Brain abscess - A rare confounding factor for diagnosis of post-traumatic epilepsy after lateral fluid-percussion injury.

OBJECTIVE: To assess the prevalence of brain abscesses as a confounding factor for the diagnosis of post-traumatic epilepsy (PTE) in a rat model of lateral fluid-percussion-induced (FPI) traumatic brain injury (TBI). METHODS: This retrospective study included 583 rats from 3 study cohorts collected over 2009-2022 in a single laboratory. The rats had undergone sham-operation or TBI using lateral FPI. Rats were implanted with epidural and/or intracerebral electrodes for electroencephalogram recordings. Brains were processed for histology to screen for abscess(es). In abscess cases, (a) unfolded cortical maps were constructed to assess the cortical location and area of the abscess, (b) the abscess tissue was Gram stained to determine the presence of gram-positive and gram-negative bacteria, and (c) immunostaining was performed to detect infiltrating neutrophils, T-lymphocytes, and glial cells as tissue biomarkers of inflammation. In vivo and/or ex vivo magnetic resonance images available from a subcohort of animals were reviewed to evaluate the presence of abscesses. Plasma samples available from a subcohort of rats were used for enzyme-linked immunosorbent assays to determine the levels of lipopolysaccharide (LPS) as a circulating biomarker for gram-negative bacteria. RESULTS: Brain abscesses were detected in 2.6% (15/583) of the rats (6 sham, 9 TBI). In histology, brain abscesses were characterized as vascularized encapsulated lesions filled with neutrophils and surrounded by microglia/macrophages and astrocytes. The abscesses were mainly located under the screw electrodes, support screws, or craniectomy. Epilepsy was diagnosed in 60% (9/15) of rats with an abscess (4 sham, 5 TBI). Of these, 67% (6/9) had seizure clusters. The average seizure frequency in abscess cases was 0.436 ± 0.281 seizures/d. Plasma LPS levels were comparable between rats with and without abscesses (p > 0.05). SIGNIFICANCE: Although rare, a brain abscess is a potential confounding factor for epilepsy diagnosis in animal models of structural epilepsies following brain surgery and electrode implantation, particularly if seizures occur in sham-operated experimental controls and/or in clusters.

Biomarkers for posttraumatic epilepsy.

A biomarker is a characteristic that can be objectively measured as an indicator of normal biologic processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. Biomarker modalities include molecular, histologic, radiographic, or physiologic characteristics. To improve the understanding and use of biomarker terminology in biomedical research, clinical practice, and medical product development, the Food and Drug Administration (FDA)-National Institutes of Health (NIH) Joint Leadership Council developed the BEST Resource (Biomarkers, EndpointS, and other Tools). The seven BEST biomarker categories include the following: (a) susceptibility/risk biomarkers, (b) diagnostic biomarkers, (c) monitoring biomarkers, (d) prognostic biomarkers, (e) predictive biomarkers, (f) pharmacodynamic/response biomarkers, and (g) safety biomarkers. We hypothesize some potential overlap between the reported biomarkers of traumatic brain injury (TBI), epilepsy, and posttraumatic epilepsy (PTE). Here, we tested this hypothesis by reviewing studies focusing on biomarker discovery for posttraumatic epileptogenesis and epilepsy. The biomarker modalities reviewed here include plasma/serum and cerebrospinal fluid molecular biomarkers, imaging biomarkers, and electrophysiologic biomarkers. Most of the reported biomarkers have an area under the receiver operating characteristic curve greater than 0.800, suggesting both high sensitivity and high specificity. Our results revealed little overlap in the biomarker candidates between TBI, epilepsy, and PTE. In addition to using single parameters as biomarkers, machine learning approaches have highlighted the potential for utilizing patterns of markers as biomarkers. Although published data suggest the possibility of identifying biomarkers for PTE, we are still in the early phase of the development curve. Many of the seven biomarker categories lack PTE-related biomarkers. Thus, further exploration using proper, statistically powered, and standardized study designs with validation cohorts, and by developing and applying novel analytical methods, is needed for PTE biomarker discovery.

Postinjury weight rather than cognitive or behavioral impairment predicts development of posttraumatic epilepsy after lateral fluid-percussion injury in rats.

