γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

Spatial omics reveals molecular changes in focal cortical dysplasia type II.

Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.

Identification of gene regulatory networks affected across drug-resistant epilepsies.

Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development.

Astroglial calcium signaling and homeostasis in tuberous sclerosis complex.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca2+) channels and intracellular Ca2+ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca2+ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca2+ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca2+ concentration and Ca2+ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca2+ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca2+ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development.

Downregulation of Ubiquitin-Specific Protease 15 (USP15) Does Not Provide Therapeutic Benefit in Experimental Mesial Temporal Lobe Epilepsy.

Structural epilepsies display complex immune activation signatures. However, it is unclear which neuroinflammatory pathways drive pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor ß, interferon α/ß, and nuclear factor erythroid 2-related factor 2 pathways. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack Usp15 gene expression underwent intrahippocampal kainate injections to induce mTLE. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wild types. Cell death, reactive gliosis, and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, these brain inflammation signatures did not differ between genotypes. Likewise, induced deletion of Usp15 in chronic epilepsy did not affect seizure generation, cell death, gliosis, or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in the form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in an mTLE mouse model and do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.

Shaping the future of European epilepsy research: Final meeting report from EPICLUSTER.

Collaboration is essential to the conduct of basic, applied and clinical research and its translation into the technologies and treatments urgently needed to improve the lives of people living with brain diseases and the health professionals who care for them. EPICLUSTER was formed in 2019 by the European Brain Research Area (EBRA) to support the coordination of epilepsy research in Europe. A key objective was to provide a platform to discuss shared research priorities by bringing together scientists and clinicians with multiple stakeholders including patient organisations and industry and the networks and infrastructures that provide healthcare and support research. Additional objectives were to facilitate access and sharing of data and biosamples, working together to ensure epilepsy is a priority for research funding, and embedding a culture of public and patient involvement (PPI) among epilepsy researchers. In this meeting report, we summarise the shared research priorities discussed by the leadership of EPICLUSTER at the recent final meeting. We also briefly review the discussion on patient and industry priorities, guidance on starting PPI for epilepsy researchers, and the sustainability of funding and infrastructures needed to ensure a comprehensive stakeholder-embedded community for epilepsy research.

Characterising seizure development, behavioural comorbidities and neuroinflammation in a self-sustained electrical status epilepticus model of mesial temporal lobe epilepsy in C57BL/6J mice.

OBJECTIVE: Status epilepticus (SE) models in rodents are commonly used to research mesial temporal lobe epilepsy (mTLE) in translational epilepsy research. However, due to differences in susceptibility of mice strains to chemoconvulsants, developing this model in mice is challenging. Mice offer experimental advantages; in particular, the ability to use transgenic strains could provide novel insights about neurobiological mechanisms or ease of genetic modification to test potential therapeutic targets. This study aimed to characterise the neuroinflammation, epileptic seizures and behavioural comorbidities after self-sustained Electrical Status Epilepticus (SSSE) in C57BL/6J mice. METHODS: SSSE was induced in C57BL/6J mice via prolonged electrical stimulation through a bipolar electrode implanted in the ventral hippocampus. Video electroencephalography (vEEG) monitoring was then performed between 1st month (acute timepoint) and 4th month (chronic timepoint). Brain tissues were collected at two timepoints for gene expression and immunohistochemical analysis: 7-days and 16-weeks post-SE. Additionally, at the chronic timepoint, animals underwent a series of neurobehavioural tests. RESULTS: Sixty percent of animals that underwent SSSE developed spontaneous seizures within the first month, and an additional 25% developed seizures at the chronic timepoint. The number of seizures per week during the chronic period ranged from 0.2 to 15.7. Mortality rate was ~9% during or after SSSE. SSSE animals displayed significant spatial memory impairment and depression-like behaviour compared to sham animals. mRNA expression of inflammatory cytokines was upregulated at 7-days following SE, but equal to sham levels at 16-weeks. SIGNIFICANCE: This study provides evidence that SSSE in C57BL/6J mice induces epileptic seizures consistent with those seen in patients with mTLE, along with cognitive and behavioural comorbidities. This model therefore has the potential to be used experimentally to uncover mechanisms to target against epileptogenesis, or to test novel treatment approaches.

Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.

