Status epilepticus (SE), especially in immature animals, is known to produce recurrent spontaneous seizures and behavioral comorbidities later in life. The cause of these adverse long-term outcomes is unknown, but it has been hypothesized that free radicals produced by SE may play a role. We tested this hypothesis by treating immature (P25) rats with the free radical scavenger N-tert-butyl-α-phenylnitrone (PBN) at the time of lithium chloride (LiCl)/pilocarpine (PILO)-induced SE. Later, long-term outcomes were assessed. Cognitive impairment (spatial memory) was tested in the Morris water maze (MWM). Emotional disturbances were assessed by the capture test (aggressiveness) and elevated plus maze's (EPM) test (anxiety). Next, the presence and severity of spontaneous seizures were assessed by continuous video/EEG monitoring for 5 days. Finally, immunochemistry, stereology and morphology were used to assess the effects of PBN on hippocampal neuropathology and neurogenesis. PBN treatment modified the long-term effects of SE in varying ways, some beneficial and some detrimental. Beneficially, PBN protected against severe anatomical damage in the hippocampus and associated spatial memory impairment. Detrimentally, PBN treated animals had more severe seizures later in life. PBN also made animals more aggressive and more anxious. Correlating with these detrimental long-term outcomes, PBN significantly modified post-natal neurogenesis. Treated animals had significantly increased numbers of mature granule cells (GCs) ectopically located in the dentate hilus (DH). These results raise the possibility that abnormal neurogenesis may significantly contribute to the development of post-SE epilepsy and behavioral comorbidities.

Department of Developmental Epileptology Institute of Physiology of the Czech Academy of Sciences Prague Czechia

Strong Epilepsy Center Department of Neurology University of Rochester Medical Center Rochester NY United States

See more in PubMed

Block F., Schwarz M. (1997). Correlation between hippocampal neuronal damage and spatial learning deficit due to global ischemia. Pharmacol. Biochem. Behav 56, 755–761. 10.1016/s0091-3057(96)00484-4 PubMed DOI

Broadbent N. J., Squire L. R., Clark R. E. (2004). Spatial memory, recognition memory, and the hippocampus. Proc. Natl. Acad. Sci. U S A 101, 14515–14520. 10.1073/pnas.0406344101 PubMed DOI PMC

Bruce A. J., Baudry M. (1995). Oxygen free radicals in rat limbic structures after kainate-induced seizures. Free Radic. Biol. Med 18, 993–1002. 10.1016/0891-5849(94)00218-9 PubMed DOI

Buckmaster P. S., Dudek F. E. (1997). Neuronal loss, granule cell axon reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J. Comp. Neurol. 385, 385–404. 10.1002/(sici)1096-9861(19970901)385:3<385::aid-cne4>3.3.co;2-y PubMed DOI

Cho K. O., Lybrand Z. R., Ito N., Brulet R., Tafacory F., Zhang L., et al. . (2015). Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline. Nat. Commun. 6:6606. 10.1038/ncomms7606 PubMed DOI PMC

Detour J., Schroeder H., Desor D., Nehlig A. (2005). A 5-month period of epilepsy impairs spatial memory, decreases anxiety, but spares object recognition in the lithium-pilocarpine model in adult rats. Epilepsia 46, 499–508. 10.1111/j.0013-9580.2005.38704.x PubMed DOI

Dorph-Petersen K. A., Nyengaard J. R., Gundersen H. J. (2001). Tissue shrinkage and unbiased stereological estimation of particle number and size. J. Microsc. 204, 232–246. 10.1046/j.1365-2818.2001.00958.x PubMed DOI

Folbergrová J., Druga R., Otáhal J., Haugvicová R., Mareš P., Kubová H. (2006). Effect of free radical spin trap N-tert-butyl-α-phenylnitrone (PBN) on seizures induced in immature rats by homocysteic acid. Exp. Neurol. 201, 105–119. 10.1016/j.expneurol.2006.03.031 PubMed DOI

