Estamos muy agradecidos por la ayuda del Dr. Li Xinyu en la preparación del manuscrito. SW fue apoyada por un fondo de inicio de la Universidad de Tulane, Consejo de Investigación Premio al Investigador Nuevo del presidente de UT Southwestern Medical Center, NIH subvención EY021862, un premio de desarrollo de la carrera de la Investigación para Prevención de fundación ceguera, y un Premio Brillante Foco en la edad degeneración macular asociada a la Investigación .

Nota: El siguiente protocolo y resultados representativos utilizan el inhibidor de HDAC SAHA como un compuesto de ejemplo. Sin embargo, este protocolo es de ningún modo limitada al uso de SAHA, y se recomienda como un método general para poner a prueba los efectos de los compuestos solubles en neovascularización de la córnea. Tendrá que ser hecha por el grado de dilución, así como la frecuencia y la duración de la aplicación modificaciones menores. Adicionalmente, los compuestos que son fácilmente solubles en agua podrán ser administrado en ausencia de DMSO. Declaración de Ética: Todos los experimentos con animales no se deben efectuar de conformidad con la legislación nacional y los reglamentos institucionales. Este protocolo fue aprobado para su uso por la Universidad de Tulane Institucional Cuidado de Animales y el empleo Comisión. 1. Preparación de los materiales (por orden de utilización) <ol><li> Corte un pedazo de papel de filtro de celulosa (11 m) en 2 círculos mm. </li><li> Preparar una solución 1 M de NaOH pordisolviendo 20 g de NaOH en 500 ml de agua destilada H 2 O. Guarde a temperatura ambiente. PRECAUCIÓN: soluciones de NaOH es corrosivo y puede causar quemaduras alcalinos. </li><li> Preparar un cóctel anestésico mediante la dilución de 10 ml de 100 mg / ml de ketamina y 2,5 ml de 20 mg / ml de xilazina en 37,5 ml de 1 x PBS para una concentración final de 20 mg / ml de ketamina y 4 mg / ml de xilazina. </li><li> Preparar una solución de 0,5% de clorhidrato de proparacaína (para la analgesia tópica) mediante la disolución de 500 mg de clorhidrato de proparacaína en 100 ml de PBS filtrada. </li><li> Preparar PBS 1x disolviendo 8 g de NaCl, 0,2 g de KCl, 1,44 g de Na 2 HPO 4, y 0,23 g de NaH 2 PO 4 en 900 ml de H2O destilada Ajuste a pH 7,4. Poner la solución en un volumen final de 1000 ml con H2O destilada y se filtra para garantizar la esterilidad. </li><li> Preparar 1.000 x soluciones madre de SAHA en ~ 100 mM en dimetil sulfóxido (DMSO) disolviendo 26,4 mg de SAHA en 1 ml de DMSO. Diluir a una 1x (~ 10 M)solución en PBS filtrada para cada uso. La solución madre, a -20 ° C durante un máximo de un mes. PRECAUCIÓN: DMSO es una toxina y mutágeno conocido. </li><li> Preparar una solución de 4% de paraformaldehído (PFA) para la fijación. Bajo una campana química, disolver 4 g del PFA en 90 ml de PBS. Llevar la mezcla a 65 ° C y valorar, a un pH de 7,4. Una vez que el PFA se ha disuelto, llevar la solución a un volumen final de 100 ml. Almacenar a 4 ° C durante un máximo de un mes. PRECAUCIÓN: PFA es una sabe que es alergénica, carcinogénico y tóxico. </li><li> Preparar tampón de bloqueo 1x diluyendo 5 ml de suero de cabra y 500 ul de Triton X-100 en 94,5 ml de PBS. </li><li> Preparar tampón de lavado 1x mediante la dilución de 500 ul de Triton X-100 en 99,5 ml de PBS. </li></ol> 2. Álcali-Tratamiento de Quemaduras Lesiones Compuesto <ol><li> Remojar una pieza circular de papel de filtro, ~ 2 mm de diámetro, en una solución de 1 M de NaOH. </li><li> Se anestesia el ratón con una inyección de 100 mg / kg de ketamina y 5 mg / kg de xilazina, WHich es de ~ 100 l del cóctel anestésico desde el paso 1.3 por cada 25 g de ratón. La anestesia debe tomar ~ 1-2 min a establecer pulg Profundidad de la anestesia puede ser determinada por pellizcar suavemente la punta o la cola del animal, si la anestesia es suficiente no debe haber ninguna respuesta. Nota: Se debe tener cuidado para asegurar el ratón no sucumbe a la anestesia hipotermia inducida. </li><li> Con un gotero, aplique tópicamente una caída del 0,5% de clorhidrato de proparacaína a la superficie de la córnea para la analgesia local. </li><li> Usando una pinza estéril, tome un pedazo de papel de filtro empapado NaOH. Si usted nota un exceso de NaOH se aferran a goteo o del papel de filtro, toque brevemente el papel de filtro empapado en un pedazo de papel de filtro seco para absorber el exceso. Coloque la pieza de papel de filtro empapado NAOH en la córnea central. Déjelo por 30 segundos para generar un álcali-quemadura aguda de ~ 2 x 2 mm 2 de superficie. Un microscopio quirúrgico es útil en colocar correctamente el papel de filtro. Nota: Sólo un ojo de la s del ratónhould lesionarse con el otro que sirve como control. </li><li> Retire el papel