Oligodendrocytes (OLs) myelinate central nervous system (CNS) axons and provide metabolic support to maintain axonal integrity. Thyroid hormone (TH) is a mitogen for oligodendroglial precursor cells (OPCs) maturation into myelinating OLs. Cellular uptake of TH is mediated by monocarboxylate transporter 8 (MCT8; encoded by slc16a2 ), and its dysfunction results in intracellular triiodothyronine (T3) deprivation, leading to hypomyelination and myelin degeneration during neuroinflammation. We showed that MCT8 expression is maintained in OPCs residing within the sub–ventricular zone (SVZ) throughout CNS development, suggesting a role during OL development. We identified MCT8 deficiency during neuroinflammatory and cuprizone demyelination models, as well as in secondary progressive multiple sclerosis (SPMS). These conditions were associated with dysregulated AKT–mTOR–PANK2 signaling and abrogated Co Enzyme A and lipid synthesis pathways in the CNS during myelin degeneration. Hence, neuroprotection during SPMS maybe achieved by overcoming MCT8 deficiencies in OLs.

Enlarged perivascular spaces (PVS) are increasingly recognized as biomarkers of cerebral small vessel disease, Alzheimer's disease, stroke, and aging-related neurodegeneration. However, manual segmentation of PVS is time-consuming and subject to moderate inter-rater reliability, while existing automated deep learning models have moderate performance and typically fail to generalize across diverse clinical and research MRI datasets. We adapted MedNeXt-L-k5, a Transformer-inspired 3D encoder-decoder convolutional network, for automated PVS segmentation. Two models were trained: one using a homogeneous dataset of 200 T2-weighted (T2w) MRI scans from the Human Connectome Project-Aging (HCP-Aging) dataset and another using 40 heterogeneous T1-weighted (T1w) MRI volumes from seven studies across six scanners. Model performance was evaluated using internal 5-fold cross validation (5FCV) and leave-one-site-out cross validation (LOSOCV). MedNeXt-L-k5 models trained on the T2w images of the HCP-Aging dataset achieved voxel-level Dice scores of 0.88+/-0.06 (white matter, WM), comparable to the reported inter-rater reliability of that dataset, and the highest yet reported in the literature. The same models trained on the T1w images of the HCP-Aging dataset achieved a substantially lower Dice score of 0.58+/-0.09 (WM). Under LOSOCV, the model had voxel-level Dice scores of 0.38+/-0.16 (WM) and 0.35+/-0.12 (BG), and cluster-level Dice scores of 0.61+/-0.19 (WM) and 0.62+/-0.21 (BG). MedNeXt-L-k5 provides an efficient solution for automated PVS segmentation across diverse T1w and T2w MRI datasets. MedNeXt-L-k5 did not outperform the nnU-Net, indicating that the attention-based mechanisms present in transformer-inspired models to provide global context are not required for high accuracy in PVS segmentation.

These preclinical trials provide the first evidence of cluster of differentiation 14 (CD14) blockade with a murine analogue of atibuclimab, a CD14-neutralizing antibody, preventing secondary immunological exacerbation of cardiac injury in a translational mouse model of reperfused ST-segment elevation myocardial infarction (STEMI), assessed using multiple clinical modalities. Multiomic studies suggest CD14 blockade downregulated macrophage-specific proinflammatory and tissue-wide remodeling processes without suppression of monocyte-macrophage infiltration or repair. These findings support a clinically practicable targeted immunomodulatory strategy of CD14 blockade initiated at reperfusion to prevent chronic immunological progression toward ischemic heart failure, and provide new insights into the pleiotropic roles of CD14 in inflammation and myocardial injury.

