Questa ricerca è stata sostenuta dal Programma di ricerca intramurale del NIH, National Institute on Aging e Baltimore Longitudinal Study on Aging. Una parte di tale sostegno è stato attraverso un contratto di R &amp; D con MedStar Research Institute. Protocollo standard e competenze tecniche sono state fornite dal AtCor.

1. Procedura <ol><li> Aprire il software tramite il collegamento sul desktop di Windows facendo doppio clic sull&#39;icona. </li><li> Clicca paziente per attivare la schermata del paziente. </li><li> Per inserire un nuovo paziente nel database, fare clic sul pulsante Nuovo nella zona di ricerca paziente, e quindi fare clic sul pulsante Sì per confermare che si desidera inserire un nuovo paziente. </li><li> Inserire i dettagli del paziente come il cognome, nome, data di nascita e sesso. Fare clic sul pulsante Salva per avanzare alla fase successiva. </li><li> Fare clic sul pulsante PWV PWA o per eseguire la misurazione. Se solo una di queste modalità è disponibile, non è necessario cliccare sul pulsante, è sufficiente procedere alla schermata studio. </li></ol> 2. Misurazione Centrale pressione sanguigna Utilizzando analisi dell&#39;onda di polso (PWA) <ol><li> Fare clic sul pulsante di studio per immettere i parametri di studio. Inserire la pressione brachiale preso da un misuratore di pressione calibrata e ogni altro dettaglio che si desidera includere. </li><li> Click sul pulsante Data Capture per procedere con una misurazione. Attaccare i tre elettrodi ECG e conduce al paziente in una configurazione II piombo modificati o utilizzando arti del paziente come indicato sui cavi. </li><li> Palpare l&#39;arteria radiale del paziente. Questo si trova sul laterale del polso, immediatamente al di sopra del polso vicino alla base del pollice. Premere delicatamente l&#39;arteria radiale contro l&#39;osso sottostante con il medio e l&#39;indice. </li><li> Posizionare il tonometro sul punto di impulso più forte. Premere delicatamente il tonometro verso il basso fino a visualizzare una forma d&#39;onda di pressione costante visualizzata sullo schermo di acquisizione dati. </li><li> Se Acquisizione automatica è attivata (vedi nota sotto), il sistema salverà automaticamente la misura. Per salvare manualmente la misurazione per l&#39;analisi, premere la barra spaziatrice sulla tastiera (o passo sulla pedaliera, se utilizzato). </li><li> Se è richiesta una misurazione ripetuta con gli stessi parametri di studio, fare clic sul pulsante Ripeti per tornare alla catturaSchermata dei dati e ripetere la misurazione come descritto nei passaggi 2,4-2,5 sopra. </li><li> Per eseguire una misurazione su un nuovo paziente, fare clic sul pulsante Paziente e tornare al punto 1.3. </li></ol> 3. La rigidità arteriosa Misurazione Utilizzando velocità dell&#39;onda di polso (PWV) <ol><li> Fare clic sul pulsante di studio per immettere i parametri di studio. </li><li> Inserisci pressione brachiale preso da uno sfigmomanometro calibrata Immettere la distanza misurata per la distale (sovra-sternale notch a polso femorale) e prossimale (sovra-sternale notch di polso carotideo) siti. Campi Farmaco, Note e operatore possono essere inseriti, se lo si desidera </li><li> Fare clic sul pulsante Capture per procedere con la misurazione. </li><li> Assicurarsi che il segnale ECG è di qualità accettabile. </li><li> Palpare il polso carotide. Questo si trova tra la laringe e il muscolo sternocleidomastoideo nel collo. Esso può essere sentito spingendo il muscolo di lato e premendo l&#39;arteria contro la laringe. </li><li&gt; Posizionare il tonometro sulla carotide e premere delicatamente il tonometro fino a quando si riceve una forma d&#39;onda di pressione costante visualizzata sullo schermo di acquisizione dati. </li><li> Quando si ha un minimo di 13 secondi delle forme d&#39;onda coerenti, fare clic sul pulsante OK o (premere sul pedale, se utilizzato). </li><li> Palpare il polso dell&#39;arteria femorale. Questo si trova sopra la coscia ventrale a metà strada tra la sinfisi pubica e la spina iliaca anteriore superiore. </li><li> Posizionare il tonometro sulla arteria femorale e premere delicatamente il tonometro fino a quando si riceve una forma d&#39;onda di pressione costante visualizzata sullo schermo di cattura. Quando si ha un minimo di 13 secondi delle forme d&#39;onda coerenti, fare clic sul pulsante OK (o premere il sul pedale, se utilizzato). </li><li> La relazione (s) può ora essere rivisto. Queste misurazioni sono tipicamente condotte in triplicato. Se l&#39;utente desidera misurare la PWV tra carotidi e arterie radiali, questi passi indicati sarà ripetuto tranne che invece di misurazione al farteria emoral, l&#39;utente dovrebbe posizionare il tonometro a livello dell&#39;arteria radiale. Per eseguire una misura su un nuovo paziente, fare clic sul pulsante Studio e ripetere questa procedura o utilizzare la funzione di ricerca per cercare un paziente esistente. </li></ol> 4. Fermare <ol><li> Il software salva automaticamente tutti i rapporti. Per chiudere il software, fare clic su Sistema dal menu principale, e quindi fare clic su Exit. Posizionare il tonometro nel vassoio modulo per la conservazione. </li></ol>

