XHTW est un WM Keck Foundation Distinguished Scholar jeunes en recherche médicale, et est également soutenu par le NIH / NHLBI subventions R01 et R01-HL089598-R01HL091947 et Muscular Dystrophy Association subvention no 69238. RJvO est le récipiendaire de la bourse américaine 2008-2010 Physiological Society postdoctorale en génomique physiologique. Ce travail est également soutenu en partie par l&#39;Alliance pour la Fondation Leducq CaMKII Signalisation Cœur.

Partie 1: Préparation du champ opératoire <ol><li> Le champ opératoire est désinfecté avec de l&#39;alcool isopropylique à 75%. </li><li> Assurez-vous que le coussin chauffant est en marche et à la bonne température. Un système recommandé est une pompe à eau circulant Gaymar connecté à un pad thérapie qui est maintenue à 37 ° C ± 1 ° C. Il est important de maintenir la température normale du corps pendant la chirurgie pour éviter une diminution rapide de la fréquence cardiaque. </li><li> Outils chirurgicaux sont stérilisés dans un stérilisateur à billes chaudes avant la chirurgie. Pour cette procédure, vous aurez besoin des instruments chirurgicaux suivants: ciseaux émoussés, bien sûr courbes pince X 2, fin 45 ° pince à angle X 2, ciseaux à ressort coudé, écarteur thoracique et porte-aiguille. </li><li> Applicateurs de coton devrait être à portée de main en cas de saignement. </li></ol> Partie 2: Préparation et l&#39;intubation des souris <ol><li> Les souris sont anesthésiées dans une chambre d&#39;induction avec 2% d&#39;isoflurane mélangé avec 0,5 à 1,0 L / min à 100% d&#39;O 2. </li><li> Tondeuses à cheveux sont utilisés pour raser le poil de l&#39;encolure au niveau de la poitrine mi. </li><li> La souris est placée dans une position couchée au sommet d&#39;un coussin chauffant afin de maintenir la température du corps. </li><li> Une bande de caoutchouc est placé sur les dents de devant de l&#39;animal de proroger le cou. En utilisant des pinces courbes dans une main, la langue est délicatement manipulé sur le côté. Avec l&#39;autre main, l&#39;intubation endotrachéale est réalisée en utilisant PE 90 tubes. Le tube endotrachéal est ensuite relié à un volume de Harvard-vélo vélo ventilateur rongeurs à 125-150 respirations / minute et un volume courant de 0,1-0.3ml. Lors de la procédure chirurgicale, l&#39;anesthésie est maintenue à 1,5-2% d&#39;isoflurane à 0,5 - 1,0 L / min à 100% d&#39;O 2. Niveau correct de l&#39;anesthésie est vérifiée en appliquant une pression sur le lit de l&#39;ongle de la souris (tep-pincement réflexe). </li><li> Le champ opératoire est désinfecté avec une solution Betadine suivie par 70% d&#39;alcool. Cette procédure est répétée trois fois. </li><li> Pour éviter la contamination du champ opératoire pendant l&#39;opération, d&#39;un champ stérile est placé sur la souris ne laissant que le champ d&#39;opération exposés. </li><li> Une paire de gants stériles sont utilisés pour chaque souris individuelle. </li></ol> Partie 3: La ligature de l&#39;aorte transverse <ol><li> Thoracotomie partielle de la deuxième côte est réalisée sous un microscope chirurgical et le sternum rétracté à l&#39;aide d&#39;un écarteur thoracique. </li><li> Pointe fine pince à angle de 45 ° sont utilisés pour séparer les tissus délicatement le thymus et la graisse de la crosse aortique. </li><li> Après identification de l&#39;aorte transverse, un petit morceau d&#39;une suture de soie 6.0 est placé entre les artères innominée et la carotide gauche (figure 1). </li><li> Deux nœuds lâches sont attachés autour de l&#39;aorte transverse et un petit morceau d&#39;une aiguille de calibre 27 ½ émoussée est placée parallèlement à l&#39;aorte transverse. Le premier nœud est rapidement lié contre l&#39;aiguille, suivie par la deuxième et l&#39;aiguille rapidement retirés afin d&#39;obtenir une constriction des 0.4mm de diamètre. Chez les souris de contrôle imposture, toute la procédure est identique sauf pour la ligature de l&#39;aorte. </li><li> L&#39;écarteur thoracique est enlevé et la sortie de l&#39;arrêt ventilateur pincé pendant 2 secondes pour regonfler les poumons. </li><li> La cage thoracique est fermée à l&#39;aide d&#39;une suture Prolène 6.0 avec un modèle de suture interrompue. </li><li> La peau est fermée à l&#39;aide d&#39;une suture Prolène 6.0 avec un modèle de surjet. </li></ol> Partie 4: récupération post-opératoire <ol><li> Pour l&#39;analgésie postopératoire, la souris est injecté avec la buprénorphine (0,1 mg / kg) par voie intrapéritonéale. S&#39;il ya des signes de déshydratation après une chirurgie, une solution saline stérile est donné par voie intrapéritonéale. </li><li> L&#39;anesthésie est progressivement abaissé à la position OFF et la sonde endotrachéale retiré lorsque des signes de respiration spontanée se produire. </li><li> La souris est déplacé à la position couchée et a permis de récupérer sur un coussin chauffant. </li></ol> Partie 5: Confirmation de la ligature réussie de l&#39;aorte transverse <ol><li> Une semaine après le TAC, la souris est à nouveau anesthésié afin de déterminer le degré de surcharge de pression induite par ligature de l&#39;aorte transverse. Comme décrit ci-dessus, l&#39;anesthésie est maintenue à 1,5-2% d&#39;isoflurane à 0,5 - 1,0 L / min à 100% d&#39;O 2 et la température corporelle à 37 ° C ± 1 ° C. </li><li> A 20 MHz Doppler sonde est placée sur les côtés gauche et droit du cou à un angle de 45 ° afin de détecter les vitesses d&#39;écoulement. Un ordinateur basé sur le processeur de signal Doppler (Indus Instruments, Houston, TX) a été utilisé pour afficher et de stocker des signaux Doppler. 5 </li><li> Selon le degré de surcharge de pression nécessaire pour que le protocole expérimental, les souris seulement avec un carotide droite (CR) / carotide gauche (LC) rapport de débit dans une certaine plage sont inclus pour analyse ultérieure. Par exemple, un degré modéré de surcharge de pression conduit à un ratio de 5-8, alors qu&#39;un resultin serré la constrictiong dans la surcharge de pression sévères conduit à un ratio de 8-10 (Figure 5.3a). Un animal factice (mais pas exploités ligaturé), cependant, on s&#39;attend à avoir un ratio de ~ 1 (figure 5.3B). </li></ol> 6: Résultats Représentant La survie typiques chirurgicales suivantes TAC souris de type sauvage est d&#39;environ 80-90%. Succès ligature chirurgicale de l&#39;aorte transverse mènera à un ratio Doppler vitesse d&#39;écoulement entre l&#39;artère carotide droite et à gauche (RC / LC) de 5-10 (figure 1). Comparé à une opération fictive souris (figure 2A), souris avec la surcharge de pression sont appelés à développer une hypertrophie cardiaque dans les 1-2 semaines, et la dilatation cardiaque après 6-8 semaines, selon l&#39;étanchéité de la constriction (figure 2B). La mortalité durant le premier mois après TAC est généralement faible (&lt;20%) dans les souris de type sauvage, bien que les souris génétiquement modifiées peuvent présenter différents taux de survie 6,7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1729/1729fig1.jpg"><img src="/files/ftp_upload/1729/1729fig1tmb.jpg" alt="Figure 1" border="0" /></a> Figure 1. Confirmation de la ligature des aorte transverse en utilisant la mesure de vitesse Doppler. Signaux représentant la vitesse Doppler de la droite (RC) et carotide gauche (LC) artères 6 jours après l&#39;ATC. Ces enregistrements montrent une ligature réussie, avec un ratio de débit RC / LC de ~ 6,8. <img src="/files/ftp_upload/1729/1729fig2.jpg" alt="Figure 2" /> Figure 2. Représentant Images cœur Après TAC. A, B. Sham-exploité cœur sauvage de souris de type.. Sauvage coeur de souris de type 16 semaines post-TAC. Chaque ligne = 1mm.

