En el presente estudio fueron apoyadas por los Estados Unidos Institutos Nacionales de Salud de subvención R01-HL092188, R01-DK083385 y R01HL098294, y la Fundación para la Educación e Investigación de Anestesia (FAER) otorga a HKE y DFG (Deutsche Forschungsgemeinschaft) Becas de Investigación (GR2121/1- 1) y la Asociación Americana del Corazón Grant AG.

Observaciones generales. Todas las operaciones deben llevarse a cabo bajo un microscopio vertical de disección (Leica) y utilizando un coagulador quirúrgico. Los ratones en los grupos experimentales y de simulación debe ser equiparado en edad y peso para asegurar la comparabilidad de los resultados. La administración de la temperatura, la presión arterial, la anestesia y el líquido debe ser estable y controlado durante todo el experimento. 1. Anestesia y Cirugía <ol><li> Ratones C57BL / 6 debe estar dentro de un rango de edad de 10 a 16 semanas y debe ser acompañado de género en los grupos respectivos. </li><li> Anestesie ratones con pentobarbital sódico (70 mg / kg de peso corporal ip) y mantener la anestesia con aprox. 10 mg / kg / h pentobarbital sódico. La sobredosis puede reducir significativamente la presión arterial y por lo tanto puede alterar los resultados. </li><li> Los ratones, colóquelas sobre una mesa calentada a temperatura controlada (temperatura ambiente, Effenberg, Munich, Alemania) con un termómetro rectal, unida a una reacción térmicacontrolador para mantener la temperatura corporal a 37 º C. </li><li> Los ratones y colóquelos en una posición supina sobre la mesa de cirugía, con las extremidades superiores e inferiores unidas a la mesa con cinta extraíble. </li><li> En el caso de los experimentos de supervivencia de técnicas adecuadas de asepsia, incluyendo el recorte de cabello, la desinfección de la piel con los yodóforos (Betadine, por ejemplo), la manipulación estéril de instrumentos esterilizados en autoclave, y usando máscaras de unos guantes estériles para el cirujano son obligatorios. </li></ol> 2. La oclusión de la tríada portal <ol><li> Cubra el área de la incisión con aceite mineral para evitar la inhalación de pelo de ratón por el corte de la piel y previniendo así el riesgo de alergias del pelo del ratón. Realizar laparotomía media y la incisión de la línea alba hasta el extremo superior de modo que usted puede exponer el hígado. Si es posible realizar la incisión con un electrodo de coagulación (Erbe, ICC50, Tuebingen, Alemania) para prevenir el sangrado. </li><li> Coloque la tabla de calefacción con el ratón en una posición que la cola es de direcciónTed para el cirujano. Esa posición te ofrece la mejor configuración para la localización de la tríada y colocar la sutura. </li><li> Desplazar el intestino en el lado derecho utilizando un hisopo de algodón húmedo para exponer la punta de la tríada portal. </li><li> Un poco cambiar el lóbulo medio e izquierdo hacia el diafragma y el lóbulo derecho hacia el intestino. Eso le dará una visión clara de la tríada portal por encima de la bifurcación de la derecha, mediana / izquierda lóbulos. </li><li> Inspeccione cuidadosamente la tríada portal y sostenga el lóbulo derecho suavemente con unas pinzas suaves. Eso le dará una bonita vista de la tríada portal de la izquierda. </li><li> Una vez identificado visualmente a colocar la aguja con el hilo de sutura (sutura de 7/0 nylon; Ethicon, Norderstedt, Alemania) a través de un soporte de la aguja en el lado izquierdo tríada portal, incluyendo la arteria hepática, la vena hepática, y el conducto biliar de la izquierda a la derecha del ratón. </li><li> La puntada tiene que estar estrechamente con la tríada. Un punto más profundo puede lesionar la vena vaca inferior que se encuentra bajola tríada. Por lo tanto mantener los lóbulos hepáticos derecho hacia abajo con las pinzas para identificar la vena cava antes de colocar la puntada. </li><li> Tome la punta de la aguja en el lado derecho con una pinza y tire suavemente de la