摘要
颞叶癫癎是一种严重的神经系统疾病,其长期的病理变化很多仍不清楚,这也是颞叶癫癎患者临床治疗的严重障碍。癫癎发生过程通常包括三个阶段:损伤→潜伏期阶段(致癎期)→自发性癫癎发作(症状性癫癎)。虽然癫癎发生和反复发作的机制在颞叶癫癎患者中很难了解,但是在动物的颞叶癫癎模型中获得了越来越多的资料。在啮齿类动物中通过匹罗卡品注射诱发癫癎持续状态(status epilepticus, SE)已广泛用于慢性癫癎的实验研究。在大鼠或小鼠中系统应用匹罗卡品诱导的中枢神经系统神经元丢失与颞叶癫癎患者的情况相似。
颞叶癫癎模型中有两种主要的神经元死亡机制。一种机制是急性死亡,与水肿或缺血有关。另一种机制是迟发性死亡,可能由细胞内浓度短暂性异常升高的游离钙介导,这也许会过度激活各种在正常情况下参与突触可塑性的酶系统如钙蛋白酶和蛋白激酶,caspases介导的程序性细胞死亡(programmed cell death, PCD)可能在迟发性损伤中起作用。神经元丢失是致癎期间环路重组的主要因素之一,因此损伤因素如SE发作诱导的细胞受损程度和时程仍是非常需要阐明的重要问题,因为这也许是慢性癫癎中发生长期病理变化和反复癫癎发作的关键性神经基础。细胞凋亡是慢性癫癎发生中神经元丢失的原因之一。凋亡是机体在一定的生理或病理条件下通过启动多种机制如死亡信号受体、线粒体功能障碍、caspase酶激活及DNA破坏而进行的程序性死亡过程。进一步阐明凋亡在慢性癫癎发生过程所起的作用仍然非常重要。
研究目的:本研究旨在阐明颞叶癫癎中可能发生的长期脑损伤特征及损伤的相关机制。研究方法:我们在颞叶癫癎的小鼠匹罗卡品模型中通过Fluoro-Jade C (FJC)染色方法探测了变性神经元的区域分布和时程变化,还结合免疫荧光技术阐明了受累神经元的类型及涉及的损伤机制。FJC是一种新发展的荧光染料,对正发生变性的神经元(包括胞体、树突、轴突和轴突末梢)有高度的亲和力,特异性标记中枢神经系统正发生变性的神经元。研究结果:(1)在本研究中,我们用FJC染色方法结合标记神经元核特异性蛋白(neuronal nuclear specific protein, NeuN)的免疫荧光技术揭示匹罗卡品诱发的SE导致了从嗅球到中脑的许多脑区发生了大量的神经元细胞变性。(2)而且,由FJC揭示的神经元变性(包括急性和迟发性神经元损伤)是时间依赖性的。FJC阳性神经元在SE后4h出现,在12h-3d时达到高峰,然后又逐渐下降,7-14d时在有些区域甚至恢复到基线水平或消失。(3)双标记资料揭示FJC与Hoechst33342双标,且大多数FJC阳性神经元也表达凋亡信号因子如cytochrome C、caspase-9和活化的caspase-3,提示这些FJC阳性神经元正经历凋亡过程且可能尚处于早期阶段,也可能提示caspases介导的程序性细胞死亡在迟发性损伤中起重要作用。(4)更有趣的是,大部分(88%)FJC阳性神经元显示GABA(gamma-aminobutyric acid)能性质,因为它们也对谷氨酸脱羧酶-67(glutamic acid decarboxylase-67, GAD-67)显示免疫反应性,这也许提示在小鼠的匹罗卡品模型中GABA能神经系统的抑制功能受到严重损害。
结论:本研究首次在小鼠匹罗卡品癫癎模型中应用FJC染色技术探测神经元死亡的区域分布、时程变化及相关机制。结合以前的研究综合考虑,在小鼠匹罗卡品颞叶癫癎模型中通过FJC标记变性的GABA能神经元的时程变化情况及所揭示的神经元死亡机制不仅有利于更好地理解颞叶癫癎中所发生的中枢神经系统长期病理变化和自发反复发作的癫癎机制,而且从治疗干预的可能性角度考虑,有助于探测治疗干预的时间窗和致癎期间的神经保护措施以预防或减轻癫癎发作。
Temporal lobe epilepsy presents a serious neurological disorder in human beings and its long-term pathological events largely remain an obscure and severe obstacle in clinical treatment of patients. The epileptic process usually consists of three phases: initial insult→latency period (epileptogenesis)→recurrent seizures (symptomatic epilepsy). The epileptogenesis and recurrent seizure mechanisms in humans are poorly understood, but growing evidences have been obtained from animal models of epilepsy. The rodent animal models of status epilepticus (SE) have been extensively utilized in experimental studies of chronic epilepsy by injection of pilocarpine. Systemic administration of pilocarpine can induce neuronal loss in central nervous system, which shows striking similarities to human temporal lobe epilepsy in rats or mice.
