Mitophagy in Refractory Temporal Lobe Epilepsy Patients with Hippocampal Sclerosis

This study aimed to determine if there is an association between mitophagy and refractory temporal lobe epilepsy (rTLE) with hippocampal sclerosis. During epilepsy surgery, we collected tissue samples from the hippocampi and temporal lobe cortexes of rTLE patients with hippocampal sclerosis (as diagnosed by a pathologist). Transmission electron microscopy (TEM) was used to study the ultrastructural features of the tissue. To probe for mitophagy, we used fluorescent immunolabeling to determine if mitochondrial and autophagosomal markers colocalized. Fourteen samples were examined. TEM results showed that early autophagosomes were present and mitochondria were impaired to different degrees in hippocampi. Immunofluorescent labeling showed colocalization of the autophagosome marker LC3B with the mitochondrial marker TOMM20 in hippocampi and temporal lobe cortexes, indicating the presence of mitophagy. Mitochondrial and autophagosomal marker colocalization was lower in hippocampus than in temporal lobe cortex (P < 0.001). Accumulation of autophagosomes and mitophagy activation are implicated in rTLE with hippocampal sclerosis. Aberrant accumulation of damaged mitochondria, especially in the hippocampus, can be attributed to defects in mitophagy, which may participate in epileptogenesis.     

伴海马硬化的颞叶癫痫患者海马组织内BDNF 表达变化

目的:研究伴海马硬化的难治性颞叶癫痫(TLE)患者海马组织内脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法:采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应(RT-PCR)法检测BDNF mRNA表达,并与3例非海马硬化TLE患者对照。结果:与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNF mRNA表达明显增加(P<0.01)。结论:伴海马硬化的难治性TLE患者海马组织中BDNF mRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

伴海马硬化的颞叶癫痫患者海马组织内BDNF表达变化

目的：研究伴海马硬化的难治性颞叶癫痫（TLE）患者海马组织内脑源性神经营养因子（brain derivedneurotrophic factor,BDNF）的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法：采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应（RT—PCR）法检测BDNFmRNA表达,并与3例非海马硬化TLE患者对照。结果：与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNFmRNA表达明显增加（P〈0．01）。结论：伴海马硬化的难治性TLE患者海马组织中BDNFmRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

Reduction of Hippocampal Collapsin Response Mediated Protein-2 in Patients with Mesial Temporal Lobe Epilepsy

Although the syndrome of mesial temporal lobe epilepsy (MTLE) associated with hippocampal sclerosis has been elaborated in recent years, pathogenesis and pathomechanisms are still elusive. Performing protein hunting in hippocampus of patients with MTLE we detected derangement of collapsin response mediated protein-2 (CRMP-2). Hippocampal tissue from controls and MTLEs was taken and two-dimensional gel electrophoresis with subsequent MALDI-MS-characterisation was applied. The proteomic approach identified 13 spots unambiguously assigned to CRMP-2. Three spots at molecular weight 55 kDa showed a significant decrease in MTLE and other 3 spots at 65 kDa showed deranged in MTLE. Immunoblotting revealed two bands at 65 and 55 kDa in the control group whereas the 55 kDa band was extremely low expressed in MTLE. CRMP-2 is required to induce axonal outgrowth and maintaining neuronal polarity in hippocampal neurons and the significant decrease of this protein may represent or underlie impaired neuronal plasticity, neurodegeneration, wiring of the brain in MTLE and may explain abnormal migration. Therefore, the decrease of CRMP-2 may well contribute to the understanding of the still unclear pathomechanisms involved in MTLE.     

Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis

Objective

Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).

Methods

We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.

Results

Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.

Conclusion

Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.     

Altered Functional Connectivity and Small-World in Mesial Temporal Lobe Epilepsy

Background

The functional architecture of the human brain has been extensively described in terms of functional connectivity networks, detected from the low–frequency coherent neuronal fluctuations that can be observed in a resting state condition. Little is known, so far, about the changes in functional connectivity and in the topological properties of functional networks, associated with different brain diseases.

Methodology/Principal Findings

In this study, we investigated alterations related to mesial temporal lobe epilepsy (mTLE), using resting state functional magnetic resonance imaging on 18 mTLE patients and 27 healthy controls. Functional connectivity among 90 cortical and subcortical regions was measured by temporal correlation. The related values were analyzed to construct a set of undirected graphs. Compared to controls, mTLE patients showed significantly increased connectivity within the medial temporal lobes, but also significantly decreased connectivity within the frontal and parietal lobes, and between frontal and parietal lobes. Our findings demonstrated that a large number of areas in the default-mode network of mTLE patients showed a significantly decreased number of connections to other regions. Furthermore, we observed altered small-world properties in patients, along with smaller degree of connectivity, increased n-to-1 connectivity, smaller absolute clustering coefficients and shorter absolute path length.

