Die Studie wurde von der Universität Linköping und der County Council von Östergötland finanziert.

1. Ovariektomie über die Dorsal-Route <ol><li> Das Tier zu betäuben, zum Beispiel unter Verwendung von 1,5% Isofluran (4% bei Induktion) in einem Sauerstoff / Lachgas 30% / 70%-Mischung. Platzieren Sie das Tier in Bauchlage auf einer Heizplatte mit einem rektalen Thermometer. Es ist wichtig, um eine dünne Schicht der Isolierung zwischen dem Tier und der Platte haben, und das Thermometer Feedback-System hält den Körper Temperatur innerhalb von 37 ± 0,5 ° C Tragen Sie das Gel zum Schutz der Augen und zu verwalten, 5 mg Rimadyl / kg Körpergewicht in Kochsalzlösung subkutan auf der lateralen Seite den Hals des Tieres für die postoperative Schmerzbehandlung verdünnt. </li><li> Shave a 4 * 4 cm (3 * 3 cm für Mäuse) Bereich cephally aus dem Beckenkamm. Waschen Sie gründlich den rasierten Bereich mit zum Beispiel vor dem Abdecken Jodopax das Tier mit einem 4 * 4 cm Öffnung Chirurgie Blatt. </li><li> Machen Sie eine 2-3 cm Medianschnitt und unverblümt sezieren die Haut von der darunter liegenden Faszie. Eines cm lateral der Mittellinie, einen weiteren Einschnitt through die Faszie. Unverblümt seitlich sezieren unter dieser Faszie als oberflächlich wie möglich bis zum Erreichen der Bauchhöhle. </li><li> Mit einer Pinzette greifen die Fettgewebe, die den Eierstock umgibt in der Bauchhöhle und ziehen Sie sie vorsichtig heraus. Identifizieren Sie den Eierstock und die Gebärmutter ligieren Hörner und Gefäße 0,5-1 cm proximal von dieser Struktur. Schneiden Sie den Eierstock und ligierten Fettgewebe und setzen Sie den restlichen Gewebe zurück in die Bauchhöhle. Machen Sie einen anderen Schnitt in der kontralateralen Faszie und den Vorgang wiederholen. </li><li> Schließen Sie die Wunden, vorzugsweise unter Verwendung eines monofilen Naht, und lassen Sie das Tier aus der Narkose erwachen. Legen Sie das Tier in einer beheizten Käfig (25-27 ° C) während mindestens 2 Stunden nach der Operation. Die Tiere sollten einzeln in den ersten Tagen nach der Operation untergebracht werden. Es ist entscheidend, dass der Käfig regelmäßig gereinigt wird / während der Erholungsphase ersetzt. Vierundzwanzig Stunden nach der Operation, den Tieren subkutan injizieren mit einer weiteren 5 mg Rimadyl / kg Körpergewicht, verdünnt inKochsalzlösung. </li></ol> 2. Subkutane Verabreichung von 17β-Östradiol durch Silastic Kapseln <ol><li> Gründlich mischen Sie die gewünschte Menge an 17β-Estradiol in Sesamöl. Für physiologischen Konzentrationen bei Ratten und Mäusen, empfehlen wir, 180 ug / ml und 18-36 pg / ml. Placebo-Kapseln sind mit reinem Sesamöl gefüllt. </li><li> Schneiden Sie 3 cm (2 cm für Mäuse) Längen von Silasticschlauch und 5 mm (3 mm für Mäuse) Längen von Holz-Tupfer zum Einstecken. Mit einer Pinzette, quetschen ein Holzpflock in die Silastic Kapsel vor dem Einspritzen des Öls 17β-estradiol/sesame Lösung, um die gesamte Länge zu füllen. Vorsichtig verschließen Sie die Silastic Kapsel in das offene Ende mit einem anderen Holz-Stecker (die Stecker sollen dauerhaft in der Kapsel). Inkubieren Sie die Kapseln in der verbleibenden 17β-Estradiol-Lösung über Nacht vor der Implantation. </li><li> Die Implantation der Kapsel kann während der Narkose bei Ovarektomie bei Ratten verwendet, durchgeführt werden. Für miCE ist jedoch eine 14-tägige Erholungsphase nach Ovariektomie dringend empfohlen, um das Risiko der Kapsel durch die hervorstehenden empfindliche Haut zu minimieren. </li><li> Nach der chirurgischen Zubereitungen im Sinne der 1.1, oder nach dem Schließen der Ovariektomie Einschnitt, rasieren und waschen ein 2 * 2 cm-Bereich auf der Dorsalseite den Hals des Tieres. Machen Sie eine 1 cm Inzision und unverblümt sezieren eine subkutane Tasche caudolaterally, mit viel Platz für die Kapsel. Wischen die Silastik Kapsel (dass bis zu diesem Zeitpunkt, in dem in 17β-Estradiol-Lösung inkubiert) und leicht zu installieren durch den Einschnitt, die anschließend durch eine Naht geschlossen. Es ist entscheidend, um sicherzustellen, dass die Kapsel-Stämme die Haut so wenig wie möglich, besonders in der Gegend in der Nähe der Wunde. Von der Herstellung von Kapseln zur Implantation ist es wichtig, dass eine Verunreinigung vermieden wird. </li></ol> 3. Perorale Verabreichung von 17β-Estradiol über die Mutter-Creme Nutella <ol><li> Gründlich aufzulösen 17β-Estradiol in Sesamöl, und dann mischen das Sesamöl mit dem Nutella. Wir empfehlen, dass jeder täglich Abschnitt 28 pg 17β-Estradiol, 5 ul Sesamöl und 1 g Nutella