Down-regulation of K+–Cl− co-transporter 2 in mouse medullary dorsal horn contributes to the formalin-induced inflammatory orofacial pain

doi:10.1016/j.neulet.2009.03.107

Neuroscience Letters

Volume 457, Issue 1, 19 June 2009, Pages 36-40

https://doi.org/10.1016/j.neulet.2009.03.107 Get rights and content

Abstract

Cation chloride co-transporters, including K⁺–Cl⁻ co-transporter 2 (KCC2) and Na⁺–K⁺–Cl⁻ co-transporter 1 (NKCC1), are of particular importance to GABAergic transmission and thus involved in the development of hyperalgesia at the spinal level. However, there are fewer relevant reports in the trigeminal system. In this study, we investigated the behavioral changes and the accompanying change in the expressions of KCC2 and NKCC1 mRNAs in mouse medullary dorsal horn (MDH) after subcutaneous injection of formalin into the left vibrissa pad. Furthermore, we observed the behavioral changes following intracisternal injection of KCC2 antisense oligodeoxynucleotides (ASO) into naïve mice. Subcutaneous injection of formalin-induced a significant increase in the number of face rubbing events which are the indicators of spontaneous pain. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that, compared to the saline control or the contralateral side, the ipsilateral mRNA level of KCC2 but not NKCC1, was significantly reduced in formalin-injected mice during phase 1 observation, followed by gradual recovery. Intracisternal injection of KCC2 ASO into naïve mice led to behavioral hypersensitivity similar to the hyperalgesia observed in formalin experiments. These findings indicate that peripheral inflammation induces down-regulation of KCC2 in the MDH, which may in turn facilitate the development of acute inflammatory pain. These results also suggest that preventing the down-regulation of KCC2 is a possible way to combat orofacial pain.

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Acknowledgements

This study was supported by the grant from the Natural Science Foundation of China (30801293, 30770694, 30600176), Program for New Century Excellent Talents in University and Innovation Research Team Program of Ministry of Education of China (IRT0560). The authors’ gratitude also goes to Prof. Jia-Yong Fan in the Department of Foreign Language of the Fourth Military Medical University for the kind help in English.

