Elsevier

Brain Research Bulletin

Volume 156, March 2020, Pages 58-66
Brain Research Bulletin

Research report
Aquaporin 4 knockout increases complete freund's adjuvant-induced spinal central sensitization

https://doi.org/10.1016/j.brainresbull.2020.01.004Get rights and content

Highlights

  • AQP4 knockout increases CFA-induced spinal central sensitization.

  • Activated spinal astrocytes with up-regulation of AQP4 in wild-type mice injected CFA.

  • AQP4 deficiency reduced CFA-induced up-regulation of GLT1 expression in the spinal cord dorsal horn.

Abstract

Growing evidence suggests a critical role of astrocytes for pain regulation. The water channel protein aquaporin 4 (AQP4), a functional regulator of astrocytes, is involved in various neurological disorders. However, the pathophysiological roles of AQP4 in pain conditions remain unclear. In the present study, we investigated the effect of AQP4 gene knockout in central sensitization induced by complete Freund's adjuvant (CFA). The behavioral analysis revealed that mechanical allodynia and thermal hyperalgesia were more severe in AQP4 null mice than those of wild-type controls over the course of 11 days following CFA intraplantar injection. CFA caused activation of astrocytes with upregulated expression levels of AQP4 and glutamate transporter 1 (GLT1) in the dorsal horn of the spinal cord. AQP4 deficiency reduced GLT1 up-regulation, causing persistent expression of the neuronal activation marker Fos within superficial dorsal horn neurons, including glutamatergic neurons. However, AQP4 deletion did not affect CFA-evoked proinflammatory cytokine expression in the spinal cord. Together, these results have shown that AQP4 absence intensifies CFA-induced spinal central sensitization, which is associated with reduced compensatory up-regulation of GLT1, subsequently increasing glutamatergic overexcitation. Therefore, targeting spinal cord AQP4 may serve as a potential strategy for treatment of peripheral inflammation-evoked hyperalgesia.

Introduction

Pain is an unpleasant subjective feeling and/or emotional experience associated with actual or potential tissue damages (Sandkühler, 2009). At present, the conduction pathway of pain has been identified, but the modulating mechanisms of pain remain elusive (Ji et al., 2016).

Transmission of nociception is controlled by numerous factors, of which glutamate (Glu), an excitatory neurotransmitter, plays a crucial role in the formation and persistence of pain (Osikowicz et al., 2013). It has become increasingly clear that strengthening of glutamatergic sensory synapses is one of major contributors to central sensitization (Dong et al., 2012; Gogas, 2005; Kanai and Hediger, 2003; Larsson and Broman, 2011). For instance, pharmacological inhibition of spinal glutamate transporters (GluTs) aggravates persistent pain (Ramos et al., 2010; Sung et al., 2003), whereas enhancing expression of GluTs by viral gene transfer mitigates chronic pain (Lin et al., 2011; Maeda et al., 2008). Therefore, modulation of glutamatergic transmission could potentially serve as a prospective therapeutic approach for the management of chronic pain (Gegelashvili and Bjerrum, 2019).

Astrocytes have been shown to modulate glutamatergic neurotransmission by Glu uptake from the synaptic cleft via their membrane-specific GluTs, including glutamate transporter 1 (GLT1) and glutamate aspartate transporter (GLAST) (excitatory amino acid transporter 1 and 2 in humans) (Pajarillo et al., 2019). Spinal astrocytes can be activated by excessive Glu released from primary sensory terminals of dorsal root ganglion (DRG) neurons and nociceptive interneurons in the spinal cord after nerve injury or peripheral inflammation (Sung et al., 2003). Therefore, enhancing astrocyte reuptake of Glu may be an effective approach for treatments of chronic pain (Gao and Ji, 2010; Ren, 2010; Yousuf and Kerr, 2016; Jin et al., 2013).

Aquaporin 4 (AQP4) is a subtype of AQPs found in the highest quantity within the central nervous system (CNS). It is specifically localized to perivascular endfeet of astrocytes, as well as lateral and basal membranes of ependymal epithelium cells (Nielsen et al., 1997). AQP4 is responsible for maintaining the homeostasis of water and ions in the CNS (Nagelhus and Ottersen, 2013). Moreover, AQP4 is implicated in regulating astrocyte uptake of Glu (Zeng et al., 2007). Early studies reported that Glu increases astrocyte water permeability, with AQP4 being the molecular target for this effect (Gunnarson et al., 2008). Further studies have revealed that AQP4 deficiency impairs hippocampal long-term potentiation, with high extracellular Glu concentration and N-methyl-d-aspartate (NMDA) receptor-mediated currents in hippocampal dentate gyrus region (Yang et al., 2013). This suggests that AQP4-mediated astrocyte reuptake of Glu is involved in the modulation of signaling pain. In the present study, we examined this possibility in a mouse model of peripheral inflammation, utilizing intraplantar injection of complete Freund's adjuvant (CFA).

Section snippets

Animals

The AQP4 gene knockout (KO) mice with a CD1 genetic background were created in our laboratory (Fan et al., 2005). Three-month old male AQP4 KO mice and wild-type (WT) mice were randomly divided into CFA-injected group and saline-injected group. The animals were housed under standard laboratory conditions, with ad libitum access to food and water. The experiment was approved by the Animal Ethical and Welfare Committee of Nanjing Medical University (NMU) and conducted in accordance with the

AQP4 deficiency increases CFA-induced mechanical allodynia and thermal hyperalgesia

The nociceptive thresholds of mechanical and thermal stimuli were comparable between AQP4 KO and WT mice at the baseline condition. Intraplantar injection of CFA reduced paw withdrawal thresholds to non-noxious mechanical stimuli in the both mouse genotypes at day 1 and day 3 (Treatment effect: F1,44 = 53.459, p < 0.001, Genotype effect: F1,44 = 1.096, p = 0.312; repeated-measures ANOVA, n = 12 per group). However, at day 7, 9 and 11, the thresholds were significantly lower in the AQP4 KO-CFA

Discussion

Chronic or persistent pain is one of the primary causes of physical and mental impairments, seriously reducing the quality of life and work efficiency (Mazereeuw et al., 2018). There are a variety of drugs, and non-pharmacological approaches, used to relieve pain (Vorobeychik et al., 2011). However, the long-term use of analgesic drugs, especially opioids, is highly addictive, and also causes serious side effects including respiratory depression and even death (Stein, 2018). Therefore, it is

Author statement

All of the authors seriously declare that neither the submitted paper nor any similar paper, either in whole or in part, has been or will be submitted to or published in any other primary scientific journal. The experiment was approved by the Animal Ethical and Welfare Committee of Nanjing Medical University (NMU) and conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of NMU. All of the authors are aware of and agree to the content of the

Declaration of Competing Interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Acknowledgements

This work was supported by a grant from the Natural Science Foundation of Jiangsu Educational Department, China (09KJA310003).

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