This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

Tutti gli esperimenti sugli animali e le procedure sono state approvate dalla cura e l&#39;uso degli animali Comitato Istituzionale della Tulane University (IACUC). 1. Procedura di impostazione chirurgica <ol><li> strumenti Autoclave, forniture chirurgiche, e le forniture di cultura prima dell&#39;intervento. forniture chirurgiche per ogni ratto sono: 1 telo, 1 telo con foro pre-cut (0,5 x 1,5 in) al centro, garze, e 1 underpad assorbente. Strumenti chirurgici includono: 1 bisturi con lama numero 10, 2 paia di pinzette, e un paio di forbici sottili. forniture Cultura includono: 1 telo, 1 paio di pinzette, e preparati inserti 6 pozzetti con filtri in policarbonato. </li><li> Sterilizzare una piattaforma plexiglass, una fase chirurgica e uno spazio banco chirurgica con il 70% di etanolo. Mantenere la fase chirurgica in una vaschetta sterile fino al momento dell&#39;uso. <ol><li> Creare una fase chirurgica perforando un approssimativamente 2 da 1 nel foro al centro di un piatto di coltura a 100 mm. Successivamente, utilizzare carta vetrata per lisciarebordi taglienti e aggiungere uno strato di colla di silicone ai bordi del foro per creare una superficie sollevata per i tessuti. </li><li> In alternativa, progettare la fase chirurgica utilizzando il software CAD e di fare del 3-D stampa (Figura 1). </li></ol></li><li> Posizionare un underpad assorbente sterile verso il basso e porre una piattaforma di plexiglas su di esso. Posizionare il telo, senza un foro pretagliato, su un tampone riscaldato accanto al underpad assorbente. </li><li> Pre-caldo sterile PBS (PBS), mezzi di comunicazione e soluzione salina a 37 ° C. Inserire i supporti e PBS in piatti della cultura separati in cima alla piastra elettrica e il luogo di soluzione salina in una provetta da 50 ml accanto alla configurazione chirurgica. </li><li> Assicurarsi che tutti i pacchetti vengono aperti prima dell&#39;inizio della chirurgia per garantire una manipolazione sterile di tutti i materiali. Un elenco completo degli strumenti comuni utilizzati in questa procedura sono elencati nella Tabella di materiali chirurgici specifici e strumenti. </li></ol> 2. Mesentery Tissue raccolta <ol><li> Usa adulti ratti maschi Wistar (350 ± 25 g, 6 - 8 settimane di età). Altri ceppi e l&#39;età dei ratti possono essere sostituiti. </li><li> Anestetizzare ratto con una iniezione intramuscolare di ketamina (80 mg / kg di peso corporeo) e xilazina (8 mg / kg di peso corporeo). Confermare il ratto è sotto anestesia da pizzicare tra le dita per controllare che la risposta riflessa; ci dovrebbe essere nessuno. analgesia preventiva per questa procedura terminale non è necessaria. </li><li> Radere la regione addominale e rimuovere i capelli rimanenti con crema depilatoria. Pulire la pelle addominale due volte con alcool isopropilico al 70% seguita da iodopovidone. Per le salviettine il chirurgo deve iniziare al centro del sito chirurgico e spostare all&#39;esterno dell&#39;area preparata in modo circolare da non sovrapporsi aree che sono stati precedentemente lavati con lo stesso pezzo di garza sterile o tampone di cotone sterile. Poi il trasferimento degli animali al setup chirurgica sterile e posizionare in cima alla piattaforma plexiglassmodulo. </li><li> Utilizzando una lama di bisturi, fare un 0.75 a - 1.25 in incisione nell&#39;intestino a partire dal 1 nel sotto lo sterno. Fare attenzione a non forare l&#39;intestino o meso (1 strato di pelle, 1 strato di tessuto connettivo, e 1 strato di muscolo). </li><li> Posizionare un drappo con un foro pre-taglio sopra l&#39;incisione e posizionare una fase chirurgica sterile in cima al telo. Assicurarsi che le allinea apertura con l&#39;incisione. Utilizzare applicatori di cotone con punta sterile per individuare ed estrarre l&#39;ileo attraverso l&#39;apertura fase chirurgica. </li><li> Tirare 6 - 8 finestre mesenterica attraverso la fase utilizzando applicatori con punta in cotone, e fare attenzione a non toccare le finestre (Figura 1). I tessuti sono tipicamente raccolti dalla regione ileo dell&#39;intestino tenue partire vicino cieco. Mantenere tessuti esposti umido con soluzione salina sterile riscaldato se necessario utilizzando una siringa sterile a gocciolare la soluzione. </li><li> Euthanize il ratto mediante iniezione intracardiaca di sodio pentobarbital (0,2 ml per topo). Prima di rimuovere mefinestre senteric, assicurano il ratto è eutanasia palpando il cuore; ci dovrebbe essere nessun impulso. </li><li> Rimuovere i tessuti mesentere desiderato utilizzando pinzette per afferrare il cuscinetto di grasso e forbici sottili per tagliare la finestra. Lasciare un bordo di grasso (2 mm) intorno alla finestra. Lavare i tessuti una volta riscaldato PBS sterile e una volta in media. </li><li> Rientro ileo esteriorizzato alla cavità addominale e smaltire animali secondo le linee guida istituzionali. </li></ol> 3. Mesentere Tissue Culture per gli Studi Time-Lapse <ol><li> Trasferimento forniture autoclave cultura (vedere paragrafo 1.1) e dei tessuti ad una cappa a flusso laminare sterile. </li><li> Utilizzare le pinzette per trasferire ogni tessuto in cima ad una membrana filtrante di policarbonato. tessuti afferrare da parte del cuscinetto di grasso per evitare di danneggiare il sistema vascolare. </li><li> si diffuse rapidamente il tessuto con il cuscinetto adiposo, facendo attenzione a non toccare la finestra. Invertire l&#39;inserto con il tessuto nel fondo di una piastra da 6 pozzetti e coprire con 3 mL di supporti (Figura 1 </strong&gt;). supporti tipici utilizzati per questa procedura include mezzi minimi essenziali (MEM) con 1% penicillina streptomicina (PenStrep) e il 10% siero fetale bovino (FBS). I supporti possono essere integrati con altri sieri e / o fattori di crescita per stimolare l&#39;angiogenesi. </li><li> Ripetere i passaggi 3,2-3,3 per ogni tessuto e la cultura in condizioni normali incubatore (5% di CO 2, 37 ° C) per un massimo di 5 giorni. </li></ol> 4. Time-Lapse Imaging di Mesentere Tissue <ol><li> Il giorno dell&#39;imaging, integrare i media in ciascun pozzetto con coniugato BSI-lectina ed incubare in condizioni standard di coltura per 30 min. Lavare i tessuti due volte con mezzi privi di lectina. BSI-lectina macchia rimarrà visibile sul tessuto mesentere per un massimo di 3 giorni in coltura. </li><li> Trasferire