Wir bedanken uns bei teilnehmenden Kinder und Familien, alle Mitarbeiter an der Kinderklinik Boston (CHB) mit besonderer Wertschätzung für die MRI-Techniker, ohne die unsere Untersuchungen nicht durchgeführt werden konnte. Wir sind besonders dankbar, Arnold Cyr für die Teilnahme und Hilfe bei unserem Videodreh und Betriebswirt Patricia Devine am CHB Waltham. Dank der Forschung und technischen Mitarbeiter am MIT Athinoula A. Martinos Imaging Center für den Austausch von Erfahrungen in MR Bildaufnahme und-analyse. Diese Arbeit wurde von der Charles H. Hood Foundation, ein Kinderkrankenhaus Boston Pilot zu gewähren und den Schweizerischen Nationalfonds (NMR) finanziert

<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. 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The authors have nothing to disclose.

Die Entstehung von funktionellen und strukturellen MRI, das menschliche Gehirn Studie hat Möglichkeiten untersucht typischen sowie atypischen Struktur des Gehirns und Funktionen erleichtert und daher ist viel versprechend für die Forschung und klinische Zwecke 6. Allerdings bleiben MRT-Studien in den jüngeren Altersgruppen weniger zahlreich, als mit denen von Erwachsenen, Jugendlichen oder Kindern, die vor allem aus technischen und praktischen Schwierigkeiten bei der Durchführung...

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

Innerhalb des letzten Jahrzehnts hat es eine Zunahme bei der Nutzung von strukturellen und funktionellen Kernspintomographie (fMRI), um die neuronalen Grundlagen der menschlichen Wahrnehmung, Kognition und Verhalten zu untersuchen wurden<sup&gt; 1, 2</sup&gt;. Darüber hinaus hat diese nicht-invasive bildgebende Verfahren in ein Werkzeug für Kliniker und Forscher geworden, typische und atypische Entwicklung des Gehirns zu erforschen. Obwohl Fortschritte in der Bildgebung und-techniken offensichtlich sind, (f) MRI in jungen pädiatrischen Populationen noch relativ selten<sup&gt; 2</sup&gt;. Praktische sowie technischen Herausforderungen bei der Abbildung Kinder Kliniker und Forscherteams präsentieren eine einzigartige Reihe von Problemen<sup&gt; 3, 2</sup&gt;. Um nur einige zu nennen, werden die untergeordneten Teilnehmer durch eine Notwendigkeit für die Motivation, Aufmerksamkeit und Zusammenarbeit in Frage gestellt. Angst kann ein zusätzlicher Faktor angesprochen werden. Forscher oder Kliniker müssen Zeitdruck, Bewegungseinschränkung, Scanner Hintergrundgeräusche und Vertrautheit mit den MR-Scanner Umgebung berücksichtigen<sup&gt; 2,4-10</sup&gt;. Eine progressive Verwendung von funktionellen und strukturellen Bildgebung in jüngeren Altersgruppen, konnte aber weiter zu unserem Verständnis der Entwicklung des Gehirns hinzu. Als Beispiel sind mehrere Forschungsgruppen arbeitet derzeit an der Früherkennung von Entwicklungsstörungen und möglicherweise sogar vor Kindern in Verbindung gebracht Verhaltensmerkmale präsentieren<sup&gt; Eg11</sup&gt;. Verschiedene Strategien und Techniken als Mittel, um Komfort und die Zusammenarbeit von kleinen Kindern beim Neuroimaging-Sitzungen zu gewährleisten berichtet worden. Play-Therapie<sup&gt; 12</sup&gt;, Verhaltenstherapeutische Ansätze<sup&gt; 13, 14,15, 16-18</sup&gt; Und Simulation<sup&gt; 19</sup&gt; Die Verwendung von Mock-Scanner Bereichen<sup&gt; 20,21</sup&gt;, Grundlegende Entspannung<sup&gt; 22</sup&gt; Und eine Kombination dieser Techniken<sup&gt; 23</sup&gt; Haben alle gezeigt, dass die Teilnehmer die Einhaltung zu verbessern und damit MRT-Daten-Qualität. Noch wichtiger ist, haben diese Strategien bewährt, um den Komfort von Familien und Kindern beteiligt zu erhöhen<sup&gt; 12</sup&gt;. Einer der wichtigsten Fortschritte solcher Techniken für die klinische Praxis ist die Möglichkeit der Vermeidung von Sedierung oder Narkose (GA) als eine Möglichkeit, Kinder-Compliance während der MR-Bildgebung Sitzungen verwalten<sup&gt; 19,20</sup&gt;. In den aktuellen Video-Bericht präsentieren wir eine pädiatrische Neuroimaging-Protokoll mit Richtlinien und Verfahren, die sich als erfolgreich erwiesen bisher in kleine Kinder haben.

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

Trotz eines Anstiegs in der Verwendung von strukturellen und funktionellen Kernspintomographie (fMRI) beim Menschen, bleibt das Studium der jungen pädiatrischen Populationen eine große Herausforderung. Wir präsentieren ein Hands-on-, Schritt-für-Schritt-Video-Protokoll einschließlich Leitlinien für die Kliniker und Forscher beabsichtigen, (f) MRI bei kleinen Kindern durchzuführen.

Erstellen MR Imaging Kinderspiel - Pediatric Neuroimaging-Protokoll, Richtlinien und Verfahren

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 Aufrufe.  Children’s Hospital Boston. Trotz eines Anstiegs in der Verwendung von strukturellen und funktionellen Kernspintomographie (fMRI) beim Menschen, bleibt das Studium der jungen pädiatrischen Populationen eine große Herausforderung. Wir präsentieren ein Hands-on-, Schritt-für-Schritt-Video-Protokoll einschließlich Leitlinien für die Kliniker und Forscher beabsichtigen, (f) MRI bei kleinen Kindern durchzuführen.

Serie: 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.

Ein Kraniotomie Chirurgie Verfahren für chronische Brain Imaging

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

Forschung

Biologie

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.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

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.

Machen Patch-Pipetten und Sharp Elektroden mit einem programmierbaren Puller

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.

Brain-Banking: Making the Most Ihrer Forschung Proben

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.

Biolumineszenz-Bildgebung von HO-1 Hochregulation in der 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 ...

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.

Machen Gynogenetic Diploid Zebrafisch von Anfang Druck

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.

Herstellung von Azinuszellen des Pankreas für die Zwecke von Calcium Imaging, Cell Verletzung Messungen und Adenovirus-Infektion

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.

Design und Analyse des Temperaturpräferenzverhaltens und seines circadianen Rhythmus in 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.

CUBIC Protokoll Visualisiert Expression Protein bei Single Cell Resolution in Ganze Berge Vorbereitung der Haut

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