There are two major mechanisms of neuronal death in models of temporal lobe epilepsy. One mechanism is acute oedemic or ischemic death of the effected neurons. The other mechanism is delayed, and is likely to be mediated by a transient but abnormal rise in intracellular free calcium concentration. This may overactivate various enzyme systems normally involved in synaptic plasticity, e.g. calpain and protein kinases. Caspase-mediated programmed cell death (PCD) can contribute to delayed damage. Neuronal loss is one of the major components of circuitry reorganisation during epileptogenesis. Thus,the injury extent and time-course of neuronal death induced by SE attack still remain crucial question to answer, which may encase key neural basis of long-term changes and recurrent seizure in chronic epilepsy. Evidences have showed that neuronal loss in the chronic epilepsy could partially result from cell apoptosis, which is a programmed physiological event but also occurred in various toxic insults and neurological diseases through multiple ways of death signaling receptors, mitochondrial dysfunction, activation of caspase enzymes, and DNA damage. It is still important to further elucidate the contribution of an apoptotic mechanism to the pathological process of chronic epilepsy.
Purpose: In the present study, we are interested in elucidating long-term brain injury and related mechanisms that may occur in the temporal lobe epilepsy. Methods: The regional distribution and time-course of degenerative neurons were examined in a mouse pilocarpine model of chronic epilepsy by Fluoro-Jade C (FJC) dye that can specifically stain degenerating neurons in the central nervous system. The type of affected neurons and mechanisms involved in cellular damage were also elucidated by combining with immunofluorescence technique. FJC is a new-developed fluorescent Fluoro-Jade (FJ) dye which has high affinity for degenerating neurons including cell body, dendrites, axons and axon terminals. Results: (1) The FJC stain combined with neuronal nuclear specific protein (NeuN) immunofluorescence revealed that pilocarpine-induced SE resulted in massive degenerative death of neuronal cells in many brain regions from olfactory bulb to midbrain. (2) Moreover, cellular degeneration including acute and delayed neuronal death revealed by FJC stain was time-dependent. The FJC-positive degenerating neurons occurred at 4h, increased into peak levels at 12h–3d, and then gradually went down, even resolved to baseline or disappeared at 7d–14d after onset of SE. (3) Double-labeling data revealed that cellular co-localization of FJC and Hoechst was abundantly observed and most of FJC-positive degenerating neurons also expressed apoptosis signaling molecules such as cytochrome C, caspase-9, and activated caspase-3, indicating that these FJC-positive cells maybe were undergoing apoptotic processes and were in an early phase of apoptosis. These may also imply that caspase-mediated PCD plays an important role in the delayed damage. (4) More interestingly, a large percentage (about 88%) of FJC-positive degenerative neurons were GABAergic as indicated with their immunoreactivity to glutamic acid decarboxylase-67 (GAD-67), implying that inhibitory function of GABAergic neural system might by seriously damaged in brains subject to SE attack in this mouse pilocarpine model.
Conclusion: This study has first applied FJC staining to demonstrate regional distribution, time-course, and related mechanisms of neuronal death in the mouse pilocarpine model. Taken together with previous studies, time-course and death mechanisms of degenerative GABAergic neurons in the mouse pilocarpine model revealed by FJC staining benefit further understanding of long-term brain pathological changes and recurrent seizure mechanism, and, from the point of possible pharmacological intervention, may also result in finding the most suitable time-window in therapeutic manipulation of the chronic epilepsy in human beings and appropriate neuroprotective treatment to prevent or lessen seizures during the epileptogenic phase.
引文
1 Freichel C, Potschka H, Ebert U, Brandt C, L?scher W.Acute changes in the neuronal expression of GABA and glutamate decarboxylase isoforms in the rat piriform cortex following status epilepticus. Neuroscience. 2006 Sep 15;141(4):2177-94.
2 Kim ST, Jeon S, Park HJ, Hong MS, Jeong WB, Kim JH, Kim Y, Lee HJ, Park HJ, Chung JH.Acupuncture inhibits kainic Acid-induced hippocampal cell death in mice. J Physiol Sci. 2008 Feb;58(1):31-8.
3 Garrido Sanabria ER, Casta?eda MT, Banuelos C, Perez-Cordova MG, Hernandez S, Colom LV. Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures.Neuroscience. 2006 Oct 27;142(3):871-83.
4 Pathak HR, Weissinger F, Terunuma M, Carlson GC, Hsu FC, Moss SJ, Coulter DA. Disrupted dentate granule cell chloride regulation enhances synaptic excitability during development of temporal lobe epilepsy. J Neurosci. 2007 Dec 19;27(51):14012-22.
5 Sastry PS, Rao KS. Apoptosis and the nervous system. J Neurochem. 2000 Jan;74(1):1-20.
6 Schmued LC, Albertson C, Slikker W Jr. Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res. 1997 Mar 14;751(1):37-46.
7 Schmued LC, Hopkins KJ. Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res. 2000a Aug 25;874(2):123-30.
8 Schmued LC, Hopkins KJ. Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration. Toxicol Pathol. 2000b Jan-Feb;28(1):91-9.
9 Schmued LC, Stowers CC, Scallet AC, Xu L. Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res. 2005 Feb 21;1035(1):24-31.
10 Ballok DA, Millward JM, Sakic B. Neurodegeneration in autoimmune MRL-lpr mice as revealed by Fluoro Jade B staining. Brain Res. 2003 Feb 28;964(2):200-10.
11 Coulter DA. Chronic epileptogenic cellular alterations in the limbic system after status epilepticus. Epilepsia. 1999;40 Suppl 1:S23-33; discussion S40-1.
12 Clifford DB, Olney JW, Maniotis A, Collins RC, Zorumski CF. The functional anatomy and pathology of lithium-pilocarpine and high-dose pilocarpine seizures. Neuroscience. 1987 Dec;23(3):953-68.
13 Oliveira AA, Nogueira CR, Nascimento VS, Aguiar LM, Freitas RM, Sousa FC, Viana GS, Fonteles MM. Evaluation of levetiracetam effects on pilocarpine-induced seizures: cholinergic muscarinic system involvement. Neurosci Lett. 2005 Sep 16;385(3):184-8.
