Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection.

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 microg in 50 microL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed's lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

[1]  S. Teixeira,et al.  Expression of neuronal isoform of nitric oxide synthase in spinal neurons of neonatal rats after sciatic nerve transection , 2001, Neuroscience Letters.

[2]  Y. Turkoz,et al.  Nitric oxide : actions and pathological roles , 1997 .

[3]  A. Kihara,et al.  Differential regulation of the neuronal isoform of nitric oxide synthase in the superior colliculus and dorsal lateral geniculate nucleus of the adult rat brain following eye enucleation , 2006, International Journal of Developmental Neuroscience.

[4]  U. Förstermann,et al.  Transcription of Different Exons 1 of the Human Neuronal Nitric Oxide Synthase Gene Is Dynamically Regulated in a Cell- and Stimulus- Specific Manner , 2003, Biological chemistry.

[5]  G. Geisslinger,et al.  No NO, no pain? The role of nitric oxide and cGMP in spinal pain processing , 2009, Trends in Neurosciences.

[6]  T. Yaksh,et al.  Quantitative assessment of tactile allodynia in the rat paw , 1994, Journal of Neuroscience Methods.

[7]  N. Calcutt,et al.  Spinal pharmacology of tactile allodynia in diabetic rats , 1997, British journal of pharmacology.

[8]  J. Levine,et al.  Neonatal capsaicin attenuates mechanical nociception in the rat , 1996, Neuroscience Letters.

[9]  G. Carmignoto,et al.  Nitric Oxide-Producing Islet Cells Modulate the Release of Sensory Neuropeptides in the Rat Substantia Gelatinosa , 1998, The Journal of Neuroscience.

[10]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[11]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[12]  G. Gebhart,et al.  Nitric oxide mediates the thermal hyperalgesia produced in a model of neuropathic pain in the rat , 1992, Neuroscience.

[13]  R. Lo,et al.  A method for measurement of analgesic activity on inflamed tissue. , 1957 .

[14]  T. Yaksh.  Spinal systems and pain processing: development of novel analgesic drugs with mechanistically defined models. , 1999, Trends in pharmacological sciences.

[15]  T. Yamamoto,et al.  Role of Nitric Oxide in the Development of Thermal Hyperesthesia Induced by Sciatic Nerve Constriction Injury in the Rat , 1995, Anesthesiology.

[16]  T. Dawson,et al.  REVIEW ■ : Nitric Oxide: Actions and Pathological Roles , 1995 .

[17]  G. Gebhart,et al.  Production of endogenous nitric oxide and activation of soluble guanylate cyclase are required for N-methyl-D-aspartate-produced facilitation of the nociceptive tail-flick reflex. , 1992, European journal of pharmacology.

[18]  B. Sung,et al.  Nitric oxide mediates behavioral signs of neuropathic pain in an experimental rat model , 1998, Neuroreport.

[19]  S. Raja,et al.  Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice , 2007, Molecular pain.

[20]  S. Maier,et al.  Snake venom phospholipase A2s (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide , 2004, Pain.

[21]  S. Maier,et al.  Snake venom components enhance pain upon subcutaneous injection: an initial examination of spinal cord mediators , 2004, Pain.

[22]  A. Beaudet,et al.  Morphine and Pain-Related Stimuli Enhance Cell Surface Availability of Somatic δ-Opioid Receptors in Rat Dorsal Root Ganglia , 2006, The Journal of Neuroscience.

[23]  T. Meert,et al.  Comparison of five different rat models of peripheral nerve injury , 2005, Pharmacology Biochemistry and Behavior.

[24]  M. Hori,et al.  Inhibition of nitric oxide synthesis increases adenosine production via an extracellular pathway through activation of protein kinase C. , 1997, Circulation.

[25]  F. DeRubertis,et al.  Nitric oxide suppresses increases in mesangial cell protein kinase C, transforming growth factor beta, and fibronectin synthesis induced by thromboxane. , 1996, Journal of the American Society of Nephrology : JASN.

[26]  Yang Wang,et al.  Translational Regulation of Human Neuronal Nitric-oxide Synthase by an Alternatively Spliced 5′-Untranslated Region Leader Exon* , 2003, The Journal of Biological Chemistry.

[27]  B. Mayer,et al.  Long-lasting increase of nitric oxide synthase immunoreactivity, NADPH-diaphorase reaction and c-JUN co-expression in rat dorsal root ganglion neurons following sciatic nerve transection , 1993, Neuroscience Letters.

[28]  O. Krizanova,et al.  The effect of N-nitro-L-arginine and aminoguanidine treatment on changes in constitutive and inducible nitric oxide synthases in the spinal cord after sciatic nerve transection. , 2008, International journal of molecular medicine.

[29]  L. O. Randall,et al.  A method for measurement of analgesic activity on inflamed tissue. , 1957, Archives internationales de pharmacodynamie et de therapie.

[30]  N. Calcutt,et al.  Neuronal Nitric Oxide Synthase mRNA Upregulation in Rat Sensory Neurons after Spinal Nerve Ligation: Lack of a Role in Allodynia Development , 1999, The Journal of Neuroscience.

[31]  Weiqi Shi,et al.  Local Nogo-66 administration reduces neuropathic pain after sciatic nerve transection in rat , 2007, Neuroscience Letters.

[32]  Lewis O. Harvey,et al.  Efficient estimation of sensory thresholds , 1986 .

[33]  J. Levine,et al.  Nitric Oxide Signaling in Pain and Nociceptor Sensitization in the Rat , 1998, The Journal of Neuroscience.

[34]  G. Pereira,et al.  mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration , 2006, Neuroscience Letters.

[35]  T. Hökfelt,et al.  Marked increase in nitric oxide synthase mRNA in rat dorsal root ganglia after peripheral axotomy: in situ hybridization and functional studies. , 1992, Proceedings of the National Academy of Sciences of the United States of America.