Este trabajo fue apoyado por subvenciones de la und Ernst Berta Grimmke Stiftung y la Deutsche Ophthalmologische Gesellschaft (el perro).

Los procedimientos experimentales de este estudio fueron aprobados por el Animal cuidado Comité de Renania-Palatinado, Alemania. Cuidado de los animales conforme a las directrices institucionales y la Asociación para la investigación en visión y Oftalmología (ARVO) declaración para el uso de animales en oftálmica y la visión. Los animales fueron tratados según la Directiva 2010/63/UE para experimentos con animales. Hombres ratones C57BL/6J (el Jackson Laboratory, Bar Harbour, ME, Estados Unidos) 3-4 meses de edad se utilizaron para los experimentos. Los animales fueron alojados bajo condiciones estándar (temperatura de 23 ± 2 ° C, humedad rango 55 ± 10% y 12 h de ciclos de luz/oscuridad) y tenían acceso a ratón estándar chow y agua ad libitum.
1. aislamiento de Retina de ratón
<ol><li>Colocar los instrumentos de disección sobre la mesa de preparación y comprobar si todos los instrumentos están completos para permitir una rápida preparación.</li>
	<li>El ratón de sacrificio por inhalación de CO2 .</li>
	<li>Decapitar el ratón con tijeras de acero, corte el cráneo sagitally en dos mitades con el mismo par de tijeras y quitar la piel y el cerebro usando las tijeras de ojo (figura 1).</li>
	<li>Cortar el hueso del cráneo para dejar solo el hueso orbital usando tijeras de ojo.</li>
	<li>La órbita de transferencia en una bandeja de disección con tampón Krebs-Henseleit helada.</li>
	<li>Mantener el plato de la disección en el hielo o cambiar el tampón cada 10 minutos.</li>
	<li>Cortar el hueso orbital usando tijeras de ojo y retire suavemente el globo ocular junto con el tejido orbital usando tipo precisión 4 pinzas y tijeras de Vannas estudiante primavera (figura 2).</li>
	<li>Retire suavemente la glándula de Harder, tejido conectivo y los músculos extraoculares con tipo precisión 5 pinzas y tijera Vannas, teniendo cuidado de no para dañar la rama principal de la arteria oftálmica.</li>
	<li>Ligar las ramas orbitarias de la arteria oftálmica y finalmente el extremo proximal de la rama principal de la arteria oftálmica con suturas de Nylon 10-0 (figura 3).</li>
	<li>Transferencia del globo del ojo en una placa Petri que contiene etanol al 70% para 10 s uso tipo 4 pinzas de precision. Coloque el globo del ojo en el plato de disección y cambiar el búfer con solución fresca de Krebs. La córnea aparece blanquecina después de la exposición de etanol.</li>
	<li>Punción de la córnea periférica con una aguja de 30 calibre y diseccionar la córnea utilizando tijera Vannas. Suavemente cortar el tejido del iris, coloque un punto agudo de la tijera Vannas entre la coroides y la retina y corte la coroides y la esclera. Dejar algún tejido escleral alrededor del nervio óptico (figura 4).</li>
</ol>2. montaje de la Retina en la cámara de perfusión
Nota: La cámara de perfusión utilizada para experimentos de arteriola retiniana es hecho en casa. Consiste en un depósito transparente con un tubo interior y la salida (figura 5). Uno de los extremos de un tubo de silicona se pega a la parte inferior de la cámara con histoacryl adhesiva y el otro extremo conectado a una llave de tres vías. Conecte una jeringa que contiene tampón Krebs fresca de la llave de tres vías y llene el tubo con tampón.
<ol><li>Tome una micropipeta de vidrio con un sostenedor de la aguja y empuje en el extremo del tubo en la parte inferior de la cámara de perfusión. Entonces rompe la punta de la micropipeta con tijeras tipo 4 para obtener una propina de 100 μm de diámetro.</li>
	<li>En la punta de la micropipeta y lavarlo a través de la jeringa conectada al tubo. Tenga cuidado de que el capilar no está obstruido por escombros. En caso de obstrucción, quitar, lavar la tubería e inserte otra micropipeta.</li>
	<li>Llene la cámara de perfusión con tampón Krebs frío y poner la cámara en un microscopio estéreo.</li>
	<li>Coloque una plataforma de plástico transparente en la cámara de perfusión. Esta plataforma tiene una sangría de 1,8 mm de ancho para el nervio óptico y la arteria oftálmica y se coloca en dos alambres de acero de 1,6 mm de diámetro. Luego, coloque un anillo de acero inoxidable de 2,8 mm de diámetro interior y 4,0 mm de diámetro exterior en la plataforma.</li>
	<li>Transferir la retina con el nervio óptico y arteria oftálmica adjuntada de la sala de disección en la cámara de perfusión con cuidado con una cuchara. Este paso es importante para evitar el potencial de estiramiento de los tejidos ya que puede ser perjudicial para el experimento.</li>
	<li>Cortar la parte suturada del extremo proximal de la arteria oftálmica, coloque dos lazos preformados de sutura de nylon 10-0 en la micropipeta y cannulate la arteria oftálmica con la micropipeta. Atar la arteria para la micropipeta (figura 6) y colocar la retina en la plataforma transparente usando pinzas de punto fino.</li>
	<li>Suavemente Abra el capsula anterior lente con dos pinzas de punta fina, tire de la lente y corte el capsula entera de la lente usando microscissors.</li>
	<li>Posteriormente, hacer cuatro incisiones radiales en la retina de mitad de la distancia de la plana de las igualdades al nervio óptico y coloque un anillo de acero inoxidable en la retina para fijarlo a la parte inferior. La retina está lista para el experimento (figura 7).</li>
</ol>3. preparación de las arteriolas retinianas para el experimento
<ol><li>Coloque la cámara de perfusión bajo un microscopio de luz. El objetivo es una lente de objetivo 100 X inmersión en agua.</li>
	<li>Conecte las mangueras de dos para en - y hacia fuera - flujo a una bomba pericíclicas y circular de la cámara con tampón Krebs oxigenada con 95% O2 y gas con 5% CO2 a 37 ° C. Utilice una tasa de flujo entre 100 y 120 mL/min.</li>
	<li>Conecte el tubo de silicona, que está conectado con la micropipeta, a un depósito de silicona manguera mediante una llave de tres vías. Luego, llenar el depósito con tampón Krebs a una altura correspondiente a 50 mm Hg pero no ha abierto la llave de tres vías.</li>
	<li>Mire a través de los objetivos del microscopio y foco en la superficie retiniana. Búsqueda de los vasos sanguíneos o células de sangre rojas. Es posible que los vasos sanguíneos se derrumbado totalmente, a veces solamente que algunas células de sangre rojas son visibles.</li>
	<li>Cuando se encuentra un vaso sanguíneo, se centran en él y abrir la llave de tres vías. El diámetro del vaso debe aumentar inmediatamente y los glóbulos rojos ya sea centrifugada, si se trata de una arteriola, o centripetally en caso de una vénula. Las arteriolas son más pequeñas en diámetro en comparación con vénulas del mismo orden.</li>
	<li>Una vez que encuentra un vaso sanguíneo con una pared bien visible, puede ser empleado para los experimentos después de un período de equilibrio de 30 min (figura 8). Durante el período de equilibrio, las arteriolas desarrollan solamente débil tono intrínseco lo que resulta en menos del 10% de reducción del diámetro luminal.</li>
</ol>4. realizar el experimento
<ol><li>Probar la viabilidad de los vasos con el tromboxano mimética 9,11-dideoxy-9α, 11α-methanoepoxy prostaglandina F2α (U46619) añadiendo a la solución del baño. Este agente constricts arteriolas retinianas de ratón de aproximadamente el 50% del diámetro de reclinación a concentraciones ≥ 10-6 M.</li>
	<li>Una vez el barco es viable lavar al agonista de la bañera por la bomba de circulación y fuente buffer Krebs fresca en el baño.</li>
</ol>