Intimate partner violence (IPV) is a serious global health concern that primarily affects women. Traumatic brain injury (TBI; e.g., concussion) and non-fatal strangulation (NFS) are common forms of IPV-related brain injuries which often occur in combination and are highly repetitive in nature. While our understanding of the consequences of repetitive TBI (rTBI) has improved due to research in sport and military settings, little is known about the repercussions of repetitive NFS (rNFS) or rTBI+rNFS. We recently developed the first rat model of NFS and found that rats exposed to a single TBI + NFS event had exacerbated deficits and pathophysiology than the injuries in isolation. In the current study we utilized these models to investigate the more clinically relevant setting of recurrent IPV-related brain injuries. Female Sprague-Dawley rats were assigned to repeated sham, rTBI, rNFS, and rTBI+rNFS groups. The rats received five exposures to their injury regime, with each exposure 24 h apart, followed by a 12-week recovery before behavioral testing and ex vivo MRI. Only rTBI+rNFS rats had persisting social deficits, while both groups with rTBI had cognitive deficits and anhedonia. Advanced diffusion MRI analysis found that the rTBI+rNFS rats had reduced fiber density and cross-section, an indication of reduced white matter integrity, compared to all other groups. Tensor-based morphometry and region of interest MRI analysis revealed that both groups with rTBI had brain areas with a statistically significant reduction in volume. These findings indicate that rTBI+rNFS results in worse social deficits and white matter damage and provides further evidence that IPV-related brain injuries can result in long-term debilitating consequences.

OBJECTIVE:To test a hypothesis that a lesion in the optic tract, a thalamocortical pathway projecting from the thalamus to the perilesional cortex, is a prognostic biomarker for the development of post-traumatic epilepsy (PTE). METHODS:A lateral fluid-percussion injury (FPI)-induced traumatic brain injury (TBI) was induced in adult male Sprague-Dawley rats. The development of PTE was assessed from 1-month-long video-electroencephalography (EEG) data acquired during the seventh post-injury month. Diffusion magnetic resonance imaging (MRI) tractography of the optic radiation was performed using data acquired at 9 days and at 5 months post-injury. The optic tract in each hemisphere was divided into segments, and tract and diffusion tensor-based metrics were obtained for use as predictor variables (a total of 451 predictors). In the training cohort, including 17 TBI rats with (TBI+) and 66 without epilepsy (TBI-) at 9 days and 18 TBI+ and 69 TBI- rats at 5 months, 20 predictor variables with the highest effect sizes (Cohen's delta) were selected. These variables were used with elastic net-regularized logistic regression to model the development of PTE. The predictor variables chosen in the training cohort were also used for model fitting in a separate validation cohort (8 TBI+ and 23 TBI- at 9 days; 9 TBI+ and 23 TBI- at 5 months). RESULTS:In the training cohort, the fitted models explained the development of PTE with (cross-validated) areas under the receiver-operating characteristic (ROC) curve (AUC) of 0.74 (p = .003) at 9 days and 0.76 (p < .001) at 5 months post-TBI. Models fitted in the validation cohort achieved AUC values of 0.50 (p = 1) at 9 days and 0.77 (p = .03) at 5 months post-TBI. SIGNIFICANCE:Our data show that white matter pathology is involved in post-traumatic epileptogenesis after lateral FPI. Moreover, imaging of thalamocortical pathway(s) to perilesional cortex could identify translational prognostic biomarkers for post-traumatic epileptogenesis.

