Ce travaux ont été soutenus par des subventions du Conseil de recherches en génie du Canada (RGPIN 238382-2011) en sciences naturelles et Instituts de recherche en santé (MOP115076) canadiennes. Heather Paul a été soutenu par une bourse d&#39;études supérieures du Conseil de recherches en génie du Canada en sciences naturelles et et une bourse de l&#39;Alberta Innovates Health Solutions. Megan Hallam a été soutenue par une bourse d&#39;études en sciences naturelles et en génie Conseil de recherches de troisième cycle, une bourse Frederick Banting et Charles Best études supérieures du Canada, et de recherche de l&#39;Hôpital Institut de formation Award en génétique, développement de l&#39;enfant, et de la santé à l&#39;enfance de l&#39;Alberta.

Ce protocole a été approuvé par l&#39;Université de Calgary Comité de protection des animaux et conforme à la Guide pour le soin et l&#39;utilisation des animaux de laboratoire. 1. Dam séparée de Offspring <ol><li> Séparer le barrage de sa progéniture pour un minimum de 5 min avant la traite 12. NOTE: Le barrage peut traire jusqu&#39;à 5-6 h après la séparation 1,6,13, mais les périodes de séparation de plus de 4 heures peuvent modifier la composition du lait 14. Alors que le temps de la séparation ne semble pas influer sur le volume de collecte de lait 12, il est conseillé que le temps de la séparation cohérente être maintenue pendant toute l&#39;étude. La composition du lait peut changer au cours de l&#39;allaitement 15, donc il faut essayer de garder le jour de la collecte du lait conforme. La production maximale de lait est suggéré de se produire le jour de lactation 14 12. </li><li> En utilisant une chambre de réchauffement, assurer les chiots sont en mesure de maintenir tempéra du corps proprere sans la présence de leur mère pendant la durée de la procédure de traite. NOTE: Dans l&#39;étude présentée ci-dessous, la traite a été effectuée au moment du sevrage, lorsque les barrages étaient âgés d&#39;environ 22 semaines et vieux les descendants 21 jours, donc pas de chambre de réchauffement a été utilisé. </li></ol> 2. Mise en place et préparation <ol><li> Recueillir tous les documents nécessaires à la procédure de traite. REMARQUE: Tous les matériaux peuvent être trouvés dans le matériel et l&#39;équipement table. </li><li> Placez un coussin chauffant sur le banc où aura lieu la traite et de couvrir le tampon avec un banc absorbante sous-pad. </li><li> Mettre en place le système anesthésique. Assurez-vous que le système dispose de l&#39;oxygène et de l&#39;isoflurane avant début suffisante. Attacher le masque anesthésique qui sera utilisé pour l&#39;anesthésie induction initiale de la machine. Placez le masque qui sera utilisé pour l&#39;entretien de l&#39;anesthésie à proximité si différente de celle du masque anesthésique initial. </li><li> Fixez une aiguille 25 G à une seringue de 1 ml de l&#39;ocytocineinjection en utilisant une technique aseptique. </li><li> Mettez le coussin chauffant afin que la température du corps de la mère est maintenue au cours de la procédure de traite. REMARQUE: Surveiller la température du coussin chauffant pour assurer le pavé ne devienne pas trop chaud et causer des brûlures. En variante, utiliser une source de chaleur, telle qu&#39;une table d&#39;opération chauffée, qui peut être réglé à une température donnée. </li></ol> 3. Anesthésier le barrage à l&#39;isoflurane <ol><li> Ouvrez le réservoir d&#39;oxygène et tourner le débit à 1 L (1000 cc) par minute. Tournez sur l&#39;écoulement de l&#39;isoflurane et mettre à 5%. ATTENTION: Éviter l&#39;inhalation directe de l&#39;anesthésie et de prévenir l&#39;accumulation de vapeurs anesthésiques. </li><li> Anesthésier le barrage. </li><li> Basculer vers le masque de maintenance si nécessaire, en plaçant le décubitus dorsal de barrage sur le pad de banc absorbant. Confirmez anesthésie par le manque de pédale réflexe. </li><li> Réduire le débit de l&#39;isoflurane à 2-3% pour l&#39;entretien de l&#39;anesthésie. Surveiller en permanence barrage long de la procédure afin d&#39;assurer depression de respiration