Riconosciamo il sapiente aiuto di Annie B. Kristensen, Elisabeth V. Andersen, Belinda B. Bringtoft ed i custodi degli animali presso il Dipartimento di Medicina Sperimentale, Frederiksberg Campus, Facoltà di Scienze della Salute e Scienze Mediche, Università di Copenhagen, Danimarca. Jacob Lønholdt viene ringraziato per l&#39;eccellente assistenza tecnica per le riprese delle procedure. Questo studio è stato sostenuto in parte dal Centro LIFEPHARM di Farmacologia in vivo e Università di Copenhagen.

Le procedure illustrate sono state eseguite secondo protocolli approvati dal danese Animal Sperimentazione Ispettorato sotto il Ministero dell&#39;alimentazione, dell&#39;agricoltura e della pesca. 1 Prelievo di sangue dal laterale safena o tarsale Vena <ol><li> Eseguire questa procedura senza anestesia e con l&#39;aiuto di un investigatore, una gestione della cavia, e una raccolta del campione. </li><li> Radere la regione tarsale della zampa posteriore con un rasoio elettrico fino a quando le vene sono visibili. Assicurarsi che la zona è abbastanza grande da evitare pelliccia interferire con il sito di puntura. </li><li> Posizionare la zampa posteriore in un bagno di acqua tiepida e strofinare delicatamente la gamba, rendendo le vene più dilatati ed evidenti. </li><li> Pulire la gamba con un asciugamano pulito e disinfettare il sito di puntura con una garza o un batuffolo di cotone imbevuto di alcool al 70%. </li><li> Trattenere la cavia ed estendere la gamba posteriore verso il basso, mentre applicando una leggera pressione immediatamente al di sopra del ginocchio to fornire stasi. </li><li> Pungere la vena con un ago 21 G o bisturi. Raccogliere esempio sangue., Utilizzando un tubo di raccolta o capillare. Manipolare delicatamente la gamba per aiutare il flusso di sangue. </li><li> Rilasciare stasi, una volta ottenuto il campione. Applicare un pezzo di garza o cotone lana con una leggera pressione sul sito di puntura finché il sanguinamento si è fermato. </li><li> Ritorna l&#39;animale nella sua gabbia e monitor per 5-10 minuti al fine di garantire l&#39;emostasi. </li></ol> 2 Prelievo di sangue dalla vena dell&#39;orecchio <ol><li> Eseguire questa procedura senza anestesia da solo o preferibilmente con l&#39;aiuto di un collega. </li><li> Disinfettare la superficie dorsale del padiglione auricolare con una garza o un batuffolo di cotone imbevuto di alcool al 70%. </li><li> Applicare una leggera pressione alla base dell&#39;orecchio per fornire stasi. </li><li> Pungere la vena con un ago 21 G o bisturi, e raccogliere il sangue. </li><li> Rilasciare stasi, una volta ottenuto il campione, Applicare un pezzo di garza o cotone idrofilo con gepressione ntle al sito di puntura finché il sanguinamento si è fermato. </li><li> Ritorna l&#39;animale nella sua gabbia e monitor per 5-10 minuti al fine di garantire l&#39;emostasi. </li></ol> 3 Prelievo di sangue dalla giugulare Vena <ol><li> Effettuare questa procedura utilizzando l&#39;aiuto di un investigatore supplementare; una gestione della cavia e una raccolta del campione. </li><li> Anestetizzare la cavia con un anestetico adatto per tutta la durata di questa procedura. Una miscela di tiletamina (0,93 mg / kg), zolazepam (0,93 mg / kg), xilazina (1.49 mg / kg) e butorfanolo (0,06 mg / kg) può essere utilizzato. </li><li> Posizionare la cavia anestetizzata sulla sua schiena e ritrarre delicatamente le zampe anteriori caudalmente. </li><li> Shave e disinfettare (ad es., Con il 70% di alcol), la parte ventrale del collo. </li><li> Rivestire la siringa sia con eparina o EDTA prima del campionamento, al fine di ridurre il rischio di coagulazione. </li><li> Palpare la clavicola e inserire un G 25 (o 23 G) ago montato su una siringa da 1 ml approximately 1 centimetro lateralmente alla linea mediana a livello dell&#39;articolazione della spalla. Tenere la siringa in una leggera inclinazione verso l&#39;alto e mediale, mentre applicando una leggera pressione negativa. Evitare di danneggiare le strutture sottostanti come la vena giugulare si trova abbastanza superficiale. Inclinare il collo della cavia leggermente all&#39;indietro per fornire maggiore stasi della vena. </li><li> Rilasciare le zampe anteriori quando si ottiene il campione di sangue, e alleviare stasi prima che l&#39;ago viene retratto. Usare un pezzo secco di garza o di cotone per applicare una leggera pressione sul sito di puntura. </li><li> Ritorna l&#39;animale in una gabbia di recupero e monitorare al fine di garantire l&#39;emostasi e il recupero dall&#39;anestesia. </li></ol>

