Morphological, cellular, and molecular basis of brain infection in COVID-19 patients.

Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. We show the spectrum of cerebral impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, ranging from long-term alterations in mildly infected individuals (orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms) to severe acute damage confirmed in brain tissue samples extracted from the orbitofrontal region (via endonasal transethmoidal access) from individuals who died of COVID-19. In an independent cohort of 26 individuals who died of COVID-19, we used histopathological signs of brain damage as a guide for possible SARS-CoV-2 brain infection and found that among the 5 individuals who exhibited those signs, all of them had genetic material of the virus in the brain. Brain tissue samples from these five patients also exhibited foci of SARS-CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a noncanonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that reduces neuronal viability. Our data support the model in which SARS-CoV-2 reaches the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients.

Patterns of subregional cerebellar atrophy across epilepsy syndromes: An ENIGMA-Epilepsy study.

OBJECTIVE: The intricate neuroanatomical structure of the cerebellum is of longstanding interest in epilepsy, but has been poorly characterized within the current corticocentric models of this disease. We quantified cross-sectional regional cerebellar lobule volumes using structural magnetic resonance imaging in 1602 adults with epilepsy and 1022 healthy controls across 22 sites from the global ENIGMA-Epilepsy working group. METHODS: A state-of-the-art deep learning-based approach was employed that parcellates the cerebellum into 28 neuroanatomical subregions. Linear mixed models compared total and regional cerebellar volume in (1) all epilepsies, (2) temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), (3) nonlesional temporal lobe epilepsy, (4) genetic generalized epilepsy, and (5) extratemporal focal epilepsy (ETLE). Relationships were examined for cerebellar volume versus age at seizure onset, duration of epilepsy, phenytoin treatment, and cerebral cortical thickness. RESULTS: Across all epilepsies, reduced total cerebellar volume was observed (d = .42). Maximum volume loss was observed in the corpus medullare (dmax = .49) and posterior lobe gray matter regions, including bilateral lobules VIIB (dmax = .47), crus I/II (dmax = .39), VIIIA (dmax = .45), and VIIIB (dmax = .40). Earlier age at seizure onset ( η ρ max 2 = .05) and longer epilepsy duration ( η ρ max 2 = .06) correlated with reduced volume in these regions. Findings were most pronounced in TLE-HS and ETLE, with distinct neuroanatomical profiles observed in the posterior lobe. Phenytoin treatment was associated with reduced posterior lobe volume. Cerebellum volume correlated with cerebral cortical thinning more strongly in the epilepsy cohort than in controls. SIGNIFICANCE: We provide robust evidence of deep cerebellar and posterior lobe subregional gray matter volume loss in patients with chronic epilepsy. Volume loss was maximal for posterior subregions implicated in nonmotor functions, relative to motor regions of both the anterior and posterior lobe. Associations between cerebral and cerebellar changes, and variability of neuroanatomical profiles across epilepsy syndromes argue for more precise incorporation of cerebellar subregional damage into neurobiological models of epilepsy.

Junctional instability in neuroepithelium and network hyperexcitability in a focal cortical dysplasia human model.

Focal cortical dysplasia is a highly epileptogenic cortical malformation with few treatment options. Here, we generated human cortical organoids from patients with focal cortical dysplasia type II. Using this human model, we mimicked some focal cortical dysplasia hallmarks, such as impaired cell proliferation, the presence of dysmorphic neurons and balloon cells, and neuronal network hyperexcitability. Furthermore, we observed alterations in the adherens junctions zonula occludens-1 and partitioning defective 3, reduced polarization of the actin cytoskeleton, and fewer synaptic puncta. Focal cortical dysplasia cortical organoids showed downregulation of the small GTPase RHOA, a finding that was confirmed in brain tissue resected from these patients. Functionally, both spontaneous and optogenetically-evoked electrical activity revealed hyperexcitability and enhanced network connectivity in focal cortical dysplasia organoids. Taken together, our findings suggest a ventricular zone instability in tissue cohesion of neuroepithelial cells, leading to a maturational arrest of progenitors or newborn neurons, which may predispose to cellular and functional immaturity and compromise the formation of neural networks in focal cortical dysplasia.

Topographic divergence of atypical cortical asymmetry and atrophy patterns in temporal lobe epilepsy.

