Seiji Hatazaki

Unilateral hippocampal CA3-predominant damage and short latency epileptogenesis after intra-amygdala microinjection of kainic acid in mice

Mesial temporal lobe epilepsy is the most common, intractable seizure disorder in adults. It is associated with an asymmetric pattern of hippocampal neuron loss within the endfolium (hilus and CA3) and CA1, with limited pathology in extra-hippocampal regions. We previously developed a model of focally-evoked seizure-induced neuronal death using intra-amygdala kainic acid (KA) microinjection and characterized the acute hippocampal pathology. Here, we sought to characterize the full extent of hippocampal and potential extra-hippocampal damage in this model, and the temporal onset of epileptic seizures. Seizure damage assessed at four stereotaxic levels by FluoroJade B staining was most prominent in ipsilateral hippocampal CA3 where it extended from septal to temporal pole. Minor but significant neuronal injury was present in ipsilateral CA1. Extra-hippocampal neuronal damage was generally limited in extent and restricted to the lateral septal nucleus, injected amygdala and select regions of neocortex ipsilateral to the seizure elicitation side. Continuous surface EEG recorded with implanted telemetry units in freely-moving mice detected spontaneous, epileptic seizures by five days post-KA in all mice. Epileptic seizure number averaged 1-4 per day. Hippocampi from epileptic mice 15 days post-KA displayed unilateral CA3 lesions, astrogliosis and increased neuropeptide Y immunoreactivity suggestive of mossy fiber rearrangement. These studies characterize a mouse model of unilateral hippocampal-dominant neuronal damage and short latency epileptogenesis that may be suitable for studying the cell and molecular pathogenesis of human mesial temporal lobe epilepsy.

Hippocampal transcriptome after status epilepticus in mice rendered seizure damage-tolerant by epileptic preconditioning features suppressed calcium and neuronal excitability pathways.

Preconditioning brain with a sub-lethal stressor can temporarily generate a damage-refractory state. Microarray analyses have defined the changes in hippocampal gene expression that follow brief preconditioning seizures, but not the transcriptome after a prolonged and otherwise injurious seizure in previously preconditioned brain. Presently, microarray analysis was performed 24 h after status epilepticus in mice that had received previously either seizure preconditioning (tolerance) or sham-preconditioning (injury). Transcriptional changes in the hippocampal CA3 subfield of >or=2 fold were detected for 1357 genes in the tolerance group compared to a non-seizure control group, with 54% up-regulated. Of these regulated genes, 792 were also regulated in the injury group. Among the remaining 565 genes regulated only in tolerance, 73% were down-regulated. Analysis of the genes differentially suppressed in tolerance identified calcium signaling, ion channels and excitatory neurotransmitter receptors, and the synapse as over-represented among pathways, functions and compartments. Finally, 12 days continuous EEG recordings determined mice with induced tolerance had fewer spontaneous electrographic seizures compared to the injury group. Our data suggest the transcriptional phenotype of neuroprotection in tolerance may be dictated by the biology of the preconditioning stressor, functions by transcriptional reduction of vulnerability to excitotoxicity, and has anti-epileptogenic effects.

Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma

The functional significance of neuronal death for pathogenesis of epilepsy and the underlying molecular mechanisms thereof remain incompletely understood. The p53 transcription factor has been implicated in seizure damage, but its target genes and the influence of cell death under its control on epilepsy development are unknown. In the present study, we report that status epilepticus (SE) triggered by intraamygdala kainic acid in mice causes rapid p53 accumulation and subsequent hippocampal damage. Expression of p53‐up‐regulated mediator of apoptosis (Puma), a proapoptotic Bcl‐2 homology domain 3‐only protein under p53 control, was increased within a few hours of SE. Induction of Puma was blocked by pharmacologic inhibition of p53, and hippocampal damage was also reduced. Puma induction was also blocked in p53‐deficient mice subject to SE. Compared to Puma‐expressing mice, Puma‐deficient mice had significantly smaller hippocampal lesions after SE. Long‐term, continuous telemetric EEG monitoring revealed a ~60% reduction in the frequency of epileptic seizures in the Puma‐deficient mice compared to Puma‐expressing mice. These are the first data showing genetic deletion of a proapoptotic protein acting acutely to influence neuronal death subsequently alters the phenotype of epilepsy in the long‐term, supporting the concept that apoptotic pathway activation is a trigger of epileptogenesis.—Engel, T., Murphy, B. M., Hatazaki, S., Jimenez‐Mateos, E. M., Concannon, C. G., Woods, I., Prehn, J. H. M., Henshall, D. C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma. FASEB J. 24, 853–861 (2010). www.fasebj.org

Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.

A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by approximately 45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.

Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus

Prolonged seizures (status epilepticus) are associated with brain region-specific regulation of apoptosis-associated signaling pathways. Bcl-2 homology domain 3-only (BH3) members of the Bcl-2 gene family are of interest as possible initiators of mitochondrial dysfunction and release of apoptogenic molecules after seizures. Previously, we showed that expression of the BH3-only protein, Bcl-2 interacting mediator of cell death (Bim), increased in the rat hippocampus but not in the neocortex after focal-onset status epilepticus. In this study, we examined Bim expression in mice and compared seizure damage between wild-type and Bim-deficient animals. Status epilepticus induced by intra-amygdala kainic acid (KA) caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and posttranslational activation of Bim, such as CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Upregulation of bim mRNA was evident after 2 h and Bim protein increased between 4 and 24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared with wild-type animals after seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death after KA treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus, and neocortex damage in Bim-deficient mice was comparable with that in wild-type animals. These results show region-specific differential contributions of Bim to seizure-induced neuronal death.

Deletion of puma protects hippocampal neurons in a model of severe status epilepticus

Prolonged seizures (status epilepticus) can activate apoptosis-associated signaling pathways. The extent to which such pathways contribute to cell death might depend on the insult intensity, whereby the programmed or apoptotic cell death component is reduced when seizures are more severe or protracted. We recently showed that mice lacking the pro-apoptotic Bcl-2 homology domain 3-only protein Puma (Bbc3) were potently protected against damage caused by status epilepticus. In the present study we examined whether Puma deficiency was protective when the seizure episode was more severe. Intra-amygdala microinjection of 1 microg kainic acid (KA) into C57BL/6 mice triggered status epilepticus that lasted about twice as long as with 0.3 microg KA prior to lorazepam termination. Hippocampal damage was also significantly greater in the higher-dose group. Over 80% of degenerating neurons after seizures were positive for DNA fragmentation assessed by terminal deoxynucleotidyl dUTP nick end labeling (TUNEL). Microscopic analysis of neuronal nuclear morphology in TUNEL-positive cells revealed the proportion displaying large rounded clumps of condensed chromatin was approximately 50% lower in the high-dose versus low-dose KA group. Nevertheless, compared to heterozygous and wild-type mice subject to status epilepticus by high-dose KA, neuronal death was reduced by approximately 50% in the hippocampus of Puma-deficient mice. These data suggest aspects of the apoptotic component of seizure-induced neuronal death are insult duration- or severity-dependent. Moreover, they provide further genetic evidence that seizure-induced neuronal death is preventable by targeting so-called apoptosis-associated signaling pathways and Puma loss likely disrupts caspase-independent or non-apoptotic seizure-induced neuronal death.

High-Risk Plaque for Carotid Artery Stenting Evaluated With 3-Dimensional T1-Weighted Gradient Echo Sequence

Background and Purpose— Preventing cerebral embolisms is a major concern with carotid artery stenting (CAS). This study evaluated 3-dimensional T1-weighted gradient echo (3D T1GRE) sequence to predict cerebral embolism related to CAS. Methods— We performed quantitative analyses of the characteristics of 47 carotid plaques before CAS by measuring the signal intensity ratio (SIR) and plaque volume using 3D T1GRE images. We used T1-weighted turbo field echo sequence to obtain 3D T1GRE images. We also evaluated diffusion-weighted images (DWI) of the brain before and after CAS to detect ischemic lesions (DWI lesions) from cerebral emboli. Results— SIR (2.17 [interquartile range 1.50–3.07] versus 1.35 [interquartile range 1.08–1.97]; P=0.010) and plaque volume (456 mm3 [interquartile range 256–696] versus 301 mm3 [interquartile range 126–433]; P=0.008) were significantly higher in the group of patients positive for DWI lesions (P-group: n=26) than DWI lesion-negative patients (N-group: n=21). In multivariate logistic regression analysis, SIR (P=0.007) and plaque volume (P=0.042) were independent predictors of DWI lesions with CAS. Furthermore, SIR (rs=0.42, P=0.005) and plaque volume (rs=0.36, P=0.012) were positively correlated with the number of DWI lesions. From analysis of a receiver-operating characteristic curve, the most reliable cutoff values of SIR and plaque volume to predict DWI lesions related to CAS were 1.80 and 373 mm3, respectively. Conclusions— Quantitative evaluation of carotid plaques using 3D T1GRE images may be useful in predicting cerebral embolism related to CAS.

Skull Meningioma Associated with Intradural Cyst: A Case Report

We present the first report of intraosseous meningioma accompanied by intradural cyst formation. A 76-year-old woman had previously undergone breast cancer treatment, so the preoperative diagnosis was metastatic breast cancer. This case reminds us that the possibility of meningioma should be kept in mind in patients with breast cancer, irrespective of neuroimaging findings.