Development of an evaluation system for analgesic drugs targeted to allodynia-specific pain using BOLD fMRI
Naoya Yuzuriha1, Sosuke Yoshinaga1, Hiroshi Sato2, Sokichi Honda3, Keisuke Tamaki3, Toshihiro Sekizawa3, Akihiko Fujikawa3, and Hiroaki Terasawa1

1Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan, 2Bruker Biospin K.K., Yokohama, Japan, 3Drug Discovery Research, Astellas Pharm. Inc., Tsukuba, Japan

Synopsis

The aim of this study is to evaluate the analgesic effect of pregabalin on allodynia-specific pain. fMRI images of fibromyalgia model rats were acquired, before and after pregabalin administration. When treated with saline, the BOLD signal intensities in the S1, IC, and TH were increased upon the laser stimulation by up to 1.7%, 1.3%, and 1.8%, respectively. In contrast, in the pregabalin treated rats, no BOLD responses were detected. It is conceivable that the pain signals were inhibited by pregabalin, and thus the stimulation-induced BOLD responses were not observed. We successfully observed the suppression of allodynia-specific pain responses by pregabalin.

Introduction

Fibromyalgia and neuropathic disorders characterized by chronic pain induce the pathological condition “allodynia”, in which a stimulus that is normally not painful causes pain sensations. One of the hypotheses about the pathogenic mechanism of allodynia suggested that the propagation of pain signals caused by the demyelination of Aδ-fibers generates the symptoms of allodynia [1]. Recent evidence has indicated that a 532 nm green laser can stimulate the TRPV1 and TRPV4 ion channels, and activate the Aδ- and C-fibers without direct contact [2]. Thus, the pain signals propagated from Aβ-fibers to Aδ- and C-fibers, which are thought to be the cause of allodynia, can be extracted (Fig.1). The aim of this study is to elucidate the allodynia-specific neural circuits activated by green laser stimulation in the reserpine-induced myalgia (RIM) rat, an animal model of fibromyalgia with the symptoms of allodynia [3], using the blood oxygenation level dependent (BOLD) technique. We previously reported the allodynia-specific pain responses evoked by the laser stimulation [4]. In this study, we tried to evaluate the analgesic effect of pregabalin, a treatment for fibromyalgia, on the allodynia-specific pain.

Methods

To produce the RIM rats, a reserpine solution was injected subcutaneously (1 mg/kg) into Sprague Dawley rats once daily, for three consecutive days [3]. Three RIM rats (272–286 g) were used for fMRI experiments. The rats were ventilated in a N2/O2 70/30 mixture, and treated with gallamine. The rats were anesthetized with urethane (1.25 g/kg i.p.). MRI experiments were performed with a 7.0 Tesla Bruker Biospec 70/20 scanner and a rat brain 4-channel phased array surface coil. Functional data were acquired with a 4-shot GRE-EPI sequence (TR 500 ms, TE 15 ms, FA 45°, 13 slices, 0.6 mm slice thickness, matrix 64 x 64, FOV 2.56 x 2.56 cm). The rats were treated with saline (10 mL/kg i.v.), and 30 minutes later the BOLD experiments were performed. The green laser with the 350 mW output power and 2 s irradiation time, which evoked the allodynia-specific pain in the RIM rats [4], was used to irradiate the left hind paws 5 times every 2 minutes, during 12 minutes of EPI continuous scans. After the first BOLD experiment, the rats were treated with pregabalin (10 mg/kg i.v.), and 30 minutes later the same BOLD experiments were performed. The Independent Component Analysis (ICA) was performed with the FSL software. The brain regions that showed periodic BOLD responses with the same frequency as the laser stimulation (8.3 mHz) were searched. The BOLD signal intensity was analyzed with the SPM8 software.

Results

When the rats were treated with saline, through the ICA, the primary somatosensory cortex (S1), insular cortex (IC), and thalamus (TH) were found to exhibit periodic BOLD responses with the same frequency as the laser stimulations (8.3 mHz) (Fig.2). Upon the laser stimulation, the BOLD signal intensities in the S1, IC, and TH were increased by up to 1.7%, 1.3%, and 1.8%, respectively (Fig.3). In contrast, when the rats were treated with pregabalin, no periodic BOLD responses were detected by the ICA. We also analyzed the BOLD responses with the SPM8 for the S1, IC, and TH, but no significant changes due to BOLD responses were detected.

Discussion

When the rats were treated with saline, S1, IC, and TH were activated, consistent with our previous study [4]. Pregabalin reportedly binds to the α2δ subunit of the voltage-dependent calcium channel in Aδ- and C-fibers and decreases the release of neurotransmitters, resulting in the analgesic effect. It is conceivable that the pain signals in Aδ- and C-fibers were inhibited by pregabalin, and thus the stimulation-induced BOLD responses were not observed.

Conclusions

We successfully observed the suppression of allodynia-specific pain responses by pregabalin, using the 532 nm green laser and BOLD-fMRI. The 532 nm green laser is transparent to water molecules and easy to use for local stimulation without direct contact, and thus is ideal for allodynia studies. Our experimental system facilitates the elucidation of the allodynia-specific neural circuits, and provides a robust clinical and preclinical evaluation system for new analgesic agents.

Acknowledgements

We gratefully acknowledge Masayasu Takahashi and Rikita Araki for fruitful discussions.

References

[1] Ueda H., Pharmacol. Ther. 109, 57–77 (2006), [2] Wang L. et al., Cells. 3, 662–673 (2014), [3] Nagakura Y. et al., PAIN 146, 26–33 (2009), [4] Yuzuriha N. et al., Proc. Intl. Soc. Mag. Reson. Med. 3036 (2014), [5] Claudia S. B. et al., J. Neurosci. 29, 4076–4088 (2009)

Figures

Fig.1 Hypothetical schematic model of allodynic pain signaling and mechanism of pregabalin.

Fig. 2 Representative statistical brain activation maps of single RIM rats treated with saline (left) or pregabalin (right). Oscillation frequencies of the MR signals in the mapped regions included the frequency of laser stimulations.

Fig.3 Time courses of the BOLD signal intensity of RIM rats (N = 3) treated with saline (left) or pregabalin (right). Green laser stimulations were performed at the green lines.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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