This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

Todos los experimentos realizados fueron de acuerdo con la Declaración de ARVO para el uso de animales en Oftálmica y Vision Research, y con la aprobación de la Universidad de Comité de Ética Animal de L&#39;Aquila y al Comité Ético de Experimentación Animal de la Universidad Nacional de Australia. Las ratas adultas (P100-200) se utilizan en todo este estudio. 1. Preparar los gránulos de resina de copolímero <ol><li> Coloque 20 pastillas Elvax 40W en un pequeño vaso de vidrio en la vitrina de gases. </li><li> Llenar el vaso de precipitados con 100% de etanol asegurando para cubrir todos los gránulos. </li><li> Cubrir el vaso con Parafilm, y tomar las pastillas en etanol al 100% a temperatura ambiente durante 7-10 días. NOTA: A partir de este paso adelante, no use metal o plástico para manejar o almacenar la resina. </li></ol> 2. Preparar la solución de fármaco para la carga <ol><li> Se disuelve el fármaco de elección en el 0,1% de dimetilsulfóxido (DMSO). Hacer la solución del fármaco hasta 40 l de volumen.Cargue la resina con tres veces la concentración de la droga que se daría en una sola dosis. </li></ol> 3. Preparar y Cargar el Complejo de resina de Drogas <ol><li> Transferir los 20 sedimentos lavados a otro pequeño vaso de vidrio. Disolver los pellets en 4 ml de diclorometano durante aproximadamente 45 min. Cubrir el vaso con Parafilm mientras que los pellets se están disolviendo. </li><li> Preparar una solución de Fast Green FCF disolviéndolo en 0,1% de DMSO a una concentración de 0,001 mg / ml. </li><li> Tome una pipeta, y elaborar 40 l de la solución de fármaco preparado con anterioridad. Cargue otra pipeta con 40 l de solución de verde rápido. </li><li> Añadir las dos soluciones simultáneamente en el vaso de precipitados. Mix rápidamente utilizando una varilla agitadora de vidrio hasta que el colorante verde se distribuye uniformemente por toda la mezcla. </li><li> Transferir inmediatamente el vaso de precipitados sobre hielo seco durante 10 min para congelar la resina ayunar. </li><li> Establecer una &quot;cámara de evaporación&quot; que se evapore el disolvente. Llene unamayor recipiente hasta un tercio con guijarros de sulfato de calcio. Crear un pozo en los guijarros, y coloque cuidadosamente el vaso de precipitados en el pozo de manera que las piedras lleguen hasta la mitad del vaso, para estabilizar el vaso en la cámara. </li><li> Cubra el recipiente fuera más grande con Parafilm. </li><li> Traslado de la cámara de evaporación al congelador. Mantener a -20 ° C durante 2-3 semanas. </li></ol> 4. preparar el complejo fármaco-resina para la implantación quirúrgica <ol><li> Retire la cámara de evaporación del congelador. Transferir el bloque de resina sólida de fármaco a un plato de vidrio se mantienen en hielo. </li><li> Con la ayuda de un microscopio o lupa, cortar un trozo de aproximadamente 0,05 mm de diámetro y 0,1 mm de longitud del bloque con una herramienta o trépano de perforación. </li><li> Envolver el resto del bloque en el material a prueba de congelación (por ejemplo, papel de aluminio) y mantener a -20 ° C hasta su utilización. </li></ol> 5. implantar quirúrgicamente el complejo fármaco-resina en la rata de ojos </p&gt; <ol><li> Preparar al animal para la cirugía. Anestesiar usando una inyección intraperitoneal de una mezcla de ketamina (100 mg / kg de peso corporal) y xilazina (12 mg / kg de peso corporal). Observar el animal hasta que se logre la anestesia completa. NOTA: El animal debe tener una pérdida de movimientos (excepto para respirar), y una pérdida de los reflejos de la córnea y de arrastre del dedo del pie. </li><li> Traslado al animal completamente anestesiado a la mesa de cirugía. Uso mydriaticum (por ejemplo, sulfato de atropina), y anestesia local (por ejemplo, clorhidrato de tetracaína) gotas en el ojo (s) sometidos a cirugía. Aplicar lágrimas artificiales para mantener la córnea húmeda durante la cirugía. Utilizar agujas desechables e instrumentos estériles durante toda la cirugía. </li><li> Estabilizar el ojo usando un par de pinzas curvas contundentes después de que el alumno esté completamente dilatado. Utilice un microscopio de operación para hacer una herida por punción de profundidad completa para llegar al vítreo, aproximadamente 2 mm desde el limbo usando una aguja de 25 G. </li><li> Inserte la pieza preparada de tél complejo fármaco-resina en la herida de punción usando un par de pinzas, puntiagudos fino (por ejemplo, pinzas) que son capaces de penetrar en el sitio de la punción. </li><li> Como profilaxis de la infección bacteriana, aplique ungüento antibiótico en el ojo, para prevenir la inflamación, así como la prevención de la sequedad de la córnea mientras el animal se está recuperando. </li><li> Observar al animal hasta la recuperación completa. No dejar al animal sin vigilancia hasta que se haya recuperado la conciencia. No devolver el animal a la compañía de otros animales hasta que se recupere completamente. Si el animal muestra signos de estrés o malestar, los analgésicos deben ser utilizados de acuerdo con el protocolo de ética aprobado. </li><li> Siga los procedimientos experimentales habituales para la recolección y el análisis de los tejidos. </li><li> En la realización de experimentos, la eutanasia al animal con una inyección intraperitoneal de una pentobarbital (60 mg / kg de peso corporal) solución de clorhidrato de lidocaína al 2%. </li></ol> NOTA: Lidocaína Clorhidrato minducir al mínimo el malestar local.

