Estamos en deuda con Eleut Hernández para la ganadería, Gabe Gaidosh de experiencia microscopía y Khue Tran y Zhenyang Zhao para edición de vídeo. Este estudio fue financiado por el Instituto Nacional del Ojo otorga R01-EY022129 de JLG y subvenciones P30 EY022589 a UCSD y EY014801 a UM; la Asociación Americana del Corazón, la Fundación James y Esther Rey, financia el programa de intercambio de estudiantes de doctorado de Fudan University Graduate School (Nº 2010033), y una subvención sin restricciones de Investigación para Prevenir la Ceguera, Inc.

Todos los animales procedimientos fueron aprobados por la Universidad de California en San Diego y la Universidad de Miami cuidado de los animales institucional y comités uso (IACUC) y realizan de acuerdo con la Declaración de ARVO para el uso de animales en oftálmica e Investigación Visual. Todos los reactivos e instrumentos utilizados en procedimientos quirúrgicos son estériles. 1. Anestesiar y Preparar la Rata de Cirugía <ol><li> Antes del procedimiento, las ratas se anestesiaron con una inyección intraperitoneal de ketamina (60 mg / kg) y xilazina (8 mg / kg) de acuerdo con el peso corporal. Profundidad adecuada de la anestesia debe ser determinada por una respuesta negativa a los estímulos pizca dedo del pie. </li><li> Una vez anestesiado, tire de la lengua hacia adelante para evitar la asfixia y aplicar lubricante pomada para ambos ojos para evitar el secado de las córneas durante la cirugía. </li><li> Afeitarse los sitios quirúrgicos utilizando una cortadora de cabello y limpie el área tres veces con solución de detergente-yodo providona 10% y 70% de etanol. </li><li> Drape tque los animales dentro de un campo estéril. Guantes estériles e instrumentos quirúrgicos se utilizan durante la cirugía supervivencia. Vuelva a esterilizar la punta de los instrumentos utilizando un esterilizador de cuentas en caliente entre los animales. </li></ol> 2. Enfoque Quirúrgico <ol><li> PION inducción <ol><li> Cateterismo vena femoral <ol><li> Preparar y limpiar el lugar de la cirugía. Afeitarse la cara interna del muslo derecho utilizando una cortadora de cabello y limpie el área tres veces cada uno con una solución de detergente providona yodo 10% y 70% de etanol. </li><li> Preparar la tubería. Cortar una longitud de 40 cm de tubo de polietileno (PE 10) esterilizados en etanol al 70%. Enjuague el tubo con solución salina y conectarlo a una jeringa de 1 ml que contiene una solución previamente medida de colorante eritrosina B 2% (1 l / mg, produciendo una dosis de 20 mg / kg de peso corporal). El montaje de jeringa en una bomba de infusión pies interruptor controlado ajustado a una velocidad de 600 l / min. </li><li> Usar una hoja No. 15, hacer una pequeña incisión horizontal enla base del muslo derecho. Cortar y separar la membrana interior y limpiar el área con hisopos de algodón estériles. </li><li> Separar el músculo con pinzas hasta que la rama de la vena femoral es visible. Una vaina rodea la arteria, vena y el nervio. Apriete y tire de esta vaina hacia arriba con fórceps (pinzas de punta fina Dumont), y cortar una pequeña incisión (2.4 mm es generalmente adecuado) cerca de la base de la cuña de forma triangular con tijeras de primavera Vannas. Expandir el corte si es necesario. </li><li> Separar la vena y la arteria con un micro-quirúrgica gancho romo paralelo a la dirección de la vena. Tenga cuidado de no dañar las membranas y las venas delicadas ramas. Luego, levante suavemente la vena y separarlo del tejido conectivo subyacente. </li><li> Obtenga una sutura de nylon sin aguja y colocarla junto a la vena femoral. Usando el gancho micro-quirúrgica, eleve la vena y pasar las pinzas de punta fina debajo de la región distal. Coge un extremo de la sutura y tire de ella debajo de lavena. Ligar la vena distal con fuerza. Pasar una segunda sutura de una manera similar en virtud de la vena proximal y hacer un nudo flojo. </li><li> Hacer un pequeño corte en la vena cerca de la ligadura distal