Global ischemic brain injury occurs after cardiac arrest or prolonged hypotensive episodes following surgery or trauma. It causes significant neurological deficits even after successful re-establishment of blood flow. It is the primary cause of death in 68% of inpatient and 23% of out-of-hospital cardiac arrest cases, but there are currently no treatments. Endothelial activation and dysfunction impairing small vessel blood flow is the cause of brain damage. Purinergic signaling is an endogenous molecular pathway, where CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia, eATP is released, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Our group developed a bifunctional compound – anti-VCAM-CD39 that targets the dysregulated endothelium and promotes adenosine generation at the infarct site, localising the antithrombotic and anti-inflammatory effects of CD39. We investigated whether anti-VCAM-CD39 could improve outcome in a murine model of global ischaemia caused by dual carotid artery ligation (DCAL). Test drugs anti-VCAM-CD39 and controls were given 3 h after 30 min ischaemia. Assessments at 24 h included neurological function, infarct volume, perfusion, and albumin extravasation to assess blood-brain barrier (BBB) permeability. We showed that there was an overall improvement in neurological deficit in anti-VCAM-CD39-treated mice after DCAL. MRI revealed that these mice had significantly smaller infarcts and reduced apoptotic activity on the side of permanent occlusion, compared to saline treated mice. There was reduced albumin extravasation in treated mice after DCAL, suggesting anti-VCAM-CD39 conferred neuroprotection in the brain through preservation of BBB permeability. In vitro findings confirmed that anti-VCAM-CD39-mediated adenosine production protected against hypoxia-induced endothelial cell death. anti-VCAM-CD39 is a novel therapeutic that can promote neuroprotection, reduce tissue damage and inflammation after hypoxic brain injury in mice. These findings suggest that anti-VCAM-CD39 could be a new avenue of cardiac arrest therapy and could potentially be used in other cerebrovascular diseases where endothelial dysfunction is a constant underlying pathology.

OBJECTIVE:There is initial evidence that the common neurotropic parasite Toxoplasma gondii is a risk factor for the development of epilepsy; however, whether it influences epileptogenesis is unknown. This study investigated whether a pre-existing chronic T. gondii infection alters epileptogenesis and neuropathology in a mouse model of mesial temporal lobe epilepsy. METHODS:Male and female C57BL/6Jax mice were intraperitoneally administered T. gondii tachyzoites or vehicle control. After 6 weeks, mice underwent self-sustained electrical status epilepticus (SSSE) through an implanted bipolar electrode, or a sham procedure. Continuous video-EEG recordings were taken 0-4- and 12-16-weeks post-SSSE to detect spontaneous seizures. Neuroinflammatory markers were assessed within 1-week post-SSSE, behavior testing was done at 8-12 weeks post-SSSE, and ex vivo MRI was conducted at 16 weeks post-SSSE. RESULTS:Male T. gondii + SSSE mice had an increased incidence of epilepsy compared to Vehicle + SSSE, while female T. gondii + SSSE mice had worse seizure severity compared to non-infected SSSE mice. There was amplified neuroinflammation in both male and female T. gondii + SSSE mice compared to Vehicle + SSSE mice. T. gondii infection in the absence of SSSE also resulted in epilepsy and neuroinflammation. MRI revealed abnormalities in brain morphology in T. gondii + SSSE male and female mice and changes in white matter integrity in male T. gondii + SSSE mice, compared to both non-infected SSSE and T. gondii control mice. SSSE and T. gondii infection impacted anxiety and spatial memory in males, and anxiety and social behavior in females. INTERPRETATION:These findings demonstrate that a chronic T. gondii infection can result in epilepsy, and that a pre-existing T. gondii infection exacerbates epileptogenesis following a brain insult, in mice.

PURPOSE:B 0 $$ {\mathrm{B}}_0 $$ and B 1 $$ {\mathrm{B}}_1 $$ inhomogeneity corrections are crucial for accurate CEST imaging, particularly at ultra-high-field MRI. WASABI provides high fidelity δ ω $$ \delta \omega $$ and B 1 $$ {\mathrm{B}}_1 $$ maps but suffers from prolonged post-processing and sensitivity to local minima. Our objective was to design an alternative to WASABI's Levenberg-Marquardt-based optimization approach to improve both post-processing speed and accuracy. METHODS:A direct relationship was derived between δ ω $$ \delta \omega $$ and B 1 $$ {\mathrm{B}}_1 $$ values and information contained in WASABI Z-spectra. Seven in vivo brain datasets were acquired at 7T. RESULTS:The proposed approach, called RAbi DIstance SearcH (RADISH), accelerated post-processing by two orders of magnitude, with improved estimation across all brain slices. Maps produced with RADISH were consistent with those produced by unartifacted areas in the original approach, with agreements within 1 Hz and 0.5% for δ ω $$ \delta \omega $$ and r B 1 $$ {\mathrm{B}}_1 $$ maps, respectively. The percentage of whole-head artifacts was reduced from 3.90% to 1.05%. CONCLUSIONS:Improvement in speed and robustness provided by RADISH allows for reliable generation of δ ω $$ \delta \omega $$ and B 1 $$ {B}_1 $$ maps, contributing to making quantitative CEST imaging at ultra-high-field more reliable and advancing its clinical feasibility.

