O financiamento foi fornecido pelo Fight for Sight. Tecidos foram obtidos do Banco de Olhos Lions Iowa.

1. A dissecção do segmento anterior. <ol><li> A córnea é removida por primeiro fazer uma incisão no limbo dentro da câmara anterior utilizando uma lâmina de 15 ° (Figura 1). Em seguida, uma lâmina curvada de córnea escleral-tesoura é inserida dentro da câmara anterior. Circunferencial cortes são feitos na córnea, apenas anterior ao limbo. </li><li> 0,12 Colibri pinças e tesoura Westcott são usados ​​para cortar a íris circunferencialmente anterior ao corpo ciliar. </li><li> O núcleo da lente foi removida com fórceps 0,12 Colibri ou uma lâmina de 15 ° (supersharp) e do córtex e da cápsula com uma pinça. </li></ol> 2. Aspiração Central vítreo. <ol><li> Uma agulha 23 gauge em uma seringa de 5 cc é inserido em meados da década de vítreo (Figura 1). </li><li> Aproximadamente 1 mL ou mais do núcleo vítreo é suavemente aspirado. </li><li> A amostra é então colocada em um tubo de microcentrífuga e depois em nitrogênio líquido. </li></ol> 3. Dissecação hialóide anterior. <ol><li> A hialóide anterior é visto como um anel semi-transparente, ajustando a luz incidente de um microscópio de luz ganso pescoço. Aspiração anteriores do núcleo vítreo se separa da hialóide anterior de outras estruturas para facilitar a identificação. </li><li> A folha de tecido elástico é cuidadosamente puxado para longe do corpo ciliar com Colibri ou fórceps vestir-se e cortou com uma tesoura Vannas. Esta manobra é repetida. </li><li> A amostra é então colocada em um tubo de microcentrífuga e depois em nitrogênio líquido. </li></ol> 4. Dissecção da Base vítreo. <ol><li> Usando 0,12 fórceps para estabilizar a taça do olho, uma tesoura Westcott são usados ​​para &quot;flor&quot; o olho fazendo quatro igualmente espaçados de aliviar o stress cortes do corpo ciliar em direção à cabeça do nervo óptico. </li><li> A pars plicata do corpo ciliar (Figura 2) é removida de cada um dos quadrantes usando uma tesoura Westcott. </li><li> A base vítrea é, então, agarrou em ambos os lados da ora serrata com uma pinça sobre o pars plana e retina (Figura 2). </li><li> Tração contínua sobre a base vítrea é aplicada com a pinça. Corte sequencial com tesoura Westcott extrai um tecido semi-transparente com um &quot;colar de pérolas&quot; aparência como é extirpado. </li><li> A amostra é então colocada em um tubo de microcentrífuga e depois em nitrogênio líquido. </li></ol> 5. Remoção Cortex vítreo. <ol><li> A pars plana é retirado de cada um dos quadrantes com tesoura Westcott cortando o folheto aproximadamente 3 mm posterior à ora serrata (Figura 2). </li><li> Entre os folhetos de tecido, o córtex do vítreo é visualizado como um filme semi transparente. </li><li> O filme elástico ou é apreendido com 0,12 forceps ou detidos pela adesão a uma esponja Weck-Cel cirúrgico. O córtex vítreo é, então, afastou-se da retina e retirado com tesoura Westcott (Figura 1). </li><li> A amostra é colocada em um tubo de microcentrífuga e depois em nitrogênio líquido. Todas as amostras são armazenadas a -80 ° C até utilizados para a experimentação. </li></ol> 6. Resultados representante Amostras de tecido pode ser processado por uma variedade de métodos para experimentos específicos. No nosso caso, as amostras foram submetidas a análise de proteínas por SDS-PAGE (Figura 3). <img src="/files/ftp_upload/2455/2455fig1.jpg" alt="Figura 1" /> Figura 1. Transversal visão do olho humano representando subestruturas diferentes do corpo vítreo. O vítreo mais anterior é uma fina camada de colágeno chamado de hialóide anterior. O núcleo vítreo compreende toda a região central do corpo vítreo. Esta parte do vítreo é mais aquosa em contraste com a base vítrea, que é viscoso o suficiente para ser captado por uma pinça e está firmemente ligado ao corpo ciliar e retina subjacente. Abrangendo o núcleo vítreo é um shell muito finas de colágeno chamada córtex vítreo. <img src="/files/ftp_upload/2455/2455fig2.jpg" alt="Figura 2" /> Figura 2. Anatomia base vítrea. A base do vítreo é uma infra-estrutura semi-transparente do corpo vítreo localizado junto à ora serrata (setas brancas), que é a linha divisória que separa o corpo ciliar e retina. A borda anterior da base do vítreo se estende sobre a pars plana (linha branca) do corpo ciliar. A borda posterior da base vítrea estende 2-3 mm posterior à ora serrata (traço branco). Para extirpar a base do vítreo, uma pinça são usados ​​para captar o tecido e puxe-o para longe do corpo ciliar e retina subjacente. Uma vez elevado, tesoura Westcott são usados ​​para cortar ao longo da base. <img src="/files/ftp_upload/2455/2455fig3.jpg" alt="Figura 3" /> Figura 3. Unidimensional SDS-PAGE de elementos do corpo vítreo. Concentrações de proteína total para a hialóide anterior, base vítrea, vitreous núcleo e córtex vítreo foram 11,24, 20,1, 16,61, 14,24 mg / mL, respectivamente. Eletroforese em gel foi realizada em 200 kV por 45 minutos, corados com Flamingo (Bio-Rad), e visualizados através de um VersaDoc Imaging System (Bio-Rad). Os perfis para os diferentes tecidos mostrar várias bandas similares, indicando quer conservada ou cross-contaminação proteínas, bem como as bandas originais (asterisco), indicando proteínas diferencialmente localizada.