con tijeras de primavera Vannas. Ampliar el agujero como sea necesario con las pinzas de punta fina. Algunos sangre puede filtrarse a través del corte. Limpie el área quirúrgica con, BSS estéril frío y bastoncillos de algodón estériles. </li><li> Sosteniendo la pared de la vena en el borde del corte, cateterizar el recipiente con el tubo de solución salina enrojecida preparado usando un soporte de aguja. Apriete el nudo proximal alrededor de la vena y la tubería. Entonces, anclar la tubería al vincularlo a la sutura distal. </li><li> Compruebe la calidad de la cateterización presionando el interruptor de pie para inyectar solución salina, &lt;1 ml es adecuada. Asegúrese de que el tubo no está obstruido y no tiene fugas. Temporalmente cerrar la incisión con puntos de sutura para proteger el cateterismo y el tejido. </li></ol></li><li> La exposición del nervio óptico <ol><li> Prepare la zona quirúrgica limpiando la zona de pre-afeitado encima del ojo izquierdo tres veces cada uno con una solución de detergente providona yodo 10% y 70% de etanol. </li><li> Hacer una incisión a lo largo de la piel 2-3 mm detrás del ojo con una cuchilla No. 15. Apriete y levante el tejido conectivo con pinzas dentadas, y hacer una pequeña incisión con tijeras de primavera Vannas. Esta pequeña incisión es generalmente cerca de 5 mm de longitud, pero puede ser más largo para proporcionar una mayor exposición para un cirujano en formación. Continuar para diseccionar sin rodeos a través del tejido conectivo a lo largo del borde superior del hueso orbital, teniendo cuidado de evitar la interrupción de los vasos sanguíneos. Limpie el área quirúrgica con hisopos de algodón. </li><li> Diseccionar hacia abajo a través de la conjuntiva hasta que el músculo recto superior es visible. Pinch y diseccionar a través del músculo; el músculo se liberará de las profundidades de la órbita. Ahora, los tejidos circundantes se pueden utilizar para ayudar en la retracción y la elevación del ojo para la facilidad de visualización. </li><li> ReTracto el colgajo de piel y tejido conjuntivo lateralmente y hacia abajo, y mantenga en su lugar con una sutura y hemostasia. Esto hará girar el ojo hacia adelante y hacia el exterior a fin de revelar la vaina que contiene grasa que rodea el nervio óptico. </li><li> Con cuidado, inserte un par de pinzas cortantes y ampliar paralelo al nervio óptico para separar el tejido conectivo que rodea la vaina. No toque el nervio óptico con las afiladas puntas de las pinzas. </li><li> Una longitud de 5 mm del nervio óptico y la vaina que rodea ahora debe ser visible. Una red de microvasos en la superficie vaina rodea el nervio óptico. Estos se orientarán durante la irradiación láser. </li></ol></li><li> La inyección intravenosa de Eritrosina B y la irradiación láser <ol><li> Use gafas de seguridad de color naranja-en todo momento durante el funcionamiento del aparato de irradiación láser para protegerte de la luz láser verde. Encienda el láser, abrir el obturador y ajustar el pico y powe promediors del láser según sea necesario. Cierre el obturador. </li><li> Coloque la rata en el aparato de irradiación con láser. Para asegurar la colocación de haz adecuado, un haz de encuadre débil es producida por espacialmente filtrar el láser a través de un agujero de 100 m de diámetro perforados en la hoja del obturador cerrado. Re-exponer el nervio óptico con un par de pinzas de punta fina, y posicionar el haz de puntería sobre el nervio óptico intraorbitario entre 3 mm y 4 mm detrás de la cabeza del nervio óptico. </li><li> Se inyecta la solución de 2% eritrosina B través de la activación de la bomba de infusión. Se puede circular durante unos segundos mientras el cirujano añade una pequeña gota de BSS para humedecer la superficie del nervio óptico. </li><li> Verifique la posición del haz de encuadre y haga clic en el interruptor de pie para iniciar la irradiación. Un filtro de seguridad de color naranja, que se añade en la trayectoria óptica del