El estudio fue financiado por la Universidad de Linköping y de la Diputación Provincial de Östergötland.

1. Ovariectomía a través de la Ruta del dorsal <ol><li> Se anestesia el animal, por ejemplo, utilizando 1,5% de isoflurano (4% para la inducción) en un oxígeno / óxido nitroso 30% / 70% mezcla. Colocar el animal en posición boca abajo en una placa de calefacción conectado a un termómetro rectal. Es importante tener una fina capa de aislamiento entre el animal y la placa, y que el termómetro de realimentación del sistema mantiene la temperatura corporal dentro de 37 ± 0,5 ° C. Aplique el gel para proteger los ojos y administrar 5 mg Rimadyl / kg de peso diluido en solución salina por vía subcutánea en la cara lateral del cuello del animal para el alivio del dolor postoperatorio. </li><li> Afeitado un 4 * 4 cm (3 * 3 cm para los ratones) cephally zona de la cresta ilíaca. Es necesario lavar completamente el área afeitada con la Jodopax ejemplo, antes de cubrir el animal con una hoja de 4 * 4 cm de la cirugía de la apertura. </li><li> Haga una incisión de 2-3 cm de la línea media y sin rodeos disección de la piel de la fascia subyacente. Uno cm lateral de la línea media, hacer otra incisión through la fascia. Francamente disecar lateralmente bajo esta fascia como superficialmente como sea posible hasta llegar a la cavidad abdominal. </li><li> Usando las pinzas, agarra el tejido adiposo que rodea el ovario en la cavidad abdominal y tire suavemente hacia afuera. Identificar el ovario y ligar las trompas de Falopio y los vasos 0.5-1 cm proximal de esta estructura. Cortar el ovario y el tejido adiposo ligado y poner la parte posterior del tejido restante en la cavidad abdominal. Hacer otra incisión en la fascia contralateral y repita el procedimiento. </li><li> Cerrar las heridas, de preferencia utilizando una sutura monofilamento, y dejar que el animal despierta de la anestesia. Colocar el animal en una jaula calentada (25-27 ° C) durante al menos 2 horas después de la cirugía. Los animales deben ser alojados por separado los primeros días del postoperatorio. Es fundamental que la jaula se limpiarán con frecuencia / sustituido durante la fase de recuperación. Veinticuatro horas después de la cirugía, los animales se inyectan por vía subcutánea con otro 5 mg Rimadyl / kg de peso corporal diluido ensolución salina. </li></ol> 2. La administración subcutánea de 17β-estradiol a través de cápsulas de Silastic <ol><li> Mezcle la cantidad deseada de 17β-estradiol en aceite de sésamo. Para las concentraciones fisiológicas en ratas y ratones, se recomienda 180 mg / ml y 18-36 mcg / ml, respectivamente. Placebo cápsulas se llenan con aceite de sésamo puro. </li><li> Corte 3 cm (2 cm para ratones) la longitud de los tubos de silastic y 5 mm (3 mm para los ratones) longitudes de palos de madera para conectar aplicadores. El uso de pinzas, apretar un tapón de madera en la cápsula de silastic antes de inyectar la solución de aceite 17β-estradiol/sesame para llenar toda la longitud. Suavemente la tapa de la cápsula de silastic en el extremo abierto con otro tapón de madera (los tapones se supone que debe permanecer permanentemente en la cápsula). Incubar las cápsulas en el restante 17β-estradiol solución durante la noche antes de la implantación. </li><li> La implantación de la cápsula se puede realizar durante la anestesia que se utiliza en ovariectomía en ratas. Para mice, sin embargo, un período de recuperación 14-días después de la ovariectomía se recomienda para minimizar el riesgo de la cápsula que sobresale a través de la piel frágil. </li><li> Después de las preparaciones quirúrgicas como en 1.1, o después de cerrar la incisión ovariectomía, afeitarse y lavarse un área de 2 * 2 cm en la cara dorsal del cuello del animal. Haga una incisión de 1 cm y sin rodeos la disección de un bolsillo subcutáneo caudolaterally, con amplio espacio para la cápsula. Limpiar la cápsula de silastic (que hasta este punto de tiempo ha sido incubadas en la solución 17β-estradiol) y suavemente instalar a través de la incisión, que posteriormente se cierra por una sutura. Es crucial para asegurarse de que las cepas de la cápsula de la piel tan poco como sea posible, particularmente en el área cercana a la herida. Desde la producción de cápsulas a la implantación, es importante que se evita la contaminación. </li></ol> 3. La administración oral de 17β-estradiol a través de la tuerca de la crema Nutella <ol><li> Se disuelven completamente 17β-estradiol en aceite de sésamo, y luego mezclar el aceite de sésamo con la Nutella. Se recomienda que cada porción diaria contiene 28 mg 17β-estradiol, 5 l de aceite de sésamo y 1 g de Nutella por kilogramo de peso corporal para las ratas y 1,12 mg 17β-estradiol, 0.312 l