14 Fujikawa DG, Shinmei SS, Cai B. Lithium-pilocarpine-induced status epilepticus produces necrotic neurons with internucleosomal DNA fragmentation in adult rats. Eur J Neurosci. 1999 May;11(5):1605-14.
15 Ben-Ari Y. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience. 1985 Feb;14(2):375-403.
16 Zhang HJ, Sun RP, Lei GF, Yang L, Liu CX. Cyclooxygenase-2 inhibitorinhibits hippocampal synaptic reorganization in pilocarpine-induced status epilepticus rats. J Zhejiang Univ Sci B. 2008 Nov;9(11):903-15
17 Ben-Ari Y, Tremblay E, Riche D, Ghilini G, Naquet R. Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy. Neuroscience. 1981;6(7):1361-91.
18 Babb TL, Kupfer WR, Pretorius JK, Crandall PH, Levesque MF. Synaptic reorganization by mossy fibers in human epileptic fascia dentata. Neuroscience. 1991;42(2):351-63.
19 Mello LE, Cavalheiro EA, Tan AM, Kupfer WR, Pretorius JK, Babb TL, Finch DM. Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: cell loss and mossy fiber sprouting. Epilepsia. 1993 Nov-Dec;34(6):985-95.
20 Wuarin JP, Dudek FE. Excitatory synaptic input to granule cells increases with time after kainate treatment. J Neurophysiol. 2001 Mar;85(3):1067-77.
21 Winokur RS, Kubal T, Liu D, Davis SF, Smith BN. Recurrent excitation in the dentate gyrus of a murine model of temporal lobe epilepsy. Epilepsy Res. 2004 Feb;58(2-3):93-105.
22 Cavalheiro EA, Santos NF, Priel MR. The pilocarpine model of epilepsy in mice. Epilepsia. 1996 Oct;37(10):1015-9.
23 Shibley H, Smith BN. Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice. Epilepsy Res. 2002 Apr;49(2):109-20.
24 Teng X, Degterev A, Jagtap P, Xing X, Choi S, Denu R, Yuan J, Cuny GD.Structure-activity relationship study of novel necroptosis inhibitors. Bioorg Med Chem Lett. 2005 Nov 15;15(22):5039-44.
25 Wang K, Li J, Degterev A, Hsu E, Yuan J, Yuan C. Structure-activity relationship analysis of a novel necroptosis inhibitor, Necrostatin-5. Bioorg Med Chem Lett. 2007 Mar 1;17(5):1455-65.
26 Nicotera P, Leist M, Ferrando-May E. Apoptosis and necrosis: different execution of the same death. Biochem Soc Symp. 1999;66:69-73.
27 Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med. 1999 Nov;107(5):489-506.
28 McNeill-Blue C, Wetmore BA, Sanchez JF, Freed WJ, Merrick BA. Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine. Brain Res. 2006 Sep 27;1112(1):1-15.
29 Pretel S, Applegate CD, Piekut D. Apoptotic and necrotic cell death following kindling induced seizures. Acta Histochem. 1997 Mar;99(1):71-9.
30 Maglóczky Z, Freund TF. Delayed cell death in the contralateral hippocampus following kainate injection into the CA3 subfield. Neuroscience. 1995 Jun;66(4):847-60.
31 Narkilahti S, Nissinen J, Pitk?nen A. Administration of caspase 3 inhibitor during and after status epilepticus in rat: effect on neuronal damage and epileptogenesis. Neuropharmacology. 2003 Jun;44(8):1068-88.
32 Bredesen DE. Apoptosis: overview and signal transduction pathways. J Neurotrauma. 2000 Oct;17(10):801-10.
33 Peter ME, Heufelder AE, Hengartner MO. Advances in apoptosis research. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12736-7.
34 Jaeschke H, Lemasters JJ .Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology. 2003 Oct;125(4):1246-57.
35 Rich T, Allen RL, Wyllie AH. Defying death after DNA damage. Nature. 2000 Oct 12;407(6805):777-83.
36 Farber E. Programmed cell death: necrosis versus apoptosis. Mod Pathol. 1994 Jun;7(5):605-9.
37 Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem. 1999;68:383-424.
38 Reed JC. Mechanisms of apoptosis. Am J Pathol. 2000 Nov;157(5):1415-30.
39 Boyd CS, Cadenas E. Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis. Biol Chem. 2002 Mar-Apr;383(3-4):411-23.
40 Mehta SL, Manhas N, Raghubir R. Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Res Rev. 2007 Apr;54(1):34-66.
41 Buja LM. Myocardial ischemia and reperfusion injury. Cardiovasc Pathol. 2005 Jul-Aug;14(4):170-5.
42 Saikumar P, Dong Z, Weinberg JM, Venkatachalam MA. Mechanisms of cell death in hypoxia/reoxygenation injury. Oncogene. 1998 Dec 24;17(25):3341-9.
43 Teng X, Keys H, Jeevanandam A, Porco JA Jr, Degterev A, Yuan J, Cuny GD.Structure-activity relationship study of [1,2,3]thiadiazole necroptosis inhibitors.Bioorg Med Chem Lett. 2007 Dec 15;17(24):6836-40.
44 Qian T, Nieminen AL, Herman B, Lemasters JJ. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am J Physiol. 1997 Dec;273(6 Pt 1):C1783-92.
45 Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12;86(1):147-57.
