Este estudio fue financiado por la Fundación Mcihael J. Fox para la Investigación del 2011 Innovaciones de Respuesta Rápida de Parkinson Programa de Premios. Los financiadores no tenía papel en el diseño del estudio, recogida y análisis de datos, decisión a publicar, o la preparación del manuscrito.

Antes de la cirugía asegúrese de todos los procedimientos que hay que hacer son aprobados por IACUC y las autoridades éticas o legales adicionales y utilizan prácticas de tratamiento de los animales sin causarles daño. Esto incluye el uso de cirugía condiciones estériles, anestesiar el ratón usando IACUC método aprobado, lubricantes ojos ratones con la pomada de veterinario durante la cirugía, y la prestación de atención posquirúrgica. No continúe con la cirugía si hay alguna cuestión de si se aprueban aspectos del procedimiento. Todos los procedimientos realizados en este documento fueron aprobados por el Comité Universitario Institucional Cuidado de Animales y Uso Boston. 1. Preparación de los animales y Suministros Quirúrgicos <ol><li> Autoclave todo instrumento quirúrgico que se utiliza durante la cirugía. </li><li> Asegúrese de que todas las técnicas que se van a realizar son aprobados por los organismos reguladores de los animales. </li></ol> 2. La recolección de la mucosa del injerto <ol><li> Elija un ratón genéticamente idéntico de edad similar a lael ratón experimental y practicar la eutanasia en un método aprobado IACUC (aquí: la asfixia isoflurano seguido por dislocación cervical). </li><li> Con unas tijeras quirúrgicas, quitar la piel alrededor de la región nasal de la cabeza del ratón exponer el cráneo. </li><li> Con un taladro neumático, marca con tres líneas dos de los cuales flanquean lateralmente la región nasal y una tercera en la línea de los ojos que conecta las dos líneas perpendiculares. </li><li> Perforar abajo ventralmente con el fin de separar el tabique nasal del tejido circundante. Un camino más amplio evitará daños a la membrana de la mucosa sin embargo, también hará que sea más difícil aislar la membrana. Se recomienda un corte estrecho cerca de la línea media. </li><li> Utilice unas tijeras para cortar el tabique libre de cualquier tejido adherido al mismo y guárdelo en una solución salina estéril. En este momento el injerto se puede limpiar para eliminar cualquier tejido conectado. La situación ideal es tener las membranas mucosas no dañadas expuestas en ambos lados del tabique cartílago. Uno grpopa se puede suministrar de membrana para dos ratones proporcionó el área de la superficie de la membrana es suficiente para cubrir los sitios de craneotomía. Se recomienda que el injerto se utiliza lo más rápidamente posible y el investigador procede a la etapa 3, tan pronto como se aísla el injerto. </li></ol> 3. Implantación quirúrgica de la mucosa del injerto <ol><li> El uso de procedimientos estándar, asépticas murinos quirúrgicos, anestesiar y montar un ratón en el marco estereológico. Utilice aproximadamente 2% de isoflurano en oxígeno puro usando una máquina de anestesia de roedores. </li><li> Inmovilizar el ratón en un aparato estereotáxico con barras de oído y un soporte de la nariz. Aplique una pomada oftálmica para los ojos y frote el cuero cabelludo con betadine y etanol al 75% durante tres rondas. El uso de cualquiera de tijeras o un cortador de pelo, quitar el pelo de la cabeza. Exponer el cráneo con una hoja de afeitar y nivelar la cabeza. Realizar una craneotomía por encima de la ubicación del cerebro que se dosifica. Por ejemplo, cuando la orientación del cuerpo estriado cortar un 1,25 mmdiámetro del orificio circular en el cráneo (con centro en AP: 1,00 mm; ML: 0,88 mm) usando un taladro neumático. Humedecer la zona perforada con solución salina estéril y utilizar una hoja de afeitar para quitar el cráneo. </li><li> Retire con cuidado la duramadre con la punta de una aguja. Además, esto se puede lograr mediante la aplicación de una cantidad mínima de adhesivo tisular a la superficie de la duramadre húmedo. Una vez que esta capa se ha endurecido, el movimiento