Gli autori desidera ringraziare Caroline Hartley e Rebeccah Slater (dipartimento di pediatria, Università di Oxford, UK) per la revisione critica la nostra carta e Walter Magerl (dipartimento di neurofisiologia, centro di biomedicina e tecnologia medica Mannheim (CBTM), Università di Heidelberg, Germania) per averci sostenuto con attrezzature tecniche e la conoscenza.

Lo studio è stato approvato del competente comitato etico della Svizzera nordoccidentale (EKNZ 2015-079) e informato consenso scritto dei genitori per il partecipante è stato ottenuto prima della misurazione.
1. preparazione
<ol><li>Assicurarsi che il bambino è depositato. Dovrebbe essere tranquillo e costante, senza succhiare movimenti durante la registrazione a causa di artefatti di movimento. Il bambino può essere addormentato9.</li>
	<li>Misurare la circonferenza della testa del neonato con un nastro di misurazione per definire le dimensioni della PAC EEG.</li>
	<li>Identificare la posizione di elettrodo attivo: Cz / punto di posizione di vertice contrassegnando il punto di mezzo tra nasion e inion e il punto di mezzo tra il preaurical destro e sinistro con una matita di marcatore di pelle. Per il posizionamento degli elettrodi Vedi anche Figura 1.</li>
	<li>Identificare le posizioni di elettrodo di terra: fronte di destra (Fp2) e l'elettrodo di riferimento: mastoide sinistro (A1).</li>
	<li>Pulire i siti di elettrodo (Cz, Fp2 e A1) con disinfettante con un batuffolo di cotone. In caso di un sacco di capelli, separare i capelli per visualizzare il cuoio capelluto.</li>
	<li>Pulire delicatamente i siti elettrodo con EEG durante la preparazione pasta, utilizzando un tampone di cotone, per abbassare l'impedenza.</li>
	<li>Posizionare il tappo di EEG con elettrodi fissati sulla testa del neonato.</li>
	<li>Iniettare il gel conduttivo EEG gli elettrodi usando una siringa con un ago di plastica corto per ottimizzare il contatto tra gli elettrodi e il cuoio capelluto.</li>
	<li>Regolare gli elettrodi fino a quando l'impedenza è inferiore a 50 kΩ come descritto nelle raccomandazioni del produttore e in linee guida pubblicate12.</li>
	<li>Posizionare una videocamera per registrare le espressioni facciali di neonato.</li>
	<li>Collegare la sonda di piatto-punta per il dispositivo di innesco a contatto, che è fissato al dispositivo di registrazione EEG. Quando la sonda piatto-punta raggiunge la forza nominale sulla pelle, viene generato un segnale di trigger dal dispositivo di innesco a contatto. Questo segnale viene inviato al computer, tagging EEG registrazione con un segno di trigger.</li>
</ol>2. misurazione
<ol><li>Selezionare un nome di studio per memorizzare i dati.</li>
	<li>
Aggiungere filtri passa banda online:
	<ol>
<li>Filtri di visualizzazione: Filtro low cut-off: frequenza: 1Hz Alto filtro cutoff: frequenza: 70 Hz Filtro notch: 50 Hz Frequenza di campionamento: 2000 Hz</li>
	</ol>
</li>
	<li>Avviare la registrazione video e EEG.</li>
	<li>Tenere la mano destra di neonato in posizione orizzontale. Lo stimolo può essere applicato anche su un sito diverso, ad esempio il piede, che si tradurrà in latenza più lunga rispetto dalla mano, per vedere dettagli 3,7.</li>
