Agradecemos a los niños participantes y sus familias, todo el personal del Hospital Infantil de Boston (CHB), con especial agradecimiento a los técnicos de la RM, sin los cuales nuestros estudios no se pudo realizar. Estamos especialmente agradecidos a Arnold Cyr para la participación y ayuda durante nuestra sesión de vídeo y Gerente de Operaciones Patricia Devine en el Waltham CHB. Gracias a la investigación y el personal técnico de Athinoula MIT A. Martino Centro de Imagen para el intercambio de experiencias en la adquisición de imagen de resonancia magnética y análisis. Esta investigación fue financiada por el Charles H. Hood Foundation, del Hospital Infantil de Boston piloto de subvención y los suizos de la Fundación Nacional (RMN)

<ol>
	<li>Boecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+stage+of+fMRI+applications+in+newborns+and+children+during+the+first+year+of+life.">Current stage of fMRI applications in newborns and children during the first year of life.</a> Rofo. 180 (8), 707-714 (2008).</li><li>Bookheimer, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+issues+in+pediatric+neuroimaging.">Methodological issues in pediatric neuroimaging.</a> Ment Retard Dev Disabil Res Rev. 6 (3), 161-165 (2000).</li><li>Souweidane, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+mapping+in+sedated+infants+and+young+children+with+passive-functional+magnetic+resonance+imaging.">Brain mapping in sedated infants and young children with passive-functional magnetic resonance imaging.</a> Pediatr Neurosurg. 30 (2), 86-92 (1999).</li><li>Poldrack, R. A., Pare-Blagoev, E. J., Grant, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+functional+magnetic+resonance+imaging:+progress+and+challenges.">Pediatric functional magnetic resonance imaging: progress and challenges.</a> Top Magn Reson Imaging. 13 (1), 61-70 (2002).</li><li>Macmaster, F. P., Rosenberg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+children+for+MRI.">Preparing children for MRI.</a> Pediatr Radiol. 38 (3), 270-270 (2008).</li><li>Wilke, M., Holland, S. K., Myseros, J. S., Schmithorst, V. J., Ball, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+magnetic+resonance+imaging+in+pediatrics.">Functional magnetic resonance imaging in pediatrics.</a> Neuropediatrics. 34 (5), 225-233 (2003).</li><li>Davidson, M. C., Thomas, K. M., Casey, B. J., J, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+developing+brain+with+fMRI.">Imaging the developing brain with fMRI.</a> Ment Retard Dev Disabil Res Rev. 9 (3), 161-167 (2003).</li><li>Sury, M. R., Harker, H., Begent, J., Chong, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+management+of+infants+and+children+for+painless+imaging.">The management of infants and children for painless imaging.</a> Clin Radiol. 60 (7), 731-741 (2005).</li><li>Hunt, R. H., Thomas, K. M. <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+methods+in+developmental+science:+a+primer.">Magnetic resonance imaging methods in developmental science: a primer.</a> Dev Psychopathol. 20 (4), 1029-1051 (2008).</li><li>O'Shaughnessy, E. S., Berl, M. M., Moore, E. N., Gaillard, W. D., D, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+functional+magnetic+resonance+imaging+(fMRI):+issues+and+applications.">Pediatric functional magnetic resonance imaging (fMRI): issues and applications.</a> J Child Neurol. 23 (7), 791-801 (2008).</li><li>Raschle, N. M., Chang, M., Lee, M., Buechler, R., Gaab, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+Behavioral+and+Neural+Pre-Markers+of+Developmental+Dyslexia+in+Children+Prior+to+Reading+Onset.">Examining Behavioral and Neural Pre-Markers of Developmental Dyslexia in Children Prior to Reading Onset.</a> , (1985).</li><li>Pressdee, D., May, L., Eastman, E., Grier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+play+therapy+in+the+preparation+of+children+undergoing+MR+imaging.">The use of play therapy in the preparation of children undergoing MR imaging.</a> Clin Radiol. 52 (12), 945-947 (1997).</li><li>Slifer, K. J., Cataldo, M. F., Cataldo, M. D., Llorente, A. M., Gerson, A. 