Los autores desean agradecer a Shawna Rideout-Gros y Alexandra Garcés, por su ayuda con el cuidado de los animales, y Ping Wu por su asistencia técnica. El apoyo a este trabajo fue proporcionado por los Institutos Nacionales de Salud bajo la subvención núm. EB003268, y el Programa Canada Research Chairs.

1. La ecografía y la resonancia magnética de configuración Una resonancia magnética compatible con tres ejes del sistema de ultrasonido focalizado se utilizó en este estudio (FUS Instruments, Inc., Toronto, Ontario, Canadá). Dos transductores de ultrasonidos se utilizaron diferentes: uno en casa construida 551,5 kHz esféricamente centrada transductor (radio de curvatura = 60 mm, diámetro externo = 75 mm, diámetro interno = 20 mm), y un 1,503 MHz, 8-sector de matriz con integrado PZT hidrófono (Imasonic Inc., Voray-sur-L&#39;Orgnon, Francia), impulsados ​​como un solo elemento transductor esférico centrado (FN = 0.8, apertura = 10 cm). Una resonancia magnética compatible con PVDF receptor 8 se utiliza para grabar las emisiones acústicas cuando el transductor kHz 551,5 se utilizó. Si se utilizan diferentes equipos, se sugieren los siguientes: <ol><li> Seleccionar un transductor adecuado, calibrado ultrasonido con una frecuencia central en la gama MHz MHz 0,25 -1,5 y un número f (radio de curvatura / abertura) de 1 o menos. </li><li> Coloque un baño de agua filled con agua tibia, desgasificado, agua desionizada en la cama de un 1,5 o 3 T T resonancia magnética. El baño de agua debe tener una placa superior, que puede contener a los animales. Monte el transductor de ultrasonido en el tanque en una resonancia magnética compatible con la etapa de posicionamiento de tres ejes o el sistema 9, frente a la superficie del agua. </li><li> Ejecute el cable del transductor en el equipo de conducción de ultrasonido a través de un panel de la penetración. </li><li> Generar la señal de excitación del transductor mediante un generador de funciones y amplificador de potencia. Utilizar un circuito de adaptación externa para minimizar la potencia reflejada eléctrica. </li><li> Conociendo la longitud focal del transductor, situar el foco del transductor en la superficie del agua y sonicar la superficie del agua para generar una fuente de agua visible. Pasos 1.6 y 1.7 se describe cómo registrar el enfoque utilizando la fuente de agua generado. Un método más preciso para localizar el foco, que implica un tratamiento con ultrasonidos de absorción de calor de gel y de imagen resultante de la elevación de temperatura en el foco con la RM, se puede encontrar en 9 &lt;/ Sup&gt;. </li><li> Marcar la localización del foco utilizando un marcador que será visible en las imágenes de RM. Esto puede hacerse utilizando una placa con un agujero central que se llenará con agua cuando se coloca sobre el foco. El agujero lleno de agua será visible en la RM y proporcionará las coordenadas focales en dos planos, mientras que la coordenada en la dirección axial del transductor se puede determinar a partir de la superficie del agua. </li><li> Usando una secuencia localizador 3-plano, la imagen del marcador de enfoque y registrar la localización del foco en el sistema de coordenadas de MRI. </li><li> Para controlar las emisiones acústicas, montar un hidrófono 8 MR-compatible que se utiliza como un detector de cavitación pasiva (PCD) en el baño de agua dirigida en el foco del transductor, o utilizar un transductor con hidrófonos integrado. </li><li> Pase los cables de PCD en una tarjeta de alcance que es lo suficientemente rápida para capturar ráfagas de hasta 10 ms en el PRF de hasta 2 Hz (por ejemplo, ATS460, AlazarTech, Pointe-Claire, Quebec, Canadá). Los cables deben estar conectados a tierra a la penetracióntración del panel o una pantalla de radiofrecuencia para minimizar el ruido. </li></ol> 2. Preparación de Animales <ol><li> Se anestesia a los animales que utilizan gas isofluorano. Desde isofluorano tiene un efecto sobre la alteración BBB 10, los animales deben ser retirados los gases de 10 minutos antes del inicio del experimento. Ocular lugar lubricante pomada en cada ojo en el comienzo de la anestesia para proteger la córnea de la