lateral con una punta de cuchilla de afeitar se puede utilizar para eliminar la membrana. </li><li> Coloque la membrana de la mucosa por encima de la superficie del cerebro teniendo cuidado extremo para mantener el lado epitelial hacia fuera de la herida. Esto se hace mejor mediante la transferencia de todo el tabique sobre la superficie del cráneo adyacente al sitio de la craneotomía con pinzas. Con la punta de un par de tijeras quirúrgicas, tire de la membrana fuera del cartílago y en la superficie del cráneo y del cerebro. No deje que la membrana se seque o toque con cualquier material absorbente. El injerto debe superponerse generosamente todos los bordes óseos de la craneotomía site. </li><li> Cubra el injerto con una pieza estéril de nitrilo. Esto actúa para impedir la adherencia de la piel al injerto durante la cicatrización. El nitrilo necesita ser lo suficientemente grande como para cubrir toda la membrana de la mucosa. Recorte membrana excesiva si es necesario. Evite cualquier movimiento del nitrilo, una vez que ha entrado en contacto con el injerto. </li><li> Cierre la piel con una sutura 5-0 estéril y dejar correr el ratón recuperar durante 3-7 días antes de proceder a la siguiente etapa. Tenga cuidado de no perturbar la barrera de nitrilo o el injerto de mucosa durante el cierre de la piel. </li></ol> 4. Administración de la solución de dosificación <ol><li> Después de asegurar el ratón anestesiado en el marco estereotáxico, cortar la sutura con unas tijeras y eliminar el exceso de piel alrededor del cráneo. </li><li> Retire la barrera de nitrilo y limpiar la superficie del cráneo. Utilice salinos y algodón hisopos estériles para limpiar el área hasta que el injerto es visible. Puede ser necesario cortar el injerto con una maquinilla de afeitar si ha crecido más grande que el desired superficie. </li><li> Si el experimento será más de unos pocos días, es conveniente implantar al menos dos tornillos del cráneo para reforzar el implante de cabeza. </li><li> Coloque el muy por encima del injerto de modo que los bordes están en contacto con el cráneo. Aplicar el adhesivo de cianoacrilato en la unión entre el pozo y el cráneo. Llene el bien con solución salina estéril y comprobar para asegurarse de que no haya fugas. Wells se hacen de agujas de jeringa corte. </li><li> Aplique cemento para huesos en el cráneo para asegurar el bien en su lugar. </li><li> Quitar la solución salina desde el pozo con una pipeta. Lave bien las varias veces para verificar que el adhesivo no se ha filtrado in Agregar la solución deseada; en este caso se utiliza 50 l de dextrano fluorescente. Se espera que los compuestos solubles en agua de una polaridad similar se comportan de la misma como dextrano. Entrega de compuestos hidrófobos o suspensiones no ha sido explorado con este método. </li><li> Se tapa la parte superior del pozo mediante el uso de una pieza circular de nitrilo asegurado a laarriba usando adhesivo de cianoacrilato. Asegúrese de que el adhesivo no entre en contacto con los contenidos del pocillo. </li></ol> 5. Análisis de transmucosa de entrega <ol><li> Una vez transcurrido el período de tiempo deseado, anestesiar el ratón y el intercambio de los contenidos del pocillo con una solución de colorante azul de Evan. Este colorante se utiliza para verificar que el injerto estaba intacto. </li><li> Después de 30 min, anestesiar el ratón en gran medida, se retira la solución colorante, y la eutanasia mediante decapitación. </li><li> Elimine manualmente el implante y retire el cerebro utilizando tijeras quirúrgicas. Tenga cuidado de mantener el injerto en su lugar. </li><li> Una vez retirado, el flash-congelar el cerebro en una solución de isobutano se enfrió en un baño de hielo seco. </li><li> Incrustar el cerebro en temperatura óptima de corte solución (OCT) y el tramo a 50 micras. </li><li> Colocar las rebanadas deseadas directamente en un portaobjetos de microscopio. </li><li> Imagen de la rebanada utilizando un microscopio de fluorescencia tan pronto como la solución de octubre se ha secado. </li></ol>

