Les travaux ont été soutenus par des subventions de la Manpei Suzuki Diabetic Foundation (YT), du Boston Children’s Hospital OFD/BTREC/CTREC Faculty Career Development Grant, de la Boston Children’s Hospital Ophthalmology Foundation, du BCH Pilot Award, de la BCH Manton Center Fellowship et de la Little Giraffe Foundation (ZF), de la German Research Foundation (DFG; à BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH CRDI (1U54HD090255), Massachusetts Lions Eye Foundation (LEHS).

Toutes les expériences sur les animaux décrites ont été approuvées par le Comité institutionnel de soins et d’utilisation des animaux du Boston Children’s Hospital (numéro de protocole ARCH 19-04-3913R).
1. Préparation
<ol><li>Ajouter 5 mL de pénicilline/streptomycine (10000 U/mL) et 5 mL et 10 mL de suppléments disponibles dans le commerce à 500 mL de milieu classique complet avec sérum. Aliquot 50 mL du milieu initialement. REMARQUE : Ne retournez aucun milieu au stock pour éviter la contamination.</li>
	<li>Mettez un aliquot de milieu classique complet sur la glace.</li>
	<li>Utilisez 70 % d’éthanol pour nettoyer le microscope, les forceps et les ciseaux disséquants.</li>
	<li>Préparer deux plats de culture cellulaire (10 cm), l’un au microscope à dissection, l’autre sur glace; mettre 10 mL de milieu classique complet dans chaque plat.</li>
</ol>2. Étapes expérimentales (Figure 1)
<ol><li>Sacrifier les souris C57BL/6J autour de la kétamine postnatale (P) 20 à l’aide de kétamine de 75 à 100 mg/kg et de 7,5 à 10 mg/kg de xylazine injectée intraperitoneally. Gardez les yeux dans le milieu classique complet sur la glace avant la dissection.</li>
	<li>Enlever le tissu conjonctif (muscle et tissu adipeux) et le nerf optique sur l’œil.</li>
	<li>Utilisez un micro-ciseaux pour couper circonférentiellement 0,5 mm postérieur au limbus cornéen. Retirer le complexe cornée/iris, vitreux et la lentille.</li>
	<li>Faire une incision de 1 mm perpendiculairement au bord de coupe vers le nerf optique et couper une bande circonférentielle de 1 mm de largeur. Séparez les régions centrales et périphériques du complexe. Utilisez des forceps pour décoller la rétine du complexe RPE/choroid/sclera.</li>
	<li>Gardez la bande choroïde périphérique dans un milieu classique complet sur la glace; isoler l’autre œil et répéter le processus pour couper une deuxième bande.</li>
	<li>Couper la bande circulaire en 6 ~morceaux carrés égaux (~1 mm x 1 mm). REMARQUE : Ne touchez jamais à aucun bord.</li>
	<li>Décongeler l’extrait de membrane basale (BME) par instruction de fabrication. Ajouter 30 μL/puits de BME au centre de chaque puits d’une plaque de culture tissulaire de 24 puits. Assurez-vous que la gouttelette de BME forme un dôme convexe au bas de la plaque sans toucher les bords. REMARQUE : Décongeler le BME toute la nuit au réfrigérateur. Le BME doit être sur la glace à tout moment après le dégel.</li>
	<li>Placez le tissu au milieu du BME. REMARQUE : N’aplatissez pas l’explantation choroïde; généralement, laissez le tissu se développer dans le BME. L’orientation du tissu (côté sclérical vers le haut ou vers le bas) n’a pas d’impact sur les résultats expérimentaux.</li>
	<li>Incuber la plaque à 37 °C pendant 10 min pour laisser le gel se solidifier.</li>
	<li>Ajouter 500 μL du milieu/puits classique complet.</li>
	<li>Changez le médium classique tous les deux jours (500 μL). La germination choroïde peut être observée après 3 jours au microscope. REMARQUE : Pour le traitement des facteurs de croissance, affamer le tissu pendant 4 h. Diluer un composé d’essai dans un milieu à facteur de croissance réduit (1:200 boost au lieu de 1:50).</li>
</ol>3.SWIFT-Choroid méthode informatisée de quantification17 (Figure 2)
REMARQUE : Une méthode informatisée pour mesurer la superficie couverte par les navires en croissance a été utilisée. Un plugin macro au logiciel ImageJ est nécessaire avant la quantification (voir informations complémentaires pour plus de détails).
<ol><li>Ouvrez l’image de germination choroïde avec ImageJ et vérifiez "Image | Le type| 8-bit" à l’échelle grise.</li>
	<li>Aller à "Image | Ajuster | Luminosité/Contraste (Ctrl/shift/C)" et optimiser le contraste.</li>
	<li>Utilisez la fonction baguette magique pour décrire et enlever de l’image le tissu choroïde qui sont présents au centre des germes (en utilisant la clé raccourcie "F1« ) (Figure 2A,B). REMARQUE : Réglez le taux de tolérance de la baguette magique à 20-30%.</li>
	<li>Supprimez l’arrière-plan de l’image avec les outils de sélectiongratuits ( Figure 2C). Aller à "Image | Ajuster | Seuil (Ctrl/shift/T)« . Utilisez la fonction seuil pour définir les germes microvasculaires en arrière-plan et en périphérie (Figure 2D).</li>
	<li>Cliquez sur" F2" et un résumé apparaîtra. Enregistrez une image de la zone sélectionnée en cliquant sur "Enregistrer« . Enregistrer dans le même dossier que l’image d’origine pour la référence future.</li>
	<li>Une fois qu’un groupe d’échantillons est mesuré, copiez l’enregistrement pour analyse de données. REMARQUE : Il est également possible de mesurer la zone (μm²) par «Analyser | Set Scale" à l’aide d’images avec barres d’échelle.</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. 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Les auteurs n’ont pas de divulgation financière. La méthode informatisée est disponible gratuitement aux établissements d’enseignement par l’intermédiaire des auteurs.