<ol>
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Este trabajo demostró el uso de un complejo de fármaco-resina para el suministro de liberación lenta de fármacos a la retina. El objetivo de presentar un método que es relativamente barato y fácil de aplicar en un modelo de pequeños animales. Dado que la función de APB es actuar como un análogo de glutamato, bloqueará la respuesta de la retina en el ojo. Los resultados muestran que APB provocó el bloqueo de la respuesta de la retina en una semana después de la implantación. Esto...

El tratamiento de enfermedades crónicas como la diabetes y la hipertensión arterial presenta muchos desafíos, ya que estas enfermedades generalmente requieren tratamiento durante largos períodos de tiempo, a menudo de por vida. Esto ha llamado para el desarrollo de sistemas de administración de fármacos de liberación lenta, que reducen la necesidad de una dosificación frecuente. La eficacia de estos métodos de liberación lenta se ha demostrado mediante el desarrollo de bombas de insulina, para reducir el número de inyecciones de insulina necesarias para tratar la diabetes. Las enfermedades crónicas de los ojos, sobre todo los que afectan a sus capas internas, requieren la administración frecuente de medicamentos a través de procedimientos invasivos. Una enfermedad crónica que afecta tal que el ojo humano es la degeneración macular relacionada con la edad (AMD). Afecta a la retina central, que es una capa de tejido neural situado en la parte posterior del ojo responsable de iniciar la visión. AMD es la principal causa de ceguera en el mundo occidental 1. Un desafío particular con el tratamiento de reenfermedades intes- es que el medicamento es necesario para llegar a esta capa profunda en el ojo, que a menudo requiere de métodos invasivos de entrega. Los fármacos se administran habitualmente a la cámara vítrea y la retina utilizando inyecciones intravítreas. Sin embargo, con cada inyección hay un riesgo de complicaciones postinyección, incluyendo endoftalmitis, desprendimiento de retina, cataratas y hemorragia del vítreo 2. Este riesgo se multiplica con cada reinyección de la droga. La reducción de la necesidad de múltiples inyecciones sería un beneficio importante en el tratamiento de la AMD. Para tratar la forma húmeda de AMD, donde el nuevo crecimiento de los vasos es un sello distintivo, la estrategia terapéutica establecida es apuntar a los factores de crecimiento endotelial (VEGF) que utilizan inhibidores del VEGF 3. En la actualidad, éstos se entregan a través de inyecciones intravítreas repetidas. Del mismo modo, en el tratamiento de edema macular, una complicación común de la retinopatía diabética, los corticosteroides se entregan a través de inyecciones repetidas <shasta&gt; 4. La entrega de estos medicamentos a través de métodos de liberación lenta de hecho podría reducir el riesgo de complicaciones inyección. La noción de un sistema de administración de fármacos de liberación lenta se describió primero usando un vehículo de caucho de silicona para entregar pequeñas moléculas en los tejidos animales 5. Desde entonces, otros métodos de liberación lenta se han desarrollado para proporcionar moléculas más grandes, varios de los cuales han sido probados en el ojo. Portadores de partículas, tales como microesferas biodegradables, poli-lactida-co-glicolida (PLGA) nanopartículas y vesículas de fosfolípidos (liposomas) pueden ser útiles como vehículos de administración 6,7. Nanopartículas y liposomas de PLGA se han comparado en un entorno in vitro por su capacidad para suministrar agentes anti-cáncer a través de la esclerótica en el tiempo 7. Ambos vehículos fueron efectivos en la liberación de los fármacos lentamente. Sin embargo, el estudio sólo se llevó a cabo en un entorno in vitro. BOCHOT et al. (2002) 8 probadosla eficacia de los liposomas para entregar moléculas a la retina in vivo. Ellos han demostrado que los liposomas entregar con éxito pequeños oligonucleótidos a la retina de conejo. Los autores sugirieron que los liposomas podrían ser beneficiosos en el tratamiento de enfermedades de la retina 8. Sin embargo, la naturaleza de estas vesículas para flotar en el humor vítreo significa que es probable que difuminar o poner en peligro la visión 9. Okabe et al. (2003) 10 no utilizan discos de polímero biodegradable compuesta por 33% de acetato de etileno-vinilo para aplicar beta-metasona en conejos. Se implantaron los discos en un bolsillo escleral y demostraron la liberación eficaz del fármaco en el humor vítreo y la retina de hasta un mes 10. Sin embargo, en este protocolo particular, el implante fue relativamente grandes y rígidos, y requiere un procedimiento quirúrgico más complejo que incluye una gran incisión escleral y la sutura. Un estudio anterior investigó el tissue respuesta a diversos vehículos de polímero por implantarlos en la córnea de los ojos del conejo, y se encontró que los copolímeros de acetato de etileno-vinilo lavadas en alcohol no causan inflamación o irritación. Estos complejos se muestran para sostener el suministro de compuestos más grandes en los tejidos animales durante largos períodos