Le financement a été fourni par Fight for Sight. Les tissus ont été obtenus auprès de la Banque Oculaire des Lions de l&#39;Iowa.

1. Dissection du segment antérieur. <ol><li> La cornée est enlevée en faisant d&#39;abord une incision au niveau du limbe dans la chambre antérieure à l&#39;aide d&#39;une lame de 15 ° (figure 1). Puis une lame incurvée de la cornée-sclérale ciseaux est inséré dans la chambre antérieure. Découpes circonférentielles sont faites dans la cornée, juste en avant du limbe. </li><li> 0,12 Colibri pinces et de ciseaux Westcott sont utilisés pour couper l&#39;iris circonférentiellement antérieure du corps ciliaire. </li><li> Le noyau du cristallin est retiré avec une pince ou d&#39;un Colibri 0,12 à 15 ° lame (supersharp) et le cortex et la capsule avec une pince. </li></ol> 2. Aspiration fondamentale vitré. <ol><li> Une aiguille de calibre 23 sur une seringue de 5 cc est inséré dans le milieu du vitré (figure 1). </li><li> Environ 1 mL ou plus du noyau du vitré est doucement aspiré. </li><li> L&#39;échantillon est ensuite placé dans un tube de centrifugeuse, puis dans l&#39;azote liquide. </li></ol> 3. Dissection antérieure hyaloïde. <ol><li> La hyaloïde antérieure est considéré comme un anneau semi-transparente en ajustant la lumière incidente à partir d&#39;un microscope à lumière col de cygne. L&#39;aspiration précédente du noyau vitreux sépare la hyaloïde antérieure auprès d&#39;autres structures pour faciliter leur identification. </li><li> La feuille de tissu élastique est soigneusement arrachée du corps ciliaire avec Colibri ou pinces à pansement et coupé avec des ciseaux Vannas. Cette manoeuvre est répétée. </li><li> L&#39;échantillon est ensuite placé dans un tube de centrifugeuse, puis dans l&#39;azote liquide. </li></ol> 4. Dissection base du vitré. <ol><li> Utiliser 0,12 pince pour stabiliser la coupe des yeux, des ciseaux Westcott sont utilisés pour «fleur» de l&#39;œil en faisant quatre équidistants-stress coupes du corps ciliaire vers la tête du nerf optique. </li><li> Le plicata pars du corps ciliaire (figure 2) est retiré de chacun des quadrants en utilisant des ciseaux Westcott. </li><li> La base du vitré est ensuite saisie de chaque côté de l&#39;ora serrata avec une pince sur la pars plana et de la rétine (figure 2). </li><li> Traction continue sur la base du vitré est appliqué avec la pince. De coupe avec des ciseaux séquentielle Westcott extraits un tissu semi-transparent avec un «collier de perles&quot; apparence car elle est excisée. </li><li> L&#39;échantillon est ensuite placé dans un tube de centrifugeuse, puis dans l&#39;azote liquide. </li></ol> 5. Retrait cortex vitréen. <ol><li> La pars plana est excisé de chacun des quadrants avec des ciseaux Westcott en coupant la notice d&#39;environ 3 mm en arrière de l&#39;ora serrata (figure 2). </li><li> Entre les feuillets de tissu, le cortex vitréen est visualisée comme un film semi-transparents. </li><li> Le film élastique est soit saisi avec une pince 0,12 ou détenus par l&#39;adhésion à une éponge Weck-Cel chirurgicale. Le cortex vitréen est alors arrachée de la rétine et excisée avec des ciseaux Westcott (figure 1). </li><li> L&#39;échantillon est placé dans un tube de centrifugeuse, puis dans l&#39;azote liquide. Tous les échantillons sont stockés à -80 ° C jusqu&#39;à leur utilisation pour l&#39;expérimentation. </li></ol> 6. Les résultats représentatifs Des échantillons de tissus peuvent être traités par une variété de méthodes pour des expériences spécifiques. Dans notre cas, les échantillons ont été soumis à l&#39;analyse des protéines par SDS-PAGE (figure 3). <img src="/files/ftp_upload/2455/2455fig1.jpg" alt="Figure 1" /> Figure 1. Vue en coupe de l&#39;œil humain dépeignant sous-structures différentes parties du corps vitré. Le vitré plus antérieure est une fine couche de collagène appelé la hyaloïde antérieure. Le noyau vitreux comprend toute la région centrale du corps vitré. Cette portion du corps vitré est plus aqueuse, contrairement à la base du vitré, ce qui est assez visqueux pour être saisi par des pinces et est fermement attaché au corps ciliaire et la rétine sous-jacente. Englobant le cœur vitré est une coquille très mince collagène appelé le cortex vitréen. <img src="/files/ftp_upload/2455/2455fig2.jpg" alt="Figure 2" /> Figure 2. Anatomie base du vitré. La base du vitré est une sous-structure semi-transparente du corps vitré situé le long de l&#39;ora serrata (flèches blanches), qui est la ligne de démarcation séparant le corps ciliaire et la rétine. Le bord antérieur de la base du vitré s&#39;étend sur la pars plana (ligne blanche) du corps ciliaire. Le bord postérieur de la base du vitré s&#39;étend 2-3-mm derrière l&#39;ora serrata (tiret blanc). Pour accise la base du vitré, forceps sont utilisés pour saisir le tissu et tirez loin du corps ciliaire et la rétine sous-jacente. Une fois élevés, ciseaux Westcott sont utilisés pour couper le long de la base. <img src="/files/ftp_upload/2455/2455fig3.jpg" alt="Figure 3" /> Figure 3. Unidimensionnelle SDS-PAGE des éléments du corps vitré. Concentrations de protéines totales de la hyaloïde antérieure, base du vitré, vitreous coeur, et cortex vitréen étaient 11,24, 20,1, 16,61, 14,24 mg / mL, respectivement. L&#39;électrophorèse sur gel a été réalisée à 200 kV pendant 45 minutes, colorées avec Flamingo (Bio-Rad), et visualisées en utilisant un système d&#39;imagerie VersaDoc (Bio-Rad). Les profils pour les différents tissus montrent plusieurs bandes similaires, indiquant soit conservée ou la contamination croisée des protéines, ainsi que des bandes uniques (astérisque), indiquant protéines différentiellement localisées.

