Inflammasomes at the crossroads of traumatic brain injury and post-traumatic epilepsy.

Post-traumatic epilepsy (PTE) is one of the most debilitating consequences of traumatic brain injury (TBI) and is one of the most drug-resistant forms of epilepsy. Novel therapeutic treatment options are an urgent unmet clinical need. The current focus in healthcare has been shifting to disease prevention, rather than treatment, though, not much progress has been made due to a limited understanding of the disease pathogenesis. Neuroinflammation has been implicated in the pathophysiology of traumatic brain injury and may impact neurological sequelae following TBI including functional behavior and post-traumatic epilepsy development. Inflammasome signaling is one of the major components of the neuroinflammatory response, which is increasingly being explored for its contribution to the epileptogenic mechanisms and a novel therapeutic target against epilepsy. This review discusses the role of inflammasomes as a possible connecting link between TBI and PTE with a particular focus on clinical and preclinical evidence of therapeutic inflammasome targeting and its downstream effector molecules for their contribution to epileptogenesis. Finally, we also discuss emerging evidence indicating the potential of evaluating inflammasome proteins in biofluids and the brain by non-invasive neuroimaging, as potential biomarkers for predicting PTE development.

Applying Proteomics and Computational Approaches to Identify Novel Targets in Blast-Associated Post-Traumatic Epilepsy.

Traumatic brain injury (TBI) can lead to post-traumatic epilepsy (PTE). Blast TBI (bTBI) found in Veterans presents with several complications, including cognitive and behavioral disturbances and PTE; however, the underlying mechanisms that drive the long-term sequelae are not well understood. Using an unbiased proteomics approach in a mouse model of repeated bTBI (rbTBI), this study addresses this gap in the knowledge. After rbTBI, mice were monitored using continuous, uninterrupted video-EEG for up to four months. Following this period, we collected cortex and hippocampus tissues from three groups of mice: those with post-traumatic epilepsy (PTE+), those without epilepsy (PTE-), and the control group (sham). Hundreds of differentially expressed proteins were identified in the cortex and hippocampus of PTE+ and PTE- relative to sham. Focusing on protein pathways unique to PTE+, pathways related to mitochondrial function, post-translational modifications, and transport were disrupted. Computational metabolic modeling using dysregulated protein expression predicted mitochondrial proton pump dysregulation, suggesting electron transport chain dysregulation in the epileptic tissue relative to PTE-. Finally, data mining enabled the identification of several novel and previously validated TBI and epilepsy biomarkers in our data set, many of which were found to already be targeted by drugs in various phases of clinical testing. These findings highlight novel proteins and protein pathways that may drive the chronic PTE sequelae following rbTBI.

Adult Born Dentate Granule Cell Mediated Upregulation of Feedback Inhibition in a Mouse Model of Traumatic Brain Injury.

Post-traumatic epilepsy (PTE) and behavioral comorbidities frequently develop after traumatic brain injury (TBI). Aberrant neurogenesis of dentate granule cells (DGCs) after TBI may contribute to the synaptic reorganization that occurs in PTE, but how neurogenesis at different times relative to the injury contributes to feedback inhibition and recurrent excitation in the dentate gyrus is unknown. Thus, we examined whether DGCs born at different postnatal ages differentially participate in feedback inhibition and recurrent excitation in the dentate gyrus using the controlled cortical impact (CCI) model of TBI. Both sexes of transgenic mice expressing channelrhodopsin2 (ChR2) in postnatally born DGCs were used for optogenetic activation of three DGC cohorts: postnatally early born DGCs, or those born just before or after CCI. We performed whole-cell patch-clamp recordings from ChR2-negative, mature DGCs and parvalbumin-expressing basket cells (PVBCs) in hippocampal slices to determine whether optogenetic activation of postnatally born DGCs increases feedback inhibition and/or recurrent excitation in mice 8-10 weeks after CCI and whether PVBCs are targets of ChR2-positive DGCs. In the dentate gyrus ipsilateral to CCI, activation of ChR2-expressing DGCs born before CCI produced increased feedback inhibition in ChR2-negative DGCs and increased excitation in PVBCs compared with those from sham controls. This upregulated feedback inhibition was less prominent in DGCs born early in life or after CCI. Surprisingly, ChR2-positive DGC activation rarely evoked recurrent excitation in mature DGCs from any cohort. These results support that DGC birth date-related increased feedback inhibition in of DGCs may contribute to altered excitability after TBI.SIGNIFICANCE STATEMENT Dentate granule cells (DGCs) control excitability of the dentate gyrus through synaptic interactions with inhibitory GABAergic interneurons. Persistent changes in DGC synaptic connectivity develop after traumatic brain injury, contributing to hyperexcitability in post-traumatic epilepsy (PTE). However, the impact of DGC neurogenesis on synaptic reorganization, especially on inhibitory circuits, after brain injury is not adequately described. Here, upregulation of feedback inhibition in mature DGCs from male and female mice was associated with increased excitation of parvalbumin-expressing basket cells by postnatally born DGCs, providing novel insights into underlying mechanisms of altered excitability after brain injury. A better understanding of these inhibitory circuit changes can help formulate hypotheses for development of novel, evidence-based treatments for post-traumatic epilepsy by targeting birth date-specific subsets of DGCs.

