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Up-regulation of brain-derived neurotrophic factor in the dorsal root ganglion of the rat bone cancer pain model.

ABSTRACT: Metastatic bone cancer causes severe pain, but current treatments often provide insufficient pain relief. One of the reasons is that mechanisms underlying bone cancer pain are not solved completely. Our previous studies have shown that brain-derived neurotrophic factor (BDNF), known as a member of the neurotrophic family, is an important molecule in the pathological pain state in some pain models. We hypothesized that expression changes of BDNF may be one of the factors related to bone cancer pain; in this study, we investigated changes of BDNF expression in dorsal root ganglia in a rat bone cancer pain model. As we expected, BDNF mRNA (messenger ribonucleic acid) and protein were significantly increased in L3 dorsal root ganglia after intra-tibial inoculation of MRMT-1 rat breast cancer cells. Among the eleven splice-variants of BDNF mRNA, exon 1-9 variant increased predominantly. Interestingly, the up-regulation of BDNF is localized in small neurons (mostly nociceptive neurons) but not in medium or large neurons (non-nociceptive neurons). Further, expression of nerve growth factor (NGF), which is known as a specific promoter of BDNF exon 1-9 variant, was significantly increased in tibial bone marrow. Our findings suggest that BDNF is a key molecule in bone cancer pain, and NGF-BDNF cascade possibly develops bone cancer pain.

SUBMITTER: Tomotsuka N

PROVIDER: S-EPMC4103927 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Up-regulation of brain-derived neurotrophic factor in the dorsal root ganglion of the rat bone cancer pain model.

Tomotsuka Naoto N Kaku Ryuji R Obata Norihiko N Matsuoka Yoshikazu Y Kanzaki Hirotaka H Taniguchi Arata A Muto Noriko N Omiya Hiroki H Itano Yoshitaro Y Sato Tadasu T Ichikawa Hiroyuki H Mizobuchi Satoshi S Morimatsu Hiroshi H

Journal of pain research 20140711

Metastatic bone cancer causes severe pain, but current treatments often provide insufficient pain relief. One of the reasons is that mechanisms underlying bone cancer pain are not solved completely. Our previous studies have shown that brain-derived neurotrophic factor (BDNF), known as a member of the neurotrophic family, is an important molecule in the pathological pain state in some pain models. We hypothesized that expression changes of BDNF may be one of the factors related to bone cancer pa ...[more]

PMID: 25050075

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Project description:Bone cancer pain is one of the most severe and intractable complications in patients suffering from primary or metastatic bone cancer and profoundly compromises the quality of life. Emerging evidence indicates that the dorsal root ganglion play an integral role in the modulation of pain hypersensitivity. However, the underlying molecular mechanisms during dorsal root ganglion-mediated bone cancer pain remain elusive. In this study, RNA-sequencing was used to detect the differentially expressed genes in dorsal root ganglion neurons of a rat bone cancer pain model established by intratibial inoculation of Walker 256 breast cancer cells. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that the differentially expressed genes (fold change > 1.5; false discovery rate < 0.05) were enriched in the bone morphogenetic protein (BMP) signaling pathway, transforming growth factor-β signaling pathway, and positive regulation of cartilage development. Importantly, serum deprivation-response protein ( Sdpr), hephaestin ( Heph), transthyretin ( Ttr), insulin receptor substrate 1 ( Irs1), connective tissue growth factor ( Ctgf ), and Bmp2 genes were associated with bone pain and degeneration. Of note, Bmp2, a pleiotropic and secreted molecule mediating pain and inflammation, was one of the most significantly upregulated genes in dorsal root ganglion neurons in this bone cancer pain model. Consistent with these data, upregulation of Bmp2 in the bone cancer pain model was validated by immunohistochemistry, real-time quantitative polymerase chain reaction, and western blotting. Importantly, intrathecal administration of siRNA significantly reduced Bmp2 transcription and ameliorated bone cancer pain in rat as shown by paw withdrawal mechanical threshold and spontaneous and movement-evoked pain-like behaviors. In conclusion, we have characterized the comprehensive gene expression profile of dorsal root ganglion from a bone cancer pain rat model by RNA-sequencing and identified Bmp2 as a potential therapeutic target for bone cancer pain treatment.