	<li>Primo record sfondo attività EEG. Mentre è tenuto in mano di neonato, annotare la registrazione EEG manualmente per registrare i periodi in cui non vengono applicati stimoli e l'infante è a riposo.</li>
	<li>Condurre la quantità necessaria di stimoli piatto-punta sonda secondo il disegno dello studio sulla mano destra di neonato. Nel nostro disegno sperimentale, impostare il numero di stimoli piatto-punta sonda a 50. Prestare attenzione a utilizzare la sonda piatto-punta perpendicolarmente alla mano di neonato, così non si piega la punta e la corretta forza è applicata. Eseguire gli stimoli di piatto-punta sonda usando un minimo di 2 a 3 s intervallo Inter-stimolo (ISI)6 per evitare la sommatoria.</li>
	<li>Interrompere tutte le registrazioni.</li>
	<li>Documentare i dettagli di impostazione sperimentale.</li>
</ol>3. analisi dei dati
<ol><li>Filtrare i dati grezzi di EEG offline utilizzando un filtro passa-alto a 1 Hz e un filtro passa-basso a 30 Hz.</li>
	<li>Segmentare i dati in epoche di 1.500 ms (500 ms prima a 1.000 ms dopo l'inizio dello stimolo).</li>
	<li>Eseguire una correzione della linea di base per l'intervallo pre-stimolo.</li>
	<li>Manualmente e rifiutare le epoche di EEG contenente manufatti, quali artefatti di movimento e rumore, dopo l'ispezione visiva. Si veda anche il contorno tutorial: https://sccn.ucsd.edu/wiki/Chapter_01:_Rejecting_Artifacts</li>
	<li>Media le epoche di EEG (per la risposta sfondo e stimolo separatamente).</li>
	<li>Legnosa filtrare i dati con un jitter massimo di ± 50 ms nella finestra di tempo 0 - 1.000 ms post inizio di stimolo. In questo modo le differenze di latenza tra i neonati. Fare questo per le risposte di sfondo e stimolo separatamente.</li>
	<li>Progetto di un modello di attività nocive-evocato cerebrale, che è stato definito un set di dati indipendenti ed è stato descritto dettaglio altrove11 ai dati per accertare l'entità della risposta evocata nocive all'interno di ogni singola prova. Il modello descrive una forma d'onda caratteristica della risposta evocata nocive e la grandezza riflette la quantità di questa risposta evocata nocive all'interno la prova individuale. Nota: L'intervallo di tempo in cui il modello di progetto dipende dall'età dell'infante e la posizione dello stimolo. Se stimoli vengono applicati alla mano, allora l'intervallo di tempo di interesse è 200-500 ms dopo stimolo11. Se gli stimoli vengono applicati al piede, poi l'intervallo di tempo di interesse è 400-700 ms dopo stimolo11. Si noti che questo modello è stato convalidato solo attualmente per gli infanti tra gestazione 34-43 weeks'. Per metodi dettagliati associati all'utilizzo di questo modello e una discussione delle limitazioni, vedere Hartley et al 201711.</li>
	<li>Controllare che l'entità dell'attività del cervello nocive-evocato calcolato utilizzando il modello è significativamente più in alto seguendo lo stimolo, confrontato con sullo sfondo EEG.</li>
	<li>Analizzare l'espressione facciale del comportamento in seguito (fotocamera è collegata al EEG registrazione) utilizzando il sistema di codificazione facciale neonatale2,13.</li>
</ol>

