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

Tutti gli occhi enucleati sono stati ottenuti da un macello e tutti gli esperimenti sono stati eseguiti in conformità con le leggi biosicurezza istituzionali. <ol><li> Fissare occhio enucleato in giù su una superficie. A tale scopo, mettendo i perni dei tessuti attraverso il tessuto in eccesso intorno all&#39;occhio e fissandolo in giù in una tavola di polistirolo. </li><li> Staccare e staccare congiuntiva perilimbare dall&#39;occhio. <ol><li> Utilizzare una pinza sottile (0,3) e pinze microscissors (forbici Westcott) per incidere la congiuntiva al limbus e senza mezzi termini sezionare fuori la sclera. Tagliare la congiuntiva lungo il limbus come la dissezione procede per consentire un&#39;ulteriore dissezione. </li><li> Rimuovere congiuntiva tutto intorno all&#39;occhio (360 gradi) per esporre il più sclera possibile. NOTA: È utile lasciare una piccola quantità di congiuntiva per facilitare fissarsi l&#39;occhio con i perni chirurgici o una pinza durante il resto della procedura. </li></ol></li><li> Creare un completo lembo sclerale spessore lungo unolato dell&#39;occhio (bisturi 69). <ol><li> Fare un ~ 5 millimetri incisione sclerale parallelamente al limbus e 3 mm posteriormente al limbus tagliando delicatamente nella sclera con un bisturi (lama da bisturi 11) fino coroide pigmentazione scura è visibile. Fare attenzione a non incidere coroide stesso. </li><li> Sezionare cautela lungo il piano tra la sclera e della coroide, sia con le forbici (utilizzando smussa) o con il bisturi, finché è possibile ampliare lo spazio potenziale tra questi tessuti spingendo coroide leggermente verso l&#39;interno. </li><li> Effettuare un&#39;altra incisione sclerale perpendicolare alla prima incisione sclerale con microscissors taglienti, creando una incisione a forma di T. </li><li> Poi continuare a sezionare bruscamente in modo circonferenziale per rimuovere il tessuto sclerale dalla coroide sottostante e spingere delicatamente la coroide dalla sclera come detto sopra. </li><li> Ingrandire il lembo sclerale, se necessario. <ol><li> Delicatamente Blunt sezionare la coroide lontano dalla scleradurante tutto il processo. </li><li> Allargare lo sportello finché l&#39;incisione fatta perpendicolarmente al limbus raggiunge il nervo ottico e incisione fatta parallelo al limbus è almeno un terzo della circonferenza dell&#39;occhio (45 °). </li><li> Utilizzare il lembo sclerale come fonte di leva per manipolare l&#39;occhio durante la dissezione smussa. Il contro-trazione sul lembo facilita scollamento. </li></ol></li><li> Ripetere il passaggio precedente sull&#39;altro lembo sclerale. </li></ol></li><li> Tagliare i lembi sclerali per esporre una vasta area della coroide. </li><li> Continuare l&#39;incisione fatta nel passaggio 3 circonferenza dell&#39;occhio (360 gradi). </li><li> Rimuovere il restante tessuto coroide-retina. <ol><li> All&#39;interno della regione esposta, usare un cotton-tip per spazzolare delicatamente il tessuto della coroide-retina rimanente. In alternativa, afferrare delicatamente il tessuto con una pinza e sbucciare fuori dal corpo vitreo sottostante. </li><li> Se necessario fare un&#39;incisione nella coroide stArting dal nervo ottico. Poi staccarsi coroide delicatamente per esporre la retina e la corteccia vitreo. </li></ol></li><li> Continuare smussato sezionare la coroide dalla sclera e poi staccarsi coroide per ottenere il tutto, vitrea intatto. </li><li> Utilizzare il cerchio sclerale attaccato alla cornea per posizionare l&#39;intero vetrosa in posizione desiderata. A questo punto, il vitreo è collegato alla sclera anteriore e lente. </li><li> Blunt sezionare il vitreo dall&#39;interno della sclera, intorno alla lente, eliminando tutti sclera. </li><li> Utilizzare uno strumento smussato per raccogliere la lente dal vitreo se necessario. Utilizzare il campione per vari esperimenti vitreodynamic. <ol><li> Mettere campione in un bicchiere di vetro con nota superficie esposta all&#39;aria. Inserire sonda sensibile all&#39;ossigeno al bordo del campione. Utilizzare un micromanipolatore per spostare la sonda ad una distanza nota (r) nel campione. </li><li> Utilizzare Leggi di Fick per ottenere l&#39;equazione di diffusione teorica. Con il data raccolta in fase di 10.1, ottenere il termine coefficiente di diffusione sperimentale / reazione 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.

