Publications by authors named "José Eduardo Peixoto-Santos"

20 Publications

  • Page 1 of 1

DNA methylation-based classification of malformations of cortical development in the human brain.

Acta Neuropathol 2022 Jan 19;143(1):93-104. Epub 2021 Nov 19.

Department of Neuropathology, Affiliated Partner of the ERN EpiCARE, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Malformations of cortical development (MCD) comprise a broad spectrum of structural brain lesions frequently associated with epilepsy. Disease definition and diagnosis remain challenging and are often prone to arbitrary judgment. Molecular classification of histopathological entities may help rationalize the diagnostic process. We present a retrospective, multi-center analysis of genome-wide DNA methylation from human brain specimens obtained from epilepsy surgery using EPIC 850 K BeadChip arrays. A total of 308 samples were included in the study. In the reference cohort, 239 formalin-fixed and paraffin-embedded (FFPE) tissue samples were histopathologically classified as MCD, including 12 major subtype pathologies. They were compared to 15 FFPE samples from surgical non-MCD cortices and 11 FFPE samples from post-mortem non-epilepsy controls. We applied three different statistical approaches to decipher the DNA methylation pattern of histopathological MCD entities, i.e., pairwise comparison, machine learning, and deep learning algorithms. Our deep learning model, which represented a shallow neuronal network, achieved the highest level of accuracy. A test cohort of 43 independent surgical samples from different epilepsy centers was used to test the precision of our DNA methylation-based MCD classifier. All samples from the test cohort were accurately assigned to their disease classes by the algorithm. These data demonstrate DNA methylation-based MCD classification suitability across major histopathological entities amenable to epilepsy surgery and age groups and will help establish an integrated diagnostic classification scheme for epilepsy-associated MCD.
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http://dx.doi.org/10.1007/s00401-021-02386-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8732912PMC
January 2022

Improving surgical outcome with electric source imaging and high field magnetic resonance imaging.

Seizure 2021 Aug 11;90:145-154. Epub 2021 Feb 11.

Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo, Brazil. Electronic address:

While most patients with focal epilepsy present with clear structural abnormalities on standard, 1.5 or 3 T MRI, some patients are MRI-negative. For those, quantitative MRI techniques, such as volumetry, voxel-based morphometry, and relaxation time measurements can aid in finding the epileptogenic focus. High-field MRI, just recently approved for clinical use by the FDA, increases the resolution and, in several publications, was shown to improve the detection of focal cortical dysplasias and mild cortical malformations. For those cases without any tissue abnormality in neuroimaging, even at 7 T, scalp EEG alone is insufficient to delimitate the epileptogenic zone. They may benefit from the use of high-density EEG, in which the increased number of electrodes helps improve spatial sampling. The spatial resolution of even low-density EEG can benefit from electric source imaging techniques, which map the source of the recorded abnormal activity, such as interictal epileptiform discharges, focal slowing, and ictal rhythm. These EEG techniques help localize the irritative, functional deficit, and seizure-onset zone, to better estimate the epileptogenic zone. Combining those technologies allows several drug-resistant cases to be submitted to surgery, increasing the odds of seizure freedom and providing a must needed hope for patients with epilepsy.
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http://dx.doi.org/10.1016/j.seizure.2021.02.006DOI Listing
August 2021

Neuropathology of the 21st century for the Latin American epilepsy community.

Seizure 2021 Aug 8;90:51-59. Epub 2021 Feb 8.

Department of Neuropathology, University Hospitals Erlangen, Erlangen, Germany. Electronic address:

Many people with epilepsy remain drug-resistant, despite continuous efforts and advances in research and treatment. It is mandatory to understand the epilepsy's underlying etiology, whether it is structural, genetic, infectious, metabolic, immune or (currently) unknown, as it contains major information about the clinical phenotype, cognitive comorbidities, (new) drug targets and also help to predict postsurgical outcome. A multimodal approach, including digital slides and multichannel immunofluorescence labelling can increase the diagnostic yield of subtle pathologies, while DNA methylation arrays could helps in the diagnosis of difficult-to-classify lesions. Such techniques are not always available, however, in low-income countries. Even without access to expensive molecular techniques, automated analysis scripts and machine learning algorithms can be developed by Latin American researchers to improve our diagnostic yield from routine Hematoxylin & Eosin stained tissue sections. The pathology community of Latin America contributed substantially to our current knowledge of etiologies related to human epilepsies and experimental epilepsy models. To further boost the impact of Latin American research, local centers should adhere to modern, multimodal neuropathology techniques, integrate different levels of knowledge, and strengthen their scientific collaborations. Dedicated teaching courses in Epileptology, such as the Latin American Summer Schools of Epilepsy (LASSE) or International Summer School for Neuropathology and Epilepsy Surgery (INES) addressing young researcher and neurologists, are most successful to promote this endeavor. In this review, we will describe the state of neuropathology at the 21st century and also highlight Latin American researchers' contributions to the current knowledge in neuropathology of epilepsy.
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http://dx.doi.org/10.1016/j.seizure.2021.02.003DOI Listing
August 2021

