Los autores agradecen a Brian Johnson, de la Histología y Imaging Core de la Universidad de Washington en busca de ayuda con la tinción de tricrómico y análisis. Este trabajo fue apoyado por el NIH subvenciones y HL098067 HL089455.

Los Comités Institucionales Cuidado y Uso de Animales (IACUC) de la Universidad de Washington y el Cedars-Sinai Medical Center han aprobado el trabajo con animales necesarios para estos estudios. 1. Preparación <ol><li> Esterilizar tanto las pinzas de extremos romos y el depresor través de autoclave. </li><li> El uso de una cabina de seguridad biológica, preparar una solución de trabajo de la BLM en PBS a partir del polvo liofilizado. Sonicar la solución durante 10 min a 35 Khz para asegurar una mezcla uniforme. Nota: Se recomienda un volumen total de entre 30 y 45 l para evitar la variación de pipeteado en el extremo inferior, y la asfixia con volúmenes más grandes. </li><li> Prepare un área de trabajo limpia que incluye aproximadamente 1 m 2 para el procedimiento en sí, así como los lugares designados para jaulas, tanto antes como después del procedimiento. </li><li> Fijar la base de la junta procedimiento para el banco inmediatamente delante del investigador poniendo 2 o 3 tiras de cinta de laboratorio a través de la base y SUBCARying banco. Ver Figura 1 para más especificaciones sobre la creación de una tabla. </li><li> Ate una sola longitud de tamaño de 4.0 hilo de sutura entre los dos tornillos de posicionamiento de la junta procedimiento. </li><li> Generar un espirómetro improvisada eliminando y desechando el émbolo de tres jeringas de 1 ml, y el depósito de 60 l de PBS en la parte superior de cada barril para formar un sello hermético. Asegurar el centro del catéter vagamente a una de las jeringas y colocarlo a un lado de la junta. </li><li> Aspirado de 300 l de aire en una jeringa de 1 ml y colocarlo a un lado de la junta. </li><li> Cortar una pieza adicional de cinta de aproximadamente 6 pulgadas de largo y el lugar de uno de los lados. Esto se utiliza para asegurar el animal a la junta en el paso 2.4. </li><li> Configurar una cámara de isoflurano. Adjuntar O2, isoflurano, y el vacío a los puertos apropiados tanto en la cámara de exposición y el vacío despacho. Alternativamente, administrar anestésico en un isoflurano-compatible biológicacabina de seguridad. </li></ol> 2. La intubación <ol><li> Anestesiar al ratón con isoflurano en la cámara hasta que se pierde la conciencia y la respiración se desacelera a un ritmo adecuado. Una exposición típica incluye 4% de isoflurano y 2% O 2 durante 3 a 4 minutos, y el resultado ideal es de 2 a 2,5 min de sedación. Esto corresponde a una tasa de respiración de 1 respiración cada 2 seg. </li><li> A la espera de la sedación a instaurarse, aspirado de entre 30 y 45 l de BLM en una pipeta y el lugar de uno de los lados. </li><li> Cuando esté listo, suspender el ratón sedado por sus incisivos superiores desde el hilo unido a los tornillos de posicionamiento de la plataforma procedimiento. Asegúrese de que el dorso del animal quede plana contra la superficie de la plataforma. </li><li> Teniendo cuidado de no restringir la ventilación, coloque un trozo de cinta sin apretar a través de la parte inferior (caudal) de la cavidad torácica, justo por encima del diafragma. La colocación debe ser lo suficientemente apretado para mantener la alineación adecuada durante el procedure, pero no tan apretado que restringe la respiración. </li><li> Encender el iluminador a entre 80% y 100% de intensidad y orientar el cuello de cisne de modo que es de 1 a 2 cm de la superficie de la piel, cerca del plexo solar. revisar periódicamente la punta del cuello de cisne para el calor para asegurarse de que no perjudique el ratón. </li><li> De pie detrás de la plataforma, utilizar las pinzas de punta roma, estériles para localizar la lengua. Teniendo cuidado de evitar los incisivos inferiores, suavemente agarre y sacar la lengua fuera de la cavidad oral. </li><li> Usando la mano restante, inserte el depresor y utilizarlo para aplanar la lengua contra el suelo de la cavidad oral. Liberar la pinza, pero deje el depresor en el lugar durante los siguientes dos pasos. </li><li> Oriente la luz para que la tráquea es visible al guiar el cuello de cisne proximalmente desde el nivel del plexo solar hasta que alcanza el nivel de la bronquios principales. Nota: La tráquea se puede distinguir fácilmente por la acción de la respiración, WHIch hace que la luz emitida a fluctuar en intensidad. Cuando está colocado correctamente, esta estructura será discernible en el plano axial como un pasador situado en el centro de la luz con la luz ambiental mínimo en la propia cavidad oral. </li><li> Ángulo de la jeringa para que siga el camino natural de la tráquea, y bajar la punta del catéter 22-G, con la jeringa unida que contiene la gota, directamente en el lumen. La burbuja PBS comenzará a subir y bajar con cada respiración sobre la colocacion exitosa. Nota: Esta acción se puede retrasar por varios segundos, como resultado de la sedación profunda. </li><li> Alimentar el catéter en un 5 mm adicionales. Retire el depresor de lengua. </li><li> Cambiar la jeringa con la mano opuesta, y agarrando el cubo, retire suavemente la jeringa. </li><li> Fuerte entre 30 y 45 l de BLM en el centro del interior del cubo del catéter, coloque la segunda jeringa y dispensar 300 l de aire en el cubo. </li><li> Vuelva a colocar la segundajeringa con la primera que contiene la burbuja de PBS. La burbuja seguirá subiendo y bajando si el procedimiento se ha realizado con éxito. </li></ol> 3. Cuidado post-procedimental <ol><li> Retirar el catéter y cinta, y colocar al animal en un lugar cálido y seco hasta que se recupere la conciencia - por lo general dentro de un par de minutos. </li></ol>