Ci sono due passaggi critici che devono essere accuratamente eseguite durante la dissezione vitreo. Fase 3, la creazione di un completo lembo sclerale spessore, è fondamentale per l&#39;intero dissezione. Si deve prestare attenzione a non tagliare in coroide quando si crea il pieno spessore lembo sclerale. L&#39;altro passaggio fondamentale è sezionando via la sclera dalla coroide. Questo passaggio deve essere fatto con attenzione per evitare la creazione di più fori nella coroide da cui vitreo può fuoriuscire. C&#3...

L&#39;obiettivo di questo metodo consiste nel dettaglio una tecnica per isolare un intero, corpo vitreo intatto, con il nucleo vitreo e corteccia intatta, da un occhio di cadavere, ai fini dell&#39;analisi vitreodynamic. Poiché il campo della fisiologia del vitreo è cresciuta, i ricercatori multidisciplinari, quali la meccanica dei fluidi ricercatori, stanno studiando le proprietà fisiche e biomeccaniche del corpo vitreo 1. A tal fine, è indispensabile dettaglio una tecnica per isolare tutto, corpo vitreo intatta per favorire ricercatori multidisciplinari. Sebag et al. 2 e altri 3 eseguiti eleganti intere dissezioni vetrose sugli occhi cadavere dell&#39;uomo e hanno mostrato le illustrazioni dei risultati. Tuttavia, la tecnica utilizzata non è stata descritta in dettaglio e non esperti, non sarebbe in grado di replicare il metodo indipendente. Altri studi hanno raccolto vitreo da occhi cadavere con metodi semplici, come l&#39;aspirazione o dissezione parziale,entrambi i quali non provocano un tutto, corpo vitreo intatto. Gisladottis et al. 4 e Xu et al. 5 indagare permeabilità nel vitreo raccolte da occhi cadavere. Tuttavia, poiché nessun metodo di estrazione vetrosa è stata descritta, si è ipotizzato che essi aspirati vitreo con una siringa. Watts et al. 6 andato un passo avanti descrivendo un metodo per isolare coniglio vitreo con una tecnica chirurgica. Tuttavia, questo metodo provoca un isolamento del solo nucleo vitreo e non la corteccia vitreo. Skeie et al. 7 poi organizzato il vitreo in 4 regioni uniche ed elegantemente descritto un metodo per sezionare fuori ogni parte per l&#39;analisi. Questa tecnica tuttavia, non comporta un vitreous intatto nel suo complesso. La tecnica attuale è stato sviluppato per facilitare esperimenti biofisici attualmente eseguite solo in occhi cadavere. I metodi precedenti, come descritto unbove, sono limitati perché 1) nessuno isolare completamente tutto il corpo vitreo, 2) raccolte nucleo vitreo e la corteccia sono omogeneizzati, 3) vitrea struttura anatomica non è mantenuta, o 4) tecniche di dissezione non sono adeguatamente dettagliate per la replica da parte dei ricercatori in altri campi . In aggiunta, a causa della opacità sclera e della coroide, visualizzazione del corpo vitreo è limitata nel bulbo oculare intatto. Questo limita la precisione e la fattibilità di misurazioni che possono essere fatte all&#39;interno occhio intero. Inoltre, le strutture anatomiche circostanti il ​​vitreo possono confondere lo studio delle proprietà biochimiche e fisiche del vitreo. Negli ultimi anni, il corpo della scienza vitreo è cresciuto enormemente e non vi è ragione di credere che tutto il corpo vitreo ha proprietà diverse rispetto le sue singole parti. Vi è un crescente interesse per lo studio delle proprietà fisiche, biomeccaniche e chimiche del vitreo per vitreodynamics research, che ha applicazioni in medicina clinica, come la somministrazione di farmaci, l&#39;ossigenazione intravitreale 8 e vitrectomia. Vitreodynamics farmacologici, che utilizza agenti farmacologici per manipolare il corpo vitreo, possono essere utilizzati per migliorare i risultati vitrectomia 9. Proprietà biomeccaniche vengono utilizzate per modellare il flusso di fluido vitreale, che può essere utilizzato per migliorare drug delivery tecnologie intravitreale 10-12. Le proprietà fisiche di diversi segmenti del vitreo sono cruciali per comprendere vitreo-retinica trasporto dell&#39;ossigeno 13. La tecnica dissezione vitreo proposto può essere usato per studiare varie proprietà del intatte vitreo. Permette da banco esperimenti da fare su tutto, corpi vetrosi intatti con una migliore visualizzazione. In sintesi, i metodi attuali per lo studio del vitreo sono o non adeguatamente descritte o causare un isolamento incompleta nucleo vitreo e corteccia. Pertanto, vi è la necessità di eseguire experiments in un modello di occhio trasparente, pur mantenendo l&#39;anatomia del vitreo che esiste negli occhi cadavere.

