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

Alle Augen enukleiert wurden von einem Schlachthaus erhalten, und alle Experimente wurden in Übereinstimmung mit institutionellen Biosicherheit Gesetze durchgeführt. <ol><li> Sichere entkernte Auge nach unten auf eine Oberfläche. Tun Sie dies, indem Gewebestifte durch das überschüssige Gewebe um das Auge herum und befestigen Sie sie nach unten in eine Styroporplatte. </li><li> Sezieren und nehmen perilimbal Bindehaut aus dem Auge. <ol><li> Verwenden feinen Pinzette (0.3 Pinzette) und Mikroschere (Westcott Schere), die Bindehaut am Limbus einzuschneiden und stumpf sezieren von der Lederhaut. Schneiden Sie die Bindehaut entlang des Limbus, wie der stumpfe Präparation schreitet weiter Dissektion ermöglichen. </li><li> Entfernen Binde ganzen Weg rund um das Auge (360 Grad), um so viel wie möglich Sklera freizulegen. HINWEIS: Es ist hilfreich, eine kleine Menge von Bindehaut zu verlassen, um zu erleichtern Fixierung des Auges mit den chirurgischen Stiften oder einer Pinzette während der Rest des Verfahrens. </li></ol></li><li> Erstellen Sie eine volle Dicke Skleralappen entlang einerSeite des Auges (Skalpellklinge 69). <ol><li> Einen ~ 5 mm Skleraeinschnitt parallel zum Limbus und 3 mm hinter dem Limbus durch leichtes Einschneiden der Sklera mit dem Skalpell (Skalpell 11) bis dunkel pigmentierten Chorioidea sichtbar. Achten Sie darauf, die Aderhaut selbst einzuschneiden. </li><li> Sorgfältig seziert entlang der Ebene zwischen der Sklera und der Choroidea, entweder mit einer Schere (unter Verwendung stumpf) oder mit dem Skalpell, bis es möglich ist, das Potential Raum zwischen diesen Geweben zu vergrößern, indem der Choroidea sanft nach innen. </li><li> Eine weitere Skleraeinschnitt senkrecht zur ersten Skleraeinschnitt mit scharfen Mikroscheren, wodurch eine T-förmige Einschnitt. </li><li> Dann weiterhin unverblümt sezieren in Umfangs Mode, um die Skleragewebe von der darunter liegenden Aderhaut zu entfernen, und schieben Sie die Aderhaut weg von der Lederhaut, wie oben erwähnt. </li><li> Vergrößern Sie die Lederhautlappen nach Bedarf. <ol><li> Sanft leckersten Blunt der Aderhaut weg von der Lederhaut zu sezierenwährend des gesamten Prozesses. </li><li> Vergrößern der Klappe bis der Schnitt senkrecht zu dem Limbus vorgenommen erreicht den Sehnerv und der Einschnitt parallel zum Limbus ist zumindest ein Drittel des Auges Umfangs (45 °). </li><li> Verwenden Sie die Lederhautlappen als Quelle der Hebelwirkung für die Manipulation des Auges während stumpf. Die Gegen Traktion auf der Klappe erleichtert es stumpf. </li></ol></li><li> Wiederholen Sie diesen Schritt auf der anderen Lederhautlappens. </li></ol></li><li> Schneiden Sie die Lederhautlappen um einen großen Bereich der Aderhaut freizulegen. </li><li> Weiterhin den Einschnitt in Schritt 3, die um das Auge des Umfangs (360 Grad) aus. </li><li> Entfernen Sie die verbleibenden Aderhaut-Retina-Gewebe. <ol><li> Innerhalb der freigelegten Bereich, mit einem Watte-Spitze vorsichtig abbürsten die verbleibende Aderhaut-Retina-Gewebe. Alternativ sanft greifen das Gewebe mit einer Pinzette und abziehen der darunter liegenden Glaskörper. </li><li> Falls erforderlich einen Einschnitt in die Aderhaut starting vom Sehnerven. Dann abziehen der Aderhaut sanft, um die Netzhaut und Glaskörper freizulegen. </li></ol></li><li> Weiter stumpfes Sezieren der Aderhaut weg von der Lederhaut und dann abziehen der Aderhaut, die ganze, intakte Glaskörper zu erhalten. </li><li> Verwenden Sie die Sklerarand an der Hornhaut angebracht, um die gesamte Glaskörper in gewünschten Stelle zu positionieren. An diesem Punkt wird der Glaskörper an der vorderen Sklera und Objektiv. </li><li> Blunt den Glaskörper von der Innenseite der Sklera zu zergliedern, um die Linse, Entfernen aller Sklera. </li><li> Verwenden Sie ein stumpfes Werkzeug, um die Linse von der Glaskörper schöpfen, wenn es sein muss. Verwenden Sie die Probe für verschiedene vitreodynamic Experimente. <ol><li> Man gibt die Probe in einem Becherglas mit bekannter Oberfläche der Luft ausgesetzt. Platzieren Sauerstoff empfindliche Sonde an der Kante der Probe. Verwenden Sie einen Mikromanipulator, um die Sonde zu einem bekannten Abstand (r) in die Probe zu bewegen. </li><li> Verwenden Fickschen Gesetzen der Diffusion, die theoretische Diffusionsgleichung erhalten. Mit dem data in Schritt 10.1 gesammelt, erhalten den experimentellen Diffusionskoeffizient / Reaktionszeit usw. </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.