<ol>
	<li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel.">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog Retin Eye Res. 19, 323-344 (2000).</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=Molecular+biology+of+pharmacologic+vitreolysis.">Molecular biology of pharmacologic vitreolysis.</a> Trans Am Ophthalmol Soc. 103, 473-494 (2005).</li><li>Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+inflammatory+immune+mediators+in+vitreoretinal+diseases.">Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases.</a> PLoS One. 4, e8158-e8158 (2009).</li><li>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P., Feener, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+vitreous+proteome+in+diabetes+without+diabetic+retinopathy+and+diabetes+with+proliferative+diabetic+retinopathy.">Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.</a> J Proteome Res. 7, 2516-2525 (2008).</li><li>Izuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+SOD+and+VEGF+are+increased+in+vitreous+bodies+from+proliferative+diabetic+retinopathy+patients.">Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients.</a> Mol Vis. 15, 2663-2672 (2009).</li><li>Azimzadeh, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formalin-fixed+paraffin-embedded+(FFPE)+proteome+analysis+using+gel-free+and+gel-based+proteomics.">Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.</a> J Proteome Res. 9, 4710-4720 (2010).</li></ol>

Le corps vitré est un gel semi-transparente, dont la composition moléculaire est mal comprise, en particulier au niveau de ses sous-structures: la base du vitré, le noyau, le cortex, et hyaloïde antérieure. Le noyau contient du vitré collagènes II, V, IX et XI, avec les protéoglycanes chondroïtine sulfate, héparane sulfate protéoglycanes, et hyaluronane 1,2. Biomarqueurs protéiques dans le coeur du vitré ont été associés à des maladies telles que la rétinopathie diabétique. 3-5 Comment ces protéines sont différentiellement exprimés dans chacune des sous-structures, et dans de nombreux cas l&#39;identité des protéines spécifiques, ne sont pas connus. Ces détails peuvent donner un aperçu de l&#39;origine des protéines associées à certaines maladies vitréo-rétinienne et d&#39;aider les thérapies futures cibles. Bien que le poste d&#39;intervalle optimal mortem pour la dissection des tissus n&#39;a pas été déterminée, la dégradation des protéines peut affecter expériences en aval. Par exemple, l&#39;immunohistochimie est affectée dans les yeux de l&#39;autopsie de 12 heures et certaines activités enzymatiques spécifiques peuvent être réduits en quelques heures (observation non publiée). Tous les tissus dans cette étude ont été recueillies entre 2 et 8 heures de la mort, sans changements importants dans l&#39;expression de protéines ou d&#39;aptitude à analsyis protéomique. La méthode de congélation de l&#39;azote liquide de conservation est choisi au cours de fixation afin d&#39;éviter de petits changements dans la structure des protéines causée par fixateur réticulation, ce qui a été démontré dans d&#39;autres tissus par LC-MS/MS. 6 études protéomiques dépendra de la capacité à bien disséquer les différents compartiments du corps vitré, comme l&#39;a démontré dans cette expérience vidéo. Nous avons validé la technique de dissection avec une dimension SDS-PAGE. Comme nos résultats suggèrent, il ya des protéines différentiellement exprimés dans les sous-structures différentes du corps vitré. L&#39;identification de ces protéines permettra une compréhension plus détaillée de cloisonnement vitré.

<table border="1"><tbody><tr><td> Nom </td><td> Société </td><td> Nombre de catalogue </td></tr><tr><td> 0,12 pince </td><td> Storz Ophthalmics </td><td> E1502 </td></tr><tr><td> 5 cc seringue </td><td> Becton-Dickinson </td><td> 309603 </td></tr><tr><td> Droit pinces à pansement avec des dentelures </td><td> Storz Ophthalmics </td><td> E1400 </td></tr><tr><td> Aiguille de calibre 23 </td><td> Becton-Dickinson </td><td> 305145 </td></tr><tr><td> Colibri pince </td><td> Storz Ophthalmics </td><td> 2 / 132 </td></tr><tr><td> Castroviejo angle des ciseaux cornéens </td><td> Storz Ophthalmics </td><td> E3223 </td></tr><tr><td> Vannas courbé ciseaux à capsulotomie </td><td> Storz Ophthalmics </td><td> E3387 </td></tr><tr><td> Weck-Cel chirurgicale lances </td><td> Medtronic </td><td> 0008680 </td></tr><tr><td> Ciseaux Westcott ténotomie courbé </td><td> Storz Ophthalmics </td><td> E3320 </td></tr></tbody></table>

dissection of human vitreous body elements for proteomic analysis

Le vitré est une optiquement clair, matrice de collagène extracellulaire qui remplit l&#39;intérieur de l&#39;œil et recouvre la rétine. 1,2 interactions anormales entre les sous-structures vitré et la rétine sous-tendent plusieurs maladies vitréo-rétinienne, y compris la déchirure de la rétine et le détachement, plissement maculaire, trou maculaire, liée à l&#39;âge de traction vitréo dégénérescence maculaire, vitréorétinopathie proliférante, rétinopathie diabétique proliférative, et vitreoretinopathies hérité. 1,2 La composition moléculaire des sous-structures vitreuses n&#39;est pas connue. Puisque le corps vitré est transparent avec accès chirurgical limité, il a été difficile à étudier ses sous-structures au niveau moléculaire. Nous avons développé une méthode permettant de séparer et de préserver ces tissus pour l&#39;analyse protéomique et biochimique. La technique de dissection dans cette vidéo expérimentale montre comment isoler base du vitré, hyaloïde antérieure, le noyau vitreux, et le cortex vitré de l&#39;autopsie yeux humains. Unidimensionnelle SDS-PAGE des analyses de chaque composant du vitré a montré que notre technique de dissection donné lieu à quatre profils protéiques unique correspondant à chaque sous-structure du vitré humain. Identification de protéines différentiellement compartimentée révélera molécules candidates qui sous-tendent diverses maladies vitréo-rétinienne.

Watch this Scientific Journal Video about Dissection of Human Vitreous Body Elements for Proteomic Analysis at JoVE.com

Dissection of Human Vitreous Body Elements for Proteomic Analysis

Cette vidéo montre une technique efficace pour différencier et disséquer les différentes structures de semi-transparente du corps humain vitreuse en post mortem yeux.

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 ...

dissection-of-human-vitreous-body-elements-for-proteomic-analysis

Research

JoVE Journal

Medicine

31.7K vues.  University of Iowa. Cette vidéo montre une technique efficace pour différencier et disséquer les différentes structures de semi-transparente du corps humain vitreuse en post mortem yeux.

Vidéo: Dissection of Human Vitreous Body Elements for Proteomic Analysis

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.

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 ...

Recherche

Médecine

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Laser-microdissection de la prostate humaine épithélium pour l&#39;analyse de l&#39;ARN

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

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 ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Composition corporelle et l’analyse mise en cage métabolique in Mice Fed graisse haute

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

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 ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

Tout le corps et Quantification régionale d’homme actif brun tissu adipeux à l’aide de 18F-FDG TEP/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of skeletal muscle mass and adipose tissues on Computed Tomography (CT) and/or Magnetic Resonance Imaging (MRI) may inform surgery risk evaluation and disease prognosis. This article describes two quantification methods originally described by Mourtzakis et al. and Avrutin et al.: tissue segmentation and linear measurement of skeletal muscle. Patients' cross-sectional image at the midpoint of the third lumbar vertebra was obtained for both measurements. For segmentation, the images were imported into Slice-O-Matic and colored for skeletal muscle, intramuscular adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue. Then, surface areas of each tissue type were calculated using the tag surface area function. For linear measurements, the height and width of bilateral psoas and paraspinal muscles at the level of the third lumbar vertebra are measured and the calculation using these four values yield the estimated skeletal muscle mass. Segmentation analysis provides quantitative, comprehensive information about the patients' body composition, which can then be correlated with disease progression. However, the process is more time-consuming and requires specialized training. Linear measurements are an efficient and clinic-friendly tool for quick preoperative evaluation. However, linear measurements do not provide information on adipose tissue composition. Nonetheless, these methods have wide applications in a variety of diseases to predict surgical outcomes, risk of disease progression and inform treatment options for patients.

Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification.

Segmentation et mesure linéaire pour l’analyse de la composition corporelle à l’aide de Slice-O-Matic et Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of ...

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 ...