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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=A+video+system+to+help+children+cooperate+with+motion+control+for+radiation+treatment+without+sedation.">A video system to help children cooperate with motion control for radiation treatment without sedation.</a> J Pediatr Oncol Nurs. 13 (2), 91-97 (1996).</li><li>Tyc, V. L., Fairclough, D., Fletcher, B., Leigh, L., Mulhern, R. K., K, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Children's+distress+during+magnetic+resonance+imaging+procedures.">Children's distress during magnetic resonance imaging procedures.</a> Child Health Care. 24 (1), 5-19 (1995).</li><li>Slifer, K. J., Koontz, K. L., Cataldo, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant-contingency-based+preparation+of+children+for+functional+magnetic+resonance+imaging.">Operant-contingency-based preparation of children for functional magnetic resonance imaging.</a> J Appl Behav Anal. 35 (2), 191-194 (2002).</li><li>Byars, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+conducting+large-scale+functional+magnetic+resonance+imaging+studies+in+children.">Practical aspects of conducting large-scale functional magnetic resonance imaging studies in children.</a> J Child Neurol. 17 (12), 885-890 (2002).</li><li>Rosenberg, D. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reviewing+the+process+of+preparing+children+for+MRI.">Reviewing the process of preparing children for MRI.</a> Pediatr Radiol. 38 (3), 271-279 (2008).</li><li>Preston, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Testing children : a practitioner's guide to the assessment of mental development in infants and young children. , (2005).</li><li>Kotsoni, E., Byrd, D., Casey, B. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+virtual+reality+distraction+to+reduce+claustrophobia+symptoms+during+a+mock+magnetic+resonance+imaging+brain+scan:+a+case+report.">Use of virtual reality distraction to reduce claustrophobia symptoms during a mock magnetic resonance imaging brain scan: a case report.</a> Cyberpsychol Behav. 10 (3), 485-488 (2007).</li><li>Cho, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+acoustic+noise+in+MRI.">Analysis of acoustic noise in MRI.</a> Magn Reson Imaging. 15 (7), 815-822 (1997).</li><li>Gaab, N., Gabrieli, J. D., Glover, G. 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An fMRI study comparing auditory processing in the absence and presence of recorded scanner noise using a sparse design.</a> Hum Brain Mapp. 28 (8), 721-732 (2007).</li><li>Bilecen, D., Radu, E. W., Scheffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MR+tomograph+as+a+sound+generator:+fMRI+tool+for+the+investigation+of+the+auditory+cortex.">The MR tomograph as a sound generator: fMRI tool for the investigation of the auditory cortex.</a> Magn Reson Med. 40 (6), 934-937 (1998).</li><li>Eden, G. F., Joseph, J. E., Brown, H. E., Brown, C. P., Zeffiro, T. 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C., Pike, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Event-related+fMRI+of+the+auditory+cortex.">Event-related fMRI of the auditory cortex.</a> Neuroimage. 10 (4), 417-429 (1999).</li><li>Gaab, N., Gaser, C., Zaehle, T., Jancke, L., Schlaug, G., G, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+pitch+memory--an+fMRI+study+with+sparse+temporal+sampling.">Functional anatomy of pitch memory--an fMRI study with sparse temporal sampling.</a> Neuroimage. 19 (4), 1417-1426 (2003).</li><li>Hall, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Sparse"+temporal+sampling+in+auditory+fMRI.">"Sparse" temporal sampling in auditory fMRI.</a> Hum Brain Mapp. 7 (3), 213-223 (1999).</li><li>Gaab, N., Gabrieli, J. D., Glover, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+in+peace+or+noise:+scanner+background+noise+suppresses+default-mode+network.">Resting in peace or noise: scanner background noise suppresses default-mode network.</a> Hum Brain Mapp. 29 (7), 858-867 (2008).</li></ol>

The authors have nothing to disclose.

La aparición de la resonancia magnética funcional y estructural para estudiar el cerebro humano ha facilitado la posibilidad de examinar típicos, así como la estructura cerebral atípica y las funciones y por lo tanto tiene un gran potencial tanto para fines de investigación y clínica 6. Sin embargo, los estudios de resonancia magnética en los grupos de edad más jóvenes siguen siendo menos numerosos en comparación con las de los adultos, adolescentes o niños, que se debe p...

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

En la última década ha habido un aumento en el uso de la resonancia estructural y magnética funcional (fMRI) para investigar las bases neurales de la percepción humana cognición y el comportamiento<sup&gt; 1, 2</sup&gt;. Además, este método de imagen no invasivas se ha convertido en una herramienta para los médicos y los investigadores a explorar el desarrollo del cerebro típicos y atípicos. A pesar de los avances en las herramientas y las técnicas de neuroimagen son evidentes, (f) de resonancia magnética en los jóvenes la población pediátrica es relativamente infrecuente<sup&gt; 2</sup&gt;. Problemas prácticos, así como técnicas de imagen cuando los niños presentan los médicos y los equipos de investigación con un conjunto único de problemas<sup&gt; 3, 2</sup&gt;. Para nombrar sólo algunos, los participantes del niño son desafiados por la necesidad de motivación, el estado de alerta y la cooperación. La ansiedad puede ser un factor adicional a tener en cuenta. Los investigadores o médicos deben tener en cuenta las limitaciones de tiempo, la restricción de movimiento, el ruido de fondo del escáner y la falta de familiaridad con el medio ambiente escáner de RM<sup&gt; 2,4-10</sup&gt;. Un uso progresivo de la neuroimagen funcional y estructural de los grupos más jóvenes, sin embargo, aún podría añadir a nuestra comprensión del desarrollo del cerebro. A modo de ejemplo, varios grupos de investigación están trabajando actualmente en la detección temprana de trastornos del desarrollo, potencialmente, incluso antes de que los niños presentan características de comportamiento asociadas<sup&gt; Eg11</sup&gt;. Diversas estrategias y técnicas se han reportado como un medio para garantizar la comodidad y la cooperación de los niños pequeños durante las sesiones de neuroimagen. Terapia de juego<sup&gt; 12</sup&gt;, Los enfoques conductuales<sup&gt; 13, 14,15, 16-18</sup&gt; Y la simulación<sup&gt; 19</sup&gt;, El uso de áreas escáner simulacro<sup&gt; 20,21</sup&gt;, La relajación básica<sup&gt; 22</sup&gt; Y una combinación de estas técnicas<sup&gt; 23</sup&gt;, Han demostrado para mejorar el cumplimiento de los participantes y calidad de los datos tanto de resonancia magnética. Aún más importante, estas estrategias han demostrado que aumentan la comodidad de las familias y los niños que participan<sup&gt; 12</sup&gt;. Uno de los principales avances de estas técnicas para la práctica clínica es la posibilidad de evitar la sedación o anestesia general (GA) como una forma de gestionar el cumplimiento de los niños durante las sesiones de imágenes MR<sup&gt; 19,20</sup&gt;. En el informe de vídeo actual, se presenta un protocolo de neuroimagen pediátrica con las directrices y procedimientos que han demostrado ser exitosa hasta la fecha en los niños pequeños.

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&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

A pesar del aumento en el uso de la resonancia estructural y magnética funcional (fMRI) en los seres humanos, el estudio de la joven población infantil sigue siendo un desafío. Se presenta un práctico, paso a paso el protocolo de video, incluyendo directrices para los médicos e investigadores con la intención de realizar (f) de resonancia magnética en niños pequeños.

Hacer jugar MR Imaging Niño - Protocolo neuroimagen pediátrica, Directrices y Procedimientos

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

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

18.0K vistas.  Children’s Hospital Boston. A pesar del aumento en el uso de la resonancia estructural y magnética funcional (fMRI) en los seres humanos, el estudio de la joven población infantil sigue siendo un desafío. Se presenta un práctico, paso a paso el protocolo de video, incluyendo directrices para los médicos e investigadores con la intención de realizar (f) de resonancia magnética en niños pequeños.

Video: 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.

Un procedimiento de cirugía craneotomía para imágenes cerebrales crónicas

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

Investigación

Biología

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.

Etiquetado y hESCs hMSCs con nanopartículas de óxido de hierro para no invasivo en el seguimiento in vivo mediante RM

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

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.

Hacer patch pipetas y electrodos de Sharp con un extractor programable

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

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Banca cerebro: Aprovechar al máximo las muestras de su investigación

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

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.

Bioluminiscencia de imágenes de hemo oxigenasa-1 Upregulation en el procedimiento 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.

Hacer pez cebra Gynogenetic diploides por presión 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.

Preparación de las células acinares pancreáticas para los fines de Imagen de calcio, Lesiones Medidas Celulares e infección viral

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.

Protocolo optimizado para Retina Preparación Wholemount de la Imagen y la inmunohistoquímica

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.

Diseño y Análisis Del Comportamiento De Preferencia De Temperatura Y Su Ritmo Circadiano En 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 CÚBICO Visualiza la expresión de la proteína en la resolución de la celda individual en su totalidad para la piel Mount

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