TSPO PET upregulation predicts epileptic phenotype at disease onset independently from chronic TSPO expression in a rat model of temporal lobe epilepsy.

Neuroinflammation is a key component of epileptogenesis, the process leading to acquired epilepsy. In recent years, with the development of non-invasive in vivo positron emission tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of neuroinflammation, it has become possible to perform longitudinal studies to characterize neuroinflammation at different disease stages in animal models of epileptogenesis. This study aimed to utilize the prognostic capability of TSPO PET imaging at disease onset (2 weeks post-SE) to categorize epileptic rats with distinct seizure burden based on TSPO levels at disease onset and investigate their association to TSPO expression at the chronic epilepsy stage. Controls (n = 14) and kainic acid-induced status epilepticus (KASE) rats (n = 41) were scanned non-invasively with [18F]PBR111 PET imaging measuring TSPO expression. Animals were monitored using video-electroencephalography (vEEG) up to chronic disease (12 weeks post-SE), at which TSPO levels ([3H]PK11195) as well as other post-mortem abnormalities (namely synaptic density ([3H]UCB-J), neuronal loss (NeuN), and neurodegeneration (FjC)) were investigated. By applying multivariate analysis, TSPO PET imaging at disease onset identified three KASE groups with significantly different spontaneous recurrent seizures (SRS) burden (defined as rare SRS, sporadic SRS, and frequent SRS) (p = 0.003). Interestingly, TSPO levels were significantly different when comparing the three KASE groups (p < 0.0001), with the frequent SRS group characterized only by a limited focal TSPO increase at disease onset. On the contrary, TSPO measured during chronic epilepsy was found to be the highest in the frequent SRS group and correlated with seizure burden (r = 0.826, p < 0.0001). Importantly, early and chronic TSPO levels did not correlate (r = -0.05). Finally, significant pathological changes in neuronal loss, synaptic density, and neurodegeneration were found not only when compared to control animals (p < 0.01), but also between the three KASE rat categories in the hippocampus (p < 0.05). Early and chronic TSPO upregulation following epileptogenic insult appear to be driven by two superimposed dynamic processes. The former is associated with epileptogenesis as measured at disease onset, while the latter is related to seizure frequency as quantified during chronic epilepsy.

What value can TSPO PET bring for epilepsy treatment?

Epilepsy is one of the most common neurological disorders and affects both the young and adult populations. The question we asked for this review was how positron emission tomography (PET) imaging with translocator protein (TSPO) radioligands can help inform the epilepsy clinic and the development of future treatments targeting neuroinflammatory processes.Even though the first TSPO PET scans in epilepsy patients were performed over 20 years ago, this imaging modality has not seen wide adoption in the clinic. There is vast scientific evidence from preclinical studies in rodent models of temporal lobe epilepsy which have shown increased levels of TSPO corresponding to neuroinflammatory processes in the brain. These increases peaked sub-acutely (1-2 weeks) after the epileptogenic insult (e.g. status epilepticus) and remained chronically increased, albeit at lower levels. In addition, these studies have shown a correlation between TSPO levels and seizure outcome, pharmacoresistance and behavioural morbidities. Histological assessment points to a complex interplay between different cellular components such as microglial activation, astrogliosis and cell death changing dynamically over time.In epilepsy patients, a highly sensitive biomarker of neuroinflammation would provide value for the optimization of surgical assessment (particularly for extratemporal lobe epilepsy) and support the clinical development path of anti-inflammatory treatments. Clinical studies have shown a systematic increase in asymmetry indices of TSPO PET binding. However, region-based analysis typically does not yield statistical differences and changes are often not restricted to the epileptogenic zone, limiting the ability of this imaging modality to localise pathology for surgery. In this manuscript, we discuss the biological underpinnings of these findings and review for which applications in epilepsy TSPO PET could bring added value.

Balloon cells promote immune system activation in focal cortical dysplasia type 2b.

AIMS: Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro-epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. METHODS: The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well-characterised FCD 2 cohort. RESULTS: Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T-lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. CONCLUSION: We conclude that, next to mutation-driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b.

WONOEP appraisal: Network concept from an imaging perspective.

