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The effects of L-NAME on neuronal NOS and SOD1 expression in the DRG–spinal cord network of axotomised Thy 1.2 eGFP mice

  • Matthew J. G. Bradman (a1), Richard Morris (a1), Anne McArdle (a1), Malcolm J. Jackson (a1) and Thimmasettappa Thippeswamy (a1)...


Nitric oxide (NO) plays an important role in pathophysiology of the nervous system. Copper/zinc superoxide dismutase (SOD1) reacts with superoxide, which is also a substrate for NO, to provide antioxidative protection. NO production is greatly altered following nerve injury, therefore we hypothesised that SOD1 and NO may be involved in modulating axotomy responses in dorsal root ganglion (DRG)–spinal network. To investigate this interaction, adult Thy1.2 enhanced membrane-bound green fluorescent protein (eGFP) mice underwent sciatic nerve axotomy and received NG-nitro- <l-arginine methylester (L-NAME) or vehicle 7–9 days later. L4–L6 spinal cord and DRG were harvested for immunohistochemical analyses. Effect of injury was confirmed by axotomy markers; small proline-rich repeat protein 1A (SPRR1A) was restricted to ipsilateral neuropathology, while Thy1.2 eGFP revealed also contralateral crossover effects. L-NAME, but not axotomy, increased neuronal NO synthase (nNOS) and SOD1 immunoreactive neurons, with no colocalisation, in a lamina-dependent manner in the dorsal horn of the spinal cord. Axotomy and/or L-NAME had no effect on total nNOS+ and SOD1+ neurons in DRG. However, L-NAME altered SOD1 expression in subsets of axotomised DRG neurons. These findings provide evidence for differential distribution of SOD1 and its modulation by NO, which may interact to regulate axotomy-induced changes in DRG–spinal network.


Corresponding author

Correspondence should be addressed to: Thimmasettappa Thippeswamy, Faculty of Health and Life Sciences, University of Liverpool, Room no. 4.306, 4th Floor, UCD Duncan Building Daulby Street, Liverpool L69 3GA, UK phone: 00 44 151 706 4048 email:


