Questa pubblicazione video è stato reso possibile da un finanziamento del National Institute of Diabetes and Digestive e malattie renali (NIDDK) per il programma Progetto &quot;Patobiologia del sistema enterico&quot; Grant DK 68055. Christopher T. Creedon è stato sostenuto dal Programma Mentori pubblico Rochester scuole. Ringraziamo il Sig. Gary Stoltz per l&#39;assistenza tecnica, la signora Kristy Zodrow segreteria per l&#39;assistenza e il Dr. Douglas Baer da Los Gatos Research, Inc (Mountain View, CA).

1. Formazione e assuefazione dei Topi <ol><li> Prima dell&#39;analisi, mettere tutti i topi nelle camere di prova per 2-4 ore con flusso d&#39;aria costante, al fine di abituarli alle condizioni di prova. Questo riduce notevolmente i livelli di stress che altrimenti potrebbero causare il rilevamento aberrante di ritardato svuotamento gastrico. Trattare i topi stesso modo se l&#39;esperimento svuotamento gastrico è in esecuzione. Preparare il tuorlo d&#39;uovo (vedi oltre), senza l&#39;aggiunta di acido ottanoico e alimentare 0,2 g per ogni mouse. </li><li> Ripetere questo processo fino a quando i topi sono sufficientemente formati (in genere 2-3 volte). I topi sono tipicamente abituano facilmente purché le condizioni ambientali sono mantenuti uguali. </li></ol> Nota: non abituati topi continuano a muoversi per circa 1 ora dopo il trasferimento alla camera, e defecare e urinare frequentemente, mentre i topi abituati rapidamente stabilirsi nel loro nuovo ambiente e distendersi. Note: Durante l&#39;esperimento: gli animali Monitor per i segni di perdita di assuefazione, come eccesso di minzione, defecazione, mancanza di interesse per mangiare l&#39;uovo. Se questo è il caso considerare nuovamente abituare in una camera vuota 1-2 volte prima di ottenere dati di svuotamento gastrico. La coerenza è molto importante mentre si fa questo esperimento. Fare le cose esattamente nello stesso modo ogni volta che è l&#39;unico modo per ottenere risultati affidabili e riproducibili. Questo include dare trattamento (ad esempio insulina) ogni giorno, allo stesso tempo, non separando i topi dai loro compagni di gabbia se non assolutamente necessario, digiuno i topi e iniziare il test di svuotamento gastrico, allo stesso tempo, e il trattamento di topi stesso modo. 2. Preparazione del Isotope contenenti farina di prova <ol><li> Inizia con peso di 5 g di tuorlo d&#39;uovo in una provetta da 50 ml falco. Ripetere questi passaggi ogni giorno sperimentale per preparare un pasto fresco di prova. </li><li> Aggiungere 10 ml di acido ottanoico con un Concentrazionezione di 2 pl / g al tubo da 50 ml falco contenente l&#39;uovo e mescolare energicamente per 1 minuto con una spatola nel tubo falcon. </li><li> L&#39;uovo viene poi trasferito in un bicchiere di vetro e riscaldato su un becco Bunsen fino coagula e la sua consistenza è adatto a fare palline. Questo richiede in genere circa 30 sec. </li></ol> Nota: Le sfere di tuorlo d&#39;uovo dovrebbe pesare 0,2 g per topo. Questo è importante per mantenere la dose cumulativa costante in tutti i topi. 3. Di iniziare l&#39;esperimento <ol><li> Una volta addestrato e pronto per lo svuotamento gastrico, veloce i topi durante la notte (12 ore) su un metallo &quot;mesh-bottom&quot; rack digiuno per evitare coprofagia. Assicurarsi che hanno libero accesso ad acqua potabile. Poiché topi diabetici sono usati nell&#39;esperimento corrente, non dovrebbero essere tenuti a digiuno per più di 16 ore. </li><li> Inizia la creazione di camere di svuotamento gastrico. Utilizzare camere pulite e le coperture che sono stati essiccati all&#39;aria. Inoltre, qualsiasitubi che collegano le camere all&#39;analizzatore o l&#39;alimentazione dell&#39;aria CO 2 deve essere priva di umidità, acqua può interferire con il segnale letto dall&#39;analizzatore </li><li> Collegare le camere per i tubi di ingresso che forniscono un flusso d&#39;aria costante. Quindi collegare i tubi di scarico dalle camere alla macchina. Chiudere le provette e accendere il flusso d&#39;aria. </li></ol> Nota: applicare una piccola quantità di vaselina al termine dei coperchi di copertura in modo da chiudere facilmente e sono sigillati in modo sicuro. Questa tenuta è necessario raccogliere tutta l&#39;anidride carbonica prodotta dai topi. 4. Procedure sperimentali <ol><li> Iniziate pesando ogni mouse. Il peso corporeo è una misura della loro salute continua bene. Poi posto ogni topo nella camera appropriata. Naturalmente è importante avere l&#39;aria che fluisce nelle camere in questo momento. </li><li> Per avviare la misurazione, lasciare che i topi per acclimatarsi alle camere prima di regolarezione dei livelli di aria. </li><li> Una volta che i topi apparire calmo, che può richiedere alcuni minuti, regolare la portata d&#39;aria per ogni camera del mouse. Questo può essere diverso per ciascun topo. Tipicamente, il flusso di aria viene regolato all&#39;inizio dell&#39;esperimento per assicurarsi che esalato CO 2 raggiunge livelli rilevabili con qualsiasi apparecchiatura viene utilizzata, e fare in modo che il livello rimane sufficientemente bassa per garantire fatturato sana aria. Usiamo iniziali livelli di CO 2 tra 1.000 e 1.500 parti per milione. </li><li> Se ha difficoltà con la regolazione, verificare la presenza di perdite d&#39;aria. Quindi ripetere il processo per ciascuna delle sezioni e guardare per un altro giro di misurazioni per vedere se le rettifiche apportate al flusso d&#39;aria hanno corretto il livello di CO 2. È importante per ottenere una linea di base stabile lettura prima di alimentare i topi. Usiamo una macchina con una funzione di auto calibrazione. Se questo non è il caso di taratura deve essere controllata. </li><li> Quando questo avviene, somministrare il pasto uovoal primo mouse e registrare il tempo di ciascun topo riceve il cibo. </li><li> Corriamo il procedimento per 4 ore per ottenere i valori sufficienti per il montaggio del CO 13 2 curva di arricchimento per ogni mouse. Controllare i topi ogni 30-60 min per assicurarsi che i livelli di CO 2 sono ancora sicuro per i topi. </li><li> Preparare nuove scatole contenenti cibo prima della fine della prova lo topi può iniziare a mangiare immediatamente dopo la prova è finita. </li></ol>

