Nous remercions le Dr William Cashdollar, et le Centre Foundation Imaging Northwestern Mutual à l&#39;Institut de recherche de sang du Wisconsin de l&#39;aide pour les études d&#39;imagerie. Nous remercions également le Dr Daria Ilatovskaya pour la lecture critique de ce manuscrit et de discussion utile. Cette étude a été soutenue par les National Institutes of Health des subventions HL108880 (AS) et DP2-OD008396 (à AMG).

Les procédures expérimentales décrites ci-dessous ont été approuvés par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) au Collège médical du Wisconsin et étaient en conformité avec les National Institutes of Health Guide pour le soin et l&#39;utilisation des animaux de laboratoire. 1. Isolement de l&#39;aorte Rat <ol><li> Anesthésier rats avec de l&#39;isoflurane (5% induction, 1,5 à 2,5% maintenance) ou d&#39;une autre méthode IACUC approuvé. </li><li> Placez le rat dans une position couchée. Afin d&#39;exposer les organes abdominaux, faire une petite incision médiane ventrale à travers la peau et du tissu sous-cutanée abdominale. Utilisation des tampons de gaze dévier doucement les intestins pour exposer l&#39;aorte et la veine cave comme représenté sur la figure 1. </li><li> En bref, l&#39;aorte Blunt disséquer du tissu conjonctif et de la veine cave. Utilisation de suture, attacher l&#39;aorte aussi proche que possible du rein. Disséquer environ 1 - 2 cm de l&#39;aorte et le placer dans un 15 ml conique tuêtre contenant physiologique normale de Salt Solution (PSS; en mm: 140 NaCl, 1,2 MgCl2, 2 CaCl2, KCl 4,5, 6 glucose, 10 HEPES) sur de la glace. </li><li> Une fois que l&#39;aorte est isolé, l&#39;euthanasie de l&#39;animal selon des protocoles approuvés IACUC. À la fin de non-survie procédures euthanasier les animaux profondément anesthésiés par thoracotomie induire pneumothorax pour assurer la disparition de l&#39;animal humain. 3 </li><li> En utilisant un microscope de dissection, pinces à pointe fine, et microciseaux dissèquent la graisse et du tissu conjonctif hors de l&#39;aorte. Une fois l&#39;aorte est propre, couper l&#39;aorte longitudinalement et le placer dans PSS sur la glace pour plus tard. </li></ol> 2. Chargement Dye et incubation <ol><li> Pipet 7 pi de 1 mM Fluo-quatre heures colorant, qui est fourni en solution à base de DMSO, en particuliers des tubes de 500 pi qui ont été couverts d&#39;une feuille d&#39;aluminium. Conserver au congélateur à -20 ° C pour préserver les propriétés de colorant au fil du temps. Remarque: si ces aliquoteslutions doivent être stables pendant au moins six mois. </li><li> Avant utilisation, permettent les solutions de colorant se réchauffer à la température ambiante avant ouverture. Préparer des solutions de travail immédiatement avant utilisation. Ne pas stocker réactif dilué pour une utilisation ultérieure. </li><li> Ajouter 7 pi de ready-made 1 mM Fluo-quatre heures colorant dissous dans une solution de DMSO à 450 ul du PSS ne contenant pas de calcium. Ajouter 40 pi de solution non de DMSO base de 10% d&#39;acide Pluronic au cocktail de chargement pour aider à disperser les acétoxyméthyliques (AM) esters et améliorer le chargement du colorant de calcium. </li><li> Placez les aortes disséqués dans les tubes 500 pi contenant le cocktail de chargement (comme décrit dans le paragraphe 2.3) pendant 1 h. Couvrir tous les tubes avec du papier et le placer sur un agitateur rotatif à la température ambiante. </li><li> Pour surveiller la production de NO dans le système vasculaire, utilisez DAF-FM diacétate colorant. Incuber l&#39;aorte dans une solution contenant 450 ul de PSS, 40 ul d&#39;acide Pluronic et 5 ul de 10 mM de diacétate de DAF-FM. Comme pour le protocole d&#39;incubation de calcium (décrite au point 2.3), placer l&#39;aortedans un tube de 500 ul contenant la solution NO-teinture couvert de feuille et le