<ol>
	<li>DeOgburn, 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=Effects+of+increased+dietary+cholesterol+with+carbohydrate+restriction+on+hepatic+lipid+metabolism+in+Guinea+pigs.">Effects of increased dietary cholesterol with carbohydrate restriction on hepatic lipid metabolism in Guinea pigs.</a> Comp.Med. 62 (2), 109-115 (2012).</li><li>Fernandez, M., Volek, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guinea+pigs+a+suitable+animal+model+to+study+lipoprotein+metabolism,+atherosclerosis+and+inflammation.">Guinea pigs a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation.</a> Nutr Metab (Lond). 3 (17), (2006).</li><li>Frikke-Schmidt, H., Tveden-Nyborg, P., Birck, M. M., Lykkesfeldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dietary+fat+and+cholesterol+exacerbates+chronic+vitamin+C+deficiency+in+guinea+pigs.">High dietary fat and cholesterol exacerbates chronic vitamin C deficiency in guinea pigs.</a> Br J Nutr. 105 (1), 54-61 (2011).</li><li>Hem, A., Smith, A. J., Solberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saphenous+vein+puncture+for+blood+sampling+of+the+mouse,+rat,+hamster,+gerbil,+guinea+pig,+ferret+and+mink.">Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink.</a> Lab Anim. 32 (4), 364-368 (1998).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+guidelines+and+recommendations.">FELASA guidelines and recommendations.</a> J Am Assoc Lab Anim Sci. 51 (3), 311-321 (2012).</li><li>Ye, P., Cheah, I. K., Halliwell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diets+and+pathology+in+the+guinea+pig+Atherosclerosis+or+liver+damage.">High fat diets and pathology in the guinea pig Atherosclerosis or liver damage.</a> Biochim Biophys Acta. 1832 (2), 355-364 (2013).</li><li>Tveden-Nyborg, 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=Maternal+vitamin+C+deficiency+during+pregnancy+persistently+impairs+hippocampal+neurogenesis+in+offspring+of+guinea+pigs.">Maternal vitamin C deficiency during pregnancy persistently impairs hippocampal neurogenesis in offspring of guinea pigs.</a> PLoS One. 7 (10), 12-18391 (2012).</li><li>Schjoldager, J. G., Tveden-Nyborg, P., Lykkesfeldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+maternal+vitamin+C+deficiency+overrides+preferential+fetal+ascorbate+transport+but+does+not+influence+perinatal+survival+in+guinea+pigs.">Prolonged maternal vitamin C deficiency overrides preferential fetal ascorbate transport but does not influence perinatal survival in guinea pigs.</a> Br J Nutr. 110 (9), 1573-1579 (2013).</li><li>Greulich, 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=Secretory+products+of+guinea+pig+epicardial+fat+induce+insulin+resistance+and+impair+primary+adult+rat+cardiomyocyte+function.">Secretory products of guinea pig epicardial fat induce insulin resistance and impair primary adult rat cardiomyocyte function.</a> J Cell Mol Med. 15 (11), 2399-2410 (2011).</li><li>Swifka, J., Weiss, J., Addicks, K., Eckel, J., Rosen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+fat+from+guinea+pig+a+model+to+study+the+paracrine+network+of+interactions+between+epicardial+fat+and+myocardium.">Epicardial fat from guinea pig a model to study the paracrine network of interactions between epicardial fat and myocardium.</a> Cardiovasc Drugs Ther. 22 (2), 107-114 (2008).</li><li>Pilny, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+hematology+of+rodent+species.">Clinical hematology of rodent species.</a> Vet.Clin North Am Exot Anim Pract. 11 (3), 523-vii (2008).</li><li>Franco, N. H., Olsson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientists+and+the+3Rs:+attitudes+to+animal+use+in+biomedical+research+and+the+effect+of+mandatory+training+in+laboratory+animal+science.">Scientists and the 3Rs: attitudes to animal use in biomedical research and the effect of mandatory training in laboratory animal science.</a> Lab Anim. 48 (1), 50-60 (2014).</li><li>Van, L. 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=eAssessing+the+application+of+the+3Rs+a+survey+among+animal+welfare+officers+in+The+Netherlands.">eAssessing the application of the 3Rs a survey among animal welfare officers in The Netherlands.</a> Lab Anim. 47 (3), 210-219 (2013).</li><li>Sloan, R. 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=High+doses+of+ketamine-xylazine+anesthesia+reduce+cardiac+ischemia-reperfusion+injury+in+guinea+pigs.">High doses of ketamine-xylazine anesthesia reduce cardiac ischemia-reperfusion injury in guinea pigs.</a> J Am Assoc Lab Anim Sci. 50 (3), 349-354 (2011).</li><li>Jacobson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+anaesthetic+regimen+for+surgical+procedures+in+guinea+pigs.">A novel anaesthetic regimen for surgical procedures in guinea pigs.</a> Lab Anim. 35 (3), 271-276 (2001).</li><li>Saha, J. K., Xia, J., Grondin, J. M., Engle, S. K., Jakubowski, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hyperglycemia+induced+by+ketamine+xylazine+anesthesia+in+rats+mechanisms+and+implications+for+preclinical+models.">Acute hyperglycemia induced by ketamine xylazine anesthesia in rats mechanisms and implications for preclinical models.</a> Exp Biol Med(Maywood). 230 (10), 777-784 (2005).</li></ol>