46 Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itoh N, Suda T, Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature. 1993 Aug 26;364(6440):806-9.
47 Lemasters JJ. V. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. Am J Physiol. 1999 Jan;276(1 Pt 1):G1-6.
48 Formigli L, Papucci L, Tani A, Schiavone N, Tempestini A, Orlandini GE, Capaccioli S, Orlandini SZ. Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol. 2000 Jan;182(1):41-9.
49 Bursch W, Ellinger A, Gerner C, Fr?hwein U, Schulte-Hermann R. Programmed cell death (PCD). Apoptosis, autophagic PCD, or others? Ann N Y Acad Sci. 2000;926:1-12.
50 Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 2001 Jun;8(6):569-81.
51 Elmore SP, Qian T, Grissom SF, Lemasters JJ. The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J. 2001 Oct;15(12):2286-7.
52 Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene. 2004 Apr 12;23(16):2881-90.
53 Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol. 1995 Jan;146(1):3-15.
54牛万成,李玺。细胞死亡方式理论的研究进展。徐州医学院学报2006,26(5):467-470。
55 Levin S. Apoptosis, necrosis, or oncosis: what is your diagnosis? A report from the Cell Death Nomenclature Committee of the Society of Toxicologic Pathologists. Toxicol Sci. 1998 Feb;41(2):155-6.
56 Asher E, Payne CM, Bernstein C. Evaluation of cell death in EBV-transformed lymphocytes using agarose gel electrophoresis, light microscopy and electron microscopy. II. Induction of non-classic apoptosis ("para-apoptosis") by tritiated thymidine. Leuk Lymphoma. 1995 Sep;19(1-2):107-19.
57 Mironova EV, Evstratova AA, Antonov SM. A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture. J Neurosci Methods. 2007 Jun 15;163(1):1-8.
58 Lakhani SA, Masud A, Kuida K, Porter GA Jr, Booth CJ, Mehal WZ, Inayat I, Flavell RA. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science. 2006 Feb 10;311(5762):847-51.
59 Brauchi S, Cea C, Farias JG, Bacigalupo J, Reyes JG. Apoptosis induced by prolonged exposure to odorants in cultured cells from rat olfactory epithelium. Brain Res. 2006 Aug 4;1103(1):114-22.
60 Golbs A, Heck N, Luhmann HJ. A new technique for real-time analysis of caspase-3 dependent neuronal cell death. J Neurosci Methods. 2007 Apr 15;161(2):234-43.
61 Davoli MA, Fourtounis J, Tam J, Xanthoudakis S, Nicholson D, Robertson GS, Ng GY, Xu D. Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat. Neuroscience. 2002;115(1):125-36.
62 Fickert P, Trauner M, Fuchsbichler A, Zollner G, Wagner M, Marschall HU, Zatloukal K, Denk H. Oncosis represents the main type of cell death in mouse models of cholestasis. J Hepatol. 2005 Mar;42(3):378-85.
63 Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA. Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases. Am J Pathol. 1997 Nov;151(5):1205-13.
64 Ishikawa Y, Satoh T, Enokido Y, Nishio C, Ikeuchi T, Hatanaka H. Generation of reactive oxygen species, release of L-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture. Brain Res. 1999 Apr 3;824(1):71-80.
65 Shimizu K, Matsubara K, Ohtaki K, Shiono H. Paraquat leads to dopaminergic neural vulnerability in organotypic midbrain culture. Neurosci Res. 2003 Aug;46(4):523-32.
66 Tenn CC, Wang Y. VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons. Neurosci Lett. 2007 May 1;417(2):155-9.
67 Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D, Yahalom J. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001 Jan 15;61(2):439-44.
68 Arthur CR, Gupton JT, Kellogg GE, Yeudall WA, Cabot MC, Newsham IF, Gewirtz DA. Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison. Biochem Pharmacol. 2007 Oct 1;74(7):981-91.
69 Monette R, Small DL, Mealing G, Morley P. A fluorescence confocal assayto assess neuronal viability in brain slices. Brain Res Brain Res Protoc. 1998 Jan;2(2):99-108.
70 Reichstein D, Ren L, Filippopoulos T, Mittag T, Danias J. Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma. Exp Eye Res. 2007 Jan;84(1):13-21.
71 Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995 Jul 17;184(1):39-51.
72 Wang L, Liu YH, Huang YG, Chen LW. Time-course of neuronal death in the mouse pilocarpine model of chronic epilepsy using Fluoro-Jade C staining. Brain Res. 2008 Nov 19;1241:157-67.
73 Elphick LM, Meinander A, Mikhailov A, Richard M, Toms NJ, Eriksson JE, Kass GE. Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct. Anal Biochem. 2006 Feb 1;349(1):148-55.
74 Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol. 1972 Mar;32(3):281-94.
75 Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates, second ed. Academic Press, New York, 2001.
76 Voutsinos-Porche B, Koning E, Kaplan H, Ferrandon A, Guenounou M, Nehlig A, Motte J. Temporal patterns of the cerebral inflammatory response in the rat lithium-pilocarpine model of temporal lobe epilepsy. Neurobiol Dis. 2004 Dec;17(3):385-402.
77 FolbergrováJ, Druga R, Otáhal J, HaugvicováR, Mares P, KubováH.Seizures induced in immature rats by homocysteic acid and the associated brain damage are prevented by group II metabotropic glutamate receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate. Exp Neurol. 2005 Apr;192(2):420-36.