Functional connectivity alterations in traumatic brain injury patients with late seizures.

PTE is a neurological disorder characterized by recurrent and spontaneous epileptic seizures. PTE is a major public health problem occurring in 2-50% of TBI patients. Identifying PTE biomarkers is crucial for the development of effective treatments. Functional neuroimaging studies in patients with epilepsy and in epileptic rodents have observed that abnormal functional brain activity plays a role in the development of epilepsy. Network representations of complex systems ease quantitative analysis of heterogeneous interactions within a unified mathematical framework. In this work, graph theory was used to study resting state functional magnetic resonance imaging (rs-fMRI) and reveal functional connectivity abnormalities that are associated with seizure development in traumatic brain injury (TBI) patients. We examined rs-fMRI of 75 TBI patients from Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) which aims to identify validated Post-traumatic epilepsy (PTE) biomarkers and antiepileptogenic therapies using multimodal and longitudinal data acquired from 14 international sites. The dataset includes 28 subjects who had at least one late seizure after TBI and 47 subjects who had no seizures within 2 years post-injury. Each subject's neural functional network was investigated by computing the correlation between the low frequency time series of 116 regions of interest (ROIs). Each subject's functional organization was represented as a network consisting of nodes, brain regions, and edges that show the relationship between the nodes. Then, several graph measures concerning the integration and the segregation of the functional brain networks were extracted in order to highlight changes in functional connectivity between the two TBI groups. Results showed that the late seizure-affected group had a compromised balance between integration and segregation and presents functional networks that are hyperconnected, hyperintegrated but at the same time hyposegregated compared with seizure-free patients. Moreover, TBI subjects who developed late seizures had more low betweenness hubs.

miRNA dysregulation in traumatic brain injury and epilepsy: a systematic review to identify putative biomarkers for post-traumatic epilepsy.

Traumatic brain injury (TBI) leads to post-traumatic epilepsy (PTE); hence, both TBI and PTE share various similar molecular mechanisms. MicroRNA (miRNA) is a small noncoding RNA that acts as a gene-silencing molecule. Notably, the dysregulation of miRNAs in various neurological diseases, including TBI and epilepsy, has been reported in several studies. However, studies on commonly dysregulated miRNAs and the regulation of shared pathways in both TBI and epilepsy that can identify potential biomarkers of PTE are still lacking. This systematic review covers the peer-review publications of TBI and database studies of epilepsy-dysregulated miRNAs of clinical studies. For TBI, 290 research articles were identified after screening, and 12 provided data for dysregulated miRNAs in humans. The compiled data suggest that 85 and 222 miRNAs are consecutively dysregulated in TBI and epilepsy. In both, 10 miRNAs were found to be commonly dysregulated, implying that they are potentially dysregulated miRNAs for PTE. Furthermore, the targets and involvement of each putative miRNA in different pathways were identified and evaluated. Additionally, clusters of predicted miRNAs were analyzed. Each miRNA's regulatory role was linked with apoptosis, inflammation, and cell cycle regulation pathways. Hence, these findings provide insight for future diagnostic biomarkers.

Discovery and Validation of Circulating microRNAs as Biomarkers for Epileptogenesis after Experimental Traumatic Brain Injury-The EPITARGET Cohort.