Dysmorphic neurons as cellular source for phase-amplitude coupling in Focal Cortical Dysplasia Type II.

Clin Neurophysiol 2021 03 20;132(3):782-792. Epub 2021 Jan 20.

Department of Neuropathology, University Hospital Erlangen, Germany.

Objective: Reliable localization of the epileptogenic zone is necessary for successful epilepsy surgery. Neurophysiological biomarkers include ictal onsets and interictal spikes. Furthermore, the epileptic network shows oscillations with potential localization value and pathomechanistic implications. The cellular origin of such markers in invasive EEG in vivo remains to be clarified.

Methods: In the presented pilot study, surgical brain samples and invasive EEG recordings of seven patients with surgically treated Focal Cortical Dysplasia (FCD) type II were coregistered using a novel protocol. Dysmorphic neurons and balloon cells were immunohistochemically quantified. Evaluated markers included seizure onset, spikes, and oscillatory activity in delta, theta, gamma and ripple frequency bands, as well as sample entropy and phase-amplitude coupling between delta, theta, alpha and beta phase and gamma amplitude.

Results: Correlations between histopathology and neurophysiology provided evidence for a contribution of dysmorphic neurons to interictal spikes, fast gamma activity and ripples. Furthermore, seizure onset and phase-amplitude coupling in areas with dysmorphic neurons suggests preserved connectivity is related to seizure initiation. Balloon cells showed no association.

Conclusions: Phase-amplitude coupling, spikes, fast gamma and ripples are related to the density of dysmorphic neurons and localize the seizure onset zone.

Significance: The results of our pilot study provide a new powerful tool to address the cellular source of abnormal neurophysiology signals to leverage current and novel biomarkers for the localization of epileptic activity in the human brain.
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http://dx.doi.org/10.1016/j.clinph.2021.01.004DOI Listing
March 2021

Histological correlates of hippocampal magnetization transfer images in drug-resistant temporal lobe epilepsy patients.

Neuroimage Clin 2020 8;28:102463. Epub 2020 Oct 8.

Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil. Electronic address:

Objective: Temporal lobe epilepsy patients (TLE) often present with hippocampal atrophy, increased T2 relaxation, and reduced magnetization transfer ratio (MTR) in magnetic resonance images (MRI). The histological correlates of the reduced hippocampal MTR are so far unknown. Since MTR is dependent on the tissue's macromolecules, our aim was to evaluate the correlations between cellular populations, extracellular matrix molecules and the MTR in TLE patients.

Methods: Patients with TLE (n = 26) and voluntaries (=20) were scanned in a 3 Tesla MRI scanner, and MTR images were calculated from 3DT1 sequences with magnetization pulse on resonance. Immunohistochemistry for neurons, reactive astrocytes, activated microglia, and extracellular matrix chondroitin sulfate were performed in formalin fixed, paraffin embedded tissues of TLE and autopsy controls (n = 10). Results were considered significant with adjusted p < 0.05.

Results: Compared to the respective controls, TLE patients had reduced hippocampal MTR, increased reactive astrocytes and activated microglia, increased extracellular chondroitin sulfate, and reduced neuron density, compares to controls. MTR correlated positively with neuron density in CA3 and with chondroitin sulfate in CA3 and CA1. Multiple linear regressions reinforced the correlations between chondroitin sulfate and MTR.

Significance: Our data indicate that extracellular matrix molecules are the most significant histological correlates of magnetization transfer ratio in the hippocampus of TLE patients.
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http://dx.doi.org/10.1016/j.nicl.2020.102463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586233PMC
June 2021

Drebrin expression patterns in patients with refractory temporal lobe epilepsy and hippocampal sclerosis.