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The authors have nothing to disclose.

En los casos en que la aerosolización es poco práctico debido a la limitada disponibilidad de reactivos, la seguridad, o el coste, la administración directa de la tráquea es un método superior para la administración de agentes exógenos en los pulmones 16 transtraqueal instilación ha sido ampliamente utilizado para lograr esto.; Sin embargo, como con cualquier intervención quirúrgica, sino que también lleva consigo la posibilidad de complicaciones causadas por el procedimiento en sí, y no necesaria...

A pesar de algunas diferencias anatómicas y fisiológicas, 1 modelos murinos continúan siendo de gran valor para el modelado de la biología humana y la patogénesis de la enfermedad. 2 Desde un punto de vista de la cría, los ratones son fáciles de manejar, tienen un tiempo de reproducción de baja, una esperanza de vida acelerada, y son relativamente baratos a casa. Con el desarrollo de diversas cepas genéticas y estrategias (por ejemplo., Knock-out condicionales, ratones reportero, enfoques de linaje de trazado, etc.), así como la amplia gama de reactivos disponibles (por ejemplo., Anticuerpos, proteínas recombinantes, inhibidores, etc.), los ratones se han convertido en un organismo modelo de vertebrados esencial para descubrir los procesos de homeostasis y enfermedades humanas. 3 Los ratones han sido especialmente valiosa para el estudio de enfermedades pulmonares, incluyendo lesión pulmonar aguda (ALI) y fibrosis pulmonar. 4 ALI en los seres humanos puede ser causado por trauma, lesión o sepsis y se caracteriza por epitelial yfuga endotelial (es decir., edema), la inflamación y la fibrosis incipiente. En muchos pacientes, ALI progresa a su forma severa, síndrome de dificultad respiratoria aguda (SDRA), que a menudo resulta en la fibrosis y la muerte por insuficiencia respiratoria. 5,6 La fibrosis pulmonar es una patología progresiva y fatal que se caracteriza por el exceso de deposición de la matriz extracelular , especialmente colágeno de tipo I, que lleva a la función pulmonar. 7,8 administración de bleomicina (BLM) es el modelo más ampliamente utilizado y mejor caracterizado para inducir ALI y fibrosis en animales de experimentación. 9 Aunque BLM inducida por la fibrosis pulmonar en roedores hace no recapitular completamente los fenotipos fibróticos humanos, 10 estudios con ratones con este modelo han llevado al descubrimiento de muchos factores importantes que influyen en la aparición y progresión de la enfermedad. 11 Aunque el mecanismo exacto (s) detrás de la fibrogénesis BLM inducida son desconocidos, la lesión iniciarse cree que surgen de la ruptura de cadenas de ADN dependientes del contacto en las células epiteliales que recubren las vías aéreas y los alvéolos, y, en particular, tipo 1 neumocitos. 12 La necesidad de un contacto directo entre la BLM y el epitelio pulmonar pone de relieve la importancia de una vía de administración robusta , y estas preocupaciones también son pertinentes para una amplia gama de tratamientos dirigidos a las vías respiratorias distales, incluyendo las proteínas recombinantes, anticuerpos, siRNA, virus, bacterias, partículas, y más. Aspiración orofaríngea (OPA) ha sido ampliamente utilizado para este fin 13, pero un importante una deficiencia de OPA es que alguna porción del agente suministrado puede ser tragado en el tracto gastrointestinal, lo que conduce a la imprecisión en la dosis administrada. Otro método ampliamente utilizado es la instilación transtraqueal, que implica la traqueostomía con anestesia fuerte como para exponer la tráquea y la instilación de un agente directamente en el tracto respiratorio. 