Los autores gracias a la Sra. Risa Otsuka de asistencia técnica y ayudar a los experimentos. Esta investigación fue financiada parcialmente por el Ministerio de Educación, Ciencia, Deportes y Cultura, Grant-en-Ayudas a Jóvenes Científicos (B), 2010, 22700476 y Suzuken Memorial Foundation 2010 para KT Y este estudio fue apoyado por la investigación y el desarrollo de la simulación integrada de próxima generación de la materia viva, una parte del desarrollo y la utilización del proyecto del superordenador de última generación de MEXT, y en parte por JST ERATO Proyecto Biológico Suematsu gas.

1. Etiquetado de eritrocitos con fluoresceína isotiocianato me isómero (FITC) Todos los protocolos experimentales que utilizamos fueron aprobados por el Comité de Cuidado Animal de la Universidad de Keio Facultad de Medicina. <ol><li> Anestesiar a un ratones donantes, y después de hacer una incisión celiotomía retirar toda la sangre de la vena cava inferior con una jeringa de 1 ml que contiene una pequeña cantidad de heparina. El ratón se sacrificó inmediatamente por inyección de pentobarbital sódico (100 mg / kg ip). </li><li> Lavar la sangre entera dos veces con PBS (pH 7,4) por centrifugación durante 5 min a 400 g en un rotor de giro-cubo. </li><li> Disolver 10 mg de FITC en 5 ml de 100 mM de Na 2 HPO 4 y filtrar con una membrana que tiene 0,22 micras-poros. </li><li> En un tubo de ensayo mezclar 1 ml de la solución FITC con 0,15 ml de 3 mM de glucosa, 0,25 ml de 180 mM de NaH 2 ml de PO 4, y 1,5 de 100 mM de Na 2 HPO 4. Añadir 0,2 ml de la RBC pellets, y toque el tubo para mezclar. </li><li> Mantener el tubo durante 2 horas a 4 ° C y luego lavar los glóbulos rojos teñidos en PBS dos veces. </li><li> Ponga una pequeña cantidad de la suspensión de glóbulos rojos en una hierba de diapositivas y ver si los glóbulos rojos aspecto saludable (es decir, que su forma y tamaño son normales) y que la intensidad de la fluorescencia es suficiente. </li><li> Suspender el 0,1 ml de glóbulos rojos en 0,9 ml de PBS a pH 7,4, y mantener la suspensión a 4 ° C hasta que la inyección. </li></ol> 2. Preparación de oxígeno sensible a Dye <ol><li> Disolver 500 mg de BSA en 10 ml de PBS a pH 7,4. </li><li> Añadir 30 mg de Pd (II)-meso-tetra (4-carboxifenil) porfirina (Pd-TCPP) a la solución de BSA y se agita durante la noche. </li><li> (Opcional) Extracto de BSA-enlazado Pd-TCPP mediante flujo por gravedad o una cromatografía en columna de centrifugación para separarlo de libre Pd-TCPP. </li><li> Centrifugar la solución para eliminar sin disolver Pd-TCPP, y filtrar el sobrenadante con un filtro de membrana que tiene 0,22 micras-poros. &lt;/ Li&gt; <li> Tienda 1-ml alícuotas en tubos a -20 ° C. Evitar una repetición de los ciclos de congelación-descongelación. </li></ol> 3. Preparación de Animales <ol><li> Por observación microscópica de la microcirculación preparar una placa de plástico con un agujero de 20 mm de diámetro, y una cinta de vidrio de 30-mm cuadrados cubierta cuadrada sobre el agujero. </li><li> Anestesiar a un ratón, quite la piel, y la preparación de la piel. Colocar un catéter en la cola vano para la inyección de drogas mediante el uso de una aguja G 30 