Questo lavoro è stato sostenuto da un finanziamento della Fondazione Simons (SFARI # 89.638 a RB).

Il seguente protocollo aderisce alle linee guida della University of Washington comitato di revisione istituzionale. 1. Assessment elettrofisiologici <ol><li> Preparazione della sessione <ol><li> Preparazione della camera: posto il manipulandum (vedi figura 1), un blocco di legno con un sensore collegato, che invia un indicatore di data e ora per software di acquisizione quando viene afferrato, sul tavolo entro cogliere portata del partecipante. Attivare il software di acquisizione EEG e cominciare &quot;nuova sessione&quot; (Figura S1). </li><li> Preparazione Net: soluzione calda di acqua distillata (1 L), cloruro di potassio (1 cucchiaio), e shampoo per bambini (1 cucchiaino) a 104 ° C. Mettere a bagno 128-elettrodo dense-array sistema EEG nella soluzione salina riscaldata. </li><li> Preparazione partecipante: assicurarsi che il partecipante è seduto comodamente circa 75 cm dal monitor presentazione dello stimolo e completamente in vista del video camepoca. Trova e segnare il vertice sulla testa del partecipante con un pennarello pelle. Misurare il vertice trovando l&#39;intersezione del punto medio tra il nasion e il inion nel punto medio tra preauriculars. </li><li> NET: posizionare il tappo EEG sulla testa del partecipante tale che l&#39;elettrodo vertice sia posizionata direttamente sopra il marchio vertice. Controllare impedenze e garantire che impedenze sono al di sotto della soglia appropriata per il sistema EEG in uso (figura S2). </li><li> Iniziare sessione di riprese video. </li></ol></li><li> Impostazione di registrazione: Segnale di riferimento all&#39;elettrodo vertice. Filtro analogico tra 0,1 e 100 Hz, amplificare il segnale, e digitalizzare a 500 campioni / sec. </li><li> Presentazione dello stimolo: presente partecipante con 3 condizioni: osservare, eseguire e di riposo, adattate dal paradigma sviluppato da Muthukumaraswamy e colleghi 5. <ol><li> Osservare condizione: Istruire partecipante a sedere in silenzio e guardareun video di una persona afferrare la manipulandum. Ogni prova deve durare 6 sec. Ora il video preregistrato per le prove osservare proprio per garantire che la portata osservata avviene esattamente 3 sec. Monitorare l&#39;attenzione visiva del partecipante durante il compito, e segnare le prove durante le quali essi non frequentano per lo schermo da scartare durante la post-elaborazione. </li><li> Esegui condizione: Istruire partecipante di sedersi tranquillamente con la mano appoggiata destra appena sotto la manipulandum e, dopo aver sentito un segnale uditivo preregistrato, di imitare l&#39;afferrare manipulandum da osservare videoclip condizione. Ogni prova deve durare 6 sec. Assicurarsi che il cue uditivo è presentato esattamente 3 secondi da preregistrazione una traccia uditiva che mantiene un costante temporizzata eseguire cue e l&#39;intervallo inter-trial. Utilizzare un sensore sulla manipulandum per registrare con precisione il tempo che si verifica comprensione del partecipante (Figura S3). </li><li> Condizione di riposo: Istruire partecipante a sedere quietemente con gli occhi aperti e passivamente osservare una piccola croce sul monitor stimolo. Record EEG continuo durante la condizione di riposo per 3 min. </li><li> Per entrambi osservare ed eseguire le condizioni, attuali blocchi randomizzati di dieci prove, per un totale di quaranta prove per ogni condizione. Assicurarsi che l&#39;immagine del manipulandum rimane sullo schermo durante i blocchi osservare ed eseguire, anche tra le prove. Somministrare la condizione di riposo al completamento delle condizioni di osservare ed eseguire. </li></ol></li><li> Elaborazione dati <ol><li> Dopo la raccolta dei dati, ricontrollare impedenze. Nota: le eventuali modifiche ai livelli di impedenza. Fine della registrazione software di acquisizione. </li><li> Post-processing: segnali EEG Rereference alla media. Segmento dati EEG continuo in quaranta 6 sec prove per ciascuna condizione (figura S4). </li><li> Condurre il rilevamento automatico artefatto. Utilizza algoritmi automatizzati per controllare segmenti di artefatti da movimento individuando veloci ampiezze medie exceeding 200 mV, differenziali ampiezze medie superiori a 100 mV, e varianza pari a zero in un determinato processo (Figura S5). </li><li> Eseguire il rilevamento artefatto manuale controllando visivamente i dati e confermando con la recensione video della sessione di rimuovere tutte le prove in condizione di osservazione contaminata con qualsiasi artefatto movimento e tutte le prove nella condizione di esecuzione contaminata con qualsiasi movimento manufatto estraneo al gesto presa. Escludi prove con artefatto significativo dall&#39;analisi. Eliminare tutte le prove che sono stati segnalati durante l&#39;acquisizione non hanno partecipato. Esaminare e notare tasso di rigetto di prova per ogni gruppo diagnostico in analisi. </li></ol></li><li> Analisi dei dati <ol><li> Per Muthukumaraswamy et al. 5, segmento prove puliti in due epoche sec, comprensivi di 1 sec di dati prima della presa e 1 sec dopo sia per le condizioni di osservare (come indicato dalla fotocellula) ed esecuzione (come indicato dal sensore manipulandum). Segmento cappoggiò 2 sec epoche dalla condizione di riposo. </li><li> Trasformata veloce di Fourier (FFT) ogni segmento. Selezionare un gruppo di otto elettrodi su ciascun emisfero circonda le posizioni standard C3 e C4 per analisi statistiche (seguenti Muthukumaraswamy et al. 5 e Bernier et al. 3) (Figura 2). Per ogni condizione, la potenza media di tutti i trial inclusi per calcolare spettri di potenza. </li><li> Calcola mu attenuazione esaminando la potenza media durante l&#39;esecuzione o osservazione di un&#39;azione motore, rispetto alla potenza media durante la condizione di riposo, in tutta la gamma 8-13 Hz. Utilizzare il registro di questo rapporto per determinare il grado di attenuazione. Nota: un valore negativo rappresenta attenuazione durante l&#39;esecuzione o l&#39;osservazione, mentre un valore positivo indica aumento. Questa metodologia prende in considerazione la variabilità tra gli individui, e la non-normalità dei valori espressi in forma di rapporto. &lt;br /&gt; Nota: Questo protocollo è stato sviluppato utilizzando una fitta-array sistema EEG 128 elettrodi con software Net Station versione 4.1. Mentre i passi di base sono simili in tutti i sistemi di EEG, protocolli di acquisizione e analisi possono variare. </li></ol></li></ol> 2. Caratterizzazione del campione <ol><li> Identificare il potenziale popolazione di pazienti per la partecipazione paradigma attraverso i registri di ricerca, elenchi dei partecipanti precedenti, o rinvii da cliniche della zona e medici. </li><li> Schermo potenziali partecipanti per probabilità di soddisfare i criteri diagnostici per il costrutto clinico (ad esempio Autism Spectrum Disorder) e di individuare eventuali criteri di esclusione, come la presenza di trauma cranico, tumore, la storia di sequestro, o l&#39;uso di farmaci anti-convulsivanti o barbiturici che possono distorcere l&#39; segnale elettrofisiologico. </li><li> Confermare lo stato di diagnostica della popolazione paziente attraverso l&#39;utilizzo di strumenti diagnostici standard d&#39;oro (es. Autism Diagnostic Interview-Reduto (ADI-R 11) e il Autism Diagnostic Observation Schedule-Generic (ADOS-G, 12) amministrati da medico esperto seguendo Manuale Diagnostico e Statistico - 5 ª Edizione (DSM-5) Criteri di 13. </li><li> Identificare campione di controllo abbinate sul rilevanti variabili di interesse, come l&#39;età, il sesso, capacità cognitive, ecc </li></ol>

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Neurosci. , 110-123 (2011).</li><li>Oberman, L., McCleery, J., Hubbard, E., Bernier, R., Pineda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+mu+suppression+to+observed+actions+in+individuals+with+autism+spectrum+disorders.">Developmental changes in mu suppression to observed actions in individuals with autism spectrum disorders.</a> Soc. Cogn. Affective Neurosci. 8, 300-304 .</li></ol>

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Il successo di acquisizione, elaborazione e analisi dei dati elettrofisiologici relativi al ritmo mu e l&#39;applicazione di popolazioni cliniche richiede 1) l&#39;applicazione di strumenti metodologici EEG, 2) rilevazione accurata artefatti e riduzione dei dati, 3) l&#39;identificazione precisa del ritmo mu, e 4) la caratterizzazione accurata della popolazione clinica e l&#39;identificazione di appropriati gruppi di controllo. Metodologia appropriata EEG richiede...

Elettroencefalografia (EEG) è un metodo efficace, efficiente e non invasivo di valutare e registrare l&#39;attività cerebrale. Come neuroni fuoco nel cervello, la tensione risultante può essere amplificato, registrato e rappresentato graficamente. La risoluzione temporale di EEG permette l&#39;analisi di anche brevi cambiamenti nei modelli di oscillazione del cervello, così come l&#39;analisi della risposta del cervello a stimoli specifici. Nonostante sia la più antica tecnica di imaging cerebrale, risalente alla fine del 19 ° secolo, EEG ha ancora ampia applicabilità. Mentre la risonanza magnetica funzionale (fMRI) ha un&#39;ottima risoluzione spaziale, ha relativamente scarsa risoluzione temporale. Ciò rappresenta un notevole limitazione di valutazione fMRI data la velocità alla quale gran processi avvengono nel cervello. EEG ha la capacità di valutare l&#39;attività elettrica cerebrale a livello millisecondo, fornendo potenziale iNsight nelle fasi di elaborazione del cervello. L&#39;evoluzione delle tecnologie hanno inoltre ampliato l&#39;applicabilità di EEG. Un aumento della densità di sistemi di registrazione ha permesso lo sviluppo di tecniche di localizzazione sorgente, attenuare alcune delle limitazioni di EEG proposito di risoluzione spaziale. Inoltre, i sistemi moderni hanno ridotto il partecipante set-up tempo individuale significativo, che consenta la valutazione delle popolazioni precedentemente non disponibili, come il neonato e campioni clinici 1-3,28-30. Data l&#39;eccellente risoluzione temporale, EEG può essere utilizzato per esaminare la risposta neurale correlate a specifici comportamenti, stati o stimoli esterni. Un esempio di questa utilità è la valutazione del sistema specchio (MNS) negli esseri umani. I neuroni specchio sono stati originariamente identificati nella scimmia con singola registrazione neurone 4, evidenziando un gruppo dineuroni che hanno risposto congiuntamente, l&#39;esecuzione e l&#39;osservazione di azioni motorie. Questo metodo di registrazione diretta di sistemare gli elettrodi nel cervello viene raramente utilizzata negli esseri umani, e solo in casi clinici disastrose. EEG ha fornito un metodo per valutare le MNS monitorando il ritmo EEG mu. Questo modello oscillazione nell&#39;intervallo 8-12 Hz ha dimostrato di attenuare potenza EEG in risposta all&#39;esecuzione e osservazione di azioni motorie, simile al pattern di attivazione osservato nelle scimmie 5-7. Allo stesso modo, la stimolazione della putative regioni cerebrali MNS attraverso la Stimolazione Magnetica Transcranica (ad esempio, il giro frontale inferiore) abolisce mu EEG ritmo 8 e la soppressione EEG mu ritmo correla con i segnali provenienti da fMRI BOLD nelle regioni dei neuroni specchio putativi all&#39;interno di soggetti 9, fornendo un sostegno supplementare che questo ritmo indici, almeno in parte, l&#39;attività MNS. La valutazione del ritmo EEG mu ha consentito una valutazione non invasiva di neuroni specchio attoivity nell&#39;uomo. EEG fornisce una metodologia unica per esaminare l&#39;attività cerebrale e quando combinato con test basati comportamentale, può essere un potente strumento per chiarire aspetti della cognizione sociale, come l&#39;imitazione, in popolazioni cliniche. Inoltre, l&#39;applicabilità di EEG per l&#39;uso con le popolazioni con disabilità cognitive o di linguaggio permette di comprensione abilità di individui per i quali altre tecniche di imaging o paradigmi comportamentali possono essere utilizzare meno successo. Il protocollo descritto fornisce una via per esaminare le funzioni cognitive sociali teoricamente legati al sistema dei neuroni specchio nei soggetti con sviluppo tipico e atipico, come il Disturbo dello Spettro Autistico.

<table border="0" cellpadding="0" cellspacing="0" width="994">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Geodesic EEG System</td>
			<td style="width:71px;">EGI</td>
			<td style="width:119px;">N/A</td>
			<td style="width:539px;">Any EEG system, not only EGI based systems, is applicable for the described study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MATLAB software</td>
			<td style="width:71px;">MATLAB</td>
			<td style="width:119px;">N/A</td>
			<td>Any mathematical, statistical software that can work with matrices is applicable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Netstation software</td>
			<td style="width:71px;">EGI</td>
			<td style="width:119px;">N/A</td>
			<td>Any EEG acquisition software is applicable for the described study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Manipulandum</td>
			<td style="width:71px;">custom</td>
			<td style="width:119px;">N/A</td>
			<td>Any object that is co-registered with data acquisition software to signal a successful grasp</td>
		</tr>
	</tbody>
</table>

