K. Wong, STC Wong durch die Finanzierung von Functional and Molecular Imaging Center, Brigham and Womens Hospital unterstützt K. Wong, STC Wong sind durch das Center for Biotechnology and Informatics, The Methodist Hospital Research Institute, Weill Cornell Medical College unterstützt. KK Kwong, I. Chen, JQ Ren werden durch die Mittel aus dem Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital unterstützt. Lenuta Klötzer, Braden Kuo werden durch Mittel aus dem Neuroenteric Research Center, Massachusetts General Hospital unterstützt Die Autoren danken Drs. Q. Zeng und X. Xu von Optical Imaging Lab, Brigham and Womens Hospital for technische Hilfe.

<ol><li> Weibliche HO-1-Luciferase transgenen Mäusen (Alter von 4-6 Wochen) von Taconic Farms, Inc. kann bei der Ankunft erworben werden, sind Mäuse in ein Tier Wohnanlage für mindestens 4 Tage platziert, um Unterkunft zu ermöglichen. </li><li> Vorbereitung für die Biolumineszenz-Bildgebung: <ol style="list-style-type:lower-alpha;"><li> Wiegen Sie jedes HO-1-Maus. Tierische Gewicht ist für die Berechnung der Dosierung von Luciferin erforderlich. </li><li> Haarentfernung: Verwenden Sie ein Wattestäbchen, um Haarentferner Nair ® über den Bauch und / oder Rücken des Tieres gelten. Warten auf 5 bis 10 Sekunden. Verwenden Sie ein sauberes Wattestäbchen abzuwischen Haar. Tauchen Sie ein Stück Gewebe in destilliertem Wasser zu reinigen restlichen Haare zu wischen. </li><li> Legen Sie eine nicht-fluoreszierenden schwarzem Papier (Strathmore Artagain ® schwarzes Papier) auf dem Imaging-Plattform eines IVIS 100 Rufnummer um Hintergrundgeräusche zu reduzieren. </li><li> Bereiten Luciferin (von Xenogen Corporation)-Lösung mit einer Konzentration von 7,5 mg / ml (gelöst in sterilem H 2 O). Die Dosis von Luciferin bei 65,5 mg / kg Körpergewicht. So erfordert ein 20-Gramm-Maus intraperitoneale Injektion von 0,175 ml Luciferin-Lösung. Um dies zu erreichen, ist eine einmalige Injektion Volumen von Luciferin-Lösung (0.175ml), um eine Spritze mit einer 26-Gauge-Nadel vorinstalliert </li></ol></li><li> Gua Sha Verfahren: Gua Sha ist nur einmal vor der Ausführung des Biolumineszenz-Bildgebung Protokoll angewendet. Da Gua Sha ist nicht schmerzhaft, muss die Maus nur zu kurz betäubt durch Isofluran, ruhig zu bleiben. Gua Sha Verfahren umfasst folgende Schritte: <ol style="list-style-type:lower-alpha;"><li> Bewerben Speiseöl oder destilliertem Wasser auf Hautbereiche durch Gua Sha gezielt ein paar Mal während der Gua Sha Verfahren zu schmieren. </li><li> Wiederholt kratzen die Haar-freie Region der Rückseite der Maus in sanfte, aber feste Kraft mit einer keramischen Suppenlöffel oder einem Plastiklöffel. </li><li> Das Schaben wird fortgesetzt, bis der Rücken Haut rot wird, ist das ein Zeichen der subkutanen Blutextravasat, in der Regel innerhalb von 2 bis 3 Minuten erreicht. </li></ol></li><li> Biolumineszenz-Bildgebung kann sofort oder einige Stunden nach Gua Sha gestartet werden als HO-1 Hochregulation wird langsam über einige Stunden hinweg gebaut. Das Verfahren für die Biolumineszenz-Bildgebung ist: <ol style="list-style-type:lower-alpha;"><li> Anesthetize der Maus in einer Narkose Kammer mit einer Mischung aus Isofluran (1,5%) und medizinischem Luft gefüllt. </li><li> Sobald die Maus narkotisiert, bewegen Sie die Maus, um die Imaging-Kammer auf der IVIS 100 optische Bildgebung Station. Bewegen Sie die Maus in Rückenlage (Bauch nach oben). Die Imaging-Kammer wird kontinuierlich mit 1,5% Isofluran infundiert. Die Imaging-Plattform wird bei 37 ° C erhitzt, damit die Maus warm. </li><li> Stellen Sie die Imaging-Akquisition &quot;medium Binning&quot; und die Belichtung auf 30 Sekunden sein. Start, um Bilder