78 Ebert U, Brandt C, L?scher W. Delayed sclerosis, neuroprotection, and limbic epileptogenesis after status epilepticus in the rat. Epilepsia. 2002;43 Suppl 5:86-95.
79 Margerison JH, Corsellis JA. Epilepsy and the temporal lobes. A clinical, electroencephalographic and neuropathological study of the brain in epilepsy, with particular reference to the temporal lobes. Brain. 1966 Sep;89(3):499-530.
80 Knopp A, Frahm C, Fidzinski P, Witte OW, Behr J. Loss of GABAergic neurons in the subiculum and its functional implications in temporal lobe epilepsy. Brain. 2008 Jun;131(Pt 6):1516-27.
81 McIntyre DC, Kelly ME. The parahippocampal cortices and kindling. Ann N Y Acad Sci. 2000 Jun;911:343-54.
82 Covolan L, Mello LE. Temporal profile of neuronal injury following pilocarpine or kainic acid-induced status epilepticus. Epilepsy Res. 2000 Apr;39(2):133-52.
83 Kobayashi M, Buckmaster PS.Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy. J Neurosci. 2003 Mar 15;23(6):2440-52.
84 Wilson CL, Isokawa M, Babb TL, Crandall PH. Functional connections in the human temporal lobe. I. Analysis of limbic system pathways using neuronal responses evoked by electrical stimulation. Exp Brain Res. 1990;82(2):279-92.
85 Goldring S, Edwards I, Harding GW, Bernardo KL. Results of anterior temporal lobectomy that spares the amygdala in patients with complex partial seizures. J Neurosurg. 1992 Aug;77(2):185-93.
86 Jutila L, Ylinen A, Partanen K, Alafuzoff I, Mervaala E, Partanen J, Vapalahti M, Vainio P, Pitk?nen A. MR volumetry of the entorhinal, perirhinal, and temporopolar cortices in drug-refractory temporal lobe epilepsy. AJNR Am J Neuroradiol. 2001 Sep;22(8):1490-501.
87 Du F, Whetsell WO Jr, Abou-Khalil B, Blumenkopf B, Lothman EW, Schwarcz R. Preferential neuronal loss in layer III of the entorhinal cortex in patients with temporal lobe epilepsy. Epilepsy Res. 1993 Dec;16(3):223-33.
88 Bertram EH, Zhang DX, Mangan P, Fountain N, Rempe D. Functional anatomy of limbic epilepsy: a proposal for central synchronization of a diffusely hyperexcitable network. Epilepsy Res. 1998 Sep;32(1-2):194-205.
89 Druga R, Mares P, Otáhal J, KubováH. Degenerative neuronal changes in the rat thalamus induced by status epilepticus at different developmental stages. Epilepsy Res. 2005 Jan;63(1):43-65.
90 Cassidy RM, Gale K. Mediodorsal thalamus plays a critical role in the development of limbic motor seizures. J Neurosci. 1998 Nov 1;18(21):9002-9.
91 Mraovitch S, Calando Y. Limbic and/or generalized convulsive seizures elicited by specific sites in the thalamus. Neuroreport. 1995 Feb 15;6(3):519-23.
92 Newberg AB, Alavi A, Berlin J, Mozley PD, O'Connor M, Sperling M. Ipsilateral and contralateral thalamic hypometabolism as a predictor of outcome after temporal lobectomy for seizures. J Nucl Med. 2000Dec;41(12):1964-8.
93 Sojkova J, Lewis PJ, Siegel AH, Siegel AM, Roberts DW, Thadani VM, Williamson PD; SPECT studies. Does asymmetric basal ganglia or thalamic activation aid in seizure foci lateralization on ictal SPECT studies? J Nucl Med. 2003 Sep;44(9):1379-86.
94 Norden AD, Blumenfeld H. The role of subcortical structures in human epilepsy. Epilepsy Behav. 2002 Jun;3(3):219-231.
95 Juhász C, Nagy F, Watson C, da Silva EA, Muzik O, Chugani DC, Shah J, Chugani HT. Glucose and [11C]flumazenil positron emission tomography abnormalities of thalamic nuclei in temporal lobe epilepsy. Neurology. 1999 Dec 10;53(9):2037-45.
96 Natsume J, Bernasconi N, Andermann F, Bernasconi A. MRI volumetry of the thalamus in temporal, extratemporal, and idiopathic generalized epilepsy. Neurology. 2003 Apr 22;60(8):1296-300.
97 Bertram EH, Mangan PS, Zhang D, Scott CA, Williamson JM. The midline thalamus: alterations and a potential role in limbic epilepsy. Epilepsia. 2001 Aug;42(8):967-78.
98 Bertram EH, Zhang D, Williamson JM. Multiple roles of midline dorsal thalamic nuclei in induction and spread of limbic seizures. Epilepsia. 2008 Feb;49(2):256-68.
99 Kuroda M, López-Mascaraque L, Price JL. Neuronal and synaptic composition of the mediodorsal thalamic nucleus in the rat: a light and electron microscopic Golgi study. J Comp Neurol. 1992 Dec 1;326(1):61-81.
100 Kuroda M, Yokofujita J, Murakami K. An ultrastructural study of the neural circuit between the prefrontal cortex and the mediodorsal nucleusof the thalamus. Prog Neurobiol. 1998 Mar;54(4):417-58.
101 Bokor H, Frère SG, Eyre MD, Slézia A, Ulbert I, Lüthi A, Acsády L. Selective GABAergic control of higher-order thalamic relays. Neuron. 2005 Mar 24;45(6):929-40.