de aceite de sésamo y 60 mg de Nutella para cada ratón de 30 g. Placebo nueces crema se prepara de forma idéntica, salvo sin el 17β-estradiol. </li><li> Capacitar a los animales para comer libre de hormonas de nuez de crema durante cinco días antes del experimento. Los primeros tres días de entrenamiento, las ratas de forma óptima, debe estar capacitado en grupos (aproximadamente 0,5 g de Nutella 2-5 animales), en sus propias jaulas. En los días cuarto y quinto de la formación, los animales se colocan en jaulas separadas antes de recibir la porción diaria para asemejarse a la situación experimental. </li><li> Durante el experimento, los animales se colocan en jaulas separadas para cada ocasión de alimentación, después de lo cual la porción de Nutella se sirve en pequeñas baldosas de cerámica. Si la formación se ha realizado el anterior, oexperiencia ur es que más del 95% de las ratas y ratones aceptar la tuerca de crema, y ​​una vez completamente habituados, la mayoría de los animales que consumen en cuestión de segundos. </li></ol> 4. Los resultados representativos Suero fisiológico 17β-estradiol concentraciones, que los métodos actuales apuntan a reproducir, es de aproximadamente 5 a 140 pg / ml para las ratas y 5-35 pg / ml para los ratones 4,6. Las cápsulas subcutáneas silastic de acuerdo con las instrucciones anteriores, cuando se administra a 300 g hembra Sprague-Dawley, producir disminuyendo lentamente 17β-estradiol concentraciones de suero de alrededor de 40 pg / ml el día 2 a 10 pg / ml el día 35. Sin embargo, inmediatamente después de la administración, un pico de concentración en suero alrededor de 1000 pg / ml se puede esperar, cayendo al rango fisiológico dentro de las 8 horas (Figura 1A) 4,5. Las cápsulas de silastic para los ratones descritos anteriormente con 17β-estradiol/mL mg de 36 años, cuando se administró a 25 mujeres C57BL g / 6 ratones, hacer que el suero concentradoentrations que poco a poco disminuyen de aproximadamente 90 pg / mL día 2 a 25 pg / mL día 35. Esto es, probablemente, precedido por un pico similar como se encontró en las ratas, aunque esto no ha sido investigado (Figura 1B) 6. El método Nutella peroral con las dosificaciones se ha descrito anteriormente, cuando se administra a 300 g hembra Sprague-Dawley, hace que los niveles fisiológicos de 17β-estradiol con las fluctuaciones diarias entre 10-70 pg / ml (Figura 2A) 4. Para ratones utilizando la dosis anterior, los niveles diarios fluctúan entre aproximadamente 20 y 120 pg / ml 6 (Figura 2B). <img alt="Figura 1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html> . rong&gt; Figura 1 se presentan los gráficos estilizados 17β-estradiol concentraciones en ratas (A, n = 10 en los puntos de tiempo hasta 24 h, n = 15 en los puntos de tiempo posteriores) 4,5 y los ratones (B, n = 10) 6 después de la implantación de cápsulas. Las cápsulas de silastic producir un pico corto inicial de las concentraciones séricas de hasta 1000 pg / ml, pero la disminución en cuestión de horas a niveles inferiores a 100 pg / ml. Para las próximas semanas 4-5, las cápsulas de rendimiento estable, aunque cada vez menor, las concentraciones séricas. Las áreas sombreadas corresponden a fisiológicos 17β-estradiol concentraciones séricas en ratas hembra intactas y ratones, respectivamente. A pesar de que visualmente no se muestra en los gráficos, hay variabilidad interindividual en las concentraciones de 17β-estradiol, lo que hace coeficientes de variación desde 27 hasta 183% en las ratas y de 28 a 115% en los ratones con el método de la cápsula de silastic (media muy baja concentraciones a menudo generan altos coeficientes de variación). iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; Figura 2 gráficos estilizados presentan 17β-estradiol concentraciones en ratas (A, n = 15). 4 y ratones (B, n = 10) 6, como consecuencia de la administración de hormonas diaria en Nutella. El método Nutella peroral hace picos diarios, sin embargo, con mucho, no tan alta como producido por vía subcutánea o métodos intravenosos. Esto imita la farmacocinética de la terapia humana sustitución hormonal, y el tratamiento por vía oral puede proceder durante tanto tiempo como sea necesario, incluso si el ejemplo anterior sólo presentan un período inicial de 96 horas. Las áreas sombreadas corresponden a fisiológicos 17β-estradiol concentraciones séricas en ratas hembra intactas y ratones, respectivamente. Aunque no se demostró visualmente en los gráficos, hay variabilidad interindividual en las concentraciones de 17β-estradiol, lo que hace coeficientes de variación desde 40 hasta 132% en las ratas y de 13 a 219% en los ratones con el método de Nutella (concentraciones medias muy bajas a menudo generan altacoeficientes de variación).