zona del hígado, sin lesionar el hígado. </li><li> Adjunte a cada extremo de la sutura un tubo Eppendorf (aproximadamente 3 g, llena de agua) y colocar ambos extremos de sutura sobre los titulares de sutura. Coloque el soporte de sutura distal de la puntada para que la sutura no interfiere con la tríada derecha. </li><li> Mediante la aplicación de las ponderaciones de la tríada será inmediatamente obstruida, provocando la interrupción del suministro de sangre a los lóbulos izquierdo y la mediana del hígado. El éxito de la oclusión puede ser fácilmente confirmado por la inspección visual de la palidez pálido de los lóbulos isquémicos. En contraste, el cambio de color desaparece inmediatamente cuando los pesos colgantes son liberados de los polos y el hígado se reperfundido. Este modelo de isquemia parcial hepática mesentérica evita la congestión mediante la preservación de Blood flujo de los lóbulos hepáticos derecho. </li><li> Vuelva a colocar el intestino en la cavidad abdominal. Mantener el hígado y el intestino tibia con una esponja húmeda empapada con agua a 37.0 ° C. Además de mantener una temperatura corporal estable de cubrir los ratones con una envoltura de los alimentos disponibles en el mercado después de terminar la cirugía. </li><li> Realizar la isquemia como se requiere según el protocolo experimental. Después de un comunicado de la isquemia de tiempo definido los pesos y la reperfusión de los lóbulos del hígado la mediana y la izquierda se iniciará inmediatamente. Esta técnica permite la producción de isquemia reversible en ratones. Si no hay tiempos de isquemia más se llevará a cabo retirar con cuidado el hilo de sutura. </li><li> Añadir aproximadamente 400 l de cloruro sódico en la cavidad abdominal 15 minutos antes y directamente después de la reperfusión. Continuar para sustituir el líquido cada 30 minutos en una cantidad de 100 l para compensar la pérdida de fluido durante el tiempo de reperfusión. </li><li> Para los experimentos de supervivencia de la herida quirúrgica se cierra con sutura continua de la pared muscular y wi pielª una sutura 4/0. </li></ol> 3. Los cambios en la perfusión del hígado Los parámetros de resultados se recomienda lo siguiente para determinar isquemia hepática daño hepático siguiente: Determinar las enzimas hepáticas (por ejemplo, ALT, AST) y la histología hepática (tinción H &amp; E) isquemia hepática siguiente y reperfusión 3,9. <img alt="Figura 1" src="/files/ftp_upload/2550/2550fig1.jpg" /> Figura 1 Modelo de un sistema de suspensión-el peso de la isquemia hepática. Un sistema de peso que cuelga se ha establecido para inducir isquemia hepática del lóbulo izquierdo y la mediana de tres. (A) Por lo tanto el lóbulo caudado y el derecho se separaron suavemente del lóbulo izquierdo. El lóbulo derecho estaba ligeramente desplazado a continuación para ver con claridad la tríada portal encima de la bifurcación de los lóbulos derecho e izquierdo, la mediana. Una sutura de nylon 8,0 se colocó bajo la tríada hepática izquierda. (B) al final de cada sutura un peso pequeño (3 g Eppendorf tuBES llenos de agua) se adjunta y aplicada. Isquemia hepática es visible por un cambio de color del lóbulo izquierdo y la mediana de rojo a amarillo pálido. Este diseño experimental permite la oclusión precisa y reperfusión de la tríada hepática izquierda por la aplicación y liberación de la carga de peso. <img alt="Figura 2" src="/files/ftp_upload/2550/2550fig2.jpg" /> Figura 2. Efecto de diferentes tiempos de isquemia en el hígado alanina (ALT) y aspartato (AST) aminotransferasa en ratones. Parcial portal de la isquemia se indujo la tríada como se indica (0 - 60 min de isquemia). Después de 2 h de la reperfusión, ALT (A) y AST (B) se midieron. Los resultados se expresan como media ± SE de 6 a 7 ratones / grupo. * P &lt;0,05 en comparación con isquemia min 0 (control simulado).

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No hay conflictos de interés declarado.