Conclusions/Significance

We suggest that the mTLE alterations observed in functional connectivity and topological properties may be used to define tentative disease markers.     

Role of Altered Expression,Activity and Sub-cellular Distribution of Various Histone Deacetylases (HDACs) in Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis

Cellular and Molecular Neurobiology - Histone deacetylases (HDACs) have been described to have both neurotoxic and neuroprotective roles, and partly, depend on its sub-cellular distribution. HDAC...     

Heritability of Subcortical Volumetric Traits in Mesial Temporal Lobe Epilepsy

Objectives

We aimed to 1) determine if subcortical volume deficits are common to mesial temporal lobe epilepsy (MTLE) patients and their unaffected siblings 2) assess the suitability of subcortical volumetric traits as endophenotypes for MTLE.

Methods

MRI-based volume measurements of the hippocampus, amygdala, thalamus, caudate, putamen and pallidium were generated using an automated brain reconstruction method (FreeSurfer) for 101 unrelated ‘sporadic’ MTLE patients [70 with hippocampal sclerosis (MTLE+HS), 31 with MRI-negative TLE], 83 unaffected full siblings of patients and 86 healthy control subjects. Changes in the volume of subcortical structures in patients and their unaffected siblings were determined by comparison with healthy controls. Narrow sense heritability was estimated ipsilateral and contralateral to the side of seizure activity.

Results

MTLE+HS patients displayed significant volume deficits across the hippocampus, amygdala and thalamus ipsilaterally. In addition, volume loss was detected in the putamen bilaterally. These volume deficits were not present in the unaffected siblings of MTLE+HS patients. Ipsilaterally, the heritability estimates were dramatically reduced for the volume of the hippocampus, thalamus and putamen but remained in the expected range for the amygdala. MRI-negative TLE patients and their unaffected siblings showed no significant volume changes across the same structures and heritability estimates were comparable with calculations from a healthy population.

Conclusions

The findings indicate that volume deficits for many subcortical structures in ‘sporadic’ MTLE+HS are not heritable and likely related to acquired factors. Therefore, they do not represent suitable endophenotypes for MTLE+HS. The findings also support the view that, at a neuroanatomical level, MTLE+HS and MRI-negative TLE represent two distinct forms of MTLE.     

Changes in the Expression of Selenoproteins in Mesial Temporal Lobe Epilepsy Patients

Selenoproteins are enzymes containing selenium in their structure and are involved in cellular processes such as defense against oxidative stress and cell survival. The aim of this study is to investigate the expression of four selenoproteins (GPX1, TRXR1, SELP and SELW) in the hippocampus of intractable mesial temporal lobe epilepsy (MTLE) patients who underwent curative surgery. The selenoproteins is investigated at the mRNA level via RT-PCR and in situ hybridization and by immunostaining at the protein level. The expression of SELW exhibited a relative induction of more than tenfold, and immunostaining findings provided evidence that this upregulation is confined to neurons. GPX1 was also upregulated 2.3-fold, and TRXR1 was downregulated between 70 and 20% in MTLE patients. The profound induction of SELW has been accompanied by GPX1 and displayed a strong correlation with BCL2 expression, suggesting a protective role for these selenoproteins, and may be an indicator of a defense mechanism in surviving neurons.     

基于纤维束追踪空间统计的内侧颞叶癫痫患者弥散张量成像研究

本研究通过分析磁共振弥散张量成像(diffusion tensor imaging,DTI)数据,观察内侧颞叶癫痫(mesial temporal lobe epilepsy,mTLE)患者大脑白质的改变。46例伴有单侧海马硬化的内侧颞叶癫痫患者(24例左侧颞叶癫痫和22例右侧颞叶癫痫),以及42例年龄和性别匹配的正常志愿者纳入本研究。采用基于纤维束追踪的空间统计分析方法(track-based spatialstatistics,TBSS),主要观察患者各向异性系数(fractionsal anisotropy,FA)的变化。结果发现,与正常志愿者相比,左侧mTLE患者FA降低的区域呈双侧分布,稍偏向患侧,胼胝体、上纵束、下纵束、内囊前肢等白质双侧都有异常,而扣带束、下额枕束只在左脑显著降低;右侧mTLE患者FA降低主要见于右脑,包括胼胝体、上纵束、下纵束和钩束等。结果表明,基于TBSS方法的DTI研究揭示了伴有海马硬化的mTLE患者的脑白质异常,有助于加深对mTLE病理生理机制的了解。     

Functional Connectivity Estimated from Intracranial EEG Predicts Surgical Outcome in Intractable Temporal Lobe Epilepsy

