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><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 &amp;micro;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&amp;nbsp;</td><td>Spectra Physics</td><td></td><td>Ti:sapphire laser 690nm &amp;mdash; 1040nm</td></tr><tr><td>Filters</td><td>Olympus</td><td>FV10-MRL/R&amp;nbsp;</td><td>495 to 540 nm&amp;nbsp;</td></tr><tr><td>25&amp;times; 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&amp;nbsp;</td><td>Warner Instrument&amp;nbsp;</td><td>SHD-27LH</td><td></td></tr><tr><td>Other basic reagents&amp;nbsp;</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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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

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Calcificazione vascolare cellule muscolari lisce e imaging di aortica calcificazioni e infiammazione

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Ricerca

Medicina

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Valutazione dei meccanismi di controllo vascolare utilizzando Video microscopia del arterie di resistenza isolata dei ratti

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolamento di microvascolare endoteliale tubi da arterie di resistenza mouse

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biologia

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Calcio dei periciti cerebrali e imaging emodinamico nei topi transgenici in vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Neuroscienze

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 ...

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 ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

L&#39;applicazione di geneticamente codificato fluorescente ossido nitrico (NO •) sonde, i geNOps, in tempo reale Imaging di NO • I segnali in singole cellule

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Isolamento delle arteriole retiniche per studi di fisiologia delle cellule Ex Vivo

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.

A due fotoni Imaging di Intracellulare Ca

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Capitoli in questo video

Calcificazione vascolare cellule muscolari lisce e imaging di aortica calcificazioni e infiammazione

Valutazione dei meccanismi di controllo vascolare utilizzando Video microscopia del arterie di resistenza isolata dei ratti

Isolamento di microvascolare endoteliale tubi da arterie di resistenza mouse

Calcio dei periciti cerebrali e imaging emodinamico nei topi transgenici in vivo

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

Misura di fluttuazioni calcio all'interno del reticolo sarcoplasmatico di coltura cellule muscolari lisce Uso FRET-based Imaging confocale

Sviluppo e caratterizzazione di

L'applicazione di geneticamente codificato fluorescente ossido nitrico (NO •) sonde, i geNOps, in tempo reale Imaging di NO • I segnali in singole cellule

Isolamento delle arteriole retiniche per studi di fisiologia delle cellule Ex Vivo

Misurazione della vasorilassazione endotelio-dipendente nell'aorta toracica del topo mediante miografia tensometrica a camera di piccolo volume