Folbergrová J., He Q. P., Li P. A., Smith M. L., Siesjö B. K. (1999). The effect of α-phenyl-N-tert-butyl nitrone on bioenergetic state in substantia nigra following flurothyl-induced status epilepticus in rats. Neurosci. Lett. 266, 121–124. 10.1016/s0304-3940(99)00279-7 PubMed DOI

Folbergrová J., Ješina P., Kubová H., Druga R., Otáhal J. (2016). Status epilepticus in immature rats is associated with oxidative stress and mitochondrial dysfunction. Front. Cell. Neurosci 10:136. 10.3389/fncel.2016.00136 PubMed DOI PMC

Gundersen H. J., Bendtsen T. F., Korbo L., Marcussen N., Møller A., Nielsen K., et al. . (1988). Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96, 379–394. 10.1111/j.1699-0463.1988.tb05320.x PubMed DOI

Hattiangady B., Rao M. S., Shetty A. K. (2004). Chronic temporal lobe epilepsy is associated with severely declined dentate neurogenesis in the adult hippocampus. Neurobiol. Dis. 17, 473–490. 10.1016/j.nbd.2004.08.008 PubMed DOI

Hattiangady B., Shetty A. K. (2008). Implications of decreased hippocampal neurogenesis in chronic temporal lobe epilepsy. Epilepsia 49, 26–41. 10.1111/j.1528-1167.2008.01635.x PubMed DOI PMC

He Q. P., Smith M. L., Li P. A., Siesjö B. K. (1997). Necrosis of the substantia nigra, pars reticulata, in flurothyl-induced status epilepticus is ameliorated by the spin trap α phenyl-N-tert-butyl nitrone. Free Radic. Biol. Med 22, 917–922. 10.1016/s0891-5849(96)00478-9 PubMed DOI

Hensley K., Carney J. M., Stewart C. A., Tabatabaie T., Pye Q., Floyd R. A. (1997). Nitrone-based free radical traps as neuroprotective agents in cerebral ischaemia and other pathologies. Int. Rev. Neurobiol 40, 299–317. 10.1016/s0074-7742(08)60725-4 PubMed DOI

Hester M. S., Danzer S. C. (2014). Hippocampal granule cell pathology in epilepsy—a possible structural basis for comorbidities of epilepsy? Epilepsy Behav. 38, 105–116. 10.1016/j.yebeh.2013.12.022 PubMed DOI PMC

Hosford B. E., Liska J. P., Danzer S. C. (2016). Ablation of newly generated hippocampal granule cells has disease-modifying effects in epilepsy. J. Neurosci. 36, 11013–11023. 10.1523/JNEUROSCI.1371-16.2016 PubMed DOI PMC

Huang X., McMahon J., Huang Y. (2012). Rapamycin attenuates aggressive behavior in a rat model of pilocarpine-induced epilepsy. Neuroscience 215, 90–97. 10.1016/j.neuroscience.2012.04.011 PubMed DOI PMC

Iyengar S. S., LaFrancois J. J., Friedman D., Drew L. J., Denny C. A., Burghardt N. S., et al. . (2015). Suppression of adult neurogenesis increases the acute effects of kainic acid. Exp. Neurol. 264, 135–149. 10.1016/j.expneurol.2014.11.009 PubMed DOI PMC

Jakubs K., Nanobashvili A., Bonde S., Ekdahl C. T., Kokaia Z., Kokaia M., et al. . (2006). Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability. Neuron 52, 1047–1059. 10.1016/j.neuron.2006.11.004 PubMed DOI

Jessberger S., Zhao C., Toni N., Clemenson G. D., Jr., Li Y., Gage F. H. (2007). Seizure-associated, aberrant neurogenesis in adult rats characterized with retrovirus-mediated cell labeling. J. Neurosci. 27, 9400–9407. 10.1523/jneurosci.2002-07.2007 PubMed DOI PMC