Traumatic brain injury (TBI) causes 10-20% of structural epilepsies and 5% of all epilepsies. The lack of prognostic biomarkers for post-traumatic epilepsy (PTE) is a major obstacle to the development of anti-epileptogenic treatments. Previous studies revealed TBI-induced alterations in blood microRNA (miRNA) levels, and patients with epilepsy exhibit dysregulation of blood miRNAs. We hypothesized that acutely altered plasma miRNAs could serve as prognostic biomarkers for brain damage severity and the development of PTE. To investigate this, epileptogenesis was induced in adult male Sprague Dawley rats by lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least one unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Cortical pathology was analyzed by magnetic resonance imaging on day 2 (D2), D7, and D21, and by histology 6 months post-TBI. Small RNA sequencing was performed from tail-vein plasma samples on D2 and D9 after TBI (n = 16, 7 with and 9 without epilepsy) or sham operation (n = 4). The most promising miRNA biomarker candidates were validated by droplet digital polymerase chain reaction in a validation cohort of 115 rats (8 naïve, 17 sham, and 90 TBI rats [21 with epilepsy]). These included 7 brain-enriched plasma miRNAs (miR-434-3p, miR-9a-3p, miR-136-3p, miR-323-3p, miR-124-3p, miR-212-3p, and miR-132-3p) that were upregulated on D2 post-TBI (p < 0.001 for all compared with naïve rats). The acute post-TBI plasma miRNA profile did not predict the subsequent development of PTE or PTE severity. Plasma miRNA levels, however, predicted the cortical pathology severity on D2 (Spearman ρ = 0.345-0.582, p < 0.001), D9 (ρ = 0.287-0.522, p < 0.001-0.01), D21 (ρ = 0.269-0.581, p < 0.001-0.05) and at 6 months post-TBI (ρ = 0.230-0.433, p < 0.001-0.05). We found that the levels of 6 of 7 miRNAs also reflected mild brain injury caused by the craniotomy during sham operation (ROC AUC 0.76-0.96, p < 0.001-0.05). In conclusion, our findings revealed that increased levels of neuronally enriched miRNAs in the blood circulation after TBI reflect the extent of cortical injury in the brain but do not predict PTE development.

Treatment of Status Epilepticus after Traumatic Brain Injury Using an Antiseizure Drug Combined with a Tissue Recovery Enhancer Revealed by Systems Biology.

We tested a hypothesis that in silico-discovered compounds targeting traumatic brain injury (TBI)-induced transcriptomics dysregulations will mitigate TBI-induced molecular pathology and augment the effect of co-administered antiseizure treatment, thereby alleviating functional impairment. In silico bioinformatic analysis revealed five compounds substantially affecting TBI-induced transcriptomics regulation, including calpain inhibitor, chlorpromazine, geldanamycin, tranylcypromine, and trichostatin A (TSA). In vitro exposure of neuronal-BV2-microglial co-cultures to compounds revealed that TSA had the best overall neuroprotective, antioxidative, and anti-inflammatory effects. In vivo assessment in a rat TBI model revealed that TSA as a monotherapy (1 mg/kg/d) or in combination with the antiseizure drug levetiracetam (LEV 150 mg/kg/d) mildly mitigated the increase in plasma levels of the neurofilament subunit pNF-H and cortical lesion area. The percentage of rats with seizures during 0-72 h post-injury was reduced in the following order: TBI-vehicle 80%, TBI-TSA (1 mg/kg) 86%, TBI-LEV (54 mg/kg) 50%, TBI-LEV (150 mg/kg) 40% (p < 0.05 vs. TBI-vehicle), and TBI-LEV (150 mg/kg) combined with TSA (1 mg/kg) 30% (p < 0.05). Cumulative seizure duration was reduced in the following order: TBI-vehicle 727 ± 688 s, TBI-TSA 898 ± 937 s, TBI-LEV (54 mg/kg) 358 ± 715 s, TBI-LEV (150 mg/kg) 42 ± 64 (p < 0.05 vs. TBI-vehicle), and TBI-LEV (150 mg/kg) combined with TSA (1 mg/kg) 109 ± 282 s (p < 0.05). This first preclinical intervention study on post-TBI acute seizures shows that a combination therapy with the tissue recovery enhancer TSA and LEV was safe but exhibited no clear benefit over LEV monotherapy on antiseizure efficacy. A longer follow-up is needed to confirm the possible beneficial effects of LEV monotherapy and combination therapy with TSA on chronic post-TBI structural and functional outcomes, including epileptogenesis.

[Predictive capability of Cys112Arg single nucleotide polymorphisms of the apolipoprotein E gene in assessing the risk of immediate and early post-traumatic seizures].

