Con el apoyo de DK065872 (PEV), una familia Smith Premio de la Fundación de Excelencia en Investigación Biomédica (PEV), F31 DA023760.

Resumen Dos meses de edad, los ratones C57BL/6J edad media o equivalente o tres meses de edad promedio de edad de las ratas Sprague Dawley o equivalente son anestesiados con ketamina (60 mg / kg ip para las ratas, 100 mg / kg ip de ratones) y xilazina (10 mg / kg, ip, ya sea para las especies). La sedación se controla mediante una pizca punta suave reflejo de retirar demostrado en <a href="/index/details.stp?ID=236">Walantus et al.</a> (JOVE, 6, 2007) y <a href="/index/details.stp?ID=404">Szot et al.</a> (JOVE, 9, 2007). Termorregulación puede ser proporcionada a través de una almohadilla eléctrica thermostatregulated (ALA Instruments Inc.) y monitoreados a través de un termómetro rectal. La cabeza rapada de pelo y limpiar con yodo antes de la incisión. Después de incisión en la piel (2 cm de largo para las ratas, 1 cm de largo para los ratones) y la eliminación de todos los tejidos blandos de la superficie del cráneo, la colocación de la cánula guía se determina en relación a bregma. Un agujero de 6 mm se perfora el cráneo con un taladro a pilas para cirugía de roedores (Herramientas de Bellas Science, Inc.). Hay que tener cuidado para que la broca no penetra a través de las membranas meníngeas o los vasos sanguíneos. Skull se implanta con bilaterales de 5 mm calibre 21 ejes de acero inoxidable guía que conduce al núcleo accumbens posterior, el cuerpo estriado dorsal o la corteza prefrontal. Las coordenadas estereotáxicas se establecen de acuerdo con Franklin y Paxinos, 1997 (el cerebro del ratón en coordenadas estereotáxica, Academic Press) o Paxinos y Watson, 2006 (el cerebro de rata en coordenadas estereotáxica, Academic Press). Los implantes se fijan con cemento dental. Un bolo de Ringer lactato de la solución salina al 0,9% se da al final de la cirugía (5ml SC en ratas y 1 ml SC en ratones después de los fluidos se calientan a la temperatura normal del cuerpo) para prevenir la deshidratación. La buprenorfina (0.1-0.5mg/kg SC) se administra dos veces al día y, luego, en una función de las necesidades, si el animal parece estar en el dolor. El tratamiento local con antibióticos (bacitracina pomada) y el tratamiento con antibióticos sistémicos (penicilina 100.000 UI / kg IM cada 12 horas durante las primeras 48 horas después de la operación) si se administran después de la operación las infecciones. Después de la cirugía, los animales son alojados individualmente con comida y agua ad libitum disponibles. Al menos una semana se permite para la recuperación antes de microdiálisis y la eutanasia. Tras la recuperación de la cirugía, los animales son trasladados a una jaula de microdiálisis y sondas de microdiálisis se insertan y se consolidó en los ejes de guía que se han instalado durante la cirugía. Sonda de inserción no causa dolor o malestar debido a que la sonda está pasando por alto tejido de la piel, los músculos y las meninges a través del eje de guía. Por lo tanto, la inserción de la sonda se realiza sin anestesia y los efectos de anestesia inducida por el comportamiento de la neuroquímica o se evitan. Dejamos que las sondas de estabilizar durante 12 horas y luego iniciar el muestreo cada 30 minutos por otro 12.08 horas, dependiendo del experimento. Hacemos un seguimiento de comportamiento locomotor del animal a través de fotocélulas o registro manual de movimiento por el experimentador. Microdialysate muestras se inyectan en una cromatografía líquida de alta con detección electroquímica (HPLC-CE), instrumento para la detección y análisis neuroquímico. Buscamos efectos sobre la neuroquímica basal y el comportamiento del aparato locomotor. Al final del experimento, el animal es sacrificado por una sobredosis de ketamina sistémica (200 mg / kg ip) y xilazina (20 mg / kg, ip). Entonces, el corazón es perfundido con solución salina al 0,9% seguido de paraformaldehído al 4%. Los cerebros se retiran, congeladas y cortadas a lo largo de las vías de la sonda de microdiálisis para verificar la colocación de la sonda precisa. Procedimiento <ol><li> Coloque el instrumento estereotáxico y todos los materiales necesarios. Asegúrese de que el área y los instrumentos se limpian y se esterilizan. </li><li> Afeitarse la piel con maquinilla de afeitar eléctrica. Van desde las orejas hasta justo entre los ojos, mover de afeitar en diferentes direcciones para limpiar con eficacia la zona de la piel. Aplicar povidona / yodo a la zona de afeitado, sino proteger los ojos de ella. </li><li> Montar el animal en el aparato estereotáxico mediante la colocación de las barras de oído en el conducto auditivo externo y el ajuste en su lugar. En primer lugar montar un bar de oído en el conducto auditivo externo, y luego