L’analyse choroïdienne de germination aide la recherche dans amd néovasculaire9,10,18,19,20. Les explantations choroïdes peuvent être isolées de souris ainsi que de rats etd’humains 17,21. L’explant choroïde comprend les CE, les macrophages et les péricytes17. Dans ...

La dysrégulation choroïde d’angiogenèse est associée à la dégénérescence maculaire relative à l’âge néoovasculaire (DME)1. Le choroïde est un lit microvasculaire présent sous l’épithélium pigmentaire rétinien (EPR). Il a été démontré que la réduction du flux sanguin dans le choroïde est associée à la progression de la D AMD2. La relation complexe entre l’endothélium vasculaire, l’EPR, les macrophages, les péricytes et d’autres cellules est responsable de l’homéostasiedu tissu 3,4,5. Par conséquent, un microenvironnement choroïdien reproductible de modélisation d’essai est critique pour l’étude de la DMO néovasculaire.
Les analyses d’angiogenèse ex vivo et les cultures cellulaires endothéliales in vitro peuvent compléter les études sur le comportement microvasculaire in vivo, pour tester de nouveaux médicaments et pour des études sur la pathogénie. Les cellules endothéliales telles que les cellules endothéliales microvasculaires rétiniennes humaines (HRMECs), les cellules endothéliales des veines ombilicales humaines (HUVEC), le cerveau animal primaire isolé ou les CE rétiniens sont souvent utilisées dans des études in vitro pour la recherche sur l’angiogenèseoculaire 6,7,8. Les HRMEC en particulier ont été largement utilisés comme modèle de néovascularisation choroïdienne in vitro (CNV)9 en évaluant la prolifération endothéliale, la migration, la formation tubulaire et les fuites vasculaires pour évaluer les interventions6,10. Cependant, les CE en culture sont limités comme modèle de CNV en raison du manque d’interactions avec d’autres types de cellules trouvés dans le choroïde et parce que la plupart des EC utilisés dans ces analyses ne proviennent pas de choroïdes. Les CE choroïdes de souris sont difficiles à isoler et à maintenir dans la culture.
L’essai aortique d’anneau est employé couramment comme modèle de prolifération vasculaire macro. Les pousses vasculaires provenant d’explants aortiques comprennent les CE, les péricytes et les macrophages11. L’essai d’anneau aortique modèle le grand angiogenèse denavire bien 12,13,14. Cependant, il a des limitations comme modèle de néovascularisation choroïdienne car les anneaux aortiques sont un tissu macrovasculaire dépourvu de l’environnement microvasculaire choroïde caractéristique, et les germes des grands vaisseaux peuvent différer des germes des réseaux capillaires impliqués dans la pathologie microvasculaire. Récemment, un groupe a publié un essai rétinien ex vivo15,16. Bien qu’il soit approprié pour la maladie néovasculaire rétinienne, il n’est pas aussi approprié pour la néovascularisation choroïde comme vu dans AMD.
L’essai choroïde de germination utilisant le RPE de souris, le choroïde, et le tissu explanté sclérical a été développé pour mieux modéliser CNV. Le tissu peut facilement être isolé des yeux de souris (ou d’autres espèces)17. Cet essai permet l’évaluation reproductible du potentiel pro- et anti-angiogenic des composés pharmacologiques et l’évaluation du rôle des voies spécifiques dans la néovascularisation choroïde utilisant le tissu des souris génétiquement modifiées et contrôle18. Cet essai choroïde de germination a été référencé dans beaucoup de publicationssuivantes 9,10,18,19,20. Ici, la méthode impliquée dans l’utilisation de cet essai sont démontrées.