This project aimed to determine if a correlation-based measure of functional connectivity can identify epileptogenic zones from intracranial EEG signals, as well as to investigate the prognostic significance of such a measure on seizure outcome following temporal lobe lobectomy. To this end, we retrospectively analyzed 23 adult patients with intractable temporal lobe epilepsy (TLE) who underwent an invasive stereo-EEG (SEEG) evaluation between January 2009 year and January 2012. A follow-up of at least one year was required. The primary outcome measure was complete seizure-freedom at last follow-up. Functional connectivity between two areas in the temporal lobe that were sampled by two SEEG electrode contacts was defined as Pearson’s correlation coefficient of interictal activity between those areas. SEEG signals were filtered between 5 and 50 Hz prior to computing this correlation. The mean and standard deviation of the off diagonal elements in the connectivity matrix were also calculated. Analysis of the mean and standard deviation of the functional connections for each patient reveals that 90% of the patients who had weak and homogenous connections were seizure free one year after temporal lobectomy, whereas 85% of the patients who had stronger and more heterogeneous connections within the temporal lobe had recurrence of seizures. This suggests that temporal lobectomy is ineffective in preventing seizure recurrence for patients in whom the temporal lobe is characterized by weakly connected, homogenous networks. This pilot study shows promising potential of a simple measure of functional brain connectivity to identify epileptogenicity and predict the outcome of epilepsy surgery.     

Scale Invariance Properties of Intracerebral EEG Improve Seizure Prediction in Mesial Temporal Lobe Epilepsy

Although treatment for epilepsy is available and effective for nearly 70 percent of patients, many remain in need of new therapeutic approaches. Predicting the impending seizures in these patients could significantly enhance their quality of life if the prediction performance is clinically practical. In this study, we investigate the improvement of the performance of a seizure prediction algorithm in 17 patients with mesial temporal lobe epilepsy by means of a novel measure. Scale-free dynamics of the intracerebral EEG are quantified through robust estimates of the scaling exponents—the first cumulants—derived from a wavelet leader and bootstrap based multifractal analysis. The cumulants are investigated for the discriminability between preictal and interictal epochs. The performance of our recently published patient-specific seizure prediction algorithm is then out-of-sample tested on long-lasting data using combinations of cumulants and state similarity measures previously introduced. By using the first cumulant in combination with state similarity measures, up to 13 of 17 patients had seizures predicted above chance with clinically practical levels of sensitivity (80.5%) and specificity (25.1% of total time under warning) for prediction horizons above 25 min. These results indicate that the scale-free dynamics of the preictal state are different from those of the interictal state. Quantifiers of these dynamics may carry a predictive power that can be used to improve seizure prediction performance.     

A Macaque Model of Mesial Temporal Lobe Epilepsy Induced by Unilateral Intrahippocampal Injection of Kainic Acid

Objective

In order to better investigate the cause/effect relationships of human mesial temporal lobe epilepsy (mTLE), we hereby describe a new non-human primate model of mTLE.

Methods

Ten macaques were studied and divided into 2 groups: saline control group (n = 4) and kainic acid (KA) injection group (n = 6). All macaques were implanted bilaterally with subdural electrodes over temporal cortex and depth electrodes in CA3 hippocampal region. KA was stereotaxically injected into the right hippocampus of macaques. All animals were monitored by video and electrocorticography (ECoG) to assess status epilepticus (SE) and subsequent spontaneous recurrent seizures (SRS). Additionally, in order to evaluate brain injury produced by SE or SRS, we used both neuroimaging, including magnetic resonance image (MRI) & magnetic resonance spectroscopy (MRS), and histological pathology, including Nissl stainning and glial fibrillary acid protein (GFAP) immunostaining.

Results

The typical seizures were observed in the KA-injected animal model. Hippocampal sclerosis could be found by MRI & MRS. Hematoxylin and eosin (H&E) staining and GFAP immunostaining showed neuronal loss, proliferation of glial cells, formation of glial scars, and hippocampal atrophy. Electron microscopic analysis of hippocampal tissues revealed neuronal pyknosis, partial ribosome depolymerization, an abnormal reduction in rough endoplasmic reticulum size, expansion of Golgi vesicles and swollen star-shaped cells. Furthermore, we reported that KA was able to induce SE followed by SRS after a variable period of time. Similar to human mTLE, brain damage is confined to the hippocampus. Accordingly, hippocampal volume is in positive correlations with the neuronal cells count in the CA3, especially the ratio of neuron/glial cell.

Conclusions

The results suggest that a model of mTLE can be developed in macaques by intra-hippocampal injection of KA. Brain damage is confined to the hippocampus which is similar to the human mTLE. The hippocampal volume correlates with the extension of the hippocampal damage.     

