Acta Neuropathologica

, Volume 112, Issue 4, pp 471–481 | Cite as

The fate of Nissl-stained dark neurons following traumatic brain injury in rats: difference between neocortex and hippocampus regarding survival rate

  • Hidetoshi Ooigawa
  • Hiroshi NawashiroEmail author
  • Shinji Fukui
  • Naoki Otani
  • Atsushi Osumi
  • Terushige Toyooka
  • Katsuji Shima
Original Paper


We studied the fate of Nissl-stained dark neurons (N-DNs) following traumatic brain injury (TBI). N-DNs were investigated in the cerebral neocortex and the hippocampus using a rat lateral fluid percussion injury model. Nissl stain, acid fuchsin stain and immunohistochemistry with phosphorylated extracellular signal-regulated protein kinase (pERK) antibody were used in order to assess posttraumatic neurons. In the neocortex, the number of dead neurons at 24 h postinjury was significantly less than that of the observed N-DNs in the earlier phase. Only a few N-DNs increased their pERK immunoreactivity. On the other hand, in the hippocampus the number of dead neurons was approximately the same number as that of the N-DNs, and most N-DNs showed an increased pERK immunoreactivity. These data suggest that not all N-DNs inevitably die especially in the neocortex after TBI. The fate of N-DNs is thus considered to differ depending on brain subfields.


Dark neuron Nissl stain Traumatic brain injury Extracellular signal-regulated protein kinase 



We are grateful to A. Yano, N. Nomura and T. Suzuki for their valuable technical contributions to this work, and N. Tsuzuki, H. Katoh, S. Ishihara, T. Miyazawa and A. Onuki for helpful suggestions.