Temporal lobe epilepsy, a common drug-resistant epilepsy in adults, is primarily a limbic network disorder associated with predominant unilateral hippocampal pathology. Structural MRI has provided an in vivo window into whole-brain grey matter structural alterations in temporal lobe epilepsy relative to controls, by either mapping (i) atypical inter-hemispheric asymmetry; or (ii) regional atrophy. However, similarities and differences of both atypical asymmetry and regional atrophy measures have not been systematically investigated. Here, we addressed this gap using the multisite ENIGMA-Epilepsy dataset comprising MRI brain morphological measures in 732 temporal lobe epilepsy patients and 1418 healthy controls. We compared spatial distributions of grey matter asymmetry and atrophy in temporal lobe epilepsy, contextualized their topographies relative to spatial gradients in cortical microstructure and functional connectivity calculated using 207 healthy controls obtained from Human Connectome Project and an independent dataset containing 23 temporal lobe epilepsy patients and 53 healthy controls and examined clinical associations using machine learning. We identified a marked divergence in the spatial distribution of atypical inter-hemispheric asymmetry and regional atrophy mapping. The former revealed a temporo-limbic disease signature while the latter showed diffuse and bilateral patterns. Our findings were robust across individual sites and patients. Cortical atrophy was significantly correlated with disease duration and age at seizure onset, while degrees of asymmetry did not show a significant relationship to these clinical variables. Our findings highlight that the mapping of atypical inter-hemispheric asymmetry and regional atrophy tap into two complementary aspects of temporal lobe epilepsy-related pathology, with the former revealing primary substrates in ipsilateral limbic circuits and the latter capturing bilateral disease effects. These findings refine our notion of the neuropathology of temporal lobe epilepsy and may inform future discovery and validation of complementary MRI biomarkers in temporal lobe epilepsy.

A systems-level analysis highlights microglial activation as a modifying factor in common epilepsies.

AIMS: The causes of distinct patterns of reduced cortical thickness in the common human epilepsies, detectable on neuroimaging and with important clinical consequences, are unknown. We investigated the underlying mechanisms of cortical thinning using a systems-level analysis. METHODS: Imaging-based cortical structural maps from a large-scale epilepsy neuroimaging study were overlaid with highly spatially resolved human brain gene expression data from the Allen Human Brain Atlas. Cell-type deconvolution, differential expression analysis and cell-type enrichment analyses were used to identify differences in cell-type distribution. These differences were followed up in post-mortem brain tissue from humans with epilepsy using Iba1 immunolabelling. Furthermore, to investigate a causal effect in cortical thinning, cell-type-specific depletion was used in a murine model of acquired epilepsy. RESULTS: We identified elevated fractions of microglia and endothelial cells in regions of reduced cortical thickness. Differentially expressed genes showed enrichment for microglial markers and, in particular, activated microglial states. Analysis of post-mortem brain tissue from humans with epilepsy confirmed excess activated microglia. In the murine model, transient depletion of activated microglia during the early phase of the disease development prevented cortical thinning and neuronal cell loss in the temporal cortex. Although the development of chronic seizures was unaffected, the epileptic mice with early depletion of activated microglia did not develop deficits in a non-spatial memory test seen in epileptic mice not depleted of microglia. CONCLUSIONS: These convergent data strongly implicate activated microglia in cortical thinning, representing a new dimension for concern and disease modification in the epilepsies, potentially distinct from seizure control.

Improving the prediction of epilepsy surgery outcomes using basic scalp EEG findings.