mantenerlo en su lugar y se deslizan en la barra de la otra oreja. Usted sabe que está en la ubicación correcta cuando la cabeza ya no puede ser movido de lado a lado. Asegurar la boca con el montaje anterior de la estereotáxica y asegúrese de que la cabeza esté a nivel con una regla. Coloque la regla en posición vertical con respecto a la plataforma del instrumento estereotáxico y comprobar si hay un ángulo de 90 ° entre el gobernante y el centro de la cabeza del animal). Confirmarlo mediante el instrumento estereotáxico si ofrece esa capacidad. </li><li> Hacer una incisión anterior / posterior en el cuero cabelludo con un bisturí estéril que se extiende desde la lambda para justo entre los ojos del animal. Use pinzas hemostáticas esterilizados para desprender la piel y mantener la incisión abierta. El uso de varios hisopos de algodón estériles, secar la superficie del cráneo al descubierto. <li> Coloque la cánula guía en su montura, encontrar bregma en el cráneo, y la posición del derecho cánula guía en este lugar. Anote las coordenadas anterior / posterior y lateral. Desde bregma, encontrar las coordenadas correctas necesarias para la colocación de la sonda con la ayuda del atlas estereotáxico. La posición de la cánula guía para las coordenadas correctas, añadiendo o restando de bregma. Traiga su cánula guía hacia abajo hasta que toque el cráneo, y luego grabar esta coordenada ventral. Haga una marca de lápiz con un lápiz estéril en este lugar en el cráneo, que es donde se le de perforación. </li><li> Retire la cánula guía y esterilizar su broca. Con cuidado, perfore un agujero en la marca del lápiz hasta llegar a través de la anchura del cráneo. Consulte con la cánula guía para ver si podía limpiar el agujero sin tocar los lados. Mantenga la perforación y el control hasta que la cánula puede borrar en un camino recto. Una vez que se hace el agujero, use una aguja estéril para golpear suavemente las meninges con el fin de permitir la inserción de la cánula sin obstrucciones. </li><li> A continuación, utilizando un taladro de mano, hacer seis agujeros para tornillos de cabeza: dos por delante del orificio de la cánula, dos laterales para el agujero de la cánula, y posterior a los lados dos. Esterilizar seis tornillos y colocarlos en el cráneo hasta que estén bien anclados en. </li><li> Limpie la cánula guía con etanol y la solución salina, montar y bajar lentamente a la coordenada correcta ventral. Asegúrese de que los lados no se toquen y que va a la perfección vertical. </li><li> Coloque el tornillo de anclaje medial y detrás de los tornillos de cabeza posterior y mantenerla en su lugar con unas pinzas. Mezcle un lote delgada de cemento dental líquido y cubrir la cánula guía, tornillos, y el resto del cráneo con una espátula estéril. Hacer otro lote, esta vez más grueso, y cubren por completo la zona y el tornillo de la cánula y el anclaje suficiente para asegurarla. </li><li> A medida que el cemento se vuelve más grueso y antes de que solidifique, separar la piel de la Copa del cemento y el molde de la copa de cemento con la espátula para asegurarse de que la tapa de cemento es lisa por todas partes y no irrita la piel después. </li><li> Deje que el cemento dental se seque por completo antes de retirar al animal del aparato. Retire la hemostatos. Aplique bacitracina todo el camino alrededor de la tapa de cemento. </li><li> Una vez que el animal está fuera del instrumento estereotáxico, que se inyectan con 0,25 ml de penicilina IM (intramuscular), seguido de 1 ml de solución salina SC (subcutánea). </li><li> Colocar el animal en su propia jaula y el monitor hasta que se tome conciencia antes de volver a su habitación para recuperarse. </li><li> Observar a los animales hasta que se recupere de la anestesia en el día de la cirugía y al día después de la operación, hasta el final del experimento, los signos de infección y la evaluación del dolor / angustia. Esto incluye los fines de semana y días festivos. Escaso movimiento espontáneo, la vocalización de socorro durante la manipulación, postura encorvada, diarrea, hinchazón y pus en el área de la headmount, y la falta de alimentación / consumo, son signos de dolor y angustia. La buprenorfina (0.1-0.5mg/kg SC) se administra dos veces al día, y luego, en una función de las necesidades, si el animal parece estar en el dolor. El tratamiento local con antibióticos (bacitracina pomada) y el tratamiento con antibióticos sistémicos (penicilina 100.000 UI / kg IM cada 12 horas durante las primeras 48 horas después de la operación) si se administran después de la operación las infecciones. Si alguno de estos síntomas persisten después de la administración de buprenorfina, el líquido adicional, y el tratamiento antibiótico en las 12 horas de cirugía, el animal es sacrificado. </li></ol>