14 Sin embargo, no sólo puede talesun procedimiento sea indeseable debido a su invasividad, pero también es mucho tiempo, requiere un poco de entrenamiento, y causa una lesión potente para el tracto respiratorio. 15,16 Varios protocolos se han desarrollado que implican la administración directa de los agentes en el tráquea sin la necesidad de intervención quirúrgica, 16,17,18,19,20 tiempos de recuperación pero estos métodos implican extendían causada por anestésicos potentes, el uso de equipo costoso (es decir., otoscopio / laringoscopio, tableros de procedimientos disponibles en el mercado, de fibra óptica cables, etc.), un exceso de manipulación en la cavidad oral, y la incertidumbre con respecto a la dosis. Este documento describe un método relativamente fácil de administración a través de intubación que permite al investigador rápida, económica y fiable a infundir un reactivo en el pulmón murino con riesgo limitado de daño residual a los tejidos circundantes.

<table><tbody><tr><td>Bleomycin For Injection, 30 units/vial</td><td>APP Pharmaceuticals, LLC</td><td>103720</td><td>For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots</td></tr><tr><td>Blunt End Forceps</td><td></td><td></td><td></td></tr><tr><td>Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4&quot;)&amp;nbsp;</td><td>Covidien</td><td>E1551G</td><td>Before use, create a 45 degree bend 1.5 cm&amp;nbsp; from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.&amp;nbsp;</td></tr><tr><td>Exel Safelet Catheter 22G X 1&quot;</td><td>Exel International</td><td>26746</td><td></td></tr><tr><td>1 ml Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)</td><td>BD</td><td>309659</td><td></td></tr><tr><td>0.2 ml Pipettor and Filter Tips</td><td></td><td></td><td></td></tr><tr><td>Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator</td><td>Dolan Jenner Industries, Inc.</td><td>181-1</td><td>Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.</td></tr><tr><td>Intubation Board</td><td></td><td></td><td>See Diagram 1.</td></tr><tr><td>Colored Label Tape: 0.5&amp;nbsp;in. Wide</td><td>Fisherbrand</td><td>15-901-15A</td><td></td></tr><tr><td>Oxygen&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Phosphate-Buffered Saline, 1X</td><td>Corning</td><td>21-040-CV</td><td>Product should be sterile</td></tr><tr><td>Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0</td><td>Harvard Apparatus</td><td>517698</td><td></td></tr><tr><td>Table Top Anesthesia Machine Isoflurane</td><td>Highland Medical Equipment</td><td></td><td>http://www.highlandmedical.net/</td></tr><tr><td>Slide Top Induction Mouse Isoflurane Chamber</td><td>MIP / Anesthesia Technologies</td><td>AS-01-0530-SM</td><td></td></tr><tr><td>FORANE (isoflurane, USP) Liquid For Inhalation 100 mL&amp;nbsp;</td><td>Baxter</td><td>1001936040</td><td></td></tr><tr><td>&amp;nbsp;Nanozoomer Digital Pathology system</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>IgM ELISA Quantification Kit&amp;nbsp;</td><td>Bethyl Laboratories</td><td>E90-101</td><td></td></tr></tbody></table>

an improved method for rapid intubation of the trachea in mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