microscopio, se desencadenó inmediatamente seguido por la apertura del obturador después de un retardo de 1 seg. </li><li> Irradiar el nervio óptico para 90 seg con una potencia pico de 135MW y potencia media de 18MW producida por un haz de chopper que gira a 250 Hz con un ciclo de trabajo de 15% (esto minimiza los efectos térmicos). Fluorescencia amarilla, visualizado como naranja brillante a través del filtro de seguridad, será emitida desde la superficie superior del nervio óptico y es suficiente para asegurar que el haz irradia el nervio simétricamente. </li><li> Después de la irradiación, el filtro de seguridad de color naranja se abrirá automáticamente. Microhemorragias se puede observar en algunos casos. </li><li> Aliviar la tracción sobre los músculos extraoculares y volver el ojo a una posición neutral. Cierre la incisión con suturas interrumpidas. Luego retirar el catéter y ligar la vena femoral con fuerza para evitar fugas; cerrar con suturas interrumpidas. Aplique un ungüento antibiótico para ambas incisiones. Compruebe el fondo de ojo para verificar la integridad vascular de la vena central de la retina y la arteria. </li></ol></li></ol></li><li> Etiquetado retrógradade las células ganglionares de la retina (CGR). Nota: Con el fin de evaluar la supervivencia de RGC, etiquetado retrógrada con Fluorogold (FG) debe ser completado una semana antes PION. El método se describe en detalle en JoVe protocolo 819 13. <ol><li> En pocas palabras: anestesiar al animal con ketamina (60 mg / kg) y xilazina (8 mg / kg) y afeitar la cabeza. </li><li> Quirúrgicamente frote el lugar de la incisión y hacer una incisión en la línea media en la cabeza para exponer el cráneo. </li><li> Perforar agujeros bilaterales a través del cráneo (ø 2x2 mm) 0,5 mm de ambas suturas sagital y las transversales. </li><li> Aspirar con cuidado el contenido cerebral que se encuentra sobre la superficie dorsal de la colículo superior (SC) utilizando una bomba de vacío. Luego, coloque un pequeño trozo de espuma de gel empapada con 4% FG sobre la superficie de la SC. </li><li> Cierre la incisión con suturas y el cuidado del animal usando los cuidados postoperatorios estándar. </li></ol></li><li> El cuidado post-operatorio y la eutanasia <ol><li> Después de la cirugía, coloque animal en una jaula limpia separada en la parte superior de una almohadilla de recirculación de agua calentada hasta que se recupere el animal. </li><li> Analgésicos post-quirúrgicas (HCl buprenorfina, 0,01 mg / kg) deben administrarse dos veces al día durante tres días consecutivos para minimizar las molestias. </li><li> Rata debe mantenerse separado y observó hasta que son capaces de mantener decúbito esternal y recuperar la conciencia suficiente. </li><li> Los signos de recuperación y buena salud son monitoreados diariamente por lo menos 5 días después de la cirugía, o hasta retirar la sutura y la curación adecuada de la zona quirúrgica, lo que ocurra último. </li><li> La eutanasia a los animales por perfusión con PFA al 4% en los puntos de tiempo científicamente apropiados después de la cirugía de acuerdo a los intereses de investigación. </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-arteritic+anterior+ischemic+optic+neuropathy:+role+of+systemic+corticosteroid+therapy..">Non-arteritic anterior ischemic optic neuropathy: role of systemic corticosteroid therapy..</a> Graefe's archive for clinical and experimental ophthalmology. 246, 1029-1046 (2008).</li><li>Prado, R., Dietrich, W. D., Watson, B. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+Induced+Graded+Spinal-Cord+Infarction+-+Behavioral,+Electrophysiological,+and+Morphological+Correlates..">Photochemically Induced Graded Spinal-Cord Infarction - Behavioral, Electrophysiological, and Morphological Correlates..</a> J Neurosurg. 67, 745-753 (1987).</li><li>Dietrich, W. D., Busto, R., Watson, B. D., Scheinberg, P., Ginsberg, M. 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The authors have nothing to disclose.