Jung K. H., Chu K., Kim M., Jeong S. W., Song Y. M., Lee S. T., et al. . (2004). Continuous cytosine-b-D-arabinofuranoside infusion reduces ectopic granule cells in adult rat hippocampus with attenuation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus. Eur. J. Neurosci. 19, 3219–3226. 10.1111/j.0953-816x.2004.03412.x PubMed DOI

Jung K. H., Chu K., Lee S. T., Kim J., Sinn D. I., Kim J. M., et al. . (2006). Cyclooxygenase-2 inhibitor, celecoxib, inhibits the altered hippocampal neurogenesis with attenuation of spontaneous recurrent seizures following pilocarpine-induced status epilepticus. Neurobiol. Dis. 23, 237–246. 10.1016/j.nbd.2006.02.016 PubMed DOI

Kotake Y. (1999). Pharmacologic properties of phenyl N-tert-butylnitrone. Antioxid Redox. Signal 1, 481–499. 10.1089/ars.1999.1.4-481 PubMed DOI

Kubová H., Mareš P. (2013). Are morphologic and functional consequences of status epilepticus in infant rats progressive? Neuroscience 235, 232–249. 10.1016/j.neuroscience.2012.12.055 PubMed DOI

Kubová H., Mareš P., Suchomelová L., Brožek G., Druga R., Pitkänen A. (2004). Status epilepticus in immature rats leads to behavioural and cognitive impairment and epileptogenesis. Eur. J. Neurosci. 19, 3255–3265. 10.1111/j.0953-816x.2004.03410.x PubMed DOI

Kubová H., Rejchrtová J., Redkozubova O., Mareš P. (2005). An outcome of status epilepticus in immature rats varies according to the paraldehyde treatment. Epilepsia 46, 38–42. 10.1111/j.1528-1167.2005.01005.x PubMed DOI

Liu S., Wang J., Zhu D., Fu Y., Lukowiak K., Lu Y. M. (2003). Generation of functional inhibitory neurons in the adult rat hippocampus. J. Neurosci. 23, 732–736. 10.1523/JNEUROSCI.23-03-00732.2003 PubMed DOI PMC

Lothman E. W., Bertram E. H., III. (1993). Epileptogenic effects of status epilepticus. Epilepsia 34, S59–S70. 10.1111/j.1528-1157.1993.tb05907.x PubMed DOI

Lowenstein D. H. (1996). Recent advances related to basic mechanisms of epileptogenesis. Epilepsy Res. Suppl. 11, 45–60. 10.1016/b978-012373961-2.00107-7 PubMed DOI

Maia G. H., Quesado J. L., Soares J. I., do Carmo J. M., Andrade P. A., Andrade J. P., et al. . (2014). Loss of hippocampal neurons after kainate treatment correlates with behavioral deficits. PLoS One 9:e84722. 10.1371/journal.pone.0084722 PubMed DOI PMC

Majak K., Pitkänen A. (2004). Do seizures cause irreversible cognitive damage? Evidence from animal studies. Epilepsy Behav. 5, S35–S44. 10.1016/j.yebeh.2003.11.012 PubMed DOI

Malberg J. E., Duman R. S. (2003). Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacology 28, 1562–1571. 10.1038/sj.npp.1300234 PubMed DOI

Malberg J. E., Eisch A. J., Nestler E. J., Duman R. S. (2000). Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J. Neurosci. 20, 9104–9110. 10.1523/jneurosci.20-24-09104.2000 PubMed DOI PMC

McCloskey D. P., Hintz T. M., Pierce J. P., Scharfman H. E. (2006). Stereological methods reveal the robust size and stability of ectopic hilar granule cells after pilocarpine-induced status epilepticus in the adult rat. Eur. J. Neurosci. 24, 2203–2210. 10.1111/j.1460-9568.2006.05101.x PubMed DOI PMC

McElroy P. B., Liang L. P., Day B. J., Patel M. (2017). Scavenging reactive oxygen species inhibits status epilepticus-induced neuroinflammation. Exp. Neurol. 298, 13–22. 10.1016/j.expneurol.2017.08.009 PubMed DOI PMC