  1. 1.
    Alessandrini A, Namura S, Moskowitz MA, Bonventre JV (1999) MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia. Proc Natl Acad Sci USA 96:12866–12869PubMedCrossRefGoogle Scholar
  2. 2.
    Atillo A, Soderfeldt B, Kalimo H, Olsson Y, Siesjo BK (1983) Pathogenesis of brain lesions caused by experimental epilepsy. Light- and electron-microscopic changes in the rat hippocampus following bicuculline-induced status epilepticus. Acta Neuropathol (Berl) 59:11–24CrossRefGoogle Scholar
  3. 3.
    Auer RN, Kalimo H, Olsson Y, Siesjo BK (1985) The temporal evolution of hypoglycemic brain damage. I. Light- and electron-microscopic findings in the rat cerebral cortex. Acta Neuropathol (Berl) 67:13–24CrossRefGoogle Scholar
  4. 4.
    Beer R, Franz G, Krajewski S, Pike BR, Hayes RL, Reed JC, Wang KK, Klimmer C, Schmutzhard E, Poewe W, Kampfl A (2001) Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury. J Neurochem 78:862–873PubMedCrossRefGoogle Scholar
  5. 5.
    Beer R, Franz G, Srinivasan A, Hayes RL, Pike BR, Newcomb JK, Zhao X, Schmutzhard E, Poewe W, Kampfl A (2000) Temporal profile and cell subtype distribution of activated caspase-3 following experimental traumatic brain injury. J Neurochem 75:1264–1273PubMedCrossRefGoogle Scholar
  6. 6.
    Cheung EC, Slack RS (2004) Emerging role for ERK as a key regulator of neuronal apoptosis. Sci STKE 2004:PE45PubMedCrossRefGoogle Scholar
  7. 7.
    Chu CT, Levinthal DJ, Kulich SM, Chalovich EM, DeFranco DB (2004) Oxidative neuronal injury. The dark side of ERK1/2. Eur J Biochem 271:2060–2066PubMedCrossRefGoogle Scholar
  8. 8.
    Conti AC, Raghupathi R, Trojanowski JQ, McIntosh TK (1998) Experimental brain injury induces regionally distinct apoptosis during the acute and delayed post-traumatic period. J Neurosci 18:5663–5672PubMedGoogle Scholar
  9. 9.
    Cooper JD, Payne JN, Horobin RW (1988) Accurate counting of neurons in frozen sections: some necessary precautions. J Anat 157:13–21PubMedGoogle Scholar
  10. 10.
    Cortez SC, McIntosh TK, Noble LJ (1989) Experimental fluid percussion brain injury: vascular disruption and neuronal and glial alterations. Brain Res 482:271–282PubMedCrossRefGoogle Scholar
  11. 11.
    Csordas A, Mazlo M, Gallyas F (2003) Recovery versus death of “dark” (compacted) neurons in non-impaired parenchymal environment: light and electron microscopic observations. Acta Neuropathol (Berl) 106:37–49Google Scholar
  12. 12.
    Dash PK, Mach SA, Moore AN (2002) The role of extracellular signal-regulated kinase in cognitive and motor deficits following experimental traumatic brain injury. Neuroscience 114:755–767PubMedCrossRefGoogle Scholar
  13. 13.
    Dineley KT, Westerman M, Bui D, Bell K, Ashe KH, Sweatt JD (2001) Beta-amyloid activates the mitogen-activated protein kinase cascade via hippocampal alpha7 nicotinic acetylcholine receptors: in vitro and in vivo mechanisms related to Alzheimer’s disease. J Neurosci 21:4125–4133PubMedGoogle Scholar
  14. 14.
    Gallyas F, Hsu M, Buzsaki G (1993) Four modified silver methods for thick sections of formaldehyde-fixed mammalian central nervous tissue: ‘dark’ neurons, perikarya of all neurons, microglial cells and capillaries. J Neurosci Methods 50:159–164PubMedCrossRefGoogle Scholar
  15. 15.
    Gallyas F, Zoltay G (1992) An immediate light microscopic response of neuronal somata, dendrites and axons to non-contusing concussive head injury in the rat. Acta Neuropathol (Berl) 83:386–393CrossRefGoogle Scholar
  16. 16.
    Gallyas F, Zoltay G, Balas I (1992) An immediate light microscopic response of neuronal somata, dendrites and axons to contusing concussive head injury in the rat. Acta Neuropathol (Berl) 83:394–401CrossRefGoogle Scholar
  17. 17.
    Hicks R, Soares H, Smith D, McIntosh T (1996) Temporal and spatial characterization of neuronal injury following lateral fluid-percussion brain injury in the rat. Acta Neuropathol (Berl) 91:236–246CrossRefGoogle Scholar
  18. 18.
    Irving EA, Bamford M (2002) Role of mitogen- and stress-activated kinases in ischemic injury. J Cereb Blood Flow Metab 22:631–647PubMedCrossRefGoogle Scholar
  19. 19.
    Ishida K, Shimizu H, Hida H, Urakawa S, Ida K, Nishino H (2004) Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive. Neuroscience 125:633–644PubMedCrossRefGoogle Scholar
  20. 20.
    Kampfl A, Posmantur RM, Zhao X, Schmutzhard E, Clifton GL, Hayes RL (1997) Mechanisms of calpain proteolysis following traumatic brain injury: implications for pathology and therapy: implications for pathology and therapy: a review and update. J Neurotrauma 14:121–134PubMedGoogle Scholar
  21. 21.
    Kampfl A, Zhao X, Whitson JS, Posmantur R, Dixon CE, Yang K, Clifton GL, Hayes RL (1996) Calpain inhibitors protect against depolarization-induced neurofilament protein loss of septo-hippocampal neurons in culture. Eur J Neurosci 8:344–352PubMedCrossRefGoogle Scholar
  22. 22.
    Keane RW, Kraydieh S, Lotocki G, Alonso OF, Aldana P, Dietrich WD (2001) Apoptotic and antiapoptotic mechanisms after traumatic brain injury. J Cereb Blood Flow Metab 21:1189–1198PubMedCrossRefGoogle Scholar
  23. 23.
    Konigsmark BW (1970) Methods for counting of neurons. In: Nauta WJH, Ebbesson SOE (eds) Contemporary research methods in neuroanatomy. Springer, Berlin Heidelberg New York, pp 315–340Google Scholar
  24. 24.
    Kulich SM, Chu CT (2001) Sustained extracellular signal-regulated kinase activation by 6-hydroxydopamine: implications for Parkinson’s disease. J Neurochem 77:1058–1066PubMedCrossRefGoogle Scholar
  25. 25.
    Lowenstein DH, Thomas MJ, Smith DH, McIntosh TK (1992) Selective vulnerability of dentate hilar neurons following traumatic brain injury: a potential mechanistic link between head trauma and disorders of the hippocampus. J Neurosci 12:4846–4853PubMedGoogle Scholar