<ol>
	<li>Yeung, C. K., McCurrie, 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=A+simple+method+to+investigate+the+inhibitory+effects+of+drugs+on+gastric+emptying+in+the+mouse+in+vivo.">A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo.</a> J. Pharmacol. Toxicol. Methods. 45, 235-240 (2001).</li><li>Osinski, M. A., Seifert, T. R., Cox, B. F., Gintant, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+evaluation+of+drug-evoked+changes+in+gastric+emptying+in+mice.">An improved method of evaluation of drug-evoked changes in gastric emptying in mice.</a> J. Pharmacol. Toxicol. Methods. 47, 115-120 (2002).</li><li>Ghoos, Y. F., 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=Measurement+of+gastric+emptying+rate+of+solids+by+means+of+a+carbon-labeled+octanoic+acid+breath+test.">Measurement of gastric emptying rate of solids by means of a carbon-labeled octanoic acid breath test.</a> Gastroenterology. 104, 1640-1647 (1993).</li><li>Symonds, E., Butler, R., Omari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+breath+tests+can+detect+alterations+in+gastric+emptying+in+the+mouse.">Noninvasive breath tests can detect alterations in gastric emptying in the mouse.</a> Eur. J. Clin. Invest. 32, 341-344 (2002).</li><li>Symonds, E. L., Butler, R. N., Omari, T. I. <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+gastric+emptying+in+the+mouse+using+the+[13C]-octanoic+acid+breath+test.">Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test.</a> Clin. Exp. Pharmacol. Physiol. 27, 671-675 (2000).</li><li>Verhulst, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ghrelin+in+the+relationship+between+hyperphagia+and+accelerated+gastric+emptying+in+diabetic+mice.">Role of ghrelin in the relationship between hyperphagia and accelerated gastric emptying in diabetic mice.</a> Gastroenterology. 135, 1267-1276 (2008).</li><li>Schoonjans, 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=The+13C-octanoic+acid+breath+test:+validation+of+a+new+noninvasive+method+of+measuring+gastric+emptying+in+rats.">The 13C-octanoic acid breath test: validation of a new noninvasive method of measuring gastric emptying in rats.</a> Neurogastroenterol. Motil. 14, 287-293 (2002).</li><li>Choi, K. 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=Determination+of+gastric+emptying+in+nonobese+diabetic+mice.">Determination of gastric emptying in nonobese diabetic mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 293, 1039-1045 (2007).</li><li>Atkinson, M. A., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOD+mouse+model+of+type+1+diabetes:+as+good+as+it+gets?.">The NOD mouse model of type 1 diabetes: as good as it gets?.</a> Nat. Med. 5, 601-604 (1999).</li><li>Camilleri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Diabetic+gastroparesis.">Clinical practice. Diabetic gastroparesis.</a> N. Engl. J. Med. 356, 820-829 (2007).</li><li>Choi, K. 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=Heme+oxygenase-1+protects+interstitial+cells+of+Cajal+from+oxidative+stress+and+reverses+diabetic+gastroparesis.">Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis.</a> Gastroenterology. 135, 2055-2064 (2008).</li><li>Kashyap, P. 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=Carbon+monoxide+reverses+diabetic+gastroparesis+in+NOD+mice.">Carbon monoxide reverses diabetic gastroparesis in NOD mice.</a> Am. J. Physiol. GI. G298, G1013-G1019 (2010).</li></ol>

