Value of ultra-high field MRI in patients with suspected focal epilepsy and negative 3 T MRI (EpiUltraStudy): protocol for a prospective, longitudinal therapeutic study.

PURPOSE: Resective epilepsy surgery is a well-established, evidence-based treatment option in patients with drug-resistant focal epilepsy. A major predictive factor of good surgical outcome is visualization and delineation of a potential epileptogenic lesion by MRI. However, frequently, these lesions are subtle and may escape detection by conventional MRI (≤ 3 T). METHODS: We present the EpiUltraStudy protocol to address the hypothesis that application of ultra-high field (UHF) MRI increases the rate of detection of structural lesions and functional brain aberrances in patients with drug-resistant focal epilepsy who are candidates for resective epilepsy surgery. Additionally, therapeutic gain will be addressed, testing whether increased lesion detection and tailored resections result in higher rates of seizure freedom 1 year after epilepsy surgery. Sixty patients enroll the study according to the following inclusion criteria: aged ≥ 12 years, diagnosed with drug-resistant focal epilepsy with a suspected epileptogenic focus, negative conventional 3 T MRI during pre-surgical work-up. RESULTS: All patients will be evaluated by 7 T MRI; ten patients will undergo an additional 9.4 T MRI exam. Images will be evaluated independently by two neuroradiologists and a neurologist or neurosurgeon. Clinical and UHF MRI will be discussed in the multidisciplinary epilepsy surgery conference. Demographic and epilepsy characteristics, along with postoperative seizure outcome and histopathological evaluation, will be recorded. CONCLUSION: This protocol was reviewed and approved by the local Institutional Review Board and complies with the Declaration of Helsinki and principles of Good Clinical Practice. Results will be submitted to international peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: www.trialregister.nl : NTR7536.

Ultra-high field magnetic resonance imaging in human epilepsy: A systematic review.

RATIONALE: Resective epilepsy surgery is an evidence-based curative treatment option for patients with drug-resistant focal epilepsy. The major preoperative predictor of a good surgical outcome is detection of an epileptogenic lesion by magnetic resonance imaging (MRI). Application of ultra-high field (UHF) MRI, i.e. field strengths ≥ 7 Tesla (T), may increase the sensitivity to detect such a lesion. METHODS: A keyword search strategy was submitted to Pubmed, EMBASE, Cochrane Database and clinicaltrials.gov to select studies on UHF MRI in patients with epilepsy. Follow-up study selection and data extraction were performed following PRISMA guidelines. We focused on I) diagnostic gain of UHF- over conventional MRI, II) concordance of MRI-detected lesion, seizure onset zone and surgical decision-making, and III) postoperative histopathological diagnosis and seizure outcome. RESULTS: Sixteen observational cohort studies, all using 7T MRI were included. Diagnostic gain of 7T over conventional MRI ranged from 8% to 67%, with a pooled gain of 31%. Novel techniques to visualize pathological processes in epilepsy and lesion detection are discussed. Seizure freedom was achieved in 73% of operated patients; no seizure outcome comparison was made between 7T MRI positive, 7T negative and 3T positive patients. 7T could influence surgical decision-making, with high concordance of lesion and seizure onset zone. Focal cortical dysplasia (54%), hippocampal sclerosis (12%) and gliosis (8.1%) were the most frequently diagnosed histopathological entities. SIGNIFICANCE: UHF MRI increases, yet variably, the sensitivity to detect an epileptogenic lesion, showing potential for use in clinical practice. It remains to be established whether this results in improved seizure outcome after surgical treatment. Prospective studies with larger cohorts of epilepsy patients, uniform scan and sequence protocols, and innovative post-processing technology are equally important as further increasing field strengths. Besides technical ameliorations, improved correlation of imaging features with clinical semiology, histopathology and clinical outcome has to be established.

Visual field deficits after epilepsy surgery: a new quantitative scoring method.

