26. Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, de Groat WC: The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 2001; 21: 8690-6
27. Laird JM, Souslova V, Wood JN, Cervero F: Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice. J Neurosci 2002; 22: 8352-6
28. Beyak MJ, Ramji N, Krol KM, Kawaja MD, Vanner SJ: Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role incolitis-induced hyperexcitability. Am J Physiol Gastrointest Liver Physiol 2004; 287: G845-55
29. Sleeper AA, Cummins TR, Dib-Hajj SD, Hormuzdiar W, Tyrrell L, Waxman SG, Black JA: Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy. J Neurosci 2000; 20: 7279-89
30. Decosterd I, Ji RR, Abdi S, Tate S, Woolf CJ: The pattern of expression of the voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models. Pain 2002; 96: 269-77
31. Zhang XF, Zhu CZ, Thimmapaya R, Choi WS, Honore P, Scott VE, Kroeger PE, Sullivan JP, Faltynek CR, Gopalakrishnan M, Shieh CC: Differential action potentials and firing patterns in injured and uninjured small dorsal root ganglion neurons after nerve injury. Brain Res 2004; 1009: 147-58
32. Porreca F, Lai J, Bian D, Wegert S, Ossipov MH, Eglen RM, Kassotakis L, Novakovic S, Rabert DK, Sangameswaran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci U S A 1999; 96: 7640-4
33. Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F: Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 2002; 95: 143-52
34. Roza C, Laird JM, Souslova V, Wood JN, Cervero F: The tetrodotoxin-resistant Na+ channel Nav1.8 is essential for the expression of spontaneous activity in damaged sensory axons of mice. J Physiol 2003; 550: 921-6
35. Black JA, Langworthy K, Hinson AW, Dib-Hajj SD, Waxman SG: NGF has opposing effects on Na+ channel III and SNS gene expression in spinal sensory neurons. Neuroreport 1997; 8: 2331-5
36. Dib-Hajj SD, Black JA, Cummins TR, Kenney AM, Kocsis JD, Waxman SG: Rescue of alpha-SNS sodium channel expression in small dorsal root ganglion neurons after axotomy by nerve growth factor in vivo. J Neurophysiol 1998; 79: 2668-76
37. Fjell J, Cummins TR, Dib-Hajj SD, Fried K, Black JA, Waxman SG: 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; 67: 267-82
38. Fjell J, Cummins TR, Fried K, Black JA, Waxman SG: In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons. J Neurophysiol 1999; 81: 803-10
39. Cummins TR, Black JA, Dib-Hajj SD, Waxman SG: 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; 20: 8754-61
40. Fang X, Djouhri L, McMullan S, Berry C, Okuse K, Waxman SG, Lawson SN: trkA is expressed in nociceptive neurons and influences electrophysiological properties via Nav1.8 expression in rapidly conducting nociceptors. J Neurosci 2005; 25: 4868-78
41. Vijayaragavan K, Powell AJ, Kinghorn IJ, Chahine M: Role of auxiliary beta1-, beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium channel. Biochem Biophys Res Commun 2004; 319: 531-40
42. Okuse K, Malik-Hall M, Baker MD, Poon WY, Kong H, Chao MV, Wood JN: Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature 2002; 417: 653-6
43. Malik-Hall M, Poon WY, Baker MD, Wood JN, Okuse K: Sensory neuron proteins interact with the intracellular domains of sodium channel NaV1.8. Brain Res Mol Brain Res 2003; 110: 298-304
44. Fang X, Djouhri L, Black JA, Dib-Hajj SD, Waxman SG, Lawson SN: The presence and role of the tetrodotoxin-resistant sodium channel Na(v)1.9 (NaN) in nociceptive primary afferent neurons. J Neurosci 2002; 22: 7425-33
45. Maruyama H, Yamamoto M, Matsutomi T, Zheng T, Nakata Y, Wood JN, Ogata N: Electrophysiological characterization of the tetrodotoxin-resistant Na+ channel, Na(v)1.9, in mouse dorsal root ganglion neurons. Pflugers Arch 2004; 449: 76-87
46. Blum R, Kafitz KW, Konnerth A: Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9. Nature 2002; 419: 687-93
47. Priest BT, Murphy BA, Lindia JA, Diaz C, Abbadie C, Ritter AM, Liberator P, Iyer LM, Kash SF, Kohler MG, Kaczorowski GJ, MacIntyre DE, Martin WJ: Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior. Proc Natl Acad Sci U S A 2005; 102: 9382-7
48. Baker MD, Chandra SY, Ding Y, Waxman SG, Wood JN: GTP-induced tetrodotoxin-resistant Na+ current regulates excitability in mouse and rat small diameter sensory neurones. J Physiol 2003; 548: 373-82
49. Rush AM, Waxman SG: PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G-proteins. Brain Res 2004; 1023: 264-71
50. Klein JP, Tendi EA, Dib-Hajj SD, Fields RD, Waxman SG: Patterned electrical activity modulates sodium channel expression in sensory neurons. J Neurosci Res 2003; 74: 192-8
51. Keizer DW, West PJ, Lee EF, Yoshikami D, Olivera BM, Bulaj G, Norton RS: Structural basis for tetrodotoxin-resistant sodium channel binding by mu-conotoxin SmIIIA. J Biol Chem 2003; 278: 46805-13