Neuronal injury and death following focal mild brain injury: The role of network excitability and seizure

Document Type: Original Article


1 Department of Neurosciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

2 Shefa Neuroscience Research Center, Khatamolanbia Hospital, Tehran , Iran

3 Department of Neurosurgery, Department of Neurology, and Epilepsy Research Center, Munster University, Germany

4 Razavi Neuroscience Center, Razavi Hospital, Mashhad, Iran

5 Quchan Higher Health Education Center, Mashhad University of Medical Sciences, Mashhad, Iran

6 Neuroscience Research Center , Shahid Beheshti University of Medical Sciences, Tehran, Iran

7 Faculty of Pharmacy, Department of Toxicology, Tehran University of Medical Sciences, Tehran, Iran


Objective(s): While traumatic brain injury (TBI) is a predisposing factor for development of post-traumatic epilepsy (PTE), the occurrence of seizures following brain trauma can infuriate adverse consequences of brain injury. However, the effect of seizures in epileptogenesis after mild TBI cannot yet be accurately confirmed. This study was designed to investigate the histopathological and molecular modifications induced by seizures on traumatized brain.
Materials and Methods: Using a new method, head was traumatized and seizures were evoked by sub-convulsive dose of pentylenetetrazole (PTZ) fifteen days after induction of focal mild TBI. Convulsion assessments were performed one hour after PTZ injection and was followed by histopathological and molecular evaluations.
Results: A significantly higher score and longer duration of seizure attacks as well as higher number of epileptiform discharges were observed in the TBI+PTZ group compared to sham and TBI groups. An elevated number of apoptotic cells was observed in the TBI+PTZ group compared to sham and TBI rats. Molecular investigations revealed higher levels of Bax/Bcl2 ratio, Caspase 3, and NF-κB in the TBI+PTZ group compared to the other animal groups. The value of Nrf2 did not change after mild TBI compared to sham and PTZ control groups. Occurrence of seizures after TBI, however, significantly decreased the level of Nrf2.
Conclusion: Our data indicated that seizure occurrence following mild TBI aggravates cell injury and death via activation of neuroinflammatory processes and may increase the risk of PTE. Additionally, our results suggest a potential protective role of Nrf2 after chemically evoked PTE.