<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>

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O corpo vítreo é um gel semi-transparente, cuja composição molecular é mal compreendida, especialmente no nível de suas subestruturas: a base do vítreo, núcleo, córtex, e hialóide anterior. O núcleo vítreo contém colágenos II, V, IX e XI, juntamente com proteoglicanos sulfato de condroitina, sulfato de heparan proteoglicanos, e. Hyaluronan 1,2 biomarcadores de proteína no núcleo vítreo têm sido associadas a doenças como a retinopatia diabética. 3-5 Como estas proteínas são diferencialmente expressos em cada uma das subestruturas, e em muitos casos, as identidades de proteínas específicas, não são conhecidos. Estas informações podem dar uma visão sobre a origem de proteínas associadas com certas doenças vítreo-retinianas e ajudar a futuras terapias-alvo. Embora o ideal intervalo post mortem para dissecção dos tecidos não foi determinado, a degradação de proteínas pode afetar experimentos a jusante. Por exemplo, imuno-histoquímica é afetado nos olhos de 12 horas pós-morte e algumas atividades de enzima específica pode ser reduzida dentro de algumas horas (observação não publicada). Todos os tecidos no presente estudo foram coletados entre 2 e 8 horas de morte, sem mudanças significativas na expressão da proteína ou da aptidão para analsyis proteômica. O método de congelamento de nitrogênio líquido de preservação é escolhido em detrimento de fixação para evitar pequenas mudanças na estrutura da proteína causada por fixador reticulação, que tem sido demonstrada em outros tecidos por LC-MS/MS. 6 estudos Proteômica dependerá da capacidade de precisão dissecar os diferentes compartimentos do vítreo, como demonstrado nesta experiência de vídeo. Temos validou a técnica de dissecção utilizando 1-dimensional SDS-PAGE. Como nossos resultados sugerem, existem proteínas diferencialmente expressos no corpo várias subestruturas vítreo. A identificação dessas proteínas irá fornecer uma compreensão mais detalhada de compartimentalização vítreo.

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> 0,12 forceps </td><td> Storz Ophthalmics </td><td> E1502 </td></tr><tr><td> 5 cc-seringa </td><td> Becton-Dickinson </td><td> 309603 </td></tr><tr><td> Pinça reta vestir Com serrilhas </td><td> Storz Ophthalmics </td><td> E1400 </td></tr><tr><td> Agulha de calibre 23 </td><td> Becton-Dickinson </td><td> 305145 </td></tr><tr><td> Colibri forceps </td><td> Storz Ophthalmics </td><td> 2 / 132 </td></tr><tr><td> Castroviejo em ângulo tesoura de córnea </td><td> Storz Ophthalmics </td><td> E3223 </td></tr><tr><td> Vannas Curved Tesoura Capsulotomia </td><td> Storz Ophthalmics </td><td> E3387 </td></tr><tr><td> Weck-Cel cirúrgica lanças </td><td> Medtronic </td><td> 0008680 </td></tr><tr><td> Westcott Curved Tesoura Tenotomia </td><td> Storz Ophthalmics </td><td> E3320 </td></tr></tbody></table>

dissection of human vitreous body elements for proteomic analysis

O vítreo é uma opticamente clara, matriz extracelular de colágeno, que preenche o interior do olho e recobre a retina. 1,2 interações anormais entre subestruturas vítreo e da retina subjacentes várias doenças vítreo-retinianas, incluindo rasgaduras da retina e descolamento, pucker macular, buraco macular, idade degeneração macular relacionada à tração vítreo, Vitreorretinopatia proliferativa, retinopatia diabética proliferativa, e vitreoretinopathies herdada. 1,2 A composição molecular do vítreo subestruturas não é conhecido. Desde que o corpo vítreo é transparente com acesso cirúrgico limitado, tem sido difícil de estudar suas subestruturas no nível molecular. Desenvolvemos um método para separar e preservar estes tecidos para análise proteômica e bioquímica. A técnica de dissecção neste vídeo experimental mostra como isolar base vítrea, hialóide anterior, o núcleo vítreo, e no córtex vítreo dos olhos pós-morte humana. Unidimensional SDS-PAGE análises de cada componente do vítreo mostrou que a nossa técnica de dissecção resultou em quatro perfis de proteína única correspondente a cada subestrutura do corpo vítreo humano. Identificação de proteínas diferencialmente compartimentada irá revelar as moléculas candidato subjacente várias doenças vítreo-retinianas.

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

Este vídeo mostra uma técnica eficaz para diferenciar e dissecando os vários semi-transparente das estruturas do corpo humano na pós vítreos olhos mortem.

Dissecção da Human Elements Corpo Vítreo para Análise Proteômica

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 visualizações.  University of Iowa. Este vídeo mostra uma técnica eficaz para diferenciar e dissecando os vários semi-transparente das estruturas do corpo humano na pós vítreos olhos mortem.

Vídeo: 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 humor vítreo Dissecção de Análise Vitreodynamic

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

Medicina

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.

Captura de Laser Microdissection de Próstata Humano Epithelium para Análise de RNA

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.

Dissecação do pâncreas Mouse para análise histológica e perfis metabólicos

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.

Dissecção e cultura de explante de murino alantoide para a análise In Vitro de fixação alantoico

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.

Composição corporal e análise de enjaulamento metabólico em ratos de alta gordura Fed

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.

Análise quantitativa de Micro-CT de Aortopathy em um modelo do rato de β-aminopropionitrile-induzida de aneurisma da aorta e dissecação

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.

Todo o corpo e quantificação Regional de ativo humano marrom tecido adiposo usando 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 ...

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.

Segmentação e Medição Linear para Análise de Composição Corporal usando Slice-O-Matic e 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.

Localização, Dissecção e Análise do Gânglio Murine Stellate

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.

Um Método Não Invasivo para Gerar o Modelo de Lesão da Cartilagem Intra-Articular Induzida por Carga Cíclica do Joelho de Rato

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