BACKGROUND: Anterior temporal lobectomy (ATL) as a treatment for drug-resistant temporal lobe epilepsy (TLE) frequently causes visual field deficits (VFDs). Reported VFD encompasses homonymous contralateral upper quadrantanopia. Its reported incidence ranges from 15 to 90%. To date, a quantitative method to evaluate postoperative VFD in static perimetry is not available. A method to quantify postoperative VFD, which allows for comparison between groups of patients, was developed. METHODS: Fifty-five patients with drug-resistant TLE, who underwent ATL with pre- and postoperative perimetry, were included. Temporal lobe resection length was measured on postoperative MRI. Percentage VFD was calculated for the total visual field, contralateral upper quadrant, or other three quadrants combined. RESULTS: Patients were divided into groups by resection size (< 45 and ≥ 45 mm) and side of surgery (right and left). We found significant higher VFD in the ≥ 45 vs. < 45 mm group (2.3 ± 4.4 vs. 0.7 ± 2.4%,p = 0.04) for right-sided ATL. Comparing VFD in both eyes, we found more VFD in the right vs. left eye following left-sided ATL (14.5 ± 9.8 vs. 12.9 ± 8.3%, p = 0.03). We also demonstrated significantly more VFD in the < 45 mm group for left- vs. right-sided surgery (6.7 ± 6.7 vs. 13.1 ± 7.0%, p = 0.016). A significant quantitative correlation between VFD and resection size for right-sided ATL was shown (r = 0.52, p < 0.01). CONCLUSIONS: We developed a new quantitative scoring method for the assessment of postoperative visual field deficits after temporal lobe epilepsy surgery and assessed its feasibility for clinical use. A significant correlation between VFD and resection size for right-sided ATL was confirmed.

In vivo assessment of the human cerebral microcirculation and its glycocalyx: A technical report.

INTRODUCTION: The cerebral microcirculation and its glycocalyx, a matrix coating the luminal endothelium, are key regulators of capillary permeability and cerebral blood flow. Microvascular abnormalities are described in several neurological disorders. However, assessment of the cerebral microcirculation and glycocalyx has mainly been performed ex vivo. NEW METHOD: Here, the technical feasibility of in vivo assessment of the human cerebral microcirculation and its glycocalyx using sidestream dark field (SDF) imaging is discussed. Intraoperative assessment requires the application of a sterile drape covering the camera (slipcover). First, sublingual measurements with and without slipcover were performed in a healthy control to assess the impact of this slipcover. Subsequently, using SDF imaging, the sublingual (reference), cortical, and hippocampal microcirculation and glycocalyx were evaluated in patients who underwent resective brain surgery as treatment for drug-resistant temporal lobe epilepsy. Finally, vessel density, and the perfused boundary region (PBR), a validated gauge of glycocalyx health, were calculated using GlycoCheck© software. RESULTS: The addition of a slipcover affects vessel density and PBR values in a control subject. The cerebral measurements in five patients were more difficult to obtain than the sublingual ones. This was probably at least partly due to the introduction of a sterile slipcover. Results on vessel density and PBR showed similar patterns at all three measurement sites. COMPARISON WITH EXISTING METHODS: This is the first report on in vivo assessment of the human cerebrovascular glycocalyx. Assessment of the glycocalyx is an additional application of in vivo imaging of the cerebral microcirculation using SDF technique. This method enables functional analysis of the microcirculation and glycocalyx, however the addition of a sterile slipcover affects the measurements. CONCLUSIONS: SDF imaging is a safe, quick, and straightforward technique to evaluate the functional cerebral microcirculation and glycocalyx. Because of their eminent role in cerebral homeostasis, this method may significantly add to research on the role of vascular pathophysiology underling various neurological disorders.

Validation of reference genes in human chordoma.

BACKGROUND: Chordoma are rare slow-growing tumors of the axial skeleton, which are thought to arise from remnants of the notochord. Little is known about the underlying mechanisms that drive this tumor. However, the assessment of gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) is hampered due to a lack of validated reference genes. Using an unstable reference gene in qRT-PCR may lead to irreproducible results. METHODS: The expression of 12 candidate reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) was analyzed by qRT-PCR in flash frozen chordoma samples from 18 patients. GeNorm and NormFinder algorithms were used to rank the stability of the genes. RESULTS: From most to least stably expressed, the top six genes found by geNorm were PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP. When analyzed by NormFinder, the top six genes were ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT. GAPDH alone, which is often used as a reference gene in chordoma gene expression studies, is not stable enough for reliable results. CONCLUSION: In gene expression studies of human chordomas, PGK1, ACTB, and YWHAZ are more stably expressed, and therefore, are preferred reference genes over the most often used reference gene so far, GAPDH.

Protocol for intraoperative assessment of the human cerebrovascular glycocalyx.