<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

L’angiogenèse choroïde pathologique, une caractéristique saillante de la dégénérescence maculaire relative à l’âge, mène à l’affaiblissement de vision et à la cécité. Les tests de prolifération des cellules endothéliales (EC) utilisant des cellules endothéliales microvasculaires rétiniennes humaines (HRMECs) ou des EC rétiniennes primaires isolées sont largement utilisés dans des modèles in vitro pour étudier l’angiogenèse rétinienne. Cependant, isoler les cellules endothéliales rétiniennes murines pures est techniquement difficile et les CE rétiniens peuvent avoir des réponses de prolifération différentes que les cellules endothéliales choroïdes et les différentes interactions cellule/cellule. Un essai choroïdien choroïde fortement reproductible de germination comme modèle de prolifération microvasculaire choroïde a été développé. Ce modèle inclut l’interaction entre la vascularisation choroïde (EC, macrophages, péricytes) et l’épithélium pigmentaire rétinien (EPR). Les explants rpe/choroïdes/scléraux de souris sont isolés et incubés dans l’extrait basal de membrane croissance-facteur-réduit (BME) (jour 0). Le milieu est changé tous les deux jours et la germination choroïde est quantifiée au jour 6. Les images de l’explantation choroïde individuelle sont prises avec un microscope de phase inversée et la zone de germination est quantifiée à l’aide d’un plug-in macro semi-automatisé au logiciel ImageJ développé dans ce laboratoire. Cet essai reproductible de germination choroïde ex vivo peut être employé pour évaluer des composés pour le traitement potentiel et pour la recherche microvasculaire de maladie pour évaluer des voies impliquées dans la prolifération choroïde de micro navire utilisant le type sauvage et le tissu génétiquement modifié de souris.

Comparaison de la croissance de la germination choroïde par jour
Nous avons disséqué le choroïde avec de la sclérienne, incrusté dans le BME et les avons cultivés pendant 6 jours( Figure 1). La germination choroïde chez les souris C57BL/6J du jour 3 au jour 6 a été examinée au microscope et quantifiée avec SWIFT-Choroid une méthode de quantification semi-automatisée dans ImageJ. Dans un cas représentatif, la zone choroïde de germin...

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An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Ce protocole présente l’essai choroïde de germination, un modèle ex vivo de prolifération microvasculaire. Cet essai peut être utilisé pour évaluer les voies impliquées dans la prolifération des micro-vaisseaux choroïdiens et évaluer les traitements médicamenteux à l’aide de tissus de souris sauvages et génétiquement modifiés.

Un ex Vivo Choroid Sprouting Assay de l’angiogenèse microvasculaire oculaire

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

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Biology

5.5K vues.  Boston Children's Hospital, Harvard Medical School. Ce protocole présente l’essai choroïde de germination, un modèle ex vivo de prolifération microvasculaire. Cet essai peut être utilisé pour évaluer les voies impliquées dans la prolifération des micro-vaisseaux choroïdiens et évaluer les traitements médicamenteux à l’aide de tissus de souris sauvages et génétiquement modifiés.

Vidéo: 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: un modèle in vitro pour l&#39;étude de l&#39;angiogenèse (Interview)

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

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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.

Optimisé fibrine billes de gel d&#39;analyse pour l&#39;étude de l&#39;angiogenèse

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 Chargement mécanique du 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 ...

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.

Assay Rat angiogenèse Mésentère

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.

Des cellules endothéliales Tube Formation test pour la<em&gt; In Vitro</em&gt; Etude de l&#39;angiogenèse

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.

Un<em&gt; Ex vivo</em&gt; Système de culture à étudier la thyroïde développement

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.

La protéine c-FOS détection immunohistologique: Un outil utile comme marqueur des voies centrales impliquées dans les réponses physiologiques spécifiques<em&gt; In Vivo</em&gt; et<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.

Isolement des artérioles rétiniennes Ex Vivo études de physiologie cellulaire

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.

Un modèle de culture de tissu d'Ex Vivo du cartilage transformant dans les explants de genou de bovine

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.

Excroissance glomerulaire en tant qu’essai Ex Vivo pour analyser les voies impliquées dans l’activation des cellules épithéliales pariétales

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