Neuroimaging techniques applied to a variety of organisms-from zebrafish, to rodents to humans-can offer valuable insights into neuronal network properties and their dysfunction in epilepsy. A wide range of imaging methods used to monitor neuronal circuits and networks during evoked seizures in animal models and advances in functional magnetic resonance imaging (fMRI) applied to patients with epilepsy were discussed during the XIV Workshop on Neurobiology of Epilepsy (XIV WONOEP) organized in 2017 by the Neurobiology Commission of the International League Against Epilepsy (ILAE). We review the growing number of technological approaches developed, as well as the current state of knowledge gained from studies applying these advanced imaging approaches to epilepsy research.

The effect of pharmacological inhibition of Serine Proteases on neuronal networks in vitro.

Neurons are embedded in an extracellular matrix (ECM), which functions both as a scaffold and as a regulator of neuronal function. The ECM is in turn dynamically altered through the action of serine proteases, which break down its constituents. This pathway has been implicated in the regulation of synaptic plasticity and of neuronal intrinsic excitability. In this study, we determined the short-term effects of interfering with proteolytic processes in the ECM, with a newly developed serine protease inhibitor. We monitored the spontaneous electrophysiological activity of in vitro primary rat cortical cultures, using microelectrode arrays. While pharmacological inhibition at a low dosage had no significant effect, at elevated concentrations it altered significantly network synchronization and functional connectivity but left unaltered single-cell electrical properties. These results suggest that serine protease inhibition affects synaptic properties, likely through its actions on the ECM.

Spatiotemporal expression and inhibition of prolyl oligopeptidase contradict its involvement in key pathologic mechanisms of kainic acid-induced temporal lobe epilepsy in rats.

OBJECTIVE: Prolyl oligopeptidase (PREP) has been implicated in neuroinflammatory processes and neuroplasticity and has been suggested as a target for the treatment of neurodegenerative disease. The aim of this investigation was to explore the involvement of PREP in the neuropathologic mechanisms relevant to temporal lobe epilepsy (TLE) using a PREP inhibitor in a well-established rat model. METHODS: PREP activity and expression was studied in Sprague-Dawley rats 2 and 12 weeks following kainic acid-induced status epilepticus (KASE). Continuous video-electroencephalography monitoring was performed for 2 weeks in the 12-week cohort to identify a relationship of PREP expression/activity with epileptic seizures. In addition, the animals included in the 2-week time point were treated with a specific inhibitor of PREP, KYP-2047, or saline continuously, starting immediately after SE. PREP activity and its expression were analyzed in rat brain by using enzyme kinetics and western blot. In addition, markers for microglial activation, astrogliosis, cell loss, and cell proliferation were evaluated. RESULTS: Enzymatic activity of PREP was unchanged following induction of SE after 2 and 12 weeks in rats. PREP activity in epileptic rats did not relate to the number of seizures/day at the 12-week time point. Moreover, continuous inhibition of PREP for 2 weeks after KASE did not alter the SE-mediated neuroinflammatory response, cell loss, or cell proliferation in the hippocampal subgranule zone measured at the 2-week time point. SIGNIFICANCE: PREP inhibition does not affect key pathologic mechanisms, including activation of glial cells, cell loss, and neural progenitor cell proliferation, in this KASE model of TLE. The results do not support a direct role of PREP in seizure burden during the chronic epilepsy period in this model.

Hypersynchronicity in the default mode-like network in a neurodevelopmental animal model with relevance for schizophrenia.

BACKGROUND: Immune activation during pregnancy is an important risk factor for schizophrenia. Brain dysconnectivity and NMDA receptor (NMDAR) hypofunction have been postulated to be central to schizophrenia pathophysiology. The aim of this study was to investigate resting-state functional connectivity (resting-state functional MRI-rsfMRI), microstructure (diffusion tension imaging-DTI) and response to NMDAR antagonist (pharmacological fMRI-phMRI) using multimodal MRI in offspring of pregnant dams exposed to immune challenge (maternal immune activation-MIA model), and determine whether these neuroimaging readouts correlate with schizophrenia-related behaviour. METHODS: Pregnant rats were injected with Poly I:C or saline on gestational day 15. The maternal weight response was assessed. Since previous research has shown behavioural deficits can differ between MIA offspring dependent on the maternal response to immune stimulus, offspring were divided into three groups: controls (saline, n = 11), offspring of dams that gained weight (Poly I:C WG, n = 12) and offspring of dams that lost weight post-MIA (Poly I:C WL, n = 16). Male adult offspring were subjected to rsfMRI, DTI, phMRI with NMDAR antagonist, behavioural testing and histological assessment. RESULTS: Poly I:C WL offspring exhibited increased functional connectivity in default mode-like network (DMN). Poly I:C WG offspring showed the most pronounced attenuation in NMDAR antagonist response versus controls. DTI revealed no differences in Poly I:C offspring versus controls. Poly I:C offspring exhibited anxiety. CONCLUSIONS: MIA offspring displayed a differential pathophysiology depending on the maternal response to immune challenge. While Poly I:C WL offspring displayed hypersynchronicity in the DMN, altered NMDAR antagonist response was most pronounced in Poly I:C WG offspring.

