Decreasing P2X3 Receptor Expression of Ipsilateral Intact DRG Neuron in Rats with SNL-induced Neuropathic Pain 

Partially Contribute to Electroacupuncture Analgesia

LIANG Yi, LI Guangwen, GU Yanping, CHEN Yong, HUANG Liyen Mae 

(1 Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province 310053, China 2 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX77555, USA)

Abstract: Background: Nerve injuries caused by surgeries, lesions or diseases often leads to the development  of  neuropathic  pain,  which  was  difficulty  to  treat  with  only  40-60%  of  people achieving partial relief by medication. Neuropathic pain often accompanies with spontaneous pain, allodynia, hyperalgesia as well as dysesthesia and chronic pain with neuropathic characteristics is more  prevalent  (6.9-10.6%)  in  the  general  population.  Electroacupuncture  (EA),  a  method combining acupuncture and electrical stimulation together, has been widely used to treat painful condition in clinics and preclinical investigations. Furthermore, accumulating studies showed that EA exerted potential analgesic effects on neuropathic pain evoked by various nerve injuries, such as spinal nerve ligation (SNL), chronic constriction injury (CCI) and spared nerve injury of sciatic nerve (SNI). However, the underlying mechanisms of EA analgesia are not fully understood. The P2X3 receptor (P2X3R), a member of the P2X purinoceptor family, is a ligand-gated cationic channel activated by the binding of allogenic neurotransmitter adenosine 5'-triphosphate (ATP). Several lines of evidence demonstrated that the P2X3R plays a key role in the pathobiology of neuropathic pain.  Upregulation  of  P2X3R  expression  or  enhanced  activity  contributes  to  the occurrence  and  maintenance  of  allodynia  and  hyperalgesia  arising  from  nerve  injury,  while administration  of  selective  antagonists  or  antisense  oligonucleotide  of  P2X3R  reverse  that phenomenon. Furthermore studies have revealed that the P2X3Rs localized on primary sensory neuron and afferent nerve endings serve as targets for extracellular ATP released from damaged cells,  to  initiate  a  nociceptive  signals.  These  findings  suggest  the  P2X3Rs  in  the  dorsal  root ganglion (DRG) are likely important targets for the treatment of neuropathic pain. EA has been shown to reduce P2X3R activation and expression in neuropathic pain evoked by CCI or chronic visceral hypersensitivity. It is still unclear whether EA analgesia is associated with the P2X3Rs in DRGs  of  SNL  rats.  SNL  rat  model  is  a  neuropathic  pain  model  produced  by  ligation  and/or transecting the L5 spinal nerve. The advantage of this  model is that uninjured (intact) ganglia neurons  can  be  distinguished  from  activating  transcription  factor  3  (ATF3)  labeled  damaged (axotomized) neurons. It had been reported that EA can reduce SNL-induced neuropathic pain by decreasing  transient  receptor  potential  vanilloid  type  1  (TRPV1)  upregulation  in  ipsilateral adjacent undamaged L4 and L6 DRGs. However, little is known about the effect of EA on P2X3Rs located in intact or damaged DRG neurons of SNL rats, and whether this EA effect contributes to its  alleviation  of  SNL-induced  neuropathic  pain.  We  therefore  studied  the  effect  of  EA  on mechanical  allodynia  or  α,β-meATP-evoked  nociceptive  flinches  and  on  the  expression  of P2X3Rs in ipsilateral intact (L4) and damaged (L5) DRG in SNL rats. Methods: The neuropathic pain model was established by ligating left L5 spinal nerve in adult male Sprague-Dawley rats. Forty-seven rats were randomly divided into Control group (n=13), Spinal Nerve Ligation (SNL) + sham EA group (n=17) and SNL + EA group (n=17). EA stimulation at ipsilateral ST36 and BL60 acupoints (frequencies: 2 Hz, three stimulating intensities: 0.5-1-1.5mA, 10 min each) was given for 30 min, once per day for 7 days. Pain behaviors were determined as changes in paw withdrawal thresholds (PWTs) on day 0 (D0), D7, D13. On D14, α,β-meATP induced finches were determined.  Following  flinch  studies,  rats  were  sacrificed.  P2X3R  and  activating  transcription factor-3  (ATF3)  immunoreactivity  (ir)  in  cells  were  studied  using  fluorescence immunohistochemistry assay.  Results:  EA stimulation alleviated SNL-induced tactile allodynia and reduced α, β-me ATP induced nocifensive flinch response. After nerve injury, the percentage of P2X3R-ir cells in ipsilateral L5 DRG significantly decreased whereas that of L4 DRG was unchanged. In addition, less than 10% of neurons were ATF3 positive in ipsilateral L4 DRG whilst more than 80% in L5 DRG. P2X3R labeled cells, which mostly P2X3R+/ATF3-cells, in ipsilateral L4  DRG of  SNL  rats  were  decreased by  EA  stimulation.  Conclusions:  EA  stimulation  exerts favorable analgesic effect on neuropathic pain, but has no effect on nerve injury recovery. Thus, decreasing  P2X3R  expression  in  ipsilateral  undamaged  neurons  of  adjacent  undamaged  DRG contributes to EA analgesia.

Key words: Electroacupuncture, Analgesia, Neuropathic pain, P2X3 receptor, ATF3