INTRODUCTION: Adequate functioning of the blood-brain barrier (BBB) is important for brain homoeostasis and normal neuronal function. Disruption of the BBB has been described in several neurological diseases. Recent reports suggest that an increased permeability of the BBB also contributes to increased seizure susceptibility in patients with epilepsy. The endothelial glycocalyx is coating the luminal side of the endothelium and can be considered as the first barrier of the BBB. We hypothesise that an altered glycocalyx thickness plays a role in the aetiology of temporal lobe epilepsy (TLE), the most common type of epilepsy. Here, we propose a protocol that allows intraoperative assessment of the cerebrovascular glycocalyx thickness in patients with TLE and assess whether its thickness is decreased in patients with TLE when compared with controls. METHODS AND ANALYSIS: This protocol is designed as a prospective observational case-control study in patients who undergo resective brain surgery as treatment for TLE. Control subjects are patients without a history of epileptic seizures, who undergo a craniotomy or burr hole surgery for other indications. Intraoperative glycocalyx thickness measurements of sublingual, cortical and hippocampal microcirculation are performed by video microscopy using sidestream dark-field imaging. Demographic details, seizure characteristics, epilepsy risk factors, intraoperative haemodynamic parameters and histopathological evaluation are additionally recorded. ETHICS AND DISSEMINATION: This protocol has been ethically approved by the local medical ethical committee (ID: NL51594.068.14) and complies with the Declaration of Helsinki and principles of Good Clinical Practice. Informed consent is obtained before study enrolment and only coded data will be stored in a secured database, enabling an audit trail. Results will be submitted to international peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: NTR5568.

Tonic GABAA Receptors as Potential Target for the Treatment of Temporal Lobe Epilepsy.

Tonic GABAA receptors are a subpopulation of receptors that generate long-lasting inhibition and thereby control network excitability. In recent years, these receptors have been implicated in various neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and epilepsy. Their distinct subunit composition and function, compared to phasic GABAA receptors, opens the possibility to specifically modulate network properties. In this review, the role of tonic GABAA receptors in epilepsy and as potential antiepileptic target will be discussed.

Morphology of the human cervical vagus nerve: implications for vagus nerve stimulation treatment.

OBJECTIVES: The vagus nerve has gained a role in the treatment of certain diseases by the use of vagus nerve stimulation (VNS). This study provides detailed morphological information regarding the human cervical vagus nerve at the level of electrode implant. RESULTS: Eleven pairs of cervical vagus nerves and four pairs of intracranial vagus nerves were analysed by the use of computer software. It was found that the right cervical vagus nerve has an 1.5 times larger effective surface area on average than the left nerve [1,089,492 ± 98,337 vs 753,915 ± 102,490 µm(2), respectively, (P < 0.05)] and that there is broad spreading within the individual nerves. At the right side, the mean effective surface area at the cervical level (1,089,492 ± 98,337 µm(2)) is larger than at the level inside the skull base (630,921 ± 105,422) (P < 0.05). This could imply that the vagus nerve receives anastomosing and 'hitchhiking' branches from areas other than the brainstem. Furthermore, abundant tyrosine hydroxylase (TH)- and dopamine ß-hydroxylase (DBH)-positive staining nerve fibres could be identified, indicating catecholaminergic neurotransmission. In two of the 22 cervical nerves, ganglion cells were found that also stained positive for TH and DBH. Stimulating the vagus nerve may therefore induce the release of dopamine and noradrenaline. A sympathetic activation could therefore be part of mechanism of action of VNS. Furthermore, it was shown that the right cervical vagus nerve contains on average two times more TH-positive nerve fibres than the left nerve (P < 0.05), a fact that could be of interest upon choosing stimulation side. We also suggest that the amount of epineurial tissue could be an important variable for determining individual effectiveness of VNS, because the absolute amount of epineurial tissue is widely spread between the individual nerves (ranging from 2,090,000 to 11,683,000 µm(2)). CONCLUSIONS: We conclude by stating that one has to look at the vagus nerve as a morphological entity of the peripheral autonomic nervous system, a composite of different fibres and (anastomosing and hitchhiking) branches of different origin with different neurotransmitters, which can act both parasympathetic and sympathetic. Electrically stimulating the vagus nerve therefore is not the same as elevating the 'physiological parasympathetic tone', but may also implement catecholaminergic (sympathetic) effects.

Inter- and intraobserver agreement of seizure behavior scoring in the amygdala kindled rat.