<ol>
	<li>Najjar, S. S., Scuteri, 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=Pulse+wave+velocity+is+an+independent+predictor+of+the+longitudinal+increase+in+systolic+blood+pressure+and+of+incident+hypertension+in+the+Baltimore+Longitudinal+Study+of+Aging.">Pulse wave velocity is an independent predictor of the longitudinal increase in systolic blood pressure and of incident hypertension in the Baltimore Longitudinal Study of Aging.</a> J. Am. Coll. Cardiol. 51 (14), 1377-1383 (2008).</li><li>Doonan, R. J., Scheffler, P., 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=Altered+arterial+stiffness+and+subendocardial+viability+ratio+in+young+healthy+light+smokers+after+acute+exercise.">Altered arterial stiffness and subendocardial viability ratio in young healthy light smokers after acute exercise.</a> PloS one. 6 (10), (2011).</li><li>Laurent, S., Cockcroft, J., 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=Expert+consensus+document+on+arterial+stiffness:+methodological+issues+and+clinical+applications.">Expert consensus document on arterial stiffness: methodological issues and clinical applications.</a> Eur. Heart J. 27 (21), 2588-2605 (2006).</li><li>Mitchell, G. F., Hwang, S. -. J., 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=Arterial+stiffness+and+cardiovascular+events:+the+Framingham+Heart+Study.">Arterial stiffness and cardiovascular events: the Framingham Heart Study.</a> Circulation. 121 (4), 505-511 (2010).</li><li>Abbatecola, A. M., Chiodini, P., 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=Pulse+wave+velocity+is+associated+with+muscle+mass+decline:+Health+ABC+study.">Pulse wave velocity is associated with muscle mass decline: Health ABC study.</a> Age. , (2011).</li><li>Watson, N. L., Sutton-Tyrrell, K., 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=Arterial+stiffness+and+gait+speed+in+older+adults+with+and+without+peripheral+arterial+disease.">Arterial stiffness and gait speed in older adults with and without peripheral arterial disease.</a> Am. J. Hypertens. 24 (1), 90-95 (2011).</li><li>Mackenzie, I. S., Wilkinson, I. B., Cockcroft, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+arterial+stiffness+in+clinical+practice.">Assessment of arterial stiffness in clinical practice.</a> QJM. 95 (2), 67-74 (2002).</li><li>O'Rourke, M. F., Pauca, A., Jiang, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse+wave+analysis.">Pulse wave analysis.</a> Br. J. Clin. Pharmacol. 51 (6), 507-522 (2001).</li><li>Stoner, L., Young, J. M., Fryer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessments+of+arterial+stiffness+and+endothelial+function+using+pulse+wave+analysis.">Assessments of arterial stiffness and endothelial function using pulse wave analysis.</a> Int. J. Vasc. Med. 2012, 903107 (2012).</li><li>Farro, I., Bia, D., 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=Pulse+wave+velocity+as+marker+of+preclinical+arterial+disease:+reference+levels+in+a+uruguayan+population+considering+wave+detection+algorithms,+path+lengths,+aging,+and+blood+pressure.">Pulse wave velocity as marker of preclinical arterial disease: reference levels in a uruguayan population considering wave detection algorithms, path lengths, aging, and blood pressure.</a> Int. J. Hypertens. 2012, (2012).</li><li>Lakatta, E. G., Wang, M., Najjar, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+aging+and+subclinical+arterial+disease+are+fundamentally+intertwined+at+macroscopic+and+molecular+levels.">Arterial aging and subclinical arterial disease are fundamentally intertwined at macroscopic and molecular levels.</a> Med. Clin. N. Am. 93 (3), 583-604 (2009).</li><li>Ambrose, 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> Clinical Manual (CVMS) Rev. 2, 1-51 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+Values+for+Arterial+Stiffness'+Collaboration+Determinants+of+pulse+wave+velocity+in+healthy+people+and+in+the+presence+of+cardiovascular+risk+factors:+'establishing+normal+and+reference+values.">Reference Values for Arterial Stiffness' Collaboration Determinants of pulse wave velocity in healthy people and in the presence of cardiovascular risk factors: 'establishing normal and reference values.</a> Eur. Heart J. 31 (19), 2338-2350 (2010).</li><li>van Bortel, L. M., Laurent, S., 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=Expert+consensus+document+on+the+measurement+of+aortic+stiffness+in+daily+practice+using+carotid-femoral+pulse+wave+velocity.">Expert consensus document on the measurement of aortic stiffness in daily practice using carotid-femoral pulse wave velocity.</a> J. Hypertens. 30 (3), 445-448 (2012).</li><li>Scuteri, A., Brancati, A., Gianni, W., Assisi, A., Volpe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+is+an+independent+risk+factor+for+cognitive+impairment+in+the+elderly:+a+pilot+study.">Arterial stiffness is an independent risk factor for cognitive impairment in the elderly: a pilot study.</a> J. Hypertens. 23 (6), 1211 (2005).</li><li>Khoshdel, A. R., Carney, S. L., Nair, B. R., Gillies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+management+of+cardiovascular+diseases+by+pulse+wave+velocity:+combining+clinical+practice+with+clinical+research+using+evidence-based+medicine.">Better management of cardiovascular diseases by pulse wave velocity: combining clinical practice with clinical research using evidence-based medicine.</a> Clin. Med. Res. 5 (1), 45-52 (2007).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione. Gli autori hanno ricevuto assistenza con la descrizione del protocollo e le prestazioni di studio SphygmoCor che produce questo prodotto descritto.

La misurazione della velocità dell&#39;onda di polso (PWV) è un metodo non invasivo per analizzare rigidità arteriosa centrale in modo relativamente semplice e riproducibile 3. Per valutare PWV, la carotide (come un surrogato per l&#39;aorta discendente) e forme d&#39;onda polso femorale vengono acquisite utilizzando un tonometro ad alta fedeltà, il tempo di ritardo in arrivo dell&#39;onda di impulso alla referenziamento tra i due siti è stimato un onde ECG simultaneamente registrati da un software proge...

A correction was made to <a href="http://www.jove.com/video/50817">Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging</a>. There was an error with the author, Majd AlGhatrif's, name. The author's name has been corrected to:
Majd AlGhatrif
instead of:
Majd al Ghatrif

A correction to the author list has been made for article <a href="http://www.jove.com/video/50817">Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging</a>.

Erratum: Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Velocità dell&#39;onda di polso (PWV) è considerata il gold standard nella valutazione della rigidità arteriosa 2. Mentre non usato clinicamente negli Stati Uniti, è stato convalidato e ampiamente utilizzato in ambito clinico in Europa 3. Cambiamenti nella PWV sono stati correlati con i fattori di rischio cardiovascolari come l&#39;ipertensione, l&#39;obesità, ridotta tolleranza al glucosio e la dislipidemia. Maggiore rigidità aortica è stata associata ad un aumentato rischio di primo evento cardiovascolare 4, muscolare declino massa 5, e più lenta velocità di andatura in soggetti con malattia arteriosa periferica 6. Valutazione della PWV è compiuta analizzando la velocità con cui un impulso di sangue viaggia dalla carotide all&#39;arteria femorale 7. Si tratta di un metodo relativamente semplice, con risultati altamente riproducibili. I progressi della tecnologia continuano a semplificare la procedura, riducendo la dipendenza dell&#39;operatore e diminuendo la quantità di tempo necessario per laesame. Il sistema utilizzato nel nostro laboratorio è prodotto da SphygmoCorand utilizza un elettrocardiogramma e un tonometro ad alta fedeltà per acquisire forme d&#39;onda dalla carotide e polsi femorali. Il software analizza queste variabili per determinare la velocità da 1) la stima del tempo che l&#39;onda di impulso impiega a percorrere tra la carotide e femorale facendo riferimento alla tempistica di arrivo dell&#39;onda di pressione in ogni sito arteriosa rispetto alle forme d&#39;onda elettrocardiografiche 2) dividendo la distanza misurata tra i due siti per il tempo previsto. Il più vicini siti di campionamento, la minore precisione della PWV, limitando l&#39;uso di questo metodo per vasi più grandi come le arterie femorali e carotidee. Oltre a determinare PWV, il sistema può calcolare la pressione in aorta ascendente applicando una funzione di trasferimento alla forma d&#39;onda prodotta dal polso radiale 8 che è la &quot;pressione centrale&quot; e la pressione che più incide direttamente il cuore in fase di espulsione. Queste informazioni possono aiutare nella valutazione dell&#39;effetto del sistema arterioso del ventricolo sinistro in modo affidabile e non invasiva 9. Tecniche alternative utilizzate per l&#39;esame di rigidità