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Albakri, Q.A. and Stuehr, D.J. (1996) Intracellular assembly of inducible NO synthase is limited by nitric oxide-mediated changes in heme insertion and availability. Journal of Biological Chemistry 271, 54145421.
Beckman, J.S., Smith, C.M. and Koppenol, W. H. (1993) ALS, SOD, and peroxynitrite. Nature 364, 584.
Beggs, S. and Salter, M. W. (2007) Stereological and somatotopic analysis of the spinal microglial response to peripheral nerve injury. Brain, Behavior, and Immunity 21, 624633.
Belle, M.D., Pattison, E.F., Cheunsuang, O., Stewart, A., Kramer, I., Sigrist, M. et al. (2007) Characterization of a thy1.2 GFP transgenic mouse reveals a tissue-specific organization of the spinal dorsal horn. Genesis 45, 679688.
Bergman, E., Carlsson, K., Liljeborg, A., Manders, E., Hokfelt, T. and Ulfhake, B.D. (1999) Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging. Brain Research 832, 6383.
Bonilla, I.E., Tanabe, K. and Strittmatter, S.M. (2002) Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth. Journal of Neuroscience 22, 13031315.
Bradman, M.J.G., Arora, D.K., Morris, R. and Thippeswamy, T. (2010) How do the satellite glia cells of the dorsal root ganglia respond to stressed neurons? Nitric oxide saga from embryonic development to axonal injury in adulthood. Neuron Glia Biology 6, 1117.
Brenman, J.E., Chao, D.S., Gee, S.H., McGee, A.W., Craven, S.E., Santillano, D.R. et al. (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alphal-syntrophin mediated by PDZ domains. Cell 84, 757767.
Browning, M.D., Garyu, J.W., Dumler, J.S. and Scorpio, D.G. (2006) Role of reactive nitrogen species in development of hepatic injury in a C57bl/6 mouse model of human granulocytic anaplasmosis. Comparative Medicine 56, 5562.
Callsen-Cencic, P., Hoheisel, U., Kaske, A., Mense, S. and Tenschert, S. (1999) The controversy about spinal neuronal nitric oxide synthase: under which conditions is it up- or downregulated? Cell and Tissue Research 295, 183194.
Chen, X.-J., Levedakou, E.N., Millen, K.J., Wollmann, R.L., Soliven, B. and Popko, B. (2007) Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene. Journal of Neuroscience 27, 1451514524.
Cheng, C., Chen, M., Shi, S., Gao, S., Niu, S., Li, X. et al. (2007) Effect of peripheral axotomy on gene expression of NIDD in rat neural tissues. Journal of Molecular Neuroscience 32, 199206.
Culotta, V.C., Yang, M. and O'Halloran, T.V. (2006) Activation of superoxide dismutases: putting the metal to the pedal. Biochimica et Biophysica Acta 1763, 747758.
Das, R., Kravtsov, G.M., Ballard, H.J. and Kwan, C.Y. (1999) L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium. British Journal of Pharmacology 128, 493499.
Deng, H.X., Hentati, A., Tainer, J.A., Iqbal, Z., Cayabyab, A., Hung, W.Y. et al. (1993) Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science 261, 10471051.
Deng, Y.S., Zhong, J.H. and Zhou, X.F. (2000) Effects of endogenous neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury. Experimental Neurology 164, 344350.
Fiallos-Estrada, C.E., Kummer, W., Mayer, B., Bravo, R., Zimmermann, M. and Herdegen, T.D. (1993) 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. Neuroscience Letters 150, 169173.
Freire, M.A.M., Guimaraes, J.S., Leal, W.G. and Pereira, A. (2009) Pain modulation by nitric oxide in the spinal cord. Frontiers in Neuroscience 3, 175181.
Gonzalez-Zulueta, M., Ensz, L.M., Mukhina, G., Lebovitz, R.M., Zwacka, R.M., Engelhardt, J.F. et al. (1998) Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity. Journal of Neuroscience 18, 20402055.
Hobbs, A.J., Fukuto, J.M. and Ignarro, L.J.D. (1994) Formation of free nitric oxide from l-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase. Proceedings of the National Academy of Sciences of the U.S.A. 91, 1099210996.
Huie, R.E. and Padmaja, S. (1993) The reaction of NO with superoxide. Free Radical Research Communications 18, 195199.
Imlay, J., Chin, S. and Linn, S. (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240, 640642.
Imlay, J.A. and Linn, S. (1988) DNA damage and oxygen radical toxicity. Science 240, 13021309.
Keilhoff, G., Fansa, H. and Wolf, G. (2002) Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy. Journal of Chemical Neuroanatomy 24, 181187.
Keilhoff, G., Fansa, H. and Wolf, G. (2004) Neuronal NOS deficiency promotes apoptotic cell death of spinal cord neurons after peripheral nerve transection. Nitric Oxide: Biology and Chemistry 10, 101111.
Kluchova, D., Klimcik, R. and Kloc, P. (2002) Neuronal nitric oxide synthase in the rabbit spinal cord visualised by histochemical NADPH-diaphorase and immunohistochemical NOS methods. General Physiology and Biophysics 21, 163174.
Lawson, S.N. (1979) The postnatal development of large light and small dark neurons in mouse dorsal root ganglia: a statistical analysis of cell numbers and size. Journal of Neurocytology 8, 275294.
Levy, D., Kubes, P. and Zochodne, D.W. (2001) Delayed peripheral nerve degeneration, regeneration, and pain in mice lacking inducible nitric oxide synthase. Journal of Neuropathology and Experimental Neurology 60, 411421.
Lindenau, J., Noack, H., Possel, H., Asayama, K. and Wolf, G. (2000) Cellular distribution of superoxide dismutases in the rat CNS. Glia 29, 2534.
Little, J.W., Doyle, T. and Salvemini, D. (2010) Reactive nitroxidative species and nociceptive processing: determining the roles for nitric oxide, superoxide, and peroxynitrite in pain. Amino Acids 42, 7594.
Maxwell, D.J., Belle, M.D., Cheunsuang, O., Stewart, A. and Morris, R. (2007) Morphology of inhibitory and excitatory interneurons in superficial laminae of the rat dorsal horn. Journal of Physiology 584, 521533.
Moncada, S. and Higgs, E.A. (2006) The discovery of nitric oxide and its role in vascular biology. British Journal of Pharmacology 147(Suppl), S193S201.
Morris, R. and Grosveld, F. (1989) Expression of Thy-1 in the nervous system of the rat and mouse. Immunology Series 45, 121148.
Murphy, M.E. and Sies, H.D. (1991) Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase. Proceedings of the National Academy of Sciences of the U.S.A. 88, 1086010864.
Murphy, S. (2000) Production of nitric oxide by glial cells: regulation and potential roles in the CNS. Glia 29, 113.
Noack, H., Lindenau, J., Rothe, F., Asayama, K. and Wolf, G. (1998) Differential expression of superoxide dismutase isoforms in neuronal and glial compartments in the course of excitotoxically mediated neurodegeneration: relation to oxidative and nitrergic stress. Glia 23, 285297.
Orr, W.C. and Sohal, R.S. (1994) Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263, 11281130.
Perry, J.J.P., Shin, D.S., Getzoff, E.D. and Tainer, J.A. (2010) The structural biochemistry of the superoxide dismutases. Biochimica et Biophysica Acta 1804, 245262.
Raoul, C.D., Estevez, A.G., Nishimune, H., Cleveland, D.W., DeLapeyriere, O., Henderson, C.E. et al. (2002) Motoneuron death triggered by a specific pathway downstream of Fas: potentiation by ALS-linked SOD1 mutations. Neuron 35, 10671083.
Rasband, W. (1997) ImageJ. U. S. National Institutes of Health, Bethesda, MD, USA.
Rexed, B. (1952) The cytoarchitectonic organization of the spinal cord in the cat. Journal of Comparative Neurology 96, 414495.
Ridger, V.C., Greenacre, S.A., Handy, R.L., Halliwell, B., Moore, P.K., Whiteman, M. et al. (1997) Effect of peroxynitrite on plasma extravasation, microvascular blood flow and nociception in the rat. British Journal of Pharmacology 122, 10831088.
Rigaud, M., Gemes, G., Barabas, M.-E., Chernoff, D.I., Abram, S.E., Stucky, C.L. et al. (2008) Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain. Pain 136, 188201.
Rodriguez-Crespo, I., Straub, W., Gavilanes, F. and Ortiz de Montellano, P.R. (1998) Binding of dynein light chain (PIN) to neuronal nitric oxide synthase in the absence of inhibition. Archives of Biochemistry and Biophysics 359, 297304.
Rogerio, F., Teixeira, S.A., de Rezende, A.C.S., de Sa, R.C., de Souza Queiroz, L., De Nucci, G. et al. (2005) Superoxide dismutase isoforms 1 and 2 in lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin treatment. Brain Research. Developmental Brain Research 154, 217225.
Rosenfeld, J., Cook, S. and James, R. (1997) Expression of superoxide dismutase following axotomy. Experimental Neurology 147, 3747.
Ryu, T.H., Jung, K.Y., Ha, M.J., Kwak, K.H., Lim, D.G. and Hong, J.G. (2010) Superoxide and nitric oxide involvement in enhancing of N-methyl-D-aspartate receptor-mediated central sensitization in the chronic post-ischemia pain model. Korean Journal of Pain 23, 110.
Sasaki, M., Gonzalez-Zulueta, M., Huang, H., Herring, W.J., Ahn, S., Ginty, D.D. et al. (2000) Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism. Proceedings of the National Academy of Sciences of the U.S.A. 97, 86178622.
Schmidt, H.H., Hofmann, H., Schindler, U., Shutenko, Z.S., Cunningham, D.D. and Feelisch, M. (1996) No. NO from NO synthase. Proceedings of the National Academy of Sciences of the U.S.A. 93, 1449214497.