Gli autori dichiarano di non avere conflitto di interessi finanziari.

La tecnica qui descritta permette di ripetuti e non invasivo per la misurazione in vivo di svuotamento gastrico solido nei topi. Questo sistema ha il vantaggio che gli animali non sono trattenuti nella camera di misura, permettendo loro di muoversi e ruotare liberamente. Dal momento che questo è un ambiente familiare, i topi hanno ancora bisogno di essere addestrati e abituati alle camere di prova per prevenire gli effetti dello stress sullo svuotamento gastrico. In generale, si assume i dati svuotame...

Questo manoscritto descrive le considerazioni tecniche e metodologiche implicate nella misurazione non invasiva di svuotamento gastrico nei topi. Seguendo il protocollo qui descritto, gli investigatori in modo affidabile e riproducibile seguire le modifiche di svuotamento gastrico a causa dello sviluppo della malattia, studiare l&#39;impatto di agenti farmacologici sullo svuotamento gastrico e seguire la risposta di svuotamento gastrico per il trattamento di malattie sottostanti o difetti 6,8, 11,12. In precedenti pubblicazioni, l&#39;applicazione di 13 C test di respiro acido ottanoico ha dimostrato di essere un modo utile per misurare svuotamento gastrico nell&#39;uomo e negli animali 3,8. Questo documento descrive in dettaglio, le procedure necessarie per ottenere dati affidabili sul 6-8 mesi necessari per uno studio longitudinale di svuotamento gastrico nei topi con diabete. I vantaggi di seguire questo protocollo rispetto ai metodi precedentemente pubblicati sono che il ricercatore può essere assicurato il richiederlo datid sarà affidabile e riproducibile. Inoltre, il sistema automatizzato per la raccolta e l&#39;analisi dei campioni di gas descritto qui aumenta il numero di animali che possono essere seguiti simultaneamente in uno studio. Nel complesso, l&#39;obiettivo di questo lavoro è quello di identificare i fattori chiave che mantengono assuefazione dei topi al test e che ridurre la variabilità nei risultati ottenuti. Per la misurazione in vivo di svuotamento gastrico, i topi sono tenuti a digiuno per una notte e messo nelle camere di prova di plastica trasparente con portata costante. Dopo i topi vengono abituati ai tubi, al basale esalato 13 CO 2 livelli sono determinati e flusso d&#39;aria adeguato di conseguenza. Successivamente, l&#39;amministrazione di un pasto di prova costituito da tuorlo d&#39;uovo mescolato con 13 C-marcato acido ottanoico. Poiché i topi a digiuno e formati, in genere mangiare il pasto di prova entro 2 minuti. Il somministrato acido ottanoico non viene assorbito nello stomaco, ma sarà ripreso nel duodeno und otterrà metabolizzato nel fegato in 13 CO 2, che viene rilasciato ed espirata, con un conseguente arricchimento di 13 CO 2 nell&#39;aria circostante. Campioni d&#39;aria sono raccolti ad intervalli di tempo determinati ed analizzati mediante l&#39;analizzatore degli isotopi di carbonio. Il rate-limiting passo in questo processo è lo svuotamento gastrico e l&#39;escrezione polmonare di 13 CO 2 corrisponde direttamente con lo svuotamento gastrico del pasto marcato. <img src="/files/ftp_upload/50301/50301fig1.jpg" alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/50301/50301fig1highres.jpg"/> Figura 1. Schema dell&#39;apparato svuotamento gastrico. Dopo il digiuno notturno, i topi sono posti in camere trasparenti, che consentano loro di muoversi e girare liberamente. Un tubo di ingresso permette afflusso di aria fresca e costante ed una uscita conduce l&#39;analizzatore per misurare l&#39;isotopo 13 C-a-12Rapporto C nel respiro esalato. La camera dispone anche di una porta centrale per la consegna di prodotti alimentari contenenti [13 C]-acido ottanoico.

