Rapid and long-term alterations of hippocampal GABAB receptors in a mouse model of temporal lobe epilepsy

Alterations of gamma-aminobutyric acid (GABA)B receptor expression have been reported in human temporal lobe epilepsy (TLE). Here, changes in regional and cellular expression of the GABAB receptor subunits R1 (GBR1) and R2 (GBR2) were investigated in a mouse model that replicates major functional and histopathological features of TLE. Adult mice received a single, unilateral injection of kainic acid (KA) into the dorsal hippocampus, and GABAB receptor immunoreactivity was analysed between 1 day and 3 months thereafter. In control mice, GBR1 and GBR2 were distributed uniformly across the dendritic layers of CA1-CA3 and dentate gyrus. In addition, some interneurons were labelled selectively for GBR1. At 1 day post-KA, staining for both GBR1 and GBR2 was profoundly reduced in CA1, CA3c and the hilus, and no interneurons were visible anymore. At later stages, the loss of GABAB receptors persisted in CA1 and CA3, whereas staining increased gradually in dentate gyrus granule cells, which become dispersed in this model. Most strikingly, a subpopulation of strongly labelled interneurons reappeared, mainly in the hilus and CA3 starting at 1 week post-KA. In double-staining experiments, these cells were selectively labelled for neuropeptide Y. The number of GBR1-positive interneurons also increased contralaterally in the hilus. The rapid KA-induced loss of GABAB receptors might contribute to epileptogenesis because of a reduction in both presynaptic control of transmitter release and postsynaptic inhibition. In turn, the long-term increase in GABAB receptors in granule cells and specific subtypes of interneurons may represent a compensatory response to recurrent seizures.     

Contribution of early Alzheimer's disease‐related pathophysiology to the development of acquired epilepsy

Aberrant epileptic activity is detectable at early disease stages in Alzheimer's disease (AD) patients and in AD mouse models. Here, we investigated in young ArcticAβ mice whether AD‐like pathology renders neuronal networks more susceptible to the development of acquired epilepsy induced by unilateral intrahippocampal injection of kainic acid (IHK). In this temporal lobe epilepsy model, IHK induces a status epilepticus followed after two weeks by spontaneous recurrent seizures (SRS). ArcticAβ mice exhibited more severe status epilepticus and early onset of SRS. This hyperexcitable phenotype was characterized in CA1 neurons by decreased synaptic strength, increased kainic acid‐induced LTP and reduced frequency of spontaneous inhibitory currents. However, no difference in neurodegeneration, neuroinflammation, axonal reorganization or adult neurogenesis was observed in ArcticAβ mice compared to wild‐type littermates following IHK‐induced epileptogenesis. Neuropeptide Y (NPY) expression was reduced at baseline and its IHK‐induced elevation in mossy fibres and granule cells was attenuated. However, although this alteration might underlie premature seizure onset, neutralization of soluble Aβ species by intracerebroventricular Aβ‐specific antibody application mitigated the hyperexcitable phenotype of ArcticAβ mice and prevented early SRS onset. Therefore, the development of seizures at early stages of AD is mediated primarily by Aβ species causing widespread changes in synaptic function.     

New‐onset musicogenic epilepsy after temporal lobe epilepsy surgery

Musicogenic epilepsy is a reflex epilepsy provoked by listening to or playing music. The epileptogenic network involves temporal regions, usually mesiotemporal structures. We present a 31‐year‐old female patient who experienced musicogenic seizures after a right temporal lobectomy with amygdalohippocampectomy that was performed in order to treat preexisting right mesio‐temporal epilepsy.     

