Die Arbeit wurde unterstützt durch Stipendien der Manpei Suzuki Diabetic Foundation (YT), Boston Children es Hospital OFD/BTREC/CTREC Faculty Career Development Grant, Boston Children es Hospital Ophthalmology Foundation, BCH Pilot Award, BCH Manton Center Fellowship, und Little Giraffe Foundation (ZF), The German Research Foundation (DFG; to BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH IDDRC (1U54HD090255), Massachusetts Lions Eye Foundation (LEHS).

Alle beschriebenen Tierversuche wurden vom Institutional Animal Care and Use Committee am Boston Children es Hospital genehmigt (ARCH-ProtokollNummer 19-04-3913R).
1. Vorbereitung
<ol><li>Fügen Sie 5 ml Penicillin/Streptomycin (10000 U/ml) und 5 ml handelsübliche Ergänzungen zu 500 ml komplettem klassischen Medium mit Serum hinzu. Aliquot 50 ml des Mediums zunächst. HINWEIS: Geben Sie kein Medium zurück an den Bestand, um eine Kontamination zu vermeiden.</li>
	<li>Setzen Sie ein Aliquot von kompletten klassischen Medium auf Eis.</li>
	<li>Verwenden Sie 70% Ethanol, um das Sezieren von Mikroskop, Zange und Schere zu reinigen.</li>
	<li>Bereiten Sie zwei Zellkultur-Gerichte (10 cm), eine auf dem Seziermikroskop, eine auf Eis; 10 ml komplettes klassisches Medium in jedes Gericht geben.</li>
</ol>2. Experimentelle Schritte (Abbildung 1)
<ol><li>Opfern Sie C57BL/6J-Mäuse um postnatale (P) 20 mit 75-100 mg/kg Ketamin und 7,5 -10 mg/kg Xylazin intraperitoneal injiziert. Halten Sie die Augen in komplettem klassischen Medium auf Eis vor der Zerlegung.</li>
	<li>Entfernen Sie das Bindegewebe (Muskel- und Fettgewebe) und den Sehnerv auf dem Auge.</li>
	<li>Verwenden Sie eine Mikroschere, um 0,5 mm nach dem Hinterteil des Hornhaut-Limbus umlaufen zu lassen. Entfernen Sie den Hornhaut-Iris-Komplex, Glaskörper und die Linse.</li>
	<li>Machen Sie einen 1 mm Schnitt senkrecht zur Schnittkante zum Sehnerv und schneiden Sie ein Umfangsband von 1 mm Breite. Trennen Sie die zentralen und peripheren Regionen des Komplexes. Verwenden Sie Zangen, um die Netzhaut aus dem RPE/Choroid/Sklera-Komplex abzuziehen.</li>
	<li>Halten Sie das periphere Aderhautband in komplettem klassischen Medium auf Eis; isolieren Sie das andere Auge und wiederholen Sie den Vorgang, um ein zweites Band zu schneiden.</li>
	<li>Schneiden Sie das Kreisförmige Band in 6 ,gleich quadratische Stücke (ca. 1 mm x 1 mm). HINWEIS: Berühren Sie niemals eine Kante.</li>
	<li>Den Basalmembranextrakt (BME) pro Herstelleranweisung auftauen. Fügen Sie 30 L/Well von BME in die Mitte jedes Brunnens einer 24 Brunnen-Gewebekulturplatte hinzu. Stellen Sie sicher, dass das Tröpfchen von BME eine konvexe Kuppel an der Unterseite der Platte bildet, ohne die Kanten zu berühren. HINWEIS: Das BME über Nacht im Kühlschrank auftauen. BME sollte jederzeit nach dem Auftauen auf dem Eis sein.</li>
	<li>Legen Sie das Gewebe in die Mitte des BME. HINWEIS: Die Aderhautexplantation nicht abflachen; lassen Sie das Gewebe im Allgemeinen innerhalb des BME ausdehnen. Die Ausrichtung des Gewebes (Skleralseite nach oben oder unten) hat keinen Einfluss auf das experimentelle Ergebnis.</li>
	<li>Inkubieren Sie die Platte bei 37 °C für 10 min, um das Gel erstarren zu lassen.</li>
	<li>Fügen Sie 500 l des kompletten klassischen Mediums/Well hinzu.</li>
	<li>Ändern Sie das klassische Medium jeden zweiten Tag (500 l). Das Choroid-Sprießen kann nach 3 Tagen mit einem Mikroskop beobachtet werden. HINWEIS: Für die Behandlung des Wachstumsfaktors verhungern Sie das Gewebe für 4 h. Verdünnen Sie eine Versuchsverbindung in wachstumsfaktorreduzierten Medien (1:200 Boost statt 1:50).</li>
</ol>3.SWIFT-Choroid computerisierteQuantifizierungsmethode 17 (Abbildung 2)
HINWEIS: Es wurde eine computergestützte Methode zur Messung der Fläche verwendet, die von wachsenden Schiffen bedeckt ist. Vor der Quantifizierung wird ein Makro-Plugin zur ImageJ-Software benötigt (weitere Informationen finden Sie unter Ergänzende Informationen).
<ol><li>Öffnen Sie das Aderhaut-Sprossenbild mit ImageJ und aktivieren Sie "Bild | Typ| 8-Bit" mit Graustufen.</li>
	<li>Gehe zu "Image | Anpassen | Helligkeit/Kontrast (Strg/Shift/C)" und optimieren Sie den Kontrast.</li>
	<li>Verwenden Sie die Zauberstab-Funktion, um das Aderhautgewebe, das in der Mitte der Sprossen vorhanden ist, umzureißen und aus dem Bild zu entfernen (mit Tastenkombination "F1") (Abbildung 2A,B). HINWEIS: Legen Sie die Toleranzrate des Zauberstabs auf 20-30% fest.</li>
	<li>Entfernen Sie den Hintergrund des Bildes mit den kostenlosen Auswahlwerkzeugen (Abbildung 2C). Gehe zu "Image | Anpassen | Schwellenwert (Strg/Shift/T)". Verwenden Sie die Schwellenfunktion, um die mikrovaskulären Sprossen vor dem Hintergrund und der Peripherie zu definieren (Abbildung 2D).</li>
	<li>Klicken Sie auf "F2" und es wird eine Zusammenfassung angezeigt. Speichern Sie ein Bild des ausgewählten Bereichs, indem Sie auf"Speichern"klicken. Speichern Sie im selben Ordner wie das Originalbild für zukünftige Referenzen.</li>
	<li>Nachdem eine Gruppe von Stichproben gemessen wurde, kopieren Sie die aufgezeichneten für die Datenanalyse. ANMERKUNG: Es ist auch möglich, die Fläche (m2) mit"Analysieren | Set Scale" mit Bildern mit Maßstabsleisten.</li>
</ol>