<ol>
	<li>Bungay, P. M., Newton-Vinson, P., Isele, W., Garris, P. A., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+dopamine+interpreted+with+quantitative+model+incorporating+probe+implantation+trauma.">Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma.</a> J. Neurochem. 86, 932-946 (2003).</li><li>Chen, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tissue+trauma+on+the+characteristics+of+microdialysis+zero-net-flux+method+sampling+neurotransmitters.">Effects of tissue trauma on the characteristics of microdialysis zero-net-flux method sampling neurotransmitters.</a> Journal of Theor. Biology. 238, 863-881 (2006).</li><li>Geiger, B. M., Behr, G. G., Frank, L., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+defective+mesolimbic+dopamine+exocytosis+in+obesity-prone+rats.">Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats.</a> FASEB Journal. 8, 2740-2746 (2008).</li><li>Pothos, E. N., Creese, I., Hoebel, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+eating+with+weight+loss+selectively+decreases+extracellular+dopamine+in+the+nucleus+accumbens+and+alters+dopamine+response+to+amphetamine,+morphine+and+food+intake.">Restricted eating with weight loss selectively decreases extracellular dopamine in the nucleus accumbens and alters dopamine response to amphetamine, morphine and food intake.</a> The Journal of Neuroscience. 15, 6640-6650 (1995).</li></ol>

The authors have nothing to disclose.

Microdiálisis in vivo es la herramienta de elección para la medición de múltiples neurotransmisores y metabolitos en sitios distintos del cerebro de un animal vivo. Sin embargo, sólo controla los niveles extracelulares de neurotransmisores y que no ofrece el tiempo de resolución de monitor de exocitosis de neurotransmisores en tiempo real. A través de una versión de la técnica llamada &quot;red de flujo&quot;, la concentración de neurotransmisores real en un lugar determinado se puede calcular, que a su vez puede dar las medidas e...