conectado a un polietileno de 10 cm (PE 10) catéter lleno con heparinizada PBS. </li><li> Tras la incisión mediana, extender un lóbulo principal del hígado en la placa de plástico, y colocar el ratón en decúbito esternal. </li><li> Preparar pequeños trozos con una envoltura de cocina (3 mm x 8 mm) y baldosa ellos alrededor del borde del lóbulo hepático para inhibir movimiento del hígado con la respiración y evitando que el secado. </li><li> Observe la microcirculación hepática en el microscopio (con luz transmitida), y confirmar que el blood flujo no tiene ninguna estasis en el campo de visión por lo menos durante 15 min. </li><li> Lentamente inyectar 0,2 ml de eritrocitos marcadas fluorescentemente para la observación del flujo sanguíneo o 0,2 ml de Pd-TCPP solución para pO 2 medición. Esta cantidad de glóbulos rojos marcados con FITC representarán el 1/50 de todos los glóbulos rojos en circulación en la región visualizada. </li></ol> 4. Visualización de flujo de la sangre <ol><li> Excitar el FITC por irradiación con luz de una lámpara de mercurio que ha pasado por un filtro de banda (450-490 nm) 1. </li><li> Grabe la imagen fluorescente con una cámara CCD. </li></ol> 5. PO 2 de Medición Pd-TCPP fosforescencia es relativamente débil y debe ser detectado con un detector de alta sensibilidad. Todos los experimentos deben realizarse en un cuarto oscuro. <ol><li> Los picos de absorción de Pd-TCPP son a 410 nm y 532 nm, de modo que el segundo armónico de longitud de onda 532 nm es recomendado para la excitación. Esta longitud de onda puede ser generada por un láser Nd: YAG de pulso del láser 2. </li><li> Pase el haz del láser de Nd: YAG en el puerto apropiado en el microscopio invertido, y ajustar el haz de luz a una posición central en el plano focal. La resolución espacial depende del tamaño del punto láser, que se cambia al pasar el haz a través de un orificio. </li><li> Para detectar la fosforescencia, conecte un filtro de paso largo (&gt; 620 nm) y un tubo fotomultiplicador (PMT) para el otro puerto del microscopio. La PMT debe ser sensible a longitudes de onda rojas, especialmente alrededor de 700 nm. </li><li> Muestra la señal de fosforescencia (500 puntos muestreados a 200 kHz) y calcular la constante de tiempo de la decadencia mediante el ajuste de una función exponencial a la decadencia. </li><li> Para la calibración de preparar dos grupos de muestras de Pd-TCPP solución con pO 2 150 mmHg y 0. Añadir ditionito de sodio al 1% en volumen de muestra para producir ausencia de oxígeno. Mantener el pH de las muestras a 7,4 y la temperaturatura a 37 ° C durante la calibración. </li><li> Fijar el tiempo de la luminiscencia de Stern-Volmer constante de desintegración q k y el oxígeno libre τ 0 en la ecuación (1) 3. En nuestro caso, con Pd-TCPP soluciones a 37 ° C y un pH de 7,4, k q es 374 mm Hg -1 s -1 y τ 0 es de 0,74 ms. Estos parámetros de medición dependerá del equipo (es decir, láser, detector, otros dispositivos ópticos) y el método de procesamiento de señales, de modo de calibración se necesita en cada sistema de medición 4. τ 0 / τ = ​​1 + k q • τ 0 • PO 2 (1) </li><li> Ponga el ratón en la placa de la platina del microscopio, observar