la lastra ad una fase microscopio. Identificare i vasi sanguigni e linfatici in base alla loro morfologia e la struttura di rete. </li><li> Individuare un&#39;area di rete desiderata su ogni tessuto e prendere le immagini. Prendere nota del dell&#39;imagingposizione per garantire la stessa regione verrà catturato per le immagini successive. Se si utilizza un palco motorizzato, documentare le coordinate. </li><li> Rientro tessuti per l&#39;incubatore e continuare alla cultura fino punto finale desiderato. Ripetere i passaggi 4,1-4,3 come necessario a seconda momenti sperimentali desiderati. </li></ol> 5. Tessuto immunomarcatura <ol><li> BSI-lectina Etichettatura <ol><li> Incubare tessuti per 30 min a 37 ° C con 01:40 lectina FITC-coniugato in mezzi (2,5 mL di soluzione di anticorpi per pozzetto nella piastra da 6 pozzetti) seguito da due risciacqui con supporti. Per risciacqui, aggiungere media e quindi sostituire immediatamente. </li></ol></li><li> Live / Etichettatura Morto <ol><li> Incubare tessuti per 10 minuti a 37 ° C con 1: 500 2 mM etidio omodimero-1 e 1: 500 1 mM calceina AM in mezzi (2,5 mL di soluzione di anticorpi per pozzetto nella piastra da 6 pozzetti) seguito da due risciacqui con supporti. </li></ol></li><li> BSI-lectina / NG2 Labeling <ol><li> tessuti Stendere su un microscopio scivolaronoe (1 - 2 tessuti / slide) e lasciare asciugare. Rimuovere il grasso in eccesso con un bisturi, premendo con forza per asportare il grasso. </li><li> Fix tessuti in metanolo freddo per 30 minuti a -20 ° C. Lavare tessuti con PBS (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo primario incubare tessuti per 1 ora a temperatura ambiente con 1: 100 di coniglio anticorpo policlonale NG2 e 5% di siero normale di capra (NGS). Lavare tessuti con PBS (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo secondario incubare tessuti per 1 ora a temperatura ambiente con 1: 100 di capra anti-coniglio coniugato CY2-anticorpi (GAR-CY2) e 5% NGS. Lavare tessuti con PBS (3 x 10 min). </li><li> Incubare tessuti per 30 min a temperatura ambiente con 01:40 lectina FITC-coniugato in PBS seguito da due lavaggi con PBS. Per risciacqui, aggiungere PBS e quindi sostituire immediatamente. </li><li> Per montare le diapositive, coprire i tessuti con 50:50 PBS e soluzione di glicerolo e posto coprioggetto sulla parte superiore. Sigillare i bordi di scorrimento con smalto. </li></ol></li><li> LYVE-1 / PECAM Labeling<ol><li> Stendere i tessuti su un vetrino da microscopio (1 - 2 tessuti / slide) e lasciare asciugare. Rimuovere il grasso in eccesso con un bisturi, premendo con forza per asportare il grasso. </li><li> Fix tessuti in metanolo freddo per 30 minuti a -20 ° C. Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo primario incubare tessuti per 1 ora a temperatura ambiente con 1: 200 monoclonale biotinilato anticorpo CD31 e 1: 100 policlonale di coniglio LYVE-1 anticorpi in PBS + 0,1% saponina + 2% di albumina sierica bovina (BSA) + 5% NGS . Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo secondario, incubare tessuti per 1 ora a temperatura ambiente con 1: anticorpi streptavidina 500 CY3 coniugato e 1: 100 GAR-CY2 in PBS + 0,1% saponina + 2% BSA + 5% NGS. Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li> Per montare scivoli, i tessuti di copertura con 50:50 soluzione PBS e glicerolo e mettere un coprioggetto sulla parte superiore. Sigillare i bordi di scorrimento con smalto. </li></ol></li><li> BrdU / BSI-lectina Etichettatura <ol><li> Aggiungere 1 mg / mL BrdU a media e sostituire il supporto del tessuto con una soluzione di BrdU. Incubare per 2 ore a 37 ° C. </li><li> Stendere i tessuti su un vetrino da microscopio (1 - 2 tessuti / slide) e lasciare asciugare. Rimuovere il grasso in eccesso con un bisturi, premendo con forza per asportare il grasso. </li><li> Fix tessuti in metanolo freddo per 30 minuti a -20 ° C. Lavare tessuti con PBS (3 x 10 min). </li><li> Denaturare il DNA del tessuto a 2 M HCl per 1 ora a 37 ° C. Lavare tessuti in PBS + 0,1% saponina (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo primario, incubare tessuti per 1 ora a temperatura ambiente con 1: 100 monoclonale anti-BrdU in PBS + 0,1% saponina + 2% BSA + 5% NGS. Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo secondario, incubare tessuti per 1 ora a temperatura ambiente con 1: anticorpi 100 di capra anti-topo Cy3-coniugati (GAM-Cy3) in PBS + 0,1% saponina + 2% BSA + 5% NGS. Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li&gt; incubare tessuti per 30 min a temperatura ambiente con 01:40 lectina FITC-coniugato in PBS seguito da due lavaggi con PBS. </li><li> Per montare le diapositive, i tessuti di copertura con 50:50 PBS e soluzione di glicerolo e luogo coprioggetto sulla parte superiore. Sigillare i bordi di scorrimento con smalto. </li></ol></li><li> BSI-lectina / etichettatura CD11b <ol><li> Stendere i tessuti su un vetrino da microscopio (1 - 2 tessuti / slide) e lasciare asciugare. Rimuovere il grasso in eccesso con un bisturi, premendo con forza per asportare il grasso. </li><li> Fix tessuti in metanolo freddo per 30 minuti a -20 ° C. Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo primario incubare tessuti per 1 ora a temperatura ambiente con 1: 100 topo anti-ratto CD11b in PBS + 0,1% saponina + 2% BSA + 5% NGS. Lavare i tessuti con PBS + 0,1% saponina (3 x 10 min). </li><li> Per l&#39;etichettatura anticorpo secondario incubare tessuti per 1 ora a temperatura ambiente con 1: 100 GAM-Cy3 in PBS + 0,1% saponina + 2% BSA + 5% NGS. Lavare i tessuti con PBS+ 0,1% saponina (3 x 10 min). </li><li> Incubare tessuti per 30 min a temperatura ambiente con 01:40 lectina FITC-coniugato in PBS seguito da due lavaggi con PBS. </li><li> Per montare le diapositive, i tessuti di copertura con 50:50 PBS e soluzione di glicerolo e luogo coprioggetto sulla parte superiore. Sigillare i bordi di scorrimento con smalto. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Questo protocollo documenta un metodo per l&#39;utilizzo del modello di cultura di ratto mesentere come uno strumento ex vivo per l&#39;imaging time-lapse di crescita della rete microvascolare. Impieghi precedenti nel nostro laboratorio ha stabilito l&#39;uso del nostro modello di 1) angiogenesi 8, 2) linfoangiogenesi 8, 3) periciti-endoteliali interazioni cellulare 8, e 4) anti-angiogenico test anti-droga 9. La poss...

