Lysophosphatidylcholine causes neuropathic pain via the increase of neuronal nitric oxide synthase in the dorsal root ganglion and cuneate nucleus Hsin-Ying Wang a , Yi-Ju Tsai b , Seu-Hwa Chen a, c , Chi-Te Lin a , June-Horng Lue a, ⁎ a Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan b School of Medicine, College of Medicine, Fu Jen Catholic University, Taipei, Taiwan c Department of Anatomy, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan abstract article info Article history: Received 11 February 2013 Received in revised form 13 March 2013 Accepted 18 March 2013 Available online 26 March 2013 Keywords: c-Fos Neuropathic pain L-NAME 7-NI Electrical stimulation In this study, we investigated the role of nitric oxide (NO) in lysophosphatidylcholine (LPC) induced periph- eral neuropathy by the use of nitric oxide synthase (NOS) inhibitors and NO donor. We found that LPC treat- ment of the median nerve induced neuropathic pain behaviors (allodynia and hyperalgesia) and nerve demyelination. Immunohistochemistry revealed that the amounts of neuronal NOS-like immunoreative (nNOS-LI) neurons in both the dorsal root ganglion (DRG) and cuneate nucleus (CN) increased and peaked at 1 week after LPC treatment. Following electrical stimulation of the LPC-treated nerve, the number of c-Fos-LI neurons in the ipsilateral CN also increased in a dose-dependent manner following LPC injection and peaked at 1 week. Administration of L-NAME (N ω -Nitro-L-arginine methyl ester) or 7-NI (7-nitroindazole) 1 week after 4% LPC injection attenuated tactile allodynia and thermal hyperalgesia. How- ever, the application of the NO donor S-Nitroso-N-acetylpenicillamine (SNAP) only exacerbated thermal hyperalgesia. After electrical stimulation of the LPC-treated median nerve, the number of c-Fos-LI neurons in the CN diminished in the L-NAME and 7-NI groups, but increased in the SNAP group. Taken together, our findings suggest that advanced NO made by the dramatically increased number of nNOS in the DRG and CN might be involved in the neuropathic sensation and boosted neuronal activity in the CN after LPC treatment. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Lysophosphatidylcholine (LPC) is one of the plasma lipid compo- nents that transfers choline and fatty acid to tissues, and is made under both physiological and pathological conditions (Yokota and Hansson, 1995; Murugesan and Fox, 1996). Previous studies revealed that LPC treatment of the sciatic nerve resulted in mechanical allodynia and thermal hyperalgesia (Wallace et al., 2003; Inoue et al., 2008). Also, LPC injection induced nerve demyelination and an in- crease in pain-related protein levels, including neuropeptide Y (NPY), Nav 1.8, Nav 1.3, chemokines, and their receptors, in the dorsal root ganglion (DRG) (Wallace et al., 2003; Bhangoo et al., 2007). However, the effect of LPC treatment to the median nerve on the development of neuropathic pain behavior remains uncertain. It is well documented that nitric oxide (NO) is synthesized and regulated by three types of nitric oxide synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Peripheral nerve injury leads to changes in the number of nNOS-like immunoreactive (nNOS-LI) neurons in the DRG, spinal cord, and gracile nucleus (GN) (Zhang et al., 1993; Gonzalez-Hernandez and Rustioni, 1999; Ma et al., 2000; Lukacova et al., 2003; Liu et al., 2005). Although nNOS-LI neurons have been reported to be present in the cuneate nucleus (CN) (Valtschanoff et al., 1995; Wang et al., 2001, 2012), it is seldom known about the changes in the number of nNOS-LI neurons in the DRG and CN after LPC injection to the median nerve. A recent study further demonstrated that NO regulated the NPY re- lease from the injured median primary afferent terminals (PATs) and upregulated c-Fos expression in the CN after electrical stimulation (Wang et al., 2012). The expression of c-Fos in cuneothalamic projec- tion neurons (CTNs) in rats induced by electrical stimulation of the in- jured median nerve was associated with behavior signs of mechanical allodynia and thermal hyperalgesia (Day et al., 2001). Several studies have demonstrated that NO might mediate mechanical allodynia and Pharmacology, Biochemistry and Behavior 106 (2013) 47–56 Abbreviations: cGMP, cyclic guanosine monophosphate; CN, cuneate nucleus; CTNs, cuneothalamic projection neurons; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglion; GN, gracile nucleus; LPC, Lysophosphatidylcholine; L-NAME, N ω -Nitro-L-arginine methyl ester; MNT, median nerve transection; NADPH-d, nicotinamide adenine dinucleotide phosphate-diaphorase; 7-NI, 7-nitroindazole; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; NPY, neuropeptide Y; PATs, primary afferent terminals; PB, phosphate buffer; SNAP, S-Nitroso-N-acetylpenicillamine. ⁎ Corresponding author at: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, 1, Section 1, Jen Ai Rd, Taipei 10018, Taiwan. Tel.: +886 2 23123456 88178; fax: +886 2 23955804. E-mail address: thomas@ntu.edu.tw (J.-H. Lue). 0091-3057/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.03.002 Contents lists available at SciVerse ScienceDirect Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh