This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

O procedimento apresentado tem lugar no âmbito de um protocolo de pesquisa aprovado pelo Conselho de Revisão do Academic Medical Center Amsterdam, número de registo NL41985.018 Institucional. O consentimento informado escrito é exigido de todos os participantes. 1. Sistema <ol><li> Para esta experiência, utilizar um sistema de outubro de domínio de Fourier, que opera com uma banda de comprimento de onda 13 nm 1,280-1,350. Domínio de Fourier interferometria de baixa coerência permite um mapeamento contínuo que aumenta a velocidade de aquisição de dados, quando comparado com os sistemas de outubro de domínio de tempo da primeira geração. Nota: O sistema de outubro é ligada a uma sonda de fibra óptica, a digitalização de forma helicoidal em ~ 90 ° de ângulo. Ele tem um diâmetro exterior de 2.7F (0,9 mm) e um comprimento inserível de 135 cm. A sonda se conecta ao console de outubro através de um motor e controlador de óptica (doca de montagem) com uma gama pullback de 54 mm. Os conjuntos de dados outubro adquiridos consistem em 541 imagens transversais (B-scans) wom uma resolução axial de 15 mm (Figura 1, 2). </li><li> Para garantir medições de atenuação precisos e reprodutíveis, calibre, medindo μ outubro de concentrações crescentes com base na percentagem em peso de uma emulsão de gordura, (por exemplo, Intralipid) como descrito anteriormente por Kodach et al. 14, 15. Em resumo: <ol><li> Dilui-se um lote padrão de 20% de emulsão de gordura com H2O desmineralizada para atingir concentrações de 0,125, 0,250, 0.5, 1.0, 2.0, 4.0, 10, 15 e 20 por cento (estoque). <ol><li> Coloque a sonda de outubro em 200 ml da mistura de emulsão de gordura e obter uma medição OCT. </li><li> Referência cruzada extraído μ outubro valores com os valores conhecidos na literatura. </li></ol></li></ol></li></ol> 2. Time Out e posicionamento do paciente <ol><li> Antes de iniciar o procedimento, execute um &quot;time out&quot; verificando nome, data de nascimento, o procedimento, procedural lado, o uso de anticoagulantes, e alergias. </li><li> Dependendo da localização do tumor, colocar o paciente em posição de decúbito ou propenso ou lateral. Proporcionar ao paciente um apoio adequado e verificar se ele / ela espera estar confortável nesta posição durante um período de 20 a 40 min. </li><li> Usando ultra-som (US) 16, localizar o tumor e marcar o ponto de entrada da agulha na pele com tinta permanente. NOTA: Ao usar a tomografia computadorizada (TC), use um modelo de orientação agulha flexível para localizar a posição preferida da agulha de acesso. </li></ol> 3. Desinfecção e estéril Draping <ol><li> Coloque em um boné e cobrir a boca cirúrgico. </li><li> Limpe a pele ao redor do local da punção, utilizando uma solução de clorexidina / álcool, tomando cuidado para não remover a marca de entrada da agulha previamente colocado (passo 2.3). Desinfecção uma vasta área evitará a necessidade de limpeza adicional em caso de inesperada reposicionamento agulha de acesso. </li><li> Com régard do conteúdo estéril, abrir o conjunto de punção percutânea contendo: uma seringa de 10 ml, uma aspiração com agulha sem corte, uma agulha de injeção G 21, um bisturi, a 15 G co-axial introdutor agulha, a 18 G trocar agulha, e um 16 G arma core biópsia. </li><li> Lave bem as mãos, aplicando desinfetante mão depois. Coloque em um avental cirúrgico e luvas estéreis. </li><li> Cubra o paciente em campos estéreis. </li><li> Aplicar uma cobertura estéril em torno da sonda de ultra-som e corrigir o guia de