OBJECTIVE:Current preclinical epilepsy drug screening relies on animal models that poorly reflect human neurophysiology, leading to high failure rates in clinical translation. We aimed to establish a human in vitro model using human-induced pluripotent stem cell (hiPSC)-derived cortical neurons cultured on multielectrode arrays (MEAs), capable of generating precisely controlled after-discharges (ADs) through electrical stimulation. We optimized stimulation parameters to evoke epileptiform-like hypersynchronous events and validated the model using six approved antiseizure medications (ASMs). METHODS:hiPSCs were rapidly differentiated into NGN2 cortical neurons and co-cultured with astrocytes on a 12-electrode, 24-well MEA. Network activity was tracked weekly. Upon maturation, biphasic voltage stimuli (400-2000 mV, 10 pulses at 100 Hz, 100 μs phase width) were applied in 100 ms trains to induce ADs. Stimulation intensity was increased until a maximum spike count per burst was reached. The timing of the stimulating inter-burst interval (IBI) was shortened from 10 to 1 s. We tested six ASMs with distinct mechanisms of action for their ability to attenuate induced ADs, as measured by the area under the curve (AUC) of spikes within bursts. RESULTS:A ±1000 mV stimulus was sufficient to evoke robust ADs; higher voltages caused network instability without enhancing response strength. The maximum hypersynchronous bursting rate was observed with 2 s IBIs, whereas attempts to induce more frequent events using 1 s IBIs led to desynchronization and a reduction in burst frequency below baseline. Phenytoin, perampanel, clonazepam, and lamotrigine significantly reduced AUC within 5 min in a concentration-dependent manner. Vigabatrin and levetiracetam required longer pre-incubations: AUC was reduced after 6 h for levetiracetam and at 24 h for vigabatrin. SIGNIFICANCE:We present a novel hiPSC-derived, electrically induced in vitro model for screening ASM candidates. This approach captures human-relevant epileptiform dynamics, allows fine control over stimulation parameters, and enables testing of diverse drug mechanisms. Its compatibility with high-throughput platforms makes it a promising tool for ASM discovery and personalized treatment strategies.

Objective To analyze the success of harmonization and standardization of plasma miRNA biomarker discovery and validation for post-traumatic epilepsy (PTE) in the EpiBioS4Rx international multicenter project. Methods Adult male Sprague-Dawley rats were randomized to lateral fluid-percussion-induced traumatic brain injury (TBI) or sham operation at three study sites (Finland, Australia, USA). Video-electroencephalogram (vEEG) was performed in the 7th post-injury month to detect spontaneous seizures. Tail vein plasma was collected at baseline and 48 h after TBI for microRNA (miRNA) analysis. Common data elements were generated to document and monitor pre-analytic activities, including housing conditions, post-injury care, blood sampling, plasma preparation, plasma quality, storage, and shipping. miRNA analysis was performed using droplet digital PCR (ddPCR) at one study site (Finland) with on-site standardized procedures. Results The 2-day miRNA levels were successfully measured in 85 % (209/245) of the rats included in the final analysis cohort. Exclusions were related to small sample volume, hemolysis, and failed RNA extraction for ddPCR. Most of the pre-analytical factors leading to sample exclusions were related to non-optimal plasma pipetting. We also recognized gaps in data entry and monitoring of personnel training. Conclusions Our study demonstrates that conducting a successful plasma miRNA biomarker analysis requires procedural harmonization between laboratories, protocol standardization, inclusion and analysis of quality controls, training of researchers, and continuous monitoring of adherence to pre-agreed protocols.