Es gibt zwei wichtige Schritte, die sorgfältig im Glaskörper Dissektion durchgeführt werden müssen. Schritt 3, der eine gesamte Dicke Lederhautlappen, ist von entscheidender Bedeutung für die gesamte Sektion. Achten Sie darauf, nicht in die Aderhaut schneiden bei der Erstellung der gesamten Dicke Lederhautlappen werden. Die andere wichtige Schritt ist Sezieren entfernt die Lederhaut von der Aderhaut. Dieser Schritt muss sorgfältig durchgeführt werden Erstellen mehrerer Löcher in der Aderhaut, aus der die Glaskö...

Das Ziel dieser Methode ist es, detaillierter eine Technik, um eine ganze, intakte Glaskörper zu isolieren, mit dem glasartigen Kern und Cortex intakt von einem kadaver Auges, für die Zwecke der vitreodynamic Analyse. Da der Bereich der Glaskörper Physiologie hat, multidisziplinären Forscher, wie der Strömungsmechanik Forscher geworden, untersuchen die physikalischen und biomechanischen Eigenschaften des Glaskörpers 1. Zu diesem Zweck ist es wesentlich, detailliert eine Technik, die ganze, intakte Glaskörper zu isolieren, um multidisziplinäre Forscher unterstützen. Sebag et al. 2 und 3 durchgeführt, andere elegant ganze Glaskörper Dissektionen auf menschliche kadaver Augen und zeigte Darstellungen der Ergebnisse. Jedoch wurde die Technik verwendet, nicht im Detail, und Nicht-Experten beschrieben nicht in der Lage ist, das Verfahren unabhängig replizieren. Andere Studien haben Glaskörper aus kadaver Augen mit einfacheren Methoden wie Aspiration oder Teilzerlegung geerntet,von denen beide nicht in eine ganze, intakte Glaskörper führen. Gisladottis et al. 4 und Xu et al. 5 zu untersuchen Lässigkeit in Glaskörper aus kadaver Augen geerntet. Da jedoch kein Verfahren aus glasartigem Extraktion wurde beschrieben, wurde angenommen, daß sie die Glaskörperflüssigkeit mit einer Spritze abgesaugt. Watts et al. 6 ging noch einen Schritt weiter, indem ein Verfahren zur Isolierung Kaninchen Glaskörper mit einem Operationstechnik beschreibt. Führt dieses Verfahren jedoch in einer Isolation von nur dem Glaskern und dem Glaskörper nicht. Skeie et al. 7 später organisierte den Glaskörper in 4 einzigartigen Regionen und ein Verfahren beschrieben, um jeden Teil zur Analyse sezieren elegant. Diese Technik jedoch nicht in einer intakten Glaskörper als Ganzes führen. Die aktuelle Technik wurde entwickelt, um die biophysikalischen Experimenten, die derzeit nur in kadaver Augen durchgeführt werden, zu erleichtern. Früheren Verfahren, beschrieben alsBove, sind begrenzt, da 1) keine die gesamte Glaskörper vollständig zu isolieren, 2) geerntet glasigen Kern und Cortex homogenisiert, 3) glasartigen anatomischen Struktur nicht beibehalten wird, oder 4) Dissektion Techniken sind nicht ausreichend für die Replikation von Forschern in anderen Bereichen detailliert . Darüber hinaus aufgrund der Opazität des Sklera und Aderhaut, die Visualisierung des Glaskörpers in der intakten Augapfels beschränkt. Dies begrenzt die Genauigkeit und Realisierbarkeit von Messungen, die innerhalb des ganzen Auges gemacht werden können. Zusätzlich können die anatomischen Strukturen umgebenden Glaskörper das Studium der biochemischen und physikalischen Eigenschaften der glasartigen verwechseln. In den letzten Jahren hat der Körper aus glasartigem Wissenschaft enorm gewachsen und es gibt Grund zu glauben, dass die gesamte Glaskörper hat andere Eigenschaften als ihre Einzelteile. Es gibt ein wachsendes Interesse bei der Untersuchung der physikalischen, biomechanische und chemischen Eigenschaften des Glaskörpers für vitreodynamics research, die Anwendungen in der klinischen Medizin, wie Arzneimittelabgabe, intravitreale Sauerstoffversorgung 8 und Vitrektomie hat. Pharmakologische vitreodynamics, das pharmakologische Mittel verwendet, um den Glaskörper zu manipulieren, können zur Vitrektomie Ergebnisse 9 zu verbessern. Biomechanischen Eigenschaften verwendet werden, um glasartige Fluidströmung, die verwendet werden können zur Verbesserung der intravitrealen Darreichungsformen 10-12 modellieren. Die physikalischen Eigenschaften der verschiedenen Segmente des Glaskörpers sind entscheidend für das Verständnis vitreoretinalen Sauerstofftransport 13. Die vorgeschlagene glasigen Dissektionstechnik verwendet, um verschiedene Eigenschaften des intakten Glaskörper zu untersuchen. Es ermöglicht Tischexperimente auf ganze, intakte Glaskörper mit besseren Visualisierung erfolgen. Zusammenfassend aktuellen Methoden zur Untersuchung des Glaskörpers sind entweder nicht hinreichend beschrieben, oder führen zu einer unvollständigen Trennung der Glaskern und Kortex. Daher besteht ein Bedarf, e auszuführenXperiments in einem transparenten Augenmodell unter Beibehaltung der Anatomie des Glaskörpers, die im kadaver Auge vorliegt.

