Este trabajo fue apoyado por Key Project Medical Science and Technology Development Foundation, Nanjing Department of Health (No.YKK16098).

Los experimentos con animales fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) del Hospital Nanjing Drum Tower.
1. Preparación de los reactivos y animales
<ol><li>Disolver el clorhidrato de doxorrubicina (Pfizer, EE.UU.) en agua esterilizada. Vórtice para obtener una solución Dox de 1 mg/ml y mantener a 4 °C.</li>
	<li>Use ratones C57BL/6 (8–10 semanas de edad; 25–30 g de peso). Para este estudio, los ratones fueron comprados en el Centro de Investigación de Animales Modelo de la Universidad de Nanjing y mantenidos en la sala de animales del Hospital nanjing Drum Tower.</li>
	<li>Las jaulas de ratones libres de patógenos se mantuvieron bajo un ciclo de luz/oscuridad de 12 h a una temperatura constante de 23 °C. Todos los animales fueron alimentados con una dieta normal de chow y recibieron comida y agua ad libitum.</li>
</ol>2. Establecimiento del modelo animal DCM
<ol><li>Aleatorizar ratones en un grupo normal (n = 5) y Dox (n = 5).</li>
	<li>Administrar la solución dox por vía intraperitoneal a una dosis de 5 mg/kg utilizando una jeringa esterilizada de 1 ml 1 veces a la semana para el grupo Dox. Trate a los ratones de control de la misma manera con la misma cantidad de solución salina.</li>
	<li>Mida el peso corporal de los dos grupos semanalmente. En consecuencia, ajuste la dosis de inyección en función del peso corporal semanalmente durante un total de 4 semanas, con una dosis acumulada de 20 mg / kg (Figura 1). NOTA: Se eligió un período de tiempo de 4 semanas porque la ecocardiografía a las 4 semanas mostró una diferencia significativa en la función cardíaca entre los dos grupos.</li>
</ol>3. Examen ecocardiográfico
<ol><li>Al final de la cuarta semana, realice un examen ecocardiográfico de los ratones.</li>
	<li>Anestesiar a los ratones con 2% de isoflurano por vía intranasal. Si los ratones no responden a una pizca de la piel con una pinza dentada o estimulando los dedos de los pies y la cola, continúe con el protocolo.</li>
	<li>Coloque el ratón en una plataforma de manipulación de animales en posición supina. Para mantener la anestesia, cubra la nariz y la boca del animal con un cono nasal y administre isoflurano al 2%. NOTA: Para la anestesia IP, inyecte hidrato de cloral al 4% a una dosis de 0,2 ml/20 g.</li>
	<li>Retire el pelaje del pecho con cuidado con una afeitadora eléctrica. Evaluar la función cardíaca in vivo mediante ecocardiografía transtorácica.</li>
	<li>Realizar el ecocardiograma del VI tanto en las vistas paraesternales de eje largo como de eje corto a una velocidad de fotogramas de 233 Hz. Las dimensiones sistólicas finales y diastólicas finales se definieron como las fases correspondientes a la onda T del ECG y a la onda R, respectivamente.</li>
	<li>En los trazados en modo M, mida el diámetro sistólico final del VI promedio (LVIDd), el diámetro diastólico final del VI (LVID), el grosor septal interventricular (IVS) y el grosor de la pared posterior del VI (LVPW) de 3 a 5 latidos cardíacos. También calcule la fracción de eyección (EF) y el acortamiento de fracción (FS) en función de la ecocardiografía.</li>
</ol>4. Tinción histológica
<ol><li>Después del análisis ecocardiográfico, sacrificar a los ratones mediante inyección intraventricular de cloruro de potasio al 10%.</li>
	<li>Perfundir el corazón con aproximadamente 30 ml de solución salina después de la disección hasta que el hígado y el pulmón se vuelvan pálidos.</li>
	<li>Extirpe el corazón y lávelo a fondo en una solución tampón de fosfato para extruir la sangre.</li>
	<li>Fije el corazón en formalina al 4% a temperatura ambiente durante 24 h y trate el tejido en una caja de parafina para que la cera de parafina se enfríe y se solidifique.</li>
	<li>Cortar los corazones en rodajas de 5 μm de grosor para la tinción patológica.</li>
	<li>Desparar y rehidratar secciones que contengan músculo papilar.<ol>
<li>Incubar portaobjetos a 55 °C durante 30 min. Luego, incubar en xileno 2x durante 2 min cada uno; 100% etanol 2x durante 2 min cada uno; 95% de etanol 2x durante 2 min cada uno; 80% de etanol durante 2 min; 75% de etanol durante 2 min; y etanol al 50% durante 2 min.</li>
	</ol>
</li>
	<li>Tinción con hematoxilina y eosina (H&E), así como la tinción de Masson.</li>
</ol>

<ol>
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G., Semsarian, C., MacDonald, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+cardiomyopathy.">Dilated cardiomyopathy.</a> Lancet. 390 (10092), 400-414 (2017).</li><li>Ichihara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+oxidative+stress+and+cardiac+dysfunction+by+bisoprolol+in+an+animal+model+of+dilated+cardiomyopathy.">Attenuation of oxidative stress and cardiac dysfunction by bisoprolol in an animal model of dilated cardiomyopathy.</a> Biochemical and Biophysical Research Communications. 350 (1), 105-113 (2006).</li><li>Fountoulakis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+the+heart+mitochondrial+proteome+in+a+desmin+null+heart+failure+model.">Alterations in the heart mitochondrial proteome in a desmin null heart failure model.</a> Journal of Molecular and Cellular Cardiology. 38 (3), 461-474 (2005).</li><li>Fairweather, D., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coxsackievirus-induced+myocarditis+in+mice:+a+model+of+autoimmune+disease+for+studying+immunotoxicity.">Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.</a> Methods. 41 (1), 118-122 (2007).</li><li>Wang, Z. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTRP3+protected+against+doxorubicin-induced+cardiac+dysfunction,+inflammation+and+cell+death+via+activation+of+Sirt1.">CTRP3 protected against doxorubicin-induced cardiac dysfunction, inflammation and cell death via activation of Sirt1.</a> Journal of Molecular and Cellular Cardiology. 114, 38-47 (2018).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TGF-beta+pathway+mediates+doxorubicin+effects+on+cardiac+endothelial+cells.">The TGF-beta pathway mediates doxorubicin effects on cardiac endothelial cells.</a> Journal of Molecular and Cellular Cardiology. 90, 129-138 (2016).</li><li>Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., Gianni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthracyclines:+molecular+advances+and+pharmacologic+developments+in+antitumor+activity+and+cardiotoxicity.">Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity.</a> Pharmacological Reviews. 56 (2), 185-229 (2004).</li><li>Vejpongsa, P., Yeh, E. 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No se declaran conflictos de intereses.

Dox es un fármaco antitumoral antitumoral inespecífico de uso común en la práctica clínica12. Su principal efecto secundario es la cardiotoxicidad, caracterizada por miocardiopatía y posterior insuficiencia cardíaca13. El mecanismo subyacente incluye el daño de la peroxidación lipídica miocárdica, la inhibición del retículo sarcoplásmico miocárdico Ca2+-ATPasa y la activación del sistema miocárdico local de renina-angiotensina, lo que resulta en ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.
Figure 1 was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v2.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www.jove.com/files/ftp_upload/61158/61158fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www.jove.com/files/ftp_upload/61158/61158fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.

Erratum: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Una de las causas más comunes de insuficiencia cardíaca, la DCM se caracteriza por la dilatación ventricular y la disminución de la función cardíaca y es la razón más común para el trasplante de corazón en todo el mundo1. Para investigar más a fondo su patogénesis y encontrar tratamientos efectivos, el acceso a modelos animales maduros es especialmente importante. El propósito de los experimentos descritos es establecer un modelo de ratón estable de DCM que se asemeje al DCM humano.
Debido a la compleja patogénesis de DCM, hay muchos métodos diferentes para hacer modelos animales correspondientes. Los modelos2 de DCM espontáneos son relativamente estables, pero son caros y no están fácilmente disponibles. Los modelos animales modificados genéticamente3 no están bien establecidos y requieren un uso más experimental. Los modelos animales DCM inducidos por infección viral4 o defectos autoinmunes5 son fáciles de obtener, pero no son totalmente representativos de DCM. Los modelos asociados con la toxicidad miocárdica incluyen modelos DCM inducidos por alcohol y modelos animales DCM inducidos por Dox.
El modelo de miocardiopatía inducida por Dox se obtiene mediante inyección intraperitoneal de Dox6. El modelo explota el efecto secundario crónico más grave de Dox: Después de la exposición a Dox, los pacientes desarrollan síntomas de DCM de inicio tardío con uniformidad clínica7. El estrés oxidativo inducido por Dox8 y el daño mitocondrial9,que conducen a la apoptosis de los cardiomiocitos, son síntomas en la patogénesis de dcM. Existen modelos de tratamiento dox agudo y crónico: una sola dosis alta de Dox (15 mg/kg) induce un modelo a corto plazo para la miocardiopatía10,mientras que las inyecciones repetitivas de Dox en dosis bajas (seis semanales, 3 mg/kg) inducen un modelo a largo plazo para la miocardiopatía11. Según el estudio presentado, los ratones de tipo salvaje inyectados por vía intraperitoneal una vez a la semana durante un mes a una dosis de 5 mg / kg muestran morfología e histología del corazón consistentes con las características de DCM al final del tratamiento, proporcionando una forma ideal de establecer un modelo de DCM.

<table>
	<tbody>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>servicebio</td>
			<td>CAS30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Model Animal Research Center of Nanjing University</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Pfizer</td>
			<td>CAS25316-40-9</td>
			<td></td>
		</tr>
		<tr>
			<td>echocardiography</td>
			<td>Visualsonics</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin staining kit</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson staining kit</td>
			<td>Solarbio</td>
			<td>G1343</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer solution</td>
			<td>Sigma</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Sigma</td>
			<td>CAS7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>sterilized syringe</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a doxorubicin-induced murine model of dilated cardiomyopathy in vivo

La miocardiopatía dilatada (MCD) se refiere a un espectro de trastornos miocárdicos heterogéneos caracterizados por dilatación ventricular y rendimiento cardíaco deprimido en ausencia de hipertensión, enfermedades cardíacas valvulares, congénitas o isquémicas, y que pueden estar relacionados con infecciones, anomalías autoinmunes o metabólicas, o herencia familiar. Puede progresar a insuficiencia cardíaca congestiva con un mal pronóstico. La doxorrubicina (Dox) se emplea ampliamente como un fármaco quimioterapéutico, pero su uso es limitado porque causa cambios similares a la DCM del miocardio. Su toxicidad miocárdica se atribuye al estrés oxidativo, la inflamación crónica y la apoptosis cardiomiocita. No se ha establecido un modelo de DCM que explote estos síntomas de DCM inducidos por Dox.

Función cardíaca La miocardiopatía dilatada se caracteriza por dilatación ventricular progresiva y disfunción contráctil. La Figura 2 muestra imágenes ecocardiográficas representativas de los dos grupos. Los ratones tratados con Dox mostraron una fracción de eyección del ventrículo izquierdo marcadamente reducida y acortamiento fraccional del ventrículo izquierdo(Figura 3A,B). El diámetro del VI también aumentó...

Watch this Scientific Journal Video about A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo at JoVE.com

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Se describe un protocolo para establecer un modelo de miocardiopatía dilatada (MCD) inducida por doxorrubicina en ratones mediante inyección intraperitoneal a largo plazo de doxorrubicina.

Un modelo murino inducido por doxorrubicina de miocardiopatía dilatada in vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

a-doxorubicin-induced-murine-model-of-dilated-cardiomyopathy-in-vivo

Research

JoVE Journal

Medicine

4.3K vistas.  Clinical College of Nanjing Medical University. Se describe un protocolo para establecer un modelo de miocardiopatía dilatada (MCD) inducida por doxorrubicina en ratones mediante inyección intraperitoneal a largo plazo de doxorrubicina.

Video: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modelo murino de Curación de Heridas

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Investigación

Medicina

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modelo murino de hemorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

A preclínicos modelo murino de metástasis hepática

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

Cardiomiopatía Taquicardia-inducida como un modelo de insuficiencia cardíaca crónica en porcino

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

Un modelo de cardiomiopatía inducida por la doxorrubicina en pez cebra adulto

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Evaluación de la angiogénesis terapéutica en un modelo murino de isquemia de las extremidades posteriores

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Modelo de ratón in vivo de infección por implante espinal

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

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 ...