This study is aimed at investigating epileptic seizures, one of the consequences of traumatic brain injury (TBI). Immediate and early post-traumatic seizures, as well as late post-traumatic epileptic seizures or post-traumatic epilepsy, can have different pathogenetic bases. The following key risk factors associated with post-traumatic epilepsy are known: duration of unconsciousness, gunshot wounds, intracranial hemorrhage, diffuse axonal injury, prolonged (more than 3 days) post-traumatic amnesia, acute subdural hematoma with surgical evacuation, immediate and early post-traumatic epileptic seizures, fracture of the skull bones. The role of genetic factors in post-traumatic seizures is poorly understood due to the complexity and multiple causal mechanisms. This paper addresses the role of genetic factors in the occurrence and severity of epileptic events in patients with TBI. In particular, we investigated the role of the Cys112Arg single nucleotide polymorphism of the apolipoprotein E gene. Apolipoprotein E is known for its role in the transport and metabolism of lipids and, therefore, the development of cardiovascular diseases; it is also associated with Alzheimer's disease and has recently been studied in the context of association with epilepsy. The study shows an association between this polymorphism and the risk of immediate and early epileptic seizures in patients with severe TBI.

Plasma Neurofilament Light Chain (NF-L) Is a Prognostic Biomarker for Cortical Damage Evolution but Not for Cognitive Impairment or Epileptogenesis Following Experimental TBI.

Plasma neurofilament light chain (NF-L) levels were assessed as a diagnostic biomarker for traumatic brain injury (TBI) and as a prognostic biomarker for somatomotor recovery, cognitive decline, and epileptogenesis. Rats with severe TBI induced by lateral fluid-percussion injury (n = 26, 13 with and 13 without epilepsy) or sham-operation (n = 8) were studied. During a 6-month follow-up, rats underwent magnetic resonance imaging (MRI) (day (D) 2, D7, and D21), composite neuroscore (D2, D6, and D14), Morris-water maze (D35−D39), and a 1-month-long video-electroencephalogram to detect unprovoked seizures during the 6th month. Plasma NF-L levels were assessed using a single-molecule assay at baseline (i.e., naïve animals) and on D2, D9, and D178 after TBI or a sham operation. Plasma NF-L levels were 483-fold higher on D2 (5072.0 ± 2007.0 pg/mL), 89-fold higher on D9 (930.3 ± 306.4 pg/mL), and 3-fold higher on D176 32.2 ± 8.9 pg/mL after TBI compared with baseline (10.5 ± 2.6 pg/mL; all p < 0.001). Plasma NF-L levels distinguished TBI rats from naïve animals at all time-points examined (area under the curve [AUC] 1.0, p < 0.001), and from sham-operated controls on D2 (AUC 1.0, p < 0.001). Plasma NF-L increases on D2 were associated with somatomotor impairment severity (ρ = −0.480, p < 0.05) and the cortical lesion extent in MRI (ρ = 0.401, p < 0.05). Plasma NF-L increases on D2 or D9 were associated with the cortical lesion extent in histologic sections at 6 months post-injury (ρ = 0.437 for D2; ρ = 0.393 for D9, p < 0.05). Plasma NF-L levels, however, did not predict somatomotor recovery, cognitive decline, or epileptogenesis (p > 0.05). Plasma NF-L levels represent a promising noninvasive translational diagnostic biomarker for acute TBI and a prognostic biomarker for post-injury somatomotor impairment and long-term structural brain damage.

Response of Voltage-Gated Sodium and Calcium Channels Subtypes on Dehydroepiandrosterone Treatment in Iron-Induced Epilepsy.

Epilepsy is a neurological disorder characterized by the occurrence of spontaneous and recurrent seizures. In post-traumatic epilepsy (PTE), the mechanism of epileptogenesis is very complex and seems to be linked with voltage-gated ion channels. Dehydroepiandrosterone (DHEA), a neurosteroid have shown beneficial effect against various neurological disorders. We investigated antiepileptic effect of DHEA with respect to expression of voltage-gated ion channels subtypes in iron-induced epilepsy. Iron (FeCl3) solution was intracartically injected to induce epilepsy in rats and DHEA was intraperitoneally administered for 21 days. Results showed markedly increased epileptiform seizures activity along with up-regulation of Nav1.1 and Nav1.6, and down-regulation of Cav2.1α at the mRNA and protein level in the cortex and hippocampus of epileptic rats. Moreover, the study demonstrated that these channels subtypes were predominantly expressed in the neurons. DHEA treatment has countered the epileptic seizures, down-regulated Nav1.1 and Nav1.6, and up-regulated Cav2.1α without affecting their cellular localization. In conclusion, the present study demonstrates antiepileptic potential of DHEA, escorted by regulation of Nav1.1, Nav1.6, and Cav2.1α subtypes in the neurons of iron-induced epileptic rats.