Ratones intubados se controlaron diariamente para la pérdida de peso y la angustia, y se sacrificaron 4, 10 o 17 días más tarde a través de inyección intraperitoneal de 2,5% 2,2,2 tribromoetanol. Se recogió el lavado broncoalveolar (BAL) en tres lavados de PBS como se describe en otra parte 21 y el pulmón derecho se fijó en formalina al 10%, embebidos en parafina, y se tiñeron con tricrómico de Masson por la Universidad de Washington Histología y I...

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An Improved Method for Rapid Intubation of the Trachea in Mice

Un método mejorado para la rápida intubación de la tráquea en ratones

En este artículo se presenta un método rápido y sencillo para la administración de bleomicina directamente en la tráquea ratón a través de la intubación. Las principales ventajas de este método son que es altamente reproducible, fácil de dominar, y no requiere equipo especializado ni largos tiempos de recuperación.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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25.1K vistas.  University of Washington.  En este artículo se presenta un método rápido y sencillo para la administración de bleomicina directamente en la tráquea ratón a través de la intubación. Las principales ventajas de este método son que es altamente reproducible, fácil de dominar, y no requiere equipo especializado ni largos tiempos de recuperación. 

Video: An Improved Method for Rapid Intubation of the Trachea in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

La presión de la ventilación controlada para inducir la lesión pulmonar aguda en ratones

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Investigación

Medicina

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Intubación endotraqueal en ratones<em&gt; Vía</em&gt; La laringoscopia directa utilizando un otoscopio

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un sencillo método de intubación ratón de pulmón

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

No Invasiva Intratraqueal Intubación para Estudiar la Patología y Fisiología de pulmón de ratón

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

A reversible, método no invasivo para las mediciones de resistencia de las vías y Toma de Muestras Líquido del Lavado broncoalveolar en ratones

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biología

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Un modelo del cerrado-pecho para inducir constricción aórtica transversal en ratones

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Un modelo de ratón de intubación orotraqueal y cirugía de reperfusión de isquemia pulmonar ventilada

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Inmunología e Infección

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Neurociencias

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

Intubación orotraqueal repetida en ratones

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

<meta charset="utf8"></head><body>Protocolo de intubación endotraqueal basado en traqueotomía para modelos de ratas que no sobreviven

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that require controlled or high ventilation pressures or access to the thoracic cavity and organs. Conventional endoorotracheal intubation using the anatomically existing route through the mouth is well suited for survival experiments. However, this procedure poses some challenges, including generally higher levels of the required experience and technical skill, more advanced equipment, and greater time effort with relevant intubation failure rates and complications such as tracheal perforation, temporary systemic hypooxygenation, and relevant aerial leakage.
This manuscript, therefore, presents a detailed step-by-step protocol for endotracheal intubation through tracheotomy in non-survival rat models when guaranteed intubation success, constant oxygenation levels, high ventilation pressures, or open thoracotomy are required.
The protocol emphasizes the importance of meticulous surgical technique to ensure consistent and reliable outcomes, especially for researchers who are inexperienced or lack routine in the technique of endoorotracheal intubation via direct laryngoscopy. This procedure is, therefore, expected to minimize animal suffering and unnecessary animal losses.

<meta charset="utf8"></head><body>Intubación endotraqueal mediante traqueotomía y posterior toracotomía en ratas para aplicaciones no relacionadas con la supervivencia

Here we introduce a standardized procedure for endotracheal intubation via tracheotomy followed by thoracotomy in rats, aimed at enhancing the precision and reproducibility of non-survival applications requiring invasive ventilation and exposure of thoracic organs in in vivo rat models.

Intubación endotraqueal mediante traqueotomía y posterior toracotomía en ratas para aplicaciones no relacionadas con la supervivencia

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that ...