Die Finanzierung wurde von Fight for Sight zur Verfügung gestellt. Die Gewebe wurden von der Iowa Lions Eye Bank erhalten.

1. Anterior Segment Dissection. <ol><li> Die Hornhaut wird zunächst durch einen Einschnitt am Limbus in die Vorderkammer mit einem 15 ° Klinge (Abbildung 1) entfernt. Dann eine Klinge von gekrümmten Hornhaut-Sklera-Schere ist in die Vorderkammer eingeführt. Umlaufende Schnitte in der Hornhaut gemacht, unmittelbar vor dem Limbus. </li><li> 0,12 Colibri Pinzette und Schere Westcott werden verwendet, um die Iris umfänglich anterior des Ziliarkörpers geschnitten. </li><li> Der Linsenkern wird mit 0,12 Colibri Zange oder einem 15 °-Klinge (superscharfen) und der Hirnrinde und Kapsel mit einer Pinzette entfernt. </li></ol> 2. Glaskörper-Core Aspiration. <ol><li> Ein 23-Gauge-Nadel auf einer 5-ml-Spritze ist in der Mitte eingesetzt Glaskörper (Abbildung 1). </li><li> Etwa 1 ml oder mehr glasigen Kern ist vorsichtig abgesaugt. </li><li> Die Probe wird dann in einem Mikrozentrifugenröhrchen und dann in flüssigem Stickstoff gelegt. </li></ol> 3. Anterior hyaloiden Dissection. <ol><li> Der vordere hyaloidea ist als semi-transparent Ring durch die Anpassung des einfallenden Lichts von einem Schwanenhals Mikroskop Licht gesehen. Vorherige Aspiration des Glaskörpers Kern trennt den vorderen hyaloiden von anderen Strukturen zur leichteren Identifizierung. </li><li> Das Blatt aus elastischem Gewebe wird vorsichtig vom Ziliarkörper mit Colibri oder Kornzange gezogen und schneiden Sie mit Vannas Schere. Dieses Manöver wird wiederholt. </li><li> Die Probe wird dann in einem Mikrozentrifugenröhrchen und dann in flüssigem Stickstoff gelegt. </li></ol> 4. Glaskörperbasis Dissection. <ol><li> Mit 0,12 Pinzette auf die Augenmuschel zu stabilisieren, werden Westcott Schere &quot;Blume&quot; das Auge, indem sie vier gleichmäßig verteilte Spannungsarmglühen Schnitte aus dem Ziliarkörper in Richtung des Sehnervenkopfes eingesetzt. </li><li> Die Pars plicata des Ziliarkörpers (Abbildung 2) ist aus jedem der Quadranten mit Westcott Schere entfernt. </li><li> Der Glaskörper Basis wird dann auf beiden Seiten der Ora serrata mit einer Pinzette über die pars plana und der Netzhaut (Abbildung 2) erfasst. </li><li> Kontinuierliche Traktion auf den Glaskörper Basis ist mit der Zange angewendet. Sequential Schneiden mit Westcott Schere extrahiert eine semi-transparente Gewebe mit einer &quot;Perlenkette&quot; Erscheinungsbild, wie es ist herausgeschnitten. </li><li> Die Probe wird dann in einem Mikrozentrifugenröhrchen und dann in flüssigem Stickstoff gelegt. </li></ol> 5. Vitreous Cortex Removal. <ol><li> Die Pars plana ist aus jedem der Quadranten mit Westcott Schere durch Abschneiden der Broschüre ca. 3-mm hinter der Ora serrata (Abb. 2) herausgeschnitten. </li><li> Zwischen dem Gewebe Flugblätter, wird der Glaskörper Kortex visualisiert als halb transparente Folie. </li><li> Die elastische Folie ist entweder mit 0,12 Pinzette gefaßt oder über die Zugehörigkeit zu einer Weck-Cel chirurgischen Schwamm. Der Glaskörper Kortex wird dann von der Netzhaut und zog ausgeschnitten mit Westcott Schere (Abbildung 1). </li><li> Die Probe wird in einem Mikrozentrifugenröhrchen und dann in flüssigem Stickstoff gelegt. Alle Proben werden bei -80 ° C bis zum Experimentieren genutzt. </li></ol> 6. Repräsentative Ergebnisse Gewebeproben können durch eine Vielzahl von Methoden für bestimmte Experimente verarbeitet werden. In unserem Fall wurden die Proben für Protein-Analyse durch SDS-PAGE (Abbildung 3) vorgelegt. <img src="/files/ftp_upload/2455/2455fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Querschnitt des menschlichen Auges darstellen verschiedenen Teilstrukturen des Glaskörpers. Die vorderen Glaskörper ist eine dünne kollagenen Schicht bezeichnet den vorderen hyaloidea. Der Glaskörper Kern umfasst das gesamte zentrale Bereich des Glaskörpers. Dieser Teil des Glaskörpers ist wässrigen im Gegensatz zu den Glaskörper Basis, die viskos genug, um mit einer Pinzette fassen und ist fest mit dem darunter liegenden Ziliarkörper und Retina befestigt ist. Umfasst den Glaskörper Kern ist eine sehr dünne kollagene Shell aufgerufen Glaskörper Kortex. <img src="/files/ftp_upload/2455/2455fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Glaskörperbasis Anatomie. Der Glaskörper Basis ist eine semi-transparente Unterbau des Glaskörpers an der Ora serrata (weiße Pfeile), die die Trennlinie Trennung des Ziliarkörpers und der Netzhaut liegt. Der vordere Rand des Glaskörpers Basis erstreckt sich über die pars plana (weiße Linie) des Ziliarkörpers. Der hintere Rand des Glaskörpers Basis erstreckt 2-3 mm hinter der Ora serrata (weiß Bindestrich). Verbrauchsteuerpflichtige die Glaskörperbasis sind Pinzette verwendet, um das Gewebe zu erfassen und ziehen Sie sie weg von der zugrunde liegenden Ziliarkörper und Retina. Einmal erhöht, Westcott Schere werden verwendet, um an der Basis abgeschnitten. <img src="/files/ftp_upload/2455/2455fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Eindimensionale SDS-PAGE des Glaskörpers Elements. Gesamt-Protein-Konzentrationen für den vorderen hyaloidea, Glaskörperbasis, vitreous Kern und Glaskörper Kortex wurden 11,24, 20,1, 16,61, 14,24 mg / ml. Gel-Elektrophorese wurde bei 200 kV für 45 Minuten durchgeführt, gefärbt mit Flamingo (Bio-Rad), und visualisiert mit Hilfe eines VersaDoc Imaging System (Bio-Rad). Die Profile für die verschiedenen Gewebe zeigen mehrere ähnliche Bands, aus der entweder konserviert oder Cross-kontaminierenden Proteinen, sowie einzigartige Bands (Stern), was darauf hinweist unterschiedlich lokalisierte Proteine.