ne se produit pas. NOTE: Une fois sous anesthésie, les yeux du barrage devraient être protégés en utilisant un lubrifiant oculaire stérile pour éviter que les yeux de sécher ou devenir rayé. </li></ol> 4. L&#39;ocytocine Injection <ol><li> Assurer l&#39;ocytocine (20 unités USP / ml) n&#39;a pas passé sa date d&#39;expiration. Désinfecter le flacon de l&#39;ocytocine avec un alcool stérile essuyez / tampon d&#39;alcool de nettoyage. </li><li> En utilisant une technique aseptique, élaborer 2 UI (0,1 ml) de l&#39;ocytocine dans la seringue. Utilisez une nouvelle aiguille et la seringue pour chaque barrage qui sera traire. NOTE: L&#39;ocytocine doses varient généralement de simples injections de 1 à 5 UI 1,6,12,16. En variante, une dose de 4 UI / kg de poids corporel 12 peut être utilisé. Une dose unique de 2 UI peut être répétée une fois si la difficulté traite est rencontré. </li><li> Injecter l&#39;ocytocine par voie intrapéritonéale. Insérez l&#39;aiguille dans le quadrant inférieur droit de l&#39;abdomen avec le pointage de l&#39;aiguille vers la tête, à un anglede 15-30 °, environ 0,5 cm de profondeur. </li><li> Tirez sur le piston pour assurer une pression négative avant l&#39;injection. Si du fluide (sang, urine, le contenu intestinal, etc.) est aspiré dans la seringue, retirer l&#39;aiguille et de tenter l&#39;injection avec une nouvelle aiguille et la seringue. Si aucun fluide est aspiré injecter l&#39;ocytocine et jeter l&#39;aiguille et la seringue immédiatement dans un container prévu. </li><li> Attendez environ 5-15 min pour le ocytocine pour stimuler la descente du lait. </li></ol> 5. Préparation des sites à traire <ol><li> Choisissez les sites / tétines du lait seront recueillis. Le lait peut être collecté de toute tétine 12. </li><li> Retirez délicatement la fourrure autour du trayon à traire avec les tondeuses, comme la fourrure peut causer des difficultés dans la collecte de l&#39;échantillon en raison de l&#39;effet de mèche du lait. Soyez doux - la peau autour des trayons est extrêmement sensible et peut être sèche et en tant que telle est sensible aux rayures et aux larmes. </li><li> La stérilisation de la tétine is pas nécessaire, mais le cas échéant, nettoyer la tétine à l&#39;eau tiède après la fourrure est enlevée. Préparer au moins deux sites que plus d&#39;un site peut être nécessaire pour la collecte du lait. NOTE: Si l&#39;analyse du lait comprend profilage microbienne, la zone de la tétine peut exiger la stérilisation avec de l&#39;iode 5. </li></ol> 6. Lait Collection <ol><li> Pressez doucement la base de la tétine, l&#39;expulsion manuellement le lait pour la collecte. NOTE: Si l&#39;analyse du lait comprend profilage microbienne, les premières gouttes de lait doivent être jetés. </li><li> Recueillir les gouttelettes de lait dans un tube capillaire, le remplissage du tube capillaire. Les tubes capillaires qui peuvent accueillir de plus grands volumes (par exemple, 50 pi) de faciliter le processus. NOTE: Si la difficulté à recueillir le lait est rencontré, une deuxième dose d&#39;ocytocine peut être administré. Il est recommandé de la dose dépasse pas 4 UI totale. </li><li> Verser le lait à partir du tube capillaire dans un tube de microcentrifugation stérile partoucher le bout du tube qui a été utilisé pour tirer du lait du trayon vers le côté du tube de microcentrifugeuse - observer le lait étant tiré hors du tube capillaire par action capillaire. NOTE: &#39;Blow out »le lait qui ne soit pas aspiré dans le tube à l&#39;aide d&#39;une aiguille 18 G attachée à une seringue de 1 ml. </li><li> Surveiller en permanence le barrage des signes de douleur ou de dépression respiratoire et de régler le débit de l&#39;isoflurane en conséquence. </li><li> Continuer à la collecte du lait comme décrit dans cette section jusqu&#39;à ce que suffisamment de lait ont été recueillis pour l&#39;analyse du lait choisi. Pour la creamatocrit et de protéines détermination de la concentration décrite ci-dessous, de recueillir 0,25 ml de lait. REMARQUE: Utilisez un site de traite différente si la descente du lait ralentit ou