Ringraziamo il Dr. William Cashdollar, e la Northwestern Mutual Foundation Imaging Center presso il Sangue Istituto di ricerca del Wisconsin per un aiuto con gli studi di imaging. Ringraziamo anche il dottor Daria Ilatovskaya per la lettura critica di questo manoscritto e utile discussione. Questo studio è stato sostenuto dal National Institutes of Health sovvenzioni HL108880 (a AS) e DP2-OD008396 (per AMG).

Le procedure sperimentali descritte qui di seguito sono stati approvati dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) presso il Medical College of Wisconsin ed erano in conformità con il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio. 1. Isolamento del Topo Aorta <ol><li> Anestetizzare ratto con isoflurano (5% di induzione, 1,5 a 2,5% di manutenzione) o un altro IACUC metodo approvato. </li><li> Posizionare il ratto in posizione supina. Al fine di mostrare gli organi addominali, fare una piccola incisione mediana ventrale attraverso la pelle e il tessuto sottocutaneo addominale. Uso garze deviano delicatamente gli intestini per esporre l&#39;aorta e vena cava come mostrato in Figura 1. </li><li> Brevemente, Blunt sezionare l&#39;aorta dal tessuto connettivo e dalla vena cava. Utilizzando sutura, legare l&#39;aorta più vicino possibile al rene. Sezionare circa 1 - 2 cm di aorta e mettetelo in una conica da 15 ml tuessere contenente normale soluzione salina fisiologica (PSS, in mm: 140 NaCl, 1.2 MgCl 2, 2 CaCl 2, 4.5 KCl, 6 Glucosio, 10 HEPES) su ghiaccio. </li><li> Una volta che l&#39;aorta è isolato, l&#39;eutanasia degli animali secondo protocolli IACUC approvati. Al termine di tutti i non-sopravvivenza procedure euthanize animali profondamente anestetizzati di toracotomia inducendo pneumotorace per garantire la scomparsa umana dell&#39;animale. 3 </li><li> Utilizzando un microscopio dissezione, pinze a punta fine, e microscissors sezionare il grasso e tessuto connettivo fuori dell&#39;aorta. Una volta che l&#39;aorta è pulito, tagliare l&#39;aorta longitudinalmente e posizionarlo nella PSS in ghiaccio per dopo. </li></ol> 2. Dye Caricamento e incubazione <ol><li> Pipettare 7 ml di 1 mM Fluo-4 del mattino tintura, che viene fornito in soluzione basata DMSO-, in singole provette microlitri 500 che sono stati rivestiti con un foglio di alluminio. Conservare in congelatore a -20 ° C per preservare le proprietà di tintura nel corso del tempo. Nota: questi aliquotati cosìluzione dovrebbe essere stabile per almeno sei mesi. </li><li> Prima dell&#39;uso, lasciare che i soluzioni coloranti per riscaldarsi a RT prima dell&#39;apertura. Preparare le soluzioni di lavoro immediatamente prima dell&#39;uso. Non conservare il reagente diluito per un uso successivo. </li><li> Aggiungere 7 ml di ready-made 1 mM Fluo-04:00 colorante sciolto in soluzione DMSO a 450 ul di PSS che non contiene calcio. Aggiungere 40 ml di soluzione al 10% di DMSO base non Acid Pluronic al cocktail di carico per contribuire a disperdere le acetossimetil (AM) esteri e migliorare il carico del colorante calcio. </li><li> Posizionare le aorte sezionato nelle provette 500 microlitri contenenti il ​​cocktail di carico (come descritto in 2.3) per 1 h. Coprire tutte le provette con pellicola e posto su un agitatore rotante a temperatura ambiente. </li><li> Per monitorare la produzione di NO nel sistema vascolare, utilizzare DAF-FM diacetato colorante. Incubare l&#39;aorta in una soluzione contenente 450 microlitri PSS, 40 microlitri di acido Pluronic e 5 ml di 10 mM DAF-FM diacetato. Simile al protocollo di incubazione di calcio (descritto al punto 2.3), posizionare l&#39;aortain un tubo contenente 500 microlitri della soluzione di NO-dye coperto in stagnola e posto su un agitatore rotante per 30 minuti a 4 ° C seguita da successiva 30 minuti a RT. </li><li> Quando la produzione di NO è misurata, utilizzare il endoteliale NO sintasi bloccante, L-NAME, per bloccare la produzione di NO come controllo come mostrato nella Figura 4B. Per tale procedura, di aggiungere cocktail (come descritto in 2.5) 100 nM L-NAME per gli ultimi 30 minuti di incubazione (a temperatura ambiente). </li></ol> 3. La scansione laser a due fotoni Microscopia Protocol <ol><li> Dopo il 1 h di incubazione, lavare l&#39;aorta 2 - 3 volte a pulito PSS per rimuovere tintura extracellulare. </li><li> Trasferire l&#39;aorta a un piatto siliconata contenente sia PSS normale o Ca 2+ tampone -free (dipende dal protocollo sperimentale). Pin l&#39;aorta giù sul rivestimento in silicone con avventizia in contatto con il silicone (cioè, lumen endoteliali esposti all&#39;apertura piatto) utilizzando perni a forma μ. In alternativa, utilizzare una fetta di ancoraggiogriglia o colla per fissare il tessuto. Nota: per ridurre lo stress e possibili artefatti meccanici, trasferimento e fissare dell&#39;aorta in Ca 2 + -free soluzione e attendere 5 - 10 minuti prima di iniziare l&#39;esperimento. </li><li> Collegare una pompa peristaltica o una siringa alla piastra siliconata per l&#39;applicazione automatica o manuale di farmaci o per cambiare la soluzione extracellulare. </li><li> Porre la capsula sotto il nasello del montante microscopio a due fotoni, e installare e posizionare un 25 × (NA 1,05 e distanza di lavoro 2 mm) acqua-immersion lente dell&#39;obiettivo sopra il campione. Nota: Se questo obiettivo specifico non è disponibile, utilizzare un obiettivo specificamente fabbricati per l&#39;imaging due fotoni, perché può aumentare la risoluzione del segnale notevolmente rispetto ad un obiettivo normale. </li><li> Attivare il