<ol>
	<li>Rockman, H. A., Ross, R. S., Harris, A. N., Knowlton, K. U., Steinhelper, M. E., Field, L. J., Ross, J., Chein, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proc. Natl. Acad. Sci. USA. 88, 8277-8281 (1991).</li><li>Heineke, J., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiac+hypertrophy+by+intracellular+signaling+pathways.">Regulation of cardiac hypertrophy by intracellular signaling pathways.</a> Nat. Rev. Mol. Cell. Biol. 7, 589-600 (2006).</li><li>Hartley, C. J., Reddy, A. K., Madala, S., Michael, L. H., Entman, M. L., Taffett, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+estimation+of+reduced+coronary+flow+reserve+in+mice+with+pressure+overload+cardiac+hypertrophy.">Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.</a> Ultrasound Med. Biol. 34, 892-901 (2008).</li><li>Reddy, A. K., Taffett, G. E., Li, Y. -. H., Lim, S. -. W., Pham, T. T., Pocius, J. S., Entman, M. L., Michael, L. H., Hartley, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+signal+processing+for+use+in+mice:+applications.">Doppler signal processing for use in mice: applications.</a> IEEE Trans. Biomed. Eng. 52, 1771-1783 (2005).</li><li>Li, Y. -. H., Reddy, A. K., Ochoa, L. N., Pham, T. T., Hartley, C. J., Micheal, L. H., Entman, M. L., Taffett, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+age+on+peripheral+vascular+response+to+transverse+aortic+banding+in+mice.">Effect of age on peripheral vascular response to transverse aortic banding in mice.</a> J. Gerontol. 58A, 895-899 (2003).</li><li>Bourajjaj, M., Armand, A. S., Martins, d. a. C. o. s. t. a., Weijts, P. A., Nagel, B. V. a. n. d. e. r., Heeneman, R., Wehrens, S., Windt, X. H. D. e., J, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFATc2+is+a+necessary+mediator+of+calcineurin-dependent+cardiac+hypertrophy+and+heart+failure.">NFATc2 is a necessary mediator of calcineurin-dependent cardiac hypertrophy and heart failure.</a> J. Biol. Chem. 28, 22295-22303 (2008).</li><li>Oort, R. J. v. a. n., Respress, J. L., Li, N., Reynolds, C., Almeida, A. C. D. e., Skapura, D. G., Windt, L. J. D. e., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+development+of+pressure+overload+induced+cardiac+hypertrophy+and+dysfunction+in+an+RyR2-R176Q+knockin+mouse+model.">Accelerated development of pressure overload induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model.</a> Hypertension. 55, (2010).</li><li>Tarnavski, O., McMullen, J. R., Schinke, M., Nie, Q., Kong, S., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cardiac+surgery:+comprehensive+techniques+for+the+generation+of+mouse+models+of+human+diseases+and+their+application+for+genomic+studies.">Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies.</a> Physiol Genomics. 16, 349-360 (2004).</li></ol>

