Dan Z. Milikovsky

Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood–brain barrier dysfunction

Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

Imaging Blood-Brain Barrier Dysfunction in Football Players

Imaging Blood-Brain Barrier Dysfunction in Football Players There has been an increasing awareness of the long-term neuropsychiatric pathologies associated with repeated mild traumatic brain injury (mTBI) and specifically sports-related concussive and subconcussive head impacts.1 While mTBI had been associated with diffusion tensor imaging evidence of diffusivity changes in soccer,2 American football, and hockey players,3 the mechanisms underlying the development of post-mTBI neurodegenerative complications are poorly understood. Accumulating evidence points to vascular pathology and dysfunction of the blood-brain barrier (BBB) as a potential link between severe TBI and neurodegeneration.4 Moreover, participation in American football has been associated with changes in blood proteins reflecting BBB leakage.5 Thus, here we set out to visualize the extent and location of BBB dysfunction in football players using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).

Differential TGF-β Signaling in Glial Subsets Underlies IL-6–Mediated Epileptogenesis in Mice

TGF-β1 is a master cytokine in immune regulation, orchestrating both pro- and anti-inflammatory reactions. Recent studies show that whereas TGF-β1 induces a quiescent microglia phenotype, it plays a pathogenic role in the neurovascular unit and triggers neuronal hyperexcitability and epileptogenesis. In this study, we show that, in primary glial cultures, TGF-β signaling induces rapid upregulation of the cytokine IL-6 in astrocytes, but not in microglia, via enhanced expression, phosphorylation, and nuclear translocation of SMAD2/3. Electrophysiological recordings show that administration of IL-6 increases cortical excitability, culminating in epileptiform discharges in vitro and spontaneous seizures in C57BL/6 mice. Intracellular recordings from layer V pyramidal cells in neocortical slices obtained from IL-6–treated mice show that during epileptogenesis, the cells respond to repetitive orthodromic activation with prolonged after-depolarization with no apparent changes in intrinsic membrane properties. Notably, TGF-β1–induced IL-6 upregulation occurs in brains of FVB/N but not in brains of C57BL/6 mice. Overall, our data suggest that TGF-β signaling in the brain can cause astrocyte activation whereby IL-6 upregulation results in dysregulation of astrocyte–neuronal interactions and neuronal hyperexcitability. Whereas IL-6 is epileptogenic in C57BL/6 mice, its upregulation by TGF-β1 is more profound in FVB/N mice characterized as a relatively more susceptible strain to seizure-induced cell death.

Imaging blood–brain barrier dysfunction as a biomarker for epileptogenesis

A biomarker that will enable the identification of patients at high-risk for developing post-injury epilepsy is critically required. Microvascular pathology and related blood-brain barrier dysfunction and neuroinflammation were shown to be associated with epileptogenesis after injury. Here we used prospective, longitudinal magnetic resonance imaging to quantitatively follow blood-brain barrier pathology in rats following status epilepticus, late electrocorticography to identify epileptic animals and post-mortem immunohistochemistry to confirm blood-brain barrier dysfunction and neuroinflammation. Finally, to test the pharmacodynamic relevance of the proposed biomarker, two anti-epileptogenic interventions were used; isoflurane anaesthesia and losartan. Our results show that early blood-brain barrier pathology in the piriform network is a sensitive and specific predictor (area under the curve of 0.96, P < 0.0001) for epilepsy, while diffused pathology is associated with a lower risk. Early treatments with either isoflurane anaesthesia or losartan prevented early microvascular damage and late epilepsy. We suggest quantitative assessment of blood-brain barrier pathology as a clinically relevant predictive, diagnostic and pharmaco!dynamics biomarker for acquired epilepsy.

Electrocorticographic dynamics as a novel biomarker in five models of epileptogenesis

