Los autores desean agradecer al personal de las instalaciones de resonancia magnética en la Clínica Semmes-Murphey, Memphis, Tennessee, así como el departamento de Neurocirugía de la Universidad de Tennessee Health Science Center en Memphis, Tennessee.

1. Preparación de Gel Phantoms y tinte <ol><li> Preparar 0,2% de gel de agarosa disolviendo 2 g de 0,1% de polvo de agarosa en 1000 ml de agua desionizada. Se agita la solución durante aproximadamente 1 min para asegurar una mezcla adecuada; y microondas inmediatamente la solución en 3 minutos durante 9 minutos o hasta que quede transparente, revolviendo entre los intervalos. </li><li> Mientras que el gel de agarosa es líquido, vierta la solución en 5 cm x 5 cm x 5 cm contenedores. Deje espacio en la parte superior del contenedor para agregar el agua y permitir que el gel de agarosa se enfríe y se asiente. </li><li> Una vez que el gel de agarosa se ha solidificado (aproximadamente 1-2 horas), añadir 1 cm de agua a la parte superior del gel y refrigerar. Lo mejor es usar el gel dentro de 24 a 48 h de la mezcla, pero puede ser almacenado hasta por una semana refrigerado 10. </li><li> Preparar un colorante de radio-contraste en una jeringa de 60 ml que consiste en 50 ml de 0,017% de bromofenol colorante azul (BPB), y 2 mM de gadoteridol medios de comunicación de radio-contraste. <ol><li> Combine 8,5 mg de tinte BPB a 50 ml de agua desionizada para crear una solución de 0,017% de BPB. </li><li> Añadir 0,2 ml de solución madre 0,5 M gadoteridol a la solución 50 ml de BPB 0,017% para crear una solución gadoteridol 2 mM. </li></ol></li></ol> 2. Preparación de Sistema de Infusión <ol><li> Sistema de infusión de la bomba de jeringa (método preferido): Para la preparación de bomba de jeringa, sujetar el catéter de infusión directamente a la jeringa a través del sensor de presión, lo que reduce el volumen muerto de la línea de infusión. La función de purga de la bomba de jeringa puede ser utilizado para limpiar la línea de aire usando un bolo mayor que el volumen de cebado del catéter a una velocidad de 10 l / min. </li><li> Sistema de infusión de la bomba de tubo (método alternativo): Conecte la jeringa que contiene el tinte de radio-contraste con la bomba de infusión. Una el sensor de presión a la salida de la bomba con el transductor conectado al monitor IV. Adjuntar un catéter de infusión 16 G para el extremo abierto del sensor de presión. Nota: La punta del G catéter de infusión 16 tiene un diámetro internometro de 0,2 mm y un diámetro exterior de 0,35 mm. La punta está hecha de sílice fundida y la longitud de la punta es de 3 mm. Aumenta a aproximadamente 0,75 mm y se prolonga durante 15 mm, el catéter luego intensifica de forma cónica de 1,6 mm o 16 G. </li><li> Preparar para perfusión purgando el sistema durante aproximadamente 15 min a 16.667 l / min para eliminar las burbujas de aire. No exceda el caudal 16.667 l / min, ya que la máquina dejará de infusión debido a la alta presión de la línea. Después de fijar el catéter de infusión a la línea de salida de la bomba de infusión, líneas de purga de aire mediante el uso de la función &quot;bolo&quot; en la bomba de infusión. </li><li> Fije el soporte del catéter de infusión y el marco de la trayectoria al contenedor fantasma gel (5 cm x 5 cm x 5 cm) y el lugar en el MRI. </li></ol> 3. CED Gel de infusión y MR escaneo <ol><li> Poner a cero el valor de la presión (mmHg) registrado por el monitor IV antes de comenzar la perfusión. </li><li> Inserte el catéter de infusión en el gel de agarosa wITH la bomba de infusión funcionando a la velocidad de flujo más bajo posible, en este caso 1.667 l / min. </li><li> Comenzar la exploración de RM, utilizando los parámetros listados en la Tabla 1, y continuar la infusión a un ritmo de 1.667 l / min. Infundir el gel a una velocidad constante hasta que el volumen total infundido alcanza 60 l (aproximadamente 38 min). </li><li> Explore el gel de forma continua en 3 minutos y los intervalos de 50 seg. Registre las lecturas de presión cada 60 seg. Una vez que el volumen infundido alcanza 60 l, apague la bomba de infusión; y la RM completa sin dejar de grabar lecturas de la presión. </li></ol> 4. Análisis de datos MR <ol><li> Para el análisis de las imágenes de RM, utilice un visor DICOM apropiada con la funcionalidad de segmentación ROI. </li><li> Seleccione el marco correcto en cada escanear marcado por la sección transversal del catéter según se ve en la Figura 1. </li><li> Usando el &quot;retorno de la inversión - rectángulo&quot; de herramientas, seleccione la porción más grande del gel que no incluye ningunaporción de la zona de infusión. La salida de la voluntad de software una densidad media de píxeles con una desviación estándar. Encuentre el valor que corresponde a tres desviaciones estándar de la media. Este valor se utiliza como umbral para determinar cuando el contraste está presente con un nivel de confianza del 99,7%. </li><li> Usando el &quot;retorno de la inversión - el círculo&quot; de la herramienta, rodear el lugar de la infusión con un gran círculo lo suficiente y dar a esto un nombre único. </li><li> Seleccione el círculo y con el &quot;retorno de la inversión - valores de los píxeles se ajusta en&quot; herramienta, valor de umbral de entrada se encuentra en el paso 4.3 en &quot;si el valor actual es mayor que:&quot; y checkmark esta línea solamente. Luego, en &quot;a este nuevo valor:&quot;, introduzca un valor grande (25.000). Reiniciar la densidad de píxeles para seleccionar el área abarcada por el umbral previamente definido. </li><li> A continuación, utilizando el &quot;retorno de la inversión - crecer región (2D/3D segmentación)&quot; de herramientas, seleccione la región 2D crece, el algoritmo de la confianza con el parámetro radio inicial = 2, y el ROI cepillo. Haga clic dentro de la zona de infusión para el software para calcular la superficie total of esta región. </li><li> Suponiendo una nube de infusión esférica, calcular el volumen de difusión desde la zona a través de la siguiente ecuación: V = 4/3π (√ (Área / π)) 3 </li></ol>