zu erwerben. Stellen Sie das Gerät zu wiederholen Imaging Acquisition alle drei Minuten, entweder manuell oder automatisch. Nach dem Erwerb des ersten Bildes, ist eine Region of Interest (ROI) erstellt, die Bauch-, Brust-und Kopfbereich zu decken. Diese ROI wird dann kopiert und eingefügt, um die folgenden Bilder mit &quot;Living Image ®&quot;, Software begleitenden IVIS 100 Rufnummer. </li><li> Die Signalintensität ist in Photonen pro Sekunde gemessen. Mark Zeit für die Signalintensität zu erreichen ihren Höhepunkt Wert und halten auf die Bildgebung mit der Maus für etwa 5 bis 10 Minuten nach dem Peak-Zeit. </li><li> Wenn die Imaging Acquisition bei der Rückenlage geschehen ist, drehen Sie die Maus über und legen Sie sie in der Bauchlage (back up). Weiter Bildgebung das Tier für 5 bis 10 Minuten mit dem ROI jetzt gezogen, um die Rücken-und Kopfbereich in die Nutzung der Software &quot;Living Image ®&quot; zu decken. Die Wahl der Bildgebung der Bauchlage nach der Rückenlage ist beliebig. Man kann wählen, um mit der Bauchlage beginnen, abhängig von der primären Organe von Interesse. Eine weitere Option ist zur Abbildung der Rücken-und Bauchlage auf getrennten Experimenten. </li><li> Wenn die Imaging Acquisition bei der Bauchlage beendet ist, schalten Sie Isofluran und bewegen Sie die Maus aus dem Imaging-Kammer zur Induktion Kammer sich zu erholen. Die Induktion Kammer wird nun mit medizinischer Luft nur die Maus in der Regel wacht in weniger als einer Minute infundiert. </li><li> Speichern von Bilddaten für die Nachbearbeitung. </li><li> Nach der ersten Imaging-Studie, sind zusätzliche bildgebende Akquisitionen für mehrere Tage nach Gua Sha durchgeführt. Eine Probe Zeitplan Bildgebung an der 12 th Stunde, 24 th Stunde, 36 th Stunden, 48 th Stunde 72. Stunde und 120. Stunde wiederholt, aber der genaue Zeitpunkt der Bildgebung Zeitplan ist flexibel. Zusätzliche Imaging-Sitzungen können hinzugefügt werden, wenn eine längere Follow-up benötigt wird. Vor Bildgebung, wiederholen Sie die Haare entfernen Verfahren, wenn die Haare wieder gewachsen ist. </li></ol></li><li> Für die Kontrollgruppe, wiederholen Sie alle Verfahren, sondern überspringen Gua Sha (Schritt 3). Wenn die gleiche Gruppe von Mäusen ist sowohl für Steuerungs-und Gua Sha, der Steuerung exp verwendeteriment sollte vorzugsweise vor dem Gua sha Verfahren durchgeführt oder mindestens einen Monat danach. </li></ol> Repräsentative Ergebnisse: Die Biolumineszenz Bilder in Abbildung 1 zeigen in vivo Hochregulation von HO-1 in Reaktion auf Gua Sha. Der Graph in Abbildung 2 zeigt die quantitative zeitliche Änderung mehr als 120 Stunden im optischen Fluss (Photonen / sec) aus dem ganzen Körper die gleiche Maus im Zusammenhang mit Gua Sha ... <img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="Abbildung 1" border="0" /> Abbildung 1. Von links nach rechts, repräsentative Bilder der Frontansicht (Rückenlage) der gleichen Maus vor Gua Sha, bei 18 Stunden, 36 Stunden und 120 Stunden nach der Gua Sha, jeweils. Nach Gua Sha, beobachtet man den Fortschritt der signifikante Anstieg der Signalintensität mehrere Organe, die Regionen des Magen-Darm-Trakt, im Genitalbereich, die Leber, Nieren (von hinten sehen, nicht gezeigt), und andere umfassen. Bitte <a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg">klicken Sie hier</a> für eine größere Version der Abbildung 1. <img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="Abbildung 2" border="0" /> Abbildung 2. Quantitative Flussänderung (Photonen / sec) aus dem ganzen Körper über 120 Stunden verfolgt folgende Gua Sha in der gleichen Maus von Abbildung 1. Bitte <a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 2 zu sehen.