TAC, qui imite une sténose aortique humaine, est une méthode commune pour induire une hypertrophie cardiaque et l&#39;insuffisance cardiaque chez la souris. Sites alternatifs pour la constriction aortique comprennent l&#39;aorte ascendante et de l&#39;abdomen. Croissant constriction aortique fournit une surcharge extrême et plus rapide sur le ventricule gauche (VG). En revanche, la constriction aortique abdominal laisse intacte une grande partie de la circulation comme moyen de compensation possible. 8 C&#39;est pourquoi, le TAC est souvent le modèle préféré, car il prévoit une surcharge LV adéquates d&#39;une façon dépendant du temps plus favorable à l&#39;enquête. Bien que cette procédure peut être techniquement difficile, avec la pratique, nous avons atteint un taux de survie de 80-90% chez les souris de type sauvage. L&#39;étanchéité de la constriction aortique transversale détermine le degré de développement de l&#39;hypertrophie et délais dans lesquels une insuffisance cardiaque et une dilatation se développe. Par ailleurs, l&#39;âge de la souris influe sur le taux de récupération et de la cinétique de développement d&#39;insuffisance cardiaque. Souris plus âgées (&gt; 12 mois d&#39;âge) prendre plus de temps pour développer une réponse adaptative des artères carotides au TAC, et sont susceptibles de développer une cardiomyopathie dilatée plus vite que les jeunes souris (3-4 mois) 5. Non-invasive intubation de souris prend une certaine pratique. Une méthode alternative est la suivante: <ol><li> Une petite incision médiane du col utérin est faite avec des ciseaux émoussés et les tissus autour de la trachée, séparés en utilisant une pince incurvée afin d&#39;exposer la trachée. </li><li> Un morceau de fil de suture est placée autour de la dent avant et doucement tiré loin du corps de l&#39;animal à étendre la nuque. </li><li> En utilisant des pinces courbes dans une main, la langue est délicatement manipulé sur le côté. </li><li> Avec l&#39;autre main, l&#39;intubation endotrachéale est réalisée en utilisant PE 90 tube biseauté sur le bord de la facilité d&#39;entrée. </li></ol> Le tube endotrachéal est ensuite relié à un volume de Harvard-vélo vélo ventilateur rongeurs à 125-150 respirations / minute et un volume courant de 0,1-0.3ml.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gaymar T/pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Model TP-500</td>
<td>Circulating water pump</td>
</tr>
<tr>
<td>Temp. therapy pads</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>60-3414</td>
<td>&nbsp;</td>
<tr>
<td>Hot bead sterilizer</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Blunt scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5980</td>
<td>&nbsp;</td>
<tr>
<td>Angled spring scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5668</td>
<td>&nbsp;</td>
<tr>
<td>Chest retractor</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>17002-02</td>
<td>&nbsp;</td>
<tr>
<td>Course curved forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5138</td>
<td>&nbsp;</td>
<tr>
<td>Dumont fine 45&deg; angled forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11253-25</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12565-14</td>
<td>&nbsp;</td>
<tr>
<td>Electric shaver</td>
<td>&nbsp;</td>
<td>General Electric</td>
<td>&nbsp;</td>
<td>For shaving mouse fur</td>
</tr>
<tr>
<td>PE 90 tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427420</td>
<td>For intubation</td>
</tr>
<tr>
<td>MiniVent (ventilator)</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Type 845</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19-027132</td>
<td>May be purchased at medical supply store</td>
</tr>
<tr>
<td>Sterile gloves</td>
<td>&nbsp;</td>
<td>Mckesson</td>
<td>20-1565</td>
<td>&nbsp;</td>
<tr>
<td>Stereo microscope</td>
<td>&nbsp;</td>
<td>Unico</td>
<td>ZM186</td>
<td>&nbsp;</td>
<tr>
<td>6-0 silk suture</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18020-60</td>
<td>&nbsp;</td>
<tr>
<td>27 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>6-0 prolene suture</td>
<td>&nbsp;</td>
<td>Mckesson</td>
<td>3286</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Institutional Animal Facility</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transverse aortic constriction in mice