desecación u otras lesiones. </li><li> Utilizando una máquina de afeitar eléctrica afeitarse la piel de la parte superior de la cabeza del animal y el cuello, a continuación, retire la piel que queda con una crema depilatoria (por ejemplo, Nair, Church &amp; Dwight Co., Princeton, NJ, EE.UU.) y enjuague el cuero cabelludo con jabón suave y agua. </li><li> Preparar la cola con un lavado de la piel betadine seguido de un lavado bridine. Realizar una wipedown final con el alcohol con el fin de visualizar la vena de la cola. </li><li> Insertar un catéter de calibre 22 con llave de paso 3-camino en la vena de la cola y enjuague con una mezcla de heparina / solución salina (33 U / ml) para evitar la formación de coágulosen el catéter. </li><li> Entregar anestésicos inyectables ketamina (40-50 mg / kg, 10 mg / kg de xilazina) por vía intramuscular, y retirar al animal del isofluorano. </li><li> Coloque el supina animales anestesiados en el sistema de posicionamiento de ultrasonido con la parte superior de la cabeza en contacto con el baño de agua a través de un agujero en la placa superior (Fig. 1). Una pequeña cantidad de gel de ultrasonido puede ser aplicado a la parte superior de la cabeza del animal para minimizar la posibilidad de burbujas de aire atrapadas. </li><li> Tape las piernas para el sistema de posicionamiento. La cabeza puede ser mantenido en su lugar utilizando una barra de bocado, si está disponible, o la cinta colocada firmemente en el mentón. </li><li> Cubra el animal con una toalla y una manta que circula el agua con el fin de mantener el calor. </li></ol> 3. Selección del Objetivo <ol><li> Adquirir referencia axial en T2 y T1-imágenes de RM del cerebro. Si se utiliza una resonancia magnética 1,5 T y dedicado la cabeza del tamaño de RF-bobina de recepción de superficie, apto scun parámetros pueden ser: T 2 ponderada: FSE, TE = 60 ms, TR = 2000 ms T 1 ponderada: FSE, TE = 10 ms, TR = 500 ms </li><li> Seleccione el destino de la T-2 ponderados exploraciones, evitando los ventrículos y la línea media del cerebro, y la selección de una profundidad media del cerebro. </li><li> Mover el enfoque del transductor a la ubicación de destino. </li></ol> 4. Preparación de microburbujas Microburbujas DEFINITY (Lantheus Medical Imaging, MA, EE.UU.) son utilizados por varios grupos de FUS mediados de microburbujas inducidos BBBD 2,5,7. Las dosificaciones apropiadas para otros tipos de microburbujas se puede encontrar en la bibliografía 11,12. <ol><li> Activar las microburbujas Definity y poco a poco establecer un pequeño volumen con una jeringa de 1 ml con una aguja de calibre 18. </li><li> Quite el aire atrapado de la jeringa moviendo suavemente el émbolo hacia atrás y hacia adelante. No golpee la jeringa ya que esto puede romper las burbujas. </li><li> Diluir el Definity en condiciones normales<html> solución salina en una proporción de 10:1 salina para Definty lentamente inyectar el volumen requerido de Definity en una jeringa de solución salina. Si una entrega de infusión se utiliza, el Definity puede ser aún más diluida, a 50:1 o 100:1. </li><li> Invierta suavemente la jeringa para mezclar bien el Definity y solución salina hasta que un aspecto aún se consigue. Inversión suave de la jeringa inmediatamente antes de la inyección también puede ser necesaria si las burbujas han empezado a flotar fuera de la suspensión. </li><li> Calcular el volumen de la dosis requerida sobre la base de 0,02 ml / kg de Definity, o 0,2 ml / kg de la solución (menos 10:1 dilución). </li></ol> 5. Ultrasonido de entrega <ol><li> Establezca los parámetros de tratamiento con ultrasonidos, con ráfagas de bajo ciclo de trabajo, no sonicaciones de onda continua. Parámetros apropiados sonicación a 0,5 MHz son MPa 0,23 de la presión in situ, 10 ráfagas ms a 1 Hz PRF, 2 min. tiempo de sonicación total. En 1,5 MHz apropiado de las presiones situ caer alrededor de 0,45-0,5 MPa. </li></ol>_content &quot;&gt; presiones adecuadas a diferentes frecuencias pueden estimarse mediante un índice mecánico de 0,46 13. <ol start="2"><li> Comprobar que la cabeza del animal está todavía acoplado al agua. </li><li> Inyectar lentamente las microburbujas