<ol>
	<li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+potential+therapeutics+based+on+in+vivo+spatiotemporal+transcription+patterns+of+heme+oxygenase-1.">Selection of potential therapeutics based on in vivo spatiotemporal transcription patterns of heme oxygenase-1.</a> J Mol Med. 80, 655-6564 (2002).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+functional+analysis+of+transgenes+in+mice+using+whole+body+imaging+of+luciferase+expression.">Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression.</a> Transgenic Res. 10, 423-434 (2001).</li><li>Contag, C. H., Stevenson, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+patterns+of+heme+oxygenase-1+transcription.">In vivo patterns of heme oxygenase-1 transcription.</a> J Perinatol. 21, 119-1124 (2001).</li><li>Nielsen, A., Knoblauch, N. T., Dobos, G. J., Michalsen, A., Kaptchuk, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Gua+Sha+treatment+on+the+microcirculation+of+surface+tissue:+a+pilot+study+in+healthy+subjects.">The effect of Gua Sha treatment on the microcirculation of surface tissue: a pilot study in healthy subjects.</a> Explore (NY). 3, 456-466 (2007).</li><li>Schwickert, M. E., Saha, F. J., Braun, M., Dobos, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gua+Sha+for+migraine+in+inpatient+withdrawal+therapy+of+headache+due+to+medication+overuse.">Gua Sha for migraine in inpatient withdrawal therapy of headache due to medication overuse.</a> Forsch Komplementmed. 14, 297-300 (2007).</li><li>Tsai, P. S., Lee, P. H., Wang, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographics,+training,+and+practice+patterns+of+practitioners+of+folk+medicine+in+Taiwan:+a+survey+of+the+Taipei+metropolitan+area.">Demographics, training, and practice patterns of practitioners of folk medicine in Taiwan: a survey of the Taipei metropolitan area.</a> J Altern Complement Med. 14, 1243-1248 (2008).</li><li>Nielsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gua+sha+research+and+the+language+of+integrative+medicine.">Gua sha research and the language of integrative medicine.</a> J Bodyw Mov Ther. 13, 63-72 (2009).</li><li>Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+gene+expression+in+living+mammals+using+a+bioluminescent+reporter.">Visualizing gene expression in living mammals using a bioluminescent reporter.</a> Photochem Photobiol. 66, 523-531 (1997).</li><li>Cui, K., Xu, X., Zhao, H., Wong, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+study+of+factors+affecting+in+vivo+bioluminescence+imaging.">A quantitative study of factors affecting in vivo bioluminescence imaging.</a> Luminescence. 23, 292-295 (2008).</li><li>Shibahara, S., Yoshida, T., Kikuchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+increase+of+heme+oxygenase+activity+induced+by+hemin+in+cultured+pig+alveolar+macrophages.">Mechanism of increase of heme oxygenase activity induced by hemin in cultured pig alveolar macrophages.</a> Arch Biochem Biophys. 197, 607-617 (1979).</li><li>Lee, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-1+mediates+transcriptional+activation+of+the+heme+oxygenase-1+gene+in+response+to+hypoxia.">Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.</a> J Biol Chem. 272, 5375-5381 (1997).</li><li>Maines, M. 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+heme+oxygenase+system:+a+regulator+of+second+messenger+gases.">The heme oxygenase system: a regulator of second messenger gases.</a> Annu Rev Pharmacol Toxicol. 37, 517-554 (1997).</li><li>Wunder, C., Potter, R. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+oxygenase-1+attenuates+interleukin-1beta-induced+nitric+oxide+synthase+expression+in+vascular+smooth+muscle+cells.">Heme oxygenase-1 attenuates interleukin-1beta-induced nitric oxide synthase expression in vascular smooth muscle cells.</a> J Biomed Sci. 11, 799-809 (2004).</li><li>Morse, D., Choi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+oxygenase-1:+from+bench+to+bedside.">Heme oxygenase-1: from bench to bedside.</a> Am J Respir Crit Care Med. 172, 660-670 (2005).</li><li>Ryter, S. 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The authors have nothing to disclose.