Epilepsia 2020 08 14;61(8):1581-1594. Epub 2020 Jul 14.

Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.

Objective: Drebrins are crucial for synaptic function and dendritic spine development, remodeling, and maintenance. In temporal lobe epilepsy (TLE) patients, a significant hippocampal synaptic reorganization occurs, and synaptic reorganization has been associated with hippocampal hyperexcitability. This study aimed to evaluate, in TLE patients, the hippocampal expression of drebrin using immunohistochemistry with DAS2 or M2F6 antibodies that recognize adult (drebrin A) or adult and embryonic (pan-drebrin) isoforms, respectively.

Methods: Hippocampal sections from drug-resistant TLE patients with hippocampal sclerosis (HS; TLE, n = 33), of whom 31 presented with type 1 HS and two with type 2 HS, and autopsy control cases (n = 20) were assayed by immunohistochemistry and evaluated for neuron density, and drebrin A and pan-drebrin expression. Double-labeling immunofluorescences were performed to localize drebrin A-positive spines in dendrites (MAP2), and to evaluate whether drebrin colocalizes with inhibitory (GAD65) and excitatory (VGlut1) presynaptic markers.

Results: Compared to controls, TLE patients had increased pan-drebrin in all hippocampal subfields and increased drebrin A-immunopositive area in all hippocampal subfields but CA1. Drebrin-positive spine density followed the same pattern as total drebrin quantification. Confocal microscopy indicated juxtaposition of drebrin-positive spines with VGlut1-positive puncta, but not with GAD65-positive puncta. Drebrin expression in the dentate gyrus of TLE cases was associated negatively with seizure frequency and positively with verbal memory. TLE patients with lower drebrin-immunopositive area in inner molecular layer (IML) than in outer molecular layer (OML) had a lower seizure frequency than those with higher or comparable drebrin-immunopositive area in IML compared with OML.

Significance: Our results suggest that changes in drebrin-positive spines and drebrin expression in the dentate gyrus of TLE patients are associated with lower seizure frequency, more preserved verbal memory, and a better postsurgical outcome.
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http://dx.doi.org/10.1111/epi.16595DOI Listing
August 2020

Long-term potentiation prevents ketamine-induced aberrant neurophysiological dynamics in the hippocampus-prefrontal cortex pathway in vivo.

Sci Rep 2020 04 28;10(1):7167. Epub 2020 Apr 28.

Department of Neuroscience and Behavioral Sciences, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, 14049-900, Brazil.

N-methyl-D-aspartate receptor (NMDAr) antagonists such as ketamine (KET) produce psychotic-like behavior in both humans and animal models. NMDAr hypofunction affects normal oscillatory dynamics and synaptic plasticity in key brain regions related to schizophrenia, particularly in the hippocampus and the prefrontal cortex. It has been shown that prior long-term potentiation (LTP) occluded the increase of synaptic efficacy in the hippocampus-prefrontal cortex pathway induced by MK-801, a non-competitive NMDAr antagonist. However, it is not clear whether LTP could also modulate aberrant oscillations and short-term plasticity disruptions induced by NMDAr antagonists. Thus, we tested whether LTP could mitigate the electrophysiological changes promoted by KET. We recorded HPC-PFC local field potentials and evoked responses in urethane anesthetized rats, before and after KET administration, preceded or not by LTP induction. Our results show that KET promotes an aberrant delta-high-gamma cross-frequency coupling in the PFC and an enhancement in HPC-PFC evoked responses. LTP induction prior to KET attenuates changes in synaptic efficiency and prevents the increase in cortical gamma amplitude comodulation. These findings are consistent with evidence that increased efficiency of glutamatergic receptors attenuates cognitive impairment in animal models of psychosis. Therefore, high-frequency stimulation in HPC may be a useful tool to better understand how to prevent NMDAr hypofunction effects on synaptic plasticity and oscillatory coordination in cortico-limbic circuits.
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http://dx.doi.org/10.1038/s41598-020-63979-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188848PMC
April 2020

Glia and extracellular matrix molecules: What are their importance for the electrographic and MRI changes in the epileptogenic zone?

Epilepsy Behav 2021 08 26;121(Pt B):106542. Epub 2019 Dec 26.

Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; Department of Neurology and Neurosurgery, Paulista School of Medicine, UNIFESP, Sao Paulo, Brazil.