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Benjamin S. Bleier MD es el inventor de plomo de los métodos de recubrimiento de patente provisional de la administración de fármacos al sistema nervioso central.

El paso más difícil del procedimiento descrito en el presente documento es la transferencia con éxito de una membrana mucosa de tamaño adecuado sobre la superficie del cerebro. Este paso se hace mucho más fácil si el tabique nasal cosechado es lo suficientemente grande y las limpiaban bien. Si se trunca la porción ventral del septum, se debe obtener un nuevo injerto. El ángulo de perforación debe ser perpendicular a la cabeza del ratón para asegurar que la membrana de la mucosa no es dañado por el taladro. Si...

El tratamiento de la enfermedad neurológica y psiquiátrica está severamente obstaculizada por la presencia de la barrera sangre-cerebro (BBB) ​​que impide que más del 95% de todos los posibles agentes farmacéuticos de alcanzar el sistema nervioso central 1-3. Por ejemplo, factor neurotrófico derivado del glial (GDNF) ha demostrado ser eficaz en el tratamiento de la enfermedad de Parkinson cuando se inyecta directamente en el cerebro, sin embargo, es ineficaz cuando se entrega sistémica, ya que no puede penetrar la acreditación 4-6. Numerosos acercado se han desarrollado para tratar de solventar este problema. Mejoras en la administración sistémica de neurotheraputics se ha demostrado mediante el uso de conjugados de fármaco que contienen anticuerpos selectivos para proteínas de transporte situados sobre el endotelio capilar cerebral; Sin embargo este método no ha demostrado ser aplicable para una amplia gama de productos farmacéuticos 7,8. Además, la apertura osmótica de la BBB ha sido utilizado clínicaaliado, sin embargo, este método adolece de la dosificación del fármaco sistémico en oposición a una entrega más directo a la región del cerebro de interés 9. Esfuerzo considerable se ha puesto en la optimización de la entrega transnasal con la esperanza de dirigirse directamente al cerebro 10-12. Aunque algo de éxito se ha logrado, resultados concluyentes sólo se han obtenido para fármacos que poseen receptores endógenos, tales como la insulina 13,14. Además, el mecanismo de entrega transnasal ha sido motivo de controversia con la evidencia que sugiere la entrada indirecta en el cerebro a través de la captación de la neurona olfatoria o por el torrente sanguíneo 11. La entrega directa, transcraneal utilizando catéteres implantables se ha conseguido, sin embargo este procedimiento es altamente invasiva y asociada con numerosas complicaciones 15,16. Hasta la fecha, no hay ningún método general, mínimamente invasivo para entregar compuestos de alto peso molecular en el cerebro. Presentado en el presente documento es un procedimiento quirúrgico murinoque crea una interfaz semipermeable con el cerebro. Esto se logra de injerto de un explante de la membrana mucosa 17 sobre un defecto craneotomía quirúrgica en un ratón. El uso de este procedimiento se ha demostrado que los compuestos solubles de hasta 500 kDa pueden ser entregados en el sistema nervioso central (directamente en el parénquima cerebral, así como en el líquido cefalorraquídeo) tanto en un tiempo y manera dependiente de peso molecular 18. Este método de pasar por la acreditación es un modelo para la reparación de defectos de base de cráneo en los seres humanos que utiliza injertos de mucosa vascularizados para reparar agujeros en el cráneo después de la cirugía endoscópica transnasal 19,20.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="849">
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	<tbody>
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			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:220px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:263px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">mice</td>
			<td style="width:220px;">Taconic</td>
			<td>C57BL/6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane</td>
			<td style="width:220px;">Piramal Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Fine Scissors</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91461-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">pneumatic drill</td>
			<td style="width:220px;">MTI Dental</td>
			<td style="width:119px;">333-CB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">drill bit</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">forcepts</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">0.9% Sodium Chloride Injection USP</td>
			<td style="width:220px;">Abbott Laboratories</td>
			<td>4925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Polystyrene Detri Dish</td>
			<td style="width:220px;">Fisher</td>
			<td>08-757-12</td>
			<td>for temporarily storing graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Sterilizer</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen/Isoflurane System</td>
			<td style="width:220px;">SurgiVet</td>
			<td>V720100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Control System</td>
			<td style="width:220px;">Physitemp</td>
			<td style="width:119px;">TCAT-2LV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Stereotaxic Instrument</td>
			<td style="width:220px;">KOPF</td>
			<td style="width:119px;">Model 940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye Ointment</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Electric shaver</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cotton-Tipped Applicators</td>
			<td style="width:220px;">Fisher</td>
			<td>23-400-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Provadone Iodine</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>Betadine surgical scrub</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical Blade Stainless</td>
			<td style="width:220px;">Feather</td>
			<td style="width:119px;">2976#10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Scalpel Handle - #3</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3% Hydogen Peroxide</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>for cleaning the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetbond Tissue Adhesive</td>
			<td style="width:220px;">3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">needles</td>
			<td>Becton, Dickinson and Company</td>
			<td>305176</td>
			<td>needle tip cut off and used as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">nitrile gloves</td>
			<td style="width:220px;">Denville Scientific Inc</td>
			<td>G4162</td>
			<td>for well closure and protection of graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Nylon Suture Thread</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Halsey Needle Holder</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cyanoacrylate adhesive</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>commecially available super glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Kit, 1 lb, Pink Opaque</td>
			<td style="width:220px;">Stoelting</td>
			<td>51458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isobutane (2-methylbutane)</td>
			<td style="width:220px;">Aldrich</td>
			<td>M32631</td>
			<td>for dry ice bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">dry ice</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