Postinjury epilepsy (PIE) is a devastating sequela of various brain insults. While recent studies offer novel insights into the mechanisms underlying epileptogenesis and discover potential preventive treatments, the lack of PIE biomarkers hinders the clinical implementation of such treatments. Here we explored the biomarker potential of different electrographic features in five models of PIE. Electrocorticographic or intrahippocampal recordings of epileptogenesis (from the insult to the first spontaneous seizure) from two laboratories were analyzed in three mouse and two rat PIE models. Time, frequency, and fractal and nonlinear properties of the signals were examined, in addition to the daily rate of epileptiform spikes, the relative power of five frequency bands (theta, alpha, beta, low gamma, and high gamma) and the dynamics of these features over time. During the latent pre-seizure period, epileptiform spikes were more frequent in epileptic compared with nonepileptic rodents; however, this feature showed limited predictive power due to high inter- and intra-animal variability. While nondynamic rhythmic representation failed to predict epilepsy, the dynamics of the theta band were found to predict PIE with a sensitivity and specificity of >90%. Moreover, theta dynamics were found to be inversely correlated with the latency period (and thus predict the onset of seizures) and with the power change of the high-gamma rhythm. In addition, changes in theta band power during epileptogenesis were associated with altered locomotor activity and distorted circadian rhythm. These results suggest that changes in theta band during the epileptogenic period may serve as a diagnostic biomarker for epileptogenesis, able to predict the future onset of spontaneous seizures. SIGNIFICANCE STATEMENT Postinjury epilepsy is an unpreventable and devastating disorder that develops following brain injuries, such as traumatic brain injury and stroke, and is often associated with neuropsychiatric comorbidities. As PIE affects as many as 20% of brain-injured patients, reliable biomarkers are imperative before any preclinical therapeutics can find clinical translation. We demonstrate the capacity to predict the epileptic outcome in five different models of PIE, highlighting theta rhythm dynamics as a promising biomarker for epilepsy. Our findings prompt the exploration of theta dynamics (using repeated electroencephalographic recordings) as an epilepsy biomarker in brain injury patients.

TGFβ signaling is associated with changes in inflammatory gene expression and perineuronal net degradation around inhibitory neurons following various neurological insults

Brain damage due to stroke or traumatic brain injury (TBI), both leading causes of serious long-term disability, often leads to the development of epilepsy. Patients who develop post-injury epilepsy tend to have poor functional outcomes. Emerging evidence highlights a potential role for blood-brain barrier (BBB) dysfunction in the development of post-injury epilepsy. However, common mechanisms underlying the pathological hyperexcitability are largely unknown. Here, we show that comparative transcriptome analyses predict remodeling of extracellular matrix (ECM) as a common response to different types of injuries. ECM-related transcriptional changes were induced by the serum protein albumin via TGFβ signaling in primary astrocytes. In accordance with transcriptional responses, we found persistent degradation of protective ECM structures called perineuronal nets (PNNs) around fast-spiking inhibitory interneurons, in a rat model of TBI as well as in brains of human epileptic patients. Exposure of a naïve brain to albumin was sufficient to induce the transcriptional and translational upregulation of molecules related to ECM remodeling and the persistent breakdown of PNNs around fast-spiking inhibitory interneurons, which was contingent on TGFβ signaling activation. Our findings provide insights on how albumin extravasation that occurs upon BBB dysfunction in various brain injuries can predispose neural circuitry to the development of chronic inhibition deficits.

Dynamic changes in murine forebrain miR-211 expression associate with cholinergic imbalances and epileptiform activity

Significance Acute traumatic stress increases the sensitivity to develop epileptic seizures in certain people. It is therefore important to discover physiological mechanisms that avoid epilepsy. To test if rapidly inducible microRNAs (miRs) could mediate such protection, we combined mouse engineering, RNA sequencing, electric recording of brain activity, and learning tests. We discovered that miR-211, originating from an epilepsy-related genomic locus, may be involved, and therefore engineered mice produce a drug-suppressible excess of brain miR-211. In these mice, suppressing miR-211 excess to the original expression levels in normal brains led to electrically recorded epilepsy and hypersensitivity to epilepsy-inducing compounds; it also modified acetylcholine receptor composition. The functional impact of miR-211 dynamics on seizure threshold may enable future development of miR-211–directed therapeutics. Epilepsy is a common neurological disease, manifested in unprovoked recurrent seizures. Epileptogenesis may develop due to genetic or pharmacological origins or following injury, but it remains unclear how the unaffected brain escapes this susceptibility to seizures. Here, we report that dynamic changes in forebrain microRNA (miR)-211 in the mouse brain shift the threshold for spontaneous and pharmacologically induced seizures alongside changes in the cholinergic pathway genes, implicating this miR in the avoidance of seizures. We identified miR-211 as a putative attenuator of cholinergic-mediated seizures by intersecting forebrain miR profiles that were Argonaute precipitated, synaptic vesicle target enriched, or differentially expressed under pilocarpine-induced seizures, and validated TGFBR2 and the nicotinic antiinflammatory acetylcholine receptor nAChRa7 as murine and human miR-211 targets, respectively. To explore the link between miR-211 and epilepsy, we engineered dTg-211 mice with doxycycline-suppressible forebrain overexpression of miR-211. These mice reacted to doxycycline exposure by spontaneous electrocorticography-documented nonconvulsive seizures, accompanied by forebrain accumulation of the convulsive seizures mediating miR-134. RNA sequencing demonstrated in doxycycline-treated dTg-211 cortices overrepresentation of synaptic activity, Ca2+ transmembrane transport, TGFBR2 signaling, and cholinergic synapse pathways. Additionally, a cholinergic dysregulated mouse model overexpressing a miR refractory acetylcholinesterase-R splice variant showed a parallel propensity for convulsions, miR-211 decreases, and miR-134 elevation. Our findings demonstrate that in mice, dynamic miR-211 decreases induce hypersynchronization and nonconvulsive and convulsive seizures, accompanied by expression changes in cholinergic and TGFBR2 pathways as well as in miR-134. Realizing the importance of miR-211 dynamics opens new venues for translational diagnosis of and interference with epilepsy.