OBJECTIVE: To identify postinjury physiologic, behavioral, and cognitive biomarkers for posttraumatic epilepsy to enrich study populations for long-term antiepileptogenesis studies. METHODS: The EPITARGET cohort with behavioral follow-up and 1-month 24/7 video-electroencephalography (vEEG) monitoring included 115 adult male Sprague-Dawley rats with lateral fluid-percussion-induced traumatic brain injury (TBI), 23 sham-operated controls, and 13 naive rats. Animals underwent assessment of somatomotor performance (composite neuroscore), anxiety-like behavior (elevated plus maze, open field), spatial memory (Morris water maze), and depression-like behavior (Porsolt forced swim, sucrose preference). Impact force, postimpact apnea time, postimpact seizure-like behavior, and body weight were monitored. RESULTS: TBI rats were impaired in the composite neuroscore (P < .001) on days (D) 2-14 and in the spatial memory test (P < .001) on D35-39 post-TBI. Differences in the elevated plus-maze (D28 and D126) and in the open field (D29 and D127) between TBI rats and controls were meager. No differences were observed in the Porsolt forced swim and sucrose preference tests as compared with sham-operated controls. Epilepsy developed in 27% of rats by the end of the sixth month. None of the behavioral or cognitive outcome measures discriminated rats with or without epilepsy. The receiver-operating characteristic analysis indicated that a decrease in body weight between D0 and D4 differentiated TBI rats with epilepsy from TBI rats without epilepsy (48% sensitivity, 83% specificity, area under the curve [AUC] 0.679, confidence interval [CI] 95% 0.56-0.80, P < .01). A 16% body weight decrease during D0-D4 could be used as a biomarker to enrich the study population from 27% (observed) to 50%. SIGNIFICANCE: Single behavioral and cognitive outcome measures showed no power as prognostic/diagnostic biomarkers for posttraumatic epilepsy. A reduction in body weight during the first postinjury week showed some prognostic value for posttraumatic epileptogenesis and could serve as a subacute measure for selectively enriching the study population for long-term preclinical biomarker and therapy discovery studies of posttraumatic epileptogenesis.

Plau/Plaur double-deficiency did not worsen lesion severity or vascular integrity after traumatic brain injury.

Binding of urokinase-type plasminogen activator receptor (uPAR) to its ligand uPA or to its plasma membrane partner, platelet-derived growth factor receptor ß (PDGFRß), promotes neuroprotection, cell proliferation, and angiogenesis. Following injury, single deficiency in uPA or uPAR leads in increased tissue loss and compromised vascular remodeling. We hypothesized that double-deficiency of uPAR (Plaur) and uPA (Plau) would result in increased lesion area and poor vascular integrity after traumatic brain injury (TBI). TBI was induced by lateral fluid-percussion injury in Plau/Plaur double-knockout (dKO) and wild-type (Wt) mice. The cortical lesion area was quantified in unfolded cortical maps prepared from thionin-stained sections at 4 d or 30 d post-TBI. The density of PDGFRß+ pericytes and blood vessels was calculated from immunostained sections. Blood-brain barrier leakage was analyzed using ImageJ® from IgG-immunostained sections. Genotype had no effect on the total area of the cortical lesion at 4 d or 30 d post-TBI (p > 0.05) or its progression as the overall lesion area was comparable at 4 d and 30 d post-TBI in both genotypes (p > 0.05). Subfield analysis, however, indicated that damage to the visual cortex at 4 d post-TBI in dKO-TBI mice was 53 % of that in Wt-TBI mice (p < 0.05). Both genotypes had a higher density of PDGFRß-positive pericytes at 4 d than at 30 d post-TBI (p < 0.05), but no genotype effect was detected between these time-points (p > 0.05). TBI-induced increase in the density of PDGFRß+ blood vessels at the region adjacent to the lesion core was comparable in both genotypes (p > 0.05). Genotype had no effect on TBI-induced IgG leakage into the perilesional cortical parenchyma (p > 0.05). Contrary to our expectations, Plau/Plaur double-deficiency did not aggravate TBI-related structural outcome.

In Vitro and In Vivo Pipeline for Validation of Disease-Modifying Effects of Systems Biology-Derived Network Treatments for Traumatic Brain Injury-Lessons Learned.