<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>

Der Glaskörper ist eine semi-transparente Gel, dessen molekulare Zusammensetzung ist weitgehend unverstanden, vor allem auf der Ebene der Unterkonstruktion: Der Glaskörperbasis, Kern, Kortex und anterioren hyaloidea. Der Glaskörper Kern enthält Kollagene II, V, IX und XI, zusammen mit Chondroitinsulfat Proteoglykane, Heparansulfat Proteoglykane und Hyaluronsäure. 1,2 Protein-Biomarkern in den Glaskörper Kern haben mit Krankheiten wie diabetische Retinopathie in Verbindung gebracht worden. 3-5 Wie Diese Proteine ​​sind unterschiedlich in den einzelnen Substrukturen ausgedrückt, und in vielen Fällen das spezifische Protein-Identitäten sind nicht bekannt. Diese Angaben können Einblick in die Herkunft der Proteine ​​mit spezifischen vitreoretinale Erkrankungen geben und helfen Ziel zukünftige Therapien. Obwohl die optimale post mortem Intervall für Gewebedissektion wurde nicht bestimmt, so kann Proteinabbau beeinträchtigen nachgeschaltete Experimente. Zum Beispiel ist die Immunhistochemie in 12-Stunden post mortem Augen betroffen und einige spezifische Enzymaktivitäten können innerhalb weniger Stunden reduziert werden (unveröffentlichte Beobachtung). Alle Gewebe in dieser Studie waren zwischen 2 und 8 Stunden nach dem Tod, ohne wesentliche Änderungen in der Proteinexpression oder der Eignung für die Proteomik analsyis gesammelt. Der flüssige Stickstoff Einfrieren Verfahren zur Haltbarmachung über Fixierung gewählt, um kleine Veränderungen in der Proteinstruktur von Fixativ Vernetzung, die in anderen Geweben wurde durch LC-MS/MS demonstriert verhindern. 6 Proteomic Studien werden sich auf die Fähigkeit, genau ab sezieren den verschiedenen Kompartimenten des Glaskörpers, wie in diesem Video Experiment gezeigt. Wir haben die Dissektionstechnik mit 1-dimensionale SDS-PAGE bestätigt. Wie unsere Ergebnisse zeigen, gibt es unterschiedlich Proteine ​​in den verschiedenen Glaskörper Unterkonstruktionen ausgedrückt. Die Identifizierung dieser Proteine ​​liefert ein detaillierteres Verständnis der Glaskörper Abschottung.