Glial cells and extracellular matrix (ECM) molecules are crucial for the maintenance of brain homeostasis. Especially because of their actions regarding neurotransmitter and ionic control, and synaptic function, these cells can potentially contribute to the hyperexcitability seen in the epileptogenic, while ECM changes are linked to synaptic reorganization. The present review will explore glial and ECM homeostatic roles and their potential contribution to tissue plasticity. Finally, we will address how glial, and ECM changes in the epileptogenic zone can be seen in magnetic resonance imaging (MRI), pointing out their importance as markers for the extension of the epileptogenic area. This article is part of the Special Issue "NEWroscience 2018".
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http://dx.doi.org/10.1016/j.yebeh.2019.106542DOI Listing
August 2021

Manual Hippocampal Subfield Segmentation Using High-Field MRI: Impact of Different Subfields in Hippocampal Volume Loss of Temporal Lobe Epilepsy Patients.

Front Neurol 2018 20;9:927. Epub 2018 Nov 20.

Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil.

In patients with temporal lobe epilepsy (TLE), presurgical magnetic resonance imaging (MRI) often reveals hippocampal atrophy, while neuropathological assessment indicates the different types of hippocampal sclerosis (HS). Different HS types are not discriminated in MRI so far. We aimed to define the volume of each hippocampal subfield on MRI manually and to compare automatic and manual segmentations for the discrimination of HS types. The T2-weighted images from 14 formalin-fixed age-matched control hippocampi were obtained with 4.7T MRI to evaluate the volume of each subfield at the anatomical level of the hippocampal head, body, and tail. Formalin-fixed coronal sections at the level of the body of 14 control cases, as well as tissue samples from 24 TLE patients, were imaged with a similar high-resolution sequence at 3T. Presurgical three-dimensional (3D) T1-weighted images from TLE went through a FreeSurfer 6.0 hippocampal subfield automatic assessment. The manual delineation with the 4.7T MRI was identified using Luxol Fast Blue stained 10-μm-thin microscopy slides, collected at every millimeter. An additional section at the level of the body from controls and TLE cases was submitted to NeuN immunohistochemistry for neuronal density estimation. All TLE cases were classified according to the International League Against Epilepsy's (ILAE's) HS classification. Manual volumetry in controls revealed that the dentate gyrus (DG)+CA4 region, CA1, and subiculum accounted for almost 90% of the hippocampal volume. The manual 3T volumetry showed that all TLE patients with type 1 HS (TLE-HS1) had lower volumes for DG+CA4, CA2, and CA1, whereas those TLE patients with HS type 2 (TLE-HS2) had lower volumes only in CA1 ( ≤ 0.038). Neuronal cell densities always decreased in CA4, CA3, CA2, and CA1 of TLE-HS1 but only in CA1 of TLE-HS2 ( ≤ 0.003). In addition, TLE-HS2 had a higher volume ( = 0.016) and higher neuronal density ( < 0.001) than the TLE-HS1 in DG + CA4. Automatic segmentation failed to match the manual or histological findings and was unable to differentiate TLE-HS1 from TLE-HS2. Total hippocampal volume correlated with DG+CA4 and CA1 volumes and neuronal density. For the first time, we also identified subfield-specific pathology patterns in the manual evaluation of volumetric MRI scans, showing the importance of manual segmentation to assess subfield-specific pathology patterns.
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http://dx.doi.org/10.3389/fneur.2018.00927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256705PMC
November 2018

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process.

PLoS One 2017 4;12(8):e0182765. Epub 2017 Aug 4.

Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceio, Alagoas, Brazil.

Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182765PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544225PMC
October 2017

Selective post-training time window for memory consolidation interference of cannabidiol into the prefrontal cortex: Reduced dopaminergic modulation and immediate gene expression in limbic circuits.

Neuroscience 2017 05 24;350:85-93. Epub 2017 Mar 24.