en el catéter vena de la cola y enjuague con 0,5 ml de solución salina normal. Comienza la sonicación simultáneamente con el inicio de la inyección. </li></ol> 6. Evaluación de resonancia magnética del resultado del tratamiento <ol><li> Tras la sonicación, inyectar un agente de contraste para RM (por ejemplo, 0,2 ml / kg Omniscan, GE Healthcare) a través del catéter vena de la cola seguida de una infusión 0,5 ml de solución salina. </li><li> Realizar en T1 imágenes hasta que el pico de contraste haya pasado. Sonicación sitios que han sido exitosamente interrumpió mostrarán una mayor mejora que el tejido circundante. </li><li> Realizar T2 imágenes para comprobar si hay edema. Señal alta en los sitios de sonicación es indicativo de edema. </li></ol> 7. RepresentanteResultados Los agentes de contraste de MRI puede ser entregado con éxito a través de la BHE mediante ultrasonidos enfocados y difusión de microburbujas. La Figura 2 muestra típica de pre y post-FUS T 1-w imágenes. La Figura 2B muestra un contraste mejorado (CE) T 1-w imagen con distintas aberturas de coordinación en cuatro lugares sonicadas. Sonicación lugares 1 y 2 muestran la mejora particularmente brillante. En Fig.3 lugares 1 y 2 también se puede ver que se corresponden con T 2-w señal alta, lo que indica el edema. El grado de T 2-w edema puede ser a veces más fácil de visualizar en rodajas sagitales. La figura 4 muestra CE-T-1 y T 2 w-w cortes sagitales a través de lugares de sonicación 1 y 3. El edema es visible en la posición 1, pero no la ubicación 3. El análisis espectral de los datos capturados de las emisiones acústicas (Fig. 5) puede mostrar las emisiones de armónicos y / o sub / ultra emisiones armónicas cuando la cavitación estable, se está produciendo. HarmonVA también puede surgir a partir de tejido no-linealidades, mientras que las emisiones y sub ultraharmonic sólo puede ocurrir como resultado de la actividad de burbujas 14. A presiones más altas emisiones de banda ancha que indican la cavitación inercial también puede ser detectada. Sin embargo, estos se han asociado con una mayor cantidad de rojo extravasación de células sanguíneas y que microdaño sonicaciones sin cavitación de inercia 11. El uso de frecuencias más altas de sonicación resultados en las aperturas más localizados, debido al tamaño de punto más pequeño focal. La Figura 6 muestra que las frecuencias más altas se pueden utilizar para crear pequeñas regiones de apertura. Esto permite la investigación de los efectos cerebrales mediados de los con menos cercanos a los efectos del cráneo. <img alt="Figura 1." src="/files/ftp_upload/3555/3555fig1.jpg" /> Figura 1. Arreglo experimental. <spuna clase = &quot;pdflinebreak&quot;&gt; <img alt="Figura 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> Figura 2. Pre (izquierda) y después (derecha) del tratamiento T 1-w imágenes de un cerebro de rata que muestra la mejora en cuatro lugares de sonicación. <img alt="Figura 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> Figura 3. Pre (izquierda) y después (derecha) del tratamiento T 2-w imágenes de un cerebro de rata (el mismo animal, como la figura 2) que muestra T 2-w edema en los lugares de sonicación 1 y 2. <img alt="Figura 4." src="/files/ftp_upload/3555/3555fig4.jpg" /> Figura 4. Tratamiento posterior sagital T 1-w (izquierda) y T-2-W (derecha) las imágenes del cerebro de rata lo mismo que las figuras. 2 y 3. La apertura en la posición 1 (izquierda) se correlaciona con la T-2 w edema (derecha). Ubicación 3 muestra edema de apertura (a la izquierda), pero no T-2 w. </p&gt; <img alt="Figura 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> Figura 5. Espectro de frecuencia de los datos capturados en una sola ráfaga de 10 ms a 551,5 kHz. La frecuencia fundamental (f 0), así como los armónicos (2 f 0) y sub / ultraharmonics (0,5 f 0, 1,5 f 0) son visibles. <img alt="Figura 6." src="/files/ftp_upload/3555/3555fig6.jpg" /> Figura 6. Tratamiento posterior CE-T 1-w axial (izquierda) y sagital (derecha) las imágenes de un cerebro de rata con ultrasonidos en cuatro lugares en los 1.503 Mhz. Aberturas de BBB en esta frecuencia se observa que son más localizadas.