OBJECTIVES:Currently there are no biomarkers to predict chronic neurobehavioral and neurocognitive deficits following a traumatic brain injury (TBI). We measured brain metabolite using magnetic resonance spectroscopy in a rat model of TBI to determine their potential as biomarkers of neurobehavioral/neurocognitive outcomes after TBI. METHODS:TBI or sham surgery was conducted in 11-12 weeks old Sprague Dawley (SD) rats. Rats underwent 1H-MRS at 1 week and 1 month following TBI. Behavioural evaluations were performed between 5 and 6 months post-TBI. RESULTS:TBI rats showed significantly elevated myoinositol as well as sustained reductions in N-acetyl aspartate (NAA), gamma-aminobutyric acid (GABA), and glutamate levels compared to the sham operated rats. Behavioural assessments revealed cognitive impairment and depression-like behaviour in the TBI rats. We categorized TBI rats with (TBI+) and without (TBI-) behavioural impairment based on their performance relative to the sham rats on each assessment. TBI+ rats with cognitive impairment showed elevated myoinositol and decreased levels of glutathione (GSH), glutamate, and total choline, compared to the TBI- rats. Furthermore, TBI+ rats with depression-like behaviour demonstrated increased myoinositol and glutamate alongside decreased NAA, total choline and total creatine levels compared to the TBI- rats. Logistic regression analysis showed that certain MRS measures had good sensitivity and specificity to distinguish between the TBI+ and TBI- rats (AUC for ROC curve = 0.75-0.9, p < 0.05). CONCLUSION:We identified specific early changes in brain metabolites that predict chronic behavioural impairments after TBI. These translational findings may help understand TBI pathophysiology and help to predict functional outcomes in the clinical settings.

Traumatic brain injury (TBI) presents a major global health concern, characterized by a variety of negative long-term neurological outcomes. Current diagnostic tools lack the sensitivity to fully capture the complex pathophysiology of TBI and predict long-term consequences, underscoring the need for robust methods for biomarker detection. This study, conducted within the multicenter Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) framework, used a standardized lateral fluid-percussion injury (FPI) model to produce TBI in the left hemisphere of adult male Sprague-Dawley rats across three sites: University of Eastern Finland, Monash University, and the University of California, Los Angeles. This study utilized a novel kernel regression method for improved estimation of fiber orientations and streamline tractography to derive diffusion tensor imaging (DTI) metrics of 36 white matter tracts which were used as features to classify TBI versus sham-operated rodents scanned at 2 days (30 sham, 87 TBI), 9 days (29 sham, 84 TBI), 1 month (28 sham, 81 TBI), and 5 months (25 sham, 65 TBI) post-injury using elastic net regression regularization. A mean area under the curve (AUC) of 0.92 was achieved in correctly classifying the TBI rats in a leave-one-out cross-validation (LOOCV) framework. The results revealed delayed, region-specific effects on the microstructure of the left fimbria and left thalamic subcortical projections at 5 months following TBI. By integrating multi-compartment modeling, tractography, and harmonization, this study advances our understanding of the temporal evolution of TBI pathogenesis, paving the way for development of translational prognostic biomarkers for the risk of post-traumatic epilepsy (PTE).