de filtro. Usando una jeringa de 10 ml, limpiar suavemente el ojo con 10 ml de 1x PBS dos veces para lavar residual de 1 M de NaOH. </li><li> Inmediatamente aplique una gota de solución de trabajo 1x SAHA (o un control de vehículo que consiste en DMSO de PBS diluido que no contiene SAHA) tópicamente a la córnea. Repita 3 veces al día la aplicación durante 14 días. Nota: durante este período no se recomienda un ungüento antibiótico tópico ya que puede interferir con el desarrollo de la lesión y la entrega del compuesto. Use una solución de antibiótico líquido, solución de gentamicina tales como el 3%, en lugar. </li><li> Proceder directamente a la Evaluación Clínica (Protocolo 3 a continuación) o sacrificar el ratón y enucleación de los ojos durante la córnea plana mount (Protocolo 4 abajo) o parafina / histología congelado convencional. </li></ol> 3. Evaluación clínica <ol><li> Realizar un examen diario de los ratones de una manera ciega con un microscopio quirúrgico y anotar corneal NV basa en la opacidad corneal, NV, y el tamaño del buque. Utilice por lo menos dos observadores y registrar una puntuación final que es el promedio de los dos. <ol><li> Puntuación opacidad de la córnea en una escala de 0-4. 0 = totalmente claras; 1 = poco nebuloso, iris y la pupila fácilmente visible; 2 = ligeramente opacos, el iris y la pupila aún detectable; 3 = opacos, los alumnos casi no detectables; y 4 = completamente opacos, sin vistas de la pupila. </li><li> Puntuación NV en una escala de 0-3. 0 = sin neovasos; 1 = neovasos en el limbo de la córnea; 2 = neovasos que atraviesan el limbo corneal y se acercan al centro de la córnea; 3 = neovasos que atraviesan el centro de la córnea. </li><li> Puntuación tamaño del vaso en una escala de 0-3. 0 = sin neovasos; 1 = neovasos detectables al microscopio quirúrgico; 2 = neovasos fácilmente visibles con microscopio quirúrgico; 3 = neovasos fácilmente visibles sin el microscopio. </li></ol></li><li> El uso de una cámara digital, tomar imágenes representativas de los ojos a los 7 días y 14 días. </li></ol> 4.Corneal Coloración y monturas Piso <ol><li> Fijar ojo enucleado en el 4% PFA durante al menos 1 hora a 4 ° C. </li><li> Transferir el ojo a 1x PBS y utilizar pinzas para quitar con cuidado el exceso de tejido. </li><li> Con la ayuda de un microscopio quirúrgico, el uso de una aguja (calibre 18) o micro-cuchillo para perforar la región pericorneal del ojo (nota: este debe liberar líquido del ojo). <ol><li> A partir de la perforación realizada en la sección 4.3.1 usar un par de tijeras quirúrgicas para cortar la parte anterior del ojo (córnea) de la porción posterior. </li></ol></li><li> Transfiera la córnea de nuevo en PFA al 4% para la fijación de la noche a 4 ° C. </li><li> Deseche el 4% PFA (PRECAUCIÓN: PFA es peligrosa y debe ser eliminado de acuerdo con las normas institucionales) y enjuagar la córnea tres veces con 1x PBS para eliminar cualquier PFA residual. </li><li> Incubar en tampón de bloqueo 1x durante al menos 2 horas a temperatura ambiente para permeabilizar el tejido y evitar la unión no específica del anticuerpo primario. <ol><li> Aplicar el anticuerpo primario en tampón de bloqueo 1x, a una dilución de plegado 100-500. (Por ejemplo, de rata anti-ratón-PECAM-1 para la detección de los vasos sanguíneos a 1:100, de conejo anti-ratón-LYVE-1 para detectar los vasos linfáticos a 1:500, y / o anti-mouse-F4/80 de rata para la detección de macrófagos a 1:100). Incubar a 4 ° C durante la noche. </li><li> Lave seis veces con 1x tampón de lavado durante 1 hora cada vez a temperatura ambiente para eliminar por completo el anticuerpo primario no unido. </li><li> Aplicar un anticuerpo secundario en 1x tampón de bloqueo a una dilución veces 500-1.000. (Por ejemplo, un fluorescente 488 nm etiquetados de cabra anti-rata-IgG a 1:800 o un fluorescente 594 nm etiquetados de cabra anti-IgG de conejo a 1:800). Incubar a 4 ° C durante la noche. </li><li> Lavar tres veces con 1x PBS durante 1 hr cada uno a temperatura ambiente para eliminar totalmente el anticuerpo secundario no unido. </li></ol></li><li> Transferencia de la córnea en 1x PBS fresco y, con la ayuda de un microscopio quirúrgico, hacer cuidadosamente cuatro incisiones desde la periferia hacia el cientoer. Esto debería dividir la córnea en cuatro cuadrantes de aproximadamente el mismo tamaño (la forma resultante debería ser más bien como una mariposa) y deje que se acueste en una diapositiva. </li><li> Montar con un medio apropiado para el montaje de imagen fluorescente. Para obtener los mejores resultados, las muestras deben ser reflejados inmediatamente y fotografías digitales tomadas, sino que se pueden almacenar durante varias semanas protegidos de la luz a 4 ° C. </li></ol>