<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.

Anlehnung an das Protokoll zu einem erfolgreichen glasigen Dissektion mit dem Kern und Cortex (Abbildung 3) intakt zu führen. Dies ergibt sich aus den Reststücken Retina in den Glas cortex haftet. Intakte ganze Glaskörper kann auf verschiedene Weise für spezifische vitreodynamic Experimente eingesetzt werden. In unserem Fall ist die Diffusionsgeschwindigkeit von Sauerstoff in intakten Glaskörper und entsprechende Zeitkonstante wurde (Figur 2) untersucht. Glaskörper, die präparier...

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.

Ganze Glaskörper Dissection für Vitreodynamic Analysis

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 Aufrufe.  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.

Serie: 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 of Human Glaskörper Elemente für Proteomanalyse

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

Forschung

Medizin

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.

Protokoll für Long Duration Ganzkörper-Hyperthermie in Mäuse

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 Whole-Herz Myocardial Gewebeanalyse

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.

Dissektion der Maus Bauchspeicheldrüse für histologische Analyse und 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 ...

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.

Dissektion und modellabhängigen Kultur der murinen Allantois zur In-vitro- Analyse der Viruswenig Anlage

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.

Gesamte Gehirn Segmentierung und Change-Point-Analyse der anatomischen Gehirn MRI-Anwendung in Prämanifesten Huntington-Krankheit

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.

Quantitative Micro-CT-Analyse der Aortopathy in einem Mausmodell der β-Aminopropionitrile-induzierte Aortenaneurysma und Dissektion

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.

Bildgebung und Analyse von ölrot-o-gefärbten ganzen Aortenläsionen in einem Aneurysma-Hyperlipidämie-Mausmodell

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.

Lage, Dissektion und Analyse des Murin-Stellat-Ganglions

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.

Eine nicht-invasive Methode zur Erzeugung des zyklischen Belastungsinduzierten intraartikulären Knorpelläsionsmodells des Rattenknies

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