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K. Hynynen Chopra y R. son co-fundadores de Instrumentos FUS, Inc. R. Chopra, Waspe A. y K. Hynynen son accionistas en instrumentos del FUS, Inc. K. Hynynen recibe apoyo a la investigación de la FUS Instruments, Inc.

Preparación de los animales y las microburbujas son los aspectos más críticos de este procedimiento. El pelo en la cabeza del animal debe ser totalmente eliminado para evitar la atenuación del haz de ultrasonidos. El BBB puede ser interrumpido bajo isofluorano anestésico, sin embargo, se hace más difícil de lograr apertura consistente. Las microburbujas siempre deben manipularse con cuidado y de pequeño calibre, agujas de gran diámetro debe ser utilizado en la elaboración, con el f...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Pequeños Animales Sistema de Ultrasonido Enfocado </td><td> FUS Instruments, Inc. </td><td> RK-100 </td></tr><tr><td> Definity </td><td> Lantheus Medical Imaging </td><td></td></tr><tr><td> Omniscan </td><td> GE Healthcare </td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

El ultrasonido focalizado (FUS) disrupción de la barrera hematoencefálica (BBB) ​​es una técnica cada vez más investigado por eludir el 1-5 BBB. La BHE es un obstáculo importante para los tratamientos farmacéuticos de los trastornos cerebrales, ya que limita el paso de moléculas de la vasculatura en el tejido cerebral de las moléculas de menos de aproximadamente 500 Da de tamaño 6. FUS inducida interrupción BBB (BBBD) es temporal y reversible 4 y tiene una ventaja sobre medios químicos de inducir BBBD por ser altamente localizada. FUS inducida BBBD proporciona un medio para investigar los efectos de una amplia gama de agentes terapéuticos en el cerebro, que de otro modo no sería entregable al tejido en concentración suficiente. Mientras que una amplia gama de parámetros de ultrasonido han demostrado su eficacia a perturbar el BBB 2,5,7, hay varios pasos críticos en el procedimiento experimental para asegurar la interrupción exitosa con precisión en el blanco. Este protoccontornos ol cómo lograr guiada por RM FUS BBBD inducidas en un modelo de rata, con un enfoque en la preparación de los animales y los pasos críticos de microburbujas de manejo del experimento.

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Microburbujas mediada por la interrupción ultrasonido focalizado de la barrera hemato-encefálica (BHE) es una técnica prometedora para no invasiva la entrega de fármacos en el cerebro<sup&gt; 1-3</sup&gt;. Este protocolo describe el procedimiento experimental para la resonancia magnética transcraneal guiada por disrupción de la BHE en un modelo de rata.

Resonancia magnética guiada por disrupción de la barrera sangre-cerebro con ultrasonido transcraneal Enfocado en un modelo de rata

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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17.8K vistas.  Sunnybrook Research Institute. Microburbujas mediada por la interrupción ultrasonido focalizado de la barrera hemato-encefálica (BHE) es una técnica prometedora para no invasiva la entrega de fármacos en el cerebro 1-3. Este protocolo describe el procedimiento experimental para la resonancia magnética transcraneal guiada por disrupción de la BHE en un modelo de rata.

Video: MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodal de imágenes de la angiogénesis en un modelo de rata desnuda de las metástasis óseas del cáncer de pecho utilizando imágenes de resonancia magnética, tomografía computarizada y la ecografía volumétrica

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Investigación

Medicina

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Modelo de rata de ruptura de la barrera sangre-cerebro para permitir dirigida Therapeutics neurovascular

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Un modelo de infusión de nutrientes crónica en la rata

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Estirar en Brain células endoteliales microvasculares (CEND) como<em&gt; In Vitro</em&gt; Lesión cerebral traumática Modelo de la barrera hematoencefálica

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Establecimiento de un<em&gt; In Vitro</em&gt; Modelo de Rata barrera hematoencefálica (BBB): Un Enfoque en la acreditación Impermeabilidad y transporte mediado por los receptores

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

Medición ascendente aórtica Rigidez<em&gt; En Vivo</em&gt; En ratones usando ultrasonido

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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 ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

RM guiada dmPFC-rTMS como tratamiento para el trastorno depresivo mayor resistente al tratamiento

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Ensayos in vitro para evaluar la formación y la interrupción de los vasos sanguíneos de barrera hematoencefálica

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...