Human brain slices for epilepsy research:pitfalls, solutions and future challenges

Increasingly, neuroscientists are taking the opportunity to use live human tissue obtained from elective neurosurgical procedures for electrophysiological studies in vitro. Access to this valuable resource permits unique studies into the network dynamics that contribute to the generation of pathological electrical activity in the human epileptic brain. Whilst this approach has provided insights into the mechanistic features of electrophysiological patterns associated with human epilepsy, it is not without technical and methodological challenges. This review outlines the main difficulties associated with working with epileptic human brain slices from the point of collection, through the stages of preparation, storage and recording. Moreover, it outlines the limitations, in terms of the nature of epileptic activity that can be observed in such tissue, in particular, the rarity of spontaneous ictal discharges, we discuss manipulations that can be utilised to induce such activity. In addition to discussing conventional electrophysiological techniques that are routinely employed in epileptic human brain slices, we review how imaging and multielectrode array recordings could provide novel insights into the network dynamics of human epileptogenesis. Acute studies in human brain slices are ultimately limited by the lifetime of the tissue so overcoming this issue provides increased opportunity for information gain. We review the literature with respect to organotypic culture techniques that may hold the key to prolonging the viability of this material. A combination of long-term culture techniques, viral transduction approaches and electrophysiology in human brain slices promotes the possibility of large scale monitoring and manipulation of neuronal activity in epileptic microcircuits

Vergleich präanalytischer Faktoren zur molekularen und serologischen Diagnostik von COVID-19

Background: The diagnosis of SARS-CoV-2 infection relies on RT-PCR from nasopharyngeal swabs. The pre-analytical value of different methods of material harvesting for SARS-CoV-2 are unknown. Methods: We conducted a comprehensive investigation of the pre-analytical performance for different pharyngeal sampling procedures in hospitalized patients with confirmed SARS-CoV-2 infection. In addition to swabs taken simultaneously from different locations, saliva and pharyngeal lavages were also analyzed using RT-PCR.Results: In 10 COVID-19 patients, standard nasopharyngeal swabs detected 8 out of 10 positive patients, whereas swabs taken from the palatoglossal arch resulted in 9 correct-positive results. Brushing the posterior pharynx wall with swabs resulted in detection of 9 out of 10 positive patients with no difference using either dry swabs or liquid Amies medium. A strong correlation between Ct values of both swab materials was observed. Pharyngeal lavages yielded 6 out of 10 positive results in concordance with 85% of nasopharyngeal swabs in late-stage COVID-19 patients. Investigating 23 patients with early SARS-CoV-2 infection, pharyngeal lavages showed a concordance rate of 100% compared to nasopharyngeal swabs.Conclusions: The diagnostic performance of swabs taken from the palatoglossal arch in detecting SARS-CoV-2 infection is similar to that of specimens taken from the nasopharyngeal region. However, the former sampling method is associated with less discomfort and much easier to perform. Pharyngeal lavages may replace swabs for mass screening in early stages of SARS-CoV-2 infection. The predictive values are comparable, and the procedure is performed without exposing healthcare workers to transmission risks.Hintergrund: Die Diagnose der SARS-CoV-2-Infektion basiert auf einer RT-PCR aus Nasen-Rachen-Abstrichen. Der präanalytische Wert der verschiedenen Methoden der Materialentnahme für die Diagnose einer SARS-CoV-2 Infektion ist unbekannt. Methoden: Wir führten eine umfassende Untersuchung der präanalytischen Wertigkeit verschiedener pharyngealer Probeentnahmeverfahren bei hospitalisierten Patienten mit bestätigter SARS-CoV-2-Infektion durch. Neben Abstrichen mit verschiedenen Materialien, die gleichzeitig an verschiedenen Orten entnommen wurden, wurden auch Speichel- und Rachenspülungen mittels RT-PCR analysiert.Ergebnisse: Von 10 COVID-19 Patienten wurden mittels standardisierten Nasen-Rachen-Abstrichen 8 von 10 Patienten korrekt erkannt, während Abstriche vom vorderen Rachenring zu 9 korrekt-positiven Ergebnissen führten. Abstriche der hinteren Rachenwand führten bei 9 von 10 Patienten zum korrekten SARS-CoV-2 Nachweis ohne Unterschiede bei Verwendung von trockenen Abstrich-Materialien oder Abstrichtupfern mit flüssigem Amies-Medium. Es wurde eine starke Korrelation zwischen den Ct-Werten bei Verwendung der beiden Abstrichmaterialien beobachtet. Bei COVID-19-Patienten im Spätstadium wurden mittels Rachenspülungen 6 von 10 Patienten korrekt zugeordnet, während dies bei 85% der Nasen-Rachenabstriche der Fall war. Bei der Untersuchung von 23 Patienten mit einer frühen SARS-CoV-2-Infektion zeigten die Rachenspülungen im Vergleich zu den Nasen-Rachen-Abstrichen eine Konkordanzrate von 100%.Schlussfolgerung: Abstriche, die vom vorderen Rachenring entnommen werden, weisen bei Patienten mit SARS-CoV-2-Infektion eine ähnliche diagnostische Sicherheit auf wie Proben aus dem Nasen-Rachenraum-Bereich. Diese Probenentnahmemethode ist jedoch deutlich weniger unangenehm und einfacher durchführbar. Rachenspülungen können in frühen Stadien einer SARS-CoV-2-Infektion Nasenrachenabstriche für das Massenscreening ersetzen. Die prädiktiven Werte sind vergleichbar und das Gesundheitspersonal wird nicht unnötig einem Übertragungsrisiko ausgesetzt

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

&lt;p&gt;West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime--boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8(+) specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.&lt;/p&gt;</p

Simulating human sleep spindle MEG and EEG from ion channel and circuit level dynamics

BackgroundAlthough they form a unitary phenomenon, the relationship between extracranial M/EEG and transmembrane ion flows is understood only as a general principle rather than as a well-articulated and quantified causal chain.MethodWe present an integrated multiscale model, consisting of a neural simulation of thalamus and cortex during stage N2 sleep and a biophysical model projecting cortical current densities to M/EEG fields. Sleep spindles were generated through the interactions of local and distant network connections and intrinsic currents within thalamocortical circuits. 32,652 cortical neurons were mapped onto the cortical surface reconstructed from subjects' MRI, interconnected based on geodesic distances, and scaled-up to current dipole densities based on laminar recordings in humans. MRIs were used to generate a quasi-static electromagnetic model enabling simulated cortical activity to be projected to the M/EEG sensors.ResultsThe simulated M/EEG spindles were similar in amplitude and topography to empirical examples in the same subjects. Simulated spindles with more core-dominant activity were more MEG weighted.Comparison with existing methodsPrevious models lacked either spindle-generating thalamic neural dynamics or whole head biophysical modeling; the framework presented here is the first to simultaneously capture these disparate scales.ConclusionsThis multiscale model provides a platform for the principled quantitative integration of existing information relevant to the generation of sleep spindles, and allows the implications of future findings to be explored. It provides a proof of principle for a methodological framework allowing large-scale integrative brain oscillations to be understood in terms of their underlying channels and synapses