Crescita della rete microvascolare e rimodellamento sono denominatori comuni per la funzione dei tessuti, la guarigione della ferita, e molteplici patologie e un processo chiave è l&#39;angiogenesi, definita come la crescita di nuovi vasi sanguigni da quelli esistenti 1, 2. Per Tissue Engineering nuovi vasi o la progettazione di terapie basate angiogenici, comprendendo l&#39;importanza delle dinamiche cellulari coinvolti nell&#39;angiogenesi è fondamentale. Tuttavia, questo processo è complesso. Può variare in punti specifici all&#39;interno di una rete microvascolare e coinvolge più tipi di cellule (ad esempio cellule endoteliali, cellule muscolari lisce, periciti, macrofagi, cellule staminali) e sistemi multipli (reti linfatici e reti neurali). Sebbene i modelli in vitro hanno contribuito enormemente ad esaminare il rapporto tra le diverse cellule coinvolte nell&#39;angiogenesi 3, la loro rilevanza fisiologica può essere compromessa a causa di thei<html> r limitata complessità e il fatto che essi non riflettono strettamente uno scenario in vivo. Per superare queste limitazioni, sistemi di coltura tridimensionale 3, ex vivo modelli di tessuto 4, sistemi microfluidici 5, 6, e 7 modelli di calcolo sono stati sviluppati e introdotti negli ultimi anni. Tuttavia, vi è ancora la necessità di un modello con funzionalità time-lapse per studiare l&#39;angiogenesi in reti microvascolari intatti ex vivo. La creazione di nuovi modelli di time-lapse per gli studi di angiogenesi con quel livello di complessità fornirà uno strumento prezioso per comprendere i meccanismi alla base che regolano l&#39;angiogenesi e per migliorare le terapie. Un modello potenziale che permette alla ex vivo indagini dell&#39;angiogenesi attraverso una rete microvascolare intatto è il modello di cultura mesentere ratto&gt; 8. Nel recente lavoro, abbiamo dimostrato che il sangue e le reti microvascolari linfatici rimangono vitali dopo la cultura. Ancora più importante, il mesentere modello di cultura del ratto può essere utilizzato per studiare le interazioni funzionali periciti-endoteliale di cellule del sangue, e le connessioni delle cellule endoteliali linfatiche, e time-lapse imaging. L&#39;obiettivo di questo lavoro è quello di fornire il nostro protocollo per il metodo time-lapse imaging. I nostri risultati rappresentativi documentano le molteplici tipi di cellule che rimangono vitali dopo la stimolazione dell&#39;angiogenesi con siero e offrono esempi di utilizzo di questo metodo per quantificare specifiche risposte angiogenici dei tessuti così come endoteliali studi di monitoraggio delle cellule.