<table border="1"><tbody><tr><td> NOME </td><td> AZIENDA </td><td> CAT NUMERO </td><td> COMMENTI </td></tr><tr><td> Una fonte di alimentazione d&#39;aria costante, il flusso e composizione </td><td> centrale di alimentazione aria nella struttura di ricerca </td><td></td><td></td></tr><tr><td> 130 ml di campionamento camera che dispone di aria in entrata, di uscita dell&#39;aria, l&#39;apertura e la gestione alimentare Tubi di plastica per l&#39;alimentazione pneumatica </td><td> In casa costruita </td><td></td><td></td></tr><tr><td> Acido ottanoico </td><td> Cambridge isotopo laboratori (Andover, MA) </td><td> CLM-293-1 </td><td></td></tr><tr><td> Per preparare il pasto uovo: <ul><li> piccolo becher </li><li> 50 ml, tubo </li><li> Becco Bunsen </li><li> uovo </li><li> spatola </li></ul></td><td> Ogni fornitore </td><td></td><td> Cercate diessere coerente con il fornitore uovo poiché il contenuto nutrizionale e appetibilità delle uova può influenzare lo svuotamento gastrico e l&#39;ingestione del pasto </td></tr><tr><td> Biossido di carbonio isotopo analizzatore </td><td> Los Gatos Research Inc. (Mountain View, CA) </td><td></td><td></td></tr></tbody></table>

assessment of gastric emptying in non-obese diabetic mice using a [13c]-octanoic acid breath test

Gastrico studi svuotamento nei topi sono stati limitati dalla incapacità di seguire i cambiamenti di svuotamento gastrico, nello stesso animale, poiché le tecniche più comunemente utilizzate richiedono l&#39;uccisione degli animali e il recupero post-mortem del 1,2 pasto. Questo approccio evita studi longitudinali per determinare le variazioni di svuotamento gastrico con l&#39;età e la progressione della malattia. Il comunemente usato [13 C]-ottanoico breath test con l&#39;acido per l&#39;uomo 3 è stato modificato per l&#39;uso nel topo e nel ratto 4-6 7 e abbiamo precedentemente dimostrato che questo test è affidabile e sensibile alle variazioni di svuotamento gastrico in risposta ai farmaci e durante progressione di malattia diabetica 8. In questo video di presentazione il principio e l&#39;attuazione pratica di questo test modificato è spiegato. Come nel precedente studio, i topi NOD LTJ vengono utilizzati, un modello di diabete di tipo 1 9. Una quota di questi topi sviluppano i sintomi di gastroparesi, una complicazione di Diabetees caratterizzata da ritardato svuotamento gastrico senza ostruzione meccanica dello stomaco 10. Questo documento dimostra come addestrare i topi per i test, come preparare il pasto di prova e ottenere 4 ore di dati di svuotamento gastrico e come analizzare i dati ottenuti. L&#39;analizzatore isotopo di carbonio utilizzati nel presente studio è adatto per il campionamento automatico dei campioni di aria da un massimo di 12 topi contemporaneamente. Questa tecnica permette di follow-up longitudinale di svuotamento gastrico da grandi gruppi di topi con diabete o altre malattie di lunga durata.

A dati rappresentativi indicati da tre topi diverse è mostrato in Figura 2. Il grafico nero rappresenta i punti dati di un mouse con svuotamento gastrico normale. Essa mostra la frazione di 13 C che viene recuperato in aria espirata espressa come percentuale della dose somministrata per ora espressa come funzione di tempo. La curva blu è da un topo con un accelerato svuotamento gastrico con un valore di mezzo T di 40 min e la curva rossa è da un mouse con svuotamento gastrico ritardato con...