laser due fotoni utilizzando il software (il laser inizia a pompare e accendere). Tune l&#39;eccitazione laser a 820 nm. </li><li> Utilizzando epifluorescenza, individuare l&#39;aorta e mettere a fuoco l&#39;obiettivo sula superficie endoteliale utilizzando la regolazione grossolana. </li><li> Accendere il microscopio in modalità di scansione laser a due fotoni, assicurando che il laser di eccitazione è la modalità di blocco, i rilevatori non descanned sono impegnati e sono installati i filtri di emissione adeguati per gli spettri di emissione previsto. Nota: Qui, il FV10-MRL / R (495-540 nM) sono stati utilizzati. </li><li> Iniziare la scansione in tempo reale utilizzando la potenza del laser circa il 5% e finemente focalizzare l&#39;obiettivo, al fine di visualizzare le cellule endoteliali. Nota: le cellule endoteliali esporranno un segnale fluorescente forte quando incubate in condizioni normali PSS. Questo sarà notevolmente più debole quando i vasi sono incubati in tampone privo di calcio. Le cellule endoteliali saranno presenti sulla parte superiore dell&#39;aorta aperto e le cellule muscolari lisce sono visibili appena sotto le cellule endoteliali (vedere Figura 2). Poiché i vasi sanguigni sono né liscia né, ci saranno luoghi in cui sono presenti sia le cellule muscolari lisce e cellule endoteliali. &lt;/ Li&gt; <li> Una volta che le cellule sono a fuoco, programmare il software del microscopio di raccogliere immagini in sequenza in modalità di scansione veloce (scansione raster, finestra 512 x 512 pixel con una frequenza di raccolta cornice di 1,1 s). In parallelo con Fluo-4:00 fluorescenza, raccogliere trasmesse immagini luce per rilevare variazioni vaso contrazione e visualizzare meglio le condizioni sperimentali. </li><li> Dopo la raccolta di immagini di base dei segnali, modificare la concentrazione di ioni Ca 2+ nella soluzione esterna o lentamente applicare agenti farmacologici stimolo utilizzando la pompa peristaltica mentre imaging. Osservare l&#39;aumento segnale fluorescente all&#39;interno delle cellule caricate. Nota: Le cellule endoteliali rispondono ai cambiamenti nel flusso, in tal modo si raccomanda di utilizzare applicazioni a bassa laminari e di prova del veicolo prima dell&#39;applicazione di attivatori farmacologiche di Ca 2+ o NO segnalazione. Per ricalcolare calcio intracellulare a valori di concentrazione Nm navi cariche di Fluo-4 (costante di dissociazione per Fluo-4 is 345 nM), intensità di fluorescenza potrebbe essere registrato all&#39;inizio dello studio e dopo l&#39;aggiunta di ionomicina e MnCl 2. 4 </li></ol> 4. Elaborazione delle immagini e calcoli <ol><li> Scaricare e installare Figi pacchetto di elaborazione delle immagini. </li><li> Aprire il file di immagine in Fiji. Dopo l&#39;importazione del file, dividere i canali (luce trasmessa e di segnale fluorescente) quando richiesto. Utilizzare solo il segnale fluorescente per l&#39;analisi dei dati. </li><li> Nell&#39;ambito del programma Fiji, fare clic sulla scheda analizzare e scorrere verso il basso fino all&#39;opzione strumenti. Sotto l&#39;opzione strumenti, trovare la scheda che dice direttore ROI e fare clic su di esso; Apparirà una nuova finestra. </li><li> Dopo questo, clicca su misurazioni impostati nella scheda analizzare. Selezionare le misure specifiche di interesse. Per le misure transitorie di calcio, utilizzare l&#39;opzione valore medio grigio. </li><li> Con lo strumento traccia circolare, iniziare a tracciare le regioni di interesse (ROI) e cliccare su &quot;aggiungi&quot; nella schermata direttore ROI per ogni ROI. Una volta che tutte le cellule di interesse sono stati rintracciati, nella finestra di gestione ROI, selezionare Misura Multi nella scheda &quot;altro&quot;. Ricordati di salvare le misurazioni sia direttamente o copiare e incollare tutte queste misure in un foglio di calcolo. </li><li> Aprire file * .txt o foglio di calcolo Excel da Fiji e trasferire i numeri per un nuovo foglio di lavoro di origine. Nota: in alternativa, usare qualsiasi programma come Sigma Plot o Prism per ulteriori analisi. </li><li> Nell&#39;ambito del programma di origine, utilizzare il menu Plot Linea + Simbolo → osservare una panoramica di tutte le risposte transitorie. Deselezionare le cellule non rispondono. </li><li> Ricalcola il numero di traccia su scala temporale (asse X), utilizzare Colonna → Impostare valori di colonna e moltiplicare la colonna numero di traccia per la frequenza di raccolta di immagini (nel nostro caso 1.1s). </li><li> Utilizzare Analisi → Statistiche su righe per tutte le registrazioni selezionate per calcolare Media (asse Y) e traccia errore standard. Tracciare Grafico finale per il gruppo attuale di cellule Plot → Linea + Symbol. </li><li> Il mean calcolo transiente di calcio è descritto come seguito. <ol><li> Per diffusione totale del calcio all&#39;interno del programma Origin, clicca su Grafico, quindi utilizzare il menu Analisi → calcolo → Integra. Integrante totale di area sotto la curva appare nel Registro dei risultati. </li><li> Per cinetiche transitori, clicca su Grafico, quindi utilizzare il menu: Curva Analisi → non lineare Fit → Select Data Set (scegliere l&#39;asse X vanno dal massimo al fine di risposta ai transienti). Inizia Fitting → Selezionare la funzione → decadimento esponenziale 1 → Fatto. Nota: raccordo esponenziale appare nella finestra di Grafico e Risultato Log. I dati ottenuti (valori) rappresentano quantità totale di calcio rilasciato, e cinetica dei canali mediate la risposta transitoria. </li></ol></li></ol>