Here we describe in detail a method for inducing PION in a rat model. The most critical part of the protocol is the exposure and irradiation of the optic nerve &#8211; to expose the nerve as long as possible while avoiding damage caused by the sharp fine tip forceps or from stretching. In rats, the ophthalmic artery enters the optic nerve &#8804;1 mm from the optic nerve head. Therefore, irradiation of the optic nerve 3-4 mm away from the optic nerve head should only result in ischemia of the capillaries feeding the nerv...

En pacientes mayores de 50 años, la neuropatía óptica isquémica (ION) es el tipo más frecuente de neuropatía óptica aguda 2. La condición puede presentarse como uno de los dos subtipos de acuerdo con la fuente de suministro específica afectada sangre y la presentación clínica: anterior (AION) o posterior (PION) 3. Aunque la patogénesis y el curso de AION se han estudiado ampliamente 4-7, PION ha quedado mal entendido debido a su baja prevalencia, presentación variables, criterios diagnósticos mal definidos y la falta de un modelo animal. Por otra parte, no hay tratamientos se han demostrado para prevenir o revertir la pérdida de la visión de AION o PION eficacia. Por lo tanto, un modelo animal reproducible y fiable de PION es de gran valor para estudiar el proceso de la enfermedad in vivo y probar nuevos regímenes terapéuticos para la neuroprotección y la regeneración del axón. Fotoquímicamente inducida lesión isquémica a la microvasculatura resultando en vasogedema enic y trombosis crea efectivamente isquemia tisular regional 08.12. Después de la inyección en la circulación vascular, el tinte fotosensible eritrosina B produce reactiva de oxígeno molecular singlete tras la activación por irradiación con láser de los vasos diana. El oxígeno singlete peroxidizes directamente el endotelio vascular, estimular la adherencia de plaquetas / agregación y que conduce a la formación de trombos oclusivos. Daño isquémico se extendió a las zonas vecinas y aún más exacerbada por la compresión microvascular debido al edema vasogénico. El objetivo general de este protocolo es inducir fotoquímicamente isquemia en el nervio óptico retrobulbar para reflejar el daño causado por PION. Para nuestro conocimiento, este es el primer modelo de lesión isquémica en el nervio óptico posterior 1. Como este modelo produce isquemia evitando al mismo tiempo el trauma físico, los procesos fisiológicos de la neuropatía óptica isquémica posterior son mejores imitaban y estudiados. Además, este modelo ofrece una nueva plataforma para la detección de agentes terapéuticos candidatos para neuropatías ópticas y otra enfermedad isquémica del SNC. Aquí, un protocolo detallado para la cateterización de la vena femoral, la exposición del nervio óptico, la inyección intravenosa de la eritrosina B y la irradiación con láser en un modelo de rata PION se describen.

<table>
	<tbody>
		<tr>
			<td>532-nm Nd:YAG laser&#160;</td>
			<td>Laser glow</td>
			<td>LRS-532-KM-200-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam chopper</td>
			<td>custom-made</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical shutter and corresponding shutter drive timer</td>
			<td>AAM Vincent Associates</td>
			<td>SD-10</td>
			<td></td>
		</tr>
		<tr>
			<td>25-cm focal length spherical lens</td>
			<td>CVI/Mellles-Griot</td>
			<td>01 LPX 293</td>
			<td>plano-convexBK7 glass lens with HEBBARTM antireflection coating</td>
		</tr>
		<tr>
			<td>Erythrosin B&#160;</td>
			<td>MP Biomedicals</td>
			<td>190449</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogold</td>
			<td>Fluorochrome,LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Cardinal Health</td>
			<td>CAH1203421</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing (PE10)</td>
			<td>BD Intramedic</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 10 Blade</td>
			<td>Miltex</td>
			<td>4-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>F.S.T.</td>
			<td>91150-20</td>
			<td>DUMONT #5 RUSTLESS NON-MAGNETIC</td>
		</tr>
		<tr>
			<td>Forceps with Teeth</td>
			<td>F.S.T.</td>
			<td>11153-10</td>
			<td>Germany stainless</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>F.S.T.</td>
			<td>18025-10&#160;</td>
			<td>Germany stainless</td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>F.S.T.</td>
			<td>2-220&#160;</td>
			<td>JJECK Stainless</td>
		</tr>
		<tr>
			<td>Polyglactin suture</td>
			<td>Ethicon</td>
			<td>J488G</td>
			<td>7-0 suture</td>
		</tr>
		<tr>
			<td>hemostat</td>
			<td>F.S.T.</td>
			<td>12075-12&#160;</td>
			<td>Germany stainless</td>
		</tr>
	</tbody>
</table>