Project description:BackgroundPrimary and metastatic cancers that affect bone are frequently associated with severe and intractable pain. The mechanisms underlying the development of bone cancer pain are largely unknown. The aim of this study was to determine whether enhanced excitability of primary sensory neurons contributed to peripheral sensitization and tumor-induced hyperalgesia during cancer condition. In this study, using techniques of whole-cell patch-clamp recording associated with immunofluorescent staining, single-cell reverse-transcriptase PCR and behavioral test, we investigated whether the intrinsic membrane properties and the excitability of small-sized dorsal root ganglion (DRG) neurons altered in a rat model of bone cancer pain, and whether suppression of DRG neurons activity inhibited the bone cancer-induced pain.ResultsOur present study showed that implantation of MRMT-1 tumor cells into the tibial canal in rats produced significant mechanical and thermal hyperalgesia in the ipsilateral hind paw. Moreover, implantation of tumor cells provoked spontaneous discharges and tonic excitatory discharges evoked by a depolarizing current pulse in small-sized DRG neurons. In line with these findings, alterations in intrinsic membrane properties that reflect the enhanced neuronal excitability were observed in small DRG neurons in bone cancer rats, of which including: 1) depolarized resting membrane potential (RMP); 2) decreased input resistance (Rin); 3) a marked reduction in current threshold (CT) and voltage threshold (TP) of action potential (AP); 4) a dramatic decrease in amplitude, overshot, and duration of evoked action potentials as well as in amplitude and duration of afterhyperpolarization (AHP); and 5) a significant increase in the firing frequency of evoked action potentials. Here, the decreased AP threshold and increased firing frequency of evoked action potentials implicate the occurrence of hyperexcitability in small-sized DRG neurons in bone cancer rats. In addiotion, immunofluorescent staining and single-cell reverse-transcriptase PCR revealed that in isolated small DRG neurons, most neurons were IB4-positive, or expressed TRPV1 or CGRP, indicating that most recorded small DRG neurons were nociceptive neurons. Finally, using in vivo behavioral test, we found that blockade of DRG neurons activity by TTX inhibited the tumor-evoked mechanical allodynia and thermal hyperalgesia in bone cancer rats, implicating that the enhanced excitability of primary sensory neurons underlied the development of bone cancer pain.ConclusionsOur present results suggest that implantation of tumor cells into the tibial canal in rats induces an enhanced excitability of small-sized DRG neurons that is probably as results of alterations in intrinsic electrogenic properties of these neurons. Therefore, alterations in intrinsic membrane properties associated with the hyperexcitability of primary sensory neurons likely contribute to the peripheral sensitization and tumor-induced hyperalgesia under cancer condition.

Project description:Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two major members of the neurotrophin family. Using immunohistochemistry and in situ hybridization histochemistry, we examined the effect of L5 spinal nerve ligation (SPNL), a neuropathic pain model, on the expression of BDNF in the uninjured L4 dorsal root ganglion (DRG). After L5 SPNL, both immunoreactivity for BDNF and the hybridization intensity for BDNF mRNA increased mainly in the small- and medium-sized neurons. The percentage of BDNF mRNA-expressing neurons increased in the ipsilateral L4 DRG compared with the contralateral DRG from the third to 28th day after ligation. A significantly greater number of BDNF-immunoreactive neurons were observed in the ipsilateral L4 DRG than contralateral side 14 d after ligation. To test the contribution of BDNF to the thermal hyperalgesia produced in this model, we intrathecally injected anti-BDNF antibody at third day after ligation. This treatment clearly attenuated thermal hyperalgesia for a few hours. Almost all BDNF mRNA-expressing neurons coexpressed trkA, a high-affinity NGF receptor, mRNA. The percentage of BDNF mRNA-expressing cells of trkA cells significantly increased in the ipsilateral L4 DRG 14 d after ligation. Furthermore, we examined the contribution of NGF on this phenotypic change using ELISA, Northern blot analysis, and anti-NGF antibody. NGF content in the ipsilateral L4 DRG linearly increased and reached a statistical significant level 14 d after L5 SPNL. Moreover, at this time point, the increase in NGF mRNA was observed in the ipsilateral L5 DRG and sciatic nerve, but not in the ipsilateral L4 DRG or L4 spinal nerve. Local application of anti-NGF antibody to the L4 spinal nerve beside the L5 spinal nerve-ligation site prevented the development of thermal hyperalgesia for 5 d after ligation. Our data suggest that BDNF, which increased in the uninjured L4 DRG neurons, acts as a sensory neuromodulator in the dorsal horn and contributes to thermal hyperalgesia in this neuropathic pain model. The contribution of locally synthesized NGF to thermal hyperalgesia was also demonstrated. These dynamic alterations in the expression and content of BDNF and NGF in the uninjured DRG neurons might be involved in the pathomechanisms of neuropathic pain.

Dataset Information

Up-regulation of brain-derived neurotrophic factor in the dorsal root ganglion of the rat bone cancer pain model.

Publications

Up-regulation of brain-derived neurotrophic factor in the dorsal root ganglion of the rat bone cancer pain model.

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