Myers C. E., Bermudez-Hernandez K., Scharfman H. E. (2013). The influence of ectopic migration of granule cells into the hilus on dentate gyrus-CA3 function. PLoS One 8:e68208. 10.1371/journal.pone.0068208 PubMed DOI PMC

Nairismägi J., Pitkänen A., Kettunen M. I., Kauppinen R. A., Kubová H. (2006). Status epilepticus in 12-day-old rats leads to temporal lobe neurodegeneration and volume reduction: a histologic and MRI study. Epilepsia 47, 479–488. 10.1111/j.1528-1167.2006.00455.x PubMed DOI

Nissinen J., Halonen T., Koivisto E., Pitkänen A. (2000). A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res. 38, 177–205. 10.1016/s0920-1211(99)00088-1 PubMed DOI

Nitecka L., Tremblay E., Charton G., Bouillot J. P., Berger M. L., Ben-Ari Y. (1984). Maturation of kainic acid seizure-brain damage syndrome in the rat: II. Histopathological sequelae. Neuroscience 13, 1073–1094. 10.1016/0306-4522(84)90289-6 PubMed DOI

Parent J. M., Elliott R. C., Pleasure S. J., Barbaro N. M., Lowenstein D. H. (2006). Aberrant seizure-induced neurogenesis in experimental temporal lobe epilepsy. Ann. Neurol. 59, 81–91. 10.1002/ana.20699 PubMed DOI

Patel M., Li Q. Y. (2003). Age dependence of seizure-induced oxidative stress. Neuroscience 118, 431–437. 10.1016/s0306-4522(02)00979-x PubMed DOI

Pauletti A., Terrone G., Shekh-Ahmad T., Salamone A., Ravizza T., Rizzi M., et al. . (2017). Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy. Brain 140, 1885–1899. 10.1093/brain/awx117 PubMed DOI PMC

Paxinos G., Watson C. (1986). The Rat Brain in Stereotaxic Coordinates. New York, NY: Academic Press.

Pearson J. N., Rowley S., Liang L. P., White A. M., Day B. J., Patel M. (2015). Reactive oxygen species mediate cognitive deficits in experimental temporal lobe epilepsy. Neurobiol. Dis. 82, 289–297. 10.1016/j.nbd.2015.07.005 PubMed DOI PMC

Pekcec A., Fuest C., Mühlenhoff M., Gerardy-Schahn R., Potschka H. (2008). Targeting epileptogenesis-associated induction of neurogenesis by enzymatic depolysialylation of NCAM counteracts spatial learning dysfunction but fails to impact epilepsy development. J. Neurochem. 105, 389–400. 10.1111/j.1471-4159.2007.05172.x PubMed DOI

Peterson S. L., Purvis R. S., Griffith J. W. (2005). Comparison of neuroprotective effects induced by α-phenyl-N-tert-butyl nitrone (PBN) and N-tert-butyl-α-(2 sulfophenyl) nitrone (S-PBN) in lithium-pilocarpine status epilepticus. Neurotoxicology 26, 969–979. 10.1016/j.neuro.2005.04.002 PubMed DOI

Pinel J. P., Treit D., Rovner L. I. (1977). Temporal lobe aggression in rats. Science 197, 1088–1089. 10.1126/science.560719 PubMed DOI

Pitkänen A., Kubová H. (2004). Antiepileptic drugs in neuroprotection. Expert Opin. Pharmacother. 5, 777–798. 10.1517/eoph.5.4.777.30162 PubMed DOI

Pitkänen A., Nissinen J., Nairismägi J., Lukasiuk K., Gröhn O. H., Miettinen R., et al. . (2002). Progression of neuronal damage after status epilepticus and during spontaneous seizures in a rat model of temporal lobe epilepsy. Prog. Brain Res. 135, 67–83. 10.1016/s0079-6123(02)35008-8 PubMed DOI