The choroid plexus plays an important role in brain homeostasis, including the active secretion of cerebrospinal fluid. Its function and structure have been reported to be affected by normal ageing. However, existing measures of choroid plexus volume may be complicated by partial volume (in vivo MRI) and tissue fixation artefacts (histology). In this study, we investigate possible changes in choroid plexus volume within the lateral ventricles of aged mice utilising two structural MRI protocols explicitly designed for time-efficient, high-resolution in vivo imaging of the choroid plexus. Two MRI sequences were utilised to examine in vivo choroid plexus volume in the lateral ventricles of young (∼ 6 months) and aged (∼ 24 months) mouse brains: (1) an ultra-long echo-time T2 weighted fast-spin-echo and (2) a multi-TE T2* mapping protocol. A test-retest study was performed on a subset of the data to examine the reproducibility of choroid plexus volume estimation based on manual segmentation. A two-way ANOVA test was performed to determine possible differences in choroid plexus volume in young and aged mouse groups across the two distinct MRI protocols. Reproducibility tests showed a low test-retest variability of the manual segmentation pipeline for both MRI protocols. A statistically significant reduction of in vivo choroid plexus volume was found in the aged mouse brain. This finding is concordant with previous histological observations of a reduction in epithelial cell height with ageing across a wide range of species. We present an in vivo investigation of changes to lateral ventricle choroid plexus volume in the mouse brain utilising a manual segmentation approach based on two bespoke MRI protocols designed for time-efficient high resolution imaging of the choroid plexus. Based on these protocols, we provide evidence for a reduction in choroid plexus volume in the aged brain. This research provides insight for studies utilising MRI measurements of choroid plexus volume as a biomarker of age-related neurologic conditions as it indicates that the ageing process itself does not result in hypertrophy of the choroid plexus, but a decrease in tissue volume.

Perivascular spaces (PVSs) form a central component of the brain’s waste clearance system, the glymphatic system. These structures are visible on MRIs when enlarged, and their morphology is associated with aging and neurological disease. Manual quantification of PVS is time consuming and subjective. Numerous deep learning methods for PVS segmentation have been developed for automated segmentation. However, the majority of these algorithms have been developed and evaluated on homogenous datasets and high resolution scans, perhaps limiting their applicability for the wide range of image qualities acquired in clinical and research settings. In this work we train a nnUNet, a top-performing task driven biomedical image segmentation deep learning algorithm, on a heterogenous training sample of manually segmented MRIs of a range of different qualities and resolutions from 7 different datasets acquired on 6 different scanners. These are compared to the two currently publicly available deep learning methods for 3D segmentation of PVS, evaluated on scans with a range of resolutions and qualities. The resulting model, PINGU (Perivascular space Identification Nnunet for Generalised Usage), achieved voxel and cluster level dice scores of 0.50(SD=0.15) and 0.63(0.17) in the white matter (WM), and 0.54 (0.11) and 0.66(0.17) in the basal ganglia (BG). Performance on unseen “external” sites’ data was substantially lower for both PINGU (0.20-0.38 [WM, voxel], 0.29-0.58 [WM, cluster], 0.22-0.36 [BG, voxel], 0.46-0.60 [BG, cluster]) and the publicly available algorithms (0.18-0.30 [WM, voxel], 0.29-0.38 [WM cluster], 0.10-0.20 [BG, voxel], 0.15-0.37 [BG, cluster]). Nonetheless, PINGU strongly outperformed the publicly available algorithms, particularly in the BG. PINGU stands out as broad-use PVS segmentation tool, with particular strength in the BG, an area of PVS highly related to vascular disease and pathology.

Dysfunctional Tar DNA binding protein-43 (TDP-43) is found in approximately 95 % of all people with amyotrophic lateral sclerosis (ALS). Recent evidence suggests that the glymphatic system, which clears the brain of waste proteins, is impaired in ALS and may contribute to the accumulation of TDP-43. This study extends this work to investigate how glymphatic function changes over time in the rNLS8 doxycycline (Dox)-dependent TDP-43 mouse model of ALS. Motor function, advanced MRI biomarkers of neurodegeneration, and cortical glymphatic pathway gene expression were assessed together with dynamic contrast-enhanced MRI (DCE-MRI) assessment of glymphatic function at 0-, 3-, 7-, and 21-days after removing mice from Dox feed to initiate cytoplasmic human TDP-43 expression. A trend toward increased glymphatic influx was observed at 3-days post-Dox, together with MRI evidence of brain changes that occurred in the absence of hind-limb clasping and motor impairment. Glymphatic flow is facilitated by aquaporin-4 (AQP4) water channels polarized to astrocytic end feet. We found that while glymphatic function normalized to control levels at 7-days post-Dox, AQP4 expression in the cortex was significantly decreased. After 3-weeks of human TDP-43 expression, glymphatic dysfunction, weight loss, neurodegeneration, motor impairments and astrogliosis were observed. Our findings highlight early glymphatic dysfunction in ALS, suggesting its potential as a therapeutic target.