El presente estudio describe una técnica de realización de la isquemia en un modelo murino utilizando un sistema de suspensión de peso para la oclusión de la tríada portal hepático izquierdo. Aunque es un método fácil de aprender las trampas siguientes deben ser tenidas en cuenta. Un error muy común al principio es un punto de profundidad que incluye la tríada portal y la vena cava. La tasa de respiración y el corazón se ralentizará un plazo de 15 minutos debido a la mayor resistencia y la disminución de devolver la sangre al corazón que resulta en una temprana muerte de los ratones. La mejor manera de prevenir la introducción de asignaturas de la vena cava es localizar la vena cava antes de la puntada a la celebración de los lóbulos derecho hacia abajo con unas pinzas. Otra cuestión importante es que los pesos no son de pesado que puede dar lugar a demasiada tensión en el juicio portal de izquierda y derecha que también detiene el flujo sanguíneo en los lóbulos derecho. Por lo tanto la cantidad de agua en los viales de Eppendorf debe ajustarse al peso del ratón y la stitulares UTURO debe colocarse distalmente desde la ubicación de puntada. Por el contrario, si los pesos son de la presión arterial no se haya detenido por completo. Ambos errores son fácilmente detectables por un cambio de color pálido, adicional a la del lóbulo derecho o el cambio de color insuficiente de los lóbulos medios e izquierdo, respectivamente. Por lo tanto el cambio de color hepática tras la aplicación de los pesos debe ser controlada cada 10 minutos. Otra complicación quirúrgica común es las heridas sufridas por el hígado con la aguja. Por lo tanto los lóbulos derecho debe ser mantenido con un fórceps suaves de modo que la punta de la aguja puede ser fácilmente visto y tomada con un soporte de aguja. Si estas trampas en el comienzo se tienen en cuenta el sistema de suspensión de peso para la isquemia hepática proporciona una lesión altamente reproducibles debido a la isquemia, reduciendo al mínimo la variabilidad de daño hepático asociado con sujeción de la tríada portal. Mediante el uso de un sistema de suspensión de peso, la tríada portal es manipula solamente una vez en toda laprocedimiento quirúrgico, causando daños significativamente menor a los lóbulos hepáticos. Además, ninguna de congestión hepática o intestinal se produce con esta técnica. Más traumatismo de los tejidos se produce mediante la eliminación y la sustitución de las abrazaderas de los buques, sobre todo con una nueva aplicación de la pinza durante los ciclos de isquemia múltiples, como en el precondicionamiento isquémico. El uso de colgar pesos que esté en un lugar alejado del tejido hepático, de acuerdo con nuestras observaciones, proporciona la ventaja de oclusión fiable al tiempo que evita el trauma del tejido debido a la manipulación de los lóbulos del hígado de la reaplicación de una abrazadera. En conjunto, el presente estudio proporciona la viabilidad del sistema de suspensión-el peso de la oclusión de la tríada portal durante la isquemia, reduciendo al mínimo la variabilidad y las limitaciones asociadas con la sujeción. Así, esta técnica podría ser de interés para otros investigadores que consideran que el estudio de la isquemia hepática.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> El pentobarbital sódico (más grave) </td><td> Ls Vortech Farmacéutica, SA </td><td> VPL 9372 </td><td> 4 mg / ml en solución salina </td></tr><tr><td> Sutura, Prolene 8.0 </td><td> Ethicon, EE.UU. </td><td> M8739 </td><td> reutilizables </td></tr><tr><td> microscopio de disección </td><td> Leica </td><td> n / a </td><td> considerar la distancia generosa de trabajo </td></tr><tr><td> Calefacción Mesa </td><td> Rt, Effenberger, Alemania </td><td> n / a </td><td> único proveedor y una sola </td></tr><tr><td> Los instrumentos quirúrgicos </td><td> IPM, Dumont </td><td></td><td> Pinzas, porta agujas </td></tr><tr><td> Coagulador </td><td> Erbe, ICC50, Tuebingen, Alemania </td><td> n / a </td><td> Distintos proveedores de coaguladores </td></tr></tbody></table> Tabla 1. Reactivos específicos y equipos.