OBJECTIVE: This study aims to evaluate the role of scalp electroencephalography (EEG; ictal and interictal patterns) in predicting resective epilepsy surgery outcomes. We use the data to further develop a nomogram to predict seizure freedom. METHODS: We retrospectively reviewed the scalp EEG findings and clinical data of patients who underwent surgical resection at three epilepsy centers. Using both EEG and clinical variables categorized into 13 isolated candidate predictors and 6 interaction terms, we built a multivariable Cox proportional hazards model to predict seizure freedom 2 years after surgery. Harrell's step-down procedure was used to sequentially eliminate the least-informative variables from the model until the change in the concordance index (c-index) with variable removal was less than 0.01. We created a separate model using only clinical variables. Discrimination of the two models was compared to evaluate the role of scalp EEG in seizure-freedom prediction. RESULTS: Four hundred seventy patient records were analyzed. Following internal validation, the full Clinical + EEG model achieved an optimism-corrected c-index of 0.65, whereas the c-index of the model without EEG data was 0.59. The presence of focal to bilateral tonic-clonic seizures (FBTCS), high preoperative seizure frequency, absence of hippocampal sclerosis, and presence of nonlocalizable seizures predicted worse outcome. The presence of FBTCS had the largest impact for predicting outcome. The analysis of the models' interactions showed that in patients with unilateral interictal epileptiform discharges (IEDs), temporal lobe surgery cases had a better outcome. In cases with bilateral IEDs, abnormal magnetic resonance imaging (MRI) predicted worse outcomes, and in cases without IEDs, patients with extratemporal epilepsy and abnormal MRI had better outcomes. SIGNIFICANCE: This study highlights the value of scalp EEG, particularly the significance of IEDs, in predicting surgical outcome. The nomogram delivers an individualized prediction of postoperative outcome, and provides a unique assessment of the relationship between the outcome and preoperative findings.

Inflammatory and neurotrophic factor plasma levels are related to epilepsy independently of etiology.

OBJECTIVE: Inflammation plays an essential role in epilepsy. Studies indicate that cytokines and neurotrophic factors can act in neuroexcitability and epileptogenesis. We aimed to investigate the association between plasma inflammatory and neurotrophic markers, seizure frequency, and chronic epilepsy subtypes. METHODS: We studied 446 patients with epilepsy and 166 healthy controls. We classified patients according to etiology and seizure frequency. We measured plasma levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, IL-17, interferon-γ (IFNγ), tumor necrosis factor α (TNFα), soluble TNF receptor 1 (sTNFr1), sTNFr2, brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT3), NT4/5, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) by enzyme-linked immunosorbent assay or cytometric bead array. RESULTS: The plasma levels of BDNF, NT3, NGF, and sTNFr2 were higher, whereas IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ, TNFα, CNTF, and sTNFr1 were lower in patients than controls. IL1, GDNF, and NT4/5 were similar between groups. These markers did not correlate with age, sex, and epilepsy duration. The molecule sTNFr2 was the best marker to discriminate patients from controls (area under the curve = .857), also differing between patients with frequent and infrequent seizures. SIGNIFICANCE: This large cohort confirmed that patients with epilepsy have abnormal levels of plasma inflammatory and neurotrophic markers independent of the underlying etiology. Plasma level of sTNFr2 was related to seizure frequency and discriminated people with or without epilepsy with good accuracy, making it a potential biomarker for epilepsy and seizure burden.

White matter abnormalities across different epilepsy syndromes in adults: an ENIGMA-Epilepsy study.

The epilepsies are commonly accompanied by widespread abnormalities in cerebral white matter. ENIGMA-Epilepsy is a large quantitative brain imaging consortium, aggregating data to investigate patterns of neuroimaging abnormalities in common epilepsy syndromes, including temporal lobe epilepsy, extratemporal epilepsy, and genetic generalized epilepsy. Our goal was to rank the most robust white matter microstructural differences across and within syndromes in a multicentre sample of adult epilepsy patients. Diffusion-weighted MRI data were analysed from 1069 healthy controls and 1249 patients: temporal lobe epilepsy with hippocampal sclerosis (n = 599), temporal lobe epilepsy with normal MRI (n = 275), genetic generalized epilepsy (n = 182) and non-lesional extratemporal epilepsy (n = 193). A harmonized protocol using tract-based spatial statistics was used to derive skeletonized maps of fractional anisotropy and mean diffusivity for each participant, and fibre tracts were segmented using a diffusion MRI atlas. Data were harmonized to correct for scanner-specific variations in diffusion measures using a batch-effect correction tool (ComBat). Analyses of covariance, adjusting for age and sex, examined differences between each epilepsy syndrome and controls for each white matter tract (Bonferroni corrected at P < 0.001). Across 'all epilepsies' lower fractional anisotropy was observed in most fibre tracts with small to medium effect sizes, especially in the corpus callosum, cingulum and external capsule. There were also less robust increases in mean diffusivity. Syndrome-specific fractional anisotropy and mean diffusivity differences were most pronounced in patients with hippocampal sclerosis in the ipsilateral parahippocampal cingulum and external capsule, with smaller effects across most other tracts. Individuals with temporal lobe epilepsy and normal MRI showed a similar pattern of greater ipsilateral than contralateral abnormalities, but less marked than those in patients with hippocampal sclerosis. Patients with generalized and extratemporal epilepsies had pronounced reductions in fractional anisotropy in the corpus callosum, corona radiata and external capsule, and increased mean diffusivity of the anterior corona radiata. Earlier age of seizure onset and longer disease duration were associated with a greater extent of diffusion abnormalities in patients with hippocampal sclerosis. We demonstrate microstructural abnormalities across major association, commissural, and projection fibres in a large multicentre study of epilepsy. Overall, patients with epilepsy showed white matter abnormalities in the corpus callosum, cingulum and external capsule, with differing severity across epilepsy syndromes. These data further define the spectrum of white matter abnormalities in common epilepsy syndromes, yielding more detailed insights into pathological substrates that may explain cognitive and psychiatric co-morbidities and be used to guide biomarker studies of treatment outcomes and/or genetic research.

