Elsevier

Neuroscience

Volume 159, Issue 4, 10 April 2009, Pages 1422-1429
Neuroscience

Pain Mechanism
Botulinum toxin type a (150 kDa) decreases exaggerated neurotransmitter release from trigeminal ganglion neurons and relieves neuropathy behaviors induced by infraorbital nerve constriction

https://doi.org/10.1016/j.neuroscience.2009.01.066Get rights and content

Abstract

Many patients with trigeminal neuropathies suffer severe chronic pain which is inadequately alleviated with centrally-acting drugs. These drugs also possess severe side effects making compliance difficult. One strategy is to develop new treatments without central side effects by targeting peripheral sensory neurons, since sensory neuron excitability and neurotransmitter release increase in chronic pain states. Such treatments may include the highly purified botulinum toxin type A 150 kDa (BoNT/A) which reportedly blocks vesicular neurotransmitter release. We set out to determine if experimental trigeminal neuropathy induced by infraorbital nerve constriction (IoNC) in rats could alter neurotransmitter release from somata of trigeminal sensory neurons and if it could be attenuated by BoNT/A. Thus, we monitored the secretory activity of acutely dissociated trigeminal ganglion (TRG) neurons from naïve and IoNC rats by measuring the fluorescence intensity of the membrane-uptake marker (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). FM4-64 staining showed that neurons possess a pool of recycled vesicles which could be released by high KCl (75 mM) application. BoNT/A pre-treatment of acutely dissociated TRG neurons from naïve rats significantly reduced the rate of FM4-64 dye release. Neurons isolated from TRG ipsilateral to IoNC exhibited significantly faster onset of FM4-64 release than neurons contralateral to IoNC (sham surgery). IoNC also produced long-lasting ipsilateral tactile allodynia, measured as large decreases of withdrawal thresholds to mechanical stimulation. Intradermal injection of BoNT/A in the area of infraorbital branch of the trigeminal nerve (IoN) innervation alleviated IoNC-induced mechanical allodynia and reduced the exaggerated FM4-64 release in TRG neurons from these rats. Our results suggest that BoNT/A decreases neuropathic pain behaviors by decreasing the exaggerated neurotransmitter release from TRG sensory neurons.

Section snippets

Trigeminal neuropathy model

All procedures regarding animal usage in this study were performed in accordance to specifications of an animal protocol approved by Okayama University (OKU-2007137). All experiments were conformed to relevant National Institutes of Health guidelines on the ethical use of animals. We minimized the number of animals used and their suffering. The unilateral constriction of the infraorbital branch of the trigeminal nerve (IoN) was made according to the method described by Vos et al. (1994). Adult

In vitro pre-treatment with BoNT decreases KCl-evoked FM4-64 release from somata of acutely isolated TRG neurons

Initially we measured FM4-64 fluorescence intensity by confocal microscopy (2.0 s intervals) before and after KCl stimulation in acutely isolated neurons from naive rats with or without BoNT pre-treatment (BoNT/A was pre-applied in vitro for 3 h). We previously reported that the basal decay rate in FM4-64 intensity is dependent on the rate of signal acquisition (photobleaching) and on background vesicular release, which varies between IB4 (+) and IB4 (−) neurons (Matsuka et al., 2007). Since it

Discussion

In this study we demonstrated that unilateral IoNC results in long-lasting (>2 weeks) tactile allodynia in the region innervated by the IoN. This is consistent with previous demonstrations that chronic constriction injury of the IoN produces behavioral alterations indicative of trigeminal neuropathic pain (Vos et al 1994, Idanpaan-Heikkila and Guilbaud 1999, Kitagawa et al 2006, Shinoda et al 2007). It is widely acknowledged that persistent hyperexcitability of primary sensory neurons is a

Conclusions

Our data show that unilateral IoNC in rats produces long-lasting behaviors of neuropathy which are concomitant with increased transmitter release from somata of TRG neurons acutely isolated from the side of the injury. We also demonstrate that peripheral injection of BoNT/A alleviates the pain behaviors of IoNC and decreases the exaggerated neurotransmitter release from acutely isolated ipsilateral TRG neurons. The data add to our understanding of neuropathic pain mechanisms and suggest that

Acknowledgments

This study was supported by a grant from the Ministry of Education, Science and Culture of Japan (No. 18390512), Ryobi Teien Memorial Foundation and Japanese Association for Dental Science.

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