Nous reconnaissons l&#39;aide habile de Annie B. Kristensen, Elisabeth V. Andersen, Belinda B. Bringtoft et les gardiens d&#39;animaux au Département de médecine expérimentale, Frederiksberg Campus, Faculté de la santé et des sciences médicales de l&#39;Université de Copenhague, Danemark. Jacob Lønholdt est remercié pour son excellente assistance technique pour filmer les procédures. Cette étude a été financée en partie par le Centre LIFEPHARM pour pharmacologie in vivo et de l&#39;Université de Copenhague.

Les procédures indiquées ont été réalisées selon des protocoles approuvés par l&#39;Inspection expérimentation animale danois sous le ministère de l&#39;Alimentation, de l&#39;Agriculture et de la Pêche. 1 prélèvement sanguin de la saphène latérale ou tarsien Vein <ol><li> Effectuez cette procédure sans anesthésie et l&#39;aide d&#39;un enquêteur, un traitement de la cochon de Guinée, et un prélèvement de l&#39;échantillon. </li><li> Raser la région du tarse de la patte arrière avec un rasoir électrique jusqu&#39;à ce que les veines sont visibles. S&#39;assurer que la zone est assez grande pour éviter fourrure interférer avec le site de ponction. </li><li> Placez la patte arrière dans un bain d&#39;eau tiède et frottez doucement la jambe, ce qui rend les veines plus dilatées et évident. </li><li> Essuyez la jambe avec une serviette propre et désinfecter le site de ponction avec de la gaze ou un coton-tige imbibé d&#39;alcool à 70%. </li><li> Retenez le cochon de Guinée et d&#39;étendre la jambe arrière vers le bas tout en exerçant une légère pression immédiatement au-dessus du genou to fournir stase. </li><li> Piquer la veine à l&#39;aide d&#39;une aiguille de 21 G ou lancette. Prélever le sang par exemple., À l&#39;aide d&#39;un tube de collecte ou capillaire. Manipuler doucement la jambe pour faciliter le flux sanguin. </li><li> Relâcher la stase, une fois que l&#39;échantillon a été obtenu. Appliquez un morceau de gaze ou de coton avec une légère pression sur le site de ponction jusqu&#39;à ce que le saignement s&#39;est arrêté. </li><li> Retour à l&#39;animal de sa cage et le moniteur pendant 5-10 min pour assurer l&#39;hémostase. </li></ol> 2. échantillonnage du sang de la veine de l&#39;oreille <ol><li> Effectuez cette procédure sans anesthésie soit seul ou de préférence avec l&#39;aide d&#39;un collègue. </li><li> Désinfecter la surface dorsale de l&#39;oreille à l&#39;aide de la gaze ou un coton-tige imbibé d&#39;alcool à 70%. </li><li> Appliquez une légère pression à la base de l&#39;oreille pour fournir stase. </li><li> Piquer la veine à l&#39;aide d&#39;une aiguille de 21 G ou lancette, et recueillir le sang. </li><li> Relâchez la stase, une fois l&#39;échantillon a été obtenu, Appliquez un morceau de gaze ou de coton hydrophile gepression ntle au site de ponction jusqu&#39;à ce que le saignement s&#39;est arrêté. </li><li> Retour à l&#39;animal de sa cage et le moniteur pendant 5-10 min pour assurer l&#39;hémostase. </li></ol> 3 Prélèvement de sang de la veine jugulaire <ol><li> Effectuer cette procédure en utilisant l&#39;aide d&#39;un enquêteur supplémentaire; une manipulation du cochon de Guinée et un prélèvement de l&#39;échantillon. </li><li> Anesthésier le cochon de Guinée avec un anesthésique approprié pour toute la durée de cette procédure. Un mélange de tilétamine (0,93 mg / kg), zolazepam (0,93 mg / kg), de xylazine (1,49 mg / kg) et le butorphanol (0,06 mg / kg) peut être utilisé. </li><li> Placez le cochon de Guinée anesthésié sur le dos et retirer délicatement les pattes de devant caudale. </li><li> Rasage et désinfecter (par exemple., Avec 70% d&#39;alcool), la partie ventrale du cou. </li><li> Enduire la seringue avec l&#39;héparine ou de l&#39;EDTA avant l&#39;échantillonnage afin de réduire le risque de formation de caillots. </li><li> Palper la clavicule et insérer un 25 G (ou 23 G) aiguille montée sur une seringue de 1 ml apron 1 cm en dehors de la ligne médiane au niveau de l&#39;articulation de l&#39;épaule. Conserver la seringue dans un léger angle vers le haut et interne tout en appliquant une légère pression négative. Éviter d&#39;endommager les structures sous-jacentes comme la veine jugulaire est situé tout à fait superficielle. Inclinez le cou du cochon de Guinée légèrement vers l&#39;arrière pour fournir stase augmentation de la veine. </li><li> Relâchez les pattes avant lorsque l&#39;échantillon de sang est obtenu, et soulager la stase avant que l&#39;aiguille est rétractée. Utilisez un morceau sec de gaze ou de coton pour appliquer une légère pression sur le site de ponction. </li><li> Retour de l&#39;animal dans une cage de récupération et de suivi pour s&#39;assurer l&#39;hémostase et la récupération après l&#39;anesthésie. </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>