In vivo measurement of brain network connectivity reflects progression and intrinsic disease severity in a model of temporal lobe epilepsy.

Different types of brain injury, such as status epilepticus (SE), trauma, or stroke may initiate the process of epileptogenesis and lead to the development of temporal lobe epilepsy. Epileptogenesis is characterized by an initial latent period during which impaired network communication and synaptic circuit alterations are occurring. Ultimately, these modifications result in the development of spontaneous recurrent seizures (SRS). Current knowledge on the functional connectivity network changes during epileptogenesis and how network alterations relate to seizure is very limited. To investigate these underlying network connectivity modifications, we imaged epileptic and control rats by means of resting-state functional MRI (rsfMRI) during epileptogenesis. A cohort of animals was video-electroencephalography (video-EEG) monitored continuously over 12 weeks to determine disease severity during the course of disease, with the first SRS appearing around 2 weeks post-SE for most of the animals. Epileptic animals displayed a significant wide-spread hyposynchrony at 2 weeks post-SE, followed by a significant increase in network synchronicity from 2 to 4 weeks post-SE. Interestingly, subjects with a delayed epilepsy onset demonstrated significantly lower synchronicity compared to controls and the epileptic group at 4 weeks post-SE. Finally, network connectivity at 4 weeks post-SE was found to correlate with seizure onset (r = 0.858, p < .0001) and disease severity measured over 12 weeks (e.g. cingulate cortex: r = 0.863, p = .002), suggesting a possible network strengthening upon seizure reoccurrence. Our findings indicate that epileptogenesis is characterized by an initial hyposynchrony of brain networks and the disease-associated progression reflects disease severity.

Neuroimaging of Subacute Brain Inflammation and Microstructural Changes Predicts Long-Term Functional Outcome after Experimental Traumatic Brain Injury.

There is currently a lack of prognostic biomarkers to predict the different sequelae following traumatic brain injury (TBI). The present study investigated the hypothesis that subacute neuroinflammation and microstructural changes correlate with chronic TBI deficits. Rats were subjected to controlled cortical impact (CCI) injury, sham surgery, or skin incision (naïve). CCI-injured (n = 18) and sham-operated rats (n = 6) underwent positron emission tomography (PET) imaging with the translocator protein 18 kDa (TSPO) radioligand [18F]PBR111 and diffusion tensor imaging (DTI) in the subacute phase (≤3 weeks post-injury) to quantify inflammation and microstructural alterations. CCI-injured, sham-operated, and naïve rats (n = 8) underwent behavioral testing in the chronic phase (5.5-10 months post-injury): open field and sucrose preference tests, two one-week video-electroencephalogram (vEEG) monitoring periods, pentylenetetrazole (PTZ) seizure susceptibility tests, and a Morris water maze (MWM) test. In vivo imaging revealed pronounced neuroinflammation, decreased fractional anisotropy, and increased diffusivity in perilesional cortex and ipsilesional hippocampus of CCI-injured rats. Behavioral analysis revealed disinhibition, anhedonia, increased seizure susceptibility, and impaired learning in CCI-injured rats. Subacute TSPO expression and changes in DTI metrics significantly correlated with several chronic deficits (Pearson's |r| = 0.50-0.90). Certain specific PET and DTI parameters had good sensitivity and specificity (area under the receiver operator characteristic [ROC] curve = 0.85-1.00) to distinguish between TBI animals with and without particular behavioral deficits. Depending on the investigated behavioral deficit, PET or DTI data alone, or the combination, could very well predict the variability in functional outcome data (adjusted R2 = 0.54-1.00). Taken together, both TSPO PET and DTI seem promising prognostic biomarkers to predict different chronic TBI sequelae.

