| 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 ratsWalker 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 cellsResults 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 painIn 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...
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