Interactions between in vivo neuronal-glial markers, side of hippocampal sclerosis, and pharmacoresponse in temporal lobe epilepsy.

OBJECTIVE: To evaluate the interactions of metabolic neuronal-glial changes with the presence and hemispheric-side of hippocampal sclerosis (HS) and its potential role in predicting pharmacoresistance in temporal lobe epilepsy (TLE). METHODS: We included structural magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1 H-MRS) metabolic data for 91 patients with unilateral TLE and 50 healthy controls. We measured the values of total N-acetyl aspartate/total creatine (tNAA/tCr), glutamate/tCr (Glu/tCr), and myo-inositol/tCr (mIns/tCr). To assess the influence of the pharmacoresponse and hemispheric-side of HS on metabolic data, the relationship between clinical and MRI data, and the predictive value of NAA/Cr, we used analysis of variance/covariance and built a logistic regression model. We used bootstrap simulations to evaluate reproducibility. RESULTS: Bilateral tNAA/tCr reduction was associated with pharmacoresistance and with left HS, a decrease of Glu/tCr ipsilateral to the seizure focus was associated with pharmacoresistance, and ipsilateral mIns/tCr increase was related to pharmacoresistance and the presence of left HS. The logistic regression model containing clinical and 1 H-MRS data discriminated pharmacoresistance (area under the curve [AUC] = 0.78). However, the reduction of tNAA/tCr was the main predictor, with the odds 2.48 greater for pharmacoresistance. SIGNIFICANCE: Our study revealed a spectrum of neuronal-glial changes in TLE, which was associated with pharmacoresistance, being more severe in left-sided HS and less severe in MRI-negative TLE. These noninvasive, in vivo biomarkers provide valuable additional information about the interhemispheric differences in metabolic dysfunction, seizure burden, and HS, and may help to predict pharmacoresistance.

Revisiting the clinical impact of variants in EFHC1 in patients with different phenotypes of genetic generalized epilepsy.

The most common form of genetic generalized epilepsy (GGE) is juvenile myoclonic epilepsy (JME), which accounts for 5 to 10% of all epilepsy cases. The gene EFHC1 has been implicated as a putative cause of JME. However, it remains debatable whether testing for EFHC1 mutations should be included in the diagnostic epilepsy gene panels. To investigate the clinical utility of EFHC1 testing, we studied 125 individuals: 100 with JME and 25 with other GGEs. We amplified and sequenced all EFHC1 coding exons. Then, we predicted the pathogenicity or benign impact of the variants using the analyses proposed by the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP). Mutation screening revealed 11 missense variants in 44 probands with JME (44%) and one of the seven individuals with generalized tonic-clonic seizures on awakening (14%). Six of the 11 variants (54%) were classified as 'benign,' and the remaining variants were considered variants of uncertain significance (VUS). There is currently a limitation to test for genes that predispose an individual to complex, nonmonogenic phenotypes. Thus, we show suggestive evidence that EFHC1 testing lacks a scientific foundation based on the disputed nature of the gene-disease relationship and should be currently limited to research purposes.

ATP Synthase Subunit Beta Immunostaining is Reduced in the Sclerotic Hippocampus of Epilepsy Patients.