placer sur un agitateur rotatif pendant 30 min à 4 ° C puis 30 min à température ambiante prochaine. </li><li> Lorsque la production de NO est mesurée, utilisez la NO synthase endothéliale bloqueur, L-NAME, de bloquer la production de NO comme un contrôle comme montré dans la figure 4B. Pour cette procédure, ajouter à cocktail (tel que décrit à 2,5) 100 nM L-NAME pour les 30 dernières minutes d&#39;incubation (à température ambiante). </li></ol> 3. balayage laser Deux Protocole photon Microscopie <ol><li> Après l&#39;incubation de 1 h, laver l&#39;aorte 2 - 3 fois en propre PSS à éliminer le colorant extracellulaire. </li><li> Transférer l&#39;aorte dans un plat recouvert de silicone contenant soit PSS normale ou Ca 2+ tampon exempt (dépend du protocole expérimental). Pin de l&#39;aorte vers le bas sur le revêtement de silicone avec l&#39;adventice en contact avec la silicone (par exemple, lumière endotheliales exposées à l&#39;ouverture de plat) à l&#39;aide des broches en forme de μ. Sinon, utilisez un ancrage Slicegrille ou d&#39;une colle pour fixer le tissu. Remarque: Pour réduire le stress et artefacts mécaniques possibles, le transfert et fixer aorte Ca 2+ exempt de solution et d&#39;attendre 5 à 10 minutes avant de commencer l&#39;expérience. </li><li> Raccorder une pompe péristaltique ou une seringue à la plaque revêtue de silicone pour une application manuelle ou automatique de la drogue ou pour modifier la solution extracellulaire. </li><li> Placer le plat sous le morceau de la verticale à deux photons microscope nez, et à installer et positionner un 25 × (NA 1,05 et distance de travail 2 mm) immersion dans l&#39;eau objectif au-dessus du spécimen. Remarque: Si cet objectif spécifique ne sont pas disponibles, utilisez un objectif spécifiquement fabriqués pour deux imagerie photonique, car il peut augmenter de façon spectaculaire la résolution par rapport à un objectif régulier de signal. </li><li> Activez le laser à deux photons en utilisant le logiciel (le laser commence à pomper et allumez). Tune l&#39;excitation laser à 820 nm. </li><li> Utilisation épifluorescence, localiser l&#39;aorte et de se concentrer sur l&#39;objectifla surface endothéliale en utilisant ajustement grossier. </li><li> Mettez le microscope en mode de balayage laser à deux photons, veiller à ce que le laser d&#39;excitation est mode verrouillé, les détecteurs non-descanned sont engagés et les filtres d&#39;émission appropriées pour les spectres d&#39;émission attendue sont installés. Remarque: Ici, l&#39;FV10-MRL / R (495 à 540 nM) ont été utilisés. </li><li> Lancer la numérisation en direct en utilisant environ 5% de la puissance laser et ajuster finement l&#39;objectif afin de visualiser les cellules endothéliales. Remarque: Les cellules endothéliales présenteront un signal fluorescent forte lorsqu&#39;elle est incubée dans PSS normale. Ce sera nettement plus faible lorsque les vaisseaux sont incubés dans un tampon sans calcium. Les cellules endothéliales sont présents sur la partie supérieure de l&#39;aorte ouvert et les cellules du muscle lisse peut être vu juste en dessous des cellules endotheliales (voir figure 2). Parce que les vaisseaux sanguins ne sont ni lisse ni même, il y aura des endroits où les deux cellules musculaires lisses et des cellules endothéliales sont présents. &lt;/ Li&gt; <li> Une fois que les cellules sont en discussion, le programme de logiciel de microscope pour recueillir des images séquentielles en mode de balayage rapide (balayage de trame, 512 x 512 pixels de fenêtre avec une fréquence de collecte de trame de 1,1 s). En parallèle avec Fluo-quatre heures fluorescence, collecter transmis des images en lumière afin de détecter des changements dans contraction des vaisseaux et de mieux visualiser les conditions expérimentales. </li><li> Après la collecte d&#39;images de signaux