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Introduzione sperimentale. Per meglio comprendere l&#39;apporto di calcio e NO alla fisiologia vascolare, un nuovo metodo è stato sviluppato per la misurazione [Ca 2+] i e NO all&#39;interno muscolari lisce e cellule endoteliali dei isolate aorte intatti. Insieme, questo protocollo è composto da questi passaggi critici: 1) l&#39;isolamento meccanico e preparazione (digestione enzimatica non) della nave. È importante mantenere il tessuto sano e intatto il più possibile avere registrazio...

Il calcio è un secondo messaggero fondamentale all&#39;interno delle cellule vascolari quali cellule endoteliali e muscolari lisce. È lo stimolo primario per la contrazione vascolare e svolge un ruolo importante nella dilatazione vascolare, compresi i suoi effetti attraverso NO generazione nel endotelio. A causa di limitazioni di tecnologie di imaging, è stato virtualmente impossibile osservare movimentazione calcio all&#39;interno del vaso intatto. Lo sviluppo dei due sistemi di imaging fotonica e la creazione di nuova di calcio o di coloranti di etichettatura, permette di immagine ad una profondità e una risoluzione sufficiente per iniziare a comprendere le dinamiche del calcio e la produzione di NO all&#39;interno del sistema vascolare. Microscopia a due fotoni è stata recentemente applicata in tessuti, organi e studi, compresi quelli interi a causa della sua superiore capacità di penetrare in profondità i tessuti con sfondo bassa fluorescenza e sensibilità segnale alto animale. 1,2 Lo spettro ristretto di due fotoni alla illuminatpunto focale ioni e l&#39;uso di rilevatori non descanned sono le ragioni per cui due fotoni microscopia è superiore alla microscopia confocale tradizionale. Microscopia confocale non può produrre immagini di alta qualità alla profondità del tessuto necessario a causa della auto-fluorescenza e la dispersione di luce fuori fuoco nella pinhole confocale. Di conseguenza, abbiamo sviluppato un metodo che utilizza un microscopio a due fotoni per misurare [Ca 2+] i segnalazione e produzione di NO in intatti, singole cellule dei vasi sanguigni con alta risoluzione ed un basso rapporto segnale-rumore.