Epilepsy is a common disease presenting with recurrent seizures. Hippocampal sclerosis (HS) is the commonest histopathological alteration in patients with temporal lobe epilepsy (TLE) undergoing surgery. HS physiopathogenesis is debatable. We have recently studied, by using mass spectrometry-based proteomics, an experimental model of TLE induced by electrical stimulation. Specifically, protein expressions of both the beta subunit of mitochondrial ATP synthase (ATP5B) and of membrane ATPases were found to be reduced. Here, we investigated tissue distribution of ATP5B and sodium/potassium-transporting ATPase subunit alpha-3 (NKAα3), a protein associated with neuromuscular excitability disorders, in human hippocampi resected "en bloc" for HS treatment (n = 15). We used immunohistochemistry and the stained area was digitally evaluated (increase in binary contrast of microscopic fields) in the hippocampal sectors (CA1-CA4) and dentate gyrus. All HS samples were classified as Type 1, according to the International League Against Epilepsy (ILAE) 2013 Classification (predominant cell loss in CA1 and CA4). ATP5B was significantly decreased in all sectors and dentate gyrus of HS patients compared with individuals submitted to necropsy and without history of neurological alterations (n = 10). NKAα3 expression showed no difference. Moreover, we identified a negative correlation between frequency of pre-operative seizures and number of neurons in CA1. In conclusion, our data showed similarity between changes in protein expression in a model of TLE and individuals with HS. ATP5B reduction would be at least in part due to neuronal loss. Future investigations on ATP5B activity could provide insights into the process of such cell loss.

Structural brain abnormalities in the common epilepsies assessed in a worldwide ENIGMA study.

Progressive functional decline in the epilepsies is largely unexplained. We formed the ENIGMA-Epilepsy consortium to understand factors that influence brain measures in epilepsy, pooling data from 24 research centres in 14 countries across Europe, North and South America, Asia, and Australia. Structural brain measures were extracted from MRI brain scans across 2149 individuals with epilepsy, divided into four epilepsy subgroups including idiopathic generalized epilepsies (n =367), mesial temporal lobe epilepsies with hippocampal sclerosis (MTLE; left, n = 415; right, n = 339), and all other epilepsies in aggregate (n = 1026), and compared to 1727 matched healthy controls. We ranked brain structures in order of greatest differences between patients and controls, by meta-analysing effect sizes across 16 subcortical and 68 cortical brain regions. We also tested effects of duration of disease, age at onset, and age-by-diagnosis interactions on structural measures. We observed widespread patterns of altered subcortical volume and reduced cortical grey matter thickness. Compared to controls, all epilepsy groups showed lower volume in the right thalamus (Cohen's d = -0.24 to -0.73; P < 1.49 × 10-4), and lower thickness in the precentral gyri bilaterally (d = -0.34 to -0.52; P < 4.31 × 10-6). Both MTLE subgroups showed profound volume reduction in the ipsilateral hippocampus (d = -1.73 to -1.91, P < 1.4 × 10-19), and lower thickness in extrahippocampal cortical regions, including the precentral and paracentral gyri, compared to controls (d = -0.36 to -0.52; P < 1.49 × 10-4). Thickness differences of the ipsilateral temporopolar, parahippocampal, entorhinal, and fusiform gyri, contralateral pars triangularis, and bilateral precuneus, superior frontal and caudal middle frontal gyri were observed in left, but not right, MTLE (d = -0.29 to -0.54; P < 1.49 × 10-4). Contrastingly, thickness differences of the ipsilateral pars opercularis, and contralateral transverse temporal gyrus, were observed in right, but not left, MTLE (d = -0.27 to -0.51; P < 1.49 × 10-4). Lower subcortical volume and cortical thickness associated with a longer duration of epilepsy in the all-epilepsies, all-other-epilepsies, and right MTLE groups (beta, b < -0.0018; P < 1.49 × 10-4). In the largest neuroimaging study of epilepsy to date, we provide information on the common epilepsies that could not be realistically acquired in any other way. Our study provides a robust ranking of brain measures that can be further targeted for study in genetic and neuropathological studies. This worldwide initiative identifies patterns of shared grey matter reduction across epilepsy syndromes, and distinctive abnormalities between epilepsy syndromes, which inform our understanding of epilepsy as a network disorder, and indicate that certain epilepsy syndromes involve more widespread structural compromise than previously assumed.