Following a potential epileptogenic insult, prolonged high rates of nonlinear dynamical regimes of intermittency type is the hallmark of epileptogenesis

The lack of a marker of epileptogenesis is an unmet medical need, not only from the clinical perspective but also from the point of view of the pre-clinical research. Indeed, the lack of this kind of marker affects the investigations on the mechanisms of epileptogenesis as well as the development of novel therapeutic approaches aimed to prevent or to mitigate the severity of the incoming epilepsy in humans. In this work, we provide evidence that in an experimental model of epileptogenesis that mimics the alteration of the blood-brain barrier permeability, a key-mechanism that contributes to the development of epilepsy in humans and in animals, the prolonged occurrence in the electrocorticograms (ECoG) of high rates of a nonlinear dynamical regimes known as intermittency univocally characterizes the population of experimental animals which develop epilepsy, hence it can be considered as the first biophysical marker of epileptogenesis.

Willingness to treat infectious diseases: what do students think?

Introduction Outbreaks of serious communicable infectious diseases remain a major global medical problem and force healthcare workers to make hard choices with limited information, resources and time. While information regarding physicians’ opinions about such dilemmas is available, research discussing students’ opinions is more limited. Methods Medical students were surveyed about their willingness to perform medical procedures on patients with communicable diseases as students and as physicians. Students were asked about their opinions regarding the duty to treat in such cases. Results 74% of respondents felt that by deciding to enter medical school they were morally obliged to treat any patient despite the risks. Students’ willingness to treat as physicians is significantly higher than their willingness to treat as students. HIV was significantly the most tolerated disease with respect to performing mouth to mouth resuscitation. Among preclinical students, we found that willingness to treat during the later years is significantly greater than during the earlier years. Among clinical students, the opposite was observed. Discussion Students’ greater willingness to treat as physicians is mostly attributed to perceptions of higher obligations as a qualified doctor. There is greater but not total willingness to perform resuscitation on patients with HIV relative to other diseases. The increased willingness of preclinical students and the decreased willingness of clinical students both emphasise the importance of patient–physician communication and ethics studies during medical school.

Differential TGF-beta signaling in glial subsets underlies IL-6-mediated epileptogenesis in mice

Histopathological studies confirm the prominent deposition of immunoglobulins (Ig) and the complement activation in acute demyelinating lesions of multiple sclerosis (MS) patients, suggesting that, at least in a subgroup of MS patients, autoantibodies could contribute to the disease. Recently, the ATP-sensitive inward rectifying potassium channel KIR4.1 has been identified as an antibody target in a small cohort of MS patients, since anti-KIR4.1 antibodies (Abs) were found in the serum of MS patients compared to those with other neurological disorders and healthy donors. Indeed, enhanced KIR4.1 expression was reported either in the brains of cuprizone-induced demyelination mouse model and in reactive astrocytes present at the borders of active MS lesions, suggesting that an abnormal KIR4.1 expression in inflamedMS brainsmight facilitate autoimmune sensitization against this channel. In the current study, we further investigated KIR4.1 expression in association with astrogliosis (GFAP), oligodendrocyte loss (MBP, CNPase) and neurodegeneration (beta-tubulin) in the cuprizone demyelinating model. C57BL/6 male mice were fed with cuprizone (0.2% w/w) for 5 weeks to induce acute demyelination. As control, agematched mice received normal chow diet. We observed a strong localization of KIR4.1 on GFAP-positive astrocytes in the cortex of mouse brain sections. Furthermore, serial brain sections from cuprizone-fed and control mice were immunostained with an MS serum resulted positive for anti-KIR4.1 Ab by ELISA assay using synthetic KIR4.1 peptides; confocal microscopy analysis showed a comparable immunolabelling pattern between MS serum and polyclonal KIR4.1 antibodies, suggesting that the KIR4.1-Abs in the MS serum indeed react with the native KIR4.1 antigen. KIR4.1 expression has also been investigated in experimental autoimmune encephalitis. Our data point further support the possible involvement of KIR4.1 as a candidate autoantigen in MS and are in line with recent reports. In this context, it might be relevant to address whether ‘danger’ signals alter KIR4.1 expression, as might occur in the inflammatory response to Theilers virus-induced demyelination model.