INTRODUCTION: The Racine scale is a 5-point seizure behavior scoring paradigm used in the amygdala kindled rat. Though this scale has been applied widely in experimental epilepsy research, studies of reproducibility are rare. The aim of the current study was, therefore, to assess its interobserver variability and intraobserver variability. MATERIAL AND METHODS: A video database set was acquired in the course of amygdala kindling of 67 Wistar rats. Six blinded observers received scoring instructions and then viewed a set of 15 random videos (session #1). Next, each observer scored 379 to 1048 additional videos (session #2) and finally scored the same set of 15 videos again (session #3). Scores included the occurrence of seizures (yes or no), the total seizure time (start of stimulus until the absence of seizure behavior), and the highest Racine stage. Interobserver variability and intraobserver variability were assessed in and between sessions #1 and #3 using a 2-way mixed intraclass correlation or Cohen's kappa depending on the variable. RESULTS: Interobserver agreement in session #1 was 0.664 for seizure occurrence, 0.861 for total seizure time, and 0.797 for the highest Racine stage. In session #3, interobserver agreement on seizure occurrence declined to 0.492, total seizure time declined to 0.625, and agreement for the highest Racine stage was 0.725. Interobserver agreement was scored insufficiently on focal R2 seizures in both sessions (0.287 and 0.182). Intraobserver agreement reached >0.80 agreement for seizure occurrence, highest seizure score, and total seizure time in 3 out of 4 observers. Racine's scale stage 2 seizure scores were only 0.135 in one observer but 0.650, 0.810, and 0.635 in the other observers. DISCUSSION AND CONCLUSION: Overall, interobserver agreement and intraobserver agreement in scoring with Racine's scale were adequate. However, because interobserver agreement declined after a period of individually scoring videos, we suggest periodic repetition of the standardized instruction in the course of evaluating videos in order to ensure reproducible results.

The influence of neuropathology on brain inflammation in human and experimental temporal lobe epilepsy.

It is unclear to what extent neuropathological changes contribute to brain inflammation observed in temporal lobe epilepsy (TLE). Here, we compared cytokine levels between histopathologically-confirmed sclerotic hippocampi and histopathologically-confirmed normal hippocampi from TLE patients. We analyzed a similar cytokine panel in the hippocampi of amygdala-kindled rats and we evaluated neuropathological changes by immunohistochemistry. In TLE patients, cytokine levels were not significantly different between sclerotic and non-sclerotic hippocampi. Though kindling resulted in increased astrocyte activation, cytokine levels and microglia activation were unchanged. These results suggest that the chronic epileptic state in TLE can also occur in the absence of intracerebral inflammation. Highlights.

Rat vagus nerve stimulation model of seizure suppression: nNOS and ΔFos B changes in the brainstem.

Vagus nerve stimulation (VNS) is a moderately effective treatment for intractable epilepsy. However, the mechanism of action is poorly understood. The effect of left VNS in amygdala kindled rats was investigated by studying changes in nNOS and ΔFos B expression in primary and secondary vagus nerve projection nuclei: the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMV), parabrachial nucleus (PBN) and locus coeruleus (LC). Rats were fully kindled by stimulation of the amygdala. Subsequently, when the fully kindled state was reached and then maintained for ten days, rats received a single 3-min train of VNS starting 1min prior to the kindling stimulus and lasting for 2min afterwards. In control animals the vagus nerve was not stimulated. Animals were sacrificed 48h later. The brainstems were stained for neuronal nitric oxide synthase (nNOS) and ΔFos B. VNS decreased seizure duration with more than 25% in 21% of rats. No VNS associated changes in nNOS immunoreactivity were observed in the NTS and no changes in ΔFos B were observed in the NTS, PBN, or LC. High nNOS immunopositive cell densities of >300cells/mm(2) were significantly more frequent in the left DMV than in the right (χ(2)(1)=26.2, p<0.01), independent of whether the vagus nerve was stimulated. We conclude that the observed nNOS immunoreactivity in the DMV suggests surgery-induced axonal damage. A 3-min train of VNS in fully kindled rats does not affect ΔFos B expression in primary and secondary projection nuclei of the vagus nerve.

Horner's syndrome: a complication of experimental carotid artery surgery in rats.