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Los autores no tienen nada que revelar.

El protocolo que se presenta aquí se traduce en niveles reproducibles de hemangiogenesis, LI, y la inflamación, por lo que es un sistema ideal para estudiar estos tres procesos (interrelacionados). Mientras que este método produce centralizado NV córnea, varios métodos que se han desarrollado para causar más dirigida NV, a saber, la sutura de la córnea 17 y implantados factor de crecimiento expresando gránulos 18, también puede ser de interés. Nuestro protocolo está diseñado para su uso...

Ceguera corneal es la cuarta causa más común de ceguera, responsable de aproximadamente el 4% de todos los casos 1. La neovascularización corneal (NV) desempeña un papel importante en muchas de estas patologías, incluyendo la queratitis herpética (la principal causa infecciosa de ceguera en Occidente) y el tracoma (la causa principal de ceguera en todo el mundo infecciosa) 2. Las terapias actuales incluyen esteroides, fármacos antiinflamatorios no esteroideos (AINEs), las terapias anti-VEGF, y ciclosporina A, así como las técnicas quirúrgicas convencionales o láser 3. Sin embargo, la naturaleza gravemente debilitante de patologías NV basado corneales, la escasez de instalaciones quirúrgicas capaces de tratar NV corneal, y la falta de una opción farmacológica encarecidamente realizar encabezó una mesa redonda de expertos reciente a la conclusión de que, a pesar de las terapias existentes, la necesidad médica fundamental presentado por estas patologías permanece insatisfecha 4. La córnea humanaconsta de 5 capas, 3 capas celulares (epitelial, del estroma y el endotelio) y 2 interfaz (membrana de Bowman y la membrana de Descemet). Funciona como una barrera mecánica y la superficie de refracción para el ojo. Su naturaleza transparente es la consecuencia de un delicado equilibrio de sus componentes, y es parte integral de su función adecuada 5. Normalmente avascular, la córnea recibe sangre de microvasos que se ejecutan a lo largo de su borde exterior que se alimenta desde la ciliar y las arterias oftálmicas. Corneal NV se produce cuando un estímulo promueve la angiogénesis de estos vasos permitiendo que crezcan hacia el centro de la córnea y por lo tanto limitar la visión 6. Angiogénesis corneal incluye hemangiogenesis y linfangiogénesis, que resultan en el crecimiento hacia el interior de los vasos sanguíneos y los vasos linfáticos de la arcada vascular del limbo hacia el centro de la córnea. Esto conduce a un desglose de la córnea &quot;privilegio angiogénico&quot;, un aumento en la opacidad de la córnea y la fibrosis, la alteración de la córnea Layers, y edema 7. Los disparadores precisos de NV corneal son numerosos, que van desde una respuesta a las enfermedades infecciosas como el tracoma en un estado inducido químicamente causado medicinas tradicionales, productos químicos industriales, o incluso agentes de guerra química. Los mecanismos moleculares de este proceso no son, por ahora, completamente caracterizados; sin embargo, se han identificado algunos jugadores clave. En condiciones normales la córnea posee un &#39;privilegio angiogénico&#39; única mantenida por una matriz redundante de factores anti-angiogénicos (tales como solubles de VEGF-R1) 8. Sin embargo, en respuesta a un estímulo externo (tal como una lesión), habrá una regulación por incremento local de factores pro-angiogénicos (por ejemplo, VEGF-A). Esto inclina la balanza de factores pro y anti-angiogénicos que subyace privilegio angiogénico de la córnea, y conduce a hemangiogenesis, lymphangeogenesis, y la inflamación, por lo tanto, causando la patología de la córnea e incluso ceguera 9. &lt;p class = &quot;jove_content&quot;&gt; Debido a la necesidad médica no satisfecha de esta patología altamente debilitante, es de interés para el campo para tener un modelo animal fiable de NV corneal. Aquí se presenta un modelo de este tipo: control de lesiones álcalis quemadura. Se han utilizado diversos modelos de ojo de lesiones basado en el uso de anillos de papel de filtro desde 1970 10. En 1989, un grupo de oftalmólogos Escuela de Medicina de Harvard caracteriza un modelo estándar de una lesión alcalino-quemadura