agulha no lugar. </li></ol> 4. outubro Preparação <ol><li> Inicie o console de outubro e digite os dados do paciente nos campos marcados paciente ID, apelido, nome próprio e DOB (data de nascimento), utilizando a interface do console. </li><li> No que diz respeito do conteúdo estéril, descompacte o pacote de outubro contendo uma sonda de outubro, uma cobertura estéril doca de montagem, e uma seringa de 5 ml luer-lock. </li><li> Aplique a cobertura estéril para a doca de montagem do console outubro. Guiando a doca de montagem não-estéril requer oajuda de um assistente. </li><li> Encha a seringa de 5 ml com 0,9% de NaCl e anexá-lo ao porto de descarga. Lave a sonda de outubro até que a água aparece na parte distal da cobertura de sonda. </li><li> Coloque a sonda de outubro no encaixe de montagem. Depois de colocar a sonda irá rodar e emitem luz vermelha, confirmando o bom funcionamento. Deixar a sonda em sua capa protetora durante a lavagem e carregar para minimizar o risco de danos. </li><li> Remova a sonda outubro de sua capa. Coloque a sonda sobre uma superfície dura e usar um bisturi para encurtar a ponta. Fixar a parte distal da sonda durante o corte, a fim de minimizar a pressão sobre a fibra óptica e prisma. Corte 5 milímetros distal do prisma, usando o (vermelho) luz emitida para orientação. </li></ol> 5. Puncture <ol><li> Anestesiar a pele e camadas profundas usando lidocaína a 2% (20 mg / ml). Aguarde alguns minutos, permitindo a lidocaína tenha efeito. Pergunte ao paciente se há alguma dor. </li><li> Usando o guia da agulha, coloque o15 G co-axial introdutor agulha verificar a posição através de imagens. Se a colocação for satisfatório, remova o obturador (core agulha fina). </li><li> Coloque a 18 G trocar agulha através da agulha de introdução, perfurando o tumor. Novamente verificar a posição da agulha com a imagem. Se a colocação é satisfatória remover o obturador. </li><li> Alimente a sonda de outubro até a agulha trocar até sentir resistência. </li><li> Enquanto que fixa a sonda de outubro, retrair a agulha trocar, expondo a sonda de outubro para o tecido do tumor. Mantendo a ponta da agulha de trocarte dentro do tumor minimiza a torção da sonda de outubro durante os ciclos respiratórios. Isto reduz o risco de dano da sonda. </li><li> Outubro de digitalização: <ol><li> Executar uma varredura outubro, com o console fixado em 541 B-scans por dataset. O sistema de outubro usada aqui vai realizar uma tração automática ao longo de um comprimento de 5,4 centímetros que não requerem ajustes de parâmetros específicos. </li><li> Confira o scan de qualidade, produtos manufacturados e o aparecimento de tecido sólido (Figura 1A). Artefactos mais comumente aparecem como faixas circulares pé para fora do padrão normal outubro (Figura 1B). </li><li> Substitua a sonda se artefactos persistir após um novo exame. </li></ol></li><li> Repita o passo 5.6 até um mínimo de 3 conjuntos de dados PTU são adquiridos. </li><li> Remova a sonda outubro e trocar agulha, deixando a agulha de introdução no local. </li><li> Arme a arma core biópsia e colocá-lo através da agulha de introdução, verificando a posição na imagem. </li><li> Se o posicionamento for satisfatório, disparar a arma biópsia. </li><li> Coloque material da biópsia em recipiente de acordo com protocolo de departamento de patologia. Aqui, lugar biópsias em uma placa de Petri com uma incrustação de papel, suficientemente saturado com NaCl 0,9%. </li><li> Verifique a qualidade core biópsia e repita o passo 5.9 e 5.10 até material suficiente é obtido. </li></ol>

