Traumatic brain injury (TBI) is a serious health concern in the United States and worldwide. Understanding the mechanisms underlying resting-state functional activity can play an important role in profiling induced disruptions, potentially enabling accurate and timely interventions. Due to its homology to the human brain, the porcine brain is a valuable translational model for investigating focal TBI. In this study, we used the porcine controlled cortical impact TBI model targeting the motor cortex to evaluate dynamic disruptions in functional activity in mild or severe TBI, including hemispheric imbalance, reorganization, and compensation mechanisms, using independent component analysis (ICA). Our whole-brain findings revealed that immediately after TBI, high-level resting-state networks (RSNs), including the executive control network (ECN) and cerebellar network (CN), showed significantly decreased activity, whereas the sensory motor network (SMN) displayed preserved functional activity. Further hemispheric findings demonstrated a significant decrease in ipsilateral functional activity, whereas contralateral functional activity increased in SMN. Additionally, cortical areas (CoAs) associated with the SMN demonstrated a pronounced hemispheric imbalance at the acute phase. Hemispheric and CoAs analyses revealed a restoration of functional activity over a two-month period. These alterations and lateral imbalance suggest potential lateral reorganization, compensation mechanisms, and temporal recovery processes. These findings are consistent with outcomes previously demonstrated in human TBI patients, further underscoring the importance of the porcine translational model for investigating changes in brain activity or connectivity due to TBI and future novel treatments.

Early identification of epileptiform activity in patients with traumatic brain injury (TBI) remains a clinical challenge, with important implications for prognosis and treatment. The primary objective of this prospective single-center study was to determine the incremental diagnostic yield of 1 h EEG (1hEEG) and continuous EEG (cEEG; 24-h long EEG segment) compared with routine EEG (rEEG; first 30 min recording) for detecting interictal epileptiform discharges (IEDs) and electrographic seizures in adults with acute TBI and acute intracranial hemorrhage and/or brain contusion(s). A secondary exploratory objective was to assess whether clinical, neuroimaging, and EEG characteristics were associated with similarity between shorter recordings and the corresponding prolonged recording. Thirty-five adults underwent cEEG during ICU admission. Monitoring began a median of 2 days after TBI (range, 0-7 days) and lasted a median of 3 days (range, 1-9 days; mean, 3.8 days), yielding 134 analyzable segments (mean 3.8 segments per patient). For each segment, the first 30 min and first 60 min were compared with the full cEEG segment. IEDs were identified in 14/134 segments (10.4%) during the first 30 min and first hour and detection was increased to 19/134 segments (14.2%) during prolonged monitoring; thus, 5/19 IED-positive segments were detected only after the first hour. Electrographic seizures were identified during cEEG in 3/35 patients (8.6%); two had seizures within the initial 30 min, whereas one had a seizure only during prolonged monitoring. All seizures were electrographic/nonconvulsive. Prolonged EEG increased the detection of IEDs in a subset of recordings and identified an electrographic seizure that was missed by shorter recordings. Reduced level of consciousness, greater TBI severity, sedative-drug exposure, and additional trauma to other body parts were associated with a greater similarity (shorter mathematical distance) between rEEG, 1hEEG and cEEG findings (all p < 0.05). Clinical variables may help prioritize patients in whom shorter EEG recordings are more representative of prolonged EEG; however, these exploratory findings should not be used to exclude high-risk patients from cEEG.

GABAergic inhibition delays oxygen-induced seizures, but whether this protection is maintained during repeated hyperbaric oxygen (HBO2) exposure is unclear. We hypothesized that tiagabine (TGB) prevents increased seizure susceptibility during repeated HBO2 by preserving presynaptic nerve-terminal function and tested whether single and repeated HBO2 alter mitochondrial bioenergetics, mitophagy/autophagy, and region-specific transcriptional responses. Mice were exposed to 4.5 ATA O2 for 60 min and re-exposed after 48 h, 72 h, or 7 d intervals. Measurements included seizure latency, synaptosomal neurotransmitters and oxygen consumption, mitophagy (mito-QC reporter)/autophagy (p62 and LC3-II/LC3-I), and spatial transcriptomics with gene ontology enrichment to define region-specific responses. Repeated HBO2 shortened seizure latency and reduced the antiseizure efficacy of TGB. Synaptosomal neurotransmitters were altered by HBO2 but not modified by TGB. Mitophagy/autophagy showed region-specific changes, with increased LC3-II/LC3-I at 48 h and region- and time-dependent changes in p62 expression, accompanied by reduced mitolysosomal signal at 7 d in hippocampal CA3 and cerebellar granular layer, while TGB increased signal in the cerebellar molecular layer. Synaptosomal mitochondrial respiration showed time-dependent changes, with early increases in oxygen consumption followed by reduced maximal respiration and spare respiratory capacity after repeated exposure. Spatial transcriptomics revealed region-specific responses, shifting from broad activation after a single exposure to more restricted intracellular and metabolic programs after repeated exposure. These findings indicate that TGB does not preserve presynaptic neurotransmitter content or prevent increased seizure susceptibility during repeated HBO2 exposure. Repeated HBO2 induces mitochondrial and transcriptional adaptations associated with reduced bioenergetic reserve, with susceptibility to oxygen toxicity influenced by recovery interval.