<ol>
	<li>Toda, N., Nakanishi-Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+ocular+blood+flow,+glaucoma,+and+diabetic+retinopathy.">Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy.</a> Prog Retin Eye Res. 26 (3), 205-238 (2007).</li><li>Schuster, A. K., Fischer, J. E., Vossmerbaeumer, C., Vossmerbaeumer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography-based+retinal+vessel+analysis+for+the+evaluation+of+hypertensive+vasculopathy.">Optical coherence tomography-based retinal vessel analysis for the evaluation of hypertensive vasculopathy.</a> Acta Ophthalmol. 93 (2), e148-e153 (2015).</li><li>Cherecheanu, A. P., Garhofer, G., Schmidl, D., Werkmeister, R., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+perfusion+pressure+and+ocular+blood+flow+in+glaucoma.">Ocular perfusion pressure and ocular blood flow in glaucoma.</a> Curr Opin Pharmacol. 13 (1), 36-42 (2013).</li><li>Faraci, F. M., Sigmund, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+biology+in+genetically+altered+mice+:+smaller+vessels,+bigger+insight.">Vascular biology in genetically altered mice : smaller vessels, bigger insight.</a> Circ Res. 85 (12), 1214-1225 (1999).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Retinal+Blood+Flow+Using+Fluorescently+Labeled+Red+Blood+Cells.">Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells.</a> eNeuro. 2 (2), (2015).</li><li>Guevara-Torres, A., Joseph, A., Schallek, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label+free+measurement+of+retinal+blood+cell+flux,+velocity,+hematocrit+and+capillary+width+in+the+living+mouse+eye.">Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.</a> Biomed Opt Express. 7 (10), 4229-4249 (2016).</li><li>Schallek, J., Geng, Y., Nguyen, H., Williams, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+topography+of+retinal+pericytes+in+the+living+mouse+retina+using+in+vivo+adaptive+optics+imaging+and+ex+vivo+characterization.">Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.</a> Invest Ophthalmol Vis Sci. 54 (13), 8237-8250 (2013).</li><li>Gericke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+muscarinic+acetylcholine+receptor+subtype+mediating+cholinergic+vasodilation+in+murine+retinal+arterioles.">Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles.</a> Invest Ophthalmol Vis Sci. 52 (10), 7479-7484 (2011).</li><li>Gericke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+alpha1-adrenoceptor+subtypes+in+murine+ophthalmic+arteries.">Functional role of alpha1-adrenoceptor subtypes in murine ophthalmic arteries.</a> Invest Ophthalmol Vis Sci. 52 (7), 4795-4799 (2011).</li><li>Bohmer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alpha(1)B+-adrenoceptor+subtype+mediates+adrenergic+vasoconstriction+in+mouse+retinal+arterioles+with+damaged+endothelium.">The alpha(1)B -adrenoceptor subtype mediates adrenergic vasoconstriction in mouse retinal arterioles with damaged endothelium.</a> Br J Pharmacol. 171 (16), 3858-3867 (2014).</li></ol>