de référence, modifier la concentration en ions Ca 2+ dans la solution externe ou lentement appliquer des agents de relance pharmacologiques utilisant la pompe péristaltique en imagerie. Observez l&#39;augmentation de signal fluorescent dans les cellules chargées. Remarque: Les cellules endothéliales de répondre aux changements dans la circulation, il est donc recommandé d&#39;utiliser des applications de laminaires bas et essais de véhicules avant l&#39;application d&#39;activateurs pharmacologiques de Ca 2+ ou NO signalisation. Pour recalculer calcium intracellulaire à des valeurs de concentration en nM navires chargés avec du Fluo-4 (constante de dissociation de Fluo-4 is 345 nM), l&#39;intensité de la fluorescence peut être enregistrée au début et après plus d&#39;ionomycine et MnCl 2. 4 </li></ol> 4. Traitement et calculs image <ol><li> Téléchargez et installez Fidji logiciel de traitement d&#39;image. </li><li> Ouvrez le fichier d&#39;image dans les îles Fidji. Après l&#39;importation du fichier, diviser les canaux (de lumière transmise et de signal fluorescent) lorsque vous êtes invité. Utilisez uniquement le signal fluorescent pour l&#39;analyse des données. </li><li> Dans le programme Fidji, cliquez sur l&#39;onglet d&#39;analyser et de faire défiler à l&#39;option d&#39;outils. Sous l&#39;option d&#39;outils, trouver l&#39;onglet qui dit gestionnaire de ROI et cliquez dessus; une nouvelle fenêtre apparaît. </li><li> Après cela, cliquez sur ensemble des mesures sous l&#39;onglet analyser. Sélectionnez les mesures spécifiques d&#39;intérêt. Pour les mesures transitoires de calcium, utilisez l&#39;option de la valeur moyenne de gris. </li><li> Avec l&#39;outil de trace circulaire, commencer à tracer les régions d&#39;intérêt (ROI) et cliquez sur &quot;ajouter&quot; sur l&#39;écran du gestionnaire de ROI pour chaque ROI. Une fois que toutes les cellules d&#39;intérêt ont été tracées, dans la fenêtre du gestionnaire de ROI, sélectionnez multi Mesure sous l&#39;onglet «plus». Pensez à sauvegarder soit les mesures directement ou copier et coller toutes ces mesures dans un tableur. </li><li> Ouvrir fichier * .txt ou un tableur Excel de Fidji et de transférer les numéros d&#39;une nouvelle feuille de calcul d&#39;origine. Remarque: Vous pouvez également utiliser un programme comme Sigma Plot ou Prism pour une analyse ultérieure. </li><li> Dans le programme d&#39;origine, utilisez le menu Plot → Ligne + Symbole d&#39;observer un aperçu de toutes les réponses transitoires. Décochez les cellules ne répondent pas. </li><li> Recalculer numéro de trace à l&#39;échelle de temps (axe X), utiliser la colonne → Définir valeurs de colonnes et de multiplier la colonne de numéro de trace à la fréquence de collecte d&#39;images (dans notre cas, 1,1 s). </li><li> Utilisez Analysis → Statistiques sur les lignes pour tous les enregistrements sélectionnés pour calculer moyenne (axe Y) et trace d&#39;erreur standard. Tracer Graphique finale pour le groupe actuel de cellules Plot → Ligne + Symbole. </li><li> Le mean calcium calcul transitoire est décrit comme suit. <ol><li> Pour diffusion totale de calcium dans le programme d&#39;origine, cliquez sur Graphique, puis utilisez le menu Analyse → → Intégrer Calcul. Intégrante totale de la zone sous la courbe apparaît dans Résultat Connexion. </li><li> Pour cinétiques transitoires, cliquez sur Graphique, puis utilisez le menu: Analyse → Curve non-linéaire Fit → Choisir Data Set (choisissent axe X vont de maximale à la fin de la réponse transitoire). Lancer Fitting → Sélectionnez → Fonction exponentielle Decay 1 → Terminé. Remarque: raccord exponentielle apparaît dans la fenêtre graphique et le résultat Connexion. Obtenu données (valeurs) représentent quantité totale de calcium libéré, et la cinétique des canaux médiés la réponse transitoire. </li></ol></li></ol>