<ol>
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Los autores declaran que no tienen intereses financieros en competencia.

<html> Los pasos críticos para garantizar el éxito de la infusión son: purgar la línea de infusión de aire, mezclando el gel de agarosa, el análisis de los datos de RM, utilizando pequeños diámetros de catéter interno, utilizando escalonada diseños de catéter para reducir al mínimo el reflujo, y la minimización de la presión sentida por el gel o tejido en el que se está infundiendo el fármaco. Como se dijo anteriormente, en detrimento principal para el éxito de la infusión es la línea de aire de infusi...

Entrega potenciada por convección (CED) se ha propuesto como una opción de tratamiento para un amplio espectro de trastornos neurológicos que incluyen tumores malignos cerebrales, epilepsia, trastornos metabólicos, enfermedades neurodegenerativas (como la enfermedad de Parkinson) 1, derrames cerebrales y trauma 2. CED emplea flujo mayor presión positiva para la distribución de un fármaco u otro líquido de infusión. CED proporciona la entrega segura, fiable y homogénea de compuestos de bajo peso molecular, que van de menor a mayor, con un volumen clínicamente relevantes 3. Administración de fármacos tradicional al tejido cerebral está severamente restringido por la barrera sangre-cerebro 4. Formado por las uniones estrechas entre las células endoteliales que forman los capilares en el cerebro, los bloques de barrera de sangre-cerebro polar y moléculas de alto peso molecular a partir de entrar en el parénquima del cerebro. Perfusión cerebral intraparenquimatosa directo vía CED puede superar las limitaciones de las modalidades de administración de fármacos terapéuticos anterioresy permite el uso de agentes terapéuticos que no cruzaría la barrera sangre-cerebro, y por lo tanto han sido previamente disponible como opciones de tratamiento viables 5. Investigadores de los Institutos Nacionales de Salud de EE.UU. (NIH) describen CED a principios de 1990 como un medio para alcanzar mayores concentraciones terapéuticas de medicamentos que por simple difusión 6-8. Los primeros métodos de CED consistió en implantar uno o más catéteres en el cerebro, la conexión de una bomba de infusión para el catéter, y el bombeo de los agentes terapéuticos directamente en la región objetivo. Se informó que el aumento de la fracción de distribución y concentración relativamente estable a ocurrir como la presión positiva creada por la bomba de infusión hace que los tejidos se dilaten y permiten la permeación del fármaco 9. La técnica fundamental para CED sigue siendo en gran medida el mismo que se describió por primera vez. Los avances en el diseño del catéter 10, técnica de infusión <sup&gt; 11, control de presión de la línea 2, y el seguimiento de MRI en tiempo real para corregir desplazamiento cerebro 12, 13, optimizar múltiples infusiones colineales 14, y vigilar para la pérdida de líquido de infusión 15 se han incrementado la seguridad y eficacia del tratamiento 10. Importancia adicional ha sido colocado en el diseño del catéter y la estrategia de infusión incluyendo la velocidad de flujo. El éxito de CED, con reflujo catéter limitada y el daño tisular, se ha correlacionado con el diseño del catéter y la velocidad de infusión. El uso de un catéter con un diámetro estrecho y una baja tasa de infusión para limitar el flujo de retorno a lo largo de la interfaz cerebro-catéter, así como limitar los daños en la punta del catéter 16. La RM proporciona una confirmación visual de la ubicación correcta para la colocación del catéter de infusión, y por lo tanto la administración de