Racine R. J. (1972). Modification of seizure activity by electrical stimulation: II. Motor seizures. Electroenceph. Clin. Neurophysiol 32, 281–294. 10.10.1016/0013-4694(72)90177-0 PubMed DOI

Rao M. S., Shetty A. K. (2004). Efficacy of doublecortin as a marker to analyse the absolute number and dendritic growth of newly generated neurons in the adult dentate gyrus. Eur. J. Neurosci. 19, 234–246. 10.1111/j.0953-816x.2003.03123.x PubMed DOI

Rejchrtová J., Kubová H., Druga R., Mareš P., Folbergrová J. (2005). Effects of a free radical scavenger N-tert-butyl-α-phenylnitrone (PBN) on short-term recovery of immature rats after status epilepticus. Physiol. Res. 54, 215–227. PubMed

Sankar R., Shin D. H., Liu H., Mazarati A., Pereira de Vasconcelos A., Wasterlain C. G. (1998). Patterns of status epilepticus-induced neuronal injury during development and long-term consequences. J. Neurosci. 18, 8382–8393. 10.1523/jneurosci.18-20-08382.1998 PubMed DOI PMC

Scharfman H. E., Goodman J., McCloskey D. (2007). Ectopic granule cells of the rat dentate gyrus. Dev. Neurosci. 29, 14–27. 10.1159/000096208 PubMed DOI PMC

Scharfman H. E., Sollas A. E., Berger R. E., Goodman J. H., Pierce J. P. (2003). Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures. Neuroscience 121, 1017–1029. 10.1016/s0306-4522(03)00481-0 PubMed DOI

Sloviter R. S. (1992). Possible functional consequences of synaptic reorganization in the dentate gyrus of kainate-treated rats. Neurosci. Lett. 137, 91–96. 10.1016/0304-3940(92)90306-r PubMed DOI

Torii M.-A., Matsuzaki F., Osumi N., Kaibuchi K., Nakamura S., Casarosa S., et al. . (1999). Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system. Development 126, 443–456. PubMed

Tuunanen J., Halonen T., Pitkänen A. (1996). Status epilepticus causes selective regional damage and loss of GABAergic neurons in the rat amygdaloid complex. Eur. J. Neurosci. 8, 2711–2725. 10.1111/j.1460-9568.1996.tb01566.x PubMed DOI

Ueda Y., Yokoyama H., Nakajima A., Tokumaru J., Doi T., Mitsuyama Y. (2002). Glutamate excess and free radical formation during and following kainic acid-induced status epilepticus. Exp. Brain Res. 147, 219–226. 10.1007/s00221-002-1224-4 PubMed DOI

Wasterlain C. G., Fujikawa D. G., Penix L., Sankar R. (1993). Pathophysiological mechanisms of brain damage from status epilepticus. Epilepsia 34, S37–S53. 10.1111/j.1528-1157.1993.tb05905.x PubMed DOI

Wasterlain C. G., Shirasaka Y., Mazarati A. M., Spigelman I. (1996). Chronic epilepsy with damage restricted to the hippocampus: possible mechanisms. Epilepsy Res. 26, 255–265. 10.1016/s0920-1211(96)00058-7 PubMed DOI

West M. J., Slomianka L., Gundersen H. J. (1991). Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231, 482–497. 10.1002/ar.1092310411 PubMed DOI

Wood J. C., Jackson J. S., Jakubs K., Chapman K. Z., Ekdahl C. T., Kokaia Z., et al. . (2011). Functional integration of new hippocampal neurons following insults to the adult brain is determined by characteristics of pathological environment. Exp. Neurol. 229, 484–493. 10.1016/j.expneurol.2011.03.019 PubMed DOI

Zhang X., Cui S. S., Wallace A. E., Hannesson D. K., Schmued L. C., Saucier D. M., et al. . (2002). Relations between brain pathology and temporal lobe epilepsy. J. Neurosci. 22, 6052–6061. 10.1523/JNEUROSCI.22-14-06052.2002 PubMed DOI PMC