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Les auteurs ont rien à révéler.

Présentation expérimentale. Pour mieux comprendre la contribution de calcium et NON à la physiologie vasculaire, une nouvelle méthode a été développée pour mesurer [Ca 2+] i et de NO dans les cellules musculaires lisses et endothéliales des aortes intactes isolées. Ensemble, ce protocole se compose de ces étapes critiques: 1) l&#39;isolement et la préparation mécanique (digestion enzymatique pas) du navire. Il est important de garder le tissu sain et intact autant que possible...

Le calcium est un second messager fondamental à l&#39;intérieur de cellules vasculaires tels que des cellules endotheliales et des muscles lisses. Il est le stimulus primaire pour la contraction vasculaire et joue un rôle majeur dans la dilatation vasculaire, y compris ses effets par le biais de la production de NO dans le endothélium. En raison des limites des technologies d&#39;imagerie, il a été pratiquement impossible d&#39;observer la manipulation de calcium dans le récipient intact. Le développement de deux systèmes d&#39;imagerie photonique et la création de nouvelles calcium ou pas de colorants d&#39;étiquetage, il est possible de l&#39;image à une profondeur et une résolution suffisantes pour commencer à comprendre la dynamique de calcium et la production de NO dans le système vasculaire. La microscopie à deux photons a récemment été appliquée dans les tissus, les organes et les études sur les animaux entiers, même en raison de sa capacité supérieure pour pénétrer profondément les tissus à faible fluorescence de fond et de la sensibilité du signal haute. 1,2 Le spectre étroit de deux excitation photonique au illumination point focal et l&#39;utilisation de détecteurs non-descanned sont les raisons pour lesquelles les deux microscopie photon est supérieure à la microscopie confocale traditionnelle. La microscopie confocale ne peut pas produire des images de haute qualité à la profondeur de tissu nécessaire en raison de l&#39;auto-fluorescence et la diffusion de la lumière hors-focus dans le sténopé confocal. Par conséquent, nous avons développé une méthode utilisant un microscope à deux photons pour mesurer [Ca 2+] i la signalisation et la production de NO dans les cellules intactes, individuels des vaisseaux sanguins avec une résolution élevée et un faible rapport signal sur bruit.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DAF-FM</td>
			<td>Life Technologies</td>
			<td>D-23842</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>Pluronic F-68 solution</td>
			<td>Sigma-Aldrich</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Tocris</td>
			<td>0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus upright microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai HP DeepSee-OL&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; water-immersion objective lens</td>
			<td>Olympus</td>
			<td>XLPL25XWMP</td>
			<td>N.A. 1.05 and working distance 2 mm</td>
		</tr>
		<tr>
			<td>Slice Anchor grid&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