<table border="1"><tbody><tr><td> Name </td><td> Firma </td><td> Catalog Number </td></tr><tr><td> 0,12 Pinzette </td><td> Storz Ophthalmics </td><td> E1502 </td></tr><tr><td> 5-ml-Spritze </td><td> Becton-Dickinson </td><td> 309603 </td></tr><tr><td> Gerade Kornzangen mit Zacken </td><td> Storz Ophthalmics </td><td> E1400 </td></tr><tr><td> 23 Gauge Nadel </td><td> Becton-Dickinson </td><td> 305145 </td></tr><tr><td> Colibri Zange </td><td> Storz Ophthalmics </td><td> 2 / 132 </td></tr><tr><td> Castroviejo abgewinkelt Hornhaut Schere </td><td> Storz Ophthalmics </td><td> E3223 </td></tr><tr><td> Vannas Curved Kapsulotomie Scissors </td><td> Storz Ophthalmics </td><td> E3387 </td></tr><tr><td> Weck-Cel chirurgischen Speere </td><td> Medtronic </td><td> 0008680 </td></tr><tr><td> Westcott Curved Tenotomie Scissors </td><td> Storz Ophthalmics </td><td> E3320 </td></tr></tbody></table>

dissection of human vitreous body elements for proteomic analysis

Der Glaskörper ist eine optisch klare kollagenen extrazellulären Matrix, die das Innere des Auges und liegt über der Netzhaut füllt. 1,2 Abnormal Interaktionen zwischen Glaskörper Unterkonstruktionen und der Netzhaut zugrunde liegen mehrere vitreoretinale Krankheiten, einschließlich Netzhauteinriss und Loslösung, Macular Pucker, Makula-Loch, altersbedingter Makula-Degeneration, vitreomacular Traktion, proliferative Vitreoretinopathie, proliferative diabetische Retinopathie, und erbte vitreoretinopathies. 1,2 Die molekulare Zusammensetzung des Glaskörpers Unterkonstruktionen ist nicht bekannt. Da der Glaskörper ist transparent mit begrenzten chirurgischen Zugang, ist es schwierig gewesen, seine Unterkonstruktionen auf molekularer Ebene zu untersuchen. Wir entwickelten ein Verfahren zur Trennung und zur Erhaltung dieser Gewebe für Proteomik und biochemische Analyse. Die Dissektionstechnik in diesem experimentellen Video zeigt, wie Glaskörperbasis, anterior hyaloidea, glasigen Kern und Glaskörper Kortex von postmortalen menschlichen Augen zu isolieren. Eindimensionale SDS-PAGE-Analysen der einzelnen Glaskörper Komponente gezeigt, dass unsere Dissektionstechnik in vier einzigartigen Protein-Profilen, die der jeweiligen Unterkonstruktion des menschlichen Glaskörpers geführt. Identifizierung von differentiell compartmentalized Proteine ​​wird zeigen, Kandidaten-Moleküle zugrunde liegenden verschiedenen vitreoretinale Krankheiten.

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

Dieses Video zeigt eine effektive Technik zur Differenzierung und seziert die verschiedenen semi-transparente Strukturen des menschlichen Glaskörper in post mortem Augen.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

31.8K Aufrufe.  University of Iowa. Dieses Video zeigt eine effektive Technik zur Differenzierung und seziert die verschiedenen semi-transparente Strukturen des menschlichen Glaskörper in post mortem Augen.

Serie: Dissection of Human Vitreous Body Elements for Proteomic Analysis

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal fluctuations over time. Temporal Lobe Epilepsy (TLE) is the most common type of adult epilepsy and involves multiple brain networks. The default mode network (DMN) is involved in conscious, resting state cognition and is thought to be affected in TLE where seizures cause impairment of consciousness. The DMN in epilepsy was examined using seed based fcMRI. The anterior and posterior hubs of the DMN were used as seeds in this analysis. The results show a disconnection between the anterior and posterior hubs of the DMN in TLE during the basal state. In addition, increased DMN connectivity to other brain regions in left TLE along with decreased connectivity in right TLE is revealed. The analysis demonstrates how seed-based fcMRI can be used to probe cerebral networks in brain disorders such as TLE.

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

Netzwerkanalyse des Default Mode Netzwerk mit Functional MRI-Konnektivität in Temporallappen-Epilepsie

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal ...

Forschung

Medizin

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.

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 ...

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-Mikrodissektion für Menschen Prostataepithel für die RNA-Analyse

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.

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 ...

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.

Körperzusammensetzung und festsetzende Stoffwechselanalyse bei hohen Fett Fed Mäusen

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.

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 ...

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.

Segmentierung und lineare Messung zur Analyse der Körperzusammensetzung mit Slice-O-Matic und 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.

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 ...