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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. [ABSTRACT FROM AUTHOR]

Progress in the preclinical and clinical development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI) necessitates the discovery of prognostic biomarkers for post-injury outcome. Our previous mRNA-seq data revealed a 1.8–2.5 fold increase in clusterin mRNA expression in lesioned brain areas in rats with lateral fluid-percussion injury (FPI)-induced TBI. On this basis, we hypothesized that TBI leads to increases in the brain levels of clusterin protein, and consequently, increased plasma clusterin levels. For evaluation, we induced TBI in adult male Sprague-Dawley rats (n = 80) by lateral FPI. We validated our mRNA-seq findings with RT-qPCR, confirming increased clusterin mRNA levels in the perilesional cortex (FC 3.3, p < 0.01) and ipsilateral thalamus (FC 2.4, p < 0.05) at 3 months post-TBI. Immunohistochemistry revealed a marked increase in extracellular clusterin protein expression in the perilesional cortex and ipsilateral hippocampus (7d to 1 month post-TBI), and ipsilateral thalamus (14d to 12 months post-TBI). In the thalamus, punctate immunoreactivity was most intense around activated microglia and mitochondria. Enzyme-linked immunoassays indicated that an acute 15% reduction, rather than an increase in plasma clusterin levels differentiated animals with TBI from sham-operated controls (AUC 0.851, p < 0.05). Our findings suggest that plasma clusterin is a candidate biomarker for acute TBI diagnosis. [ABSTRACT FROM AUTHOR]

We assessed the effect of antioxidant therapy using the Food and Drug Administration-approved respiratory drug N-acetylcysteine (NAC) or sulforaphane (SFN) as monotherapies or duotherapy in vitro in neuron-BV2 microglial co-cultures and validated the results in a lateral fluid-percussion model of TBI in rats. As in vitro measures, we assessed neuronal viability by microtubule-associated-protein 2 immunostaining, neuroinflammation by monitoring tumor necrosis factor (TNF) levels, and neurotoxicity by measuring nitrite levels. In vitro, duotherapy with NAC and SFN reduced nitrite levels to 40% (p < 0.001) and neuroinflammation to –29% (p < 0.001) compared with untreated culture. The treatment also improved neuronal viability up to 72% of that in a positive control (p < 0.001). The effect of NAC was negligible, however, compared with SFN. In vivo, antioxidant duotherapy slightly improved performance in the beam walking test. Interestingly, duotherapy treatment decreased the plasma interleukin-6 and TNF levels in sham-operated controls (p < 0.05). After TBI, no treatment effect on HMGB1 or plasma cytokine levels was detected. Also, no treatment effects on the composite neuroscore or cortical lesion area were detected. The robust favorable effect of duotherapy on neuroprotection, neuroinflammation, and oxidative stress in neuron-BV2 microglial co-cultures translated to modest favorable in vivo effects in a severe TBI model. [ABSTRACT FROM AUTHOR]

• Fluid-percussion injury induces patchy loss of parvalbumin positive interneurons in the perilesional cortex. • Interneuron loss is accompanied by a robust regulation of genes related to GABA A receptor-mediated inhibition. • Traumatic brain injury induces both presynaptic and postsynaptic deficiency in GABAergic markers in the perilesional cortex. Traumatic brain injury (TBI) causes 10–20% of structural epilepsy, with seizures typically originating in the cortex. Alterations in the neuronal microcircuits in the cortical epileptogenic zone, however, are poorly understood. Here, we assessed TBI-induced changes in perisomatic gamma aminobutyric acid (GABA)-ergic innervation in the perilesional cortex. We hypothesized that TBI will damage parvalbumin (PV)-immunoreactive inhibitory neurons and induce regulation of the associated GABAergic molecular interactome. TBI was induced in adult male Sprague-Dawley rats by lateral fluid-percussion injury. At 1-month post-TBI, the number of PV-positive somata was plotted on unfolded cortical maps and the distribution and density of immunopositive terminals analyzed. Qualitative analysis revealed either patchy microlesions of several hundred micrometers in diameter or diffuse neuronal loss. Quantitative analysis demonstrated a reduction in the number of PV-positive interneurons in patches down to 0% of that in sham-operated controls in the perilesional cortex. In the majority of patches, the cell numbers ranged from 71% to 90% that of the controls. The loss of PV-positive somata was accompanied by decreased axonal labeling. In situ hybridization revealed downregulated PV mRNA expression in the perilesional cortex. Gene Set Enrichment Analysis indicated a robustly downregulated expression profile of PV-related genes, which was confirmed by quantitative reverse transcriptase polymerase chain reaction. Specifically, we found that genes encoding postsynaptic GABA-A receptor genes, Gabrg2 and Gabrd, were downregulated in TBI animals compared with controls. Our data suggests that patchy reduction in PV-positive perisomatic inhibitory innervation contributes to the development of focal cortical inhibitory deficit after TBI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are small vesicles involved in intercellular communication. Data is emerging that EVs and their cargo have potential as diagnostic biomarkers and treatments for brain diseases, including traumatic brain injury and epilepsy. Here, we summarize the current knowledge regarding changes in EV numbers and cargo in status epilepticus (SE) and traumatic brain injury (TBI), which are clinically significant etiologies for acquired epileptogenesis in animals and humans. We also review encouraging data, which suggests that EVs secreted by stem cells may serve as recovery-enhancing treatments for SE and TBI. Using Gene Set Enrichment Analysis, we show that brain EV-related transcripts are positively enriched in rodent models of epileptogenesis and epilepsy, and altered in response to anti-seizure drugs. These data suggest that EVs show promise as biomarkers, treatments and drug targets for epilepsy. In parallel to gathering conceptual knowledge, analytics platforms for the isolation and analysis of EV contents need to be further developed. [ABSTRACT FROM AUTHOR]