la microcirculación hepática, y ajustar la posición de la microvasculatura de destino hasta el punto de que el punto láser. </li></ol> 6. La hepatitis Modelo <ol><li> Los ratones rápidos con acceso libre al agua durante la noche. </li><li> Prepare una20 mg por ml de solución de acetaminofeno (APAP) en DMSO. </li><li> Inyéctela intraperitonealmente a 1 ml/100 g de peso corporal (es decir, 200 mg / kg de peso corporal). Después de la inyección, el ratón se puede dar acceso libre a comida y agua 5. </li><li> 24 horas después de la inyección, anestesiar el ratón y exponer el hígado, al hacer una incisión mediana. Si se logra la hepatitis, necrosis en la región pericentral será fácilmente visible para el ojo desnudo. </li></ol> 7. Los resultados representativos Imágenes de ejemplo de la microcirculación hepática se muestra en la Figura 1, donde (a) es una imagen de luz transmitida y (b) es una imagen de fluorescencia. Individuales de los eritrocitos FITC marcado se observan en la película de vídeo, y vénulas portales, sinusoides y vénulas centrales son reconocidos por la dirección del flujo de sangre. Como se muestra en la Figura 2, el punto láser irradiar una vénula central a través de una lente de objetivo x100 era aproximinmediatamente 10 micras de diámetro. Intravascular pO 2 en la microcirculación hepática se midió en C57BL macho / 6 ratones, y la Figura 3 (a) muestra las relaciones entre pO 2 y el diámetro del vaso en vénulas portales y central. La PO 2 gradiente desde el portal de vénulas centrales indica que los glóbulos rojos liberan oxígeno de manera eficiente al pasar por sinusoides. Como se muestra en la Figura 3 (b), el promedio de pO 2 fue de 59,8 mmHg en los vasos portales, 48,2 mmHg en sinusoides, y 38,9 mmHg en vénulas centrales. Cuando se produce una hepatitis aguda mediante la inyección de APAP intraperitoneal, intravascular pO 2 fue significativamente elevado en cada parte de la microcirculación hepática: p &lt;0,05 en las venas porta y p &lt;0,01 en los sinusoides y venas centrales (Figura 3 (c)). <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="Figura 1" /> Figura 1. Baja magnificación iLos magos que muestra la microcirculación hepática con (a) de luz transmitida y fluorescencia (b). <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="Figura 2" /> Figura 2. Imagen del punto láser irradiar una vénula dirigido central de PO 2 de medición. <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="Figura 3" /> Figura 3. PO 2 gradiente en la microcirculación hepática de ratones sanos y ratones con lesión hepática inducida por paracetamol. (A) muestra PO 2 en comparación con el diámetro del vaso para el portal de vénulas (PV) y el centro de las vénulas (CV), y (b) muestra el gradiente de oxígeno del PV a la CV a través de los sinusoides (S). La PO 2 diferencia entre PV y el CV refleja el consumo de oxígeno de los hepatocitos y otras células del parénquima. (C) muestra que en las condiciones de la hepatitis inducida por la administración de APAP, pO 2 fue significativamente elevado en Puerto Vallarta, S, y el CV y la pO 2gradiente de disminución, lo que indica que el consumo de oxígeno hepático se ha visto comprometida.