Is inpatient ictal video-electroencephalographic monitoring mandatory in mesial temporal lobe epilepsy with unilateral hippocampal sclerosis? A prospective study.

OBJECTIVE: To compare surgical outcome in mesial temporal lobe epilepsy (MTLE) patients with unilateral hippocampal sclerosis (MTLE-HS) who had or did not have preoperative video-electroencephalographic monitoring (VEEG). METHODS: A prospective study was undertaken with 166 consecutive pharmacoresistant unilateral MTLE-HS patients. All patients were investigated with detailed seizure semiology, serial routine outpatient EEG, magnetic resonance imaging, neuropsychological evaluation, and if necessary, other examinations. Postoperative follow-up ranged between 2 and 16 years. Patients were divided into: (1) patients operated on based on routine outpatient EEG information, with >80% of EEGs with unilateral interictal epileptiform discharges (IEDs) ipsilateral to HS or ictal events (n = 71); and (2) patients submitted to preoperative VEEG (n = 95). To avoid the bias generated by ictal recordings, we performed a subanalysis of: (1) patients without preoperatively ictal recordings (n = 80) and (2) patients with ictal recordings in VEEG or routine outpatient EEG (n = 86). RESULTS: Groups were similar regarding gender, age at surgery, seizure onset, preoperative seizure frequency, and duration of follow-up. Overall, 136/166 (81.92%) were classified as Engel I seizure outcome, with no difference between groups; 76.84% and 88.73% of patients with and without VEEG, respectively, had Engel I postoperative seizure outcome (P = .77). The time lag until surgery was shorter in the group without VEEG (80 vs 38 months; P = .01). Considering ictal recordings, 76.74% of patients with seizures recorded and 87.50% without ictal recordings had Engel I outcome (P = .11). SIGNIFICANCE: We performed the first prospective study in a tertiary epilepsy center comparing surgical outcomes in unilateral MTLE-HS patients investigated preoperatively with and without VEEG. Based on the surgical outcome, VEEG is not imperative in patients with unilateral MTLE-HS who have compatible semiology and clearly ipsilateralized IEDs evaluated by a multidisciplinary and experienced epilepsy group.

Progression of gray matter atrophy in seizure-free patients with temporal lobe epilepsy.

OBJECTIVES: To investigate the presence and progression of gray matter (GM) reduction in seizure-free patients with temporal lobe epilepsy (TLE). METHODS: We enrolled 39 consecutive TLE patients, seizure-free for at least 2 years--20 with magnetic resonance imaging (MRI) signs of hippocampal sclerosis (TLE-HS), 19 with normal MRI (TLE-NL), and 74 healthy controls. For longitudinal analysis, we included individuals who had a second MRI with minimum interval of 18 months: 21 patients (10 TLE-HS, 11 TLE-NL) and 11 controls. Three-dimensional (3D) T1 -weighted images acquired in 3 Tesla MRI were analyzed with voxel-based morphometry (VBM). The images of patients with right-sided interictal epileptogenic zone (EZ) were right-left flipped, as well as a comparable proportion of controls. Cross-sectional analysis: The patients' images from each group were compared to controls to investigate differences in GM volumes. Longitudinal analysis: The first and second images were compared in each group to look for decreased GM volume. RESULTS: Cross-sectional analysis: Patients with TLE-HS had diffuse GM atrophy, including hippocampus and parahippocampal gyrus, insula, frontal, and occipital lobes ipsilateral to EZ, bilateral thalamus and contralateral orbitofrontal gyrus, and caudate. In contrast, TLE-NL group did not present significant differences compared to controls. Longitudinal analysis: TLE-HS presented progressive GM reduction in ipsilateral insula and occipital lobe, contralateral motor area, and bilateral temporal and frontal lobes. TLE-NL had GM progression in ipsilateral hypothalamus and parietal lobe, contralateral cerebellum, and bilateral temporal lobe. Controls did not show changes in GM volume between MRIs. SIGNIFICANCE: Diffuse extrahippocampal GM atrophy is present in seizure-free patients with TLE-HS. In addition, there is progressive GM atrophy in patients with and without HS. These results demonstrate that not only ongoing seizures are involved in the progression of GM atrophy. An underlying pathologic mechanism could be responsible for progressive brain volume loss in TLE patients even in seizure-free periods.