vascolare, come la distensibilità carotidea attraverso il monitoraggio eco, richiedono una grande quantità di competenze tecniche e sono più tempo rispetto alla PWV. Monitoraggio Echo generalmente utilizza analisi video-immagine e quindi l&#39;accuratezza di tali misurazioni sono limitate 3. Altre tecniche per esaminare i cambiamenti vascolari includono la valutazione della carotide spessore intima-mediale (IMT), che fornisce risultati più strettamente correlati alla progressione aterosclerotica anche se le immagini in sezione trasversale e trasversali ottenuti possono fornire una approssimazione grossolana di carotide elastance. Questo metodo, tuttavia, è afflitto da alcuni degli stessi vincoli studi distensibilità sopra descritti. Come i ricercatori continuano a guardare i giochi di rigidità ruolo arteriosi in vari stati di malattia,Misurazione PWV probabilmente continuerà a fornire un endpoint surrogato quantitativa e riproducibile.

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pulse wave velocity testing in the baltimore longitudinal study of aging

Velocità dell&#39;onda di polso carotideo-femorale è considerata il gold standard per la misurazione della rigidità arteriosa centrale ottenuti con metodi non invasivi 1. I soggetti sono posti in posizione supina e lasciati riposare tranquillamente per almeno 10 min prima dell&#39;inizio della prova. La dimensione corretta bracciale è selezionato e una pressione sanguigna è ottenuta utilizzando un dispositivo oscillometrico. Una volta ottenuto un riposo pressione sanguigna, le forme d&#39;onda di pressione vengono acquisiti dalla destra arterie carotidi comuni femorali e destro. Il sistema calcola automaticamente il tempo di transito degli impulsi tra questi due siti (usando l&#39;arteria carotide come surrogato per l&#39;aorta discendente). Misure di superficie del corpo vengono utilizzati per determinare la distanza percorsa dall&#39;onda impulsi tra i due siti di campionamento. Questa distanza viene quindi divisa per il tempo di transito dell&#39;impulso conseguente velocità dell&#39;onda di polso. Le misurazioni sono eseguite in triplice copia e la media viene utilizzato per analelisi.

<html><img alt="Tabella 1" fo:content-width="5in" fo:src="/files/ftp_upload/50817/50817table1highres.jpg" src="/files/ftp_upload/50817/50817table1.jpg" /> Tabella 1. Risultati PWV sono prodotte in metri al secondo. In soggetti sani, queste velocità possono variare da una media di 6,2 m / sec (in soggetti sotto i 30 anni) a 10,9 m / sec (in soggetti di 70 anni e più). Questi valori sono stati stabiliti nell&#39;articolo &quot;determinanti della velocità dell&...

Watch this Scientific Journal Video about Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging at JoVE.com

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Velocità dell&#39;onda di polso (PWV) misurazione valuta rigidità arteriosa da un sistema basato tonometria-che misura la velocità con cui un&#39;onda di pressione arteriosa viaggia lungo l&#39;albero arterioso, tipicamente approssimare il tempo che ci vuole questa onda a partire dall&#39;oggetto dell&#39;aorta discendente (usando l&#39;carotide arteria come surrogato) per l&#39;arteria femorale. Carotideo-femorale PWV aumenta da due a tre volte nel corso della vita adulta.