PURPOSE: To report on the occurrence of iatrogenic Horner's syndrome (HS) in epileptic rats after implantation of an electrode for vagus nerve stimulation and to describe the possible consequences of this new complication of carotid artery surgery in rats. METHODS: A bipolar circular electrode was placed around the left carotid artery and vagus nerve of 31 rats. The incidence of HS was evaluated by visual inspection within 24 h after surgery. RESULTS: 68% of rats suffered from HS immediately after surgery. This complication did not affect epileptogenesis. CONCLUSION: The occurrence of HS in the rat is a frequent complication of vagus nerve electrode implantation, which does not affect epileptogenesis in this study. However, rats affected by HS may suffer from damage to the sympathetic innervation of the gut, due to rat-specific neuroanatomy. Therefore, caution towards other research questions is warranted.

The role of interleukin-1 in seizures and epilepsy: a critical review.

Interleukin-1 (IL-1) has a multitude of functions in the central nervous system. Some of them involve mechanisms that are related to epileptogenesis. The role of IL-1 in seizures and epilepsy has been investigated in both patients and animal models. This review aims to synthesize, based on the currently available literature, the consensus role of IL-1 in epilepsy. Three lines of evidence suggest a role for IL-1: brain tissue from epilepsy patients and brain tissue from animal models shows increased IL-1 expression after seizures, and IL-1 has proconvulsive properties when applied exogeneously. However, opposing results have been published as well. More research is needed to fully establish the role of IL-1 in seizure generation and epilepsy, and to explore possible new treatment strategies that are based on interference with intracellular signaling cascades that are initiated when IL-1 binds to its receptor.

Surgery for temporal lobe epilepsy after cerebral malaria.

The most common indication for epilepsy surgery is temporal lobe epilepsy (TLE) which usually is divided into two categories, mesial and lateral TLE. The commonest pathology underlying mesial temporal lobe epilepsy (MTLE) is mesial temporal sclerosis (MTS); we report on a 50-year-old male patient, who contracted cerebral malaria and developed MTLE shortly thereafter. Magnetic resonance imaging (MRI) showed MTS. Surgical treatment was an anteromedial temporal lobe resection with amygdalohippocampectomy. The patient is seizure free, 36 months after surgical treatment. This is the first report describing MTLE-onset subsequent to cerebral malaria and discussing the potential pathophysiological relationship between cerebral malaria and MTS.

Increased glutamine synthetase but normal EAAT2 expression in platelets of ALS patients.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and glutamate excitotoxicity has been implicated in its pathogenesis. Platelets contain a glutamate uptake system and express components of the glutamate-glutamine cycle, such as the predominant glial excitatory amino acid transporter 2 (EAAT2). In several neurological diseases platelets have proven to be systemic markers for the disease. We compared properties of key components of the glutamate-glutamine cycle in blood platelets of ALS patients and healthy controls. Platelets were analyzed for (3)H-glutamate uptake in the presence or absence of thrombin and for EAAT2 and glutamine synthetase protein expression by Western blotting. Platelets of ALS patients showed a 37% increase in expression of glutamine synthetase, but normal expression of glutamate transporter EAAT2. Glutamate uptake in resting or thrombin-stimulated platelets did not differ significantly between platelets from ALS patients and controls. Thrombin-stimulation resulted in about a seven-fold increase in glutamate uptake. Our data suggest that glutamine synthetase may be a peripheral marker of ALS and encourage further investigation into the role of this enzyme in ALS.

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2.

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.

Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients.

RATIONALE: Altered expression of glutamate transporter EAAT2 protein has been reported in the hippocampus of patients with temporal lobe epilepsy (TLE). Two alternative EAAT2 mRNA splice forms, one resulting from a partial retention of intron 7 (I7R), the other from a deletion of exon 9 (E9S), were previously implicated in the loss of EAAT2 protein in patients with amyotrophic lateral sclerosis. METHODS: By RT-PCR we studied the occurrence of I7R and E9S in neocortical and hippocampal specimens from TLE patients and non-neurological controls. RESULTS: Both splice forms were found in all neocortical specimens from TLE patients (100% I7R, 100% E9S). This was significantly more than in controls (67% I7R, 60% E9S; P < 0.05). We also detected I7R and E9S in all seven motor cortex post-mortem samples from patients with amyotrophic lateral sclerosis. Within the TLE patient group, both splice variants appeared significantly more in non-sclerotic (100%), than in sclerotic hippocampi (69%, P < 0.05). CONCLUSION: These data indicate that the epileptic brain, especially that of TLE patients without hippocampal sclerosis, is highly prone to alternative EAAT2 mRNA splicing. Our data confirm that the presence of alternative EAAT2 splice forms is not disease specific.