We developed a pipeline for the discovery of transcriptomics-derived disease-modifying therapies and used it to validate treatments in vitro and in vivo that could be repurposed for TBI treatment. Desmethylclomipramine, ionomycin, sirolimus and trimipramine, identified by in silico LINCS analysis as candidate treatments modulating the TBI-induced transcriptomics networks, were tested in neuron-BV2 microglial co-cultures, using tumour necrosis factor α as a monitoring biomarker for neuroinflammation, nitrite for nitric oxide-mediated neurotoxicity and microtubule associated protein 2-based immunostaining for neuronal survival. Based on (a) therapeutic time window in silico, (b) blood-brain barrier penetration and water solubility, (c) anti-inflammatory and neuroprotective effects in vitro (p < 0.05) and (d) target engagement of Nrf2 target genes (p < 0.05), desmethylclomipramine was validated in a lateral fluid-percussion model of TBI in rats. Despite the favourable in silico and in vitro outcomes, in vivo assessment of clomipramine, which metabolizes to desmethylclomipramine, failed to demonstrate favourable effects on motor and memory tests. In fact, clomipramine treatment worsened the composite neuroscore (p < 0.05). Weight loss (p < 0.05) and prolonged upregulation of plasma cytokines (p < 0.05) may have contributed to the worsened somatomotor outcome. Our pipeline provides a rational stepwise procedure for evaluating favourable and unfavourable effects of systems-biology discovered compounds that modulate post-TBI transcriptomics.

Deficiency of urokinase-type plasminogen activator and its receptor affects social behavior and increases seizure susceptibility.

Extracellular proteolysis initiated by the binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) regulates the development of inhibitory neuronal circuits in the cerebral cortex and tissue remodeling after epileptogenic brain injury. To study the function of different components of the uPA-uPAR system on behavior and epileptogenesis, and to complement our previous studies on naïve and injured mice deficient in the uPA-encoding gene Plau or the uPAR-encoding gene Plaur, we analyzed the behavioral phenotype, seizure susceptibility, and perineuronal nets surrounding parvalbumin-positive inhibitory interneurons in Plau and Plaur (double knockout dKO) mice. In a climbing test, dKO mice showed reduced interest towards the environment as compared with Wt mice (p < 0.01). In a social approach test, however, dKO mice spent more time than Wt mice exploring the compartment containing a stranger mouse than the empty compartment (p < 0.05). Moreover, in a social interaction test, dKO mice exhibited increased contact time (p < 0.01). Compared with Wt mice, the dKO mice also had a longer single contact duration (p < 0.001) with the stranger mouse. In the elevated plus-maze, grooming, and marble burying tests, the anxiety level of dKO mice did not differ from that of Wt mice. Rearing time in an exploratory activity test, and spatial learning and memory in the Morris swim navigation task were also comparable between dKO and Wt mice. In the pentylenetetrazol (PTZ) seizure-susceptibility test, dKO mice had a shorter latency to the first epileptiform spike (p = 0.0001) and a greater total number of spikes (p < 0.001) than Wt mice. The dKO genotype did not affect the number of cortical perineuronal nets. Our findings indicate that Plau/Plaur-deficiency leads to a more social phenotype toward other mice with diminished interest in the surrounding environment, and increased seizure susceptibility.

Epilepsy biomarkers - Toward etiology and pathology specificity.

A biomarker is a characteristic that is measured as an indicator of normal biologic processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. Biomarker modalities include molecular, histologic, radiographic, or physiologic characteristics. In 2015, the FDA-NIH Joint Leadership Council developed the BEST Resource (Biomarkers, EndpointS, and other Tools) to improve the understanding and use of biomarker terminology in biomedical research, clinical practice, and medical product development. The BEST biomarker categories include: (a) susceptibility/risk biomarkers, (b) diagnostic biomarkers, (c) monitoring biomarkers, (d) prognostic biomarkers, (e) predictive biomarkers, (f) pharmacodynamic/response biomarkers, and (g) safety biomarkers. Here we review 30 epilepsy biomarker studies that have identified (a) diagnostic biomarkers for epilepsy, epileptogenesis, epileptogenicity, drug-refractoriness, and status epilepticus - some of the epileptogenesis and epileptogenicity biomarkers can also be considered prognostic biomarkers for the development of epilepsy in subjects with a given brain insult, (b) predictive biomarkers for epilepsy surgery outcome, and (c) a response biomarker for therapy outcome. The biomarker modalities include plasma/serum/exosomal and cerebrospinal fluid molecular biomarkers, brain tissue molecular biomarkers, imaging biomarkers, electrophysiologic biomarkers, and behavioral/cognitive biomarkers. Both single and combinatory biomarkers have been described. Most of the reviewed biomarkers have an area under the curve >0.800 in receiver operating characteristics analysis, suggesting high sensitivity and specificity. As discussed in this review, we are in the early phase of the learning curve in epilepsy biomarker discovery. Many of the seven biomarker categories lack epilepsy-related biomarkers. There is a need for epilepsy biomarker discovery using proper, statistically powered study designs with validation cohorts, and the development and use of novel analytical methods. A strategic roadmap to discuss the research priorities in epilepsy biomarker discovery, regulatory issues, and optimization of the use of resources, similar to those devised in the cancer and Alzheimer's disease research areas, is also needed.

