Effects of Bradykinin B2 Receptor Ablation from Tyrosine Hydroxylase Cells on Behavioral and Motor Aspects in Male and Female Mice.

The kallikrein-kinin system is a versatile regulatory network implicated in various biological processes encompassing inflammation, nociception, blood pressure control, and central nervous system functions. Its physiological impact is mediated through G-protein-coupled transmembrane receptors, specifically the B1 and B2 receptors. Dopamine, a key catecholamine neurotransmitter widely distributed in the CNS, plays a crucial role in diverse physiological functions including motricity, reward, anxiety, fear, feeding, sleep, and arousal. Notably, the potential physical interaction between bradykinin and dopaminergic receptors has been previously documented. In this study, we aimed to explore whether B2R modulation in catecholaminergic neurons influences the dopaminergic pathway, impacting behavioral, metabolic, and motor aspects in both male and female mice. B2R ablation in tyrosine hydroxylase cells reduced the body weight and lean mass without affecting body adiposity, substrate oxidation, locomotor activity, glucose tolerance, or insulin sensitivity in mice. Moreover, a B2R deficiency in TH cells did not alter anxiety levels, exercise performance, or motor coordination in female and male mice. The concentrations of monoamines and their metabolites in the substantia nigra and cortex region were not affected in knockout mice. In essence, B2R deletion in TH cells selectively influenced the body weight and composition, leaving the behavioral and motor aspects largely unaffected.

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

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.

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

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".

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

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.

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

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.

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

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.

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

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.

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

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.

Dysfunctional hippocampal-prefrontal network underlies a multidimensional neuropsychiatric phenotype following early-life seizure.

Brain disturbances during development can have a lasting impact on neural function and behavior. Seizures during this critical period are linked to significant long-term consequences such as neurodevelopmental disorders, cognitive impairments, and psychiatric symptoms, resulting in a complex spectrum of multimorbidity. The hippocampus-prefrontal cortex (HPC-PFC) circuit emerges as a potential common link between such disorders. However, the mechanisms underlying these outcomes and how they relate to specific behavioral alterations are unclear. We hypothesized that specific dysfunctions of hippocampal-cortical communication due to early-life seizure would be associated with distinct behavioral alterations observed in adulthood. Here, we performed a multilevel study to investigate behavioral, electrophysiological, histopathological, and neurochemical long-term consequences of early-life Status epilepticus in male rats. We show that adult animals submitted to early-life seizure (ELS) present working memory impairments and sensorimotor disturbances, such as hyperlocomotion, poor sensorimotor gating, and sensitivity to psychostimulants despite not exhibiting neuronal loss. Surprisingly, cognitive deficits were linked to an aberrant increase in the HPC-PFC long-term potentiation (LTP) in a U-shaped manner, while sensorimotor alterations were associated with heightened neuroinflammation, as verified by glial fibrillary acidic protein (GFAP) expression, and altered dopamine neurotransmission. Furthermore, ELS rats displayed impaired HPC-PFC theta-gamma coordination and an abnormal brain state during active behavior resembling rapid eye movement (REM) sleep oscillatory dynamics. Our results point to impaired HPC-PFC functional connectivity as a possible pathophysiological mechanism by which ELS can cause cognitive deficits and psychiatric-like manifestations even without neuronal loss, bearing translational implications for understanding the spectrum of multidimensional developmental disorders linked to early-life seizures.

A controversial question: Can morphometry and clinical history be enough to diagnose hippocampal dysplasia?

The presence of dysmorphic neurons with strong cytoplasmatic accumulation of heavy non-phosphorylated neurofilament is crucial for the diagnostics of focal cortical dysplasia type II (FCDII). While ILAE's classification describes neocortical dysplasias, some groups have reported patients with mesial t abnormal neurons in the hippocampus of mesial temporal lobe epilepsy. Here we report a patient with such abnormal neurons in the hippocampus and compared it with previous reports of hippocampal dysplasia. Finally, we discuss the need for diagnostic criteria of hippocampal dysplasia.

Minimum effective sodium valproate dose in genetic generalized epilepsies.

INTRODUCTION: Sodium valproate (VPA) is the most effective antiseizure medication (ASM) in genetic generalized epilepsies (GGEs). However, the frequent adverse effects and the high risk inflicted on the exposed offspring make it imperative to search for the lowest daily VPA dose able to control seizures for most patients. In the current published series, the VPA value of <1000 mg was the most adopted. OBJECTIVE: This study aims to provide a cutoff VPA value below which a given daily dose can be considered a low dose in patients with GGEs. METHODS: This retrospective, observational cohort study included patients with clinical and electroencephalographic diagnoses of GGEs based on the ILAE criteria. Patients were followed up for at least two years using VPA in mono- or polytherapy. Clinical data, VPA dose, and associated ASMs were analyzed. Adverse effects were also evaluated. We related seizure control to VPA doses through uni- and multivariate statistical analyses. RESULTS: From 225 patients, 169 (75%) had good seizure control, with most (60%) receiving monotherapy. The cutoff daily VPA dose capable of distinguishing these patients from those without seizure control was up to 1000 mg (p = 0.006) in univariate analyses and up to 700 mg in multivariate analyses. For patients in polytherapy, the cutoff was up to 1750 mg and 1800 mg in uni- and multivariate analyses, respectively. CONCLUSIONS: The lowest daily VPA dose in monotherapy able to control seizures for most GGE patients was up to 700 mg, a value that can be used as a low dose criterion in studies assessing the therapeutic VPA ranges. Patients using higher VPA doses or in polytherapy present a lower probability of seizure control.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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 (p ≤ 0.038). Neuronal cell densities always decreased in CA4, CA3, CA2, and CA1 of TLE-HS1 but only in CA1 of TLE-HS2 (p ≤ 0.003). In addition, TLE-HS2 had a higher volume (p = 0.016) and higher neuronal density (p < 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.

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

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.