Mild traumatic brain injury (mTBI) is a prevalent yet often overlooked public health concern due to the absence of detectable abnormalities on CT or conventional MRI scans. Approximately 18.3%-31.3% of mTBI patients experience persistent symptoms 3-6 months post-injury, despite normal imaging results, making diagnosis and treatment challenging. In recent years, advanced neuroimaging modalities have emerged with the potential to reveal subtle physiological and structural brain changes that are invisible to traditional imaging. Diffusion MRI (dMRI), for instance, is particularly valuable for detecting white matter injury; perfusion MRI assesses alterations in cerebral blood flow; sodium MRI (23Na MRI) provides insights into ionic homeostasis; and functional MRI (fMRI) detects disruptions in functional brain network connectivity. In this review, we first explore the underlying mechanisms of mTBI and then summarize current evidence supporting the use of advanced MRI techniques to detect injury signatures associated with these mechanisms. Finally, we highlight populations at heightened risk for repeated injuries-underscoring the urgent need for more sensitive diagnostic tools that can identify injury early, guide return-to-activity decisions, and prevent cumulative brain damage. EVIDENCE LEVEL: N/A. TECHNICAL EFFICACY: Stage 3.

OBJECTIVE:To test a hypothesis that acutely regulated plasma microRNAs (miRNAs) can serve as prognostic biomarkers for the development of post-traumatic epilepsy (PTE). METHODS:Adult male Sprague-Dawley rats (n = 245) were randomized to lateral fluid-percussion-induced traumatic brain injury (TBI) or sham operation at three study sites (Finland, Australia, United States). Video-electroencephalography (vEEG) was performed on the seventh post-injury month to detect spontaneous seizures. Tail vein plasma collected 48 h after TBI for miRNA analysis was available from 209 vEEG monitored animals (45 sham, 164 TBI [32 with epilepsy]). Based on small RNA sequencing and previous data, the seven most promising brain enriched miRNAs (miR-183-5p, miR-323-3p, miR-434-3p, miR-9a-3p, miR-124-3p, miR-132-3p, and miR-212-3p) were validated by droplet digital polymerase chain reaction (ddPCR). RESULTS:All seven plasma miRNAs differentiated between TBI and sham-operated rats. None of the seven miRNAs differentiated TBI rats that did and did not develop epilepsy (p > .05), or rats with ≥3 vs <3 seizures in a month (p > .05). However, miR-212-3p differentiated rats that developed epilepsy with seizure clusters (i.e., ≥3 seizures within 24 h) from those without seizure clusters (.34 ± .14 vs .60 ± .34, adj. p < .05) with an area under the curve (AUC) of .81 (95% confidence interval [CI] .65-.97, p < .01, 64% sensitivity, 95% specificity). Lack of elevation in miR-212-3p also differentiated rats that developed epilepsy with seizure clusters from all other TBI rats (n = 146, .34 ± .14 vs .55 ± .31, p < .01) with an AUC of .74 (95% CI .61-.87, p < .01, 82% sensitivity, 62% specificity). Glmnet analysis identified a combination of miR-212-3p and miR-132-3p as an optimal set to differentiate TBI rats with vs without seizure clusters (cross-validated AUC .75, 95% CI .47-.92, p < .05). SIGNIFICANCE:miR-212-3p alone or in combination with miR-132-3p shows promise as a translational prognostic biomarker for the development of severe PTE with seizure clusters.