Gli autori non hanno nulla da rivelare. Tutte le procedure sono state approvate dalla legge Sperimentazione Animale della Danimarca, che è in accordo con il Consiglio d&#39;Europa Convenzione ETS 123.

Rispetto ai topi e ratti, cavie sono timorosi per natura e devono essere affrontati con calma e gestite correttamente, al fine di ridurre lo stress. Da un punto di vista del benessere degli animali, agendo con cura è naturalmente una priorità; tuttavia, questo sarà anche ridurre il rischio di effetti di ansia associati sui dati raccolti ad es., aumento dei livelli ematici di corticosteroidi e di glucosio 12. Allo stesso modo, la gestione quotidiana sarà abituare gli animali a procedure normali e ...

La cavia è un modello valido e convalidato animale sperimentale a causa di un numero di somiglianze biologiche per l&#39;uomo, come l&#39;esigenza di un approvvigionamento alimentare di vitamina C, lipoproteine ​​plasmatiche paragonabili enzimi che metabolizzano e profili delle lipoproteine, così come similitudini in comune con placentazione umana e sviluppo prenatale 1-3,7,8. Questo rende la cavia un modello attraente e adatto per studiare gli effetti delle malattie dieta associata come la malattia non alcolica steatosi epatica, malattie cardiovascolari e gli effetti putativi di carenza di vitamina C, ma anche l&#39;effetto di esempio., Intervento dietetico materno sulla via di sviluppo prole. Ripetuto prelievo di sangue è spesso richiesto sia lungo e breve termine in studi in vivo. A causa delle differenze anatomiche specie-specifici è necessaria la modulazione della tecnica di campionamento al fine di ottenere campioni di sangue di cavie rispetto ad altri modelli di roditori <sup&gt; 4,5. Questo include la variazione nel corso di vene e arterie, e anche che le cavie non hanno una coda, quindi hanno i requisiti e le diverse opzioni sia per il campionamento e la manipolazione. Campionamento dalla safena o vena tarso, induce il minimo disagio e lo stress negli animali e non richiede anestesia 3,9. Questa tecnica permette ripetuta campioni di piccole quantità di sangue (100-400 ml), ad es., Per determinare i marcatori biochimici nel plasma. Se un campione ancora più piccolo è sufficiente (50-100 ml), questo può facilmente essere ottenuto pungere la vena dell&#39;orecchio. Questa tecnica può anche essere effettuata in animali coscienti ed è utile ad esempio quando si misura la glicemia 10,11. Campionamento dalla vena giugulare consente la raccolta di grandi quantità di sangue (1-2 ml), ma richiede l&#39;anestesia. Qui, vi presentiamo quattro diverse tecniche di campionamento del sangue sia per cavie coscienti o anestetizzati. Il procedures sono tutte le procedure non-terminali a condizione che i volumi di campione e il numero di campioni non superino le linee guida per la raccolta del sangue negli animali da laboratorio 6. E &#39;nell&#39;interesse della buona scienza, nonché di benessere degli animali che lo stress dovrebbe essere ridotto al minimo. Di conseguenza, solo ben addestrato e personale competente deve eseguire le procedure sugli animali.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:295px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:315px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electric shaver</td>
			<td style="width:295px;">Wella (Contura), Denmark</td>
			<td style="width:119px;">273207</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringes&#160;</td>
			<td style="width:295px;">B.Braun-Omnifix-F, Germany</td>
			<td style="width:119px;">9161406V</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Needles</td>
			<td style="width:295px;">BD Microlance,Spain</td>
			<td style="width:119px;">301156</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microvette collection tube&#160;</td>
			<td style="width:295px;">Sarstedt, D-51588N&#252;mbrecht, Germany</td>
			<td style="width:119px;">201,288,100</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Accu-Check analyzer</td>
			<td style="width:295px;">Roche A/S Diagnostics, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Heparin 5000 IE/ml</td>
			<td style="width:295px;">LEO Pharma, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Zoletil 50, Chemvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Narcoxyl vet 20 mg/ml, Intervet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Torbugesic vet 10 mg/ml, Scanvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Neutral eye ointment</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gauze or a cotton swab&#160;</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disinfectant (70% alcohol)</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
	</tbody>
</table>