Los autores tienen intereses financieros que compiten.

La medición de las respuestas vasculares en la retina del ratón es difícil debido al pequeño tamaño de los vasos sanguíneos retinianos. Con la técnica presentada, arteriolas retinianas se visualizan por microscopia de luz transmitida. Esto es posible, porque la retina aislada es translúcida. La ventaja de la técnica es la alta resolución óptica. La resolución espacial calculada es 11 px/μm. Sin embargo, la resolución real de este sistema óptico que utiliza la luz blanca es entre 200 y 300 nm, que se explic...

Alteraciones en la perfusión retiniana han sido implicados en la patogenia de varias enfermedades oculares, como retinopatía diabética, retinopatía hipertensiva y el glaucoma1,2,3. Así, estudios para medir la reactividad vascular en la retina son importantes para entender la fisiopatología de estas enfermedades y desarrollar el nuevo tratamiento se acerca.
Debido a la posibilidad de manipulación de genes en el genoma del ratón, el ratón se ha convertido en un modelo animal ampliamente utilizado para los estudios del sistema cardiovascular4. Sin embargo, debido al pequeño tamaño de los vasos sanguíneos retinianos (≤ 30 μm), medición de la reactividad vascular en la retina del ratón es difícil. Por ejemplo, técnicas estereoscópica para la medición en vivo están limitadas en su resolución óptica y por lo tanto, sólo permiten para detectar exactamente cambios en diámetro o sangre flujo en sangre pequeñas de menos de ≤ 30 μm de diámetro cuando está equipado con sofisticado dispositivos adicionales, tales como un microscopio confocal usando colorantes fluorescentes o la adaptación óptica escaneo luz oftalmoscopio5,6. Por otra parte, la interpretación de en vivo medidas encaminadas a identificar locales señalización mecanismos en los vasos sanguíneos retinianos pueden confundirse por anestésicos, cambios en la presión arterial sistémica y la influencia de los vasos sanguíneos retrobulbares.
Por lo tanto, hemos desarrollado un método para medir las respuestas de los vasos sanguíneos retinianos ratón con óptica de alta resolución ex vivo. La técnica aquí presentada permite la visualización de arteriolas retinianas mediante transmisión microscopia ligera. Este método, que también puede ser utilizado en ratas, proporciona acceso a las ventajas del gene targeting tecnología en investigación vascular ocular.