eeg mu rhythm in typical and atypical development

Elettroencefalografia (EEG) è un metodo efficace, efficiente e non invasivo di valutare e registrare l&#39;attività cerebrale. Data l&#39;eccellente risoluzione temporale, EEG può essere utilizzato per esaminare la risposta neurale correlate a specifici comportamenti, stati o stimoli esterni. Un esempio di questa utility è la valutazione del sistema dei neuroni specchio (MNS) negli esseri umani attraverso l&#39;esame del ritmo EEG mu. Il ritmo EEG mu, attività oscillatoria nella gamma di frequenza 8-12 Hz registrata dagli elettrodi in posizione centrale, viene soppressa quando un individuo esegue, o semplicemente osserva, azioni dirette obiettivo. Come tale, è stato proposto di riflettere attività del MNS. E &#39;stato teorizzato che la disfunzione del sistema dei neuroni specchio (MNS) svolge un ruolo che contribuisce in deficit sociali del disturbo dello spettro autistico (ASD). MNS possono essere esaminate in modo non invasivo popolazioni cliniche utilizzando EEG attenuazione ritmo mu come indice per la sua attività. Il prot descrittoocol fornisce una via per esaminare le funzioni cognitive sociali teoricamente legati ai MNS in individui con sviluppo tipico e atipico, come ASD.

Adulti tipici, bambini e neonati hanno sempre dimostrato mu ritmo sia durante l&#39;esecuzione e l&#39;osservazione di azioni attraverso una varietà di paradigmi e di stimoli 5, 14-30. Attenuazione in questa banda di frequenza è costantemente localizzato tra gli elettrodi centrali (Figura 3) che indicano che questa non è la riduzione di potenza alfa che viene registrata in altre regioni del cuoio capelluto. Allo stesso modo, l&#39;attenuazione in questa frequenza durante l&#39;osservazione...

Watch this Scientific Journal Video about EEG Mu Rhythm in Typical and Atypical Development at JoVE.com

EEG Mu Rhythm in Typical and Atypical Development

La valutazione del ritmo EEG mu fornisce una metodologia unica per l&#39;esame dell&#39;attività cerebrale e quando combinato con test basati comportamentale, può essere uno strumento potente per chiarire aspetti della cognizione sociale, come l&#39;imitazione, in popolazioni cliniche.

EEG Mu Ritmo nello sviluppo tipico e atipico

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal ...

eeg-mu-rhythm-in-typical-and-atypical-development

Research

JoVE Journal

Medicine

25.4K Views.  University of Washington.  La valutazione del ritmo EEG mu fornisce una metodologia unica per l'esame dell'attività cerebrale e quando combinato con test basati comportamentale, può essere uno strumento potente per chiarire aspetti della cognizione sociale, come l'imitazione, in popolazioni cliniche. 

Video: EEG Mu Rhythm in Typical and Atypical Development

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Pressure Monitoring diretto predice accuratamente occlusione della vena polmonare durante l&#39;ablazione Cryoballoon

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

Ricerca

Medicina

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Sviluppo di un audio-based ambiente di gioco virtuale per assistere con doti di navigazione in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

Protocollo di esame clinico per rilevare la scrapie atipica e classica negli ovini

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

In tempo reale citotossicità saggi in Human Sangue intero

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

Cervello Imaging Source in modelli preclinici Rat dell&#39;epilessia focale utilizzando ad alta risoluzione EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an anti-TNF&#945; mAb. In addition, this protocol can be used to evaluate other anti-TNF&#945; molecules, such as fusion proteins. The cellular model employed here is sensitive to TNF&#945;-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

Determinazione della potenza relativa di un Anti-TNF anticorpo monoclonale (mAb) di neutralizzante TNF utilizzando un metodo di Bioanalytical In Vitro

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Valutazione di alternanza spontanea, riconoscimento novello di oggetti e attacco degli arti nei modelli di topo transgenico Modelli di neuropathologia di Amiloide-β e Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Il modello di pilocarpina di epilessia del lobo temporale e monitoraggio EEG utilizzando il sistema di radiotelemetria in topi

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Contrazioni di umano-iPSC-derivato del Cardiomyocyte sincizi misurata con un colorante fluorescente Ca sensibile a temperatura controllata piastre da 384 pozzetti

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Un sistema di formazione e test per l'esecuzione della ricostruzione vascolare in vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...