Regular ArticleInvolvement of medullary GABAergic system in extraterritorial neuropathic pain mechanisms associated with inferior alveolar nerve transection
Introduction
It is well known that the IAN transection, such as an extraction of a mandibular third molar tooth and a mandibular advancement surgery, sometimes causes chronic neuropathic pain in the maxillary region innervated by the infraorbital nerve (Renton et al., 2012). However, the detailed mechanisms underlying such extraterritorial neuropathic pain in the orofacial region are still unknown. We have developed the rats model with trigeminal nerve injury (inferior alveolar transection: IANX) which is a branch of the 3rd division of the trigeminal nerve. The model induced mechanical allodynia and hyperalgesia in the whisker pad area innervated by the 2nd division of the trigeminal nerve (Okada-Ogawa et al., 2009, Ogawa et al., 2005, Tsuboi et al., 2004, Nomura et al., 2002, Iwata et al., 2001). Although glial mechanisms in the trigeminal spinal subnucleus caudalis (Vc, also known as the medullary dorsal horn) contribute to this extraterritorial neuropathic pain in IANX rats (Kiyomoto et al., 2013, Shibuta et al., 2012, Tsuboi et al., 2011, Okada-Ogawa et al., 2009), it is still unclear whether other mechanisms are involved. It has been widely considered that excitatory amino acid mechanisms associated with the enhancement of primary afferent inputs to dorsal horn nociceptive neurons are involved in the development of neuropathic pain following peripheral nerve injury (Yan et al., 2013, Kayser et al., 2011, Thompson et al., 1994, Garrison et al., 1993, Mao et al., 1992, Meldrum, 1992), but that functional changes in dorsal horn inhibitory system may also contribute to the enhancement of dorsal horn nociceptive neuronal excitability (Fukuoka et al., 1998, Dubner, 1997, Sommer and Myers, 1995).
GABAergic neurons are known to be present and exert inhibitory effects on spinal dorsal horn (SDH) nociceptive neurons and contribute to pain relief (Daniele and MacDermott, 2009, Dougherty and Hochman, 2008, Wiesenfeld-Hallin et al., 1997, Hiura et al., 1996, Otsuka and Yanagisawa, 1990). Recently, it has been reported that spinal nerve injury causes a downregulation of the potassium-chloride co-transporter 2 (KCC2) and a depolarizing shift in the reversal potential of GABA-mediated currents in SDH or Vc neurons, contributing to the development of neuropathic pain in the foot and orofacial area (Wei et al., 2013, Austin and Delpire, 2011, Miletic and V. M., 2008, Coull et al., 2003). Furthermore, apoptotic loss of the GABAergic neurons in the SDH is also thought to be involved in neuropathic pain (Gwak and Hulsebosch, 2011, Meisner et al., 2010, Moore et al., 2002) following spinal nerve injury. In addition, the GABA transporter inhibition in SDH neurons has been reported to contribute to neuropathic and inflammatory pain (Yamada et al., 2012, Miletic et al., 2003), although there are some reports that an increased expression of GABA transporters contributes to hyperalgesia (Xu et al., 2008, Hu et al., 2003, Ng and Ong, 2001), and that GABA transporter inhibition causes analgesic effect in a neuropathic pain model (Li et al., 2010, Daemen et al., 2008). As a result, the involvement of GABAergic inhibitory neurons in development of neuropathic pain is unclear.
Extracellular signal-regulated kinase (ERK) is known to be phosphorylated in SDH neurons following peripheral noxious stimulation, and the number of phosphorylated ERK (pERK)-immunoreactive (IR) cells increases as the noxious stimulus intensity increases (Suzuki et al., 2013, Ji et al., 1999). ERK phosphorylation is also known to be involved in the enhancement of the excitability of SDH and Vc nociceptive neurons following nerve injury or tissue inflammation (Noma et al., 2008, Shimizu et al., 2006, Ji et al., 1999). Therefore, pERK has been used as a marker of the excitability of nociceptive neurons in the SDH and Vc (Noma et al., 2008, Shimizu et al., 2006, Ji et al., 1999). However, it is unclear whether ERK phosphorylation occurs in GABAergic inhibitory neurons as well as excitatory neurons in the SDH or Vc following noxious stimulation.
We hypothesized that the activation of GABAergic neurons after the downregulation of KCC2 via the pERK pathway in the Vc contributes to the development of orofacial neuropathic pain following trigeminal nerve injury. To evaluate this hypothesis, we used the rats IANX model and transgenic rats expressing Venus, a yellow fluorescent protein, with the vesicular GABA transporter (VGAT) in order to determine if the functional changes in the GABAergic system in Vc are involved in the mechanisms underlying extraterritorial neuropathic pain in the orofacial region following IANX.
Section snippets
Materials and methods
This study was approved by the Animal Experimentation Committee at Nihon University. The procedures were performed according to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
VGAT-VenusA positive cells following IANX
The VGAT-VenusA positive cells were widely observed in the Vc, and most of them also showed NeuN-IR (yellow staining in Fig. 1A), none of them showed GFAP- or Iba1-IR (Figs. 1B, C).
The Vc was dorso-ventrally subdivided into 3 divisions, dorsal, middle and ventral portions innervated by the 3rd, 2nd and 1st branches of the trigeminal nerve, respectively (Noma et al., 2008). A large number of VGAT-VenusA positive cells were observed in the Vc receiving afferent input from the 2nd branch regions
Discussion
This study has demonstrated for the first time that the GABA transporter VGAT neurons was reduced at 7 days after IANX, and phosphorylation of ERK in VGAT-VenusA-positive neurons increased at 21 days after IANX in Vc. The HWT to mechanical stimulation of the whisker pad skin was significantly decreased in IANX rats compared with sham rats on days 7 and 21 after IANX. The background activity and mechanical evoked responses of Vc nociceptive neurons were significantly depressed after muscimol
Conclusion
In summary, the number of GABAergic neurons decreased in Vc at the early period after IANX, and the functional change from inhibitory to excitatory may be induced in GABAergic interneurons via downregulation of KCC2 in Vc at the late period after IANX. These morphological and functional changes of the GABAergic neuronal network in Vc at different time periods following IANX may be a mechanism contributing to extraterritorial orofacial neuropathic pain associated with trigeminal nerve injury.
Conflict of interest
The authors declare no financial or other conflicts of interest concerning the present study.
Acknowledgments
VGAT-Venus transgenic rats were generated by Drs. Y. Yanagawa, M. Hirabayashi, and Y. Kawaguchi in National Institute for Physiological Sciences, Okazaki, Japan, using pCS2-Venus provided by Dr. A. Miyawaki. This study was supported in part by JSPS KAKENHI Grant Number 25463151 to A.O.
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