Le calcium est un régulateur important de nombreux processus physiologiques dans les tissus vasculaires. La plupart des fonctions endothéliales et musculaires lisses dépendent fortement des changements de calcium intracellulaire ([Ca 2+] i) et de l&#39;oxyde nitrique (NO). Afin de comprendre comment [Ca 2+] i, NO et des molécules en aval sont traitées par un vaisseau sanguin en réponse à des vasoconstricteurs et vasodilatateurs, nous avons développé une nouvelle technique qui applique calcium-étiquetage (ou NO-étiquetage) colorants comportant deux microscopie photonique à mesurer la manipulation de calcium (ou de la production de NO) dans les vaisseaux sanguins isolés. Décrit ici est une procédure détaillée étape par étape qui montre comment isoler une aorte d&#39;un rat, l&#39;étiquette de calcium ou de NO dans les cellules endothéliales ou musculaires lisses, et l&#39;image transitoires de calcium (ou la production de NO) à l&#39;aide d&#39;un microscope à deux photons suivante physiologique ou des stimuli pharmacologiques. Les avantages de l&#39;utilisation de la méthode sont multiples: 1) il est possible d&#39;simultanély mesurer les transitoires de calcium dans les cellules endothéliales et les cellules des muscles lisses en réponse à différents stimuli; 2) elle permet de cellules endotheliales de l&#39;image et des cellules musculaires lisses dans leur milieu d&#39;origine; 3) cette méthode est très sensible au calcium intracellulaire ou pas de changements et génère des images à haute résolution pour des mesures précises; et 4) approche décrite peut être appliquée à la mesure d&#39;autres molécules, telles que les espèces réactives de l&#39;oxygène. En résumé, l&#39;application de deux microscopie biphotonique d&#39;émission laser pour contrôler les transitoires de calcium et la production de NO dans les endothéliales et musculaires lisses des cellules d&#39;un vaisseau sanguin isolé a fourni des données quantitatives de haute qualité et de la promotion de notre compréhension des mécanismes de régulation de la fonction vasculaire.

Afin d&#39;évaluer avec précision la contribution de calcium à la physiologie vasculaire (vasodilatation et la vasoconstriction), un protocole a été conçu pour charger colorants de calcium dans les cellules endothéliales et les cellules musculaires lisses dans isolé aorte intacte. Le dispositif expérimental général mis en place le montre la figure 1, montre la stratégie de base pour l&#39;isolement et la préparation du navire avant l&#39;imagerie. Brièvement, après isolement de l&#39;aort...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Deux photons imagerie intracellulaire de Ca<sup&gt; 2+</sup&gt; Manipulation et production d&#39;oxyde nitrique endothélial et en cellules de muscle lisse d&#39;un Rat aorte isolée

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Research

JoVE Journal

Biology

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

11.2K vues.  Medical College of Wisconsin. Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta. 

Vidéo: Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Deux photons axotomie et time-lapse imagerie confocale dans embryons de poissons zèbres vivent

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Recherche

Biologie

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Ca focale 2 + Détection de transitoires dans le muscle lisse

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

L&#39;isolement de l&#39;homme de l&#39;artère ombilicale cellules musculaires lisses (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Mesure de la fonction musculaire lisse dans les tissus isolés Bath-applications à la pharmacologie de recherche

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagerie Ca locale<sup&gt; 2+</sup&gt; Signaux de cellules de mammifères cultivées

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En détection de visage de l&#39;oxyde nitrique et de superoxyde dans la couche endothéliale des artères intactes

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Imagerie confocale à base de FRET mesure des fluctuations de calcium à l&#39;intérieur du réticulum sarcoplasmique de Cultured cellules musculaires lisses Utilisation

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Isolement de murin coronaire vasculaires cellules de muscle lisse

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Développement et caractérisation de<em&gt; In vitro</em&gt; Microvaisseaux réseau et des mesures quantitatives de l&#39;endothélium [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Et production d&#39;oxyde nitrique

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Blessure à la gorge aortique murine: une efficacité<em&gt; In vivo</em&gt; Modèle de prolifération des cellules musculaires lisses et fonction endothéliale

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...