1.    Frye CA, Scalise TJ. Anti-seizure effects of progesterone and 3alpha,5alpha-THP in kainic acid and perforant pathway models of epilepsy. Psychoneuroendocrinology 2000;25:407-420.
2.    Asikainen I, Kaste M, Sarna S. Early and late posttraumatic seizures in traumatic brain injury rehabilitation patients: brain injury factors causing late seizures and influence of seizures on long-term outcome. Epilepsia 1999;40:584-589.
3.    Angeleri F, Majkowski J, Cacchio G, Sobieszek A, D’Acunto S, Gesuita R, et al. Posttraumatic epilepsy risk factors: one-year prospective study after head injury. Epilepsia  1999;40:1222-12230.
4.    Englander J, Bushnik T, Duong TT, Cifu DX, Zafonte R, Wright J, et al. Analyzing risk factors for late posttraumatic seizures: a prospective, multicenter investigation. Arch Phys Med Rehabil 2003;84:365-373.
5.    Eftekhar B, Sahraian MA, Nouralishahi B, Khaji A, Vahabi Z, Ghodsi M, et al. Prognostic factors in the persistence of posttraumatic epilepsy after penetrating head injuries sustained in war. J Neurosurg 2009;110:319-326.
6.    Salazar AM, Jabbari B, Vance SC, Grafman J, Amin D, Dillon JD. Epilepsy after penetrating head injury. I. Clinical correlates: a report of the Vietnam Head Injury Study. Neurology 1985;35:1406-1414.
7.    Bao YH, Bramlett HM, Atkins CM, Truettner JS, Lotocki G, Alonso OF, et al. Post-traumatic seizures exacerbate histopathological damage after fluid-percussion brain injury. J Neurotrauma 2010;28:35-42.
8.    D’Ambrosio R, Perucca E. Epilepsy after head injury. Curr Opin Neurol 2004;17:731-735.
9.    Pitkanen A, Bolkvadze T. Head Trauma and Epilepsy. Jasper’s Basic Mechanisms of the Epilepsies [Internet]. 2012;4th edition.
10.    Khodaie B, Lotfinia AA, Ahmadi M, Lotfinia M, Jafarian M, Karimzadeh F, et al. Structural and functional effects of social isolation on the hippocampus of rats with traumatic brain injury. Behav Brain Res 2015;278:55-65.
11.    Jafarian M, Karimzadeh F, Alipour F, Attari F, Lotfinia AA, Speckmann EJ, et al. Cognitive impairments and neuronal injury in different brain regions of a genetic rat model of absence epilepsy. Neuroscience.298:161-170.
12.    Vespa PM, McArthur DL, Xu Y, Eliseo M, Etchepare M, Dinov I, et al. Nonconvulsive seizures after traumatic brain injury are associated with hippocampal atrophy. Neurology 2010;75:792-798.
13.    Gao X, Deng-Bryant Y, Cho W, Carrico KM, Hall ED, Chen J. Selective death of newborn neurons in hippocampal dentate gyrus following moderate experimental traumatic brain injury. J Neurosci Res 2008;86:2258-2270.
14.    Grady MS, Charleston JS, Maris D, Witgen BM, Lifshitz J. Neuronal and glial cell number in the hippocampus after experimental traumatic brain injury: analysis by stereological estimation. J Neurotrauma 2003;20:929-941.
15.    Conti AC, Raghupathi R, Trojanowski JQ, McIntosh TK. Experimental brain injury induces regionally distinct apoptosis during the acute and delayed post-traumatic period. J Neurosci 1998;18:5663-5672.
16.    Bigford GE, Alonso OF, Dietrich D, Keane RW. A novel protein complex in membrane rafts linking the NR2B glutamate receptor and autophagy is disrupted following traumatic brain injury. J Neurotrauma 2009;26:703-720.
17.    Herman ST. Clinical trials for prevention of epileptogenesis. Epilepsy Res 2006;68:35-38.
18.    Golarai G, Greenwood AC, Feeney DM, Connor JA. Physiological and structural evidence for hippocampal involvement in persistent seizure susceptibility after traumatic brain injury. J Neurosci 2001;21:8523-8537.
19.    Zanier ER, Lee SM, Vespa PM, Giza CC, Hovda DA. Increased hippocampal CA3 vulnerability to low-level kainic acid following lateral fluid percussion injury. J Neurotrauma 2003;20:409-420.
20.    Sherer M, Struchen MA, Yablon SA, Wang Y, Nick TG. Comparison of indices of traumatic brain injury severity: Glasgow Coma Scale, length of coma and post-traumatic amnesia. J Neurol Neurosurg Psychiatry 2008;79:678-685.
21.    Coulter DA, Rafiq A, Shumate M, Gong QZ, DeLorenzo RJ, Lyeth BG. Brain injury-induced enhanced limbic epileptogenesis: anatomical and physiological parallels to an animal model of temporal lobe epilepsy. Epilepsy Res 1996;26:81-91.
22.    Nilsson P, Ronne-Engstrom E, Flink R, Ungerstedt U, Carlson H, Hillered L. Epileptic seizure activity in the acute phase following cortical impact trauma in rat. Brain Res 1994;637:227-232.
23.    Reeves TM, Lyeth BG, Phillips LL, Hamm RJ, Povlishock JT. The effects of traumatic brain injury on inhibition in the hippocampus and dentate gyrus. Brain Res 1997;757:119-132.
24.    Statler KD, Swank S, Abildskov T, Bigler ED, White HS. Traumatic brain injury during development reduces minimal clonic seizure thresholds at maturity. Epilepsy Res 2008;80:163-170.
25.    Ghadiri T, Sharifzadeh M, Khodagholi F, Modarres Mousavi SM, Hassanzadeh G, Zarrindast MR, et al. A novel traumatic brain injury model for induction of mild brain injury in rats. J Neurosci Methods 2014;233:18-27.
26.    Yuan XQ, Prough DS, Smith TL, Dewitt DS. The effects of traumatic brain injury on regional cerebral blood flow in rats. J Neurotrauma 1988;5:289-301.