<ol>
	<li>Bonica, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+of+a+taxonomy.">The need of a taxonomy.</a> Pain. 6 (3), 247-248 (1979).</li><li>Duhn, L. J., Medves, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+integrative+review+of+infant+pain+assessment+tools.">A systematic integrative review of infant pain assessment tools.</a> Adv Neonatal Care. 4 (3), 126-140 (2004).</li><li>Witt, N., Coynor, S., Edwards, C., Bradshaw, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Pain+Assessment+and+Management+in+the+Neonate.">A Guide to Pain Assessment and Management in the Neonate.</a> Curr Emerg Hosp Med Rep. 4, 1-10 (2016).</li><li>Hummel, P., van Dijk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+assessment:+current+status+and+challenges.">Pain assessment: current status and challenges.</a> Semin Fetal Neonatal Med. 11 (4), 237-245 (2006).</li><li>Slater, R., Fitzgerald, M., Meek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+cortical+responses+following+noxious+stimulation+inform+us+about+pain+processing+in+neonates?.">Can cortical responses following noxious stimulation inform us about pain processing in neonates?.</a> Semin Perinatol. 31 (5), 298-302 (2007).</li><li>Hartley, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+nociceptive+brain+activity,+spinal+reflex+withdrawal+and+behaviour+in+newborn+infants.">The relationship between nociceptive brain activity, spinal reflex withdrawal and behaviour in newborn infants.</a> Sci Rep. , 31 (2015).</li><li>Iannetti, G. D., Baumg&#228;rtner, U., Tracey, I., Treede, R. D., Magerl, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pinprick-evoked+brain+potentials:+a+novel+tool+to+assess+central+sensitization+of+nociceptive+pathways+in+humans.">Pinprick-evoked brain potentials: a novel tool to assess central sensitization of nociceptive pathways in humans.</a> J Neurophysiol. 110 (5), 1107-1116 (2013).</li><li>Ziegler, E. A., Magerl, W., Meyer, R. A., Treede, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+hyperalgesia+to+punctate+mechanical+stimuli.+Central+sensitization+to+A-fibre+nociceptor+input.">Secondary hyperalgesia to punctate mechanical stimuli. Central sensitization to A-fibre nociceptor input.</a> Brain. 122 (Pt 12), 2245-2257 (1999).</li><li>Slater, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evoked+potentials+generated+by+noxious+stimulation+in+the+human+infant+brain.">Evoked potentials generated by noxious stimulation in the human infant brain.</a> Eur J Pain. 14 (3), 321-326 (2010).</li><li>Fabrizi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shift+in+sensory+processing+that+enables+the+developing+human+brain+to+discriminate+touch+from+pain.">A shift in sensory processing that enables the developing human brain to discriminate touch from pain.</a> Curr Biol. 21 (18), 1552-1558 (2011).</li><li>Hartley, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+brain+activity+as+a+measure+of+analgesic+efficacy+in+infants.">Nociceptive brain activity as a measure of analgesic efficacy in infants.</a> Sci Transl Med. 9 (388), (2017).</li><li>Keil, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Committee+report:+publication+guidelines+and+recommendations+for+studies+using+electroencephalography+and+magnetoencephalography.">Committee report: publication guidelines and recommendations for studies using electroencephalography and magnetoencephalography.</a> Psychophysiology. 51 (1), 1-21 (2014).</li><li>Grunau, R. V., Johnston, C. C., Craig, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+facial+and+cry+responses+to+invasive+and+non-invasive+procedures.">Neonatal facial and cry responses to invasive and non-invasive procedures.</a> Pain. 42 (3), 295-305 (1990).</li><li>van den Broeke, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+pinprick-evoked+brain+potentials+before+and+after+experimentally+induced+secondary+hyperalgesia.">Characterizing pinprick-evoked brain potentials before and after experimentally induced secondary hyperalgesia.</a> J Neurophysiol. 114 (5), 2672-2681 (2015).</li><li>Verriotis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+activity+evoked+by+inoculation+needle+prick+in+infants+up+to+one-year+old.">Cortical activity evoked by inoculation needle prick in infants up to one-year old.</a> Pain. 156 (2), 222-230 (2015).</li><li>Rolke, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+sensory+testing+in+the+German+Research+Network+on+Neuropathic+Pain+(DFNS):+standardized+protocol+and+reference+values.">Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): standardized protocol and reference values.</a> Pain. 123 (3), 231-243 (2006).</li><li>Moultrie, F., Slater, R., Hartley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+treatment+of+infant+pain.">Improving the treatment of infant pain.</a> Curr Opin Support Palliat Care. 11 (2), 112-117 (2017).</li><li>Hu, L., Zhang, Z. G., Mouraux, A., Iannetti, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+linear+regression+to+estimate+time-frequency+electrophysiological+responses+in+single+trials.">Multiple linear regression to estimate time-frequency electrophysiological responses in single trials.</a> Neuroimage. 111, 442-453 (2015).</li><li>Hartley, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+Balance+of+Spinal+Cord+Excitability+and+Nociceptive+Brain+Activity+in+Early+Human+Development.">Changing Balance of Spinal Cord Excitability and Nociceptive Brain Activity in Early Human Development.</a> Curr Biol. 26 (15), 1998-2002 (1998).</li><li>Evers, K. S., Wellmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+Vasopressin+and+Copeptin+in+Perinatology.">Arginine Vasopressin and Copeptin in Perinatology.</a> Front Pediatr. 4 (75), (2016).</li><li>Slater, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+sucrose+as+an+analgesic+drug+for+procedural+pain+in+newborn+infants:+a+randomised+controlled+trial.">Oral sucrose as an analgesic drug for procedural pain in newborn infants: a randomised controlled trial.</a> Lancet. 376 (9748), 1225-1232 (2010).</li></ol>

