Bilateral downregulation of Nav1.8 in dorsal root ganglia of rats with bone cancer pain induced by inoculation with Walker 256 breast tumor cells

Abstract

BackgroundRapid and effective treatment of cancer-induced bone pain remains a clinical challenge and patients with bone metastasis are more likely to experience severe pain. The voltage-gated sodium channel Nav1.8 plays a critical role in many aspects of nociceptor function. Therefore, we characterized a rat model of cancer pain and investigated the potential role of Nav1.8.MethodsAdult female Wistar rats were used for the study. Cancer pain was induced by inoculation of Walker 256 breast carcinosarcoma cells into the tibia. After surgery, mechanical and thermal hyperalgesia and ambulation scores were evaluated to identify pain-related behavior. We used real-time RT-PCR to determine Nav1.8 mRNA expression in bilateral L4/L5 dorsal root ganglia (DRG) at 16-19 days after surgery. Western blotting and immunofluorescence were used to compare the expression and distribution of Nav1.8 in L4/L5 DRG between tumor-bearing and sham rats. Antisense oligodeoxynucleotides (ODNs) against Nav1.8 were administered intrathecally at 14-16 days after surgery to knock down Nav1.8 protein expression and changes in pain-related behavior were observed.ResultsTumor-bearing rats exhibited mechanical hyperalgesia and ambulatory-evoked pain from day 7 after inoculation of Walker 256 cells. In the advanced stage of cancer pain (days 16-19 after surgery), normalized Nav1.8 mRNA levels assessed by real-time RT-PCR were significantly lower in ipsilateral L4/L5 DRG of tumor-bearing rats compared with the sham group. Western-blot showed that the total expression of Nav1.8 protein significantly decreased bilaterally in DRG of tumor-bearing rats. Furthermore, as revealed by immunofluorescence, only the expression of Nav1.8 protein in small neurons down regulated significantly in bilateral DRG of cancer pain rats. After administration of antisense ODNs against Nav1.8, Nav1.8 protein expression decreased significantly and tumor-bearing rats showed alleviated mechanical hyperalgesia and ambulatory-evoked pain.ConclusionsThese findings suggest that Nav1.8 plays a role in the development and maintenance of bone cancer pain.

Increased Expression of Protease-Activated Receptor 2 and 4 Within Dorsal Root Ganglia in a Rat Model of Bone Cancer Pain

“…), and the ipsilateral lumbar 4-5 DRGs were quickly removed at the endpoint of each group for further detection of mRNA and protein as previously described (Miao et al 2010) with some modification.…”

“…and perfused with heparinized normal saline followed by 10 % neutral formaldehyde via the left ventricle. Ipsilateral lumbar 4-5 DRGs were removed and fixed as previously described (Miao et al 2010). Each slide contained seven to eight sections (7 μm) at different levels of a DRG.…”

“…), and the ipsilateral lumbar 4-5 DRGs were quickly removed at the endpoint of each group for further detection of protein as previously described (Miao et al 2010). Total protein extract was separated on 8 % SDS-polyacrylamide gels and transferred to a nitrocellulose membrane.…”

New insights of nociceptor sensitization in bone cancer pain

“…Moreover, intrathecal injection of the antisense oligodeoxynucleotides (ODNs) to Nav1.8 can alleviate the established pain-related behavior in tumor-bearing rats. After administration of antisense ODNs to Nav1.8, the Nav1.8 expression in DRG neurons was significantly decreased, resulting in a reduction of nociceptive sensitivity [78]. A decrease paw withdrawal threshold, due to mechanical and thermal hyperalgesia, and up-regulated expression of Nav1.8 and Nav1.9 mRNA and protein within lumbar 4 --5 DRGs have been found by behavioral measurement and molecular biological assays in an animal model of bone cancer pain [79].…”

Enhanced SCN7A/Nax expression contributes to bone cancer pain by increasing excitability of neurons in dorsal root ganglion

“…In previous studies, experimental models of BCP were developed and provided seminal insight in understanding the pathophysiology of BCP (Schweizerhof et al, 2009), but the cellular and molecular mechanisms underlying the development and maintenance of cancer-evoked pain are not well understood (Clohisy and Mantyh, 2003). Recently, it has been suggested that the hypersensitivity of nociceptors may play a role on the induction of BCP (Miao et al, 2010).…”