<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

Il calcio è un regolatore molto importante di molti processi fisiologici nei tessuti vascolari. La maggior parte delle funzioni endoteliali e muscolari lisce altamente dipendono dalle variazioni dei calcio intracellulare ([Ca 2+] i) e l&#39;ossido nitrico (NO). Al fine di comprendere come [Ca 2 +] i, NO e molecole a valle sono gestite da un vaso sanguigno in risposta a vasocostrittori e vasodilatatori, abbiamo sviluppato una nuova tecnica che si applica calcio etichettatura (o NO-etichettatura) coloranti con due fotoni microscopia per misurare la movimentazione di calcio (o la produzione di NO) nei vasi sanguigni isolati. Descritto qui è una procedura dettagliata passo-passo che illustra come isolare un dell&#39;aorta da un topo, etichetta di calcio o di NO nelle cellule endoteliali o muscolari lisce, e l&#39;immagine del calcio transienti (o la produzione di NO) utilizzando un microscopio a due fotoni seguente fisiologico o stimoli farmacologici. I vantaggi di utilizzare il metodo sono multi-fold: 1) è possibile simultanealy misurare transienti di calcio in entrambe le cellule endoteliali e cellule muscolari lisce in risposta a stimoli diversi; 2) che permette di cellule un&#39;immagine endoteliali e cellule muscolari lisce nel loro ambiente nativo; 3) questo metodo è molto sensibile al calcio intracellulare o nessuna modifica e genera immagini ad alta risoluzione per misurazioni precise; e 4) approccio descritto può essere applicato alla misura di altre molecole, quali le specie reattive dell&#39;ossigeno. In sintesi, l&#39;applicazione della microscopia a due fotoni emissione laser per monitorare transienti di calcio e la produzione di NO nelle endoteliali e muscolari lisce cellule di un vaso sanguigno isolato ha fornito dati quantitativi di alta qualità e promuovere la nostra comprensione dei meccanismi che regolano la funzione vascolare.

Per valutare con precisione il contributo di calcio per la fisiologia vascolare (vasodilatazione e vasocostrizione), un protocollo è stato progettato per caricare coloranti calcio in entrambe le cellule endoteliali e le cellule muscolari lisce in isolato aortae intatto. Il sperimentale generale attuato illustrato nella figura 1, mostra la strategia di base per l&#39;isolamento e la preparazione della nave prima di imaging. Brevemente, dopo l&#39;isolamento dell&#39;aorta dal ratto, deve essere pulito d...

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.

A due fotoni Imaging di Intracellulare Ca<sup&gt; 2+</sup&gt; Manipolazione e l&#39;ossido nitrico Produzione in endoteliale e della muscolatura liscia cellule di un isolato Rat Aorta

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

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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 Views.  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. 

Video: 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.

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish vivo

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

Ricerca

Biologia

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.

Focale Ca 2 + Rilevamento transitoria nel muscolo liscio

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.

Isolamento di cellule umane ombelicale muscolatura liscia arteriosa (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.

Misura di Smooth funzione muscolare nei isolato carta igienica-applicazioni Farmacologia Research

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.

Imaging locale Ca<sup&gt; 2+</sup&gt; Segnali in cellule di mammifero coltivate

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 Face Detection di ossido nitrico e superossido in strato endoteliale delle arterie Intact

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.

Misura di fluttuazioni calcio all&#39;interno del reticolo sarcoplasmatico di coltura cellule muscolari lisce Uso FRET-based Imaging confocale

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.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

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.

Sviluppo e caratterizzazione di<em&gt; In Vitro</em&gt; Microvasi Rete e misurazioni quantitative di endoteliale [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E ossido nitrico Produzione

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.

Lesione aortica di crusca a murina: un efficace<em&gt; In Vivo</em&gt; Modello di proliferazione cellulare liscia e funzione endoteliale

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