Unfolded Maps for Quantitative Analysis of Cortical Lesion Location and Extent after Traumatic Brain Injury.

We aimed to generate two-dimensional (2D) unfolded cortical maps from magnetic resonance (MR) images to delineate the location of traumatic brain injury (TBI)-induced cortical damage in functionally diverse cytoarchitectonic areas of the cerebral cortex, and to predict the severity of functional impairment after TBI based on the lesion location and extent. Lateral fluid-percussion injury was induced in adult rats and T2 maps were acquired with magnetic resonance imaging (MRI) at 3 days post-TBI. Somatomotor deficits were assessed based on the composite neuroscore and beam balance test, and spatial learning was assessed in the Morris water maze. Animals were perfused for histology at 13 days post-injury. A 2D template was generated by unfolding the cerebral cortex from 26 sections of the rat brain atlas, covering the lesion extent. Next, 2D unfolded maps were generated from T2 maps and thionin-stained histological sections from the same animals. Unfolding of the T2 maps revealed the lesion core in the auditory, somatosensory, and visual cortices. The unfolded histological lesion at 13 days post-injury was 12% greater than the MRI lesion at 3 days post-TBI, as the lesion area increased laterally and caudally; the larger the MRI lesion area, the larger the histological lesion area. Further, the larger the MRI lesion area in the barrel field of the primary somatosensory cortex (S1BF), upper lip of the primary somatosensory cortex (S1ULp), secondary somatosensory division (S2), and ectorhinal (Ect) and perirhinal (PRh) cortices, the more impaired the performance in the beam balance and Morris water maze tests. Subsequent receiver operating characteristic analysis indicated that severity of the MRI lesion in S1ULp and S2 was a sensitive and specific predictor of poor performance in the beam balance test. Moreover, MRI lesions in the S1ULp, S2, S1BF, and Ect and PRh cortices predicted poor performance in the Morris water maze test. Our findings indicate that 2D-unfolded cortical maps generated from MR images delineate the distribution of cortical lesions in functionally different cytoarchitectonic regions, which can be used to predict the TBI-induced functional impairment.

Dynamics of PDGFRß expression in different cell types after brain injury.

Platelet-derived growth factor receptor ß (PDGFRß) is upregulated after brain injury and its depletion results in the blood-brain barrier (BBB) damage. We investigated the time-window and localization of PDGFRß expression in mice with intrahippocampal kainic acid-induced status epilepticus (SE) and in rats with lateral fluid-percussion-induced traumatic brain injury (TBI). Tissue immunohistochemistry was evaluated at several time-points after SE and TBI. The distribution of PDGFRß was analyzed, and its cell type-specific expression was verified with double/triple-labeling of astrocytes (GFAP), NG2 cells, and endothelial cells (RECA-1). In normal mouse hippocampus, we found evenly distributed PDGFRß+ parenchymal cells. In double-labeling, all NG2+ and 40%-60% GFAP+ cells were PDGFRß+. After SE, PDGFRß+ cells clustered in the ipsilateral hilus (178% of that in controls at fourth day, 225% at seventh day, P < 0.05) and in CA3 (201% at seventh day, P < 0.05), but the total number of PDGFRß+ cells was not altered. As in controls, PDGFRß-immunoreactivity was detected in parenchymal NG2+ and GFAP+ cells. We also observed PDGFRß+ structural pericytes, detached reactive pericytes, and endothelial cells. After TBI, PDGFRß+ cells clustered in the perilesional cortex and thalamus, particularly during the first post-injury week. PDGFRß immunopositivity was observed in NG2+ and GFAP+ cells, structural pericytes, detached reactive pericytes, and endothelial cells. In some animals, PDGFRß vascular staining was observed around the cortical glial scar for up to 3 months. Our data revealed an acute accumulation of PDGFRß+ BBB-related cells in degenerating brain areas, which can be long lasting, suggesting an active role for PDGFRß-signaling in blood vessel and post-injury tissue recovery. GLIA 2017;65:322-341.