Temporal lobe epilepsy (TLE) is linked to progressive alterations in brain network dynamics, leading to behavioral comorbidities and emergence of drug resistance; yet the underlying mechanistic and synaptic substrates remain incompletely understood. Here, we performed longitudinal phase-specific characterization of network dysfunction in TLE employing a lithium-pilocarpine-induced model in 7-week male Wistar rats. We integrated behavioral and electroencephalographic (EEG) analyses at five timepoints: baseline, acute, latent, chronic phases, and after assessment of drug-resistant epilepsy (DRE). After the chronic phase, responsiveness to phenobarbital (PB), a standardized approach for DRE characterization, was assessed via video monitoring to identify drug-resistant (DRUG-R) and drug-sensitive (DRUG-S) subpopulations. Our data show that epileptic rats exhibited progressive and phase-dependent behavioral and EEG alterations. Behavioral profiling revealed a hypermotor phenotype, impairment in burrowing natural behavior, altered phase-specific response to anxiety and depressive-associated behavioral paradigms, and progressive memory impairment. Following PB treatment, the identified DRUG-R subpopulation displayed specific hyperactive behavioral traits compared with DRUG-S, especially in anxiety-associated and burrowing behaviors. Spectral EEG analysis revealed modulation of frequency bands across disease stages, particularly in Theta and Delta power, and descriptive analyses attempted to stratify animals based on different responsiveness to PB. Lastly, correlation analyses supported associations between EEG, frequency of seizures and behavioral measures, especially in the anxiety domain and declarative memory. This study, moving beyond a seizure-centric perspective, evidences that TLE induces progressive and phase-specific reorganization of cortical activity that relapse on distinct electrophysiological and behavioral features, offering a novel framework to identify translational stage-related signatures of epileptogenesis and DRE.

Ischemic stroke is a leading cause of death and disability. Administration of the lipid mediators elovanoids (ELVs) is protective in human neuronal-glia cultures and in experimental ischemic stroke. We now report using a single-cell multiome approach that intranasally-delivered (IN) ELV34 or its precursor reduced the loss of neuronal markers and upregulated homeostatic microglia signatures after stroke. Thus, ELV reduces disease-associated microglia (expressing Spp1, Gpnmb, Lgals3, Clec7a) and the expression of neuroinflammatory signaling genes. In astrocytes, ELV decreased reactive astrocytes (expressing Gfap, Vim, Nes, Lcn2) and upregulated genes involved in synaptic organization. Also, ELV reduced abundance of oligodendrocytes and OPCs expressing immune markers. ELV induced a phenotype shift from pro-inflammatory microglia, astrocytes, oligodendrocytes, and OPCs in response to ischemic stroke damage. ELV upregulated gene pathways promoting synaptic integrity, reducing immune cell activation and neuronal loss.

Glioblastoma (GBM) exhibits profound metabolic and redox adaptation that supports tumor progression and therapeutic resistance. Here, we identify the glutamate transporter EAAT1 (SLC1A3) as a critical regulator of glutamate-dependent redox homeostasis in GBM. Analysis of TCGA, GTEx, and CGGA datasets showed that EAAT1 expression is elevated in GBM and that higher EAAT1 expression is associated with poor patient survival. Using CRISPR/Cas9-mediated EAAT1 knockout together with biochemical, imaging, transcriptomic, and in vivo approaches, we found that loss of EAAT1 altered extracellular and intracellular glutamate homeostasis, reduced intracellular glutamate, glutamine, and glutathione levels, and increased reactive oxygen species (ROS) accumulation. EAAT1 deficiency also suppressed oxidative phosphorylation and ROS-related programs and attenuated the Keap1/Nrf2/HO-1 antioxidant axis, accompanied by reduced GPX4 expression and increased lipid peroxidation. Furthermore, EAAT1 ablation downregulated glutamine synthetase and glutaminase, suggesting impaired glutamine-dependent anaplerotic metabolism. Glutamate supplementation partially restored Keap1/Nrf2/HO-1 pathway protein expression in EAAT1-knockout cells. Functionally, EAAT1 loss inhibited GBM cell proliferation and migration, enhanced sensitivity to oxidative stress and temozolomide (TMZ), and reduced tumor growth in xenograft models. Collectively, our findings establish EAAT1 as a key metabolic regulator linking glutamate transport to antioxidant defense and therapeutic response in GBM. Targeting EAAT1 may therefore represent a metabolic vulnerability for overcoming metabolic and redox adaptation and improving TMZ responsiveness in GBM.