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L'approccio qui presentato Mostra come nocive-evocato cervello attività nei neonati può essere misurata in modo oggettivo, mediante registrazione EEG e piatto-punta sonda stimolatori di applicare stimoli nocivi sperimentali. Questa tecnica può essere utilizzata in varie regolazioni cliniche per rilevare la nocicezione, ad esempio in persone non verbali quali i neonati. Lo studio completo può essere fatto entro 15 min, compresa l'immissione del bambino, individuazione, preparazione e montaggio di elettrodi e ...

Il dolore è una spiacevole esperienza sensoriale ed emotiva associata a danno tissutale reale o potenziale, o descritta in termini di tale danno1. L'incapacità di comunicare verbalmente non nega la possibilità che un individuo sta vivendo il dolore ma lo rende molto difficile da valutare la terapia antidolorifica, per esempio in infanti neonati2. Diversi indicatori comportamentali e fisiologici sono utilizzati per valutare il dolore in pazienti non-verbale. Diverse scale sono state sviluppate nel corso degli anni, la scelta dipende dal tipo di stimolo, l'età gestazionale e l'ambiente in cui i neonati sono embeded3,4,5. Questi strumenti di valutazione del dolore neanche si basano sull'interpretazione di rater o chiedono indicatori fisiologici secondari.
In questo video, vi presentiamo un metodo per valutare la nocicezione con registrazioni elettrofisiologiche ottimizzate per l'uso nei neonati. Nocicezione è definita come il processo neurale di codifica stimoli nocivi. Così, quantificazione nocicezione è un metodo elegante e oggettivo per determinare l'input neurale in una persona non-verbale. Inoltre, l'attività corticale rilevata da elettroencefalografia (EEG) è correlato con l'intensità di eventi nocivi5,6.
Il metodo presentato qui combina la registrazione EEG con stimoli nocivi producerat da una stimolazione meccanica con una sonda di piatto-punta, chiamata anche una puntura di spillo7, che non è pelle-rottura e non provoca disagio comportamentale6. È stato dimostrato, che segue di nocicezione punteggiano la stimolazione è principalmente mediata da fibre Aδ, fa non ha bisogno di una pelle rottura potenziale lesione8 e la grandezza della nocicettivo specifico non è dipendente da dormire stato9. La sonda è ben accettata anche dai genitori quando si applica questo metodo in un ambiente di studio con neonati. La sonda è legata elettronicamente per il sistema di registrazione di EEG che permette la registrazione EEG di essere taggati con precisione quando la sonda di contatto con la pelle. Questo notevolmente semplifica il processo di chiusura di tempo ed è la premessa per tutte le successive analisi di EEG. Al fine di minimizzare i tempi di preparazione della registrazione EEG neonato abbiamo usato un sistema di posizionamento internazionale 10/20 elettrodo modificato dove abbiamo ridotto il numero di elettrodi per il requisito minimo di tre elettrodi (Figura 1). L'elettrodo Cz vertice centrale, dove l'attività cerebrale nocive-evocato è massima9,10,11, è stato usato insieme a un riferimento e un elettrodo a terra.

<table>
	<tbody>
		<tr>
			<td>Easycap</td>
			<td>EASYCAP GmbH</td>
			<td>AC-32-C</td>
			<td>EEG caps for infants sizes 34 and 36</td>
		</tr>
		<tr>
			<td>actiCAP</td>
			<td>Brain Products GmbH</td>
			<td>BP-04243-SIG</td>
			<td>active electrodes</td>
		</tr>
		<tr>
			<td>ImpBox</td>
			<td>Brain Products GmbH</td>
			<td></td>
			<td>impedance measurement</td>
		</tr>
		<tr>
			<td>V-Amp</td>
			<td>Brain Products GmbH</td>
			<td></td>
			<td>EEG recording device</td>
		</tr>
		<tr>
			<td>Contact trigger for pinprick stimulation</td>
			<td>MRC Systems GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PinPrick stimulator set</td>
			<td>MRC Systems GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EEG prepping paste</td>
			<td>USB Pharmacy</td>
			<td></td>
			<td>contains sodium chloride, pumice stone, propylene glycol</td>
		</tr>
		<tr>
			<td>SuperVisc</td>
			<td>EASYCAP GmbH</td>
			<td></td>
			<td>Electrolyte-Gel for active electrodes</td>
		</tr>
		<tr>
			<td>Brain Vision Recorder</td>
			<td>Brain Products GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Vision Analyzer</td>
			<td>Brain Products GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB using EEGLAB
			
			
			
			
			