le site choisi ne pas expulser le lait suffisamment. Un maximum d&#39;environ 2,5 ml de lait par animal peut être recueillie à 17, ou jusqu&#39;à 0,5 ml par trayon. Les auteurs recommandent que le temps total sous anesthésie être limitée à environ 45-60 min, or 45 min de traite. </li><li> La collecte du lait dans un tube de microhématocrite pour la mesure de creamatocrit à partir d&#39;échantillons de lait frais, et sceller l&#39;extrémité du tube avec de l&#39;argile étanchéité. Etiqueter le tube de microhématocrite avec l&#39;ID de barrage et stocker l&#39;échantillon de lait debout. </li><li> Lorsque la traite est terminée, éteignez le flux de l&#39;isoflurane et de l&#39;oxygène. Retirer le masque à partir du barrage et continuer à surveiller barrage jusqu&#39;au réveil. Il est recommandé de ne pas pleinement conscient, le barrage doit être placé sur un coussin de banc absorbant au cours de la période de récupération, plutôt que directement sur la literie de la cage, pour empêcher la litière d&#39;être aspiré ou de griffer les yeux du barrage lors de la récupération. REMARQUE: Ne laissez pas le barrage sans surveillance tant qu&#39;il a repris conscience suffisante pour maintenir décubitus sternale. </li><li> Si aucune autre analyse sont nécessaires, congeler le lait à -80 ° C. D&#39;autres ont suggéré que le lait peut être conservé jusqu&#39;à 3 h à 4 ° C ou 5 mois à -20 ° C 18. </li></ol> 7. Creamatocrit mesure <ol><li> Estimer la teneur en matières grasses dans le lait en calculant la creamatocrit de lait (de la crème en pourcentage de l&#39;échantillon de lait) 19. REMARQUE: Les mesures avec le lait humain ont démontré que le lait frais ou congelé peut être utilisé pour une mesure de creamatocrit cependant lait frais est plus fortement corrélée (r = 0,92 par rapport r = 0,90) avec la concentration lipidique 11. L&#39;utilisation de lait frais ou congelés pour les mesures de creamatocrit doit être maintenue uniforme dans l&#39;étude, que le lait décongelé est associée à une faible diminution de creamatocrit 11 valeurs. </li><li> Pour le lait frais, de recueillir un échantillon de lait du trayon dans un tube de microhématocrite; remplir au moins ¾ pleine (environ 15-20 pi). Alternativement, tirer du lait frais de l&#39;échantillon prélevé dans le tube capillaire après mélangeant bien. Sceller la fin avec de l&#39;argile étanchéité. </li><li> Placer le tube capillaire dans la centrifugeuse de fibrage de l&#39;hématocrite, de laextrémité fermée orientée vers l&#39;extérieur, en assurant la centrifugeuse est équilibré. </li><li> Commencez le spin de l&#39;hématocrite (120 secondes à 13 700 xg). REMARQUE: Le temps de Spin ou la vitesse peuvent varier selon le modèle de centrifugeuse utilisée. </li><li> Retirer le tube de la centrifugeuse après l&#39;essorage est terminé et effectuer les mesures pour calculer la creamatocrit. Respecter la séparation de l&#39;échantillon dans une couche de crème et une couche transparente. </li><li> Mesurer et enregistrer la longueur totale du fluide dans le tube et la longueur de la matière grasse (crème) ou couche à l&#39;aide d&#39;une règle étriers. REMARQUE: La creamatocrit est exprimé comme le pourcentage de la couche de crème à l&#39;intérieur de l&#39;échantillon de lait le 19, comme calculé (longueur de couche de crème / longueur totale de la colonne de lait) x 100 (figure 1A). Calculer la concentration et de l&#39;énergie des valeurs lipidiques provenant de la mesure de creamatocrit comme suit: la concentration de graisse (g / L) = (creamatocrit (%) - 0,59) /0.146 (figure 1B) 19; Valeur énergétique (kcal / L) = 290 + (66,8 creamatocrit * (%) (Figure 1C) 19. </li></ol> 8. Protéine Concentration Détermination <ol><li> Utilisation de la sérumalbumine bovine comme étalon de protéines, de déterminer la concentration totale en protéines du lait en utilisant un essai de protéine standard, telle qu&#39;une protéine dosage de Lowry 6. REMARQUE: La dilution du lait peut être nécessaire pour les mesures de protéines du lait à l&#39;intérieur de la courbe tombent norme de l&#39;essai. </li></ol>