rat model of photochemically-induced posterior ischemic optic neuropathy

Neuropatía óptica isquémica posterior (PION) es una enfermedad devastadora vista en la práctica clínica. Sin embargo, su patogénesis y la historia natural han permanecido poco conocidos. Recientemente, hemos desarrollado un modelo animal fiable, reproducible de PION y probamos el efecto del tratamiento de algunos factores neurotróficos en este modelo 1. El propósito de este video es demostrar nuestro modelo fotoquímica inducida de la neuropatía óptica isquémica posterior, y para evaluar sus efectos con el etiquetado retrógrada de las células ganglionares de la retina. Después de la exposición quirúrgica del nervio óptico posterior, un colorante fotosensibilizante, eritrosina B, se inyecta por vía intravenosa y un haz de láser se enfoca sobre la superficie del nervio óptico. Interacción fotoquímica de eritrosina B y el láser durante daños irradiación del endotelio vascular, lo que provocó la oclusión microvascular mediada por trombosis plaquetaria y la compresión edematosa. La lesión isquémica resultante produce una gradual pero pronounced muerte regresiva de células ganglionares de la retina, debido a una pérdida de entrada axonal - un mando a distancia, lesión inducida y el resultado clínicamente relevante. Por lo tanto, este modelo proporciona una nueva plataforma para estudiar el curso fisiopatológico de PION, y puede optimizarse aún más para probar enfoques terapéuticos para las neuropatías ópticas, así como otras enfermedades isquémicas del SNC.

La lesión isquémica resultante inducida por esta técnica produce una muerte gradual pero pronunciada de las células ganglionares de la retina después de la lesión isquémica axón. Este es un resultado clínicamente relevante similar a la observada en la enfermedad humana. Etiquetado retrógrada FG se utiliza para cuantificar la supervivencia RGC después PION. El mismo método se emplea para validar una creación modelo de éxito, así como para evaluar los efectos de diferentes regímenes terapéuticos. ...

Watch this Scientific Journal Video about Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy at JoVE.com

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

El objetivo de este protocolo es inducir fotoquímicamente lesión isquémica en el nervio óptico posterior, en la rata. Este modelo es fundamental para los estudios de la fisiopatología de la neuropatía óptica isquémica posterior, y los enfoques terapéuticos para esta y otras neuropatías ópticas, así como de otras enfermedades isquémicas del SNC.

Rata Modelo de neuropatía óptica isquémica posterior inducida por Fotoquímicamente

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

rat-model-photochemically-induced-posterior-ischemic-optic

Research

JoVE Journal

Medicine

8.5K vistas.  University of Miami Miller School of Medicine.  El objetivo de este protocolo es inducir fotoquímicamente lesión isquémica en el nervio óptico posterior, en la rata. Este modelo es fundamental para los estudios de la fisiopatología de la neuropatía óptica isquémica posterior, y los enfoques terapéuticos para esta y otras neuropatías ópticas, así como de otras enfermedades isquémicas del SNC. 

Video: Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Creation of Reversible Cholestatic Rat Model

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and affect the nutritional status and surgical outcomes. Study of the pathophysiological changes occurring in the liver and other organs is of importance. Various studies have been done in cholestatic rat models.
We used a reversible cholestatic rat model in our recent study looking at the role of methylprednisolone in the ischemia reperfusion injury. Various techniques for creation of a reversible cholestatic model have been described. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.
This model can be used in various studies, such as looking at the changes in nutritional, physiological, pathological, histological and immunological changes in the gastrointestinal tract. This model can also be used to see the effects of cholestasis and various therapeutic interventions on major hepatic surgeries.

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.

Creación de un modelo reversible Rata colestásica

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and ...

Investigación

Medicina

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferencia génica para la insuficiencia cardiaca isquémica en un modelo preclínico

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

Una técnica simplificada para fabricar un modelo de lesión isquémica

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

La endotelina-1 inducida por la arteria cerebral media oclusión Modelo para el ictus isquémico con Orientación flujometría láser Doppler en la rata

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

La angiogénesis en el pulmón de rata isquémico

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Un modelo del cochinillo de Neonatología encefalopatía hipóxico-isquémica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Demostración de la rata isquémica de la piel herida Modelo

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

El modelo de roedor de la anterior no arterítica neuropatía óptica isquémica (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

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 ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

Modelo de retinopatía inducida por oxígeno para enfermedades retinianas isquémicas en roedores

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...