			</td>
			<td>Swartz Center for Computational Neuroscience, University of California San Diego</td>
			<td></td>
			<td>For EEG processing, including averaging of all EEG epochs
			
			
			
			</td>
		</tr>
	</tbody>
</table>

electrophysiological measurement of noxious-evoked brain activity in neonates using a flat-tip probe coupled to electroencephalography

Il dolore è una spiacevole esperienza sensoriale ed emozionale. In pazienti non-verbale, è molto difficile misurare il dolore, anche con strumenti di valutazione del dolore. Tali strumenti sono soggettivi o determinano gli indicatori fisiologici secondari che hanno anche alcune limitazioni, in particolare se si desidera esplorare l'efficacia dell'analgesia. Di elaborazione corticale è essenziale per la percezione del dolore, misure di attività del cervello possono fornire un utile approccio per valutare il dolore nei neonati. Qui presentiamo un metodo per valutare la nocicezione con registrazioni di attività elettrofisiologica cervello ottimizzate per l'uso nei neonati. Per produrre stimoli nocivi altamente standardizzati e riproducibili abbiamo applicato lo stimolo meccanico con una sonda di piatto-punta, ad esempio, puntura di spillo, che non è pelle-rottura e non causare l'afflizione del comportamento. Il potenziale evocato nocive permette la misurazione oggettiva della nocicezione in pazienti non-verbale. Questo metodo può essere utilizzato nei neonati fin da 34 settimane di età gestazionale. Inoltre, potrebbe essere applicato in situazioni diverse come misurare l'efficacia dei farmaci anestetici o analgesici.

Figura 1 è la rappresentazione schematica dell'elettrodo posizionamento utilizzando il sistema di posizionamento elettrodo di 10/20 internazionale modificato. La figura 2 Mostra l'attività EEG registrato poco prima e dopo l'applicazione di un singolo stimolo nocivo utilizzando una sonda di piatto-punta con 32 mN forza, stimolazione si è verificato come descritto nel protocollo alla mano destra del neonato (<strong class="xfig"...

Watch this Scientific Journal Video about Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography at JoVE.com

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Elettrofisiologici misurazione dell'attività cerebrale nocive-evocato in neonati usando una sonda di piatto-punta accoppiata per elettroencefalografia

Misurazione del dolore in pazienti non-verbale è una sfida. In questo studio registrazione EEG viene combinato con stimolazione usando una sonda piatto-punta per rilevare attività cerebrale nocive-evocato in maniera oggettiva.

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

electrophysiological-measurement-noxious-evoked-brain-activity

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

6.5K Views.  University of Basel Children's Hospital. Misurazione del dolore in pazienti non-verbale è una sfida. In questo studio registrazione EEG viene combinato con stimolazione usando una sonda piatto-punta per rilevare attività cerebrale nocive-evocato in maniera oggettiva.

Video: Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Segmentazione e misurazione dei volumi FAT nei modelli murini di obesità Utilizzo di X-ray Tomografia Computerizzata

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

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Medicina

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Epidurale Misurazione della pressione intracranica in ratto utilizzando una fibra ottica trasduttore di pressione

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La misurazione e trattamento di soppressione in Ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

Stato dell&#39;arte cranica Ultrasound Imaging in neonati

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Molecular Probe Optimization per determinare mortalità delle cellule in un organismo fotosintetico (<em&gt; Microcystis aeruginosa</em&gt;) In citometria a flusso

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Transcutanea microcircolatoria Imaging in pretermine neonati

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Utilizzando Saccadometry con stimolazione cerebrale profonda per studiare normali e patologiche funzione cerebrale

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Metalli di imaging in tessuto cerebrale di Laser Ablation - plasma accoppiato induttivamente - spettrometria di massa (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (A&#946;) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled A&#946; peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF A&#946;1-42 between 150-4,000 pg/mL.

A reference measurement procedure for the absolute quantification of A&#946;1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Quantificazione assoluta di A?<sub&gt; 1-42</sub&gt; In CSF Utilizzando un riferimento alla procedura di misurazione di massa spettrometrica

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Misurazione ripetuta di attività muscolare respiratoria e ventilazione in modelli murini di malattie neuromuscolari

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Elettrofisiologici misurazione dell'attività cerebrale nocive-evocato in neonati usando una sonda di piatto-punta accoppiata per elettroencefalografia

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