Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00.000+00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

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>

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer+and+other+diseases.">Angiogenesis in cancer and other diseases.</a> Nature. 407 (6801), 249-257 (2000).</li><li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438 (7070), 932-936 (2005).</li><li>Kaunas, R., Kang, H., Bayless, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+Regulation+of+Angiogenic+Sprouting+by+Biochemical+Factors+and+Wall+Shear+Stress.">Synergistic Regulation of Angiogenic Sprouting by Biochemical Factors and Wall Shear Stress.</a> Cell Mol Bioeng. 4 (4), 547-559 (2011).</li><li>Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+the+neonatal+vasculature+and+analysis+of+retinal+angiogenesis+in+mice.">Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.</a> Nat Protoc. 5 (9), 1518-1534 (2010).</li><li>Song, J. W., Munn, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+forces+control+endothelial+sprouting.">Fluid forces control endothelial sprouting.</a> Proc Natl Acad Sci U S A. 108 (37), 15342-15347 (2011).</li><li>Chan, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+in+vitro+3D+capillary+beds+by+self-directed+angiogenic+sprouting.">Engineering of in vitro 3D capillary beds by self-directed angiogenic sprouting.</a> PLoS One. 7 (12), e50582 (2012).</li><li>Peirce, S. M., Mac Gabhann, F., Bautch, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+experimental+and+computational+approaches+to+sprouting+angiogenesis.">Integration of experimental and computational approaches to sprouting angiogenesis.</a> Curr Opin Hematol. 19 (3), 184-191 (2012).</li><li>Stapor, P. C., Azimi, M. S., Ahsan, T., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+angiogenesis+model+for+investigating+multicellular+interactions+across+intact+microvascular+networks.">An angiogenesis model for investigating multicellular interactions across intact microvascular networks.</a> Am J Physiol Heart Circ Physiol. 304 (2), H235-H245 (2013).</li><li>Azimi, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+model+for+anti-angiogenic+drug+testing+on+intact+microvascular+networks.">An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.</a> PLoS One. 10 (3), e0119227 (2015).</li><li>Nicosia, R. F., Ottinetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+microvessels+in+serum-free+matrix+culture+of+rat+aorta.+A+quantitative+assay+of+angiogenesis+in+vitro.">Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.</a> Lab Invest. 63 (1), 115-122 (1990).</li><li>Hutter-Schmid, B., Kniewallner, K. M., Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+as+a+model+to+study+angiogenesis+of+brain+vessels.">Organotypic brain slice cultures as a model to study angiogenesis of brain vessels.</a> Front Cell Dev Biol. 3, 52 (2015).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat Protoc. 5 (10), 1659-1665 (2010).</li><li>Unoki, N., Murakami, T., Ogino, K., Nukada, M., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+retinal+angiogenesis+reveals+decreased+development+and+progression+of+neovascular+sprouting+by+anecortave+desacetate.">Time-lapse imaging of retinal angiogenesis reveals decreased development and progression of neovascular sprouting by anecortave desacetate.</a> Invest Ophthalmol Vis Sci. 51 (5), 2347-2355 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10 (3), 588-612 (2006).</li><li>Kelly-Goss, M. R., Sweat, R. S., Azimi, M. S., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+islands+during+microvascular+regression+and+regrowth+in+adult+networks.">Vascular islands during microvascular regression and regrowth in adult networks.</a> Front Physiol. 4, 108 (2013).</li><li>Kelly-Goss, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+along+vascular+islands+during+microvascular+network+growth.">Cell proliferation along vascular islands during microvascular network growth.</a> BMC Physiol. 12, 7 (2012).</li><li>Skalak, T. C., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanical+stresses+in+microvascular+remodeling.">The role of mechanical stresses in microvascular remodeling.</a> Microcirculation. 3 (2), 143-165 (1996).</li><li>Kadohama, T., Nishimura, K., Hoshino, Y., Sasajima, T., Sumpio, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+types+of+fluid+shear+stress+on+endothelial+cell+proliferation+and+survival.">Effects of different types of fluid shear stress on endothelial cell proliferation and survival.</a> J Cell Physiol. 212 (1), 244-251 (2007).</li><li>Milkiewicz, M., Brown, M. D., Egginton, S., Hudlicka, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+shear+stress,+angiogenesis,+and+VEGF+in+skeletal+muscles+in+vivo.">Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo.</a> Microcirculation. 8 (4), 229-241 (2001).</li><li>Corliss, B. A., Azimi, M. S., Munson, J. M., Peirce, S. M., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages:+An+Inflammatory+Link+Between+Angiogenesis+and+Lymphangiogenesis.">Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis.</a> Microcirculation. 23 (2), 95-121 (2016).</li></ol>

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><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</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 ...

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

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

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modello per studiare l&#39;adesione batterica alla parete del serbatoio in condizioni di flusso

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Ricerca

Immunologia e infezioni

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrofili e monociti in mesenterica Veins intravitale Microscopia su topi anestetizzati in tempo reale

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

Rat mesentere angiogenesi Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biologia

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Rat Esteriorizzazione mesentere: un modello per indagare le dinamiche cellulari coinvolti nell&#39;angiogenesi

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

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 ...

Medicina

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Mouse fetale Whole Intestino Sistema Cultura per<em&gt; Ex Vivo</em&gt; La manipolazione delle vie di segnalazione e tridimensionale Imaging in diretta di Sviluppo del villo

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Misurazione dinamica e imaging di capillari, arterioli e periciti nel cuore del topo

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

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

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

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 ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

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 ...

Un

Riepilogo

Esplora Altri Video

Capitoli in questo video

In vitro e in vivo modello per studiare l'adesione batterica alla parete del serbatoio in condizioni di flusso

Imaging neutrofili e monociti in mesenterica Veins intravitale Microscopia su topi anestetizzati in tempo reale

Rat mesentere angiogenesi Assay

Rat Esteriorizzazione mesentere: un modello per indagare le dinamiche cellulari coinvolti nell'angiogenesi

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

Mouse fetale Whole Intestino Sistema Cultura per

Misurazione dinamica e imaging di capillari, arterioli e periciti nel cuore del topo

A due fotoni Imaging di Intracellulare Ca

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

Sviluppo e caratterizzazione di