pro Kilogramm Körpergewicht für Ratten und 1,12 pg 17β-Östradiol, 0,312 ul Sesamöl und 60 mg Nutella für jedes 30-g-Maus enthält. Placebo-Creme Nuss ist identisch mit Ausnahme der ohne 17β-Estradiol vorbereitet. </li><li> Trainieren Sie die Tiere frei von Hormonen Nuss-Creme für fünf Tage vor dem Experiment zu essen. Die ersten drei Tage der Ausbildung sollten die Ratten in Gruppen optimal trainiert werden (ca. 0,5 g Nutella auf 2-5 Tiere), in ihre Käfige. Auf der vierten und fünften Tag der Ausbildung werden die Tiere in getrennten Käfigen, bevor ihm der tägliche Portion an die experimentelle Situation ähneln setzen. </li><li> Während des Experiments werden die Tiere in getrennten Käfigen für jeden Anlass Fütterung setzen, wonach die Nutella Teil auf kleinen Keramikfliesen serviert wird. Wenn Training ist wie oben, o durchgeführt wordenur Erfahrung ist, dass mehr als 95% von Ratten und Mäusen die Nuss-Creme zu akzeptieren, und einmal vollständig gewöhnt, konsumieren die meisten Tiere es innerhalb von Sekunden. </li></ol> 4. Repräsentative Ergebnisse Physiologischer Kochsalzlösung 17β-Östradiol-Konzentrationen, die die aktuellen Methoden zielen darauf ab, zu reproduzieren, sind ca. 5-140 pg / mL für Ratten und 5-35 pg / ml für Mäuse 4,6. Die subkutane Silastic Kapseln nach den Anweisungen oben, wenn bis 300 g weiblichen Sprague-Dawley Ratten verabreicht, produzieren nur langsam abklingende 17β-Estradiol-Serumkonzentrationen von etwa 40 pg / ml Tag 2 bis 10 pg / ml Tag 35. Doch unmittelbar nach der Applikation kann ein Peak-Serumkonzentration um 1000 pg / ml erwartet werden, fallen an den physiologischen Bereich innerhalb von 8 Stunden (Abbildung 1A) 4,5. Die Kapseln für Silastic oben beschriebenen Mäusen mit 36 ​​ug 17β-estradiol/mL, wenn sie 25 g weibliche C57BL / 6 Mäusen verabreicht, machen Serum konzentrations, die sich langsam von etwa 90 pg / ml Tag 2 bis 25 pg / ml an Tag 35 zu verringern. Dies wird wahrscheinlich durch eine ähnliche Peak vorausgeht, wie in den Ratten gefunden wurde, obwohl dies wurde nicht untersucht (Abbildung 1B) 6. Die perorale Nutella Verfahren mit den Dosen oben beschrieben, wenn sie 300 g weibliche Sprague-Dawley-Ratten verabreicht, macht physiologischen Konzentrationen von 17β-Östradiol mit täglichen Schwankungen zwischen 10-70 pg / ml (2A) 4. Bei Mäusen unter Verwendung des oben Dosierung, schwanken die täglichen Ebenen zwischen etwa 20 und 120 pg / ml (2B) 6. <img alt="1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html> . rong&gt; 1 stilisierte Darstellung Graphen 17β-Östradiol-Konzentrationen in Ratten (A, n = 10 in Zeitpunkten bis zu 24 h, n = 15 in späteren Zeitpunkten) 4,5 und Mäusen (B, n = 10) 6 nach der Implantation Kapsel. Die Silastic Kapseln produzieren einen kurzen anfänglichen Spitzenwert von Serum-Konzentrationen von bis zu 1000 pg / ml, aber Rückgang innerhalb von Stunden auf Werte unter 100 pg / ml. Für die kommenden 4-5 Wochen liefern die Kapseln stabil, obwohl stetig ab, Serum-Konzentrationen. Die schraffierten Flächen auf physiologische 17β-Estradiol-Serumkonzentrationen in intakten weiblichen Ratten und Mäusen jeweils entsprechen. Obwohl nicht visuell in den Diagrammen gezeigt haben, dass die interindividuelle Variabilität in den 17β-Östradiol-Konzentrationen, wodurch Variationskoeffizienten von 27 bis 183% bei Ratten und von 28 bis 115% bei Mäusen mit dem Silicongummi Kapselverfahrens (sehr niedrige mittlere Konzentrationen erzeugen oft hohe Variationskoeffizienten). iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; 2 stilisierte Darstellung Graphen 17β-Östradiol-Konzentrationen in Ratten (A, n = 15). 4 und Mäusen (B, n = 10) 6 als Folge der täglichen Verabreichung von Hormonen in Nutella. Die perorale Nutella Verfahren macht Tagesspitzen jedoch bei weitem nicht so hoch wie durch subkutane oder intravenöse Verfahren hergestellt. Dies ahmt die Pharmakokinetik von menschlichen Hormonersatztherapie und die perorale Behandlung kann für so lange wie nötig gehen, selbst wenn das obige Beispiel nur einen anfänglichen 96 Stunden vor. Die schraffierten Flächen auf physiologische 17β-Estradiol-Serumkonzentrationen in intakten weiblichen Ratten und Mäusen jeweils entsprechen. Obwohl nicht visuell in den Diagrammen gezeigt haben, dass die interindividuelle Variabilität in den 17β-Östradiol-Konzentrationen, wodurch Variationskoeffizienten von 40 bis 132% bei Ratten und von 13 bis 219% bei Mäusen mit dem Nutella Verfahren (sehr niedrigen mittleren Konzentrationen erzeugen oft hoheVariationskoeffizienten).