Watch this Scientific Journal Video about Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test at JoVE.com

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determinazione di svuotamento gastrico con un non-invasiva [<sup&gt; 13</sup&gt; C]-ottanoico breath test con acido per gastroparesi inseguimento in femmine di topo NOD LTJ.

La valutazione di svuotamento gastrico in non-obesi topi diabetici utilizzando un [<sup&gt; 13</sup&gt; C]-ottanoico Breath Test Acid

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

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Medicine

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

13.5K Views.  Mayo Clinic. Determinazione di svuotamento gastrico con un non-invasiva [ 13 C]-ottanoico breath test con acido per gastroparesi inseguimento in femmine di topo NOD LTJ.

Video: Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

Making Sense di Ascolto: La batteria di test IMAP

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

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Medicina

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

Veloce e preciso espirata Ammoniaca misura

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

Valutazione ecografica della funzione endoteliale: una Direttiva tecnica del test dilatazione flusso-mediata

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Aprire tracheostomia gastrico acido aspirazione Modello murino di lesioni polmonari acute Risultati in massima acuta non letale Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

Un emoderivato di Test per la rilevazione di ROS1 e RET trascrizioni di fusione da acido ribonucleico usando reazione a catena della polimerasi digitale di circolazione

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Protocollo per creare ferite croniche nei topi diabetici

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Valutazione non invasiva della funzione Dorsiflexor muscolare nei topi

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Utilizzo di una biopsia chimica per la valutazione della qualità dell'innesto

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and metabolic changes are not well understood. Multiple factors are thought to play a role, including reduced caloric intake, decreased nutrient absorption, increased satiety, the release of satiety-promoting hormones, shifts in bile acid metabolism, and alterations in the gut microbiota.
The rat RYGB model presents an ideal framework to study these mechanisms. Prior work on mouse models have had high mortality rates, ranging from 17 to 52%, limiting their adoption. Rat models demonstrate more physiologic reserve to surgical stimulus and are technically easier to adopt as they allow for the use of surgical staplers. One challenge with surgical staplers, however, is that they often leave a large gastric pouch which is not representative of RYGB in humans. 
In this protocol, we present a RYGB protocol in rats that result in a small gastric pouch using surgical staplers. Utilizing two stapler fires which remove the forestomach of the rat, we obtain a smaller gastric pouch similar to that following a typical human RYGB. Surgical stapling also results in better hemostasis than sharp division. Additionally, the forestomach of the rat does not contain any glands and its removal should not alter the physiology of RYGB.
Weight loss and metabolic changes in the RYGB cohort were significant compared to the sham cohort, with significantly lower glucose tolerance at 14 weeks. Furthermore, this protocol has an excellent survival of 88.9% after RYGB. The skills described in this protocol can be acquired without previous microsurgical experience. Once mastered, this procedure will provide a reproducible tool for studying the mechanisms and effects of RYGB.

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

Un protocollo per il bypass gastrico Roux-en-Y nei ratti che utilizza cucitrici lineari

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and ...

Valutazione della riserva alveolo-capillare polmonare durante l'esercizio

Dual Test Gas Pulmonary Diffusing Capacity Measurement During Exercise in Humans Using the Single-Breath Method

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure pulmonary alveolar-capillary reserve in both healthy and patient populations. The measurement provides an estimate of the participant&#39;s ability to recruit and distend pulmonary capillaries. The method has recently been reported to exhibit a high test-retest reliability in healthy volunteers during exercise of light to moderate intensity. Of note, this technique permits up to 12 repeated maneuvers and only requires a single breath with a relatively short breath-hold time of 5 s. Representative data are provided showing the gradual changes in DL,NO and DL,CO from rest to exercise at increasing intensities&#160;of up to 60% of maximal workload. The measurement of diffusing capacity and evaluation of alveolar-capillary reserve is a useful tool to evaluate the lung&#39;s ability to respond to exercise both in the healthy population as well as in patient populations such as those with chronic lung disease.

Autore in primo piano: Integrazione delle misurazioni della riserva alveolare-capillare nell'adattamento all'esercizio e nelle strategie terapeutiche

This protocol presents a method to assess pulmonary alveolar-capillary reserve measured by combined single-breath measurement of the diffusing capacity to carbon monoxide (DL,CO) and nitric oxide (DL,NO) during exercise. Assumptions and recommendations for using the technique during exercise form the foundation of this article.

Misurazione della capacità di diffusione polmonare del gas a doppio test durante l'esercizio fisico nell'uomo utilizzando il metodo del respiro singolo

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure ...