Acta Neurochirurgica

, Volume 154, Issue 4, pp 689–697 | Cite as

iNOS-mediated secondary inflammatory response differs between rat strains following experimental brain contusion

  • Mattias Günther
  • Faiez Al Nimer
  • Caroline Gahm
  • Fredrik Piehl
  • Tiit Mathiesen
Experimental Research



Nitric oxide is a key mediator of post-traumatic inflammation in the brain. We examined the expressions of iNOS, nNOS, and eNOS in inbred DA and PVGa rat strains where DA is susceptible to autoimmune neuroinflammation and PVGa-resistant.


Parietal contusions using a weight drop model were produced in five rats per genotype. After 24 h, the brains were removed and analyzed using a range of immunohistochemical methods.


PVGa presented significantly increased iNOS expression in infiltrating inflammatory cells in the perilesional area compared to DA (p < 0.05). The amount of w3/13-positive infiltrating inflammatory cells did not differ between strains. eNOS and nNOS expression did not differ between strains. iNOS-positive cells coexpressed neuronal (NeuN), macrophage (ED-1), and leucocyte (w3/13) markers. MnSOD was significantly increased in PVGa (p < 0.05). 3-Nitrotyrosine, a measure of peroxynitrite levels, and fluoro-jade stained neuronal degeneration, did not differ between strains.


Two inbred rat strains with genetically determined differences in susceptibility to develop autoimmune disease displayed different levels of the inflammatory and anti-inflammatory mediators iNOS and MnSOD, indicating genetic regulation. Interestingly, the increased levels of iNOS did not lead to elevated expression of the neuronal cell-death marker fluoro-jade. The increased iNOS expression was correlated with increased expression of superoxide scavenger MnSOD. Excessive peroxynitrite formation was probably prevented by limitation of available superoxide. Subsequently, the higher expression of potentially deleterious iNOS in PVGa did not result in increased neuronal death.


Neurotrauma Neuroinflammation TBI Nitric oxide synthase Peroxynitrite Manganese superoxide dismutase Dark Agouti Piebald Virol Glaxo 


Conflicts of interest



  1. 1.
    (1999) Consensus conference. Rehabilitation of persons with traumatic brain injury. NIH Consensus Development Panel on Rehabilitation of Persons With Traumatic Brain Injury. JAMA 282:974–983Google Scholar
  2. 2.
    Ahn ES, Robertson CL, Vereczki V, Hoffman GE, Fiskum G (2008) Normoxic ventilatory resuscitation following controlled cortical impact reduces peroxynitrite-mediated protein nitration in the hippocampus. J Neurosurg 108:124–131PubMedCrossRefGoogle Scholar
  3. 3.
    Al Nimer F, Beyeen AD, Lindblom R, Strom M, Aeinehband S, Lidman O, Piehl F (2011) Both MHC and non-MHC genes regulate inflammation and T-cell response after traumatic brain injury. Brain Behav Immun 25:981–990PubMedCrossRefGoogle Scholar
  4. 4.
    Arteel GE, Briviba K, Sies H (1999) Protection against peroxynitrite. FEBS Lett 445:226–230PubMedCrossRefGoogle Scholar
  5. 5.
    Bayir H, Kagan VE, Borisenko GG, Tyurina YY, Janesko KL, Vagni VA, Billiar TR, Williams DL, Kochanek PM (2005) Enhanced oxidative stress in iNOS-deficient mice after traumatic brain injury: support for a neuroprotective role of iNOS. J Cereb Blood Flow Metab 25:673–684PubMedCrossRefGoogle Scholar
  6. 6.
    Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87:1620–1624PubMedCrossRefGoogle Scholar
  7. 7.
    Bellander BM, Lidman O, Ohlsson M, Meijer B, Piehl F, Svensson M (2010) Genetic regulation of microglia activation, complement expression, and neurodegeneration in a rat model of traumatic brain injury. Exp Brain Res 205:103–114PubMedCrossRefGoogle Scholar
  8. 8.
    Braden AW (1958) Strain differences in the incidence of polyspermia in rats after delayed mating. Fertil Steril 9:243–246PubMedGoogle Scholar
  9. 9.
    Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4:1313–1317PubMedCrossRefGoogle Scholar
  10. 10.
    Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG (1981) Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res 211:67–77PubMedCrossRefGoogle Scholar
  11. 11.
    Floyd RA (1999) Antioxidants, oxidative stress, and degenerative neurological disorders. Proc Soc Exp Biol Med 222:236–245PubMedCrossRefGoogle Scholar
  12. 12.
    Friedman G, Froom P, Sazbon L, Grinblatt I, Shochina M, Tsenter J, Babaey S, Yehuda B, Groswasser Z (1999) Apolipoprotein E-epsilon4 genotype predicts a poor outcome in survivors of traumatic brain injury. Neurology 52:244–248PubMedGoogle Scholar
  13. 13.
    Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15:1583–1606PubMedCrossRefGoogle Scholar
  14. 14.
    Gahm C, Danilov A, Holmin S, Wiklund PN, Brundin L, Mathiesen T (2005) Reduced neuronal injury after treatment with NG-nitro-L-arginine methyl ester (L-NAME) or 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) following experimental brain contusion. Neurosurgery 57:1272–1281, discussion 1272–1281PubMedCrossRefGoogle Scholar
  15. 15.
    Gahm C, Holmin S, Mathiesen T (2000) Temporal profiles and cellular sources of three nitric oxide synthase isoforms in the brain after experimental contusion. Neurosurgery 46:169–177PubMedCrossRefGoogle Scholar
  16. 16.
    Gahm C, Holmin S, Mathiesen T (2002) Nitric oxide synthase expression after human brain contusion. Neurosurgery 50:1319–1326PubMedGoogle Scholar
  17. 17.
    Gahm C, Holmin S, Rudehill S, Mathiesen T (2005) Neuronal degeneration and iNOS expression in experimental brain contusion following treatment with colchicine, dexamethasone, tirilazad mesylate and nimodipine. Acta Neurochir (Wien) 147:1071–1084, discussion 1084CrossRefGoogle Scholar
  18. 18.
    Gahm C, Holmin S, Wiklund PN, Brundin L, Mathiesen T (2006) Neuroprotection by selective inhibition of inducible nitric oxide synthase after experimental brain contusion. J Neurotrauma 23:1343–1354PubMedCrossRefGoogle Scholar
  19. 19.
    Hlatky R, Lui H, Cherian L, Goodman JC, O'Brien WE, Contant CF, Robertson CS (2003) The role of endothelial nitric oxide synthase in the cerebral hemodynamics after controlled cortical impact injury in mice. J Neurotrauma 20:995–1006PubMedCrossRefGoogle Scholar
  20. 20.
    Holmin S, Almqvist P, Lendahl U, Mathiesen T (1997) Adult nestin-expressing subependymal cells differentiate to astrocytes in response to brain injury. Eur J Neurosci 9:65–75PubMedCrossRefGoogle Scholar
  21. 21.
    Holmin S, Mathiesen T (1996) Dexamethasone and colchicine reduce inflammation and delayed oedema following experimental brain contusion. Acta Neurochir (Wien) 138:418–424CrossRefGoogle Scholar
  22. 22.
    Hooper DC, Scott GS, Zborek A, Mikheeva T, Kean RB, Koprowski H, Spitsin SV (2000) Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis. FASEB J 14:691–698PubMedGoogle Scholar
  23. 23.
    Inman D, Guth L, Steward O (2002) Genetic influences on secondary degeneration and wound healing following spinal cord injury in various strains of mice. J Comp Neurol 451:225–235PubMedCrossRefGoogle Scholar
  24. 24.
    Jafarian-Tehrani M, Louin G, Royo NC, Besson VC, Bohme GA, Plotkine M, Marchand-Verrecchia C (2005) 1400W, a potent selective inducible NOS inhibitor, improves histopathological outcome following traumatic brain injury in rats. Nitric Oxide 12:61–69PubMedCrossRefGoogle Scholar
  25. 25.
    Keller JN, Kindy MS, Holtsberg FW, St Clair DK, Yen HC, Germeyer A, Steiner SM, Bruce-Keller AJ, Hutchins JB, Mattson MP (1998) Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J Neurosci 18:687–697PubMedGoogle Scholar
