Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1

https://doi.org/10.1016/j.neuint.2006.08.017Get rights and content

Abstract

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED50 value of 10.6 mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.

Section snippets

Introductory statement

The capsaicin or vanilloid receptor VR1 is a member of the transient receptor potential (TRP) family and is thus also referred to as TRPV1 (Montell et al., 2002). It is predominantly expressed by nociceptive sensory neurons and functions as a cation channel (Caterina et al., 1997, Tominaga et al., 1998). TRPV1 is activated by a wide array of pain-producing stimuli including capsaicin, noxious heat and low pH (for reviews, see Cortright and Szallasi, 2004, Tominaga and Tominaga, 2005) and is

Oligonucleotides

Unmodified DNA oligonucleotides as well as phosphorothioates for in vitro and cell culture experiments were obtained from MWG Biotech AG (Ebersberg, Germany). Oligonucleotides for in vivo applications were purchased from IBA GmbH (Goettingen, Germany).

The sequences of oligonucleotides V15 and V15MM were:

  • V15: CAT GTC ATG ACG GTT AGG

  • V15MM: CAT GCT ATG AGC GTT GAG.

Cell culture and transfection

Cos-7 cells (African green monkey kidney fibroblasts) were grown in a humidified atmosphere at 37 °C with 5% CO2 in Dulbecco's modified

Results

To investigate the functional role of the vanilloid receptor TRPV1 in neuropathic pain processes in more detail, the analgesic effects of a low molecular weight receptor antagonist and an AS ODN directed against the TRPV1 mRNA were investigated and compared in vitro and in vivo. The plasticity of TRPV1 expression after the neuropathic lesion and under TRPV1 antisense treatment was monitored by immunocytochemistry.

Discussion

The aim of the present study was to investigate the involvement of TRPV1 in neuropathic pain of rats subjected to SNL. Two complementary approaches have been employed to this end: we used a low molecular weight compound that acts as a receptor antagonist as well as AS ODNs against the TRPV1 mRNA. The AS ODN has previously been shown to induce RNase H mediated cleavage of TRPV1 mRNA in vitro (Kurreck et al., 2002a) and to silence TRPV1-GFP expression in cell culture (Grünweller et al., 2003).

Acknowledgments

We thank Sabine Hees, Petra Lattermann, Elke Rodenberg-Frank, Petra Sack, Heidemarie Schneider, Elke Schumacher, Marion Zibuschka, Thomas Vanderbrück and Birgit Bieber for excellent technical assistance. This study was supported by the German Ministry for Research and Technology (Grant 01GG9818/0) and the RiNA network for RNA technologies (financed by the City of Berlin, the Federal Ministry of Education and Research and the European Regional Development Fund).

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