The authors acknowledge the following funding sources, Whittier Foundation, Harrington Foundation, National Institutes of Health and Research to Prevent Blindness.

Tous les yeux énucléés ont été obtenus à partir d&#39;un abattoir et toutes les expériences ont été réalisées en conformité avec les lois institutionnelles en matière de biosécurité. <ol><li> Fixez œil énucléé sur une surface. Pour ce faire, en plaçant les repères de tissus à travers le tissu en excès autour de l&#39;œil et la sécurisation vers le bas dans un conseil de styromousse. </li><li> Disséquer et détachez la conjonctive de l&#39;œil périlimbique. <ol><li> Utilisez une pince fine (0,3 forceps) et microciseaux (ciseaux Westcott) à inciser la conjonctive au limbe et carrément disséquer au large de la sclérotique. Couper la conjonctive long du limbe que la dissection procède à la poursuite de la dissection. </li><li> Retirer conjonctive tout autour de l&#39;oeil (360 degrés) afin d&#39;exposer autant que possible sclérotique. NOTE: Il est utile de laisser une petite quantité de la conjonctive de faciliter l&#39;œil fixateur avec les broches chirurgicales ou une pince pendant le reste de la procédure. </li></ol></li><li> Créez un volet scléral pleine épaisseur le long d&#39;uncôté de l&#39;oeil (lame de scalpel 69). <ol><li> Faire un ~ 5 mm incision scléral parallèlement au limbe et 3 mm derrière le limbe en coupant doucement dans la sclérotique avec un scalpel (lame de scalpel 11) jusqu&#39;à ce que la choroïde pigmentation foncée est visible. Veillez à ne pas inciser la choroïde lui-même. </li><li> Disséquer soigneusement le long du plan entre la sclérotique et la choroïde, soit avec l&#39;aide de ciseaux (dissection) ou avec le scalpel, jusqu&#39;à ce qu&#39;il soit possible d&#39;agrandir l&#39;espace potentiel entre ces tissus en poussant doucement vers l&#39;intérieur de la choroïde. </li><li> Faire une autre incision sclérale perpendiculaire à la première incision sclérale avec microciseaux pointus, ce qui crée une incision en forme de T. </li><li> Ensuite, continuer à disséquer sans ménagement dans un mode périphérique pour enlever le tissu scléral de la choroïde sous-jacente et pousser doucement la choroïde loin de la sclérotique, comme mentionné ci-dessus. </li><li> Agrandir le volet scléral au besoin. <ol><li> Doucement Blunt disséquer la choroïde loin de la sclérotiquetout au long du processus. </li><li> Agrandir le rabat jusqu&#39;à ce que l&#39;incision pratiquée perpendiculairement au limbe atteint le nerf optique et l&#39;incision faite parallèle au limbe est au moins un tiers de la circonférence de l&#39;oeil (45 °). </li><li> Utiliser le volet scléral en tant que source d&#39;effet de levier pour manipuler l&#39;oeil lors de la dissection émoussé. Le contre-traction sur le rabat rend plus facile pour dissection. </li></ol></li><li> Répétez l&#39;étape précédente de l&#39;autre volet scléral. </li></ol></li><li> Coupez les volets scléral pour exposer une grande surface de la choroïde. </li><li> Continuer l&#39;incision pratiquée dans l&#39;étape 3 autour de la circonférence de l&#39;oeil (360 degrés). </li><li> Retirer le tissu choroïde-rétine restant. <ol><li> Dans la région exposée, utilisez un coton-tige pour brosser délicatement le tissu choroïde-rétine restant. Sinon, prenez doucement le tissu avec une pince et le retirer le corps vitré sous-jacent. </li><li> Si nécessaire faire une incision dans la choroïde stArting du nerf optique. Puis décoller délicatement la choroïde pour exposer la rétine et le cortex vitré. </li></ol></li><li> Continuer émoussée disséquer la choroïde loin de la sclérotique, puis décoller la choroïde d&#39;obtenir l&#39;ensemble, vitreux intacte. </li><li> Utilisez le rebord scléral attaché à la cornée afin de positionner l&#39;ensemble vitré à l&#39;endroit désiré. À ce stade, le corps vitré est fixée à la sclérotique antérieure et le cristallin. </li><li> Blunt disséquer le corps vitré de l&#39;intérieur de la sclère, autour de la lentille, en supprimant tous sclérotique. </li><li> Utilisez un outil contondant pour ramasser la lentille loin de le vitré si besoin est. Utilisez l&#39;échantillon pour diverses expériences vitreodynamic. <ol><li> Placer l&#39;échantillon dans un récipient en verre avec une surface connue exposée à l&#39;air. Placez l&#39;oxygène sonde sensible au bord de l&#39;échantillon. Utilisation d&#39;un micro-manipulateur pour déplacer la sonde à une distance connue (r) dans l&#39;échantillon. </li><li> Utiliser les lois de diffusion de Fick pour obtenir l&#39;équation de diffusion théorique. Avec le data recueilli à l&#39;étape 10.1, obtenir le terme coefficient de diffusion expérimentale / réaction, etc. </li></ol></li></ol>