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The authors of this article have nothing to disclose.

Nesta publicação, um relatório sobre a viabilidade de percutânea, com base agulha, OCT do rim. Este é um primeiro passo essencial no desenvolvimento de outubro em uma técnica clinicamente aplicável para a diferenciação do tumor, denominado como um &quot;Optical Biópsia&quot;. Os nossos primeiros 25 pacientes demonstraram percutânea outubro de ser um procedimento fácil e segura. Uma biópsia óptica tem duas vantagens sobre biópsias convencionais. Em primeiro lugar, a aquisição em tempo real e análise de ...

As últimas décadas têm demonstrado um aumento constante na incidência de massas renais 1,2. Até agora, as decisões de tratamento em massa renais foram feitas principalmente com base em características de imagem de ressonância magnética e tomografia computadorizada, idade e comorbidades. No entanto, estes métodos diagnósticos e parâmetros clínicos falta a finesse para detectar realmente o potencial maligno de uma massa renal. A biópsia aspirativa por agulha fina ou com tecido suficiente para a avaliação patológica (diagnóstico) proporciona diferenciação tumor objetivo com a sensibilidade e especificidade na faixa de 95-100% 3. Portanto biópsia está ganhando aceitação na avaliação de massas renais suspeitas 4,5. No entanto, as biópsias sem tecido suficiente para estabelecer um diagnóstico ou com o normal parênquima renal (não-diagnóstico) ocorrem a uma taxa de 10-20% do total, e mesmo até 30% em pequenas massas renais (&lt;4 cm, MRE), retardando o processo de diagnóstico, devido à necessidade freqüente de adicionalprocedimentos de biópsia de 3,5. Tomografia de coerência óptica (OCT) é uma modalidade de imagem romance que tem o potencial para superar os obstáculos acima mencionadas na diferenciação de massa renal. Com base na retroespalhamento de luz no infravermelho próximo, outubro fornece imagens com uma resolução axial 15 um a uma penetração eficaz do tecido de 2-3 mm (Figura 1, 2). A perda da intensidade do sinal por cada milímetro de penetração no tecido, resultante de um tecido específico de dispersão da luz, é expressa como o coeficiente de atenuação (μ OCT: -1 mm) como descrito por Faber et al 6.. As características histológicas podem ser correlacionados com μ valores outubro fornecendo um parâmetro quantitativo para a diferenciação de tecidos (Figura 3). Durante a carcinogênese, as células malignas exibir um número aumentou, núcleos maiores e mais uma forma irregular com<html> um índice de refracção mais elevado e mais mitocôndrias activas. Devido a esta sobre-expressão de componentes celulares, uma mudança de outubro μ é para ser esperado quando se compara a tumores malignos ou tumores benignos de tecido afectada 7. Recentemente, nós estudamos a capacidade da TCO superficial para diferenciar entre massas renais benignas e malignas 8,9. Em 16 pacientes, medições intra-operatórias outubro do tecido tumoral foram obtidas utilizando uma sonda outubro colocado externamente. O braço de controle, composto por medidas de outubro de tecido afetado nos mesmos pacientes. Tecido normal mostraram um coeficiente de atenuação média significativamente mais baixa em comparação com tecido maligno, confirmando o potencial de outubro para a diferenciação do tumor. Esta análise quantitativa foi aplicada de um modo semelhante ao grau de outros tipos de tecido maligno, tal como o carcinoma urotelial 10,11 e diferenciação neoplasia epitelial da vulva 12. ent &quot;&gt; Nosso objetivo é desenvolver outubro em uma biópsia óptica, fornecendo imagens em tempo real combinada com diferenciação tumor on-the-spot. O objetivo do presente estudo é descrever um percutânea, com base agulha, outubro abordagem em pacientes diagnosticados com um massa renal reforço sólido. Esta descrição do método é, a nosso conhecimento, o primeiro a avaliar a possibilidade de agulha outubro base dos tumores renais.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

Entre os primeiros 25 tumores (23 pacientes), foram realizados um total de 24 procedimentos de outubro de sucesso. Num caso de uma avaria sonda levou à incapacidade de adquirir uma varredura OCT. Dois eventos adversos (AE) ocorreram, que são descritos em detalhe na secção discussão. As características gerais do paciente são encontrados na Tabela 1. O console de outubro tem software pré-instalado fornecendo em tempo real imagens outubro para análise qualitativa imedi...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Na Vivo, percutânea, Agulha Based, Tomografia de Coerência Óptica de massas renais

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

8.8K visualizações.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Vídeo: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Doppler Tomografia de Coerência Óptica de circulação retiniana

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Medicina

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

Perto Tomografia Projeção óptico infravermelho para avaliações de β-célula distribuição em massa em Pesquisa do Diabetes

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Geração e quantificação 3-Dimensional de Arterial lesões em ratos por meio de tomografia óptica de Projeção

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparação e Avaliação respirométrico de mitocôndrias isoladas de tecido muscular esquelético Obtido por meio de agulha de biópsia percutânea

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Avaliação transcutânea da função renal em roedores conscientes

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Tomografia de coerência óptica Mouse ultra alta resolução para auxiliar a injeção intra-ocular em pesquisa de terapia de genes da retina

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Na Vivo Imagem de Cx3cr1gfp/gfp repórter ratos com a tomografia de coerência óptica espectral-domínio e digitalização Laser Oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Avaliação da função renal em modelos do rato da doença Glomerular

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...