<ol>
	<li>Strom, J. O., Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-related+neuroprotective+versus+neurodamaging+effects+of+estrogens+in+rat+cerebral+ischemia:+a+systematic+analysis.">Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia: a systematic analysis.</a> J. Cereb. Blood Flow Metab. 29, 1359-1372 (2009).</li><li>Strom, J. 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Blood Flow Metab. 24, 298-304 (2004).</li><li>Yong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17beta-estradiol+potentiates+ischemia-reperfusion+injury+in+diabetic+ovariectomized+female+rats.">17beta-estradiol potentiates ischemia-reperfusion injury in diabetic ovariectomized female rats.</a> Brain Res. 1054, 192-199 (2005).</li><li>Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+increases+brain+lesions+in+the+cortex+and+lateral+striatum+after+transient+occlusion+of+the+middle+cerebral+artery+in+rats:+no+effect+of+ischemia+on+galanin+in+the+stroke+area+but+decreased+levels+in+the+hippocampus.">Estradiol increases brain lesions in the cortex and lateral striatum after transient occlusion of the middle cerebral artery in rats: no effect of ischemia on galanin in the stroke area but decreased levels in the hippocampus.</a> Peptides. 26, 2257-2264 (2005).</li><li>Levin-Allerhand, J. 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Die Autoren haben nichts zu offenbaren.