Excessive activation of microglia exacerbates secondary brain injury after ischemic stroke, yet the upstream mechanisms governing this response remain incompletely understood. This study aimed to investigate the role of RIO kinase 3 (RIOK3) in microglia-mediated neuroinflammation after ischemic stroke and to explore the underlying molecular mechanisms. RIOK3 expression was examined in mice subjected to transient middle cerebral artery occlusion and in primary microglia exposed to lipopolysaccharide or oxygen-glucose deprivation/reoxygenation. Microglia-targeted RIOK3 knockdown was achieved using a Cre-dependent adeno-associated virus-shRNA strategy in Tmem119-CreERT2 mice. RIOK3 was markedly upregulated in microglia after cerebral ischemia and in primary microglia following LPS or OGD/R stimulation. Microglia-targeted RIOK3 knockdown reduced infarct volume, improved early neurological and sensorimotor outcomes, preserved microglial process complexity, and reduced post-ischemic inflammation. In vitro, RIOK3 knockdown reduced microglial inflammatory responses and microglia-mediated neurotoxicity. Transcriptomic and biochemical analyses further showed that RIOK3 knockdown suppressed NF-κB-related transcriptional programs and decreased the phosphorylation of IκBα and p65. Mechanistically, immunoprecipitation assays identified Y-box-binding protein 1 (YBX1) as a RIOK3-interacting protein and mapped this interaction to the C-terminal kinase domain-containing region of RIOK3. RIOK3 enhanced YBX1 Ser102 phosphorylation and nuclear accumulation, whereas YBX1 knockdown attenuated NF-κB activation and the pro-inflammatory effects of RIOK3 overexpression. These findings identify microglial RIOK3 as an important driver of post-ischemic neuroinflammation and highlight the RIOK3-YBX1 axis as a potential therapeutic target for ischemic stroke.

Huntington's disease (HD) is marked by progressive neuronal loss and atrophy of grey matter structures, particularly the caudate and putamen. Brain imaging studies reveal that the white matter starts to decay in the brain of individuals bearing the HD mutation many years before symptomatic onset. However, the mechanism by which the HD mutation causes white matter loss remains to be further defined. In this study, we examined white matter pathology and explored the underlying mechanism in the HDQ140 knock-in mouse model of HD. Western blot analysis of proteins localized at different layers of the myelin sheath showed that myelin-associated glycoprotein (MAG), which is localized at the innermost myelin layer, was decreased earlier than proteins localized at outer layers of the myelin. The loss of MAG occurred at fully myelinated axons and was progressive with age. In postmortem symptomatic human HD brains, the level of MAG as well as other myelin proteins was also decreased. In HD mouse brains, MAG labeling was reduced at fiber bundles but accumulated in perinuclear structures of cells that expressed breast carcinoma amplified sequence 1, a marker for new oligodendrocytes. While their abundance was normal, new oligodendrocytes in HD brains were impeded in acquiring the expression of MAG. Compared with those in wild-type mouse brains, oligodendrocytes in HD mouse brains had a reduced frequency of MAG-bearing small vesicles and an increased abundance and enlargement of MAG-containing perinuclear structures. Further studies suggest that the MAG-accumulating perinuclear structures were derived from the late endosomal lysosomal compartment. Our study suggests that white matter decay in HD brains involves an early progressive loss of MAG in myelin membranes.

Neonatal hypoxia-ischemia (HI) remains a major global cause of neonatal morbidity and mortality. To develop effective therapeutic strategies, a deeper understanding of the acute inflammatory processes following HI is required. As the role of peripheral immune cell infiltration is poorly understood we used single nuclei RNA sequencing to investigate acute sequalae post-HI. This revealed a distinct neutrophil population characterized by increased expression of markers associated with neutrophil extracellular trap (NET) formation. The study evaluated the therapeutic potential of inhibiting NETosis formation using the peptidylarginine-deiminase-4 (PAD4) inhibitor Cl-amidine. We demonstrated that while oxygen-glucose deprivation (OGD) induces NET-formation and matrix-metalloproteinase-9 release in neutrophils, this is significantly reduced by the PAD4 inhibitor Cl-amidine. Second, in a co-culture model, inhibition of NETosis during OGD conditions improved neuronal survival. Third, Cl-amidine treatment reduced brain tissue loss and decreased expression levels of NETosis-related proteins 48 h post-HI in vivo. Moreover, behavioral testing performed six weeks after HI revealed improved motor function in the treatment group. This study highlights the detrimental role of neutrophil activation and NETosis in exacerbating brain injury following neonatal HI. Targeting NET formation in the acute phase after HI may represent a promising therapeutic approach to improve both immediate and long-term neurological outcomes.