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			<td>0.3 forceps</td>
			<td>Storz Opthalmics</td>
			<td>E1793</td>
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			<td>Westcott Tenotomy Scissors Curved Right</td>
			<td>Storz Opthalmics</td>
			<td>E3320 R</td>
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			<td>Scalpel Handle No. 3</td>
			<td>VWR</td>
			<td>25607-947</td>
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			<td>Scalpel Blade, #11, for #3 Handle</td>
			<td>VWR</td>
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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.

Seguendo il protocollo porterà ad una dissezione vetrosa di successo con il nucleo e la corteccia (figura 3) intatto. Questo è evidente dai pezzi residui di retina aderito alla corteccia vitreo. Intatto intera vitreo può essere utilizzato in diversi modi per specifici esperimenti vitreodynamic. Nel nostro caso, il tasso di diffusione di ossigeno intatta vitreo ed è costante di tempo corrispondente è stato studiato (Figura 2). Vetroso che è stato dissezionato (nucleo e la corteccia...

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.

Tutta vitreo Umore dissezione per l&#39;analisi 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 Views.  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.

Video: 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.

Dissezione di Human Elements corpo vitreo per l&#39;analisi proteomica

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

Ricerca

Medicina

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.

Protocollo per lungo Ipertermia Corpo intera durata in Mouse

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-cuore Analisi tessuto miocardico

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.

Dissezione del Pancreas del Mouse per l'analisi istologica e Profiling metabolico

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.

Dissezione e cultura Explant della Allantois murino per l'analisi In Vitro di allantoico allegato

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.

Segmentazione del intero-cervello e analisi del punto di svolta di anatomico del cervello MRI — applicazione in premanifesti sulla malattia di 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.

Analisi quantitativa di Micro-CT di Aortopathy in un modello murino di indotta da β-aminopropionitrile aneurisma e dissezione

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.

Imaging e analisi delle lesioni dell'aorta intera O-macchiata di olio rosso in un modello murino di iperlipidemia da aneurisma

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.

Posizione, dissezione e analisi del Ganglio Stellato Murine

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.

Un metodo non invasivo per generare il modello di lesione cartilaginea intra-articolare indotta dal carico ciclico del ginocchio di ratto

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