  26. 26.
    Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185PubMedCrossRefGoogle Scholar
  27. 27.
    Matsushita Y, Shima K, Nawashiro H, Wada K, Tsuzuki N, Miyazawa T (2000) Real time monitoring of glutamate following fluid percussion brain injury with hypoxia in the rat. Acta Neurochir Suppl 76:207–212PubMedGoogle Scholar
  28. 28.
    Maxwell WL, Povlishock JT, Graham DL (1997) A mechanistic analysis of nondisruptive axonal injury: a review. J Neurotrauma 14:419–440PubMedGoogle Scholar
  29. 29.
    McIntosh TK, Vink R, Noble L, Yamakami I, Fernyak S, Soares H, Faden AL (1989) Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model. Neuroscience 28:233–244PubMedCrossRefGoogle Scholar
  30. 30.
    Midha R, Fehlings MG, Tator CH, Saint-Cyr JA, Guha A (1987) Assessment of spinal cord injury by counting corticospinal and rubrospinal neurons. Brain Res 410:299–308PubMedCrossRefGoogle Scholar
  31. 31.
    Mori T, Wang X, Jung JC, Sumii T, Singhal AB, Fini ME, Dixon CE, Alessandrini A, Lo EH (2002) Mitogen-activated protein kinase inhibition in traumatic brain injury: in vitro and in vivo effects. J Cereb Blood Flow Metab 22:444–452PubMedCrossRefGoogle Scholar
  32. 32.
    Namura S, Iihara K, Takami S, Nagata I, Kikuchi H, Matsushita K, Moskowitz MA, Bonventre JV, Alessandrini A (2001) Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia. Proc Natl Acad Sci USA 98:11569–11574PubMedCrossRefGoogle Scholar
  33. 33.
    Nawashiro H, Shima K, Chigasaki H (1995) Selective vulnerability of hippocampal CA3 neurons to hypoxia after mild concussion in the rat. Neurol Res 17:455–460PubMedGoogle Scholar
  34. 34.
    Newcomb JK, Kampfl A, Posmantur RM, Zhao X, Pike BR, Liu SJ, Clifton GL, Hayes RL (1997) Immunohistochemical study of calpain-mediated breakdown products to alpha-spectrin following controlled cortical impact injury in the rat. J Neurotrauma 14:369–383PubMedCrossRefGoogle Scholar
  35. 35.
    Otani N, Nawashiro H, Fukui S, Nomura N, Shima K (2002) Temporal and spatial profile of phosphorylated mitogen-activated protein kinase pathways after lateral fluid percussion injury in the cortex of the rat brain. J Neurotrauma 19:1587–1596PubMedCrossRefGoogle Scholar
  36. 36.
    Otani N, Nawashiro H, Tsuzuki N, Katoh H, Miyazawa T, Shima K (2003) Mitogen-activated protein kinases phosphorylation in posttraumatic selective vulnerability in rats. Acta Neurochir Suppl 86:287–289PubMedGoogle Scholar
  37. 37.
    Pei JJ, Braak H, An WL, Winblad B, Cowburn RF, Iqbal K, Grundke-Iqbal I (2002) Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer’s disease. Brain Res Mol Brain Res 109:45–55PubMedCrossRefGoogle Scholar
  38. 38.
    Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edn. Academic, San DiegoGoogle Scholar
  39. 39.
    Petito CK, Pulsinelli WA (1984) Sequential development of reversible and irreversible neuronal damage following cerebral ischemia. J Neuropathol Exp Neurol 43:141–153PubMedGoogle Scholar
  40. 40.
    Pettus EH, Povlishock JT (1996) Characterization of a distinct set of intra-axonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability. Brain Res 722:1–11PubMedCrossRefGoogle Scholar
  41. 41.
    Poirier JL, Capek R, De Koninck Y (2000) Differential progression of Dark neuron and Fluoro-Jade labelling in the rat hippocampus following pilocarpine-induced status epilepticus. Neuroscience 97:59–68PubMedCrossRefGoogle Scholar
  42. 42.
    Posmantur RM, Newcomb JK, Kampfl A, Hayes RL (2000) Light and confocal microscopic studies of evolutionary changes in neurofilament proteins following cortical impact injury in the rat. Exp Neurol 161:15–26PubMedCrossRefGoogle Scholar
  43. 43.
    Raghupathi R, Muir JK, Fulp CT, Pittman RN, McIntosh TK (2003) Acute activation of mitogen-activated protein kinases following traumatic brain injury in the rat: implications for posttraumatic cell death. Exp Neurol 183:438–448PubMedCrossRefGoogle Scholar
  44. 44.
    Saatman KE, Graham DI, McIntosh TK (1998) The neuronal cytoskeleton is at risk after mild and moderate brain injury. J Neurotrauma 15:1047–1058PubMedGoogle Scholar
  45. 45.
    Sato M, Chang E, Igarashi T, Noble LJ (2001) Neuronal injury and loss after traumatic brain injury: time course and regional variability. Brain Res 917:45–54PubMedCrossRefGoogle Scholar
  46. 46.
    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
  47. 47.
    Schmued LC, Hopkins KJ (2000) Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874:123–130PubMedCrossRefGoogle Scholar
  48. 48.
    Subramaniam S, Strelau J, Unsicker K (2003) Growth differentiation factor-15 prevents low potassium-induced cell death of cerebellar granule neurons by differential regulation of Akt and ERK pathways. J Biol Chem 278:8904–8912PubMedCrossRefGoogle Scholar
  49. 49.
    Taft WC, Yang K, Dixon CE, Clifton GL, Hayes RL (1993) Hypothermia attenuates the loss of hippocampal microtubule-associated protein 2 (MAP2) following traumatic brain injury. J Cereb Blood Flow Metab 13:796–802PubMedGoogle Scholar
  50. 50.
    Taft WC, Yang K, Dixon CE, Hayes RL (1992) Microtubule-associated protein 2 levels decrease in hippocampus following traumatic brain injury. J Neurotrauma 9:281–290PubMedGoogle Scholar
  51. 51.
    Zhu JH, Kulich SM, Oury TD, Chu CT (2002) Cytoplasmic aggregates of phosphorylated extracellular signal-regulated protein kinases in Lewy body diseases. Am J Pathol 161:2087–2098PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Hidetoshi Ooigawa
    • 1
  • Hiroshi Nawashiro
    • 1
    Email author
  • Shinji Fukui
    • 1
  • Naoki Otani
    • 1
  • Atsushi Osumi
    • 1
  • Terushige Toyooka
    • 1
  • Katsuji Shima
    • 1
  1. 1.Department of NeurosurgeryNational Defense Medical CollegeSaitamaJapan

Personalised recommendations