Hippocampal neurodegeneration, spontaneous seizures, and mossy fiber sprouting in the F344 rat model of temporal lobe epilepsy

The links among the extent of hippocampal neurodegeneration, the frequency of spontaneous recurrent motor seizures (SRMS), and the degree of aberrant mossy fiber sprouting (MFS) in temporal lobe epilepsy (TLE) are a subject of contention because of variable findings in different animal models and human studies. To understand these issues further, we quantified these parameters at 3-5 months after graded injections of low doses of kainic acid (KA) in adult F344 rats. KA was administered every 1 hr for 4 hr, for a cumulative dose of 10.5 mg/kg bw, to induce continuous stages III-V motor seizures for >3 hr. At 4 days post-KA, the majority of rats (77%) exhibited moderate bilateral neurodegeneration in different regions of the hippocampus; however, 23% of rats exhibited massive neurodegeneration in all hippocampal regions. All KA-treated rats displayed robust SRMS at 3 months post-KA, and the severity of SRMS increased over time. Analyses of surviving neurons at 5 months post-KA revealed two subgroups of rats, one with moderate hippocampal injury (HI; 55% of rats) and another with widespread HI (45%). Rats with widespread HI exhibited greater loss of CA3 pyramidal neurons and robust aberrant MFS than rats with moderate HI. However, the frequency of SRMS (approximately 3/hr) was comparable between rats with moderate and widespread HI. Thus, in comparison with TLE model using Sprague-Dawley rats (Hellier et al. [1998] Epilepsy Res. 31:73-84), a much lower cumulative dose of KA leads to robust chronic epilepsy in F344 rats. Furthermore, the occurrence of SRMS in this model is always associated with considerable bilateral hippocampal neurodegeneration and aberrant MFS. However, more extensive hippocampal CA3 cell loss and aberrant MFS do not appear to increase the frequency of SRMS. Because most of the features are consistent with mesial TLE in humans, the F344 model appears ideal for testing the efficacy of potential treatment strategies for mesial TLE.     

Cyclicity of spontaneous recurrent seizures in pilocarpine model of temporal lobe epilepsy in rat

Pilocarpine administration to rats results in status epilepticus (SE) and after a latency period to the occurrence of spontaneous seizures. The model is commonly used to investigate mechanisms of epileptogenesis as well as the antiepileptic effects of novel compounds. Surprisingly, there have been no video-EEG studies determining the duration of latency period from SE to the appearance of the first spontaneous seizures or the type and frequency of spontaneous seizures at early phase of pilocarpine-induced epilepsy even though such information is critical for design of such studies. To address these questions, we induced SE with pilocarpine in 29 adult male Wistar rats with cortical electrodes. Rats were continuously video-EEG monitored during SE and up to 23 days thereafter. The first spontaneous seizures occurred 7.2+/-3.6 days after SE. During the follow-up, the mean daily seizure frequency was 2.6+/-1.9, the mean seizure duration 47+/-7 s, and the mean behavioral seizure score 3.2+/-0.9. Typically first seizures were partial (score 1-2). Interestingly, spontaneous seizures occurred in clusters with cyclicity, peaking every 5 to 8 days. These data show that in the pilocarpine model of temporal lobe epilepsy the latency period is short. Because many of the early seizures are partial and the seizures occur in clusters, the true phenotype of epilepsy triggered by pilocarpine-induced SE may be difficult to characterize without continuous long-term video-EEG monitoring. Finally, our data suggest that the model can be used for studies aiming at identifying the mechanisms of seizure clustering.     

匹罗卡品致痫小鼠自发性癫痫发作模型的建立及癫痫后海马新生神经细胞增生的研究

目的探讨匹罗卡品致痫小鼠自发性癫痫发作(SS)模型的特点,并观察癫痫发作后其齿状回颗粒细胞下层新生神经细胞(NPC)增生变化。方法建立SS小鼠模型,进行行为学检测。将SS发作小鼠在癫痫持续状态(SE)后不同时间点进行Doublecortin(DCX)免疫组化,Western blot观察海马NPC的分布和表达改变。结果 80%的模型小鼠出现持续SS,发作时程约10～40 s,发作频率为2.18±0.45次/周。DCX+细胞及其蛋白表达在模型组中显著增多(P<0.05),SE后4周最为明显,SE后8周开始下降,至SE后12周显著减少。增生的DCX+细胞呈簇状分布,随时间推移出现迁移分散。存在SS的模型小鼠相对于不存在SS的模型小鼠DCX+细胞增生更为明显。结论匹罗卡品致痫后SS小鼠模型存在持续的NPC增生,这可能在慢性期癫痫持续发作的病理生理机制中有着重要作用。     