  26. 26.
    Khan M, Im YB, Shunmugavel A, Gilg AG, Dhindsa RK, Singh AK, Singh I (2009) Administration of S-nitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact. J Neuroinflamm 6 Google Scholar
  27. 27.
    Lebovitz RM, Zhang H, Vogel H, Cartwright J Jr, Dionne L, Lu N, Huang S, Matzuk MM (1996) Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci USA 93:9782–9787PubMedCrossRefGoogle Scholar
  28. 28.
    Li L, Lu J, Tay SS, Moochhala SM, He BP (2007) The function of microglia, either neuroprotection or neurotoxicity, is determined by the equilibrium among factors released from activated microglia in vitro. Brain Res 1159:8–17PubMedCrossRefGoogle Scholar
  29. 29.
    Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ, Yoshimura MP, Berger C, Chan PH, Wallace DC, Epstein CJ (1995) Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet 11:376–381PubMedCrossRefGoogle Scholar
  30. 30.
    Lindholm D, Castren E, Kiefer R, Zafra F, Thoenen H (1992) Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation. J Cell Biol 117:395–400PubMedCrossRefGoogle Scholar
  31. 31.
    Lorentzen JC, Andersson M, Issazadeh S, Dahlman I, Luthman H, Weissert R, Olsson T (1997) Genetic analysis of inflammation, cytokine mRNA expression and disease course of relapsing experimental autoimmune encephalomyelitis in DA rats. J Neuroimmunol 80:31–37PubMedCrossRefGoogle Scholar
  32. 32.
    Lu J, Goh SJ, Tng PY, Deng YY, Ling EA, Moochhala S (2009) Systemic inflammatory response following acute traumatic brain injury. Front Biosci 14:3795–3813PubMedCrossRefGoogle Scholar
  33. 33.
    Lundberg C, Lidman O, Holmdahl R, Olsson T, Piehl F (2001) Neurodegeneration and glial activation patterns after mechanical nerve injury are differentially regulated by non-MHC genes in congenic inbred rat strains. J Comp Neurol 431:75–87PubMedCrossRefGoogle Scholar
  34. 34.
    Marklund N, Bakshi A, Castelbuono DJ, Conte V, McIntosh TK (2006) Evaluation of pharmacological treatment strategies in traumatic brain injury. Curr Pharm Des 12:1645–1680PubMedCrossRefGoogle Scholar
  35. 35.
    Miljkovic D, Trajkovic V (2004) Inducible nitric oxide synthase activation by interleukin-17. Cytokine Growth Factor Rev 15:21–32PubMedCrossRefGoogle Scholar
  36. 36.
    Noack H, Lindenau J, Rothe F, Asayama K, 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 23:285–297PubMedCrossRefGoogle Scholar
  37. 37.
    Popovich PG, Wei P, Stokes BT (1997) Cellular inflammatory response after spinal cord injury in Sprague–Dawley and Lewis rats. J Comp Neurol 377:443–464PubMedCrossRefGoogle Scholar
  38. 38.
    Schmued LC, Albertson C, Slikker W Jr (1997) Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 751:37–46PubMedCrossRefGoogle Scholar
  39. 39.
    Sinz EH, Kochanek PM, Dixon CE, Clark RS, Carcillo JA, Schiding JK, Chen M, Wisniewski SR, Carlos TM, Williams D, DeKosky ST, Watkins SC, Marion DW, Billiar TR (1999) Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice. J Clin Invest 104:647–656PubMedCrossRefGoogle Scholar
  40. 40.
    Ste-Marie L, Hazell AS, Bemeur C, Butterworth R, Montgomery J (2001) Immunohistochemical detection of inducible nitric oxide synthase, nitrotyrosine and manganese superoxide dismutase following hyperglycemic focal cerebral ischemia. Brain Res 918:10–19PubMedCrossRefGoogle Scholar
  41. 41.
    Steward O, Schauwecker PE, Guth L, Zhang Z, Fujiki M, Inman D, Wrathall J, Kempermann G, Gage FH, Saatman KE, Raghupathi R, McIntosh T (1999) Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models. Exp Neurol 157:19–42PubMedCrossRefGoogle Scholar
  42. 42.
    Szabo C, Ischiropoulos H, Radi R (2007) Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662–680PubMedCrossRefGoogle Scholar
  43. 43.
    Teasdale GM, Nicoll JA, Murray G, Fiddes M (1997) Association of apolipoprotein E polymorphism with outcome after head injury. Lancet 350:1069–1071PubMedCrossRefGoogle Scholar
  44. 44.
    Terpolilli NA, Zweckberger K, Trabold R, Schilling L, Schinzel R, Tegtmeier F, Plesnila N (2009) The novel nitric oxide synthase inhibitor 4-amino-tetrahydro-L-biopterine prevents brain edema formation and intracranial hypertension following traumatic brain injury in mice. J Neurotrauma 26:1963–1975PubMedCrossRefGoogle Scholar
  45. 45.
    Wada K, Chatzipanteli K, Busto R, Dietrich WD (1998) Role of nitric oxide in traumatic brain injury in the rat. J Neurosurg 89:807–818PubMedCrossRefGoogle Scholar
  46. 46.
    Weissert R, Wallstrom E, Storch MK, Stefferl A, Lorentzen J, Lassmann H, Linington C, Olsson T (1998) MHC haplotype-dependent regulation of MOG-induced EAE in rats. J Clin Invest 102:1265–1273PubMedCrossRefGoogle Scholar
  47. 47.
    Wilson DB (1965) Quantitative Studies on the Behavior of Sensitized Lymphocytes in Vitro: I. Relationship of the Degree of Destruction of Homologous Target Cells to the Number of Lymphocytes and to the Time of Contact in Culture and Consideration of the Effects of Isoimmune Serum. J Exp Med 122:143–166PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Mattias Günther
    • 1
  • Faiez Al Nimer
    • 1
  • Caroline Gahm
    • 1
  • Fredrik Piehl
    • 1
  • Tiit Mathiesen
    • 1
  1. 1.Department of Clinical Neuroscience, Section of Neurosurgery and NeuroimmunologyKarolinska InstitutetStockholmSweden

Personalised recommendations