27.    Xiong Y, Mahmood A, Meng Y, Zhang Y, Qu C, Schallert T, et al. Delayed administration of erythropoietin reducing hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome following traumatic brain injury in rats: comparison of treatment with single and triple dose. J Neurosurg 113:598-608.
28.    Bao YH, Bramlett HM, Atkins CM, Truettner JS, Lotocki G, Alonso OF, et al. Post-traumatic seizures exacerbate histopathological damage after fluid-percussion brain injury. J Neurotrauma 2011;28:35-42.
29.    Iannone M, Cosco D, Cilurzo F, Celia C, Paolino D, Mollace V, et al. A novel animal model to evaluate the ability of a drug delivery system to promote the passage through the BBB. Neurosci Lett 2010;469:93-96.
30.    Kong S, Qian B, Liu J, Fan M, Chen G, Wang Y. Cyclothiazide induces seizure behavior in freely moving rats. Brain Res 2010;1355:207-213.
31.    Otsuki Y, Li Z, Shibata MA. Apoptotic detection methods--from morphology to gene. Prog Histochem Cytochem 2003;38:275-339.
32.    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.
33.    Vakilzadeh G, Khodagholi F, Ghadiri T, Darvishi M, Ghaemi A, Noorbakhsh F, et al. Protective effect of a cAMP analogue on behavioral deficits and neuropathological changes in Cuprizone model of demyelination. Mol Neurobiol  2014;52:130-141.
34.    Atkins CM, Truettner JS, Lotocki G, Sanchez-Molano J, Kang Y, Alonso OF, et al. Post-traumatic seizure susceptibility is attenuated by hypothermia therapy. Eur J Neurosci 2010;32:1912-1920.
35.    Kharatishvili I, Pitkanen A. Association of the severity of cortical damage with the occurrence of spontaneous seizures and hyperexcitability in an animal model of posttraumatic epilepsy. Epilepsy Res 2010;90:47-59.
36.    Mukherjee S, Zeitouni S, Cavarsan CF, Shapiro LA. Increased seizure susceptibility in mice 30 days after fluid percussion injury. Front Neurol 2013;4:28.
37.    D’Ambrosio R, Fairbanks JP, Fender JS, Born DE, Doyle DL, Miller JW. Post-traumatic epilepsy following fluid percussion injury in the rat. Brain 2004;127:304-314.
38.    Jayakumar AR, Tong XY, Ruiz-Cordero R, Bregy A, Bethea JR, Bramlett HM, et al. Activation of NF-kappaB mediates astrocyte swelling and brain edema in traumatic brain injury. J Neurotrauma 2014;31:1249-1257.
39.    van Vliet EA, Aronica E, Gorter JA. Role of blood-brain barrier in temporal lobe epilepsy and pharmacoresistance. Neuroscience 2014;277:455-473.
40.    van Vliet EA, da Costa Araujo S, Redeker S, van Schaik R, Aronica E, Gorter JA. Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy. Brain 2007;130:521-534.
41.    van Vliet EA, Otte WM, Wadman WJ, Aronica E, Kooij G, de Vries HE, et al. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats I: Magnetic resonance imaging. Epilepsia 2016;57:59-69.
42.    van Vliet EA, van Schaik R, Edelbroek PM, Voskuyl RA, Redeker S, Aronica E, et al. Region-specific overexpression of P-glycoprotein at the blood-brain barrier affects brain uptake of phenytoin in epileptic rats. J Pharmacol Exp Ther 2007;322:141-147.
43.    Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, et al. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci U S A 1995;92:7834-7838.
44.    Naseer MI, Shupeng L, Kim MO. Maternal epileptic seizure induced by pentylenetetrazol: apoptotic neurodegeneration and decreased GABAB1 receptor expression in prenatal rat brain. Mol Brain 2009;2:20-31.
45.    Mazzini L, Cossa FM, Angelino E, Campini R, Pastore I, Monaco F. Posttraumatic epilepsy: neuroradiologic and neuropsychological assessment of long-term outcome. Epilepsia 2003;44:569-574.
46.    Henshall DC, Simon RP. Epilepsy and apoptosis pathways. J Cereb Blood Flow Metab 2005;25:1557-1572.
47.    Yan W, Wang HD, Hu ZG, Wang QF, Yin HX. Activation of Nrf2-ARE pathway in brain after traumatic brain injury. Neurosci Lett 2008;431:150-154.
48.    Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature 2001;412:641-647.
49.    Kaltschmidt B, Kaltschmidt C. NF-kappaB in the nervous system. Cold Spring Harb Perspect Biol 2009;1:a001271.
50.    Mattson MP, Camandola S. NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest 2001;107:247-254.
51.    Lubin FD, Ren Y, Xu X, Anderson AE. Nuclear factor-kappa B regulates seizure threshold and gene transcription following convulsant stimulation. J Neurochem 2007;103:1381-1395.
52.    Mazzuferi M, Kumar G, van Eyll J, Danis B, Foerch P, Kaminski RM. Nrf2 defense pathway: Experimental evidence for its protective role in epilepsy. Ann Neurol 74:560-568.
53.    Miller DM, Singh IN, Wang JA, Hall ED. Nrf2-ARE activator carnosic acid decreases mitochondrial dysfunction, oxidative damage and neuronal cytoskeletal degradation following traumatic brain injury in mice. Exp Neurol 2014;264:103-110.
54.    Xiong W, MacColl Garfinkel AE, Li Y, Benowitz LI, Cepko CL. NRF2 promotes neuronal survival in neurodegeneration and acute nerve damage. J Clin Invest 2015;125:1433-1445.
55.    Mazzuferi M, Kumar G, van Eyll J, Danis B, Foerch P, Kaminski RM. Nrf2 defense pathway: Experimental evidence for its protective role in epilepsy. Ann Neurol 2013;74:560-568.
56.    Patel M. Nrf2 to the rescue. Epilepsy Curr 2015;15:39-40.