Les auteurs n&#39;ont rien à divulguer. Toutes les procédures ont été approuvées par la Loi sur l&#39;expérimentation animale du Danemark, qui est en accord avec le Conseil de l&#39;Europe Convention STE 123.

Par rapport à des souris et des rats, cochons d&#39;Inde sont craintifs par nature et doivent être abordés calmement et correctement traitées afin de réduire le stress. D&#39;un point de vue de la protection des animaux, agissant avec soin est naturellement une priorité; Toutefois, ce sera aussi réduire le risque d&#39;effets de l&#39;anxiété associée sur les données recueillies par ex., l&#39;augmentation des taux de corticostéroïdes dans le sang et glucose 12. De même, la gestion quot...

Le cochon de Guinée est un modèle animal expérimental précieux et validé en raison d&#39;un certain nombre de similitudes biologiques pour les humains tels que l&#39;exigence d&#39;un apport alimentaire en vitamine C, lipoprotéines plasmatiques comparables enzymes métabolisant et profils de lipoprotéines, ainsi que les similitudes partagées avec placentation humaine et développement prénatal 1-3,7,8. Cela rend le cochon de Guinée un modèle attrayant et adapté aux effets de maladies liés à l&#39;alimentation tels que la non-stéatose hépatique, les maladies cardiovasculaires et les effets supposés de la carence en vitamine C, mais aussi l&#39;effet par exemple de l&#39;étude., Intervention alimentaire de la mère sur le développement progéniture. Prélèvement de sang répétée est souvent nécessaire à la fois court et long terme dans les études in vivo. En raison des différences anatomiques spécifiques à l&#39;espèce modulation de la technique d&#39;échantillonnage est nécessaire afin d&#39;obtenir des échantillons de sang chez les cobayes par rapport à d&#39;autres modèles de rongeurs <sup&gt; 4,5. Cela inclut la variation au cours des veines et des artères, et aussi que les cochons d&#39;Inde n&#39;ont pas de queue, ont donc des besoins différents et des options à la fois pour l&#39;échantillonnage et la manipulation. L&#39;échantillonnage de la saphène ou veine du tarse, induit un minimum d&#39;inconfort et de stress chez l&#39;animal et ne nécessite pas d&#39;anesthésie 3,9. Cette technique permet d&#39;échantillons de petites quantités de sang (100 à 400 ul), par exemple, répété., Pour déterminer les marqueurs biochimiques dans le plasma. Si un échantillon de taille encore plus petite est suffisante (50-100 pi), ce qui peut très facilement être obtenu par ponction de la veine de l&#39;oreille. Cette technique peut également être réalisée chez des animaux conscients et est utile par exemple lorsque l&#39;on mesure la glycémie 10,11. L&#39;échantillonnage de la veine jugulaire permet la collecte de plus grandes quantités de sang (1-2 ml) mais nécessite une anesthésie. Ici, nous présentons quatre techniques différentes de prélèvement sanguin soit pour des cobayes conscients ou anesthésiés. Le procedures sont toutes les procédures non-terminaux à condition que les volumes d&#39;échantillon et le nombre d&#39;échantillons ne pas dépasser les lignes directrices pour la collecte de sang chez les animaux de laboratoire 6. Il est dans l&#39;intérêt de la bonne science, ainsi que de bien-être animal que l&#39;accent devrait être maintenu à un minimum. Par conséquent, seul bien formé et personnel compétent doit effectuer les procédures sur les animaux.

