Agradecemos a participação das crianças e famílias, todos os funcionários do Hospital Infantil de Boston (CHB), com especial apreço para os técnicos de ressonância magnética, sem o qual os nossos estudos não poderia ser realizado. Estamos especialmente gratos a Arnold Cyr para a participação e ajuda durante a nossa sessão de vídeo e operação de gerente de Patricia Devine no Waltham CHB. Graças à pesquisa e equipe técnica da Athinoula do MIT A. Martinos Imaging Center para troca de experiências no MR aquisição e análise de imagem. Esta pesquisa foi financiada pela Fundação H. Charles capa, um Hospital Infantil de Boston piloto subvenção ea Fundação Nacional Suíço (NMR)

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+children+without+sedation:+preparation+with+simulation.">Magnetic resonance imaging of children without sedation: preparation with simulation.</a> J Am Acad Child Adolesc Psychiatry. 36 (6), 853-859 (1997).</li><li>de Amorim e Silva, C. J., Mackenzie, A., Hallowell, L. M., Stewart, S. E., Ditchfield, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practice+MRI:+reducing+the+need+for+sedation+and+general+anaesthesia+in+children+undergoing+MRI.">Practice MRI: reducing the need for sedation and general anaesthesia in children undergoing MRI.</a> Australas Radiol. 50 (4), 319-323 (2006).</li><li>Epstein, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+prevention+of+head+motion+during+imaging+of+patients+with+attention+deficit+hyperactivity+disorder.">Assessment and prevention of head motion during imaging of patients with attention deficit hyperactivity disorder.</a> Psychiatry Res. 155 (1), 75-82 (2007).</li><li>Lukins, R., Davan, I. G., Drummond, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cognitive+behavioural+approach+to+preventing+anxiety+during+magnetic+resonance+imaging.">A cognitive behavioural approach to preventing anxiety during magnetic resonance imaging.</a> J Behav Ther Exp Psychiatry. 28 (2), 97-104 (1997).</li><li>Hallowell, L. M., Stewart, S. E., Amorim, E. S. C. T. d. e., Ditchfield, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+considerations+for+functional+magnetic+resonance+imaging+of+pediatric+populations.">Special considerations for functional magnetic resonance imaging of pediatric populations.</a> J Magn Reson Imaging. 23 (6), 877-886 (2006).</li><li>Armstrong, T. S., Aitken, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+role+of+play+preparation+in+paediatric+anaesthesia.">The developing role of play preparation in paediatric anaesthesia.</a> Paediatr Anaesth. 10 (1), 1-4 (2000).</li><li>Garcia-Palacios, A., Hoffman, H. G., Richards, T. R., Seibel, E. J., Sharar, S. 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The authors have nothing to disclose.

O surgimento de ressonância magnética funcional e estrutural para estudar o cérebro humano tem facilitado possibilidades de examinar típicas, bem como a estrutura do cérebro e as funções atípicas e, portanto, é uma grande promessa para os dois fins de investigação e clínica 6. No entanto, exames de ressonância magnética em grupos etários mais jovens permanecem menor quantidade quando comparados aos de adultos, adolescentes ou crianças, que é principalmente devido a d...

making mr imaging child's play - pediatric neuroimaging protocol, guidelines and procedure