A WORLDWIDE ENIGMA STUDY ON EPILEPSY-RELATED GRAY AND WHITE MATTER COMPROMISE ACROSS THE ADULT LIFESPAN.

Objectives: Temporal lobe epilepsy (TLE) is commonly associated with mesiotemporal pathology and widespread alterations of grey and white matter structures. Evidence supports a progressive condition although the temporal evolution of TLE is poorly defined. This ENIGMA-Epilepsy study utilized multimodal magnetic resonance imaging (MRI) data to investigate structural alterations in TLE patients across the adult lifespan. We charted both grey and white matter changes and explored the covariance of age-related alterations in both compartments. Methods: We studied 769 TLE patients and 885 healthy controls across an age range of 17-73 years, from multiple international sites. To assess potentially non-linear lifespan changes in TLE, we harmonized data and combined median split assessments with cross-sectional sliding window analyses of grey and white matter age-related changes. Covariance analyses examined the coupling of grey and white matter lifespan curves. Results: In TLE, age was associated with a robust grey matter thickness/volume decline across a broad cortico-subcortical territory, extending beyond the mesiotemporal disease epicentre. White matter changes were also widespread across multiple tracts with peak effects in temporo-limbic fibers. While changes spanned the adult time window, changes accelerated in cortical thickness, subcortical volume, and fractional anisotropy (all decreased), and mean diffusivity (increased) after age 55 years. Covariance analyses revealed strong limbic associations between white matter tracts and subcortical structures with cortical regions. Conclusions: This study highlights the profound impact of TLE on lifespan changes in grey and white matter structures, with an acceleration of aging-related processes in later decades of life. Our findings motivate future longitudinal studies across the lifespan and emphasize the importance of prompt diagnosis as well as intervention in patients.

Identifying cellular markers of focal cortical dysplasia type II with cell-type deconvolution and single-cell signatures.

Focal cortical dysplasia (FCD) is a brain malformation that causes medically refractory epilepsy. FCD is classified into three categories based on structural and cellular abnormalities, with FCD type II being the most common and characterized by disrupted organization of the cortex and abnormal neuronal development. In this study, we employed cell-type deconvolution and single-cell signatures to analyze bulk RNA-seq from multiple transcriptomic studies, aiming to characterize the cellular composition of brain lesions in patients with FCD IIa and IIb subtypes. Our deconvolution analyses revealed specific cellular changes in FCD IIb, including neuronal loss and an increase in reactive astrocytes (astrogliosis) when compared to FCD IIa. Astrogliosis in FCD IIb was further supported by a gene signature analysis and histologically confirmed by glial fibrillary acidic protein (GFAP) immunostaining. Overall, our findings demonstrate that FCD II subtypes exhibit differential neuronal and glial compositions, with astrogliosis emerging as a hallmark of FCD IIb. These observations, validated in independent patient cohorts and confirmed using immunohistochemistry, offer novel insights into the involvement of glial cells in FCD type II pathophysiology and may contribute to the development of targeted therapies for this condition.

Patterns of subregional cerebellar atrophy across epilepsy syndromes: An ENIGMA-Epilepsy study.