heterotopic mucosal engrafting procedure for direct drug delivery to the brain in mice

Entrega de agentes terapéuticos en el cerebro se ve impedida por la presencia de la barrera sangre-cerebro (BBB), que restringe el paso de compuestos polares y de alto peso molecular de la sangre y en el tejido cerebral. Algún éxito la entrega directa en los seres humanos se ha logrado a través de la implantación de catéteres transcraneal; Sin embargo este método es altamente invasivo y asociada con numerosas complicaciones. Una alternativa menos invasiva sería para dosificar el cerebro a través de un implantado quirúrgicamente, membrana semipermeable tal como la mucosa nasal que se utiliza para reparar defectos de la base del cráneo siguiente transnasal tumor de la cirugía de extracción endoscópica en los seres humanos. Transferencia del fármaco a pesar de esta membrana evitaría eficazmente la acreditación y difundirse directamente en el cerebro y líquido cefalorraquídeo. Inspirado por este enfoque, un enfoque quirúrgico en ratones fue desarrollado que utiliza una membrana donante de la mucosa septal injertado sobre un extracraneal quirúrgica acreditación defecto. Este modelo se ha demostrado que efectivamentepermitir el paso de compuestos de alto peso molecular en el cerebro. Puesto que numerosos fármacos candidatos son incapaces de cruzar la BHE, este modelo es valiosa para la realización de los ensayos preclínicos de nuevas terapias para enfermedades neurológicas y psiquiátricas.

La obtención de una lo suficientemente grande como explante tabique nasal es crucial para las etapas subsiguientes. Esto se puede lograr mediante la perforación en el lugar en el cráneo del ratón donante se muestra en la Figura 1a. Corte a lo largo de este camino producirá un explante de suficiente tamaño como se muestra en la Figura 1b. Si la profundidad de la perforación no es lo suficientemente profunda, el injerto será truncado y que será difícil obtener una membrana lo su...

Watch this Scientific Journal Video about Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice at JoVE.com

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

Un modelo de ratón de la reconstrucción endoscópica de base de cráneo humano se ha desarrollado que crea una interfaz semipermeable entre el cerebro y la nariz con injertos de mucosa nasal. Este método permite a los investigadores estudiar la entrega al sistema nervioso central de la terapéutica de alto peso molecular que se excluyen de otra manera por la barrera sangre-cerebro cuando se administra sistémicamente.

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 ...

heterotopic-mucosal-engrafting-procedure-for-direct-drug-delivery-to

Research

JoVE Journal

Medicine

10.7K vistas.  Boston University.  Un modelo de ratón de la reconstrucción endoscópica de base de cráneo humano se ha desarrollado que crea una interfaz semipermeable entre el cerebro y la nariz con injertos de mucosa nasal. Este método permite a los investigadores estudiar la entrega al sistema nervioso central de la terapéutica de alto peso molecular que se excluyen de otra manera por la barrera sangre-cerebro cuando se administra sistémicamente. 

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

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 ...

Investigación

Medicina

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

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 diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

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 ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Ratón directa Trauma / Burn modelo de osificación heterotópica

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotópico cervical trasplante cardiaco en ratones

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

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 ...

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 ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

En Vivo Photolabeling de las células en el Colon para evaluar potencial migratorio de las células hematopoyéticas en ratones neonatales

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Nuevos métodos para la administración Intranasal bajo inhalación de anestesia para evaluar el suministro de medicamentos de la nariz al cerebro

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...