Temporal Lobe Epilepsy: A Clinical Study of 47 Cases

Clinical features of 47 cases of temporal lobe epilepsy are analyzed and treatment of this disorder is outlined. Twenty-four per cent of all cases of epilepsy seen by one of the authors over a two-year period were of this type. Fifteen of these 47 patients had a history of birth injury. Care must be taken to distinguish the symptoms of temporal lobe epilepsy from those of acute anxiety or hysteria and to differentiate the short-lived temporal lobe attack from centrencephalic petit mal.Interictal personality disturbances were identified in 11 of 24 persons with temporal lobe epilepsy, four of 35 with focal epilepsy from all other areas, and one of 17 with centrencephalic epilepsy. Personality deviations most frequently encountered were irritability, aggressiveness, bouts of depression, paranoid tendencies and exhibitionism. Medical or surgical treatment often improves the personality abnormalities concomitantly with control of seizures.     

Ictal Depth EEG and MRI Structural Evidence for Two Different Epileptogenic Networks in Mesial Temporal Lobe Epilepsy

Hypersynchronous (HYP) and low voltage fast (LVF) activity are two separate ictal depth EEG onsets patterns often recorded in presurgical patients with MTLE. Evidence suggests the mechanisms generating HYP and LVF onset seizures are distinct, including differential involvement of hippocampal and extra-hippocampal sites. Yet the extent of extra-hippocampal structural alterations, which could support these two common seizures, is not known. In the current study, preoperative MRI from 24 patients with HYP or LVF onset seizures were analyzed to determine changes in cortical thickness and relate structural changes to spatiotemporal properties of the ictal EEG. Overall, onset and initial ipsilateral spread of HYP onset seizures involved mesial temporal structures, whereas LVF onset seizures involved mesial and lateral temporal as well as orbitofrontal cortex. MRI analysis found reduced cortical thickness correlated with longer duration of epilepsy. However, in patients with HYP onsets, the most affected areas were on the medial surface of each hemisphere, including parahippocampal regions and cingulate gyrus, whereas in patients with LVF onsets, the lateral surface of the anterior temporal lobe and orbitofrontal cortex showed the greatest effect. Most patients with HYP onset seizures were seizure-free after resective surgery, while a higher proportion of patients with LVF onset seizures had only worthwhile improvement. Our findings confirm the view that recurrent seizures cause progressive changes in cortical thickness, and provide information concerning the structural basis of two different epileptogenic networks responsible for MTLE. One, identified by HYP ictal onsets, chiefly involves hippocampus and is associated with excellent outcome after standardized anteromedial temporal resection, while the other also involves lateral temporal and orbitofrontal cortex and a seizure-free surgical outcome occurs less after this procedure. These results suggest that a more extensive tailored resection may be required for patients with the second type of MTLE.     

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.     

Loss of Resting-State Posterior Cingulate Flexibility Is Associated with Memory Disturbance in Left Temporal Lobe Epilepsy

The association between cognition and resting-state fMRI (rs-fMRI) has been the focus of many recent studies, most of which use stationary connectivity. The dynamics or flexibility of connectivity, however, may be seminal for understanding cognitive functioning. In temporal lobe epilepsy (TLE), stationary connectomic correlates of impaired memory have been reported mainly for the hippocampus and posterior cingulate cortex (PCC). We therefore investigate resting-state and task-based hippocampal and PCC flexibility in addition to stationary connectivity in left TLE (LTLE) patients. Sixteen LTLE patients were analyzed with respect to rs-fMRI and task-based fMRI (t-fMRI), and underwent clinical neuropsychological testing. Flexibility of connectivity was calculated using a sliding-window approach by determining the standard deviation of Fisher-transformed Pearson correlation coefficients over all windows. Stationary connectivity was also calculated. Disturbed memory was operationalized as having at least one memory subtest score equal to or below the 5^th percentile compared to normative data. Lower PCC flexibility, particularly in the contralateral (i.e. right) hemisphere, was found in memory-disturbed LTLE patients, who had up to 22% less flexible connectivity. No significant group differences were found with respect to hippocampal flexibility, stationary connectivity during both rs-fMRI and t-fMRI, or flexibility during t-fMRI. Contralateral resting-state PCC flexibility was able to classify all but one patient with respect to their memory status (94% accuracy). Flexibility of the PCC during rest relates to memory functioning in LTLE patients. Loss of flexible connectivity to the rest of the brain originating from the PCC, particularly contralateral to the seizure focus, is able to discern memory disturbed patients from their preserved counterparts. This study indicates that the dynamics of resting-state connectivity are associated with cognitive status of LTLE patients, rather than stationary connectivity.