de la córnea central en conejo basado en remojo un pedazo de papel de filtro circular con hidróxido de sodio (NaOH) y su aplicación a la córnea en un rango específico de concentraciones 11. Desde entonces, esta técnica se ha adaptado para su uso en el 12-14 ratón. Recientemente, el laboratorio de Wang estudió los efectos terapéuticos de la histona deacetilasa (HDAC) inhibidor de SAHA en la patogénesis de NV de la córnea utilizando un modelo de lesión de la córnea del ratón alcalino-quemadura 15. La metodología del modelo de lesión de la córnea del ratón alcalino-quemadura presentado aquí fue construidoprincipalmente en el trabajo previo de otros dos trabajos 14,16.

<table border="0" cellpadding="0" cellspacing="0" width="598">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 mL Syringe</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">309659</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18 Guage Needle</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">305918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">306575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25 Guage Needle</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">305916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-F4/80 (rat anti-mouse)</td>
			<td style="width:96px;">AbD Serotech</td>
			<td>MCA497RT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-LYVE-1 (rabbit anti-mouse)</td>
			<td style="width:96px;">Abcam</td>
			<td style="width:119px;">ab14917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-PECAM-1 (rat anti-mouse)</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">553370</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-IgG Alexa488 (goat anti-rat)</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">A11006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-IgG Alexa594 (goat anti-rabbit)</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">A11012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:96px;">Tucsen</td>
			<td style="width:119px;">TCC 5.0 ICE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">12-548-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">D4540-1L</td>
			<td>Caution: Mutagenic, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps (Blunt), Iris</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps (Sharp), Dumont #4</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">500340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">P217-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Solution</td>
			<td style="width:96px;">MedVet</td>
			<td>RXKETAMINE</td>
			<td>Controlled substance, proper license required for use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Source for Microscope</td>
			<td style="width:96px;">AmScope</td>
			<td style="width:119px;">LED-14M-YA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope (Stereo 7X-45X)</td>
			<td style="width:96px;">AmScope</td>
			<td style="width:119px;">SM-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mounting Medium, Vectashield</td>
			<td style="width:96px;">Vector</td>
			<td style="width:119px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S397-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S318-1</td>
			<td>Caution: Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:96px;"></td>
			<td style="width:119px;">P6148-500G</td>
			<td>Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proparacaine Hydrochloride</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">P4554-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors (5mm blade), Vanas</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat Serum</td>
			<td style="width:96px;">MPBio</td>
			<td align="right" style="width:119px;">92939249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope Slides</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">T8787-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Whatman Grade 1 Filter Paper</td>
			<td style="width:96px;">Whatman</td>
			<td style="width:119px;">1001-6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine Solution</td>
			<td style="width:96px;">MedVet</td>
			<td style="width:119px;">RXANASED-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