de tiempo, algunos superior hasta 100 días dependiente de la droga 11. Uno de tales tipos de resina de copolímero se ha desarrollado industrialmente en forma de Elvax 40W (40% en peso de etileno-acetato de vinilo contenido de comonómero con un aditivo de amida de &quot;W&quot; para mejorar el manejo de pellets). Esta resina de copolímero es una sustancia inerte que es estable tanto a temperatura ambiente y corporal. No se ha demostrado que causa alergia o toxicidad en el tejido biológico. Esta resina efectivamente ha entregado una gran variedad de drogas en diferentes modelos experimentales que investigan las funciones de varios sistemas, como el sistema ductal mamaria 12, la corteza auditiva primaria 13,14 &lt;/ Sup&gt;, y el sistema visual 15 rana. Esta resina también se ha utilizado en el ojo para suministrar fármacos para el desarrollo de tortuga 16,17, 18,19 embrión de pollo, y hurón adulto retinas 20. En el sistema nervioso central de la rata, la resina sólo se ha utilizado en el cerebro 21-23, pero su uso en el ojo de la rata no se ha documentado. Las ventajas del uso de esta resina de copolímero para suministrar fármacos lentamente a la retina con respecto a otros métodos, es que es un compuesto estable que no causa inflamación o irritación en el ojo. A diferencia de los portadores de partículas, el complejo fármaco-resina no perjudicar la visión después de la implantación, ya que normalmente permanece en el sitio de la entrega en lugar de flotar en el humor vítreo. Sólo se requeriría un proceso de implantación sencilla en la cavidad vítrea cerca del limbo del ojo, y no requeriría la sutura después de la implantación. Recientemente, ha habido un surgimiento en varios nuevos sistemas de suministro, tales comola tecnología de células encapsuladas (ECT) 24,25, hidrogeles 26 y 27 microfilms. Sin embargo, el método utilizado en el estudio actual para preparar y entregar el complejo fármaco-resina es a la vez fácil de seguir y de bajo costo, siendo por tanto más ventajoso para su uso en un entorno de investigación básica. El reto de la utilización de este complejo para ofrecer tratamientos farmacológicos a largo plazo es determinar la concentración óptima de la droga que va a maximizar los beneficios terapéuticos de tener un menor número de inyecciones intravítreas. Este trabajo tiene como objetivo demostrar el uso del complejo fármaco-resina para el tratamiento a largo plazo de la retina de rata adulta. La eficacia de este modo de entrega se prueba utilizando el análogo de glutamato 2-amino-4-fosfonobutirato (APB) que el medicamento. APB bloquea la respuesta a la luz de las células bipolares ON imitando el glutamato, un neurotransmisor endógeno en la retina 28. Cuando APB compite con glutamato por su receptor, bloquea el light respuesta. APB se ha utilizado en estudios fisiológicos para controlar la función de la retina y medir su efecto usando métodos electrofisiológicos como el electrorretinograma (ERG). En estudios anteriores, APB se ha utilizado durante tanto a corto plazo 29 y el tratamiento a largo plazo de la retina en desarrollo; En ella se registra dar una dosis única vía inyección intraocular al día durante 30 días 30. La misma cantidad de APB (0,092 mg en solución salina estéril, a una concentración de 50 mM) se utilizó para todas las inyecciones como se sugiere en el trabajo anterior 28,29,31. Elegimos APB para poner a prueba el complejo fármaco-resina como un vehículo de liberación lenta para administrar fármacos en el ojo. Los métodos descritos en este estudio son similares a los métodos descritos anteriormente que implican la preparación de la droga-resina compleja 16,32; Sin embargo, también detalle su uso específicamente en el ojo de rata adulta. Después de la implantación quirúrgica de la resina cargada-APB en el ojo, ERG se realizó para determinar si APB suprime la rrespuesta a la luz etinal, y por lo tanto si APB ha sido lanzado con éxito en el humor vítreo y la retina.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Elvax 40W Pellets</td>
			<td>Du Pont, DE, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-4-phosphonobutyric acid (APB)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>A1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, calcium sulfate</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>238910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine, Ilium Ketamil</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, Ilium Xylazil-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulphate, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol, Chlorsig ointment</td>
			<td>Aspen Pharma Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital, Lethabarb</td>
			<td>Virbac Australia Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocaine Hydrochloride, Ilium Lignocaine-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uni-Core Punch Tool</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Microscope, Zeiss OPMI 99</td>
			<td>Zeiss, West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G Insulin Needle</td>
			<td>Terumo Corp., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

