<p style="text-align: center;">Decreasing P2X3 Receptor Expression of Ipsilateral Intact DRG Neuron in Rats with SNL-induced Neuropathic Pain&nbsp;</p><p style="text-align: center;">Partially Contribute to Electroacupuncture Analgesia</p><p style="text-align: center;">LIANG Yi, LI Guangwen, GU Yanping, CHEN Yong, HUANG Liyen Mae&nbsp;</p><p style="text-align: justify;">(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)</p><p style="text-align: justify;"><strong>Abstract</strong>: <strong>Background</strong>: Nerve injuries caused by surgeries, lesions or diseases often leads to the development&nbsp; of&nbsp; neuropathic&nbsp; pain,&nbsp; which&nbsp; was&nbsp; difficulty&nbsp; to&nbsp; treat&nbsp; with&nbsp; only&nbsp; 40-60%&nbsp; of&nbsp; 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&nbsp; prevalent&nbsp; (6.9-10.6%)&nbsp; in&nbsp; the&nbsp; general&nbsp; population.&nbsp; Electroacupuncture&nbsp; (EA),&nbsp; a&nbsp; 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&#39;-triphosphate (ATP). Several lines of evidence demonstrated that the P2X3R plays a key role in the pathobiology of neuropathic pain.&nbsp; Upregulation&nbsp; of&nbsp; P2X3R&nbsp; expression&nbsp; or&nbsp; enhanced&nbsp; activity&nbsp; contributes&nbsp; to&nbsp; the occurrence&nbsp; and&nbsp; maintenance&nbsp; of&nbsp; allodynia&nbsp; and&nbsp; hyperalgesia&nbsp; arising&nbsp; from&nbsp; nerve&nbsp; injury,&nbsp; while administration&nbsp; of&nbsp; selective&nbsp; antagonists&nbsp; or&nbsp; antisense&nbsp; oligonucleotide&nbsp; of&nbsp; P2X3R&nbsp; reverse&nbsp; 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,&nbsp; to&nbsp; initiate&nbsp; a&nbsp; nociceptive&nbsp; signals.&nbsp; These&nbsp; findings&nbsp; suggest&nbsp; the&nbsp; P2X3Rs&nbsp; in&nbsp; the&nbsp; dorsal&nbsp; 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&nbsp; of&nbsp; SNL&nbsp; rats.&nbsp; SNL&nbsp; rat&nbsp; model&nbsp; is&nbsp; a&nbsp; neuropathic&nbsp; pain&nbsp; model&nbsp; produced&nbsp; by&nbsp; ligation&nbsp; and/or transecting the L5 spinal nerve. The advantage of this&nbsp; model is that uninjured (intact) ganglia neurons&nbsp; can&nbsp; be&nbsp; distinguished&nbsp; from&nbsp; activating&nbsp; transcription&nbsp; factor&nbsp; 3&nbsp; (ATF3)&nbsp; labeled&nbsp; damaged (axotomized) neurons. It had been reported that EA can reduce SNL-induced neuropathic pain by decreasing&nbsp; transient&nbsp; receptor&nbsp; potential&nbsp; vanilloid&nbsp; type&nbsp; 1&nbsp; (TRPV1)&nbsp; upregulation&nbsp; in&nbsp; 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&nbsp; alleviation&nbsp; of&nbsp; SNL-induced&nbsp; neuropathic&nbsp; pain.&nbsp; We&nbsp; therefore&nbsp; studied&nbsp; the&nbsp; effect&nbsp; of&nbsp; EA&nbsp; on mechanical&nbsp; allodynia&nbsp; or&nbsp; α,β-meATP-evoked&nbsp; nociceptive&nbsp; flinches&nbsp; and&nbsp; on&nbsp; the&nbsp; expression&nbsp; of P2X3Rs in ipsilateral intact (L4) and damaged (L5) DRG in SNL rats. <strong>Methods</strong>: 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.&nbsp; Following&nbsp; flinch&nbsp; studies,&nbsp; rats&nbsp; were&nbsp; sacrificed.&nbsp; P2X3R&nbsp; and&nbsp; activating&nbsp; transcription factor-3&nbsp; (ATF3)&nbsp; immunoreactivity&nbsp; (ir)&nbsp; in&nbsp; cells&nbsp; were&nbsp; studied&nbsp; using&nbsp; fluorescence immunohistochemistry assay.&nbsp; <strong>Results</strong>:&nbsp; 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&nbsp; DRG of&nbsp; SNL&nbsp; rats&nbsp; were&nbsp; decreased by&nbsp; EA&nbsp; stimulation.&nbsp;<strong> Conclusions</strong>:&nbsp; EA&nbsp; stimulation&nbsp; exerts favorable analgesic effect on neuropathic pain, but has no effect on nerve injury recovery. Thus, decreasing&nbsp; P2X3R&nbsp; expression&nbsp; in&nbsp; ipsilateral&nbsp; undamaged&nbsp; neurons&nbsp; of&nbsp; adjacent&nbsp; undamaged&nbsp; DRG contributes to EA analgesia.</p><p><strong>Key words</strong>: Electroacupuncture, Analgesia, Neuropathic pain, P2X3 receptor, ATF3</p><p><br/></p>