电针对癫痫模型大鼠自发性反复发作及海马CA3和DG区谷氨酸脱羧酶67表达的影响

背景由于对药物疗法不良反应的担忧,近几年人们对使用针灸治疗癫痫越来越感兴趣。尽管现已报道了不少针灸抗癫痫作用的临床证据,但其确切机制仍不清楚。目的 研究电针(EA)对癫痫大鼠自发性复发性癫痫(SRS),以及海马CA3和齿状回(DG)区中谷氨酸脱羧酶67(GAD67)表达的影响。方法 将50只Sprague-Dawley(SD)大鼠随机分为对照组、癫痫组、假刺激组、非穴位电针组和穴位电针组,每组10只。除对照组外,其余4组均制备氯化锂—匹罗卡品颞叶癫痫大鼠模型。造模成功的大鼠采用针刺或电针穴位治疗,8周后观察自发性复发性癫痫发作的次数。分别取5组大鼠海马组织,采用荧光定量PCR和Western blotting分别检测各组大鼠海马组织中GAD67 mRNA水平和蛋白水平表达变化;采用免疫组化法检测各组大鼠海马CA3和DG区GAD67蛋白水平表达变化。结果 治疗8周后,穴位电针组与癫痫组、假刺激组、非穴位电针组比较,减少了自发性复发性癫痫发作的次数,差异有统计学意义(P<0.05)。qRT-PCR结果显示：与对照组相比,癫痫组、假刺激组、非穴位电针组和穴位电针组大鼠海马组织中GAD6...     

Increased expression of caspase 2 in experimental and human temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is often caused by a neurodegenerative brain insult that triggers epileptogenesis, and eventually results in spontaneous seizures, i.e., epilepsy. Understanding the mechanisms of cell death is a key for designing new drug therapies for preventing the neurodegeneration associated with TLE. Here, we investigated the expression of caspase 2, a protein involved in programmed cell death, during the course of epilepsy. We investigated caspase 2 expression in hippocampal samples derived from patients operated on for drug refractory TLE. To understand the evolution of altered-caspase 2 expression during the epileptic process, we also examined caspase 2 expression and activity in the rat hippocampus after status epilepticus-induced acute damage, during epileptogenesis, and after the onset of epilepsy. Caspase 2 expression was enhanced in the hippocampal neurons in chronic TLE patients. In rats, status epilepticus-induced caspase 2 labeling paralleled the progression of neurodegeneration. Proteolytic activation and cleavage of caspase 2 was also detected in the rat brain undergoing epileptogenesis. Our data suggest that caspase 2-mediated programmed cell death participates in the seizure-induced degenerative process in experimental and human TLE. The work was supported by The Academy of Finland, The Kuopio University Foundation, The Neurology Foundation, The North-Savo Regional Fund of the Finnish Cultural Foundation, Sigrid Juselius Foundation, and The Vaajasalo Foundation.     

Modeling epileptogenesis and temporal lobe epilepsy in a non-human primate

Here we describe a new non-human primate model of temporal lobe epilepsy (TLE) to better investigate the cause/effect relationships of human TLE. Status epilepticus (SE) was induced in adult marmosets by pilocarpine injection (250mg/kg; i.p.). The animals were divided in 2 groups: acute (8h post-SE) and chronic (3 and 5 months post-SE). To manage the severity of SE, animals received diazepam 5min after the SE onset (acute group: 2.5 or 1.25mg/kg; i.p.; chronic group/; 1.25mg/kg; i.p). All animals were monitored by video and electrocorticography to assess SE and subsequent spontaneous recurrent seizures (SRS). To evaluate brain injury produced by SE or SRS we used argyrophil III, Nissl and neo-Timm staining techniques. Magnetic resonance image was also performed in the chronic group. We observed that pilocarpine was able to induce SE followed by SRS after a variable period of time. Prolonged SE episodes were associated with brain damage, mostly confined to the hippocampus and limbic structures. Similar to human TLE, anatomical disruption of dentate gyrus was observed after SRS. Our data suggest that pilocarpine marmoset model of epilepsy has great resemblance to human TLE, and could provide new tools to further evaluate the subtle changes associated with human epilepsy.     