Enfermedades de la retina son difíciles de tratar como la retina se encuentra profundamente dentro del ojo. Los métodos invasivos de administración de fármacos a menudo son necesarios para el tratamiento de estas enfermedades. Enfermedades de retina crónico tales como edema retinal o la neovascularización por lo general requieren múltiples inyecciones intraoculares para tratar eficazmente la enfermedad. Sin embargo, los riesgos asociados con estas inyecciones aumentan con la entrega repetida de la droga. Por lo tanto, los métodos de entrega alternativos necesitan ser establecidos con el fin de minimizar los riesgos de reinyección. Varias otras investigaciones han desarrollado métodos para administrar fármacos a través del tiempo extendido, a través de materiales capaces de liberar sustancias químicas lentamente en el ojo. En esta investigación, se describe el uso de Elvax 40W, una resina de copolímero, para actuar como un vehículo para la administración de fármacos a la retina de rata adulta. La resina se hace y se carga con el fármaco. El complejo fármaco-resina se implanta en la cavidad vítrea, en donde será liberar el fármaco lentamente sobre timí. Este método fue probado usando 2-amino-4-fosfonobutirato (APB), un análogo de glutamato que bloquea la luz de respuesta de la retina. Se demostró que la APB se libera lentamente de la resina, y fue capaz de bloquear la respuesta de la retina por 7 días después de la implantación. Esto indica que la administración de fármacos de liberación lenta usando esta resina de copolímero es eficaz para el tratamiento de la retina, y podría ser utilizado terapéuticamente con más pruebas.

Electrorretinograma de campo completo (ERG) se utilizó para detectar el efecto de APB en la retina. Para más detalles sobre cómo se realizó el ERG, consulte los siguientes estudios 33,34. En resumen, después de anestesiar al animal, un electrodo de oro fue colocado en la córnea del ojo, y el electrodo de referencia se colocó en el cuero cabelludo anterior entre los ojos, para registrar la actividad eléctrica de la retina. Los estímulos se generaron utilizando una unidad de flash electrónico y la int...

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Este documento detalla cómo Elvax 40W se puede utilizar como un método de liberación lenta para la administración de fármacos a la retina de rata adulta. El protocolo para la preparación, carga, y entrega el complejo fármaco-resina para el ojo se describe.

De liberación lenta Drug Delivery través Elvax 40W a la Rata Retina: Implicaciones para el tratamiento de condiciones crónicas

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Research

JoVE Journal

Medicine

11.5K vistas.  University of L'Aquila.  Este documento detalla cómo Elvax 40W se puede utilizar como un método de liberación lenta para la administración de fármacos a la retina de rata adulta. El protocolo para la preparación, carga, y entrega el complejo fármaco-resina para el ojo se describe. 

Video: Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La medición y tratamiento de la represión en la ambliopía

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Investigación

Medicina

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  ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Osmótica Drug Delivery de miembros posteriores isquémicos y la perfusión de la vasculatura con Microfil para Micro-tomografía computarizada

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Intraluminal entrega de la droga a la fístula arteriovenosa ratón endotelio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Intravascular de entrega de los productos biológicos para el riñón de rata

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Análisis de la red de foramen oval de electrodos Grabaciones en epilepsia del lóbulo temporal pacientes resistentes a los medicamentos

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Método optimizado de LC-MS/MS para el análisis de alto rendimiento de las muestras clínicas de Ivacaftor, sus principales metabolitos y Lumacaftor en fluidos biológicos de pacientes con Fibrosis quística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

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) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Trans-timpánica fármaco para el tratamiento de la ototoxicidad

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...