<table>
	<tbody>
		<tr>
			<td>Steel Scissors</td>
			<td>Carl Roth GmbH</td>
			<td>3576.1</td>
			<td>1x 140 mm</td>
		</tr>
		<tr>
			<td>Eye Scissors</td>
			<td>Geuder</td>
			<td>G-19390</td>
			<td>1x straight, 10.5 cm</td>
		</tr>
		<tr>
			<td>Precision tweezers, straight with fine tips</td>
			<td>Carl Roth GmbH</td>
			<td>LH68.1</td>
			<td>2x type 4</td>
		</tr>
		<tr>
			<td>Precision tweezers, straight with extra fine tips</td>
			<td>Carl Roth GmbH</td>
			<td>LH53.1</td>
			<td>2x type 5</td>
		</tr>
		<tr>
			<td>Vannas capsulotomy scissors</td>
			<td>Geuder</td>
			<td>19760</td>
			<td>1x straight, 77 mm</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>91501-09</td>
			<td>1x curved,</td>
		</tr>
		<tr>
			<td>Barraquer Needle Holder</td>
			<td>Geuder</td>
			<td>G-17500</td>
			<td>1x curved, 120 mm</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton, Dickinson and Company</td>
			<td>305128</td>
			<td>1x 30 G</td>
		</tr>
		<tr>
			<td>Glass Capillaries (for producing micropipettes)</td>
			<td>Drummond Scientific Company</td>
			<td>9-000-1211</td>
			<td>1x (1.2 x 0.8 mm; outer/inner diameter)</td>
		</tr>
		<tr>
			<td>Nylon Suture</td>
			<td>Alcon</td>
			<td>198001</td>
			<td>1x 10-0</td>
		</tr>
		<tr>
			<td>Nunclon cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>153066</td>
			<td>1x 35 mm diameter</td>
		</tr>
		<tr>
			<td>Nunclon cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>172931</td>
			<td>1x 100 mm diameter</td>
		</tr>
		<tr>
			<td>Discofix C</td>
			<td>Braun</td>
			<td>16500C</td>
			<td>10 cm</td>
		</tr>
		<tr>
			<td>Histoacryl adhesive</td>
			<td>B. Braun Surgical, S.A.</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pericyclic pump&#160; (CYCLO II)</td>
			<td>Carl Roth GmbH</td>
			<td>EP76.1</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Vertical Pipette Puller Model 700C</td>
			<td>David Kopf Instruments</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Microscope (Vanox-T AH-2)</td>
			<td>Olympus</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Water immersion objective LUMPlanFL, 1.0 NA</td>
			<td>Olympus</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Digital camera (TK-C1381)</td>
			<td>JVC</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Perfusion chamber</td>
			<td>self-made</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Drugs and Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth GmbH</td>
			<td>K928.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2)</td>
			<td>Carl Roth GmbH</td>
			<td>5239.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalium chloride (KCl)</td>
			<td>Carl Roth GmbH</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalium dihydrogen phosphate (KH2PO4)</td>
			<td>Carl Roth GmbH</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulphate (MgSO4)</td>
			<td>Carl Roth GmbH</td>
			<td>261.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Carl Roth GmbH</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Carl Roth GmbH</td>
			<td>0965.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-(D)-(+)- Glucose monohydrate</td>
			<td>Carl Roth GmbH</td>
			<td>6780.1</td>
			<td></td>
		</tr>
		<tr>
			<td>9,11-dideoxy-9&#945;,11&#945;-methanoepoxy prostaglandin F2&#945; (U-46619)</td>
			<td>Cayman Chemical</td>
			<td>16450</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A6625-25G</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo