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No tenemos nada que revelar.

Entrega de oxígeno a los tejidos es una función esencial de la microcirculación, y un montón de artículos describen las enfermedades relacionadas con la anatomía y la reología de los microvasos 6. Flujo sanguíneo hepático es una combinación de la sangre arterial y venosa. Aproximadamente el 30% del flujo de sangre hacia el hígado es bien oxigenada sangre proporcionada por la arteria hepática y la otra 70%, suministrado por la vena porta, es la salida de sangre venosa mal oxigenada desde el bazo y ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Pd (II) meso-tetra (4-carboxifenil) porfirina </td><td> Frontier Scientific, Inc. </td><td> PdT790 </td></tr><tr><td> Isotiocianato de fluoresceína que isómero </td><td> Sigma-Aldrich Co. </td><td> F7250 </td></tr><tr><td> El acetaminofeno </td><td> Sigma-Aldrich Co. </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> Nombre del equipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td></td><td></td><td></td><td> Equipo </td></tr><tr><td> tubo fotomultiplicador </td><td> Hamamatsu Photonics KK </td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

Hay una discrepancia considerable entre la oferta y la demanda de oxígeno en el hígado, porque el consumo de oxígeno hepático es relativamente alto, pero aproximadamente el 70% del suministro sanguíneo hepático no está bien oxigenado sangre de la vena portal de deriva en el tracto gastrointestinal y bazo. El oxígeno es transportado a los hepatocitos por la sangre que fluye de una rama terminal de la vena porta con una vénula central a través de los sinusoides, y esto hace que un gradiente de oxígeno en los lóbulos hepáticos. El gradiente de oxígeno es un parámetro importante físico que implica la expresión de las enzimas de aguas arriba y aguas abajo de la microcirculación hepática, pero la falta de técnicas para medir el consumo de oxígeno en la microcirculación hepática ha retrasado la elucidación de los mecanismos relacionados con el metabolismo del oxígeno en el hígado. Por lo tanto, utiliza FITC-etiquetados eritrocitos para visualizar la microcirculación hepática y utilizado asistida por láser fosforimetría para medir la presión parcial de oxígeno en los microvasos allí. Noncontact y medición óptica continua puede cuantificar velocidades del flujo sanguíneo, los diámetros de los vasos, y los gradientes de oxígeno relacionados con el consumo de oxígeno en el hígado. En un modelo de hepatitis aguda hemos hecho mediante la administración de paracetamol a los ratones que hemos observado aumento de la presión de oxígeno en los dos vénulas portales y la central, sino un gradiente de disminución de oxígeno en los sinusoides, lo que indica que la necrosis de hepatocitos en la zona pericentral podría cambiar la presión de oxígeno y afectar la expresión de enzimas en la zona periportal. En conclusión, nuestros métodos ópticos para la medición hemodinámica hepática y el consumo de oxígeno puede revelar los mecanismos relacionados con la enfermedad hepática.

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Un sistema óptico fue desarrollado para visualizar la microcirculación hepática con eritrocitos de FITC-etiquetados y para medir la presión parcial de oxígeno en los microvasos con asistida por láser fosforimetría. Este método puede ser utilizado para investigar los mecanismos fisiológicos y patológicos mediante el análisis de la estructura microvascular, el diámetro, la velocidad del flujo de sangre, y la tensión de oxígeno.

Visualización y análisis de flujo de la sangre y del consumo de oxígeno en la microcirculación hepática: Aplicación a un Modelo de cuadro de hepatitis aguda

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

visualization-analysis-blood-flow-oxygen-consumption-hepatic

Research

JoVE Journal

Medicine

12.5K vistas.  Keio University. Un sistema óptico fue desarrollado para visualizar la microcirculación hepática con eritrocitos de FITC-etiquetados y para medir la presión parcial de oxígeno en los microvasos con asistida por láser fosforimetría. Este método puede ser utilizado para investigar los mecanismos fisiológicos y patológicos mediante el análisis de la estructura microvascular, el diámetro, la velocidad del flujo de sangre, y la tensión de oxígeno.

Video: Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murino hepática Bioluminiscente modelo tumoral

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Investigación

Medicina

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Heterotópico Rata auxiliar Trasplante de Hígado Con Flujo regulado Portal Vein Arterialización en aguda Insuficiencia Hepática

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

El Conejo Modelo Blood-shunt para el Estudio de la aguda y secuelas tardías de Hemorragia subaracnoidea: Aspectos Técnicos

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Contraste mejorado las imágenes por ultrasonido para la Evaluación de la Médula Espinal flujo sanguíneo en Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxígeno-Glucosa Privación y Reoxigenación como<em&gt; In Vitro</em&gt; La isquemia-reperfusión modelo para el estudio de la disfunción barrera hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Un modelo agudo de retina para evaluar sanguíneos de la retina barrera violación, así como potenciales fármacos para el tratamiento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análisis optimizada de<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; La esteatosis hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Un modelo de isquemia cardíaca crónica en cerdos usando un Constrictor Ameroid

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Analizando la tasa de consumo de oxígeno en los cardiomiocitos neonatales primario de ratón cultivadas utilizando un analizador de flujo extracelular

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.