use of a hanging-weight system for liver ischemia in mice

Lesión hepática aguda debido a la isquemia puede ocurrir durante varios procedimientos clínicos de trasplante hepático, por ejemplo, la resección del tumor hepático o reparación de trauma y puede resultar en insuficiencia hepática que tiene una alta tasa de mortalidad 1-2. Por lo tanto los estudios murinos de isquemia hepática se han convertido en un importante campo de la investigación, proporcionando la oportunidad de utilizar los estudios farmacológicos y genéticos 3.9. En concreto, los ratones condicionales con la eliminación específica de tejido de un gen (CRE, flox sistema) proporciona una visión de la función de las proteínas en tejidos específicos 10-13. Debido a la dificultad técnica asociada con manual de sujeción de la tríada portal en ratones, se realizó una evaluación sistemática mediante un sistema de suspensión de peso para el portal de la oclusión tríada que se ha descrito previamente 3. Mediante el uso de un sistema de suspensión de peso que colocar una sutura alrededor de la rama izquierda de la tríada portal sin causar ningún daño a los lóbulos hepáticos, ya que también lamejores abrazaderas disponibles puede causar daño hepático del tejido debido a la cercanía de tejido hepático para los vasos. Además, la rama derecha de la tríada hepática todavía se perfundieron tanto, no se produce congestión intestinal con esta técnica como el flujo de sangre a los lóbulos hepáticos derecho se conserva. Por otra parte, la tríada portal es manipula solamente una vez durante todo el procedimiento quirúrgico. Como resultado, los procedimientos como pre-acondicionamiento, con tiempos cortos de isquemia y reperfusión, se pueden realizar fácilmente. La evaluación sistemática de este modelo mediante la realización de la isquemia diferente y los tiempos de reperfusión reveló una correlación estrecha del tiempo de isquemia hepática con daño hepático, medido por la alanina (ALT) y aspartato (AST), los niveles séricos de aminotransferasas 3,9. En conjunto, estos estudios confirman la lesión hepática altamente reproducible cuando se utiliza el sistema de suspensión-el peso de la isquemia y la reperfusión hepática intermitente. Así, esta técnica podría ser útil para el interés de otros investigadoresdisfunción eréctil en los estudios de isquemia del hígado en ratones. Por lo tanto el clip de vídeo ofrece un detallado paso a paso la descripción de esta técnica.

Watch this Scientific Journal Video about Use of a Hanging-weight System for Liver Ischemia in Mice at JoVE.com

Use of a Hanging-weight System for Liver Ischemia in Mice

Hemos establecido un nuevo modelo murino de un sistema de peso que cuelga de la oclusión de la tríada portal. Esta técnica puede ser útil para futuras investigaciones en modelos de isquemia hepáticas de ratón.

El uso de un sistema de suspensión de peso para la isquemia del hígado en ratones

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

use-of-a-hanging-weight-system-for-liver-ischemia-in-mice

Research

JoVE Journal

Medicine

15.8K vistas.  University of Colorado, Denver. Hemos establecido un nuevo modelo murino de un sistema de peso que cuelga de la oclusión de la tríada portal. Esta técnica puede ser útil para futuras investigaciones en modelos de isquemia hepáticas de ratón.

Video: Use of a Hanging-weight System for Liver Ischemia in Mice

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

El uso de un sistema de peso que cuelga de la oclusión coronaria en ratones

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Investigación

Medicina

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

El uso de un sistema de suspensión de peso para la oclusión de la arteria renal aislada

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

El uso de modelos animales de sepsis para evaluar nuevas terapias a base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis inducida por isquemia focal como un modelo de lesión de médula espinal en ratones

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

El uso de un sistema de bajo flujo Anestesia digital para ratones y ratas

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Guiado por ecografía intracardiaca inyección de células madre mesenquimales humanas para aumentar autoguiado hacia el blanco en el intestino para su uso en modelos murinos de enfermedades de intestino inflamatorias experimentales

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Rebanadas de hígado cortadas con precisión de la murino como modelo Ex Vivo de biología hepática

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lesión de isquemia-reperfusión de órganos mediante la simulación de cambios hemodinámicos en el modelo de trasplante de hígado de rata

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Trasplante hepático en ratón y análisis de la permeabilidad del aloinjerto

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Autor destacado: Mejora de la investigación de trasplantes a través de la angiografía por microTC en modelos murinos 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Trasplante hepático ortotópico exitoso en ratones mediante angiografía por microtomografía computarizada

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...