Velocità dell&#39;onda di polso testing a Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

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Vital Signs: Pulse

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16.9K Views.  National Institute of Aging.  Velocità dell'onda di polso (PWV) misurazione valuta rigidità arteriosa da un sistema basato tonometria-che misura la velocità con cui un'onda di pressione arteriosa viaggia lungo l'albero arterioso, tipicamente approssimare il tempo che ci vuole questa onda a partire dall'oggetto dell'aorta discendente (usando l'carotide arteria come surrogato) per l'arteria femorale. Carotideo-femorale PWV aumenta da due a tre volte nel corso della vita a...

Video: Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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Medicina

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Attraverso lo specchio: Time-lapse Microscopia e longitudinale monitoraggio di singole cellule allo Studio Therapeutics anti-cancro

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Un regime Fenotipizzazione per topi geneticamente modificati utilizzati per studiare geni implicati in malattie umane di invecchiamento

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

Ultrasound-based velocità dell&#39;onda di polso di valutazione nei topi

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Il modello di roditore di nonarteritic Neuropatia ottica ischemica anteriore (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinale<em&gt; In Vivo</em&gt; Imaging del Cerebrovasculature: Rilevanza per le malattie del sistema nervoso centrale

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Sviluppo di un modello diretto Pulp-capping di valutazione dei polpa dentale guarigione delle ferite e riparativa dentina formazione nei topi

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Usando la formazione immagine retinica per studiare la demenza

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Measuring the Carotid to Femoral Pulse Wave Velocity (Cf-PWV) to Evaluate Arterial Stiffness

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively by the measurement of carotid to femoral pulse wave velocity (cf-PWV) (Class I; Level of Evidence A). In literature, numerous community-based or disease-specific studies have reported that higher cf-PWV is associated with increased cardiovascular risk. Here, we discuss strategies to evaluate arterial stiffness with cf-PWV. Following the well-defined steps detailed here, e.g., proper position operator, distance measurement, and tonometer position, we will obtain a standard cf-PWV value to evaluate arterial stiffness. In this paper, a detailed stepwise method to record a good quality PWV and pulse wave analysis (PWA) using a non-invasive tonometry-based device will be discussed.

Measuring the Carotid to Femoral Pulse Wave Velocity Cf-PWV to Evaluate Arterial Stiffness

This protocol describes a method to standardize the measurements of carotid-femoral pulse wave velocity to evaluate arterial stiffness.

La carotide a femorale Pulse Wave Velocity (Cf-PWV) per valutare la rigidezza arteriosa di misurazione

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively ...

Integration of Brain Tissue Saturation Monitoring in Cardiopulmonary Exercise Testing in Patients with Heart Failure

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction (HF). However, in clinical cardiopulmonary exercise testing (CPET), cerebral hemodynamics is not assessed. NIRS is used to measure cerebral tissue oxygen saturation (SctO2) in the frontal lobe. This method is reliable and valid and has been utilized in several studies. SctO2 is lower during both rest and peak exercise in patients with HF than in healthy controls (66.3 &#177; 13.3% and 63.4 &#177; 13.8% vs. 73.1 &#177; 2.8% and 72 &#177; 3.2%). SctO2 at rest is significantly linearly correlated with peak VO2 (r = 0.602), oxygen uptake efficiency slope (r = 0.501), and brain natriuretic peptide (r = -0.492), all of which are recognized prognostic and disease severity markers, indicating its potential prognostic value. SctO2 is determined mainly by end-tidal CO2 pressure, mean arterial pressure, and hemoglobin in the HF population. This article demonstrates a protocol that integrates SctO2 using NIRS into incremental CPET on a calibrated bicycle ergometer.

This protocol integrated near-infrared spectroscopy into conventional cardiopulmonary exercise testing to identify the involvement of the cerebral hemodynamic response in exercise intolerance in patients with heart failure.

Integrazione del monitoraggio della saturazione dei tessuti cerebrali nei test di esercizio cardiopolmonare in pazienti con insufficienza cardiaca

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction ...