Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Los autores agradecen a Liang Zhang por ayudar a recopilar los datos de video durante el experimento. Este estudio fue apoyado por la Fundación Nacional de Ciencias Naturales de China (número de subvención: 82070479) y los Fondos de Investigación Fundamental para las Universidades Centrales (número de subvención: 3332022128).

Los protocolos se sometieron a una revisión institucional y recibieron la aprobación del Comité Institucional de Cuidado y Uso de Animales, Hospital Fuwai, Academia China de Ciencias Médicas (FW-2021-0005). Todos los procedimientos experimentales se realizaron de acuerdo con la Guía para el Cuidado y Uso de Animales de Laboratorio publicada por los Institutos Nacionales de Salud.
NOTA: Las ratas macho Sprague-Dawley (peso: 500-600 g, edad: 12-14 semanas) se mantuvieron en condiciones est?...

<ol>
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C., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+chlorogenic+acid+treatment+on+neuroinflammation+after+deep+hypothermic+circulatory+arrest+may+be+mediated+through+CYLD/NF-&#954;B+signaling.">Beneficial effects of chlorogenic acid treatment on neuroinflammation after deep hypothermic circulatory arrest may be mediated through CYLD/NF-&#954;B signaling.</a> Brain Research. 1767, 147572 (2021).</li><li>Li, Y. A., 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=Differential+expression+profiles+of+circular+RNAs+in+the+rat+hippocampus+after+deep+hypothermic+circulatory+arrest.">Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.</a> Artificial Organs. 45 (8), 866-880 (2021).</li><li>Linardi, D., 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=Slow+versus+fast+rewarming+after+hypothermic+circulatory+arrest:+effects+on+neuroinflammation+and+cerebral+oedema.">Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.</a> European Journal of Cardiothoracic Surgery. 58 (4), 792-780 (2020).</li><li>Engelman, R., 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+Society+of+Thoracic+Surgeons,+The+Society+of+Cardiovascular+Anesthesiologists,+and+The+American+Society+of+ExtraCorporeal+Technology:+Clinical+practice+guidelines+for+cardiopulmonary+bypass--Temperature+management+during+cardiopulmonary+bypass.">The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of ExtraCorporeal Technology: Clinical practice guidelines for cardiopulmonary bypass--Temperature management during cardiopulmonary bypass.</a> Annals of Thoracic Surgery. 100 (2), 748-757 (2015).</li><li>Jenke, A., 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=AdipoRon+attenuates+inflammation+and+impairment+of+cardiac+function+associated+with+cardiopulmonary+bypass-induced+systemic+inflammatory+response+syndrome.">AdipoRon attenuates inflammation and impairment of cardiac function associated with cardiopulmonary bypass-induced systemic inflammatory response syndrome.</a> Journal of the American Heart Association. 10 (6), 018097 (2021).</li></ol>

La canulación es el procedimiento más fundamental para establecer DHCA en ratas. Antes de la canulación , remojar la arteria con 0,5 ml de lidocaína al 2% facilitará la canulación de la lobra. Después de la canulación es necesaria la heparinización con 500 UI/kg de heparina vía vena yugular externa para evitar la formación de microtrombos17. Hemos encontrado repetidamente que esta dosis de heparina puede lograr el objetivo de un tiempo de coagulación activado (ACT) >480 s. El ...

El paro circulatorio hipotérmico profundo (DHCA) se ha utilizado en cirugía cardíaca desde 19531. DHCA implica reducir la temperatura central del paciente a niveles profundamente hipotérmicos (15-22 ° C) antes de interrumpir globalmente el flujo sanguíneo al cuerpo2. El paro circulatorio puede proporcionar un campo operativo relativamente incruento. La hipotermia profunda disminuye el metabolismo, especialmente en el cerebro y el miocardio, que es un método eficaz de protección contra la isquemia3. DHCA se aplica comúnmente durante cirugías para cardiopatías congénitas complejas, enfermedad del arco aórtico e incluso tumores renales o suprarrenales con trombo de vena cava 4,5. Por lo tanto, el establecimiento de modelos animales de DHCA proporciona una referencia importante para el refinamiento del procedimiento y la prevención de complicaciones en entornos clínicos.
Aunque se pueden establecer modelos con caninos6, conejos7 y otros animales, es preferible usar ratas debido a su operatividad y bajo costo. El modelo de rata DHCA fue descrito por primera vez en 2006 por Jungwirth et al.8. Se encontró que la duración del paro circulatorio tuvo un impacto en los resultados neurológicos. Desde entonces, los modelos de ratas DHCA se han investigado ampliamente. Se ha aclarado que la DHCA podría provocar el síndrome de respuesta inflamatoria sistémica (SRIS)9. En estudios posteriores, los farmacólogos encontraron que la neuroinflamación relacionada con DHCA inducida por SIRS podría ser atenuada por resveratrol10 y triptolida11. Nuestro equipo también encontró que la neuroinflamación relacionada con DHCA podría atenuarse al inhibir la proteína de unión al ARN12 inducible por el frío. En el sistema cardiovascular, la superóxido dismutasa tiene un efecto cardioprotector sobre las lesiones por isquemia/reperfusión (I/R) durante la DHCA13. Estos resultados ampliaron la comprensión de los procesos fisiopatológicos relacionados con la DHCA y ofrecieron nuevas direcciones para mejorar los resultados de la DHCA. Sin embargo, los resultados con respecto a la endotoxemia, el estrés oxidativo y la autofagia después de DHCA no son concluyentes. La DHCA utiliza la misma tecnología operacional que el bypass cardiopulmonar (BPC)14, pero su estrategia de manejo es diferente, y los pasos para generar DHCA difieren entre varios equipos 8,9,10,11. El presente estudio tiene como objetivo proporcionar un método para establecer el procedimiento DHCA en ratas.

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			<td>Heat Exchanger</td>
			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</td>
			<td>Animal-M</td>
			<td></td>
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			<td>Membrane Oxygenator</td>
			<td>Dongguan Kewei Medical Instrument Co., Ltd.</td>
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			<td>Chengdu Techman Co., Ltd</td>
			<td>BL-420s</td>
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			<td>Changzhou Prefluid Technology Co.,Ltd</td>
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			<td>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
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			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
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			<td>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
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			<td>SPACECABS</td>
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			<td>Shanghai Alcott Biotech Co., Ltd</td>
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			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
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establishment of deep hypothermic circulatory arrest in rats

El paro circulatorio hipotérmico profundo (DHCA) se aplica rutinariamente durante las cirugías para la cardiopatía congénita compleja y la enfermedad del arco aórtico. El presente estudio tiene como objetivo proporcionar un método para establecer DHCA en ratas. Para evaluar el impacto del proceso DHCA en los signos vitales, se utilizó como control un modelo de rata de derivación cardiopulmonar (CEC) a temperatura normal sin paro circulatorio. Como era de esperar, DHCA condujo a una disminución significativa de la temperatura corporal y la presión arterial media. El análisis de gases en sangre indicó que el DHCA aumentó los niveles de ácido láctico, pero no influyó en el pH sanguíneo y las concentraciones de hemoglobina, hematocrito, Na+, Cl-, K+ y glucosa. Además, en comparación con las ratas CPB de temperatura normal, los resultados de la microscopía electrónica de transmisión mostraron un leve aumento de los autofagosomas del hipocampo en las ratas DHCA.

Como grupo control, las ratas CPB de temperatura normal (NtCPB) sin paro circulatorio mostraron una presión arterial media (PAM) y temperatura corporal estables durante todo el procedimiento, mientras que la PAM de las ratas DHCA disminuyó durante el paro cardíaco (p < 0,01, Figura 3A). La temperatura de las ratas DHCA disminuyó rápidamente durante la fase de enfriamiento y se recuperó gradualmente durante la fase de recalentamiento. Al destetar a las ratas de los circuitos de...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Este protocolo presenta el establecimiento de una parada circulatoria hipotérmica profunda en ratas, que puede aplicarse para investigar el síndrome de respuesta inflamatoria sistémica, lesión por isquemia/reperfusión, estrés oxidativo, neuroinflamación, etc.

Establecimiento de un paro circulatorio hipotérmico profundo en ratas

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

Establecimiento de paro circulatorio hipotérmico profundo en rata. Paro circulatorio hipotérmico profundo, DHCA se aplica rutinariamente durante cirugías de cardiopatía congénita compleja y enfermedad del arco aórtico. El presente estudio tiene como objetivo proporcionar un método para establecer DHCA en ratas.Los protocolos recibieron una revisión institucional y obtuvieron la aprobación del Comité Institucional de Cuidado y Uso de Animales, Hospital Fuwai, Academia China de Ciencias Médicas. Trabajos preparatorios. Asegúrese de que los circuitos CPB contengan un depósito modificado de gotero morfo, bomba de rodillos, intercambiador de calor, oxigenador de membrana, tubo de conexión y un tanque de agua conectado al circuito y produzca 12 mililitros de hidroxialmidón con un mililitro de heparina sódica y un mililitro de solución salina que prepare el circuito con una solución de cebado de 40 mililitros con la bomba de rodillos girando suavemente.Dos, anestesia y canulación Conecte el tubo con el ventilador y los parámetros laterales. Coloque una manta calefactora eléctrica y la derecha y mire hacia la derecha con cinta adhesiva.Aplique ungüento oftálmico en los ojos para evitar la sequedad. Afeite el pelo en la región inguinal derecha, la región cervical derecha y la cola con la afeitadora. Luego desinfecte la piel con aldehído y alcohol.Corte la piel y la región inguinal izquierda y disecte el músculo y el tejido blando del hueso para exponer la vena femoral izquierda y la arteria. Separe la arteria cuidadosamente. Catéter intravenoso calibre 22 en la arteria femoral izquierda.Arteria ligada y catéter con seda. Use solución salina que contenga heparina para enjuagar la cánula y evitar la coagulación. Conecte el catéter con el sensor de prensa para controlar la presión arterial.Corte la piel de la cola y luego use un bisturí para cortar la especie superficial de la arteria de la cola para exponer la arteria de la cola que se encuentra en el medio del campo quirúrgico. Canular la arteria de la cola con el catéter intravenoso calibre 22. Arteria de liga y catéter con seda.Use solución salina que contenga heparina para enjuagar el catéter y evitar la coagulación. Corte la piel de la yugular derecha de la vena y luego separe el músculo y el tejido para exponer la vena. Inserte un catéter intravenoso multiedad casero calibre 16 en la yugular externa derecha de la vena y colóquelo cuidadosamente en la vena cava inferior derecha o en la aurícula derecha.Administre heparina sódica donde la vena externa derecha cubra cada región canulada con bolas húmedas para evitar la contaminación. Tres, conecte el circuito con el catéter en la arteria de la cola y mantenga la bomba baja de uno a dos mililitros por minuto. Luego conecte el reservorio con el catéter en la vena yugular externa derecha.Asegúrese de que siempre haya un nivel sanguíneo de aproximadamente un centímetro en el depósito. Encienda el tanque de agua y ajuste la temperatura del agua a 37 grados centígrados primero. Después de que la presión arterial sea estable, aumente suavemente la bomba de flujo hasta 80 a 100 mililitros por minuto.Cuatro, enfriamiento. Ajuste la temperatura ambiente a alrededor de 20 grados centígrados. Ponga cubitos de hielo en estos guantes desechables y luego colóquelos en la cabeza y los lados correctos.La temperatura del tanque se sugirió en tiempo real de acuerdo con la temperatura rectal del arroz. Cambie los parámetros relevantes del ventilador correctamente de acuerdo con los resultados del análisis de gases en sangre. Cinco, hipotermia profunda paro circulatorio.Luego, la temperatura rectal desciende de 15 a 20 grados centígrados. Cambie la bolsa de hielo y deseche los guantes para garantizar el mantenimiento de la hipotermia profunda durante el paro circulatorio. Detenga la bomba de rodillos.Mantenga el reservorio en contacto con el medio ambiente y drene la sangre lentamente desde la vena yugular externa hasta el reservorio. Preste atención a la reforma de la presión arterial. Después de que no haya presión arterial ni latidos cardíacos, detenga el drenaje y mantenga el reservorio cerrado.Apague el ventilador. Seis, calentamiento y reperfusión. Retire todos los guantes desechables y aumente la temperatura ambiente a 25 grados centígrados.Restaure la interventilación de oxígeno de memoria mientras mantiene el recorte del tubo de drenaje venoso. Encienda la bomba de rodillos para asegurarse de que la sangre en el depósito regrese lentamente al cuerpo correcto. Encienda el ventilador después de que el nivel de sangre en el depósito permanezca un centímetro.Usar el tubo de drenaje y drenar la sangre desde la aurícula derecha hasta el reservorio lentamente. Encienda la lámpara de calefacción, la almohadilla térmica y el tanque de agua. Ajuste la temperatura del tanque de agua a 25 grados centígrados.Luego ajústelo a la temperatura izquierda oportunamente. Retire la lámpara de calefacción después de que la temperatura rectal haya vuelto a 34 grados centígrados. Siete, destete de CPB.Reduzca lenta y gradualmente el rodillo del caudal de la bomba y ajuste la velocidad del género de la vena en la velocidad de flujo reducida a un mililitro por minuto. Mantener el depósito en contacto con el medio ambiente. Infundir la sangre restante en un circuito con un caudal de un mililitro por minuto.Detenga la oxigenación de la memoria y la bomba de rodillos. Eutanasia después de un período de ventilación mecánica y anestesia profunda. Resultados representativos.Como grupo de control, la temperatura normal CPB, NtCPB, rata. Sin el paro circulatorio mostró una presión arterial media estable PAM y temperatura corporal durante todo el procedimiento. Mientras que la PAM de las ratas DHCA disminuyó durante el paro cardíaco.La temperatura de las ratas DHCA bajó rápidamente durante la fase de enfriamiento y se recuperó gradualmente durante la fase giratoria. El destete del circuito de DHCA la temperatura de las ratas DHCA volvió a la normalidad. Los datos anteriores indicaron que nuestro procedimiento DHCA imitó la temperatura real y los cambios de PAM de los pacientes en las clínicas.El efecto del procesador DHCA y el aumento se investigó mediante análisis de gases en sangre. Después del contacto de sangre total con la solución primaria, la concentración de hemoglobina fue superior a seis gramos por decilitter en ambos grupos. El destete de la superficie de DHCA, aumentó a nueve gramos por decilitter debido a la reinfusión de la sangre restante en el circuito CPB hacia la derecha.El hematocrito, HCT, mostró una tendencia similar a HB. Después de la reperfusión de DHCA, el ácido lactato aumentó rápidamente, lo que fue más pronunciado en el grupo de DHCA. El pH disminuyó después del procedimiento DHCA, que probablemente fue el resultado de la acumulación de ácido lactato. Durante todo el experimento, la concentración de sodio, cloruro, iones de potasio y glucosa no mostró diferencias significativas en cada punto 10.Este resultado sugiere que DHCA solo indujo el aumento de ácido lactato, pero no influyó en el pH de la sangre y la concentración de hemoglobina, hematocrito, iones de sodio, iones cloruro, iones de potasio y glucosa. Para evaluar el impacto de DHCA en la autofagia neuronal, utilizamos el electromicroscopio de transmisión y sorprendentemente encontramos y aumentamos el número de autofagosomas en el hipocampo de ratas DHCA. Los datos anteriores indicaron que nuestro modelo DHCA imitó la temperatura real y los cambios de MAP de los pacientes en las clínicas.Este modelo se puede aplicar para investigar el síndrome de respuesta inflamatoria sistémica, la neuroinflamación, etc.

establishment-of-deep-hypothermic-circulatory-arrest-in-rats

Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Ortotópico patas traseras trasplante en ratas

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Investigación

Inmunología e Infección

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

El diagnóstico de ecto y endoparásitos en ratas y ratones de laboratorio

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Establecimiento de Epstein-Barr transformado al crecimiento líneas celulares linfoblastoides

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

La timectomía completo en ratas adultas con no invasiva La intubación endotraqueal

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Prevención de Estrés Térmico efectos adversos en ratas<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Estenosis de la Vena Cava Inferior: un modelo murino de trombosis venosa profunda

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Trombosis venosa profunda inducida por estasis en los ratones controlados por sonografía de alta frecuencia

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Profundo dérmico inyección como un modelo de infección de la piel Candida albicans para el análisis histológico

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Establecimiento de la infección Viral y análisis de la interacción huésped-Virus en Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...