<ol>
	<li>Siggers, J. H., Ethier, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+Mechanics+of+the+Eye.">Fluid Mechanics of the Eye.</a> Annual Review of Fluid Mechanics. 44 (1), 347-372 (2012).</li><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+human+vitreous+structure.">Age-related changes in human vitreous structure.</a> Graefes Arch Clin Exp Ophthalmol. 225 (2), 89-93 (1987).</li><li>Grignolo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrous+components+of+the+vitreous+body.">Fibrous components of the vitreous body.</a> AMA Arch Ophthalmol. 47 (6), 760-774 (1952).</li><li>Gisladottir, S., Loftsson, T., Stefansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+vitreous+humour+and+saline+solution+follow+the+Stokes+Einstein+equation.">Diffusion characteristics of vitreous humour and saline solution follow the Stokes Einstein equation.</a> G Graefes Arch Clin Exp Ophthalmol. 247 (12), 1677-1684 (2009).</li><li>Xu, J., Heys, J. J., Barocas, V. H., Randolph, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+diffusion+in+vitreous+humor:+implications+for+drug+delivery.">Permeability and diffusion in vitreous humor: implications for drug delivery.</a> Pharm Res. 17 (6), 664-669 (2000).</li><li>Watts, F., Tan, L. E., Wilson, C. G., Girkin, J. M., Tassieri, M., Wright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+micro-rheology+of+the+vitreous+humor+using+an+optically+trapped+local+probe.">Investigating the micro-rheology of the vitreous humor using an optically trapped local probe.</a> Journal of Optics. 16 (1), 015301 (2014).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+human+vitreous+body+elements+for+proteomic+analysis.">Dissection of human vitreous body elements for proteomic analysis.</a> J Vis Exp. (47), e2455 (2011).</li><li>Abdallah, W., Ameri, 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=Vitreal+oxygenation+in+retinal+ischemia+reperfusion.">Vitreal oxygenation in retinal ischemia reperfusion.</a> Invest Ophthalmol Vis Sci. 52 (2), 1035-1042 (2011).</li><li>Goldenberg, D., Trese, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+vitreodynamics:+what+is+it?+Why+is+it+important.">Pharmacologic vitreodynamics: what is it? Why is it important.</a> Expert Review of Ophthalmology. 3 (3), 273-277 (2008).</li><li>Choonara, Y. E., Pillay, V., Danckwerts, M. P., Carmichael, T. R., du Toit, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+implantable+intravitreal+drug+delivery+technologies+for+the+treatment+of+posterior+segment+eye+diseases.">A review of implantable intravitreal drug delivery technologies for the treatment of posterior segment eye diseases.</a> J Pharm Sci. 99 (5), 2219-2239 (2010).</li><li>Balachandran, R. K., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+drug+delivery+to+the+posterior+eye:+effect+of+active+transport+and+loss+to+choroidal+blood+flow.">Computer modeling of drug delivery to the posterior eye: effect of active transport and loss to choroidal blood flow.</a> Pharm Res. 25 (11), 2685-2696 (2008).</li><li>Smith, C. a., Newson, T. a., 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=A+framework+for+modeling+ocular+drug+transport+and+flow+through+the+eye+using+micro-CT.">A framework for modeling ocular drug transport and flow through the eye using micro-CT.</a> Phys Med Biol. 57 (19), 6295-6307 (2012).</li><li>Quiram, P. A., Leverenz, V. R., Baker, R. M., Dang, L., Giblin, F. J., Trese, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplasmin-induced+posterior+vitreous+detachment+affects+vitreous+oxygen+levels.">Microplasmin-induced posterior vitreous detachment affects vitreous oxygen levels.</a> Retina. 27 (8), 1090-1096 (2007).</li><li>Shui, Y., Holekamp, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+state+of+the+vitreous+and+ascorbate-dependent+oxygen+consumption:+relationship+to+the+etiology+of+nuclear+cataracts.">The gel state of the vitreous and ascorbate-dependent oxygen consumption: relationship to the etiology of nuclear cataracts.</a> Arch Ophthalmol. 127 (4), 475-482 (2009).</li><li>Burk, S. E., Da Mata, A. P., Snyder, M. E., Schneider, S., Osher, R. H., Cionni, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+vitreous+using+kenalog+suspension.">Visualizing vitreous using kenalog suspension.</a> J Cataract Refract Surg. 29 (4), 645-651 (2003).</li><li>Spaide, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Posterior+Vitreous+with+Dynamic+Focusing+and+Windowed+Averaging+Swept+Source+Optical+Coherence+Tomography.">Visualization of the Posterior Vitreous with Dynamic Focusing and Windowed Averaging Swept Source Optical Coherence Tomography.</a> Am J Ophthalmol. S0002-9394 (14), 00537-00536 (2014).</li><li>Domalpally, A., Gangaputra, S., Danis, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diseases of the Vitreo-Macular Interface. 21, 21-27 (2014).</li><li>Stocchino, R., Repetto, A., Cafferata, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+vitreous+humour+motion+within+a+human+eye+model.">Experimental investigation of vitreous humour motion within a human eye model.</a> Phys Med Biol. 50 (19), 4729-4743 (2005).</li></ol>