fármacos, además de facilitar la corrección del reflujo infusión o entrega aberrante 17. Las imágenes de RM también se pueden utilizar para aproximar y realizar un seguimiento de los volúmenes de distribución (Vd) Del fármaco infundido. El Vd se calcula utilizando un valor de intensidad de la señal RM mayor que tres desviaciones estándar por encima de la media del gel no infusa que rodea como un umbral para la segmentación 18. El Vd es una medida útil para CED, ya que representa el volumen del fármaco distribuido en el cerebro. Junto con el volumen infundido (VI), en una relación puede ser generada (Vd / Vi) cuantificar el volumen cubierto por el fármaco infundido. Fantasmas en gel de agarosa imitan varias propiedades mecánicas cruciales del cerebro humano importantes para la comprensión de CED tales como: Vd, interacciones de gel en catéter, propiedades poroelásticos, y la morfología de infusión nube 10. Las mezclas de 0,2% de gel de agarosa se ​​ha demostrado para imitar los cambios en vivo en la fracción de poros locales causadas por la dilatación de gel debido a la CED. Una fracción de poro similar a cerebro humano promueve interacciones similares y mediciones precisas de Vd 19. Adicionalmente, concentraciones similares de ungeles garose tales como 0,6% y 0,8% han demostrado perfiles de presión de infusión similares al cerebro 20. Además, los geles de agarosa translúcidas proporcionan la ventaja de visualización en tiempo real de la colocación del catéter de infusión y el reflujo. Fantasmas en gel de agarosa son relativamente baratos de producir. El costo de los fantasmas de gel de agarosa puede ser clave para la formación generalizada en toda futura cirugía neurológica. Debido a estas propiedades, geles de agarosa proporcionan un sustituto útil, la replicación de muchos de los atributos clave de infusiones cerebrales humanos sin el uso de tejido cerebral. Como se indicó anteriormente, guiado imagen-CED en modelos de gel de agarosa proporciona un beneficioso método in vitro para la prueba, la investigación y la capacitación. El propósito de este artículo es describir cómo recrear fantasmas gel de agarosa, para delinear los protocolos de ensayos y análisis CED sean apropiados, y para hacer frente a los errores comunes que enfrentan durante las infusiones CED para el tratamiento de enfermedades neurológicas.

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Prohance</td>
			<td style="width:71px;">Bracco</td>
			<td style="width:119px;"></td>
			<td style="width:195px;">Gadoteridol radio contrast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Bromophenol Blue Dye</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-0404</td>
			<td>Dye for infusate visualization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Agarose Gel Powder</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-3101EDU</td>
			<td>Agarose powder for creating gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Veris MR Vital Signs Monitor</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pressure monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">16 Gauge SmartFlow Catheter</td>
			<td style="width:71px;">SurgiVision</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Continuum MR Infusion System</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">SMART Frame MRI Guided Trajectory Frame</td>
			<td style="width:71px;">ClearPoint</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Osirix Imaging Software and DICOM Viewer</td>
			<td style="width:71px;">Osirix Imaging Software</td>
			<td style="width:119px;"></td>
			<td>OsiriX 32-bit DICOM Viewer</td>
		</tr>
	</tbody>
</table>