Schuppe, H.G., Araki, M., Aonuma, H., Nagayama, T. and Newland, P.L. (2004) Effects of nitric oxide on proprioceptive signaling. Zoological Science 21, 15.
Schuppe, H.R. and Newland, P.L. (2004) Nitric oxide modulates presynaptic afferent depolarization of mechanosensory neurons. Journal of Neurobiology 59, 331342.
Shi, T.J., Holmberg, K., Xu, Z.Q., Steinbusch, H., de Vente, J. and Hokfelt, T. (1998) Effect of peripheral nerve injury on cGMP and nitric oxide synthase levels in rat dorsal root ganglia: time course and coexistence. Pain 78, 171180.
Stamler, J.S., Singel, D.J. and Loscalzo, J. (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258, 18981902.
Starkey, M.L., Davies, M., Yip, P.K., Carter, L.M., Wong, D.M., McMahon, S.B. et al. (2009) Expression of the regeneration-associated protein SPRR1A in primary sensory neurons and spinal cord of the adult mouse following peripheral and central injury. Journal of Comparative Neurology 513, 5168.
Sunico, C.R., Portillo, F., Gonzalez-Forero, D., Kasparov, S. and Moreno-Lupez, B. (2008) Evidence for a detrimental role of nitric oxide synthesized by endothelial nitric oxide synthase after peripheral nerve injury. Neuroscience 157, 4051.
Tajti, J., Fischer, J., Knyiher-Csillik, E. and Csillik, B. (1988) Transganglionic regulation and fine structural localization of lectin-reactive carbohydrate epitopes in primary sensory neurons of the rat. Histochemistry 88, 213218.
Tang, Q., Svensson, C.I., Fitzsimmons, B., Webb, M., Yaksh, T.L. and Hua, X.-Y. (2007) Inhibition of spinal constitutive NOS-2 by 1400W attenuates tissue injury and inflammation-induced hyperalgesia and spinal p38 activation. European Journal of Neuroscience 25, 29642972.
Terenghi, G., Riveros-Moreno, V., Hudson, L.D., Ibrahim, N.B. and Polak, J.M. (1993) Immunohistochemistry of nitric oxide synthase demonstrates immunoreactive neurons in spinal cord and dorsal root ganglia of man and rat. Journal of the Neurological Sciences 118, 3437.
Thippeswamy, T., Haddley, K., Corness, J.D., Howard, M.R., McKay, J.S., Beaucourt, S.M. et al. (2007) NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons. Journal of Neurochemistry 100, 790801.
Thippeswamy, T., Jain, R.K., Mumtaz, N. and Morris, R. (2001) Inhibition of neuronal nitric oxide synthase results in neurodegenerative changes in the axotomised dorsal root ganglion neurons: evidence for a neuroprotective role of nitric oxide in vivo. Neuroscience Research 40, 3744.
Thippeswamy, T., McKay, J.S., Morris, R., Quinn, J., Wong, L.-F. and Murphy, D. (2005a) Glial-mediated neuroprotection: evidence for the protective role of the NO-cGMP pathway via neuron-glial communication in the peripheral nervous system. Glia 49, 197210.
Thippeswamy, T., McKay, J.S., Quinn, J. and Morris, R. (2005b) Either nitric oxide or nerve growth factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development. Brain Research. Developmental Brain Research 154, 153164.
Verge, V.M., Xu, Z., Xu, X.J., Wiesenfeld, H.Z. and Hokfelt, T. (1992) Marked increase in nitric oxide synthase mRNA in rat dorsal root ganglia after peripheral axotomy: in situ hybridization and functional studies. Proceedings of the National Academy of Sciences of the U.S.A. 89, 1161711621.
Wink, D.A. and Mitchell, J.B. (1998) Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology and Medicine 25, 434456.
Yu, W.H.A. (2002) Spatial and temporal correlation of nitric oxide synthase expression with CuZn-superoxide dismutase reduction in motor neurons following axotomy. Annals of the New York Academy of Sciences 962, 111121.
Zhang, X., Verge, V., Wiesenfeld-Hallin, Z., Ju, G., Bredt, D., Synder, S.H. et al. (1993) Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy. Journal of Comparative Neurology 335, 563575.
Zochodne, D.W., Levy, D., Zwiers, H., Sun, H., Rubin, I., Cheng, C. and Lauritzen, M. (1999) Evidence for nitric oxide and nitric oxide synthase activity in proximal stumps of transected peripheral nerves. Neuroscience 91, 15151527.


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The effects of L-NAME on neuronal NOS and SOD1 expression in the DRG–spinal cord network of axotomised Thy 1.2 eGFP mice

  • Matthew J. G. Bradman (a1), Richard Morris (a1), Anne McArdle (a1), Malcolm J. Jackson (a1) and Thimmasettappa Thippeswamy (a1)...


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