Die Verwaltungs-Modi oben haben in früheren Studien präsentiert sich als überlegen erwiesen an gewerbliche Slow-Release-Tabletten und Injektionen in Bezug auf die Herstellung physiologischer Serum-Konzentrationen von 17β-Estradiol. Während die Slow-Release-Pellets sehr hohe und anhaltende Konzentration im Serum Spitzen produzieren, verursachen die extremen Injektionen, täglich Hormonschwankungen und erfordert wiederholte stressigen Umgang mit den Tieren 6.4. Die Bedeutung der Wahl geeigneter Verabreichu...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Rimadyl, 50 mg / ml </td><td> Pfizer </td><td> 462986 </td><td> Postoperative Analgesie nach Ovarektomie. </td></tr><tr><td> Lubrithal Augengel </td><td> Leo Laboratories </td><td></td><td> Augenschutz während der Operation. </td></tr><tr><td> Jodopax Tierarzt </td><td> Pharmaxim </td><td></td><td> Chirurgie Desinfektion. </td></tr><tr><td> 4-0 Vicryl Naht </td><td> Johnson &amp; Johnson </td><td></td><td> Wundnaht und Eierstock-Ligation. </td></tr><tr><td> 17β-Estradiol,&gt; 98% </td><td> Sigma </td><td> E8875-1G </td><td> In Sesamöl für Silastic Kapseln und Nutella. </td></tr><tr><td> Sesamöl </td><td> Sigma </td><td> S3547-1L </td><td> Das Auflösen der 17β-Estradiol. </td></tr><tr><td> Silasticschlauch, ID / OD: 1.575/3.175 mm </td><td> Dow Corning </td><td> 508-008 </td><td> Produktion von Silastic Kapseln. </td></tr><tr><td> Holzstäbchen </td><td> Selefatrade </td><td> 12-0783 </td><td> Capping von Silastic Kapseln. </td></tr><tr><td> Nutella </td><td> Ferrero </td><td></td><td> Mittel für die perorale 17β-Estradiol. </td></tr></tbody></table>

ovariectomy and 17&beta;-estradiol replacement in rats and mice: a visual demonstration

Östrogene sind eine Familie von weiblichen Sexualhormone mit einem außergewöhnlich breiten Spektrum von Effekten. Bei Ratten und Mäusen in Östrogen Forschung dienen sie allgemein ovariektomiert, um die rasch proliferierender Hormonproduktion abgetragen und ersetzt das 17β-Östradiol exogen. Es gibt jedoch keinen Konsens darüber, wie das Hormon verabreicht werden sollte, um physiologische Serumkonzentration zu erhalten. Dies ist entscheidend, da die 17β-Estradiol Niveau / Verwaltung Methode zutiefst beeinflusst die experimentellen Ergebnisse 1-3. Wir haben in einer Reihe von Studien charakterisiert die verschiedenen Modi des 17β-Estradiol Verwaltung stellte fest, dass subkutane Silastic Kapseln und pro-Nuss-Creme oral Nutella überlegen kommerziell erhältlichen Slow-Release-Pellets (hergestellt von der Firma Innovative Research of America) sind und tägliche Injektionen in Bezug auf die Herstellung physiologischer Serum-Konzentrationen von 17β-Östradiol 4-6. Unter den advantages der Mutter-Sahne-Methode, die zuvor für Buprenorphin Verwaltung 7 verwendet wurde, dh wenn für die Verabreichung von Estrogen verwendet es peroralen Hormonersatztherapie ähnelt und ist nicht-invasiv. Die subkutane Silastic Kapseln sind bequem und produzieren die stabilsten Serumkonzentrationen. Dieses Video Artikel enthält Schritt-für-Schritt-Demonstrationen von Ovariektomie und 17β-Estradiol Hormonersatz durch Silastic Kapseln und perorale Nutella bei Ratten und Mäusen, von einer Diskussion über wichtige Aspekte der Verwaltung gemacht wurden.

Watch this Scientific Journal Video about Ovariectomy and 17Î²-estradiol Replacement in Rats and Mice: A Visual Demonstration at JoVE.com

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovarektomie und anschließender Austausch von 17β-Östradiol durch Silastic Kapseln und perorale Nutella sind bei Ratten und Mäusen nachgewiesen.

Ovariektomie und 17β-Estradiol Ersatz bei Ratten und Mäusen: Die bildliche Darstellung

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

ovariectomy-17-estradiol-replacement-rats-mice-visual

Research

JoVE Journal

Biology

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

56.7K Aufrufe.  Linköping University. Ovarektomie und anschließender Austausch von 17β-Östradiol durch Silastic Kapseln und perorale Nutella sind bei Ratten und Mäusen nachgewiesen.

Serie: Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

A Magnetic Tether System zur visuellen und olfaktorischen Mediated Flight Control in Drosophila Untersuchen

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Forschung

Biologie

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Demonstration von Kutane Allodynie In Vereinigung mit chronischen Unterbauchschmerzen

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark und Track Cells In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Manuelle Restraint und Common Compound Verabreichungswege bei Mäusen und Ratten

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

Diagnostic Necropsy und ausgewählte Gewebe und Probenentnahme bei Ratten und Mäusen

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

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 ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Physiology Lab Demonstration: glomeruläre Filtrationsrate in einem Ratten-

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Validierung eines Maus-Modell zu LINC Komplexe Disrupt in einem zellspezifisch

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

In Vivo Detektion und Analyse von Rb Protein Sumoylierung in menschlichen Zellen

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...