OBJECTIVE:E2730, an uncompetitive γ-aminobutyric acid (GABA) transporter-1 (GAT-1) inhibitor, has potent anti-seizure effects in a rodent model of chronic temporal lobe epilepsy, the kainic acid status epilepticus (KASE) rat model. In this study, we examined purported neuroimaging and physiological surrogate biomarkers of the effect of E2730 on brain GABAergic function. METHODS:We conducted a randomized cross-over study, incorporating 1-week treatments with E2730 (100 mg/kg/day subcutaneous infusion) or vehicle in epileptic post-KASE rats. KASE rats underwent serial 9.4 T magnetic resonance spectroscopy (MRS) measuring GABA and other brain metabolites, [18F]Flumazenil positron emission tomography (PET) quantifying GABAA receptor availability, quantitative electroencephalography (qEEG) and transcranial magnetic stimulation (TMS)-mediated motor activity, as well as continuous video-EEG recording to measure spontaneous seizures during each treatment. Age-matched, healthy control animals treated with E2730 or vehicle were also studied. RESULTS:E2730 treatment significantly reduced spontaneous seizures, with 8 of 11 animals becoming seizure-free. MRS revealed that E2730-treated animals had significantly reduced taurine levels. [18F]Flumazenil PET imaging revealed no changes in GABA receptor affinity or density during E2730 treatment. The power of gamma frequency oscillations in the EEG was decreased significantly in the auditory cortex and hippocampus of KASE and control rats during E2730 treatment. Auditory evoked gamma frequency power was enhanced by E2730 treatment in the auditory cortex of KASE and healthy controls, but only in the hippocampus of KASE rats. E2730 did not influence motor evoked potentials triggered by TMS. SIGNIFICANCE:This study identified clinically relevant changes in multimodality imaging and functional purported biomarkers of GABAergic activity during E2730 treatment in epileptic and healthy control animals. These biomarkers could be utilized in clinical trials of E2730 and potentially other GABAergic drugs to provide surrogate endpoints, thereby reducing the cost of such trials.

Global ischemic brain injury occurs after cardiac arrest or prolonged hypotensive episodes following surgery or trauma. It causes significant neurological deficits even after successful re-establishment of blood flow. It is the primary cause of death in 68% of inpatient and 23% of out-of-hospital cardiac arrest cases, but there are currently no treatments. Endothelial activation and dysfunction impairing small vessel blood flow is the cause of brain damage. Purinergic signaling is an endogenous molecular pathway, where CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia, eATP is released, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Our group developed a bifunctional compound – anti-VCAM-CD39 that targets dysregulated endothelium and promotes adenosine generation at the infarct site, localising antithrombotic and anti-inflammatory effects of CD39. We investigated whether anti-VCAM-CD39 could improve outcome in a murine model of global ischaemia caused by dual carotid artery ligation (DCAL). Test drugs anti-VCAM-CD39 and controls were given 3h after 30min ischaemia. Assessments at 24h included neurological function, infarct volume, perfusion, albumin extravasation to assess blood-brain barrier (BBB) permeability. We showed that there was an overall improvement in neurological deficit in αVCAM-CD39-treated mice after DCAL. MRI revealed that these mice had significantly smaller infarcts and reduced apoptotic activity on the side of permanent occlusion, compared to saline treated mice. There was reduced albumin extravasation in treated mice after DCAL, suggesting anti-VCAM-CD39 conferred neuroprotection in the brain through preservation of blood brain barrier (BBB) permeability. In vitro findings confirmed that αVCAM-CD39-mediated adenosine protected against hypoxia-induced endothelial cell death. anti-VCAM-CD39 is a novel therapeutic that can promote neuroprotection, reduce tissue damage and inflammation in the brain after hypoxic brain injury in mice. These findings suggest that anti-VCAM-CD39 could be a new avenue of cardiac arrest therapy and could potentially be used in other cerebrovascular diseases where endothelial dysfunction is a constant underlying pathology.