A companion to the preclinical common data elements for physiologic data in rodent epilepsy models. A report of the TASK3 Physiology Working Group of the ILAE/AES Joint Translational Task Force.

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force created the TASK3 working groups to create common data elements (CDEs) for various aspects of preclinical epilepsy research studies, which could help improve standardization of experimental designs. This article concerns the parameters that can be measured to assess the physiologic condition of the animals that are used to study rodent models of epilepsy. Here we discuss CDEs for physiologic parameters measured in adult rats and mice such as general health status, temperature, cardiac and respiratory function, and blood constituents. We provide detailed CDE tables and case report forms (CRFs), and with this companion manuscript we discuss the monitoring of different aspects of physiology of the animals. The CDEs, CRFs, and companion paper are available to all researchers, and their use will benefit the harmonization and comparability of translational preclinical epilepsy research. The ultimate hope is to facilitate the development of biomarkers and new treatments for epilepsy.

Kainic Acid-Induced Post-Status Epilepticus Models of Temporal Lobe Epilepsy with Diverging Seizure Phenotype and Neuropathology.

The aim of epilepsy models is to investigate disease ontogenesis and therapeutic interventions in a consistent and prospective manner. The kainic acid-induced status epilepticus (KASE) rat model is a widely used, well-validated model for temporal lobe epilepsy (TLE). As we noted significant variability within the model between labs potentially related to the rat strain used, we aimed to describe two variants of this model with diverging seizure phenotype and neuropathology. In addition, we evaluated two different protocols to induce status epilepticus (SE). Wistar Han (Charles River, France) and Sprague-Dawley (Harlan, The Netherlands) rats were subjected to KASE using the Hellier kainic acid (KA) and a modified injection scheme. Duration of SE and latent phase were characterized by video-electroencephalography (vEEG) in a subgroup of animals, while animals were sacrificed 1 week (subacute phase) and 12 weeks (chronic phase) post-SE. In the 12 weeks post-SE groups, seizures were monitored with vEEG. Neuronal loss (neuronal nuclei), microglial activation (OX-42 and translocator protein), and neurodegeneration (Fluorojade C) were assessed. First, the Hellier protocol caused very high mortality in WH/CR rats compared to SD/H animals. The modified protocol resulted in a similar SE severity for WH/CR and SD/H rats, but effectively improved survival rates. The latent phase was significantly shorter (p < 0.0001) in SD/H (median 8.3 days) animals compared to WH/CR (median 15.4 days). During the chronic phase, SD/H rats had more seizures/day compared to WH/CR animals (p < 0.01). However, neuronal degeneration and cell loss were overall more extensive in WH/CR than in SD/H rats; microglia activation was similar between the two strains 1 week post-SE, but higher in WH/CR rats 12 weeks post-SE. These neuropathological differences may be more related to the distinct neurotoxic effects of KA in the two rat strains than being the outcome of seizure burden itself. The divergences in disease progression and seizure outcome, in addition to the histopathological dissimilarities, further substantiate the existence of strain differences for the KASE rat model of TLE.

Decreased levels of active uPA and KLK8 assessed by [111 In]MICA-401 binding correlate with the seizure burden in an animal model of temporal lobe epilepsy.

OBJECTIVE: Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [111 In]MICA-401. As the first step in exploring the applicability of [111 In]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [111 In]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). METHODS: In the KASE model, in vitro autoradiography with [111 In]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. RESULTS: At 7 days post-SE, in vitro autoradiography revealed significantly decreased [111 In]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [111 In]MICA-401 binding (r = -0.76, p ≤ 0.05). Similarly, at 4 days post-TBI, in vitro [111 In]MICA-401 binding was significantly decreased in medial and lateral perilesional cortex and ipsilateral dentate gyrus. Ex vivo autoradiography at 7 days post-TBI, however, revealed increased tracer uptake in perilesional cortex and hippocampus, which was likely related to tracer leakage due to blood-brain barrier (BBB) disruption. SIGNIFICANCE: Strong association of reduced [111 In]MICA-401 binding with seizure burden in the KASE model suggests that analysis of reduced levels of active uPA/KLK8 represents a novel biomarker candidate to be explored as a biomarker for epilepsy severity. However, limited BBB permeability of [111 In]MICA-401 currently limits its application in vivo.