P2X7 Receptor Mediates Spinal Microglia Activation of Visceral Hyperalgesia in a Rat Model of Chronic Pancreatitis

Pei-Yi Liu; I-Hui Lee; Ping-Heng Tan; Yen-Po Wang; Chia-Fen Tsai; Han-Chieh Lin; Fa-Yauh Lee; Ching-Liang Lu

doi:10.1016/j.jcmgh.2015.07.008

P2X7 Receptor Mediates Spinal Microglia Activation of Visceral Hyperalgesia in a Rat Model of Chronic Pancreatitis

Cell Mol Gastroenterol Hepatol. 2015 Jul 22;1(6):710-720.e5. doi: 10.1016/j.jcmgh.2015.07.008. eCollection 2015 Nov.

Authors

Pei-Yi Liu¹, I-Hui Lee², Ping-Heng Tan³, Yen-Po Wang¹, Chia-Fen Tsai⁴, Han-Chieh Lin⁵, Fa-Yauh Lee⁵, Ching-Liang Lu⁶

Affiliations

¹ Institute of Brain Science School of Medicine, National Yang-Ming University, Taipei, Taiwan; Division of Gastroenterology, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
² Institute of Brain Science School of Medicine, National Yang-Ming University, Taipei, Taiwan; Department of Neurology, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
³ Department of Anesthesia and Critical Care and Department of Biomedical Engineering, E-DA Hospital/I-Shou University, Kaohsiung, Taiwan.
⁴ Institute of Brain Science School of Medicine, National Yang-Ming University, Taipei, Taiwan; Department of Psychology, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
⁵ Division of Gastroenterology, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; Department of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
⁶ Institute of Brain Science School of Medicine, National Yang-Ming University, Taipei, Taiwan; Division of Gastroenterology, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; Department of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

Abstract

Background & aims: Molecular mechanisms underlying the activated spinal microglia in association with the pain in chronic pancreatitis (CP) remain unknown. We tested whether P2X7R on spinal microglia mediates the pathogenesis of visceral pain using a CP rat model.

Methods: The CP model was induced via intraductal injection of 2% trinitrobenzene sulfonic acid into male Sprague-Dawley rats. Hyperalgesia was assessed based on the mechanical sensitivity to Von-Frey filaments (VFFs), and nocifensive behaviors were measured in response to electrical stimulation of the pancreas. Three weeks after CP induction, spinal cord samples were harvested for immunostaining, immunoblot, and real-time polymerase chain reaction analyses of the P2X7R. Changes in nocifensive behaviors and associated molecular effectors were assessed by blocking spinal cord P2X7R pharmacologically using the selective P2X7R antagonist brilliant blue G (BBG) or genetically using short interfering RNA (siRNA).

Results: CP induced a significant up-regulation of spinal P2X7R expression, which colocalized with a microglial marker (OX-42). Intrathecal administration of BBG significantly attenuated CP-related visceral hyperalgesia in response to VFF-mediated or electrical stimulation of the pancreas, which was associated with suppressed spinal expression of P2X7R and inhibited activation of spinal microglia. Intrathecal injection of siRNA to knock down P2X7R expression in the spinal cord would suppress the nociceptive behaviors in CP rats.

Conclusions: Spinal microglia P2X7R mediates central sensitization of chronic visceral pain in CP. BBG may represent an effective drug for the treatment of chronic pain in CP patients.

Keywords: ANOVA, analysis of variance; ATP, adenosine triphosphate sulfonic acid; BBG, brilliant blue G; Brilliant Blue G; CNS, central nervous system; CP, chronic pancreatitis; Chronic Visceral Pain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IT, intrathecal; P2X7R, P2X7 receptor; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Purinergic Receptors; TBS, Tris-HCl buffer solution; TNBS, trinitrobenzene sulfonic acid; VFF, von Frey filament; siRNA Knockdown; siRNA, small-interfering RNA.