The authors have nothing to disclose.

Il ya deux étapes essentielles qui doivent être soigneusement exécutées lors de la dissection vitreux. Étape 3, la création d&#39;un volet scléral pleine épaisseur, est cruciale pour l&#39;ensemble de la dissection. Il faut prendre soin de ne pas couper dans la choroïde lors de la création de la pleine épaisseur volet scléral. L&#39;autre étape essentielle est loin disséquait la sclérotique de la choroïde. Cette étape doit être effectuée avec soin pour éviter de créer de multiples trous dans la chor...

Le but de cette méthode est de définir une technique pour isoler un ensemble de corps vitré, intact, avec le noyau et le cortex intact vitreux, à partir d&#39;un œil de cadavre, aux fins de l&#39;analyse vitreodynamic. Comme le domaine de la physiologie vitreux a augmenté, de chercheurs multidisciplinaires, telles que la mécanique des fluides chercheurs, enquêtent sur ​​les propriétés physiques et biomécaniques du corps vitré 1. À cette fin, il est essentiel de détail une technique pour isoler l&#39;ensemble, corps vitré intacte pour aider les chercheurs multidisciplinaires. Sebag et al. 2 et 3 autres dissections réalisées élégantes vitreux entiers sur des yeux de cadavres humains et ont montré des illustrations des résultats. Cependant, la technique utilisée n&#39;a pas été décrite en détail et non-experts ne serait pas capable de se répliquer indépendamment de la méthode. D&#39;autres études ont récolté vitré de yeux de cadavres à l&#39;aide de méthodes plus simples telles que l&#39;aspiration ou la dissection partielle,deux qui ne donnent pas, en tout un corps vitreux, intacte. Gisladottis et al. 4 et Xu et al. 5 enquêter perméabilité dans l&#39;humeur vitrée récoltées à partir de cadavres yeux. Cependant, étant donné qu&#39;aucune méthode d&#39;extraction vitreux a été décrit, on a supposé qu&#39;ils le corps vitré aspiré avec une seringue. Watts et al. 6 est allé plus loin en décrivant un procédé d&#39;isolement de lapin humeur vitrée avec une technique chirurgicale. Cependant, cette méthode conduit à un isolement de seulement le noyau vitreux et non le cortex vitréen. Skeie et al. 7 organisé plus tard le corps vitré en 4 régions uniques et élégamment décrit une méthode pour disséquer chaque partie pour analyse. Cette technique toutefois, ne donne pas lieu à un vitreux intact dans son ensemble. La technique actuelle a été développé pour faciliter les expériences biophysiques qui ne sont actuellement effectuées dans les yeux de cadavres. Les méthodes antérieures, comme décrit uneBove, sont limitées parce que 1) aucune isoler complètement l&#39;ensemble du corps vitré, noyau vitreux 2) récoltés et le cortex sont homogénéisés, 3) structure anatomique vitré est pas maintenue, ou 4) techniques de dissection ne sont pas suffisamment détaillées pour la réplication par les chercheurs dans d&#39;autres domaines . En outre, en raison de l&#39;opacité de la sclérotique et la choroïde, la visualisation du corps vitré est limitée dans le globe oculaire intact. Ceci limite la précision et la faisabilité de mesures qui peuvent être faites à l&#39;intérieur de l&#39;œil entier. En outre, les structures anatomiques environnantes du vitré peuvent confondre l&#39;étude des propriétés physiques et biochimiques du corps vitré. Au cours des dernières années, le corps de la science vitreux a considérablement augmenté et il ya des raisons de croire que l&#39;ensemble du corps vitré a des propriétés différentes de celles de ses parties individuelles. Il ya un intérêt croissant dans les enquêtes sur les propriétés physiques, biomécaniques et chimiques du corps vitré pour vitreodynamics research, ce qui a des applications en médecine clinique, tels que l&#39;administration de médicaments, oxygénation intravitréenne 8 et vitrectomie. Vitreodynamics pharmacologiques, qui utilise des agents pharmacologiques pour manipuler le corps vitré, peuvent être utilisés pour améliorer les résultats de vitrectomie 9. Propriétés biomécaniques sont utilisés pour modéliser l&#39;écoulement de fluide vitré, qui peut être utilisé pour améliorer les technologies d&#39;administration de médicaments par voie intravitréenne 10-12. Les propriétés physiques des différents segments du corps vitré sont cruciales pour comprendre vitréo-rétinienne transport de l&#39;oxygène 13. La technique proposée de dissection vitreux peut être utilisée pour étudier les différentes propriétés de l&#39;humeur vitrée intacte. Il permet de paillasse expériences à faire sur les corps vitreux, entières intactes avec une meilleure visualisation. En résumé, les méthodes actuelles pour l&#39;étude du corps vitré soit ne sont pas décrits de manière adéquate, ou aboutissent à un isolement incomplète du noyau vitreux et le cortex. Par conséquent, il est nécessaire d&#39;effectuer eXperiments dans un modèle oculaire transparent tout en conservant l&#39;anatomie du corps vitré qui existe dans l&#39;œil de cadavre.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.3 forceps</td>
			<td>Storz Opthalmics</td>
			<td>E1793</td>
			<td></td>
		</tr>
		<tr>
			<td>Westcott Tenotomy Scissors Curved Right</td>
			<td>Storz Opthalmics</td>
			<td>E3320 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle No. 3</td>
			<td>VWR</td>
			<td>25607-947</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade, #11, for #3 Handle</td>
			<td>VWR</td>
			<td>470174-844</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole vitreous humor dissection for vitreodynamic analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