image-guided convection-enhanced delivery into agarose gel models of the brain

Entrega potenciada por convección (CED) se ha propuesto como una opción de tratamiento para una amplia gama de enfermedades neurológicas. Neuroinfusion catéter CED permite un flujo mayor presión positiva para ofrecer una mayor cantidad de productos terapéuticos para lograr un objetivo intracraneal que los métodos de administración de fármacos tradicionales. La utilidad clínica de la resonancia magnética en tiempo real guiado CED (rCED) se encuentra en la capacidad de localizar con precisión, seguimiento de la terapia, e identificar complicaciones. Con el entrenamiento, rCED es eficiente y las complicaciones puede ser minimizado. El modelo en gel de agarosa del cerebro proporciona una herramienta accesible para las pruebas de CED, la investigación y la capacitación. Cerebro simulado rCED permite la práctica de la cirugía simulada mientras que también proporciona una retroalimentación visual de la infusión. Análisis de infusión permite el cálculo de la fracción de distribución (Vd / Vi) permitiendo que el aprendiz para verificar la similitud del modelo en comparación con el tejido cerebral humano. En este artículo se describe nuestra fantasma cerebro en gel de agarosa y se esbozan importante metrics durante una infusión y análisis CED protocolos mientras que abordan problemas más comunes que enfrentan durante la CED de infusión para el tratamiento de enfermedades neurológicas.

Interpretar y analizar infusiones CED implican varios factores importantes, como la fracción de distribución y el reflujo líquido de infusión. El cálculo de la fracción de distribución depende en gran medida en el cálculo de la Vd. Por lo tanto, la correcta interpretación de las imágenes de RM es fundamental. Se propone un método semi-automatizado para reproducir de forma fiable estas mediciones como se indica anteriormente. Estos métodos de determinar objetivamente el área de la sección transversal de la ...

Watch this Scientific Journal Video about Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain at JoVE.com

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Entrega potenciada por convección (CED) se ha propuesto como una opción de tratamiento para una amplia gama de enfermedades neurológicas. Con el fin de preparar a los profesionales de atención de salud para la adopción del CED, se necesitan modelos de formación accesibles. Se describe el uso de gel de agarosa como tal modelo del cerebro humano para la prueba, la investigación y la capacitación.

Guiada por imagen potenciada por convección Entrega en Modelos en gel de agarosa del Cerebro

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

image-guided-convection-enhanced-delivery-into-agarose-gel-models

Research

JoVE Journal

Medicine

11.3K vistas.  University of Tennessee Health Science Center.  Entrega potenciada por convección (CED) se ha propuesto como una opción de tratamiento para una amplia gama de enfermedades neurológicas. Con el fin de preparar a los profesionales de atención de salud para la adopción del CED, se necesitan modelos de formación accesibles. Se describe el uso de gel de agarosa como tal modelo del cerebro humano para la prueba, la investigación y la capacitación. 

Video: Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Resonancia magnética y PET en modelos de ratón de infarto de miocardio

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Investigación

Medicina

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Heterotópico mucosas Procedimiento La incorporación del trasplante de Drogas entrega directa al cerebro en ratones

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Ratón Trasplante de Riñón: Modelos de rechazo del aloinjerto

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Transposón mediada Integración de ADN plásmido en la zona subventricular de ratones recién nacidos para generar modelos novedosos de glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

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) ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Estudio de la señal de insulina hipotalámica a la intolerancia de la glucosa periférica con un sistema de infusión continua de medicamentos en el cerebro de ratón

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Resección guiada por la imagen de Glioblastoma e implantación intracraneal de andamios sembrado células madre terapéuticas

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Predicción de respuesta al tratamiento tratamientos guiados por imágenes mediante aprendizaje automático: un ejemplo para el Trans-Arterial tratamiento de Carcinoma Hepatocelular

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Un prospecto de válvula mitral transapical guiado por imágenes Modelo de punción de sobrecarga de volumen controlado de la insuficiencia mitral en la rata

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

Administración Intratraqueal De La Formulación De Polvo Seco En Ratones

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...