脑内局部给药建立颞叶癫疒间模型的研究

目的研究脑内局部给药建立颞叶癫疒间(EP)模型的方法。方法通过立体定向技术向大鼠海马局部注射海人酸(KA)4μg/kg,观察注射后大鼠的行为学、脑电图和病理学改变。结果KA注入海马后大鼠表现为凝视、湿狗样抖动、咀嚼运动、点头、肢体阵挛等;随后出现阵发性旋转、向上窜跳、四肢抽搐,反复间歇性发作,约10h后发作停止。以后每周约自发性发作1～3次,主要为Ⅱ～Ⅳ级发作。EEG记录到大脑皮质丛集放电,棘波或尖波持续发放。光镜下KA致疒间鼠海马结构的CA1区和CA3区锥体细胞变性及坏死,实验侧CA3区神经元丢失尤为明显。结论大鼠脑内局部注射KA可成功建立颞叶EP模型,其行为症状、电生理及海马病理改变类似人颞叶EP,是很好的研究颞叶EP的工具。     

Temporal lobe volumetric cell densities in temporal lobe epilepsy

Volumetric cell densities in 13 different subfields of the temporal lobe were calculated to test various hypotheses about mesial and lateral temporal lobe sclerosis in patients with complex partial epilepsy. In patients benefitting (primary group) from anterior temporal lobectomy (ATL), sclerosis was greater (fewer cells) in anterior than in posterior hippocampus. By contrast, the patients lacking full benefit (nonprimary group) from ATL had decreased numbers of neurons equally distributed from anterior to posterior hippocampus, indicating that zones of mesial temporal cell loss are linked to zones of epileptogenicity. These data support a model of focal hippocampal epilepsy originating from zones of cell loss and synaptic reorganization that is epileptic. There were no differences in cell densities in gyrus hippocampi or in lateral temporal gyri when patients with temporal lobe epilepsy and controls were compared. Hippocampal cell densities in mesial temporal lobe were not reduced in psychomotor epileptic patients with extrahippocampal foci consisting of foreign tissue. Variables in seizure histories were not correlated with Ammon's horn cell densities, indicating that most of the sclerosis preceded the seizures, which did virtually no significant further damage to hippocampus with repeated partial or generalized seizures.     

Age of onset of mesial temporal lobe epilepsy with hippocampal sclerosis: the effect of apolipoprotein E and febrile seizures

Purpose: Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is the most frequent pharmaco-resistant epilepsy. It has been associated with febrile seizures (FS) in childhood. Its aetiology remains unclear but genetic factors are involved. Apolipoprotein E (ApoE) is the main lipoprotein secreted in brain. It has a critical immunomodulatory function, influences neurotransmission and it is involved in repairing damaged neurons. ApoE ?4 is an isoform of ApoE with altered protein function, previously associated with refractoriness and early onset epilepsy. This study was undertaken to determine if ApoE isoforms are risk factors for MTLE-HS and influence clinical characteristics.Methods: A group of 188 MTLE-HS patients (101 F, 87 M, mean age = 44.7 ± 11.6 years, 100 with FS antecedents) was studied and compared with a group of 342 healthy individuals in a case-control genetic association study. Data were analysed with Pearson Chi-squared Test or Student's t test, as appropriated.Results: No differences in ApoE ?4 allelic frequencies between MTLE-HS patients and controls or between MTLE-HS subgroups were observed. Nevertheless, ApoE ?4 carriers had an earlier MTLE-HS onset (11.0 ± 7.9 years in ApoE ?4 carriers vs. 14.4 ± 11.2 years in ApoE ?4 non-carriers p < 0.05). Additionally, we observed that MTLE-HS patients with FS antecedents had a statistically significant early disease onset (11.5 ± 8.7 years in FS⁺ vs. 16.0 ± 12.1 years in FS^?, p < 0.01).Conclusions: Our data show that ApoE ?4 and FS may not participate directly in etiopathogenic mechanisms of MTLE-HS but could hasten the disease development in predisposed individuals.     