Transkription von HO-1, eine induzierbare Form von Häm-Oxygenase, wird von vielen Faktoren ab, einschließlich Häm, Wasserstoffperoxid, UV-Bestrahlung, Hypoxie und körperlichen Belastungen hochreguliert. Ganzkörper-Bildgebung, wie die gemeldeten HO-1 Biolumineszenz-Protokoll bietet einen schnellen Überblick über systemische HO-1-Expression in mehreren Organen. Bei Kleintieren erlaubt Biolumineszenz-Bildgebung mit hoher Empfindlichkeit in-vivo Echtzeit quantitative Längs-optische Beurteilung der Veränderungen in ...

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<td>Luciferin</td>
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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

Gua Sha ist eine traditionelle chinesische Volksmusik Therapie, die Haut beschäftigt Schaben zur subkutanen mikrovaskuläre Blutextravasat und Prellungen verursachen. Das Protokoll für Biolumineszenz optische Bildgebung von HO-1-<em&gt; Luciferase</em&gt; Transgenen Mäusen in diesem Manuskript berichtet bietet eine schnelle<em&gt; In vivo</em&gt; Test der Hochregulation des HO-1 (HO-1) Gen-Expression als Reaktion auf die Gua Sha Verfahren. HO-1 ist seit langem bekannt, Zellschutz gegen oxidativen Stress sorgen. Die Hochregulation von HO-1, durch die Biolumineszenz-Ausgang beurteilt, wird angenommen, dass eine antioxidative Reaktion auf zirkulierende Hämoglobin Produkte Gua Sha freigegeben darstellen. Gua Sha wurde durch wiederholte Schläge für einen reibungslosen Löffel Vorteil gegenüber geschmiert Haut auf dem Rücken oder andere gezielte Körperteil der transgenen Maus, bis Petechien (splinter Blutungen) oder Hautblutungen (blaue Flecken) bezeichnend für Blutaustritt aus dem subkutanen Kapillaren beobachtet wurde verabreicht. Nach Gua Sha, wurden Biolumineszenz-Bildgebung Sessions werden täglich über mehrere Tage durchgeführt, um die Dynamik der HO-1-Expression in mehreren inneren Organen zu folgen.

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

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

Gua Sha, traditionelle chinesische therapeutische Hautgeschabsel, verursacht subkutane mikrovaskuläre Blutextravasat. Wir berichten über einen Protokoll der Biolumineszenz-Bildgebung von HO-1-<em&gt; Luciferase</em&gt; Transgenen Mäusen zeigen, dass Gua Sha Häm hochreguliert Oxygenase-1 (HO-1) in mehreren Organen.

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 ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

Research

JoVE Journal

Biology

13.9K Aufrufe.  Massachusetts General Hospital, Harvard Medical School. Gua Sha, traditionelle chinesische therapeutische Hautgeschabsel, verursacht subkutane mikrovaskuläre Blutextravasat. Wir berichten über einen Protokoll der Biolumineszenz-Bildgebung von HO-1- Luciferase Transgenen Mäusen zeigen, dass Gua Sha Häm hochreguliert Oxygenase-1 (HO-1) in mehreren Organen.

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

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro und in vivo Biolumineszenz Reporter Gene Imaging von humanen embryonalen Stammzellen

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murine Renal Transplantation

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Funktionelle Bildgebung Brown Fat in Mäusen mit FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Überwachung Zell-autonome circadiane Uhr Rhythms der Genexpression mittels Luciferase Biolumineszenz Reporter

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Die Messung der Hämsynthese Levels in Säugerzellen

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Bildgebung von mitochondrialem Peroxid in lebenden Hefezellen mit mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Autor im Rampenlicht: Advancing Mitochondrial Research - mtHyper7 Biosensor für die subzelluläre Analyse 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Bildgebung von mtHyPer7, einem ratiometrischen Biosensor für mitochondriales Peroxid, in lebenden Hefezellen

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...