Na última década tem havido um aumento no uso da ressonância magnética estrutural e funcional (fMRI) para investigar as bases neurais da percepção humana, cognição e comportamento<sup&gt; 1, 2</sup&gt;. Além disso, este método de imagem não invasivos tem crescido em uma ferramenta para os médicos e pesquisadores para explorar o desenvolvimento do cérebro típicos e atípicos. Apesar de avanços nas ferramentas e técnicas de neuroimagem são aparentes, (f) MRI em jovens da população pediátrica permanece relativamente pouco frequentes<sup&gt; 2</sup&gt;. Desafios práticos bem como assistência técnica, quando as crianças apresentam imagens de médicos e equipes de pesquisa com um único conjunto de problemas<sup&gt; 3, 2</sup&gt;. Para citar apenas alguns, os participantes são desafiados criança por uma necessidade de atenção, motivação e cooperação. A ansiedade pode ser um fator adicional a ser abordado. Pesquisadores ou clínicos precisam considerar restrições de tempo, restrição de movimento, ruído de fundo scanner e pouca familiaridade com o ambiente scanner MR<sup&gt; 2,4-10</sup&gt;. A progressiva utilização de técnicas de neuroimagem funcional e estrutural em grupos etários mais jovens, no entanto, poderia aumentar ainda mais a nossa compreensão do desenvolvimento do cérebro. Como exemplo, vários grupos de pesquisa estão atualmente trabalhando para a detecção precoce de transtornos de desenvolvimento, potencialmente mesmo antes de as crianças presentes características comportamentais associados<sup&gt; Eg11</sup&gt;. Diversas estratégias e técnicas têm sido relatados como um meio para assegurar o conforto ea cooperação das crianças durante as sessões de neuroimagem. Ludoterapia<sup&gt; 12</sup&gt;, Abordagens comportamentais<sup&gt; 13, 14,15, 16-18</sup&gt; E simulação<sup&gt; 19</sup&gt;, O uso de áreas scanner simulada<sup&gt; 20,21</sup&gt; Relaxamento, básico<sup&gt; 22</sup&gt; E uma combinação destas técnicas<sup&gt; 23</sup&gt; Têm sido mostrados para melhorar a adesão do participante e, portanto, MRI qualidade dos dados. Mais importante ainda, estas estratégias têm provado para aumentar o conforto das famílias e crianças envolvidas<sup&gt; 12</sup&gt;. Um dos principais avanços de tais técnicas para a prática clínica é a possibilidade de evitar sedação ou anestesia geral (AG) como uma forma de gerenciar a conformidade das crianças durante as sessões de ressonância magnética<sup&gt; 19,20</sup&gt;. No relatório de vídeo atual, apresentamos um protocolo de neuroimagem pediátricos com diretrizes e procedimentos que provaram ser bem sucedido até à data em crianças pequenas.

Watch this Scientific Journal Video about Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure at JoVE.com

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Apesar do aumento no uso de imagens de ressonância magnética estrutural e funcional (fMRI) em humanos, o estudo de jovens populações pediátricas ainda é um desafio. Nós apresentamos um hands-on, protocolo de vídeo passo-a-passo e orientações para os médicos e pesquisadores que pretendam realizar (f) a RM em crianças pequenas.

Jogue Criança fazendo MR Imaging - Protocolo de neuroimagem Pediátrica, Diretrizes e Procedimento

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

making-mr-imaging-child-s-play-pediatric-neuroimaging-protocol

Research

JoVE Journal

Biology

18.1K visualizações.  Children’s Hospital Boston. Apesar do aumento no uso de imagens de ressonância magnética estrutural e funcional (fMRI) em humanos, o estudo de jovens populações pediátricas ainda é um desafio. Nós apresentamos um hands-on, protocolo de vídeo passo-a-passo e orientações para os médicos e pesquisadores que pretendam realizar (f) a RM em crianças pequenas.

Vídeo: Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Um procedimento de cirurgia Craniotomia para Brain Imaging crônica

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Biologia

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Rotulagem e hESCs hMSCs com óxido de ferro nanopartículas para Não-invasiva em Rastreamento vivo com ressonância magnética

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Isolamento de neutrófilos Protocolo

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Tornando-Patch pipetas e Eletrodos Sharp com um extrator programável

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Imagem de bioluminescência de heme oxigenase-1 Upregulation no Procedimento de Gua Sha

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Fazendo Zebrafish Diplóide Gynogenetic por pressão inicial

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

Preparação de células pancreáticas acinares para Fins de cálcio Imaging, celulares Medidas ferimento e infecção adenoviral

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Protocolo CUBIC Visualiza Protein Expression na resolução única célula em preparações e peles, inteiros Mount

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...