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H., 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=Endothelial+microRNA-150+is+an+intrinsic+suppressor+of+pathologic+ocular+neovascularization.">Endothelial microRNA-150 is an intrinsic suppressor of pathologic ocular neovascularization.</a> Proceedings of the National Academy of Science U. S. A. 112 (39), 12163-12168 (2015).</li><li>Zhou, Q., 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=LncEGFL7OS+regulates+human+angiogenesis+by+interacting+with+MAX+at+the+EGFL7/miR-126+locus.">LncEGFL7OS regulates human angiogenesis by interacting with MAX at the EGFL7/miR-126 locus.</a> Elife. 8, 40470 (2019).</li><li>Kobayashi, S., Fukuta, M., Kontani, H., Yanagita, S., Kimura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+angiogenesis+of+cultured+choroidal+tissues+in+streptozotocin-diabetic+Wistar+and+spontaneously+diabetic+GK+rats.">A quantitative assay for angiogenesis of cultured choroidal tissues in streptozotocin-diabetic Wistar and spontaneously diabetic GK rats.</a> Japanese Journal of Pharmacology. 78 (4), 471-478 (1998).</li><li>Kobayashi, 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=Inhibitory+effects+of+tetrandrine+and+related+synthetic+compounds+on+angiogenesis+in+streptozotocin-diabetic+rodents.">Inhibitory effects of tetrandrine and related synthetic compounds on angiogenesis in streptozotocin-diabetic rodents.</a> Biological and Pharmaceutical Bulletin. 22 (4), 360-365 (1999).</li><li>Kobayashi, S., Shinohara, H., Tsuneki, H., Nagai, R., Horiuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N(epsilon)-(carboxymethyl)lysine+proliferated+CD34(+)+cells+from+rat+choroidal+explant+in+culture.">N(epsilon)-(carboxymethyl)lysine proliferated CD34(+) cells from rat choroidal explant in culture.</a> Biological and Pharmaceutical Bulletin. 27 (9), 1382-1387 (2004).</li><li>Kobayashi, 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=Overproduction+of+N(epsilon)-(carboxymethyl)lysine-induced+neovascularization+in+cultured+choroidal+explant+of+streptozotocin-diabetic+rat.">Overproduction of N(epsilon)-(carboxymethyl)lysine-induced neovascularization in cultured choroidal explant of streptozotocin-diabetic rat.</a> Biological and Pharmaceutical Bulletin. 27 (10), 1565-1571 (2004).</li><li>Bergers, G., Song, S. <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+pericytes+in+blood-vessel+formation+and+maintenance.">The role of pericytes in blood-vessel formation and maintenance.</a> Neuro-Oncology. 7 (4), 452-464 (2005).</li><li>Browning, A. 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Die Autoren haben keine finanziellen Angaben. Die computergestützte Methode steht akademischen Einrichtungen über die Autoren kostenlos zur Verfügung.