<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

Les cochons d&#39;Inde possèdent plusieurs similitudes biologiques pour les humains et sont validés modèles animaux expérimentaux 1-3. Cependant, l&#39;utilisation de cobayes représente actuellement une zone relativement étroite des données de recherche et descriptives sur la méthodologie spécifique est proportionnellement rares. Les caractéristiques anatomiques de cobayes sont un peu différent des autres modèles de rongeurs, d&#39;où la modulation des techniques d&#39;échantillonnage pour tenir compte des différences spécifiques de l&#39;espèce, par exemple., Par rapport à des souris et des rats, sont nécessaires pour obtenir des échantillons de qualité suffisantes et élevés. Comme la fois court et long terme dans les études in vivo souvent besoin de sang répété échantillonner le choix de la technique doit être bien pris en compte afin de réduire le stress et l&#39;inconfort chez les animaux, mais aussi pour assurer la survie ainsi que la conformité avec les exigences de taille de l&#39;échantillon et de l&#39;accessibilité. Des prélèvements sanguins peuvent être obtenus à un certain nombre de sites de cobayes par exemple., La saphène et jugulaireveines, chaque technique contenant à la fois des avantages et des inconvénients de 4,5. Ici, nous présentons quatre techniques différentes de prélèvement sanguin soit pour des cobayes conscients ou anesthésiés. Les procédures sont toutes les procédures non-terminaux à condition que les volumes d&#39;échantillon et le nombre d&#39;échantillons ne pas dépasser les lignes directrices pour la collecte de sang chez les animaux de laboratoire 6. Toutes les méthodes décrites ont été soigneusement testés et demandé répétée dans le prélèvement de sang dans les études in vivo au sein de notre centre de recherche.

Quatre approches différentes techniques in vivo non terminaux dans de prélèvement sanguin soit dans les cobayes conscients ou anesthésiés ont été présentés. Figure 1 illustre l&#39;évolution de la saphène et la veine latérale du tarse dans le cochon de Guinée et sites de ponction possibles. les veines de l&#39;oreille, visibles sur la face dorsale d&#39;une oreille de porc Guinée sont illustrés à la Figure 2 Ces veines peuvent être utilisés pour le pr...

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

Même si un modèle connu, le cochon de Guinée représente actuellement un créneau en sciences des animaux de laboratoire et des données limitées sont disponibles sur l&#39;exécution de la plupart des procédures. Ici, nous présentons quatre approches différentes pour non-terminal dans les techniques de prélèvement sanguin in vivo chez soit des cobayes conscients ou anesthésiés.

Non-Terminal techniques d&#39;échantillonnage de sang chez les cobayes

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

37.2K vues.  University of Copenhagen.  Même si un modèle connu, le cochon de Guinée représente actuellement un créneau en sciences des animaux de laboratoire et des données limitées sont disponibles sur l'exécution de la plupart des procédures. Ici, nous présentons quatre approches différentes pour non-terminal dans les techniques de prélèvement sanguin in vivo chez soit des cobayes conscients ou anesthésiés. 

Vidéo: 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.

L&#39;échantillonnage d&#39;eau de mer et de Collection

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

Recherche

Biologie

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.

Enregistrement optique de l&#39;activité électrique en Guinée-cochon Réseaux entérique en utilisant la tension colorants sensibles

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

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 et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

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.

Isolement et culture de cellules endothéliales pulmonaires des souris néonatales

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.

Techniques pour l&#39;analyse des vésicules extracellulaire par cytométrie en flux

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.

Taches de sang séché - préparer et traiter pour utilisation dans les immunoessais et Techniques moléculaires

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.

L&#39;échantillonnage de sang de la veine caudale latérale de la Rat

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

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

Une Administration simple et efficace et la visualisation de microparticules dans l’appareil circulatoire des petits poissons à l’aide d’Injection de rein

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

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.

Techniques de phénotypage micron-échelle de maïs faisceaux vasculaires issu de tomographie à rayons x Microcomputed

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

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Gonadectomie et procédures de prélèvement sanguin dans le modèle de téléostéen de petite taille Medaka japonais (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...