Brain Institute, Federal University of Rio Grande do Norte, Natal - RN, Brazil. Electronic address:

The prefrontal cortex (PFC), amygdala and hippocampus display a coordinated activity during acquisition of associative fear memories. Evidence indicates that PFC engagement in aversive memory formation does not progress linearly as previously thought. Instead, it seems to be recruited at specific time windows after memory acquisition, which has implications for the treatment of post-traumatic stress disorders. Cannabidiol (CBD), the major non-psychotomimetic phytocannabinoid of the Cannabis sativa plant, is known to modulate contextual fear memory acquisition in rodents. However, it is still not clear how CBD interferes with PFC-dependent processes during post-training memory consolidation. Here, we tested whether intra-PFC infusions of CBD immediately after or 5h following contextual fear conditioning was able to interfere with memory consolidation. Neurochemical and cellular correlates of the CBD treatment were evaluated by the quantification of extracellular levels of dopamine (DA), serotonin, and their metabolites in the PFC and by measuring the cellular expression of activity-dependent transcription factors in cortical and limbic regions. Our results indicate that bilateral intra-PFC CBD infusion impaired contextual fear memory consolidation when applied 5h after conditioning, but had no effect when applied immediately after it. This effect was associated with a reduction in DA turnover in the PFC following retrieval 5days after training. We also observed that post-conditioning infusion of CBD reduced c-fos and zif-268 protein expression in the hippocampus, PFC, and thalamus. Our findings support that CBD interferes with contextual fear memory consolidation by reducing PFC influence on cortico-limbic circuits.
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http://dx.doi.org/10.1016/j.neuroscience.2017.03.019DOI Listing
May 2017

Edaravone reduces astrogliosis and apoptosis in young rats with kaolin-induced hydrocephalus.

Childs Nerv Syst 2017 Mar 17;33(3):419-428. Epub 2016 Dec 17.

Department of Surgery and Anatomy, Division of Anatomy, Ribeirao Preto Medical School, University of Sao Paulo, 3900 Av. dos Bandeirantes, Ribeirao Preto, SP, 14049-900, Brazil.

Purpose: We investigated the possible neuroprotective effects of the free radical scavenger edaravone in experimental hydrocephalus.

Methods: Seven-day-old Wistar rats were divided into three groups: control group (C), untreated hydrocephalic (H), and hydrocephalic treated with edaravone (EH). The H and EH groups were subjected to hydrocephalus induction by 20% kaolin intracisternal injection. The edaravone (20 mg/kg) was administered daily for 14 days from the induction of hydrocephalus. All animals were daily weighed and submitted to behavioral test and assessment by magnetic resonance imaging. After 14 days, the animals were sacrificed and the brain was removed for histological, immunohistochemical, and biochemical studies.

Results: The gain weight was similar between groups from the ninth post-induction day. The open field test performance of EH group was better (p < 0.05) as compared to untreated hydrocephalic animals. Hydrocephalic animals (H and EH) showed ventricular ratio values were higher (p < 0.05), whereas magnetization transfer values were lower (p < 0.05), as compared to control animals. Astrocyte activity (glial fibrillary acidic protein) and apoptotic cells (caspase-3) of EH group were decreased on the corpus callosum (p > 0.01), germinal matrix (p > 0.05), and cerebral cortex (p > 0.05), as compared to H group.

Conclusions: We have demonstrated that administration of edaravone for 14 consecutive days after induction of hydrocephalus reduced astrocyte activity and that it has some beneficial effects over apoptotic cell death.
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http://dx.doi.org/10.1007/s00381-016-3313-xDOI Listing
March 2017

Individual hippocampal subfield assessment indicates that matrix macromolecules and gliosis are key elements for the increased T2 relaxation time seen in temporal lobe epilepsy.

Epilepsia 2017 01 18;58(1):149-159. Epub 2016 Nov 18.

Department of Neurosciences and Behavioral Sciences, Ribeirao Preto School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil.

Objective: Increased T2 relaxation time is often seen in temporal lobe epilepsy (TLE) with hippocampal sclerosis. Water content directly affects the effective T2 in a voxel. Our aim was to evaluate the relation between T2 values and two molecules associated with brain water homeostasis aquaporin 4 (AQP4) and chondroitin sulfate proteoglycan (CSPG), as well as cellular populations in the hippocampal region of patients with TLE.

Methods: Hippocampal T2 imaging and diffusion tensor imaging (DTI) were obtained from 42 drug-resistant patients with TLE and 20 healthy volunteers (radiologic controls, RCs). A similar protocol (ex vivo) was applied to hippocampal sections from the same TLE cases and 14 autopsy control hippocampi (histologic and radiologic controls, HRCs), and each hippocampal subfield was evaluated. Hippocampal sections from TLE cases and HRC controls were submitted to immunohistochemistry for neurons (neuron nuclei [NeuN]), reactive astrocytes (glial fibrillary acidic protein [GFAP]), activated microglia (human leukocyte antigen-D-related [HLA-DR]), polarized AQP4, and CSPG.