non-terminal blood sampling techniques in guinea pigs

Le cavie possiedono diverse analogie biologiche per l&#39;uomo e sono convalidati modelli animali sperimentali 1-3. Tuttavia, l&#39;uso di cavie rappresenta attualmente una zona relativamente ristretta dei dati di ricerca e descrittivi sulla metodologia specifica è corrispondentemente scarsa. Le caratteristiche anatomiche di cavie sono leggermente diversi da altri modelli di roditori, quindi la modulazione di tecniche di campionamento per ospitare per specie-specifiche differenze, ad es., Rispetto ai topi e ratti, sono necessari per ottenere campioni di qualità sufficiente e di alta. Per quanto sia lungo e breve termine in studi in vivo spesso richiedono ripetuto sangue campionamento la scelta della tecnica deve essere ben considerato, al fine di ridurre lo stress e il disagio negli animali, ma anche per garantire la sopravvivenza, nonché la conformità con i requisiti di dimensione del campione e accessibilità. Campioni di sangue venoso possono essere ottenuti ad un certo numero di siti in cavie ad es., La safena e giugularevene, ogni tecnica contenente sia vantaggi che svantaggi 4,5. Qui, vi presentiamo quattro diverse tecniche di campionamento del sangue sia per cavie coscienti o anestetizzati. Le procedure sono tutte le procedure non-terminali a condizione che i volumi di campione e il numero di campioni non superino le linee guida per la raccolta del sangue negli animali da laboratorio 6. Tutti i metodi descritti sono stati accuratamente testati e applicati per ripetute in vivo prelievo di sangue in studi all&#39;interno della nostra struttura di ricerca.

Quattro diversi approcci alla non-terminali vivo tecniche di campionamento del sangue in entrambi in cavie coscienti o anestetizzati sono stati presentati. Figura 1 illustra il corso della safena laterale e vena tarsale nella cavia e possibili siti di puntura. Vene dell&#39;orecchio visibili sulla superficie dorsale di un orecchio cavia sono illustrati nella Figura 2 Queste vene possono essere usate per il prelievo di sangue quando è richiesto solo un volume molto pic...

Watch this Scientific Journal Video about Non-Terminal Blood Sampling Techniques in Guinea Pigs at JoVE.com

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Anche se un modello noto, la cavia rappresenta attualmente una nicchia in scienze animali sperimentali e dati limitati sono disponibili sul esecuzione della maggior parte delle procedure. Qui vi presentiamo quattro diversi approcci alla non-terminale in tecniche di campionamento del sangue in vivo su cavie sia coscienti o anestetizzati.

Non terminale Tecniche di prelievo di sangue in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

non-terminal-blood-sampling-techniques-in-guinea-pigs

Research

JoVE Journal

Biology

37.3K Views.  University of Copenhagen.  Anche se un modello noto, la cavia rappresenta attualmente una nicchia in scienze animali sperimentali e dati limitati sono disponibili sul esecuzione della maggior parte delle procedure. Qui vi presentiamo quattro diversi approcci alla non-terminale in tecniche di campionamento del sangue in vivo su cavie sia coscienti o anestetizzati. 

Video: Non-Terminal Blood Sampling Techniques in Guinea Pigs

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

Campionamento di acqua di mare e di raccolta

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Ricerca

Biologia

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

La registrazione ottica di attività elettrica in Guinea-maiale Reti enterici utilizzando tensione sensibile Coloranti

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Tecniche di Imaging Ca 2 + Segnalazione di sperma umano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Tecniche per l&#39;Analisi dei extracellulare vescicole citometria a flusso

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

Macchie di sangue essiccato - preparazione ed elaborazione per l'uso in test immunologici e tecniche molecolari

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Prelievo di sangue dalla vena della coda laterale del Topo

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Ripetuto il prelievo di sangue per esami del sangue in Zebrafish adulti

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

Micron-scala Phenotyping tecniche di mais fasci vascolari basato su x-Ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...