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Les auteurs ont aucun conflit d&#39;intérêt à divulguer. Toutes les expériences animales ont été menées conformément aux protocoles du CCPA approuvé.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

Le lait est la seule source de nutrition pour les mammifères nouveau-nés, la fourniture d&#39;énergie et de nutriments pour la croissance du nourrisson et le développement 1,2. Bien que le lait se compose principalement de cellules, des lipides et des protéines 1, il contient également une pléthore de composés bioactifs qui modulent le développement du début de la vie de la progéniture y compris des enzymes, des glucides, des hormones, des anticorps, des facteurs de croissance, des cytokines, des exosomes, des microvésicules et de petits ARN tels comme microARN 1,2. Le rôle fondamental du lait maternel dans l&#39;établissement de la progéniture immunitaire et la santé intestinale 3, couplée avec la preuve que allaités nourrissons sont moins sensibles à la maladie 2, met en évidence l&#39;importance d&#39;identifier les constituants du lait associés aux processus de la maladie en début de vie et les mécanismes moléculaires impliqués dans leurs actions. Le rat est le développement d&#39;un modèle populaire pour étudier l&#39;effet de divers nutritionnelle, physiologique, et les interventions chimiques sur débutdéveloppement -life 4. L&#39;analyse du lait de rat peut donc fournir de nouvelles précisions sur la santé maternelle et la progéniture. Avancées scientifiques actuelles prévoient maintenant accroître les possibilités d&#39;enquêtes approfondies sur les effets des constituants du lait spécifiques sur la santé et la maladie. Par exemple, le séquençage des profils bactériennes du lait a élucidé leur rôle dans la colonisation intestinale au début de l&#39;intestin du nourrisson 5, masse analyse par spectrométrie d&#39;oligosaccharides de lait ont donné un aperçu de la modification des profils d&#39;oligosaccharides de lait par l&#39;alimentation maternelle 6, et de séquençage profond des microARN sécrétée dans les globules de lait maternel de graisse met en évidence les rôles possibles dans la transcription des gènes, le métabolisme et la fonction immunitaire 7. Des modèles de rats représentent l&#39;un des organismes modèles les plus populaires utilisés dans les études maternels 8,9. Un avantage est leur période de gestation et de lactation courts, qui ne durent que approximatEly 21 jours chacune; par conséquent, le temps total depuis le début de la grossesse de lactation représente un court laps de temps dans lequel les données de valeur peuvent être générés. La plus grande taille de rats par rapport à des souris, dans le contexte de la collecte du lait, peut fournir un avantage significatif par rapport au volume de lait et de la facilité de collecte du lait; la production de lait chez la souris, par exemple, semble dépendre du poids total du corps avec des souris lourds produisant plus de lait 10. Ici, une description générale pour la collecte manuelle de lait de rates allaitantes est fourni. Ce protocole nécessite un minimum d&#39;équipement, est non invasif, peu coûteux, et peut être utilisé pour collecter des volumes adéquats de lait pour d&#39;autres analyses en aval. En bref, le barrage est anesthésié avec de l&#39;isoflurane, la descente du lait est stimulée par l&#39;ocytocine, et le lait est recueilli dans des tubes capillaires par l&#39;intermédiaire de l&#39;expression manuelle du lait. Enfin, comme deux composantes majeures de lait sont grasses et des protéines, une brève description de l&#39;estimation de la teneur en graisse du lait en utilisant des mesures de creamatocrit 11 et la quantification de la concentration totale en protéines en utilisant un dosage de protéine standard est présenté.

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Lait a été recueilli comme décrit au sevrage des barrages Wistar (âgés d&#39;environ 22 semaines, pesant 350 à 400 g) qui a consommé un contrôle (AIN-93G, n = 5), riche en protéines (40% de caséine poids / poids, n = 5) , ou de la fibre prébiotique élevé (21,6% en poids / poids, ratio 1: 1 d&#39;oligofructose et l&#39;inuline, n = 4) régime alimentaire pendant la grossesse et l&#39;allaitement. La dose d&#39;ocytocine était de 2 UI. Le lait a été collecté en utilisant des tubes capillaires, et un tube ...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

La collecte du lait dans le rat en utilisant tubes capillaires et estimation de la teneur du lait en matières grasses en Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K vues.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Vidéo: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Recherche

Biologie

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolement des fibroblastes de rat par portail<em&gt; In situ</emPerfusion du foie&gt;

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagerie fonctionnelle de la graisse brune chez les souris avec FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Méthodes de collecte de lait pour souris et Muntjac cerfs de Reeves

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Collecte et analyse des<em&gt; Arabidopsis</em&gt; Phloème exsudats Utilisation de la méthode EDTA-animé

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Profilage Redox cellulaire utilisant une microscopie haute teneur

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Détermination des acides sialiques dans les échantillons de foie et de lait de type sauvage et<em&gt; CMAH</em&gt; Souris knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Analyse de l’autophagie induite par la famine dans le corps adipeux larvaire de Drosophila melanogaster

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Quantification de l’infiltration graisseuse des muscles squelettiques par décellularisation

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Pleins feux sur l’auteur : Quantification de l’infiltration graisseuse des muscles squelettiques basée sur la décellularisation  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.