Insuficiencia vascular y alteraciones en la perfusión retiniana normal están entre los principales factores para la patogenesia de varios avistar-que amenaza enfermedades oculares, como retinopatía diabética, retinopatía hipertensiva y, posiblemente, glaucoma. Por lo tanto, las preparaciones microvasculares retinianas son herramientas fundamentales para estudios fisiológicos y farmacológicos delinear los mecanismos fisiopatológicos subyacentes y diseño de terapias para las enfermedades. A pesar del amplio uso de modelos murinos en investigación oftálmica, escasean los estudios sobre la reactividad vascular retiniana en esta especie. Una de las principales razones para esta discrepancia es los procedimientos de aislamiento difícil debido al pequeño tamaño de estos vasos sanguíneos retinianos, que es ~ ≤ 30 μm de diámetro luminal. Para evitar el problema de aislamiento directo de estos microvasos retinianas para estudios funcionales, se estableció una técnica de aislamiento y preparación que permite estudios ex vivo de la vasoactividad de retina de ratón bajo condiciones fisiológicas cerca . Aunque la preparación experimental actual se refiere específicamente a las arteriolas retinianas de ratón, esta metodología puede ser fácilmente empleada para microvasos de ratas.

U-46619 produjo vasoconstrictor dependiente de la concentración de respuestas en arteriolas retinianas de tipo salvaje ratones C57Bl/6J del fondo de la. En una concentración de 10-6 M, reducción del diámetro luminal fue ≈50% de diámetro de descanso. Figura 9A muestra una curva de respuesta de concentración representativa de una arteriola retiniana. En las arteriolas preconstricted con U46619, administración acumulativa de acetilcolina evoc...

Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Muchas enfermedades oculares visión-que amenaza se asocian a microvasos retiniana disfuncional. Por lo tanto, la medición de las respuestas de la arteriola retiniana es importante investigar los mecanismos fisiopatológicos subyacentes. Este artículo describe un protocolo detallado para ratón arteriola retiniana aislamiento y preparación para evaluar los efectos de sustancias vasoactivas en el diámetro vascular.

Pasos de preparación para la medición de la reactividad en ratón arteriolas retinianas Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

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Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

7.1K vistas.  University Medical Center of the Johannes Gutenberg University Mainz. Muchas enfermedades oculares visión-que amenaza se asocian a microvasos retiniana disfuncional. Por lo tanto, la medición de las respuestas de la arteriola retiniana es importante investigar los mecanismos fisiopatológicos subyacentes. Este artículo describe un protocolo detallado para ratón arteriola retiniana aislamiento y preparación para evaluar los efectos de sustancias vasoactivas en el diámetro vascular.

Video: Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex infección in vivo del tejido vivo con virus oncolíticos

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Investigación

Medicina

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Medición in vivo de la capa de ratón pulmonar superficie endotelial

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo MRI de 19F para el seguimiento de la célula

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

En Vivo Dinámica de Retina Microglial Activación Durante Neurodegeneración: Confocal oftalmoscópico Imaging y Morfometría celular en el ratón Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Medición ecocardiográfica de parámetros diastólicos ventriculares derecho en ratón

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Un modelo de cultivo de tejidos Ex Vivo para las complicaciones Fibrovascular en la retinopatía diabética proliferativa

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Preparación de gránulos Gushukang (GSK) para experimentos in Vivo e In Vitro

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Pasos de preparación para la medición de la reactividad en ratón arteriolas retinianas Ex Vivo

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Capítulos en este video

Ex infección in vivo del tejido vivo con virus oncolíticos

Medición in vivo de la capa de ratón pulmonar superficie endotelial

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

In vivo MRI ^{de 19F} para el seguimiento de la célula

Una

En Vivo Dinámica de Retina Microglial Activación Durante Neurodegeneración: Confocal oftalmoscópico Imaging y Morfometría celular en el ratón Glaucoma

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

Medición ecocardiográfica de parámetros diastólicos ventriculares derecho en ratón

Un modelo de cultivo de tejidos Ex Vivo para las complicaciones Fibrovascular en la retinopatía diabética proliferativa

Preparación de gránulos Gushukang (GSK) para experimentos in Vivo e In Vitro