Der choroidal sprossende Assay unterstützt die Forschung in neovaskulären AMD9,10,18,19,20. Aderhautexplantationen können sowohl von Mäusen als auch von Ratten und Menschen isoliert werden17,21. Die Aderhautexplantation umfasst ECs, Makrophagen und Pericyten17. In diesem Tes...

Die Dysregulation der Choroidalangiogenese ist mit der neovaskulären altersbedingten Makuladegeneration (AMD)1verbunden. Die Aderhaut ist ein mikrovaskuläres Bett unter dem retinalen Pigmentepithel (RPE). Es hat sich gezeigt, dass ein reduzierter Blutfluss in der Aderhaut mit der Progression von AMD2verbunden ist. Die komplizierte Beziehung zwischen vaskulärem Endothel, RPE, Makrophagen, Pericyten und anderen Zellen ist verantwortlich für die Homöostase des Gewebes3,4,5. Daher ist eine reproduzierbare Assaymodellierung der aderatorischen Mikroumgebung für die Untersuchung der neovaskulären AMD von entscheidender Bedeutung.
Ex-vivo-Angiogenese-Assays und in vitro endotheliale Zellkulturen können Studien über mikrovaskuläres Verhalten in vivo, zur Erprobung neuer Medikamente und für Studien zur Pathogenese ergänzen. Endothelzellen wie menschliche mikrovaskuläre Endothelzellen (HRMECs), Human Umbilical Venin Endothelial Cells (HUVEC), isolierte primäre tierische Gehirne oder netzretinale ECs werden häufig in In-vitro-Studien zur okulären Angiogeneseforschung6,7,8verwendet. Insbesondere HRMECs wurden weithin als Modell der in vitro choroidalen Neovaskularisation (CNV)9 verwendet, indem die endotheliale Proliferation, Migration, tubuläre Bildung und vaskuläre Leckage bewertet wurden, um die Interventionen6,10zu bewerten. Jedoch, ECs in der Kultur sind als Modell der CNV begrenzt, weil es an Wechselwirkungen mit anderen Zelltypen in der Aderhaut gefunden und weil die meisten EC in diesen Assays verwendet stammen nicht von Derader. Maus-Aderhaut-ECs sind in der Kultur schwer zu isolieren und zu pflegen.
Der Aortenring-Assay wird weithin als Modell der makrovaskulären Proliferation verwendet. Zu den Gefäßsprossen von Aortenexpflanzen gehören ECs, Pericyte und Makrophagen11. Der Aortenring-Assay modelliert große Gefäßangiogenese gut12,13,14. Es hat jedoch Einschränkungen als Modell der choroidalen Neovaskularisation, da Aortenringe ein makrovaskuläres Gewebe sind, das nicht die charakteristische choroidale mikrovaskuläre Umgebung aufweist, und Sprossen aus großen Gefäßen können sich von Sprossen aus Kapillarnetzwerken unterscheiden, die an mikrovaskulärer Pathologie beteiligt sind. Kürzlich veröffentlichte eine Gruppe einen Ex-vivo-Retina-Assay15,16. Obwohl, Es ist geeignet für retinale neovaskuläre Erkrankungen, Es ist nicht so geeignet für choroidale Neovaskularisation wie bei AMD gesehen.
Der aderodiensprosende Assay mit Maus-RPE, Aderhaut und skleral explantiertem Gewebe wurde entwickelt, um CNV besser zu modellieren. Das Gewebe kann leicht von Maus (oder anderen Arten) Augen isoliert werden17. Dieser Test ermöglicht eine reproduzierbare Bewertung des pro- und antiangiogenetischen Potenzials von pharmakologischen Verbindungen und die Bewertung der Rolle spezifischer Wege bei der choroidalen Neovaskularisation mit Gewebe von genetisch veränderten Mäusen und Kontrollen18. Dieser aderhautspaltnde Assay wurde in vielen nachfolgenden Publikationen9,10,18,19,20erwähnt. Hier wird die Methode demonstriert, die bei der Verwendung dieses Assays zum Einsatz kommt.