Constriction aortique transverse (TAC) chez la souris est un modèle couramment utilisé pour la pression de surcharge expérimentale induite par l&#39;hypertrophie cardiaque et l&#39;insuffisance cardiaque.<sup&gt; 1</sup&gt; TAC conduit d&#39;abord à l&#39;hypertrophie compensée du cœur, qui est souvent associée à un accroissement temporaire de la contractilité cardiaque. Au fil du temps, cependant, la réponse à la surcharge chronique hémodynamique devient inadapté, entraînant une dilatation cardiaque et insuffisance cardiaque.<sup&gt; 2</sup&gt; Le modèle murin TAC a été d&#39;abord validé par Rockman<em&gt; Et al</em&gt;.<sup&gt; 1</sup&gt;, Et a depuis été largement utilisé comme un outil précieux pour imiter humaine des maladies cardiovasculaires et d&#39;élucider les processus fondamentaux de signalisation impliquées dans la réponse hypertrophique cardiaque et le développement d&#39;insuffisance cardiaque. Lorsque comparé à d&#39;autres modèles expérimentaux d&#39;insuffisance cardiaque, comme l&#39;occlusion complète de la descendante antérieure gauche (DAL) des artères coronaires, l&#39;ATC fournit un modèle plus reproductible de l&#39;hypertrophie cardiaque et un parcours de plus de temps dans le développement progressif d&#39;une insuffisance cardiaque. Ici, nous décrivons une procédure étape par étape pour effectuer chirurgicale TAC chez les souris. Pour déterminer le niveau de surcharge de pression produite par la ligature de l&#39;aorte, une haute fréquence Doppler sonde est utilisée pour mesurer le rapport entre les vitesses d&#39;écoulement de sang dans les artères carotides droite et gauche.<sup&gt; 3, 4</sup&gt; Avec les taux de survie de chirurgie de 80-90%, l&#39;aorte transverse baguage est une technique efficace d&#39;induire une hypertrophie ventriculaire gauche et une insuffisance cardiaque chez la souris.

Watch this Scientific Journal Video about Transverse Aortic Constriction in Mice at JoVE.com

Transverse Aortic Constriction in Mice

Constriction aortique transverse (TAC) chez la souris est un modèle couramment utilisé expérimental pour étudier les mécanismes qui sous-tendent l&#39;hypertrophie cardiaque et le développement d&#39;insuffisance cardiaque. Ici, nous décrivons les procédures de la constriction de l&#39;aorte afin de créer un diplôme reproductible de l&#39;hypertrophie cardiaque chez la souris.

Constriction aortique transverse chez la souris

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Nanyang Technological University

Research

JoVE Journal

Biology

66.7K vues.  Baylor College of Medicine (BCM). Constriction aortique transverse (TAC) chez la souris est un modèle couramment utilisé expérimental pour étudier les mécanismes qui sous-tendent l'hypertrophie cardiaque et le développement d'insuffisance cardiaque. Ici, nous décrivons les procédures de la constriction de l'aorte afin de créer un diplôme reproductible de l'hypertrophie cardiaque chez la souris.

Vidéo: Transverse Aortic Constriction in Mice

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Transplantation de l&#39;intestin chez les souris

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Recherche

Biologie

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Assay anneau aortique

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Ambulatoires enregistrement de l&#39;ECG chez des souris

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Les injections intraveineuses de souriceaux nouveau-nés

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

Induction de l'échec ventriculaire droit par constriction pulmonaire d'artère et évaluation de la fonction ventriculaire droite chez les souris

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

Induire la parodontite apicale chez les souris

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture transversale du fémur de la souris avec broche stabilisatrice

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Une technique modifiée pour la constriction aortique transversale chez la souris

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

Modèle d’anévrisme de l’aorte abdominale de souris induit par le phosphate de calcium

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...