Objective: The intricate neuroanatomical structure of the cerebellum is of longstanding interest in epilepsy, but has been poorly characterized within the current cortico-centric models of this disease. We quantified cross-sectional regional cerebellar lobule volumes using structural MRI in 1,602 adults with epilepsy and 1,022 healthy controls across twenty-two sites from the global ENIGMA-Epilepsy working group. Methods: A state-of-the-art deep learning-based approach was employed that parcellates the cerebellum into 28 neuroanatomical subregions. Linear mixed models compared total and regional cerebellar volume in i) all epilepsies; ii) temporal lobe epilepsy with hippocampal sclerosis (TLE-HS); iii) non-lesional temporal lobe epilepsy (TLE-NL); iv) genetic generalised epilepsy; and (v) extra-temporal focal epilepsy (ETLE). Relationships were examined for cerebellar volume versus age at seizure onset, duration of epilepsy, phenytoin treatment, and cerebral cortical thickness. Results: Across all epilepsies, reduced total cerebellar volume was observed (d=0.42). Maximum volume loss was observed in the corpus medullare (dmax=0.49) and posterior lobe grey matter regions, including bilateral lobules VIIB (dmax= 0.47), Crus I/II (dmax= 0.39), VIIIA (dmax=0.45) and VIIIB (dmax=0.40). Earlier age at seizure onset (ηρ2max=0.05) and longer epilepsy duration (ηρ2max=0.06) correlated with reduced volume in these regions. Findings were most pronounced in TLE-HS and ETLE with distinct neuroanatomical profiles observed in the posterior lobe. Phenytoin treatment was associated with reduced posterior lobe volume. Cerebellum volume correlated with cerebral cortical thinning more strongly in the epilepsy cohort than in controls. Significance: We provide robust evidence of deep cerebellar and posterior lobe subregional grey matter volume loss in patients with chronic epilepsy. Volume loss was maximal for posterior subregions implicated in non-motor functions, relative to motor regions of both the anterior and posterior lobe. Associations between cerebral and cerebellar changes, and variability of neuroanatomical profiles across epilepsy syndromes argue for more precise incorporation of cerebellum subregions into neurobiological models of epilepsy.

Benchmarking the proteomic profile of animal models of mesial temporal epilepsy.

OBJECTIVES: We compared the proteomic signatures of the hippocampal lesion induced in three different animal models of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE+HS): the systemic pilocarpine model (PILO), the intracerebroventricular kainic acid model (KA), and the perforant pathway stimulation model (PPS). METHODS: We used shotgun proteomics to analyze the proteomes and find enriched biological pathways of the dorsal and ventral dentate gyrus (DG) isolated from the hippocampi of the three animal models. We also compared the proteomes obtained in the animal models to that from the DG of patients with pharmacoresistant MTLE+HS. RESULTS: We found that each animal model presents specific profiles of proteomic changes. The PILO model showed responses predominantly related to neuronal excitatory imbalance. The KA model revealed alterations mainly in synaptic activity. The PPS model displayed abnormalities in metabolism and oxidative stress. We also identified common biological pathways enriched in all three models, such as inflammation and immune response, which were also observed in tissue from patients. However, none of the models could recapitulate the profile of molecular changes observed in tissue from patients. SIGNIFICANCE: Our results indicate that each model has its own set of biological responses leading to epilepsy. Thus, it seems that only using a combination of the three models may one replicate more closely the mechanisms underlying MTLE+HS as seen in patients.

EEG Signal Connectivity for Characterizing Interictal Activity in Patients With Mesial Temporal Lobe Epilepsy.

Over the last decade, several methods for analysis of epileptiform signals in electroencephalography (EEG) have been proposed. These methods mainly use EEG signal features in either the time or the frequency domain to separate regular, interictal, and ictal brain activity. The aim of this work was to evaluate the feasibility of using functional connectivity (FC) based feature extraction methods for the analysis of epileptiform discharges in EEG signals. These signals were obtained from EEG-fMRI sessions of 10 patients with mesial temporal lobe epilepsy (MTLE) with unilateral hippocampal atrophy. The connectivity functions investigated were motif synchronization, imaginary coherence, and magnitude squared coherence in the alpha, beta, and gamma bands of the EEG. EEG signals were sectioned into 1-s epochs and classified according to (using neurologist markers): activity far from interictal epileptiform discharges (IED), activity immediately before an IED and, finally, mid-IED activity. Connectivity matrices for each epoch for each FC function were built, and graph theory was used to obtain the following metrics: strength, cluster coefficient, betweenness centrality, eigenvector centrality (both local and global), and global efficiency. The statistical distributions of these metrics were compared among the three classes, using ANOVA, for each FC function. We found significant differences in all global (p < 0.001) and local (p < 0.00002) graph metrics of the far class compared with before and mid for motif synchronization on the beta band; local betweenness centrality also pointed to a degree of lateralization on the frontotemporal structures. This analysis demonstrates the potential of FC measures, computed using motif synchronization, for the characterization of epileptiform activity of MTLE patients. This methodology may be helpful in the analysis of EEG-fMRI data applied to epileptic foci localization. Nonetheless, the methods must be tested with a larger sample and with other epileptic phenotypes.