102 Turski L, Cavalheiro EA, Schwarz M, Turski WA, De Moraes Mello LE, Bortolotto ZA, Klockgether T, Sontag KH. Susceptibility to seizures produced by pilocarpine in rats after microinjection of isoniazid or gamma-vinyl-GABA into the substantia nigra. Brain Res. 1986 Apr 9;370(2):294-309.
103 Fabene PF, Marzola P, Sbarbati A, Bentivoglio M. Magnetic resonance imaging of changes elicited by status epilepticus in the rat brain: diffusion-weighted and T2-weighted images, regional blood volume maps, and direct correlation with tissue and cell damage. Neuroimage. 2003 Feb;18(2):375-89.
104 Costa MS, Rocha JB, Perosa SR, Cavalheiro EA, Naffah-Mazzacoratti Mda G. Pilocarpine-induced status epilepticus increases glutamate release in rat hippocampal synaptosomes. Neurosci Lett. 2004 Feb 6;356(1):41-4.
105 Van der Werf YD, Witter MP, Groenewegen HJ. The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence for participation in processes of arousal and awareness. Brain Res Brain Res Rev. 2002 Sep;39(2-3):107-40.
106 Ishikawa A, Nakamura S. Convergence and interaction of hippocampal and amygdalar projections within the prefrontal cortex in the rat. J Neurosci. 2003 Nov 5;23(31):9987-95.
107 Reep RL, Corwin JV, King V. Neuronal connections of orbital cortex in rats: topography of cortical and thalamic afferents. Exp Brain Res. 1996Sep;111(2):215-32.
108 Zhang DX, Bertram EH. Midline thalamic region: widespread excitatory input to the entorhinal cortex and amygdala. J Neurosci. 2002 Apr 15;22(8):3277-84.
109 Vaudano E, Legg CR, Glickstein M. Afferent and Efferent Connections of Temporal Association Cortex in the Rat: A Horseradish Peroxidase Study. Eur J Neurosci. 1991;3(4):317-330.
110 Doron NN, Ledoux JE. Organization of projections to the lateral amygdala from auditory and visual areas of the thalamus in the rat. J Comp Neurol. 1999 Sep 27;412(3):383-409.
111 Scorza FA, Arida RM, Priel M, Calderazzo L, Cavalheiro EA. The contribution of the lateral posterior and anteroventral thalamic nuclei on spontaneous recurrent seizures in the pilocarpine model of epilepsy. Arq Neuropsiquiatr. 2002 Sep;60(3-A):572-5.
112 Pitk?nen A, Jolkkonen E, Kemppainen S. Anatomic heterogeneity of the rat amygdaloid complex. Folia Morphol (Warsz). 2000;59(1):1-23.
113 Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience. 1992;46(4):755-84.
114 Lerner-Natoli M, de Bock F, Bockaert J, Rondouin G. NADPH diaphorase-positive cells in the brain after status epilepticus. Neuroreport. 1994 Dec 20;5(18):2633-7.
115 Leranth C, Carpi D, Buzsaki G, Kiss J. The entorhino-septo-supramammillary nucleus connection in the rat: morphological basis of a feedback mechanism regulating hippocampal theta rhythm. Neuroscience. 1999;88(3):701-18.
116 Kiss J, Csáki A, Bokor H, Kocsis K, Kocsis B. Possibleglutamatergic/aspartatergic projections to the supramammillary nucleus and their origins in the rat studied by selective [(3)H]D-aspartate labelling and immunocytochemistry. Neuroscience. 2002;111(3):671-91.
117 Gaykema RP, Luiten PG, Nyakas C, Traber J. Cortical projection patterns of the medial septum-diagonal band complex. J Comp Neurol. 1990 Mar 1;293(1):103-24.
118 Bland BH, Oddie SD, Colom LV. Mechanisms of neural synchrony in the septohippocampal pathways underlying hippocampal theta generation. J Neurosci. 1999 Apr 15;19(8):3223-37.
119 Colom LV, García-Hernández A, Casta?eda MT, Perez-Cordova MG, Garrido-Sanabria ER. Septo-hippocampal networks in chronically epileptic rats: potential antiepileptic effects of theta rhythm generation. J Neurophysiol. 2006 Jun;95(6):3645-53.
120 Jinno S, Kosaka T. Immunocytochemical characterization of hippocamposeptal projecting GABAergic nonprincipal neurons in the mouse brain: a retrograde labeling study. Brain Res. 2002 Aug 2;945(2):219-31.
121 Tóth K, Borhegyi Z, Freund TF. Postsynaptic targets of GABAergic hippocampal neurons in the medial septum-diagonal band of broca complex. J Neurosci. 1993 Sep;13(9):3712-24.
122 Pedemonte M, Barrenechea C, Nu?ez A, Gambini JP, García-Austt E. Membrane and circuit properties of lateral septum neurons: relationships with hippocampal rhythms. Brain Res. 1998 Jul 27;800(1):145-53.
123 Alonso JR, Frotscher M. Hippocampo-septal fibers terminate on identified spiny neurons in the lateral septum: a combined Golgi/electron-microscopic and degeneration study in the rat. Cell TissueRes. 1989 Nov;258(2):243-6.
124 Benkovic SA, O'Callaghan JP, Miller DB. Sensitive indicators of injury reveal hippocampal damage in C57BL/6J mice treated with kainic acid in the absence of tonic-clonic seizures. Brain Res. 2004 Oct 22;1024(1-2):59-76.