<table border="0" cellpadding="0" cellspacing="0" width="679">
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			<td height="34" style="height:34px;width:165px;">Drape</td>
			<td style="width:183px;">Cardinal Health</td>
			<td style="width:132px;">4012</td>
			<td style="width:199px;">12&#8221;x12&#8221; Bio-Shield Regular Sterilization Wraps</td>
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			<td>Roboz Surgical Instrument</td>
			<td>RS-9843</td>
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			<td>Thermo Scientific</td>
			<td>130181</td>
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			<td>Roboz Surgical Instrument</td>
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			<td>Roboz Surgical Instrument</td>
			<td>RS-5130</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4&#34; Length</td>
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			<td height="86" style="height:86px;width:165px;">Noyes Micro Scissor</td>
			<td style="width:183px;">Roboz Surgical Instrument</td>
			<td style="width:132px;">RS-5677</td>
			<td style="width:199px;">Noyes Micro Dissecting Spring Scissors; 
			Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2&#34; Overall Length</td>
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			<td>FisherBrand</td>
			<td>13-761-52</td>
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			<td>FisherBrand</td>
			<td>23-400-124</td>
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			<td height="52" style="height:52px;">6-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-49</td>
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			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td>Luer-Lok Tip</td>
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			<td>Medical Action Industries Inc.</td>
			<td>01232</td>
			<td style="width:199px;">32 oz. Peel Pouch; Blue; Sterile Single Use</td>
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			<td height="35" style="height:35px;width:165px;">6-Well Plate Inserts (CellCrown Inserts)</td>
			<td>SIGMA</td>
			<td>Z681792-3EA</td>
			<td>6-Well Plate Inserts; Non-Sterile</td>
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			<td height="52" style="height:52px;width:165px;">Polycarbonate Filter Membrane</td>
			<td>SIGMA</td>
			<td>TMTP04700</td>
			<td style="width:199px;">Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&#160;&#181;m, 47&#160;mm, White Plain</td>
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			<td>Comments/Description</td>
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			<td style="width:183px;">Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 011168</td>
			<td style="width:199px;">Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium&#160;</td>
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			<td>MWI #: 000680</td>
			<td>Kateset 100 mg/ml</td>
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			<td height="35" style="height:35px;">Xylazine</td>
			<td style="width:183px;">LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Anased 100 mg/ml</td>
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			<td height="21" style="height:21px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td>2F7122</td>
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			<td height="21" style="height:21px;">PBS</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
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			<td height="21" style="height:21px;">MEM</td>
			<td>Invitrogen</td>
			<td>11095080</td>
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			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
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			<td>Invitrogen</td>
			<td>16000-044</td>
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			<td height="21" style="height:21px;">BSA</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
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			<td height="21" style="height:21px;width:165px;">Saponin&#160;</td>
			<td>SIGMA</td>
			<td>S7900-100G</td>
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			<td>Fisher Scientific</td>
			<td>S25372</td>
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			<td>SIGMA</td>
			<td>320331</td>
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			<td>Fisher Scientific</td>
			<td>67-56-1</td>
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			<td>Fisher Scientific</td>
			<td>56-81-5</td>
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			<td>SIGMA</td>
			<td>AB5320</td>
			<td></td>
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			<td height="21" style="height:21px;">PECAM (CD31) Antibody</td>
			<td>BD Biosciences</td>
			<td>555026</td>
			<td></td>
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			<td height="21" style="height:21px;">LYVE-1 Antibody</td>
			<td>AngioBio Co.</td>
			<td>11-034</td>
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			<td height="21" style="height:21px;">Goat Anti-Rabbit Cy2-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-585-144</td>
			<td></td>
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			<td height="21" style="height:21px;">Goat Anti-Mouse Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-227-003</td>
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			<td height="21" style="height:21px;">Streptavidin Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
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			<td>Invitrogen</td>
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			<td height="21" style="height:21px;">Normal Goat Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
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			<td height="21" style="height:21px;">5-Bromo-2&#39;-Deoxyuridine</td>
			<td>SIGMA</td>
			<td>B5002</td>
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		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Monoclonal Mouse Anti-Bromodeoxyuridine&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Clone Bu20a</td>
			<td>Dako</td>
			<td style="width:132px;">M074401-8</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Rat CD11b&#160;</td>
			<td>AbD Serotec</td>
			<td>MCA275R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