<table>
	<tbody>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>AKORN</td>
			<td>59339-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal membrane extract (BME) Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td>10cm</td>
		</tr>
		<tr>
			<td>Complete classic medium with serum and CultureBoost</td>
			<td>Cell systems</td>
			<td>4Z0-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 Proof</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td>use for 70%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>Tissue culture plate (24-well)</td>
			<td>Olympus</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>VetaKet CIII (Ketamine)</td>
			<td>AKORN</td>
			<td>59399-114-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

Die pathologische Aderhautangiogenese, ein markantes Merkmal der altersbedingten Makuladegeneration, führt zu Sehstörungen und Erblindung. Endotheliale Zell(E-Zell-Assays mit humanen mikrovaskulären Endothelzellen (HRMECs) oder isolierten primären retinalen ECs sind weit verbreitet in vitro-Modelle zur Untersuchung der retinalen Angiogenese. Die Isolierung reiner muriner retinaler Endothelzellen ist jedoch technisch anspruchsvoll und netzhautförmige ECs können andere Proliferationsreaktionen haben als aderhautförmige Endothelzellen und verschiedene Zell-/Zell-Wechselwirkungen. Es wurde ein hochreproduzierbarer ex vivo aderhautspessender Assay als Modell der choroidalen mikrovaskulären Proliferation entwickelt. Dieses Modell umfasst die Wechselwirkung zwischen Derhautvaskulatur (EC, Makrophagen, Pericyten) und retinalem Pigmentepithel (RPE). Maus-RPE/Choroid/Skleralexplanten werden isoliert und in wachstumsfaktorreduziertem Basalmembranextrakt (BME) (Tag 0) inkubiert. Medium wird jeden zweiten Tag verändert und Die Aderhautsprossen wird an Tag 6 quantifiziert. Die Bilder einzelner Aderhautexplantationen werden mit einem invertierten Phasenmikroskop aufgenommen und der Keimbereich wird mit einem halbautomatischen Makro-Plug-in zur in diesem Labor entwickelten ImageJ-Software quantifiziert. Dieser reproduzierbare Ex-vivo-Chorsponnen-Sprossen-Assay kann verwendet werden, um Verbindungen für eine mögliche Behandlung und für die Forschung an mikrovaskulären Erkrankungen zu bewerten, um Wege zu bewerten, die an der Verbreitung von Aderhautmikrogefäßen mit Wildtyp und genetisch verändertem Mausgewebe beteiligt sind.

Vergleich des Aderhautsprießens pro Tag
Wir sezierten die Aderhaut mit Sklera, eingebettet in BME und kultivierten sie für 6 Tage (Abbildung 1). Die Aderhautsprossen in C57BL/6J-Mäusen vom 3. bis 6. Tag wurden mit einem Mikroskop untersucht und mit SWIFT-Choroid, einer halbautomatischen Quantifizierungsmethode in ImageJ, quantifiziert. In einem repräsentativen Fall betrug der Aderhautsprossenbereich (die Gefäße, die sich von der Expflanze au...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Dieses Protokoll stellt einen Aderhautkeim-Assay dar, ein Ex-vivo-Modell der mikrovaskulären Proliferation. Dieser Test kann verwendet werden, um Wege zu bewerten, die an sich vermehrenden aderatorischen Mikrogefäßen beteiligt sind, und medikamentöse Behandlungen mit Wildtyp und genetisch verändertem Mausgewebe zu bewerten.

Ein Ex Vivo Choroid Sprouting Assay der ocular Microvascular Angiogenese

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

an-ex-vivo-choroid-sprouting-assay-ocular-microvascular

Research

JoVE Journal

Biology

5.5K Aufrufe.  Boston Children's Hospital, Harvard Medical School. Dieses Protokoll stellt einen Aderhautkeim-Assay dar, ein Ex-vivo-Modell der mikrovaskulären Proliferation. Dieser Test kann verwendet werden, um Wege zu bewerten, die an sich vermehrenden aderatorischen Mikrogefäßen beteiligt sind, und medikamentöse Behandlungen mit Wildtyp und genetisch verändertem Mausgewebe zu bewerten.

Serie: An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: Eine In-vitro-Modell für das Studium der Angiogenese (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Forschung

Biologie

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Optimierte Fibringel Bead Assay für das Studium der Angiogenese

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo mechanische Belastung der Sehnen

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

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 Mesenterium Angiogenese Assay

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

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Endothelzellen Schlauchbildung Assay für die<em&gt; In-vitro-</em&gt; Studium der Angiogenese

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Ein<em&gt; Ex-vivo-</em&gt; Kultur-System, um Schilddrüsenentwicklungsstudie

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Die c-fos-Protein Immunohistologische Nachweis: Ein nützliches Werkzeug als Marker der Mittelwegen beteiligt sind in spezifischen physiologischen Antworten<em&gt; In Vivo</em&gt; und<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

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

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

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

Isolierung der retinalen Arteriolen für Ex Vivo Zelle Physiologie Studien

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

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Ein Ex-Vivo-Gewebekulturmodell des Knorpel-Remodels in Rinderknie-Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Glomeruläres Outgrowth als Ex-Vivo-Assay zur Analyse von Pfaden, die an der Parietalepithel Cell Activation beteiligt sind

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...