Our objective was to define the chronically altered gene expression signature of traumatic brain injury (TBI-sig), and apply it to discover novel treatments in order to reverse pathologic gene expression or reinforce the expression of recovery-related genes. TBI was induced with lateral fluid-percussion injury in adult male rats. Genome-wide RNA-seq of the perilesional cortex, ipsilateral thalamus and ipsilateral hippocampus was performed at 3 months post-TBI. Data highlighted chronic transcriptional changes, particularly, in the perilesional cortex (4 964 regulated genes) and thalamus (1 966)(FDR < 0.05). Gene set enrichment analysis (GSEA) indicated down-regulation of ion channel and mitochondrial gene sets and upregulation of inflammatory gene sets (GSEA q-value < 0.01). Transcriptomics data were used to design the TBI-sig which was used in LINCS analysis. We identified 18 of 1064 compounds which regulated gene expression in three cell lines with strong connectivity score with TBI-sig. Eleven of these 18 compounds showed a strong connectivity with TBI-sig in neuronal cell lines. Two of 11 compounds (celecobix, sirolimus) were recently shown to have a disease-modifying effect in in vivo animal models of epilepsy. The nine other compounds revealed by the analysis were (BRD-K91844626; BRD-A11009626; NO-ASA; BRD-K55260239; SDZ-NKT-343; STK-661558; BRD-K75971499; ionomycin; desmethylclomipramine). Transcriptomics signatures of the 11 compounds were compared in more details with TBI-sig. IPA analysis of overlapping genes revealed the effects of these compounds on tubulins (Tubb2a, Tubb3, Tubb4b), Nfe2l2, S100a4, Cd44, and Nfkb2, which have been linked to TBI-relevant outcomes, including epileptogenesis and tissue repair. From all compounds revealed by the LINCS analysis, desmetylclomipramine, an active metabolite of antidepressant clomipramine, modulated most of the gene targets considered favorable for the TBI outcome. Our data demonstrate long-lasting transcriptomics changes after TBI in the perilesional cortex and thalamus, suggesting a long-lasting therapeutic window. LINCS analysis predicted that these changes can be modulated by various compounds, some of which are already in clinical use. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) induces a wide variety of cellular and molecular changes that can continue for days to weeks to months, leading to functional impairments. Currently, there are no pharmacotherapies in clinical use that favorably modify the post-TBI outcome, due in part to limited understanding of the mechanisms of TBI-induced pathologies. Our system biology analysis tested the hypothesis that chronic transcriptomics changes induced by TBI are controlled by altered DNA-methylation in gene promoter areas or by transcription factors. We performed genome-wide methyl binding domain (MBD)-sequencing (seq) and RNA-seq in perilesional, thalamic, and hippocampal tissue sampled at 3 months after TBI induced by lateral fluid percussion in adult male Sprague-Dawley rats. We investigated the regulated molecular networks and mechanisms underlying the chronic regulation, particularly DNA methylation and transcription factors. Finally, we identified compounds that modulate the transcriptomics changes and could be repurposed to improve recovery. Unexpectedly, DNA methylation was not a major regulator of chronic post-TBI transcriptomics changes. On the other hand, the transcription factors Cebpd, Pax6, Spi1, and Tp73 were upregulated at 3 months after TBI (False discovery rate < 0.05), which was validated using digital droplet polymerase chain reaction. Transcription regulatory network analysis revealed that these transcription factors regulate apoptosis, inflammation, and microglia, which are well-known contributors to secondary damage after TBI. Library of Integrated Network-based Cellular Signatures (LINCS) analysis identified 118 pharmacotherapies that regulate the expression of Cebpd, Pax6, Spi1, and Tp73. Of these, the antidepressant and/or antipsychotic compounds trimipramine, rolipramine, fluspirilene, and chlorpromazine, as well as the anti-cancer therapies pimasertib, tamoxifen, and vorinostat were strong regulators of the identified transcription factors, suggesting their potential to modulate the regulated transcriptomics networks to improve post-TBI recovery. [ABSTRACT FROM AUTHOR]

• Plau/Plaur deficiency is mildly neuroprotective after TBI. • Plau/Plaur deficiency does not regulate PDGFRβ+ pericyte expression or location. • Plau/Plaur deficiency does not reduce PDGFRβ+ blood vessel density. • Plau/Plaur deficiency does not increase blood-brain barrier dysfunction after TBI. Binding of urokinase-type plasminogen activator receptor (uPAR) to its ligand uPA or to its plasma membrane partner, platelet-derived growth factor receptor β (PDGFRβ), 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 PDGFRβ+ 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 PDGFRβ-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 PDGFRβ+ 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. [ABSTRACT FROM AUTHOR]