125 Poirier JL, Capek R, De Koninck Y. Differential progression of Dark Neuron and Fluoro-Jade labelling in the rat hippocampus following pilocarpine-induced status epilepticus. Neuroscience. 2000;97(1):59-68.
126 Sater RA, Nadler JV. On the relation between seizures and brain lesions after intracerebroventricular kainic acid. Neurosci Lett. 1988 Jan 11;84(1):73-8.
127 Pitk?nen A, Nissinen J, Nairism?gi J, Lukasiuk K, Gr?hn OH, Miettinen R, Kauppinen R. Progression of neuronal damage after status epilepticus and during spontaneous seizures in a rat model of temporal lobe epilepsy. Prog Brain Res. 2002;135:67-83.
128 Gorter JA, Gon?alves Pereira PM, van Vliet EA, Aronica E, Lopes da Silva FH, Lucassen PJ. Neuronal cell death in a rat model for mesial temporal lobe epilepsy is induced by the initial status epilepticus and not by later repeated spontaneous seizures. Epilepsia. 2003 May;44(5):647-58.
129 Cendes F. Progressive hippocampal and extrahippocampal atrophy in drug resistant epilepsy. Curr Opin Neurol. 2005 Apr;18(2):173-7.
130 Bian GL, Wei LC, Shi M, Wang YQ, Cao R, Chen LW. Fluoro-Jade C can specifically stain the degenerative neurons in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine-treated C57BL/6 mice. Brain Res. 2007 May 30;1150:55-61.
131 Sperk G, Furtinger S, Schwarzer C, Pirker S. GABA and its receptors in-epilepsy. Adv Exp Med Biol. 2004;548:92-103.
132 Cossart R, Bernard C, Ben-Ari Y. Multiple facets of GABAergic neurons and synapses: multiple fates of GABA signalling in epilepsies. Trends Neurosci. 2005 Feb;28(2):108-15.
133 Soghomonian JJ, Martin DL. Two isoforms of glutamate decarboxylase: why? Trends Pharmacol Sci. 1998 Dec;19(12):500-5.
134 Duncan JS, Sander JW, Sisodiya SM, Walker MC. Adult epilepsy. Lancet. 2006 Apr 1;367(9516):1087-100.
135 Ribak CE, Dashtipour K. Neuroplasticity in the damaged dentate gyrus of the epileptic brain. Prog Brain Res. 2002;136:319-28.
136 Houser CR, Esclapez M. Vulnerability and plasticity of the GABA system in the pilocarpine model of spontaneous recurrent seizures. Epilepsy Res. 1996 Dec;26(1):207-18.
137 Wittner L, Maglóczky Z, Borhegyi Z, Halász P, Tóth S, Eross L, SzabóZ, Freund TF.Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus. Neuroscience. 2001;108(4):587-600.
138 Dinocourt C, Petanjek Z, Freund TF, Ben-Ari Y, Esclapez M. Loss of interneurons innervating pyramidal cell dendrites and axon initial segments in the CA1 region of the hippocampus following pilocarpine-induced seizures. J Comp Neurol. 2003 May 12;459(4):407-25.
139 Bertrand S, Lacaille JC. Unitary synaptic currents between lacunosum-moleculare interneurones and pyramidal cells in rat hippocampus. J Physiol. 2001 Apr 15;532(Pt 2):369-84.
140 Fujiwara-Tsukamoto Y, Isomura Y, Kaneda K, Takada M. Synaptic interactions between pyramidal cells and interneurone subtypes during seizure-like activity in the rat hippocampus. J Physiol. 2004 Jun 15;557(Pt3):961-79.
141 Somogyi P, Klausberger T. Defined types of cortical interneurone structure space and spike timing in the hippocampus. J Physiol. 2005 Jan 1;562(Pt 1):9-26.
142 Esclapez M, Hirsch JC, Khazipov R, Ben-Ari Y, Bernard C. Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):12151-6.
143 Cossart R, Dinocourt C, Hirsch JC, Merchan-Perez A, De Felipe J, Ben-Ari Y, Esclapez M, Bernard C. Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy. Nat Neurosci. 2001 Jan;4(1):52-62.
144 Jefferys JG, Whittington MA. Review of the role of inhibitory neurons in chronic epileptic foci induced by intracerebral tetanus toxin. Epilepsy Res. 1996 Dec;26(1):59-66.
145 Schousboe A, Sarup A, Larsson OM, White HS. GABA transporters as drug targets for modulation of GABAergic activity. Biochem Pharmacol. 2004 Oct 15;68(8):1557-63.
146 Maglóczky Z, Wittner L, Borhegyi Z, Halász P, Vajda J, Czirják S, Freund TF.Changes in the distribution and connectivity of interneurons in the epileptic human dentate gyrus. Neuroscience. 2000;96(1):7-25.
147 AndréV, Marescaux C, Nehlig A, Fritschy JM. Alterations of hippocampal GAbaergic system contribute to development of spontaneous recurrent seizures in the rat lithium-pilocarpine model of temporal lobe epilepsy. Hippocampus. 2001;11(4):452-68.
148 Lothman EW, Stringer JL, Bertram EH. The dentate gyrus as a controlpoint for seizures in the hippocampus and beyond. Epilepsy Res Supp. 1992;7:301–313.
149 Behr J, Lyson KJ, Mody I. Enhanced propagation of epileptiform activity through the kindled dentate gyrus. J Neurophysiol. 1998 Apr;79(4):1726-32.
150 Halasy K, Somogyi P. Subdivisions in the multiple GABAergic innervation of granule cells in the dentate gyrus of the rat hippocampus. Eur J Neurosci. 1993 May 1;5(5):411-29.