Expression of glutamine synthetase and glutamate dehydrogenase in the latent phase and chronic phase in the kainate model of temporal lobe epilepsy

It has been suggested that astrocytic glutamate release or perturbed glutamate metabolism contributes to the proneness to epileptic seizures. Here we investigated whether astrocytic contents of the major glutamate degrading enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) decreases on moving from the latent phase (prior to seizures) to the chronic phase (after onset of seizures) in the kainate (KA) model of temporal lobe epilepsy. Western blotting and immunogold analysis of hippocampal formation indicated similar levels of GDH in the latent and chronic phases of KA injected rats and in corresponding controls. In contrast, the level of GS was increased in the latent phase compared with controls, as assessed by Western blots of whole hippocampal formation and subregions. The increase in GS paralleled that of glial fibrillary acidic protein (GFAP). Compared with the latent phase, the chronic phase revealed a lower level of GS (approaching control levels) but an unchanged GFAP content. The decrease in GS from latent to chronic phase was significant in whole hippocampal formation, dentate gyrus and CA3. It is concluded that kainate treated rats show an initial increase in GS, pari passu with the increase in GFAP, and a secondary decrease in GS that is not accompanied by a similar loss of GFAP. In a situation where glutamate catabolism is in high demand the secondary reduction in GS level may be sufficient to contribute to the seizure proneness that develops between the latent and chronic phases.     

Adoptive transfer of T lymphocytes in immunodeficient mice influences epileptogenesis and neurodegeneration in a model of temporal lobe epilepsy

Intra-hippocampal injection of kainic acid (KA) in adult mice causes a focal lesion in the CA1 area and hilus of the dentate gyrus, as well as pronounced granule cell hypertrophy and dispersion. The lesion results in chronic focal seizures, with a two-week delay following KA-induced status epilepticus. Furthermore, seizures are preceded by infiltration of T lymphocytes into the lesioned tissue and of macrophage-like cells, strongly immunopositive for the monocyte marker F4/80, into the dentate gyrus, where they regulate granule cell dispersion and survival. Unexpectedly, depletion of CD4(+) and/or CD8(+) T lymphocytes by targeted gene deletion results in a marked shortening of the delay prior to seizure onset, suggesting a role of adaptive immunity in epileptogenesis (Zattoni et al. 2011, J. Neurosci. 31, 4037). Here, we investigated the specific role of adaptive immunity in this TLE model by adoptive i.v. transfer of immunopurified T cells in mutant mice lacking either CD4(+) T cells (MHCII-knockout), CD8(+) T cells (β2-microglobulin-knockout), or both populations (RAG1-knockout mice). EEG analysis in mutants mice injected with KA two days after the T cell transfer revealed that grafting of the missing T cell population had no influence on seizure onset, but strongly influenced F4/80(+) macrophage-like cell infiltration in the dentate gyrus. Specifically, CD8(+) T cells in β2-microgloblin-knockout mice enhanced macrophage recruitment, whereas CD4(+) T cells transferred in MHCII-knockout and in RAG1-knockout mice blocked macrophage infiltration, leading to reduced granule cell dispersion and survival, thereby worsening the KA-induced lesion. These results suggest that intact adaptive immunity is required for delayed seizure onset in this mouse model of TLE and unravel complex interactions between T cells and mononuclear phagocytes for the control of neuronal integrity and survival in the lesioned brain.