an alkali-burn injury model of corneal neovascularization in the mouse

En condiciones normales, la córnea es avascular, y esta transparencia es esencial para mantener una buena agudeza visual. La neovascularización (NV) de la córnea, que puede ser causada por un traumatismo, queratoplastia o enfermedad infecciosa, se rompe el llamado &quot;privilegio angiogénico &#39;de la córnea y constituye la base de múltiples patologías visuales que pueden incluso conducir a la ceguera. Aunque hay varias opciones de tratamiento disponibles, la necesidad médica fundamental presentado por patologías corneales neovasculares permanece insatisfecha. Con el fin de desarrollar terapias seguras, eficaces y selectivos, se requiere un modelo fiable de NV de la córnea y la intervención farmacológica. Aquí se describe un modelo de neovascularización corneal lesiones álcalis quemadura en el ratón. Este protocolo proporciona un método para la aplicación de una lesión alcalino-quema controlada a la córnea, la administración de un compuesto farmacológico de interés, y la visualización del resultado. Este método podría ser instrumentalTal para el estudio de los mecanismos y oportunidades para la intervención en NV corneal y otros trastornos neovasculares.

Después de la lesión alcalino-quemadura, NV córnea se produce en una, de la manera dependiente del tiempo predecible. La Figura 1 demuestra la gran diferencia tanto en la neovascularización y la opacidad de la córnea entre un animal sin tratar (Figura 1A) y un animal tratado con el inhibidor de HDAC SAHA (Figura 1B ) en el punto de tiempo de 7 días. Las Figuras 2A y 2B demuestran una córnea plana de m...

Watch this Scientific Journal Video about An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse at JoVE.com

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

La neovascularización (NV) de la córnea puede complicar múltiples patologías visuales. Utilizando un modelo de lesión de álcali-quema controlada, un nivel cuantificable de NV corneal puede ser producido para el estudio mecanicista de la NV corneal y la evaluación de posibles terapias para trastornos neovasculares.

Una Lesión álcalis quemar modelo de neovascularización corneal en el ratón

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

an-alkali-burn-injury-model-of-corneal-neovascularization-in-the-mouse

Research

JoVE Journal

Medicine

16.5K vistas.  Tulane University.  La neovascularización (NV) de la córnea puede complicar múltiples patologías visuales. Utilizando un modelo de lesión de álcali-quema controlada, un nivel cuantificable de NV corneal puede ser producido para el estudio mecanicista de la NV corneal y la evaluación de posibles terapias para trastornos neovasculares. 

Video: An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

La isquemia reperfusión renal: un modelo de ratón de la lesión y regeneración

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Investigación

Medicina

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Un ratón neonatal lesión de médula espinal de compresión Modelo

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Un modelo de ratón para inducida por láser neovascularización coroidea

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Un Modelo de Ratón de Único y Repetitivo Lesión Cerebral Traumática Leve

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Neovascularization del diafragma como un modelo de ratón de iridis del rubeosis inducida por la punción

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Cicatrización de heridas de abrasión epitelial corneal con rebabas Ocular como un modelo de córnea

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Un nuevo enfoque de supervisión Neovascularization del injerto en la encía humana

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Un modelo de cultivo de órgano endotelial Corneal porcino usando botones de córnea divididos

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...