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Its decrease in ventral and dorsal horns causes disinhibition leading to motor (spasticity, hyperreflexia; Boulenguez et al., 2010; Modol et al., 2014) and sensory (neuropathic pain; Hasbargen et al., 2010; Lu et al., 2008) impairments, respectively. It is also reduced by inflammation through a microglia-dependent mechanism (Lin et al., 2017; Wu et al., 2009; Zhang et al., 2008), suggesting that the presence of inflammation during rehabilitation after SCI could further attenuate recovery by reducing KCC2 expression. Moreover, previous evidence showed that KCC2 expression in the lumbar spinal cord is upregulated by early exercise in motoneurons' membrane after SCI (Côté et al., 2014) and in dorsal horns after a peripheral nerve injury (López-Álvarez et al., 2015; Mòdol et al., 2014; Sánchez-Brualla et al., 2017).
Spinal cord injury (SCI) is associated with damage to musculoskeletal tissues of the spine. Recent findings show that pain and inflammatory processes caused by musculoskeletal injury mediate plastic changes in the spinal cord. These changes could impede the adaptive plastic changes responsible for functional recovery. The underlying mechanism remains unclear, but may involve the microglia-BDNF-KCC2 pathway, which is implicated in sensitization of dorsal horn neurons in neuropathic pain and in the regulation of spinal excitability by step-training. In the present study, we examined the effects of step-training and lumbar muscle inflammation induced by complete Freund's adjuvant (CFA) on treadmill locomotion in a mouse model of complete spinal transection. The impact on locomotor recovery of each of these interventions alone or in combination were examined in addition to changes in microglia and KCC2 expression in the dorsal and ventral horns of the sublesional spinal cord. Results show that angular motion at the hip, knee and ankle joint during locomotion were decreased by CFA injection and improved by step-training. Moreover, CFA injection enhanced the expression of the microglial marker Iba1 in both ventral and dorsal horns, with or without step-training. However, this change was not associated with a modulation of KCC2 expression, suggesting that locomotor deficits induced by inflammation are independent of KCC2 expression in the sublesional spinal cord. These results indicate that musculoskeletal injury hinders locomotor recovery after SCI and that microglia is involved in this effect.
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Trigeminal neuroplasticity underlies allodynia in a preclinical model of mild closed head traumatic brain injury (cTBI)
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These transporters are often involved in the central mechanism of inflammatory or neuropathic pain at the spinal level. Peripheral pain has been shown to alter K+-Cl− co-transporter 2 (KCC2) in neuropathic pain following spinal cord injury (Boulenguez et al., 2010; Cramer et al., 2008) in rat and in the formalin induced orofacial pain in mouse (Wu et al., 2009). The inhibition of KCC2 expression has been shown to directly cause a positive shift in the reversal potential of the GABAA response that establishes a depolarizing effect of GABA, which is excitatory (Rivera et al., 1999).
Post-traumatic headache (PTH) following TBI is a common and often persisting pain disability. PTH is often associated with a multimodal central pain sensitization on the skin surface described as allodynia. However, the particular neurobiology underlying cTBI-induced pain disorders are not known. These studies were performed to assess trigeminal sensory sensitization and to determine if sensitization measured behaviorally correlated with detectable changes in portions of the trigeminal sensory system (TSS), particularly trigeminal nucleus, thalamus, and sensory cortex. Thermal stimulation is particularly well suited to evaluate sensitization and was used in these studies. Recent advances in the use of reward/conflict paradigms permit use of operant measures of behavior, versus reflex-driven response behaviors, for thermal sensitization studies. Thus, to quantitate facial thermal sensitization (allodynia) in the setting of acute TBI, the current study utilized an operant orofacial pain reward/conflict testing paradigm to assess facial thermal sensitivity in uninjured control animals compared with those two weeks after cTBI in a rodent model. Significant reductions in facial contact/lick behaviors were observed in the TBI animals using either cool or warm challenge temperatures compared with behaviors in the normal animals. These facial thermal sensitizations correlated with detectable changes in multiple levels of the TSS. The immunohistochemical (IHC) studies revealed significant alterations in the expression of the serotonin (5-HT), neurokinin 1 receptor (NK1R), norepinephrine (NE), and gamma-aminobutyric acid (GABA) in the caudal trigeminal nucleus, thalamic VPL/VPM nucleus, and sensory cortex of the orofacial pain pathways. There was a strong correlation between increased expression of certain IHC markers and increased behavioral markers for facial sensitization. The authors conclude that TBI-induced changes observed in the TSS are consistent with the expression of generalized facial allodynia following cTBI. To our knowledge, this is the first report of orofacial sensitization correlated with changes in selected neuromodulators/neurotransmitters in the TSS following experimental mild TBI.
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In order to determine if the functional changes in the GABAergic system in the trigeminal spinal subnucleus caudalis (Vc) are involved in the mechanisms underlying extraterritorial neuropathic pain in the orofacial region following inferior alveolar nerve transection (IANX), mechanical noxious behavior, phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry and single neuronal activity were analyzed in vesicular GABA transporter (VGAT)-VenusA rats expressing fluorescent protein and the VGAT in Vc neurons.
The number of VGAT-VenusA positive neurons was significantly reduced in IANX rats than naive and sham rats at 7 days after nerve transection. The number of VGAT-VenusA positive pERK-immunoreactive (IR) cells was significantly increased in IANX rats at 21 days after IAN transection compared with naive and sham rats. The background activity and mechanical-evoked responses of Vc nociceptive neurons were significantly depressed after intrathecal application of the GABA receptor agonist muscimol in sham rats but not in IANX rats. Furthermore, the expression of potassium-chloride co-transporter 2 (KCC2) in the Vc was significantly reduced in IANX rats compared with sham rats. The head-withdrawal threshold (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 significant reduction of the HWT and significant increase in the number of VGAT-VenusA negative pERK-IR cells were observed in KCC2 blocker R-DIOA-injected rats compared with vehicle-injected rats on day 21 after sham treatment.
These findings revealed that GABAergic Vc neurons might be reduced in their number at the early period after IANX and the functional changes might occur in GABAergic neurons from inhibitory to excitatory at the late period after IANX, suggesting that the neuroplastic changes occur in the GABAergic neuronal network in the Vc due to morphological and functional changes at different time periods following IANX and resulting in the extraterritorial neuropathic pain in the orofacial region following trigeminal nerve injury.
Neural communication in the trigeminal ganglion contributes to ectopic orofacial pain
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Ectopic orofacial pain develops with local inflammation in remote orofacial structures. However, the mechanism underlying the spreading of pain to remote orofacial areas after local inflammation is still unknown. We investigated the functional significance of neural communication within the trigeminal ganglion (TG) via nerve growth factor (NGF) or calcitonin gene-related peptide (CGRP) in relation to whisker pad skin hyperalgesia following complete Freund's adjuvant (CFA) injection into the lower lip. Heat hyperalgesia and mechanical allodynia were induced in the ipsilateral whisker pad skin following lower lip inflammation, and reversed by local injection of transient receptor potential vanilloid 1 (TRPV1) or P2X₃ receptor (P2X₃R) antagonist, respectively. The heat hyperalgesia was diminished by neutralizing anti-NGF antibody administration into the lower lip and intraganglionic administration of tyrosine kinase receptor inhibitor. TG neurons that innervate the whisker pad skin and lower lip both expressed labeled NGF, which was administrated into the lower lip. Moreover, NGF and CGRP expression in the TG was increased following lower lip inflammation. The number of TRPV1-positive neurons that innervate the whisker pad skin was increased following lower lip inflammation and this increase was annulled by anti-NGF administration. P2X₃R- and CGRP-positive TG neurons innervating whisker pad skin were also increased by lower lip inflammation. The present findings suggest that induced NGF and CGRP following local inflammation in the lower lip increases TRPV1 and P2X₃R in TG neurons, which may result in the development of the whisker pad ectopic pain.

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¹: These authors have contributed equally to this article.

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Down-regulation of K+–Cl− co-transporter 2 in mouse medullary dorsal horn contributes to the formalin-induced inflammatory orofacial pain

Abstract

Section snippets

Acknowledgements

Pharmacol. Res.

Trends Neurosci.

Pain

Neuroscience

Pain

Prog. Neurobiol.

Trends Neurosci.

Trends Pharmacol. Sci.

Neurosci. Res.

Trends Neurosci.

Brain Res.

Neuron

Down-regulation of K⁺–Cl⁻ co-transporter 2 in mouse medullary dorsal horn contributes to the formalin-induced inflammatory orofacial pain