Results: Patients with TLE had higher in vivo and ex vivo hippocampal T2 relaxation time. Hippocampi from epilepsy cases had lower neuron density, higher gliosis, decreased AQP4 polarization, and increased CSPG immunoreactive area. In vivo relaxation correlated with astrogliosis in the subiculum and extracellular CSPG in the hilus. Ex vivo T2 relaxation time correlated with astrogliosis in the hilus, CA4, and subiculum, and with microgliosis in CA1. The difference between in vivo and ex vivo relaxation ratio correlated with mean diffusivity and with the immunopositive area for CSPG in the hilus.

Significance: Our data indicate that astrogliosis, microgliosis, and CSPG expression correlate with the increased T2 relaxation time seen in the hippocampi of patients with TLE.
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http://dx.doi.org/10.1111/epi.13620DOI Listing
January 2017

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis.

PLoS One 2016 3;11(10):e0163855. Epub 2016 Oct 3.

Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceio, Alagoas, Brazil.

The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naïve group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163855PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047645PMC
June 2017

Decreased neuron loss and memory dysfunction in pilocarpine-treated rats pre-exposed to hypoxia.

Neuroscience 2016 09 29;332:88-100. Epub 2016 Jun 29.

Department of Neuroscience and Behavioral Sciences, Ribeirão Preto School of Medicine, University of São Paulo, Ribeiãro Preto 14049-900, Brazil. Electronic address:

Preconditioning can induce a cascade of cellular events leading to neuroprotection against subsequent brain insults. In this study, we investigated the chronic effects of hypoxic preconditioning on spontaneous recurrent seizures (SRS), neuronal death, and spatial memory performance in rats subjected to pilocarpine (Pilo)-induced status epilepticus (SE). Rats underwent a short hypoxic episode (7% O2+93% N2; 30min on two consecutive days) preceding a 4-h SE (HSE group). Control groups were rats submitted to SE only (SE), rats subjected to hypoxia only (H) or normoxia-saline (C). Animals were monitored for the occurrence of SRS, and spatial memory performance was evaluated in the radial-arm maze. Hippocampal sections were analyzed for cell death and mossy fiber sprouting at 1 or 60days after SE. Compared to SE group, HSE had increased SE latency, reduced number of rats with SRS, reduced mossy fiber sprouting at 60days, and reduced cell death in the hilus and the CA3 region 1 and 60days after SE. Additionally, HSE rats had better spatial memory performance than SE rats. Our findings indicated that short hypoxic preconditioning preceding SE promotes long-lasting protective effects on neuron survival and spatial memory.
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http://dx.doi.org/10.1016/j.neuroscience.2016.06.047DOI Listing
September 2016

Temporal lobe epilepsy patients with severe hippocampal neuron loss but normal hippocampal volume: Extracellular matrix molecules are important for the maintenance of hippocampal volume.

Epilepsia 2015 Oct 27;56(10):1562-70. Epub 2015 Jul 27.

Department of Neurosciences and Behavior, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirao Preto, Sao Paulo, Brazil.

Objective: Hippocampal sclerosis is a common finding in patients with temporal lobe epilepsy (TLE), and magnetic resonance imaging (MRI) studies associate the reduction of hippocampal volume with the neuron loss seen on histologic evaluation. Astrogliosis and increased levels of chondroitin sulfate, a major component of brain extracellular matrix, are also seen in hippocampal sclerosis. Our aim was to evaluate the association between hippocampal volume and chondroitin sulfate, as well as neuronal and astroglial populations in the hippocampus of patients with TLE.

Methods: Patients with drug-resistant TLE were subdivided, according to hippocampal volume measured by MRI, into two groups: hippocampal atrophy (HA) or normal volume (NV) cases. Hippocampi from TLE patients and age-matched controls were submitted to immunohistochemistry to evaluate neuronal population, astroglial population, and chondroitin sulfate expression with antibodies against neuron nuclei protein (NeuN), glial fibrillary acidic protein (GFAP), and chondroitin sulfate (CS-56) antigens, respectively.