151 Han ZS, Buhl EH, L?rinczi Z, Somogyi P. A high degree of spatial selectivity in the axonal and dendritic domains of physiologically identified local-circuit neurons in the dentate gyrus of the rat hippocampus. Eur J Neurosci. 1993 May 1;5(5):395-410.
152 Esclapez M, Houser CR. Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy. J Comp Neurol. 1999 Sep 27;412(3):488-505.
153 Ma DL, Tang YC, Chen PM, Chia SC, Jiang FL, Burgunder JM, Lee WL, Tang FR. Reorganization of CA3 area of the mouse hippocampus after pilocarpine induced temporal lobe epilepsy with special reference to the CA3-septum pathway. J Neurosci Res. 2006 Feb 1;83(2):318-31.
154 Chen S, Buckmaster PS. Stereological analysis of forebrain regions in kainate-treated epileptic rats. Brain Res. 2005 Sep 28;1057(1-2):141-52.
155 Sanabria ER, Su H, Yaari Y. Initiation of network bursts by Ca2+-dependent intrinsic bursting in the rat pilocarpine model of temporal lobe epilepsy. J Physiol. 2001 Apr 1;532(Pt 1):205-16.
156 Wellmer J, Su H, Beck H, Yaari Y. Long-lasting modification of intrinsic discharge properties in subicular neurons following status epilepticus. EurJ Neurosci. 2002 Jul;16(2):259-66.
157 Kumar SS, Buckmaster PS. Hyperexcitability, interneurons, and loss of GABAergic synapses in entorhinal cortex in a model of temporal lobe epilepsy. J Neurosci. 2006 Apr 26;26(17):4613-23.
158 Stief F, Zuschratter W, Hartmann K, Schmitz D, Draguhn A.Enhanced synaptic excitation-inhibition ratio in hippocampal interneurons of rats with temporal lobe epilepsy. Eur J Neurosci. 2007 Jan;25(2):519-28.
159 Liu YH, Wang L, Wei LC, Huang YG, Chen LW. Up-regulation of D: -serine Might Induce GABAergic Neuronal Degeneration in the Cerebral Cortex and Hippocampus in the Mouse Pilocarpine Model of Epilepsy. Neurochem Res. 2009 Jan 3.
160 Fujikawa DG. Prolonged seizures and cellular injury: understanding the connection. Epilepsy Behav. 2005 Dec;7 Suppl 3:S3-11.
161 Fernandes AM, Maurer-Morelli CV, Campos CB, Mello ML, Castilho RF, Langone F. Fluoro-Jade, but not Fluoro-Jade B, stains non-degenerating cells in brain and retina of embryonic and neonatal rats. Brain Res. 2004 Dec 10;1029(1):24-33.
162 Langmeier M, FolbergrováJ, HaugvicováR, Pokorny J, Mares P. Neuronal cell death in hippocampus induced by homocysteic acid in immature rats. Epilepsia. 2003 Mar;44(3):299-304.
163 Collins JA, Schandi CA, Young KK, Vesely J, Willingham MC. Major DNA fragmentation is a late event in apoptosis. J Histochem Cytochem. 1997 Jul;45(7):923-34.
164 Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci. 1997 Aug;22(8):299-306.
165 Allen JW, Eldadah BA, Huang X, Knoblach SM, Faden AI. Multiplecaspases are involved in beta-amyloid-induced neuronal apoptosis. J Neurosci Res. 2001 Jul 1;65(1):45-53.
166 Morais Cardoso S, Swerdlow RH, Oliveira CR. Induction of cytochrome c-mediated apoptosis by amyloid beta 25-35 requires functional mitochondria. Brain Res. 2002 Mar 29;931(2):117-25.
167 Adrain C, Martin SJ. The mitochondrial apoptosome: a killer unleashed by the cytochrome seas. Trends Biochem Sci. 2001 Jun;26(6):390-7.
168 Marín-Teva JL, Dusart I, Colin C, Gervais A, van Rooijen N, Mallat M. Microglia promote the death of developing Purkinje cells. Neuron. 2004 Feb 19;41(4):535-47.
169 Ban JY, Cho SO, Koh SB, Song KS, Bae K, Seong YH. Protection of amyloid beta protein (25-35)-induced neurotoxicity by methanol extract of Smilacis chinae rhizome in cultured rat cortical neurons. J Ethnopharmacol. 2006 Jun 30;106(2):230-7.
170 Le DA, Wu Y, Huang Z, Matsushita K, Plesnila N, Augustinack JC, Hyman BT, Yuan J, Kuida K, Flavell RA, Moskowitz MA. Caspase activation and neuroprotection in caspase-3- deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation. Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):15188-93.
171 Li M, Ona VO, Guégan C, Chen M, Jackson-Lewis V, Andrews LJ, Olszewski AJ, Stieg PE, Lee JP, Przedborski S, Friedlander RM. Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model. Science. 2000 Apr 14;288(5464):335-9.
172 Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell. 1997 Nov 14;91(4):443-6.
173 Baille V, Clarke PG, Brochier G, Dorandeu F, Verna JM, Four E, LallementG, Carpentier P. Soman-induced convulsions: the neuropathology revisited. Toxicology. 2005 Nov 5;215(1-2):1-24.
174 Tokuhara D, Sakuma S, Hattori H, Matsuoka O, Yamano T. Kainic acid dose affects delayed cell death mechanism after status epilepticus. Brain Dev. 2007 Jan;29(1):2-8.