En suivant le protocole conduira à une dissection vitreux réussie avec le noyau et le cortex (Figure 3) intact. Ceci est évident à partir des morceaux résiduels de rétine adhéré au cortex vitréen. Vitré toute Intact peut être utilisé de plusieurs façons pour les expériences vitreodynamic spécifiques. Dans notre cas, le taux de diffusion de l&#39;oxygène dans le corps vitré intacte et il est la constante de temps correspondant a été étudiée (Figure 2). Vitreux qui a ...

Watch this Scientific Journal Video about Whole Vitreous Humor Dissection for Vitreodynamic Analysis at JoVE.com

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Whole Vitré Dissection d&#39;analyse Vitreodynamic

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

whole-vitreous-humor-dissection-for-vitreodynamic-analysis

Research

JoVE Journal

Medicine

11.3K vues.  University of Southern California. The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Vidéo: Whole Vitreous Humor Dissection for Vitreodynamic Analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Dissection de l&#39;homme des éléments du corps vitré de l&#39;analyse protéomique

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Recherche

Médecine

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocole d&#39;hyperthermie Durée du corps entier long chez la souris

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D entier coeur Myocardial analyse des tissus

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissection du pancréas de souris pour faire une analyse histologique et profilage métabolique

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

Dissection et la Culture Explant allantoïde murin pour l’analyse In Vitro de l’attachement allantoïdien

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Ensemble-cerveau Segmentation et analyse des points de changement d’IRM anatomique cérébrale — Application dans Premanifest la maladie de Huntington

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &amp;#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Analyse quantitative de Micro-CT de Aortopathy dans un modèle murin d’anévrisme de l’aorte induite par la β-aminopropionitrile et Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

Imagerie et analyse de lésions de l’aorte entière o-colorée à l’huile dans un modèle murin d’hyperlipidémie d’anévrisme

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

Emplacement, dissection et analyse du ganglion radié murin

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

Méthode non invasive pour générer le modèle de lésion cartilagineuse intra-articulaire induite par la charge cyclique du genou du rat

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Methods Article

Whole Vitré Dissection d'analyse Vitreodynamic