Results: Both TLE groups were clinically similar. NV cases had higher hippocampal volume, both ipsilateral and contralateral, when compared to HA. Compared to controls, NV and HA patients had reduced neuron density, and increased GFAP and CS-56 immunopositive area. There was no statistical difference between NV and HA groups in neuron density or immunopositive areas for GFAP and CS-56. Hippocampal volume correlated positively with neuron density in CA1 and prosubiculum, and with immunopositive areas for CS-56 in CA1, and negatively with immunopositive area for GFAP in CA1. Multiple linear regression analysis indicated that both neuron density and CS-56 immunopositive area in CA1 were statistically significant predictors of hippocampal volume.

Significance: Our findings indicate that neuron density and chondroitin sulfate immunopositive area in the CA1 subfield are crucial for the hippocampal volume, and that chondroitin sulfate is important for the maintenance of a normal hippocampal volume in some cases with severe neuron loss.
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http://dx.doi.org/10.1111/epi.13082DOI Listing
October 2015

Mesial temporal lobe epilepsy with psychiatric comorbidities: a place for differential neuroinflammatory interplay.

J Neuroinflammation 2015 Feb 25;12:38. Epub 2015 Feb 25.

Department of Neurosciences and Behavior, Ribeirao Preto Medical School, University of Sao Paulo (USP), Av Bandeirantes 3900, CEP 14049-900, Ribeirao Preto, SP, Brazil.

Background: Despite the strong association between epilepsy and psychiatric comorbidities, few biological substrates are currently described. We have previously reported neuropathological alterations in mesial temporal lobe epilepsy (MTLE) patients with major depression and psychosis that suggest a morphological and neurochemical basis for psychopathological symptoms. Neuroinflammatory-related structures and molecules might be part of the altered neurochemical milieu underlying the association between epilepsy and psychiatric comorbidities, and such features have not been previously investigated in humans.

Methods: MTLE hippocampi of subjects without psychiatric history (MTLEW), MTLE + major depression (MTLE + D), and MTLE + interictal psychosis (MTLE + P) derived from epilepsy surgery and control necropsies were investigated for reactive astrocytes (glial fibrillary acidic protein (GFAP)), activated microglia (human leukocyte antigen, MHC class II (HLA-DR)), glial metallothionein-I/II (MT-I/II), and aquaporin 4 (AQP4) immunohistochemistry.

Results: We found an increased GFAP immunoreactive area in the molecular layers, granule cell layer, and cornus ammonis region 2 (CA2) and cornus ammonis region 1 (CA1) of MTLEW and MTLE + P, respectively, compared to MTLE + D. HLA-DR immunoreactive area was higher in cornus ammonis region 3 (CA3) of MTLE + P, compared to MTLE + D and MTLEW, and in the hilus, when compared to MTLEW. MTLEW cases showed increased MT-I/II area in the granule cell layer and CA1, compared to MTLE + P, and in the parasubiculum, when compared to MTLE + D and MTLE + P. Differences between MTLE and control, such as astrogliosis, microgliosis, increased MT-I/II, and decreased perivascular AQP4 in the epileptogenic hippocampus, were in agreement to what is currently described in the literature.

Conclusions: Neuroinflammatory-related molecules in MTLE hippocampus show a distinct pattern of expression when patients present with a comorbid psychiatric diagnosis, similar to what is found in the pure forms of schizophrenia and major depression. Future studies focusing on inflammatory characteristics of MTLE with psychiatric comorbidities might help in the design of better therapeutic strategies.
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http://dx.doi.org/10.1186/s12974-015-0266-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347571PMC
February 2015

Increased metallothionein I/II expression in patients with temporal lobe epilepsy.

PLoS One 2012 18;7(9):e44709. Epub 2012 Sep 18.

Department of Neuroscience and Behavior, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto-São Paulo, Brazil.

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and patients with TLE associated with tumor or dysplasia (TLE-TD) were evaluated for expression of MT-I/II, for the vesicular zinc levels, and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis, microgliosis and reduced neuronal population. In TLE-TD, the same changes were observed, except that were mainly confined to fascia dentata. Increased vesicular zinc was observed only in the inner molecular layer of MTLE patients, when compared to control cases. Correlation and linear regression analyses indicated an association between increased MT-I/II and increased astrogliosis in TLE. MT-I/II levels did not correlate with any clinical variables, but MTLE patients with secondary generalized seizures (SGS) had less MT-I/II than MTLE patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from TLE patients and our data suggest that it is associated with astrogliosis and may be associated with different seizure spread patterns.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044709PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445538PMC
March 2013
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