Materiales se describen en el documento del protocolo

survivable stereotaxic surgery in rodents 

La capacidad de medir los niveles extracelulares basales de los neurotransmisores en el cerebro de los animales despiertos permite la determinación de los efectos de los diferentes retos sistémicos (farmacológica o fisiológica) para el sistema nervioso central. Por ejemplo, uno puede medir directamente cómo las proyecciones de la dopamina del cerebro medio animal responde a la dopamina-la liberación de fármacos, como los estímulos d-anfetamina o naturales como la comida. En este video, te mostramos cómo implantar cánulas guía de orientación sitios específicos en el cerebro de la rata, la forma de insertar e implantar una sonda de microdiálisis y el uso de cromatografía líquida de alto rendimiento, junto con detección electroquímica (HPLC-CE) para medir los niveles extracelulares de oxidables neurotransmisores y metabolitos. Introducción Local precisa de fármacos a través de la sonda de microdiálisis permite un trabajo refinado en la especificidad del sitio en el mecanismo de un compuesto s de la acción. Esta técnica ha anatómica excelente y la resolución de química, pero sólo el tiempo de resolución modesta como muestras de microdiálisis se procesan cada 20-30 minutos para asegurar niveles detectables de neurotransmisores. Complementarias ex herramientas vivo (es decir, cortar y de cultivo celular, electrofisiología) puede ayudar con la monitorización en tiempo real de la neurotransmisión.

Watch this Scientific Journal Video about Survivable Stereotaxic Surgery in Rodents at JoVE.com

Survivable Stereotaxic Surgery in Rodents

El seguimiento de los niveles de neurotransmisores extracelular en las regiones cerebrales distintas de animales libres de movimiento además de informaciones sobre el vínculo entre la liberación de neurotransmisores y la conducta. Microdiálisis in vivo, junto con detección electroquímica ofrece anatómica excelente y la resolución de químicos, así como información sobre cómo la neurotransmisión basal se ve alterada por las manipulaciones farmacológicas o fisiológicas.

La cirugía estereotáxica de supervivencia en los roedores

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

survivable-stereotaxic-surgery-in-rodents

Research

JoVE Journal

Biology

Survivable Stereotaxic Surgery in Rodents 

68.3K vistas.  Tufts University. El seguimiento de los niveles de neurotransmisores extracelular en las regiones cerebrales distintas de animales libres de movimiento además de informaciones sobre el vínculo entre la liberación de neurotransmisores y la conducta. Microdiálisis in vivo, junto con detección electroquímica ofrece anatómica excelente y la resolución de químicos, así como información sobre cómo la neurotransmisión basal se ve alterada por las manipulaciones farmacológicas o fisi...

Video: Survivable Stereotaxic Surgery in Rodents

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

El trasplante cardíaco heterotópico en ratones

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Investigación

Biología

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Trasplante de intestino delgado en ratones

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Un procedimiento de cirugía craneotomía para imágenes cerebrales crónicas

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

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

La disección de cerebro de los primates no humanos en el espacio estereotáxica

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

El establecimiento de cultivos primarios de fibroblastos de adultos de los roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

Principios de la cirugía de roedores para el cirujano de Nueva

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Grabación de la presión intracavernosa para evaluar la función eréctil en roedores

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Inhibición de la formación de la epidermis de la herida a través de la cirugía de colgajo cutáneo completo durante la regeneración de las extremidades del ajolote

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Aplicación del movimiento pasivo de la cabeza para generar aceleraciones definidas en las cabezas de roedores

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and disease. EIM is obtained by applying a low-intensity, directionally focused, electrical current to a muscle of interest across a range of frequencies (i.e., from 1 kHz to 10 MHz) and recording the resulting voltages. From these, several standard impedance components, including the reactance, resistance, and phase, are obtained. When performing ex vivo measurements on excised muscle, the inherent passive electrical properties of the tissue, namely the conductivity and relative permittivity, can also be calculated. EIM has been used extensively in animals and humans to diagnose and track muscle alterations in a variety of diseases, in relation to simple disuse atrophy, or as a measure of therapeutic intervention. Clinically, EIM offers the potential to track disease progression over time and to assess the impact of therapeutic interventions, thus offering the opportunity to shorten the clinical trial duration and reduce sample size requirements. Because it can be performed noninvasively or minimally invasively in living animal models as well as humans, EIM offers the potential to serve as a novel translational tool enabling both preclinical and clinical development. This article provides step-by-step instructions on how to perform in vivo and ex vivo EIM measurements in mice and rats, including approaches to adapt the techniques to specific conditions, such as for use in pups or obese animals.

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals, pups).

Realización de miografía de impedancia eléctrica in vivo y ex vivo en roedores

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...