L&#39;angiogenesi, definita come la crescita di nuovi vasi sanguigni da vasi preesistenti, coinvolge le cellule endoteliali, periciti, cellule muscolari lisce, cellule del sistema immunitario, e il coordinamento con i vasi linfatici e nervi. Il multi-cella, interazioni multi-sistema comportano la necessità di ricerca di angiogenesi in un ambiente fisiologicamente rilevanti. Così, mentre l&#39;uso di modelli di coltura cellulare in vitro hanno fornito intuizioni meccanicistici, una critica comune è che essi non riassumono la complessità associata con una rete microvascolare. L&#39;obiettivo di questo protocollo è quello di dimostrare la possibilità di fare confronti time-lapse di reti microvascolari intatte, prima e dopo la stimolazione dell&#39;angiogenesi nei tessuti di ratto coltivate mesentere. tessuti coltivati ​​contengono reti microvascolari che mantengono la loro gerarchia. etichettatura immunoistochimica conferma la presenza di cellule endoteliali, cellule muscolari lisce, periciti, vasi sanguigni e vasi linfatici. In unddition, etichettatura dei tessuti con BSI-lectina consente un confronto time-lapse di aree di rete locali prima e dopo la stimolazione siero o fattore di crescita caratterizzato da un aumento della germinazione capillare e densità dei vasi. Rispetto ai modelli comuni di coltura cellulare, questo metodo fornisce uno strumento per gli studi di lignaggio delle cellule endoteliali e specifica valutazione dei farmaci angiogenico tessuto in fisiologicamente rilevanti reti microvascolari.

