Decoy strategy targeting the brain-derived neurotrophic factor exon I to attenuate tactile allodynia in the neuropathic pain model of rats

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Abstract

The mechanism underlying neuropathic pain is still largely unclear. Recently, much attention has been focused on the role of brain-derived neurotrophic factor (BDNF) as a neuromodulator in the spinal cord. We previously reported that the expression of Bdnf exon I mRNA was remarkably up-regulated in the dorsal root ganglion (DRG) neurons with the rat L5 spinal nerve ligation (SNL) model. In the present study, we investigated whether neuropathic pain response would be reduced by the inhibition of the Bdnf exon I in the rat SNL model. We identified the promoter region of exon I and synthesized the decoy ODNs targeting the region. Reverse transcription-polymerase chain reaction analysis confirmed that the decoy ODN treatment reduced SNL-induced Bdnf exon I mRNA up-regulation in ipsilateral L4 and L5 DRGs. Furthermore, post-treatment with the decoy ODNs significantly attenuated SNL-induced tactile allodynia. This study suggested that decoy ODNs targeting the Bdnf exon I might provide a novel analgesic strategy for the treatment of neuropathic pain.

Highlights

► The regulatory region of the Bdnf exon I was identified using luciferase assay. ► The promoter activity was found in a 35-bp sequence in this region. ► We adopted the 35-bp fragment for the decoy ODN strategy to treat neuropathic pain. ► The Bdnf exon I mRNA level was reduced by the decoy ODN treatment. ► Intrathecal injection of the decoy ODN attenuated neuropathic pain behavior in rats.

Introduction

Neuropathic pain is a debilitating pain that is characterized by spontaneous pain, allodynia, and hyperalgesia. Despite considerable research during the past decades, the mechanism of neuropathic pain is still largely unknown. Furthermore, neuropathic pain responds poorly to most of the commonly used analgesic drugs.

The brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, may act as a survival factor for the dorsal root ganglion (DRG) neurons [1] and may also serve as a neurotransmitter and/or neuromodulator within the dorsal horn of the spinal cord [2]. Some evidence has revealed that the Bdnf mRNA expression in DRG neurons was up-regulated in animal pain models, in conditions such as sciatic axotomy [3], inflammation [4] and spinal nerve ligation (SNL) [5]. In addition, increased BDNF levels are transported anterogradely to the central terminals in the spinal dorsal horn [6] and involved in spinal plasticity and central sensitization [7].

The rat Bdnf gene consists of nine exons (exons I through IX), which are transcribed into splice-variant mRNAs separately, and all exons are regulated by distinct promoters [8]. In our previous study, among all variants, the Bdnf exon I variant showed the greatest response to peripheral inflammation in rats and nerve growth factor (NGF) stimulation in cultured rat DRG neurons [9]. Subsequently, we examined the expression profiles of the Bdnf transcripts in bilateral L4 and L5 DRGs two weeks after an L5 SNL with rats [10]. The L5 SNL induced increased expression of the Bdnf exon I variant in the ipsilateral DRG, indicating that the Bdnf exon I variant is likely to play an important role in processing SNL-induced pain information. Therefore, we hypothesized that inhibition of the Bdnf exon I variant would block the neuropathic pain response to peripheral nerve injury.

For the specific knockdown of the Bdnf exon I variant, we used the decoy oligodeoxynucleotide (ODN) strategy. This strategy involves the intracellular delivery of double-stranded ODNs corresponding to a specific promoter region, resulting in the inhibition of sequence-specific transcription factors and the transactivation of the target gene [11]. In this study, we aimed to detect the promoter region of exon I, to synthesize decoy ODNs and to investigate the effect of the decoy ODNs on tactile allodynia in a rat neuropathic pain model.

Section snippets

Animals

Male Wistar rats (CLEA Japan Inc., Tokyo, Japan) weighing 190–220 g at the time of surgery were used. Animals were housed individually in cages under a 12–12 h light–dark cycle with free access to food and water. The experimental rats were allowed to acclimatize to the facility for 5–7 days prior to surgery. All animals were treated in accordance with the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals issued by the International Association for the Study of Pain [12]

Identification of the upstream promoter region of the Bdnf exon I

To identify the proximal promoter region of the Bdnf exon I, we generated three promoter deletion constructs: S1–AS1, S2–AS1 and S3–AS1 (Table 1). The highest transcriptional activity was observed with the S1–AS1 promoter region, 7.7-fold higher than the promoterless pGL4.14 basic vector. In contrast, deletion of the S1–S2 region dramatically decreased transcriptional activity, indicating that this region probably contained positive regulatory elements essential for the activation of the Bdnf

Discussion

In the present study, we found significant promoter activity in a 219-bp fragment including the 5′ flanking region of the Bdnf exon I. Dividing the region into seven fragments, we synthesized decoy ODNs targeting the Bdnf exon I variant. RT-PCR analysis showed that the up-regulated exon I mRNA level in the ipsilateral DRGs after the SNL was reduced by intrathecal administration of the decoy ODN. We also investigated the effects of the decoy ODN treatments on mechanical allodynia in the rat L5

Conflict of interest

None declared.

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    Present address: Department of Anesthesiology, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.

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