Synaptosomal glutamate and GABA transport in patients with temporal lobe epilepsy.

High-affinity glutamate and GABA transporters found in the plasma membrane of neurons and glial cells terminate neurotransmission by rapidly removing extracellular transmitter. Impairment of transporter function has been implicated in the pathophysiologic mechanisms underlying epileptogenesis. We characterized glutamate and gamma-aminobutyric acid (GABA) transport in synaptosomes, isolated from neocortical and hippocampal biopsies of patients with temporal lobe epilepsy (TLE). We analyzed K(+)-evoked release in the presence and absence of Ca(2+) to determine vesicular and transporter-mediated release, respectively. We also analyzed (3)H-glutamate and (3)H-GABA uptake, the effect of glutamate uptake inhibitors L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and DL-threo-beta-benzyloxyaspartate (TBOA), and GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SK&F 89976-A). Neocortical synaptosomes from TLE patients did not show vesicular glutamate release, strongly reduced transporter-mediated release, and an increased basal release compared to that in rat synaptosomes. Furthermore, basal release was less sensitive to tPDC, and (3)H-glutamate uptake was reduced compared to that in rat synaptosomes. Vesicular GABA release from neocortical synaptosomes of TLE patients was reduced compared to that in rat synaptosomes, whereas transporter-mediated release was hardly affected. Furthermore, basal GABA release was more than doubled, but neither basal nor stimulated release were increased by SK&F 89976-A, which did significantly increase both types of GABA release in rat synaptosomes. Finally, (3)H-GABA uptake by synaptosomes from TLE patients was reduced significantly in hippocampus (0.19 +/- 0.04%), compared to that in neocortex (0.32 +/- 0.04%). Control experiments with human peritumoral cortical tissue suggest that impaired uptake of glutamate, but not of GABA, was caused in part by the hypoxic state of the biopsy. Our findings provide evidence for impaired function of glutamate and GABA transporters in human TLE.

Distribution of glutamate transporters in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy.

In patients suffering from temporal lobe epilepsy (TLE), increased extracellular glutamate levels in the epileptogenic hippocampus both during and after clinical seizures have been reported. These increased glutamate levels could be the result of malfunctioning and/or downregulation of glutamate transporters (also known as EAATs; excitatory amino acid transporters). In this study, the distribution of protein and mRNA of EAAT subtypes was examined in the hippocampus of TLE patients with hippocampal sclerosis (HS group) and without hippocampal sclerosis (non-HS group), and in autopsy controls without neurological disorders. EAAT protein localization was studied by immunohistochemistry on paraffin sections using specific poly- and monoclonal antibodies against the glial glutamate transporters EAAT1 and EAAT2 and the neuronal glutamate transporter EAAT3. Antibody specificity was shown by immunoblotting. In the HS group, a small decrease in EAAT1-immunoreactivity (IR) was observed in CA4 and in the polymorphic and supragranular layer of the dentate gyrus, compared with the control group. The strongest changes were found for EAAT2 levels. In the non-HS group, increased EAAT2-IR was detected in the CA1 and CA2 field, compared with non-epileptic controls. EAAT2-IR was decreased in the HS compared with the non-HS group. Fewer EAAT3-positive cells were found in the HS group than in the non-HS and control group. In both TLE groups, increased EAAT3 levels were observed in individual neurones. In the HS group, the percentage of EAAT3-IR neurones was increased in CA2 and in the granule cell layer of the dentate gyrus. Radioactive in situ hybridization for EAAT1-3 confirmed our immunohistochemical results. Non-radioactive in situ hybridization showed that not only astrocytes, but also neurones express EAAT2 mRNA. Taken together, differences in both mRNA and protein levels of glutamate transporter subtypes were found in specific regions in the TLE hippocampus, with most severe changes found for EAAT2 and EAAT3 levels. The results indicate an upregulation of EAAT2 protein expression in CA1 and CA2 in neurones in the non-HS group. This is in line with decreased EAAT2 protein levels in the HS group, since these hippocampi are characterized by severe neuronal cell loss. The functional consequences (glutamate transport capacity) of the reported changes in EAAT2 and EAAT3 remain to be determined.