Dopo 3 giorni in coltura, tessuti sono stati etichettati con un / morti kit di sopravvivenza / citotossicità diretta per dimostrare la fattibilità del microcircolo nel ratto mesentere cultura modello (Figura 2A). La maggior parte delle cellule presenti nel mesentere è rimasto vitale nella cultura in cui le cellule endoteliali sono stati individuati in base alla loro posizione nei segmenti microvascolari. Proliferazione delle cellule endoteliali è stata confermata anc...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Un<em&gt; Ex Vivo</em&gt; Metodo per Time-Lapse Imaging di coltura Rat mesenteriche microvascolari Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

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Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K Views.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

Video: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Catturare Tissue Repair in Zebrafish Larve con Time-lapse Brightfield stereomicroscopia

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Ricerca

Biologia dello sviluppo

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

analizzare<em&gt; In Vivo</em&gt; Migrazione cellulare utilizzando Trapianti cellulari e Time-lapse imaging in embrioni di zebrafish

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Analisi dello sviluppo del rene Zebrafish con time-lapse imaging Utilizzando una dissezione microscopio dotato di sezionamento ottico

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Un sommerso Sandwich carta da filtro per il lungo termine<em&gt; Ex Ovo</em&gt; Time-lapse imaging di Chick primi embrioni

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Multi-fotone Time Lapse Imaging per visualizzare lo sviluppo in tempo reale: visualizzazione della migrazione di cellule neurali della cresta negli embrioni di Zebrafish

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Coltura delle cellule epiteliali del pigmento retinico su un modello Ex Vivo della membrana di Bruch umano invecchiato

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Un metodo In Vivo per studiare l'integrità della barriera ematotesticolare di Mouse

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Analisi delle dinamiche di Actomyosin su scale cellulari e tissutali locali utilizzando film time-lapse di camere d'uovo di Drosophila coltivate

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

Perfusione ex vivo della placenta del roditore

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Un metodo di montaggio a strati per la microscopia confocale Time-Lapse estesa di embrioni di pesci zebra interi

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

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