<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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Los autores no tienen nada que revelar.

Los modos de administración presentados anteriormente tienen en estudios anteriores demostró ser superior a comerciales gránulos de liberación lenta y las inyecciones en términos de producir fisiológicas concentraciones séricas de 17β-estradiol. Mientras gránulos de liberación lenta, producen picos séricos muy elevados y prolongados de concentración, las inyecciones causar fluctuaciones extremas, la hormona del día y requieren un manejo estresante repetida de los animales de 4-6. La importancia d...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Rimadyl, 50 mg / ml </td><td> Pfizer </td><td> 462986 </td><td> La analgesia postoperatoria después de la ovariectomía. </td></tr><tr><td> Lubrithal gel para los ojos </td><td> Laboratorios LEO Pharma </td><td></td><td> Protección de los ojos durante la cirugía. </td></tr><tr><td> Jodopax veterinario </td><td> Pharmaxim </td><td></td><td> Cirugía desinfección. </td></tr><tr><td> Sutura de Vicryl 4-0 </td><td> Johnson &amp; Johnson </td><td></td><td> Sutura de la herida y la ligadura de ovario. </td></tr><tr><td> 17β-estradiol,&gt; 98% </td><td> Sigma </td><td> E8875-1G </td><td> En aceite de ajonjolí para las cápsulas de silastic y Nutella. </td></tr><tr><td> Aceite de sésamo </td><td> Sigma </td><td> S3547-1L </td><td> Disolución de la 17β-estradiol. </td></tr><tr><td> Tubos de Silastic, ID / OD: 1.575/3.175 mm </td><td> Dow Corning </td><td> 508-008 </td><td> La producción de cápsulas de silastic. </td></tr><tr><td> Aplicador de madera </td><td> Selefatrade </td><td> 12-0783 </td><td> La nivelación de las cápsulas de silastic. </td></tr><tr><td> Nutella </td><td> Ferrero </td><td></td><td> Medio para la vía oral 17β-estradiol. </td></tr></tbody></table>

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

Los estrógenos son una familia de hormonas sexuales femeninas con un espectro excepcionalmente amplia de efectos. Cuando las ratas y los ratones son utilizados en la investigación de los estrógenos que son comúnmente ovariectomizadas con el fin de realizar la ablación de la producción de hormonas de ciclo rápido, en sustitución de la exógena 17β-estradiol. Hay, sin embargo, la falta de consenso respecto a cómo la hormona debe ser administrado para obtener las concentraciones fisiológicas de suero. Esto es crucial ya que el nivel de 17β-estradiol / método de administración influye profundamente en los resultados experimentales 1-3. Tenemos en una serie de estudios que se caracterizan los diferentes modos de administración de 17β-estradiol, encontrando que subcutáneos cápsulas de silastic y por vía oral Nutella nuez de crema son superiores a las disponibles en el mercado pastillas de liberación lenta (producido por la compañía Research innovador de América) y inyecciones diarias en términos de producción fisiológicas las concentraciones séricas de 17β-estradiol 6.4. Entre los advantages del método tuerca-crema, que previamente ha sido utilizado para la buprenorfina administración 7, es que cuando se utiliza para la administración de estrógenos que se asemeja a la terapia de sustitución hormonal por vía oral y no es invasivo. Las cápsulas subcutáneas silastic son convenientes y producir las concentraciones séricas más estables. Este artículo video contiene paso a paso las demostraciones de la ovariectomía y 17β-estradiol reemplazo de la hormona por las cápsulas de silastic y Nutella por vía oral en ratas y ratones, seguidos por una discusión de aspectos importantes de los procedimientos de gestión.

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

Ovariectomía y la consiguiente sustitución de 17β-estradiol por medio de cápsulas de Silastic y Nutella por vía oral se ha demostrado en ratas y ratones.

Ovariectomía y 17β-estradiol reemplazo en ratas y ratones: una demostración visual

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 vistas.  Linköping University. Ovariectomía y la consiguiente sustitución de 17β-estradiol por medio de cápsulas de Silastic y Nutella por vía oral se ha demostrado en ratas y ratones.

Video: 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.

Un Sistema de Anclaje magnética para investigar control mediado de vuelo visual y olfativa en 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. ...

Investigación

Biología

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.

Demostración de La alodinia cutánea En asociación con el dolor pélvico crónico

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.

Una sola célula grabaciones de succión de los conos del ratón

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.

Un enfoque práctico de elaboración de mapas genéticos inducible destino: una guía visual para marcar y rastrear las células En 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.

Sujeción manual y de las rutas de la administración del compuesto en ratones y ratas

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.

La necropsia de diagnóstico y selecta colección de tejidos y muestras en ratas y ratones

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.

Diseño y Análisis Del Comportamiento De Preferencia De Temperatura Y Su Ritmo Circadiano En Drosophila

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

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.

Demostración Laboratorio Fisiología: tasa de filtración glomerular en una rata

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.

Validación de un modelo de ratón para interrumpir LINC Complejos en una celda de manera específica

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.

En Vivo Detección y análisis de la sumoilación de la proteína Rb en células humanas

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